1

Chapter 1

Natural Products

1.1 Introduction

The term ‘natural product’ means molecules of life1

but in general it refers to

distinct low molecular weight organic compounds produced by living organisms such as

bacteria, fungi, lichens, marine invertebrates, plants, insects, mammals etc2 which are

recognized for their pharmacological and biological activities. Natural products are also

known as secondary metabolites as they are not crucial for basic life processes like

growth and reproduction but assist the host organisms in their survival, facilitate

interaction and communication, help in adaption to varied environments3 and they may

have evolved to provide defence mechanism against pathogens and predators.4 They

possess diverse and complex chemical structures which are distinctive of the species or

producing organism and are formed at specific stage of the morphological development

of the host organism,5 for example during sporulation or pigment production. Secondary

metabolites are synthesized from simple intermediate products from primary

metabolism, the main precursors are acetyl Coenzyme A, shikimic acid, mevalonic

acids and amino acids.6 Their ability to bind biological targets and stimulate bioactive

effects has attracted the attention of pharmaceutical industries, natural-product chemists

and biologists to explore different natural habitats, terrestrial, aquatic and the microbial

world for the presence of natural products and for many years they have been a wealthy

source of potential drugs.7

Since primitive times many different plant species have been documented to be

used as medicines for human ailments and later many natural product compounds

purified from plants prove to be among initial lead drugs. For example, acetyl salicyclic

acid 1 (aspirin) is based upon the natural product salicin 2, isolated form the bark of the

willow tree Salix alba L.7, 8

The pain killer codeine was synthesized from the alkaloid

morphine7 3 obtained from the plant Papaver somniferum L.; digitoxin 11 from

Digitalis purpurea L. is used as a cardiotonic to ease congestive heart failure,8

and the

antimalarial drug quinine 4 has been used for years to treat fever, malaria and mouth and

throat diseases. There are a number of plant derived anti-tumor drugs, for example

paclitaxel 9 and baccatin 10 from Taxus brevifolia9 and numerous other anti-tumor

2

compounds are in clinical trials. Various plants’ secondary metabolites display

inhibitory activity against microbes for example phenols, quinone 6, coumarin 5,

catechin 7, terpenoids and essential oils possess antimicrobial effects, flavones 8 inhibit

multiple viruses including HIV and catechin 7 is also used as an anti-coagulant.10

The discovery of the antibiotics penicillin11

161 (see section 3.3) and

cephalosporin12

16 from the antibacterial fungi Penicillium notatum and

Cephalosporium acremonium respectively, led to the exploration of various

microorganisms in search of assorted bioactive metabolites. Fungi from the phyla

Basidiomycota and Ascomycota delivered many pharmacological active compounds,

75% were antimicrobial and many others verified to possess antiviral, cytotoxic,

antineoplastic, cardiovascular, anti-inflammatory, antitumor and immune-stimulating

metabolites.13

The bacterial group of Actinomycetes is also rich with novel metabolites,

mainly antibacterial, antiviral, antifungal and antitumor activities.14

The antibiotics

vancomycin 15 from Amycolatopsis orientalis7,15

and erythromycin 13 from

Saccharopolyspora erythraea are active against a wide range of bacteria and are used in

treatment of various infections.7 Doxorubicin 14 isolated from the Streptomyces

peucetius is used in the treatment leukaemia, bone sarcomas and lung and thyroid

cancer.7,15

A number of immune-suppressive agents such as cyclosporin 17 from the

3

fungus Tolyplocladium inflatum and fujimycin 12 from the bacterium Streptomyces

tsukubaensis have enhanced the field of organ transplants.2 Marine natural products

also provide a rich source of biodiversity with about 15 bioactive compounds approved

from Food and Drug Administration (FDA) and many in clinical trials.14,16

Hence, a

number of surveys conducted on drug sources for the treatment of diseases like cancer

and infections revealed that 60% of approved drugs are of natural origin.17

These

include natural products directly used as drugs as well as semi-synthetic drugs derived

from or modelled on natural products.18,19

There are major revolutions in natural product research and drug design. Large

libraries of natural product analogues and structural mimics are synthesized through

combinatorial chemistry18

and are examined by ‘High Throughput Screening

Technology’ against wide range of important biological targets.19

With present-day

genomics, cost-effective DNA sequencing of microorganisms helps in annotation of

secondary metabolites gene clusters in their respective genomes. This has motivated

researchers to express the silent secondary metabolite gene clusters whose chemical

4

products are unidentified by applying different genetic engineering techniques (see

section 3.3). Biosynthetic pathways of a large number of novel metabolites have been

elucidated from their particular biosynthetic gene clusters by different molecular

biology techniques,5 such as gene knock out, RNAi silencing, cloning, homologous

recombination and heterologous gene expression in surrogate host. These studies have

broadened the knowledge of biosynthetic genes and their encoded enzymes and

proteins. These discoveries pave the way for proteins to be used as biocatalysts20

for in

vivo synthesis of complex natural products and resolve increasing demand for new drug

products.

The main classes of natural products are terpenoids, alkaloids, nonribosomal

peptides and polyketides. They are briefly introduced in the following sections.

1.2 Terpenoids

Terpenoids are made from a five carbon unit (isoprene), which exists in two

active structural forms: dimethylallyl diphosphate 18; and isopentenyl diphosphate 19.21

These simple precursors make diverse structures and about 25,000 structures22

of

terpenes are reported. The name terpenes was named after the volatile oil of pine tree,

turpentine which is composed, among others, of the terpene compound α-pinene 20.1

Terpenoids are identified by their strong aroma, chiefly in essential oils. They have a

wide range of functions: they protect plants against attack of pests and some are used as

insecticides;22

they serve as a means of communication among organisms; they function

as attractants for insects for pollination; and many play a part as mating pheromones

and reproductive hormones.21

Many terpenes have a number of bioactivities, for

example terpenes from the genus Rubia are reported to possess antitumor, anti-

inflammatory, antimicrobial, anti-malarial and antidiabetic effects.23

Examples of

terpenoids include camphor 21, limonene 22 and gibberelllin GA4 23.

5

1.3 Alkaloids

Alkaloids, like terpenoids, are a large and diverse class of compounds, with

more than 12,000 examples known at present.24

They contain a basic amine group in

their structure and are derived biosynthetically from amino acids. Examples are

morphine 3 and the antimalarial drug quinine 4 (see section 1.1).

1.4 Nonribosomal peptides

Microorganisms, particularly fungi and bacteria, produce an assorted group of

peptide secondary metabolites called nonribosomal peptides (NRP) formed by

multifunctional enzymes known as nonribosomal peptide synthetases (NRPS),

independent of ribosomal pathway.25

NRP are formed from a wide range of substrates

(amino acids) which include both D and L proteiogenic and non-proteiogenic amino

acids which explains the numerous complex structures present in this class of natural

products.26

The NRP serve as antibiotics, immunosuppressants, cytostatins and

siderophores.25

Nonribosomal peptide synthetases (NRPS) are made up of set of

modules, each module consists of basic set of catalytic or enzymatic units called

domains. Modules are distinct sections in nonribosomal peptides and each module

incorporates single amino acids to the growing peptide chain.28

There are three core

domains in each module; the adenylation domain (A), the peptidyl carrier protein (PCP)

and the condensation domain (C).29

The last module normally contains a termination

domain which may be a thiolesterase (TE) or a reductase (R). The adenylation domain

selects and activates an amino acid by adenyaltion with ATP, the PCP serves as a

transporter of the activated substrate between catalytic domains by binding the substrate

to a 4’phosphopantetheine cofactor with a thioester bond. The condensation (C) domain

is responsible for peptide bond formation between the amino group of one amino acid

6

and the acyl group of amino acid of the adjacent module. At the end of last module, the

peptide chain is usually terminated and released by the thiolesterase domain. Many

NRPS may contain additional tailoring domains mainly the epimerization domain, N-

methylation and glycosylation which may further modify the structure of the NRP.30

Examples of NRP include vancomycin 15 and cyclosporin 17 (see section 1.1).

1.5 Fatty acid biosynthesis

Before describing the next class of natural products, the polyketides, it is

necessary to first discuss the biosynthesis of fatty acids. The biosynthesis of fatty acids

has many similar features to polyketides’ pathway and it has always remained a model

system to study the latter.

Fatty acids are primary metabolites present in all living cells. Structurally they

are carboxylic acids with a long saturated chain. They are important sources of fuel for

an organism and when metabolized they liberate large quantities of ATP. They are

essential components of phospholipids and play a basic structural role in assembly of

the cell membrane.31

Fatty acids are synthesized from simple building blocks

particularly the two carbon containing acetate in the form of acetyl Coenzyme A (CoA)

and malonyl CoA. The former acts as a starter unit and the latter as an extender unit.32

Fatty acid synthesis involves a number of key enzymes in each step. In a general

fatty acid pathway an acetyl group from the starter acetyl CoA 24 is transferred by an

acyl transferase enzyme (AT) to the thiol group of the 4’-phosphopantetheine arm of

acyl carrier protein (ACP). This is followed by the acetyl being transferred to another

enzyme β-ketoacyl synthase (KS) at the thiol of the active site cysteine. Meanwhile the

extender malonyl CoA 25 is attached to the ACP and condensation between acetyl

thiolester 27 and malonyl thiolester 26 is catalysed by the KS in a decarboxylative

Claisen condensation with liberation of CO2 and a β-keto thiol ester 28 bound to an

ACP is formed.33

ACP serves as a ‘handle’ to carry the growing acyl chain throughout

the whole process of fatty acid synthesis (Scheme 1.1).

The β-keto thiol ester 28 is then reduced to a secondary alcohol 30 by a β-

ketoacyl reductase (KR) followed by dehydration by a dehydratase enzyme (DH) to

from an unsaturated thiolester 31 and finally an enoyl reductase (ER) performs a final

reduction to form fully saturated thiolester 32.34

The fully saturated thiolester 32 can

7

enter another cycle of fatty acid chain extension with addition of a second malonyl

thiolester by ACP and this process continues until the fatty acid reaches its specific

length, for example a sixteen carbon chain in case of palmitic acid and an eighteen

carbon chain for stearic acid. The last cycle of the fatty acid is terminated by a

thiolesterase enzyme (TE) which liberates the free fatty acid 33 by a hydrolytic release

and a long chain carboxylic acid is formed. Thus fatty acid synthases are large

multifunctional proteins containing KS, ACP, AT, KR, DH, ER and TE, activities.

Scheme 1.1 Series of steps in fatty acid and polyketide biosynthesis.

1.6 Polyketides

Polyketides form a large group of secondary metabolites comprising of

structurally complex compounds, produced by plants, bacteria, fungi, lichens and

marine organisms.35

They have been extensively studied by natural product researchers

because of their fascinating biosynthetic pathways and wide range of important

biological and pharmaceutical properties. Polyketides have contributed about 50

approved drugs in the pharma industry.36

Some important bioactive compounds of this

8

class include the antibiotics erythromycin 13, rifamycin 38,37

monensin 42,38

doxycycline 43, 39

the antihelminthics avermectin 39,40

the antitumor compounds

geldanamycin 4041

and macbecin 41,42

some antifungal polyenes amphotericin 4543

and

primaricin 44, the immunosuppressant tacrolimus (FK506) 46,44

the cholesterol

lowering agent lovastatin 4745

and many others. Some polyketides are also used as

insecticides like spinosad 49,46

while some are toxins, for example aflatoxins 48.47

The basic concept of polyketides biosynthetic origin was presented in 1907 by

John Norman Collie who was a professor at University College London and also a

mountaineer.48

He proposed that ‘Keten’ groups (CH2=C=O) in the form of aldehydes

and ketones can condense and polymerise by simple reactions with liberation of CO2 to

form a range of complex compounds, many of them are present in plants. Hence they

were named polyketenes and subsequently ‘polyketides’.49

9

During the 1940s, when labelled acetate incorporation was established in fatty

acids,50

Robinson supported the polyketide theory presented by Collie.51

Arthur J. Birch

worked on the biosynthetic origin of many aromatic polyketides. He showed

experimentally the incorporation of radio labelled 14

C-acetic acids in alternating

labelling pattern in 6-methyl salicylic acid 51 in Penicillium ariseofuluum (Scheme 1.2).

He endorsed that β-keto chain are formed from condensation of acetate units which

folds to form aromatic polyketides.49

Scheme 1.2: Biosynthetic steps of 6-MSA 51 showing incorporation of 14

C acetate units.

Similar studies also demonstrated that methyl groups in fungal polyketides are

incorporated from L-methionine.52

When techniques like Mass spectrometry (MS) and

Nuclear Magnetic Resonance (NMR) were introduced, the biosynthetic investigations

on polyketides were accelerated. Many precursor compounds, mainly acetates were

used in the form of singly or doubly labelled 13

C or 2H isotopes and fed to the cultures

of polyketides producing organisms. As 13

C or 2H enriched compounds are easily

studied by NMR, they helped to understand the acetate incorporation patterns and mode

of cyclization in many polyketides.53,54

10

Polyketides are formed by a similar biosynthetic pathway to that of fatty acids.

They are formed by repetitive decarboxylative condensation of simple acetate units in

the form of acetyl CoA 24 and malonyl CoA 25 (in some cases propionate and butyrate

units) in a head to tail manner. In polyketide pathway, the first β-keto thiolester 28 is

formed by a decarboxylative Claisen condensation of acetyl 27 and malonyl thioloester

26 by the same catalytic units KS, AT and ACP (Scheme 1.1). In fungi, the polyketide

chain can also be methylated, receiving a methyl group from S-adenosyl methionine

(SAM) to form α-methyl-β-keto thiolester 29 by a unique CMeT domain not active in

the FAS pathway. The β keto chain can be further processed by the KR, DH and ER

domains.34

The cycle again continues until PKS chain reaches it specific length and the

thiolester is hydrolysed from the peptide and released by a termination domain.

In fatty acid biosynthesis, the β-keto chain is uniformly reduced from a β-keto

group to an alcohol 30, then forms an α, β- double bond in 31 by elimination of water

and at the end is fully reduced to a methylene as observed in 32 (scheme 1.1). In the

polyketide pathway the reduction process is more controlled, selective and variable.

There may not be any reduction in the poly β-keto chain of PKS chain (Scheme 1.1, 35)

and is common in aromatic polyketides. The β-keto chain may only be reduced once to

remain as a hydroxyl group, or dehydrated to remain as a enoyl group (scheme 1.1, 36),

or may be fully reduced to methylene groups to form 34 (Scheme 1.1).33

The polyketide

chain may also hold a pendant methyl or ethyl group by incorporation of methyl

malonyl or ethyl malonyl units as observed in 37 (Scheme 1.1). Polyketide biosynthesis

also sets different stereocentres during reduction of the β-keto chain (Scheme 1.1, 36

and 37). Other factors which determine the structure of the polyketides are choice of the

starter units, number of chain extensions and pattern of cyclization.31,55

The choice of all

these structural functionalities are governed by the above described enzymes (or

domains) and these are collectively called Polyketide synthases (PKS). David Hopwood

used the term ‘programming’34

for the way PKS directs different variables or structural

features in the synthesis of the diverse polyketides structures.56

These directions are

ultimately encoded with in the sequences of PKS gene clusters.

Lots of efforts have been served by researchers to understand the core of

programming of polyketide synthases and have achieved a considerable success.

Advanced genetics and molecular biology tools like sequencing, cloning, gene

expression, and enzymes purification have helped to understand the nature and structure

11

of PKS and how they catalyse different reactions or steps in biosynthesis. Many

biosynthetic pathways of polyketide metabolites and their respective gene clusters are

known.5 Hopwood and co-workers have achieved some pioneering work in PKS

genetics and construction of cloning vectors.56

His group discovered the first PKS

genes for the antibiotic actinorhordin.53

Different degenerate primers were developed

from the initial PKS known genes and were used to screen genome libraries of many

bacteria and fungi and pave way to discovery of numerous new PKS genes on the basis

of similarities in gene sequences. After allocation of gene clusters many PKS enzymes

and proteins were purified and a number of crystal structures were solved, which also

gave awareness of different catalytic sites in enzymes. The functions of many enzymes

are proved with cell free extracts and in vitro studies. The quest for understanding the

Polyketide synthases and their programming behaviour is still in progress and many

gene clusters and organisms need to be explored for their exclusive biosynthetic and

bioactive properties.57

1.7 Types of Polyketide Synthases

PKS enzymes are divided into three main groups according to the protein

assembly and arrangement of domains with in the polypeptide. The three main classes

are type I, type II and type III PKS.53

1.7.1 Type I PKS

In type I PKS, all the multifunctional domains required for polyketide chain

elongation and β-keto group processing are located on a single large polypeptide. Type I

PKS is further categorized into three types. They are modular PKS, trans-AT modular

PKS and iterative PKS.58

1.7.1(a) Modular PKS

In modular PKS, the different catalytic units are arranged in the form of sets of

domains, called modules. The domains present in each module performs a single chain

extension and β-keto processing and then passed it on to the next module for another

carbon chain addition and β-keto group processing. Each domain is used once during

the cycle and the linear order of the modules and their respective domains can define the

structure of the polyketide chain. Modular PKS are common in bacteria.59

12

The best studied example of modular PKS is erythromycin A 13,60

the gene

cluster of which was studied in late 1980s.61

It is produced by the Gram positive

bacteria Saccharopolyspora erythraea. Erythromycin A 13 biosynthesis consists of an

intermediate compound 6-deoxyerythronolide B 52 (6-DEB), which is a 14-membered

macrolactone ring and a putative polyketide synthase product.53,62

The polyketide

synthase responsible for the biosynthesis of 6-DEB 52 was named as

deoxyerythronolide B synthase (DEBS) encoded by three genes eryAI, eryAII and

eryAIII, each 10 kb in length (scheme 1.3).59

Scheme 1.3 Biosynthesis of erythromycin by modular Type I PKS.

The erythromycin PKS (EryPKS) was among the first PKS to be sequenced

among the complex polyketides and formed a model for studying modular PKS. The

three genes encode three proteins DEBS1, DEBS2 and DEBS 3. Each DEBS protein

holds two modules and each module contains the three basic domains KS-AT-ACP with

13

different combinations of KR, DH and ER depending upon the extent of β-group

processing. The last module ends with the thiolesterase (TE) domain. In the first cycle

the AT domain of the first module in DEBS1 binds a propionyl CoA and transfers it to

the pantotheine arm of the adjacent ACP and then to the next KS domain. The AT then

binds the extender unit, a methyl malonyl CoA to the ACP. This results in formation of

a diketide by the combination of the starter and the extender unit by the KS, followed by

keto-reduction by KR to form a β-hydroxy. The ACP in module 1 then transfers the

diketide to the module 2 where it is joined by a second extender unit, followed by keto

reduction by KR in module 2. The triketide is passed to the module 3 with condensation

with a third extender unit. The β-keto group in module 3 remains unreduced because of

a non-functional KR in module 3 as apparent in C-9 in 52, in the fourth cycle the β-keto

group undergoes subsequent reduction, dehydration and enoyl reduction to form a

methylene functionality at C-7 because of presence of KR, DH and ER in module 4.

Similar keto group condensation and reduction continues in cycle 5 and 6 depending on

the domains present in the respective modules. In the last, a 15 carbon PKS chain is

released from the ACP of DEBS 3 by the action of the last TE domain in module 6 and

forms a macrolide 52 structure by the combination of C-1 to carboxylate of C-13

hydroxyl. The post PKS steps includes attachment of a 6-deoxy sugars D-desosamine at

position 5, a L-cladinose at position 3 and P450 mediated hydroxylations at C-6 and C-

12 to from erythromycin A 13.59

Trans-AT PKS

There are a number of modular type I PKS gene clusters reported, which lack

the regular cis-AT, a domain found in association with ACP and KS and other domains

in a standard module.63

In trans-AT modular PKS, the prescribed function of acyl

transferase is accomplished by stand-alone AT domain(s) which act in trans with all the

other modules and serves the same function of supplying acyl building blocks to all the

respective modules in the PKS. There are a number of polyketide metabolites

biosynthesized by a trans-AT modular PKS pathway for example leinamycin 55, 64

pseudomonic acid A 54 65

and lankacidin C 53. 65

Scheme 1.4 shows the gene cluster of

leinamycin (LNM) produced by Streptomyces atroolivaceus S-140. 64

The gene cluster

consists of six PKS, one NRPS module encoded by the genes lnmI and lnmJ, among

these all the six PKS modules lack the key AT domain. The AT function is provided by

14

LnmG which loads the malonyl CoA units to all the PKS modules to biosynthesize the

compound leinamycin 55 (scheme 1.4).

Scheme 1.4 Biosynthesis of leinamycin 55 by trans-modular PKS.

1.7.1(b) Iterative Type IPKS

Type I iterative PKSs (IPKS) are made up of a single set of multifunctional

domains found in a large polypeptide. The sole set of domains carry out all cycles of

carbon chain extension and the respective β-keto chain processing, and many domains

are used repeatedly, hence the name iterative. IPKS have long been the attention of

biosynthetic investigations because the single set of enzymes can act differently in every

cycle of chain extension and processing, portraying a high level of complex

programming. IPKS are found most commonly in fungi. IPKSs are divided into three

classes according to the extent of β-keto group reduction and on the presence of KR,

DH and ER domains in the protein architecture. They are non-reducing (NR-PKS),

15

partially reducing (PR-PKS) and highly reducing (HR-PKS). This classification was

first presented by Simpson.34

Non-reducing IPKS

As the name indicates, non-reduced polyketides are formed from the

condensation of intact and non-reduced poly β-keto chain which cyclise forming

aromatic compounds with a mono or multiple ring structures. Some common non-

reduced polyketides are orsellinic acid 56, emodin 57 and norsolonic acid 58.57

Orsellinic acid 56, a tetraketide, was among the early discovered fungal PKS from

Penicillium madriti in 1968.34,66

A typical NR-PKS is composed of an N-terminal

loading component, a chain extension component and a C-terminal processing

component.

The loading component is made of a starter unit-acyl transferase (SAT). It can

load acyl CoA and in many cases complex FAS or PKS elements as starter units. For

example norsolorinic acid 58 incorporates a hexanoate unit,34

dehydrocurvularin 59

accepts a HR-tetraketide as starter unit,67

zearalenone 60 uses a HR-hexaketide as a

starter unit.68

The chain extension component consists of KS, followed by AT. The AT

is specifically a malonyl loading domain. Following the AT, is the product template

domain (PT). It is believed to be involved in chain length control34

and in some reports

it also takes part in PKS chain cyclization.57

PT is followed by ACP. Some NR-PKS

may end with ACP but many possess a distinct C-terminal processing component. The

C-terminal components may end in a Claisen cylcase-thiolesterase (CLC/TE) or may

further consist of methyl transferase (CMeT), 69

additional ACPs or thiolester reductase

(R) domains. The CLC/TE is involved in chain length decision, chain release and

cyclisation via intramolecular Claisen condensation.57,34

An example of NR-PKS with

an active CMeT domain is 3-methylorcinaldehyde 61 synthase where a methyl group is

provided by SAM (S-adenosyl methionine) (Scheme 1.5).69

The PKS structure of 3-

methyl orcinaldehyde synthase (MOS) consists of N-terminal (NT) domain responsible

for selecting the starter unit, followed by KS, AT, PT, an ACP, C-MeT domain and a C-

terminus NADPH dependent thiolester reductase (R) domain.

16

Fig 1.1 Examples of non-reduced polyketides, the complex starter units are highlighted in green in norsolorinic acid 58,

dehydrocurvularin 59 and zearalenone 60.

Scheme 1.5 Non-reduced PKS gene cluster of 3-methylorcinaldehyde 61.

Partially reducing IPKS

The protein architecture of PR-PKS consists of KS, AT, DH, a unique core

domain, followed by KR domain and at the end an ACP domain.34

PR-PKS compounds

are formed by successive condensation of acetyl starter and malonyl extender units,

forming poly β-keto chain, while β-keto processing does not necessarily occur in every

cycle; therefore they are termed as partially reduced PKS. The core domain is believed

to maintain integration and functional stability between the domains. A well-studied

PR-PKS is 6-methylsalicylic acid (6-MSA) 51. 70

The 6-MSA was originally obtained

from Penicillium patulum biosynthesized by 6-methylsalicylic synthase (MSAS).

During biosynthesis of 51, one acetate 24 and 3 malonyl extender units 25 condense in

subsequent extensions to form a tetraketide 62. The KR domain functions after the

second extension in the presence of NADPH forming an alcohol group. After the third

17

cycle, the PKS chain undergoes cyclisation and dehydration to form 6-MSA 51

(Scheme 1.6).53

Scheme 1.6 Biosynthetic pathway of 6-MSA 51.

Highly Reducing IPKS

A typical HR PKS consists of KS, AT, DH followed by a C-MeT domain in

most cases. The next domain is the ER, but many HR-PKS may not contain a functional

ER. The ER is followed by a KR and the PKS most normally terminates with an ACP

domain. With the presence of all three β-keto processing domains used iteratively, HR

PKS synthesize structures with high level of complexity and advanced programming.34

For example lovastatin 47, is a HR polyketide produced by Aspergillus terreus.

It is biosynthesized by two PKS proteins, LovB and LovF.71,105

lovB encodes lovastatin

nonaketide synthase (LNKS) and lovF encodes lovastatin diketide synthase (LDKS).

LNKS with the assistance of LovC, a trans acting ER (the ER domain in LNKS is non-

functional) synthesize a nonaketide PKS intermediate compound Monacolin J 63.

LDKS then produces a methylated diketide 64, which is loaded on to Monacolin J 63 at

C-10 hydroxy by a specialized acyltransferase encoded by a gene lovD to form 47

(Scheme 1.7). Squalestatin S172

65 is also made by two PKS chains, a main hexaketide

and a tetraketide sidechain. Other examples of HR PKS include the longest polyketides

Fumonisin B1 68 produced by Gibberella fujikuroi 73

and T-toxin 74

69 produced by

18

maize pathogen Cochliobolus heterostrophus. Solanopyrone A 66 and alternaric acid 67

produced by the plant pathogen Alternaria solani are also produced by HR PKS. 34

Scheme 1.7 Lovastatin biosynthetic pathway.

Hybrid IPKS-NRPS

Hybrid highly reduced polyketide synthases fused with nonribosomal peptide

synthetase (HRPKS-NRPS) are an important class of synthetases often found in fungi.

The protein architecture consists of the HRPKS domains (KS, AT, DH, MT, inactive

ER, KR, ACP) and nonribosomal peptide catalytic units (C, A, T and terminal R

domain) forming a megasynthase.75

PKS-NRPS have been extensively studied in recent

years because of their intriguing iterative programming code and important biological

19

activities demonstrated by the hybrid compounds.57,34

All PKS-NRPS discovered up till

now possess non-functional ER sequence and if ER activity is required, it is provided by

a distinct trans-acting ER encoding gene homologous to LovC. This property makes

them closely related to the lovastatin 47 gene cluster and presents a common origin.76

The HRPKS synthesizes a polyketide chain from an acetyl starter unit and subsequent

malonyl extender units with methyl groups delivered from SAM by a methyltransferase.

The adenylation domain of the NRPS selects and activates an amino acid and transfers

it to the thiolation domain. The condensation domain binds the amino acid and the

polyketide chain by an amide bond. The hybrid polyketide and peptide chain is released

from the megasynthase by the terminal R domain by either of two release

mechanisms.75

It can either be released in the form of an aldehyde forming pyrrolinone

70 by a Knoevenagel condensation (Scheme 1.8, A) reported in pseurotin A 78,

isoflavipucine 79 and chaetoglobosin A 80 biosynthesis or as a tetramic acid

(pyrrolidone 71) by direct Dieckmann cyclisation (Scheme 1.8, B) detected during

tenellin 87, desmethylbassianin (DMB) 88, equisetin 81 and cyclopiazonic acid 85

biosynthesis.75,57

The hybrid polyketide-peptide compound is further modified by

tailoring enzymes encoded by genes clustered near the megasynthase.

A

B

Scheme 1.8 A, Release mechanism by a Knoevenagel condensation by reductase domain; B, release mechanism by

Dieckmann cyclisation.

The first PKS-NRPS gene cluster was identified for fusarin C 77 from Fusarium

moniliforme and Fusarium venenatum in an attempt to search for C-methyltransferase

domains.77

The fusA gene encodes the synthesis of a tetramethylated heptaketide 72

fused by an amide bond to L-homoserine 73 by the C domain to form a covalently

20

bound intermediate 74 and is probably released by an R domain to form the aldehyde

75.78

Further Knoevenagel condensation forms the putative prefusarin 76. Subsequent

modifying steps of carboxylation, epoxidation and hydroxylation forms Fusarin C 77

(Scheme 1.9).75

Other examples of hybrid PKS-NRPS includes aspyridone A 84,

xyrrolin 86, militarinone C 82 and pramanicin 83.75

Scheme 1.9 Fusarin C biosynthesis.

Figure 1.2 Examples of hybrid PKS-NRPS compounds, the NRPS part is highlighted in red.

21

1.7.2 Type II PKS

In contrast to type I PKS, the enzymatic activities for the β- keto chain

elongation and processing in type II PKSs are present in separate polypeptides, and each

domain is used iteratively.53,58

A well-studied model of type II PKS is actinorhodin 89

biosynthesis (Scheme 1.10).

Scheme 1.10 Actinorhordin pathway showing Type II PKS system.

1.7.3 Type III PKS

Type III PKSs (Scheme 1.11) were originally identified in plants but recently

have also been isolated from several bacteria. In contrast to type I and type II polyketide

biosynthesis, the β-keto chain is elongated and processed at a single multifunctional

active site in type III PKSs.53

It does not require an acyl carrier protein and distinctively

accepts acyl coenzyme A building units, for example in chalcone 90 biosynthetic

pathway.

Scheme 1.11 Chalcone synthase biosynthesis.

22

1.8 AIMS

The main objective of our research is to contribute in understanding the

programming code veiled in the iterative protein structure of the hybrid PKS-NRPS

pathways. We aimed to study the PKS-NRPS systems of three compounds; tenellin 87,

desmethylbassianin 88 and aspyridone A 84.

Tenellin 87 and desmethylbassianin 88 are hybrid polyketide-peptide

metabolites produced by two different strains of enthomopathogenic fungi B. bassiana.

The tenellin 87 pathway has been elaborately studied in the Bristol Polyketide Group

with different genetic and chemical analysis and it helped understand the basic

biosynthetic pathway of tenellin 87. We aimed to continue the biosynthetic studies by

carrying out further heterologous expression of tenellin 87 genes or its components in a

heterologous host. These include expression of PKS-NRPS encoding gene in A. oryzae

without the tailoring genes and expression of tenellin polyketide synthase alone in A.

oryzae without its NRPS counterpart. The objective was to determine the product as

well as the intricate role of each component of the megasynthase in tenellin 87

biosynthesis.

Desmethyl bassianin 88 (DMB) and tenellin 87 gene clusters have 90%

sequence identity but DMB 88 differs in structure from tenellin 87 in having an

additional carbon chain extension and a methyl group less than tenellin 87. We next

intended to study the co-expression of tenellin 87 genes together with DMB 88 tailoring

genes in different combinations in A. oryzae. This was designed to see whether the two

similar yet different biosynthetic genes are compatible to work together in one

expression system, to obtain new engineered natural products and determine the

programming role of the particular genes.

The function of trans-acting enoyl reductase enzyme in tenellin 87 is encoded

by a discrete gene, tenC. The RNAi silencing of tenC has been successfully achieved

before in the native fungus using a strong constitutive promoter.79

We planned to

perform the RNAi silencing of tenC, again, this time using an inducible promoter and

grow the silenced transformants in different growth conditions. We desired to

investigate whether we can control the level of gene silencing and obtain new

compounds reflecting varying degree of silencing.

23

The last objective was to study aspyridone 84 biosynthesis. Aspyridone A 84, a

PKS-NRPS compound is produced from a silent gene cluster in Aspergillus nidulans

and its pathway has been proposed but not proved experimentally.80

We aimed to

investigate aspyridone pathway using an effective heterologous expression system in A.

oryzae and analyse the transformants. The objective was to determine the order of

different biosynthetic steps, role of each gene in the pathway and discover potential of

the megasynthase in synthesizing new bioactive natural products. The detail description

of the aims has been separately described in each chapter with the respective analytical

methods.

24

Chapter 2

Elucidation of new compounds from different genetic studies in

Beauveria bassiana

2.1 Introduction

Beauveria bassiana belongs to a group of entomopathogenic fungi. These fungi

are parasitic to insects and kill or disable them completely. They invade the insects

initially by their microscopic spores called conidia.81

These spores attach to the insect

cuticle and the conidia swells by secretion of lytic enzymes which helps it to breach and

penetrate the outer layer of cuticle. This is followed by development of morphological

structures such as appressorium on the cuticle, infection pegs and penetrant hyphae in

the epicuticle and procuticle helping the conidia hyphae to reach the body cavity of the

insect (hoemocoel). The fungal hyphae continue to proliferate causing damage to the

host tissue and nutrient exhaustion eventually leaves the insect body dead or

destroyed.82

Figure 2.1: Insect infected by fungus Beauveria bassiana. 83

Beauveria bassiana (Balsamo) Vuillemin has a long taxonomic history.84,85

Agostino Bassi (1835) first described this fungus as the causal agent of ‘mark disease’

also known as white muscardine disease in France.86

This disease caused destruction of

silkworm larvae in Southern Europe during the 18th

and 19th

centuries resulting in huge

economic losses to the silk industry. Bassi discovered that microbes can act as

contagious pathogens of animals and this formed the basic fundamentals of the ‘germ

theory of disease’.87

The first taxonomic recognition of the muscardino fungus was

proposed by Balsamo-Crivelli. He recognized Bassi’s discovery by naming this fungus

25

Botrytis bassiana. Beauverie stated that the fungus should belong to an undescribed

genus and Vuillmen established in 1912 the genus as Beauveria in his honour and

Botrytis bassiana Bals.Criv as the type species.86,88

Beauveria is distributed worldwide. They are soil borne hygomycetes (having

naked spores)89

and are pathogenic towards several different orders of insects including

Lepidoptera, Coleoptera, Hemiptera, Hymenoptera and Orthoptera.85

They are easy to

culture. There are no toxic metabolites from beauveria reported to enter the food chain

or accumulating in the environment.90

These features make B. bassiana a model system

for studying entomogenetics and effective biological control of pests.

B. bassiana produces several secondary metabolites of varied structures but the

contribution of these metabolites to pathogenesis is mostly unknown.91

Some

metabolites reported from B. bassiana include bassianin92

91, beauvericin93

93,

bassianolide91

92, beauverolide A94

94, oosporein95

95 and tenellin96

87.

Among these, bassianolide 92 has been identified in dead silkworm larvae

infected by B. bassiana.91

Beauvericin 93 is known to have mycotoxic properties.93

A

high molecular weight protein toxin Bassiacridin is stated to be isolated from B.

bassiana strain obtained from a locust.97

The Bristol polyketide group have identified

that tenellin 87 is not involved in pathogenicity.98

The present chapter involves work on

tenellin 87 and its biosynthesis.

26

Tenellin 87 is a prominent secondary metabolite of B. bassiana and its structure

was elucidated by Wat and co-workers, together with another similar compound

bassianin 91.96

Both compounds are known by their distinctive yellow colour apparent

during fermentation and in organic extracts. Tenellin 87 possesses a 5- substitiuted 2

pyridone ring with an acylated moiety at C-3.99

Tenellin 87 has been the focus of many biosynthetic studies over a period of

many years. The most distinctive reason has been that it is formed from a combination

of an amino acid and a polyketide chain.99

The key work to determine the precursors of tenellin 87 was reported by

McInnes and co-workers.100

They used [1, 2-13

C2]-acetate 96, L-[methyl-13

C]

methionine 97, (±)-[1-13

C] phenylalanine and (±)-[2-13

C] phenylalanine 98. The results

analysed by 13

C-NMR indicated that C-2, C-3 and C-7 to C-14 of tenellin 87 were

alternately enriched with doubly labelled acetate 96 and both methyl groups at C-15 and

C-16 showed enhanced peaks for labelled 13

C-methionine 97. The carboxy carbon C-1

of phenylalanine 98 forms C-4 of tenellin 87 and C-2 of phenyl alanine 98 becomes C-6

of tenellin 87. There is an intramolecular rearrangement of phenylalanine which causes

migration of the carboxy carbon adjacent to the aromatic ring forming C-4 of 87 and the

alpha carbon of phenylalanine separates to form C-6 of tenellin 87 (Scheme 2.1). They

confirmed that tenellin 87 is formed by condensation of methylated polyketide chain

having five acetates with an entity comprising all carbons from phenylalanine.100

Scheme 2.1: Incorporation of labelled acetate, methionine and phenylalanine in tenellin 87.

27

Another similar study by Leete and coworkers101

supported the intramolecular

rearrangement of phenylalanine in tenellin 87. They fed phenylalanine labelled at two

carbon positions [1, 3-13

C2]. They indicated in 13

C-NMR spectrum additional satellite

peaks adjacent to singlet peaks of C-4 and C-5 due to spin spin coupling confirming

intramolecular rearrangement of the phenylalanine side chain in tenellin 87. They

argued that had there been ‘intermolecular’ movement of carboxyl group, the 13

C-NMR

would only show singlets peaks for both C-5 and C-4.

Further isotope feeding on tenellin 87 was carried out by Wright et al.102

They

also used singly and doubly labelled sodium acetate, labelled methionine and three

different labelled forms DL-[carboxy-13

C], DL-[α-13

C] and L-[15

N] phenylalanine. Their

results supported previous feeding studies by McInnes.100

Incorporation of N-labelled

phenylalanine established that it is fused to the polyketide chain with no loss of

nitrogen. In addition they also showed severely reduced incorporation of radioactive L-

[U-14

C] tyrosine suggesting that tyrosine is not a direct precursor of tenellin 87.

Wright and coworkers proposed a route for tetramic acid formation. First,

condensation of phenylalanine 99 with the ten carbon polyketide chain 100 and then

hydroxylation of the aromatic ring by an oxygenase enzyme give quinomethine 101.

This would then undergo rearrangement of the tetramic acid to form the pyridone of

tenellin 87 (Scheme 2.2).

28

Scheme 2.2: Proposed tenellin 87 biosynthesis by Wright et al.102

Another biosynthetic proposal suggested by Cox and O’Hagan was that

phenylalanine 99 rearranges early and then condenses with a polyketide 100 to give the

six membered pyridone directly (Scheme 2.3).103

They synthesized and fed DL-[3-13

C]

and [3-14

C]-3-amino-2-phenylpropionic acids 102 to the cultures of B. bassiana.

However, the 13

C NMR analysis of this experiment did not show that 3-amino-2-

phenylpropionic acid 102 is a genuine intermediate in tyrosine 87 biosynthesis.

Scheme 2.3: Proposed biosynthesis by Cox and O’Hagan.103

29

On the basis of the Wright et al.102

hypothesis, Moore et al.99

synthesized acyl

tetramic acid in two isotopically labelled forms [4-13

C] in 103 and [phenyl-2H5] in 104

and carried out feeding experiments with B. bassiana fermentations. They found that

these compounds were not incorporated into tenellin 87 and there was no proof for its

presence in B. bassiana extracts. They observed a single minor metabolite 105, the

purified sample of metabolite on 1H NMR showed it to be para-substituted aromatic

moiety without N-hydroxylation. They concluded that para-hydroxylated acyl tetramic

acid emerged as a late intermediate and a precursor or a reduced metabolite in tenellin

biosynthetic pathway (Scheme 2.4). They also carried out isotopic feeding of tyrosine

DL-[3-13

C] and [1-13

C] phenylalanine and indicated that both tyrosine and phenylalanine

are efficiently incorporated into tenellin 87.

By this result, in contrast to the hypothesis of Wright et al.,102

Moore and co-

workers proposed that phenolic hydroxylation in tenellin 87 is introduced by tyrosine

and not at a later stage modification of the aryl ring.

Scheme 2.4: Labelled acyl tetramic acid feeding in tenellin 87 by Moore et al.99

In recent years with advancement in molecular biology and discovery of new

tools to manipulate DNA, investigations of biosynthetic routes of natural compounds

from their specific gene clusters have been very remarkable. Lately the Bristol

Polyketide Group have carried out quite a number of successful genetic studies on

polyketides genes of tenellin 87 from B. bassiana 110.25. They discovered specific

30

genes responsible for different stages and intermediate steps in tenellin biosynthesis. All

these stages reflect cryptic programming of PKS genes.

Eley et al. identified a hybrid PKS-NRPS gene cluster from the genomic DNA

of B. bassiana and showed by gene knock out experiments that this cluster is involved

in tenellin 87 biosynthesis.98

Analysis of this cluster revealed four open reading frames

(ORF) (Figure 2.2). BLAST analysis revealed that ORF1 and 2 were homologous to

cytochrome P450 enzymes. ORF3, a putative Zn dependent oxidoreductase,104

showed

high homology to enoyl-reductase (ER) enzymes. ORF4 consists of an approximately

12-kb biosynthetic gene that encodes β-ketoacyl synthase (KS), acyl transferase (AT),

dehydratase (DH), CMeT (methyltransferase), β-ketoacyl-reductase (KR) and acyl

carrier protein (ACP) domains typical of a fungal iterative type I PKS, followed by

condensation (C), adenylation (A), thiolation (T) and putative thiol (R ester reduction)

domains, characteristic of an NRPS module. A directed gene knockout (KO) experiment

confirmed ORF4 to be involved in tenellin production, and ORF4 was therefore

renamed tenS (tenellin synthetase). In this work they also proved that B. bassiana tenS

KO and WT strains are equally pathogenic towards insect larvae suggesting that tenellin

87 is not involved in insect pathogenesis of B. bassiana.98

Figure 2.2: Tenellin gene cluster identified by Eley et al.98

They proposed a pathway for the early stages of tenellin 87 biosynthesis from its

respective PKS and NRPS enzymes (Scheme 2.5). The double methylated pentaketide

106 bound to ACP is synthesised by TENS PKS and the A domain of TENS NRPS first

activates tyrosine 107 by adenylation and transfer to the thiol group of the T domain.

The polyketide and the amino acid are fused by the C domain to form N-β-ketoacyl

amino thiolester 108. The reduction domain R carries out reduction of the thiol ester

108 with the help of NADPH and release in the form of a peptide aldehyde 109. The

aldehyde can cyclise to form pre-tenellin 110, which was assumed to be a precursor of

tenellin 87 (Scheme 2.5).

31

Scheme 2.5: Tenellin 87 pathway proposed by Eley et al.98

Halo et al. expressed the tenS gene encoding tenellin synthetase (TENS), in

Aspergillus oryzae M-2-3.104

It led to the production of three new compounds, identified

as acyl tetramic acids, prototenellin A 111, prototenellin B 112 and prototenellin C 113.

Protenellin C 113 was not fully characterized due to the low concentration of the

purified compound (Table 2.1). These compounds didn’t have the methylation pattern

of tenellin 87 and the polyketide chain length in prototenellin B 112 was shorter than

tenellin 87. In addition there were double bonds between C-11 and C-12 of prototenellin

A 111 and between C-9 and C-10 of prototenellin B 112 whereas this bond is always

saturated in 87. This shows that the enoyl reductase domain present within the TENS

protein is defective and fails to carry out reduction in the first cycle of the polyketide

chain formation. These results depict that enzymes in tenS gene when expressed on its

own lose the ‘fidelity’ in the programming of the polyketide side chain of the

compound.

Halo et al. also carried out another important experiment which was co-

expression of tenS with the gene encoding the enoyl reductase enzyme (ORF3) which

led to the production of single acyl tetramic acid compound, pretenellin A 114 (Table

2.1). This compound was concluded to be a genuine precursor to 87, possessing the

32

same methylation and chain length. Pretenellin A 114 has a saturated bond in the first

keto chain extension similar to tenellin 87 proving that the enoyl reduction is carried out

by ORF3 and not by ER in the TENS protein. This result showed that in the presence of

the ORF3, the tenellin gene cluster undergoes its normal pattern of bond formation and

methylation of the polyketide unit leading to correctly programmed compound

structure. Similar stand-alone enoyl-reductase encoding genes like lovC and apdC in

PKS gene clusters are reported from lovastatin 47105

and aspyridone A80

84

respectively, where ER domain present in the PKS synthase is inoperative. The ORF3

gene in tenellin synthase is known as tenC.

The first precursor compound pretenellin A 114 produced from coexpression of

TENS and TENC was different than pre-tenellin 110 hypothesized by Eley et al.98

in

being hydroxylated at C-4 (Table 2.1). Halo et al. described that the C-terminal R

domain of the NRPS after releasing thiolester 108, does not undergo a reductive

reaction at this step but must catalyse a Dieckmann cyclisation of the N-β-ketoacyl

amino thiolester 108 directly to form the tetramic acid, pretenellin A 114 (Scheme 2.6).

This concept was further supported by similar result reported by Sims and Schmidt.106

They carried out in vitro experiments, reacting purified proteins from R domains of

equisetin synthetase (EqiS) with synthetic substrate analogues. They obtained equisetin

tetramic acids and did not observe any reduced or aldehyde intermediates thus giving

evidence for Dieckmann cyclisation activity of R domains of EqiS. Tang et al. also

produced in vitro a 3-acyltetramic acid preaspyridone A 224 by incubating Aspergillus

nidulans PKS-NRPS encoding gene apdA with its enoyl reducatse encoding gene apdC

in the absence of any oxidative enzymes, proving that its R domain also actually

catalyses a Dieckmann cyclisation.107

Scheme 2.6: Release and formation of pretenellin A 114 by Dieckmann cyclase domain of TENS.

33

In another study Halo et al. revealed late stage oxidations during the

biosynthesis of the 2-pyridone tenellin 87 in B. bassiana by a combination of gene

knockout, gene silencing by antisense RNA and gene coexpression studies.108

They

concluded that the putative cytochrome P450 oxidase encoded by tenA catalyzes the

oxidative ring expansion required to convert the tetramic acid of pretenellin A 114

(Table 2.2) to the pyridone of tenellin 87. Gene knockout and silencing of tenB

produced pretenellin B 115, which confirmed that tenB catalyzes N- hydroxylation of

115 (Table 2.2) to form 87. The tenB gene encodes a rare kind of cytochrome p450

which N-hydroxylates only 2-pyridones and not tetramic acids. Another experiment

confirmed the N-hydroxylation function of tenB when (tenA + tenB + tenS) were

coexpressed in A. oryzae producing pretenellin B 115.

The above studies on the tenellin gene cluster revealed the highly programmed

nature of the PKS-NRPS proteins. After successful studies on the tenellin 87 gene

cluster, Heneghan et al.109

carried out screening of several other species of Beauveria

bassiana to search for compounds similar to tenellin 87. Their aim was to compare new

gene clusters and their sequences of proteins to that of tenellin 87. After analysing

various B. bassiana species they found and characterized the similar compound

Desmethyl bassianin 88 (DMB) from B. bassiana strain 992.

Desmethylbassianin (DMB) 88 differs from tenellin 87 in having a single

methylation in its side chain and it also has an additional chain extention. Heneghan et

al. identified the DMB gene cluster by Southern Blot by using tenS probe (Figure 2.3).

The biosynthetic gene cluster of DMB 88 had 90% sequence identity to the tenellin 87

gene cluster.109

It has the same four open reading frames dmbA, dmbB, dmbC and dmbS

(which is the PKS-NRPS).

Figure 2.3: DMB gene cluster identified by Heneghan et al.109

34

To evaluate and confirm that dmbS is responsible for DMB 88 production,109

they carried out a double silencing and knockout strategy to disrupt the dmbS gene and

the results showed total loss of production of any DMB 88 or related compound. Similar

results to tenellin 87 were obtained when dmbS was expressed in A. oryzae (M-2-3)

producing protoDMB-B 116 and protoDMB-C 117 which cannot be regarded as

precursor of DMB 88. When dmbS was co-transformed with dmbC, it gave the preDMB

A 118 (Table 2.3) giving the same methylation and chain length pattern as DMB 88. It

confirmed that in the presence of dmbC, dmbS undergo correct reduction in the

polyketide chain of the DMB compounds, show high conformity in programming and

the yield of compounds is also increased.

In both tenellin 87 and the DMB 88 gene clusters there is more than 90%

similarity between the tenellin and DMB PKS proteins but still both these compounds

were different in their PKS chain length and methylation pattern. After successful

expression studies in both individual gene clusters, it was possible to create hybrid

tenellin and dmb genes expression in Aspergillus oryzae to know which proteins is

responsible for difference in programming in closely related gene clusters.

Heneghan et al. carried out some co-expression experiments. These showed that

DMBC and TENC are interchangeable; the programme of the PKS is influenced by tenS

and dmbS. This led to the idea of ‘fidelity’- the extent to which the PKS makes

‘mistakes’. When dmbS and dmbC were co-transformed in A. oryzae, it gave preDMB

A 118, while cotransformation of dmbS with tenC again gave preDMB A 118 (Table

2.4). This indicated that tenC and dmbC had identical effects in assisting correct

programmed compounds production. Coexpression of tenS with dmbC gave pretenellin

A 114 which is identical when tenS is expressed with tenC. These results showed that

the PKS controls the programming of polyketide chain in the presence of either of the

trans-acting ER proteins.109

In the absence of the trans acting ER encoded by tenC or dmbC the PKS

displays low fidelity. But when the trans-ER is present the PKS displays high fidelity

and high productivity.

In another experiment Heneghan and coworkers created a hybrid gene consisting

of tenSPKS with dmbNRPS to know the effect of NRPS in programming.109

This swap

produced prototenellin A 111, prototenellin B 112 and prototenellin C 113 (Table 2.4).

This means that the NRPS does not have an effect in PKS programming but it only

35

functions to connect the amino acid to the polyketide chain and plays role as off-loading

mechanism for the PKS.109

This swap produced the same compounds when tenSPKS

was expressed in A. oryzae (M-2-3).104

The productive heterologous gene expression, silencing and gene knockout

studies in DMB 88 and tenellin 87 PKS-NRPS genome resolved the characteristic

function of their respective genes. The oxidative enzymes encoded by tenA, tenB and

dmbA, dmbB carry out important ring expansion and N-hydroxylation steps. The enoyl

reductase enzymes tenC and dmbC were observed to play crucial part in production of

correctly programmed precursor compounds pretenellinA 114 and preDMB 118

(Scheme 2.7). Knocking out the hybrid PKS-NRPS genes dmbS and tenS completely

eliminated the production of tenellin and DMB in their respective fungi.

Scheme 2.7: Individual domains in TENS and DMBS proteins producing precursor compounds.

Recently Fisch and colleagues revealed the catalytic role undergone by

individual domains in DMBS and TENS proteins. Their work also solved the important

queries about the methylation and chain length difference in tenellin 87 and DMB 88

structure.110

They took tenS as a host gene and exchanged its constituent domains with

domains from dmbS one at a time in each experiment and expressed it in A. oryzae with

36

tenC. They started initially by replacing the KS-AT domain of tenS by dmbS and then

the DH in a second experiment until the entire tenS PKS gene was swapped over with

domains from dmbS PKS.

Scheme 2.8: Key metabolites desmethyl pretenellin A 119, preDMB A 118 and prebassianin 120 produced in different

domain swaps between TENS and DMBS proving catalytic role of CMeT and KR domains in programming.

No change in programming was detected by including KS-AT-DH domains

from DMBS and the clones still produced pretenellin A 114. Variations in programming

37

were observed when CMeT and KR domains were included in TENS. (KS-AT-DH-

CMeT) domains from DMBS produced a new compound desmethyl pretenellin A 119

possessing pentaketide side chain length of pretenellin A 114 but a single methylation in

the PKS chain (Scheme 2.8). This indicated that the CMeT from DMBS brings about

single methylation pattern of DMB 88. In another swap by adding KR in the TENS host

together with DMBS (KS-AT-DH-CMeT-ER) the clone produced the hexaketide

predmbA 118, proving that KR is the chain length determining enzyme. Another swap

supporting this result was observed when only KR from DMBS was located in TENS.

This clone produced a compound prebassianin 120 possessing hexaketide chain length

of predmbA 118 but with double methylation in PKS side chain (Scheme 2.8). This

study revealed that KR and CMeT domains exhibit the major part in selecting number

of methylation and chain elongation in iterative HR-PKS NRPS.110

The potential and diversity of secondary metabolites production from B.

bassiana was further examined by treating this fungus with epigenetic modifying

chemicals.111

B. bassiana strain 110.25 fungus was grown in the presence of genetic

modifiers, 5-azacytidine (5AC) 121 and suberoyl bis-hydroxamic acid (SBHA) 122

(Scheme 2.9).

Epigenetic modifiers are small chemicals that bring about modification of gene

activation and expression without modifying its nucleotide sequence.

Histones are the main protein component of chromatin providing a framework

for the DNA around which it winds and forms a structure. In addition chromatin also

exhibits an important role in gene regulation. It affects gene expression by removing

acetyl groups from its N-acetyl lysine amino acid with the help of deacetylase enzymes.

This deacetylation makes the DNA wrap around histones more firmly leading to

compressed chromatin which makes the genes inactive. Some chemicals can act as

histone deacetylase inhibitors such as suberoyl bis-hydroxamic acid (SBHA) 122. These

inhibitors block this action of deacetylase enzymes which increase lysine acetylation

leading to activation of silenced genes.

The expression of genes in cells is also dependent on DNA methylation. This

involves addition of methyl group to cytosine or adenine of DNA nucleotide. This

methylation inactivates the expression of certain genes. 5-azacytidine (5AC) 121 is a

chemical analogue of cytidine which is a nucleoside present in DNA and RNA. 5AC

38

can bring about changes in the cell genome and its behaviour by removing methyl

groups from the DNA thus activating the silenced genes. There a number of studies in

which epigenetic modifiers stimulated silent gene clusters in fungi producing new

compounds and adding variety in the library of natural products.112

Scheme 2.9: New compounds produced by B. bassiana WT after growing with epigenetic chemicals 5AC and SBHA.

Yakasai et al. reported that B. bassiana showed three times higher production of

tenellin related compounds when B. basssian WT cultures were grown in the presence

of 5AC 121 and SBHA 122.111

They also produced new compounds 3’,4’-anti-

pyridomacrolidin-A 123, 3’,4’-syn-prepyridomacrolidin-A 124, 3’,4’-syn-

prepyridomacrolidin-B 125, and reprogrammed compounds prototenellin A 111 and

protenellin E 126 possessing different methylation pattern than pretenellin A 114

(Scheme 2.9).

In the same study two different clones tenA-silenced strain and tenB-silenced

strain were grown in the presence of epigenetic chemicals. The tenA-silenced strain

39

produced new compounds 12-hydroxy pretenellin A 127 and 128 (both syn and anti

diastereomer), 14-hydroxy pretenellin A 129 (Scheme 2.10) and reprogrammed

compound protenellin E 126 but not any pyridone compounds. This showed that

silenced genes of tenA were not switched on by these chemicals.

Scheme 2.10 New compounds produced by tenA aRNA transformant after growing with epigenetic chemicals 5AC and

SBHA.

tenB silenced clones in the presence of 5AC 121 and SBHA 122 produced a

variety of different new compounds (10, 11-Z ) pyridovericin 130, (10, 11-Z )-syn-13-

hydroxy pretenellin B 131, (10, 11-Z )-anti-13-hydroxy pretenellin B 132, prototenellin

F 133, anti-13, 15-dihydroxy pretenellin B 134, syn-13-hydroxy pretenellin B 135 and

anti-13 hydroxy pretenellin B 136. In this, epigenetic modifiers overcome the silencing

factor of tenB producing tenellin 87 which is an N-hydroxylated compound. These

chemicals demonstrated the rich tendency of PKS-NRPS genes for crafting new

compounds (Scheme 2.11).

40

Scheme 2.11: New compounds produced by tenB aRNA strain after growing with epigenetic chemicals 5AC and SBHA.

2.2 Aims and Objective of the Chapter

Tenellin 87 is an example of a secondary metabolite in entomopathogenic

fungus Beauveria bassiana, the biosynthesis of which is composed of highly

programmed steps directed by an organized enzyme complex. This enzyme complex is

encoded by a hybrid gene cluster of iterative polyketide synthases-non ribosomal

peptide synthetases (PKS-NRPS).

The modern tools of biotechnology help to manipulate and study genes in fungi.

These techniques include different gene expression in host fungi and silencing or

knock-out of target genes in native organisms. The results of these techniques are

examined with a number of the latest laboratory instruments particularly Liquid

Chromatography-Mass Spectrometry. New chemical compounds are detected and their

structures are elucidated. Different molecular genetic techniques combined with

chemistry apparatuses help to explore the relation between compounds and their

41

corresponding proteins and genes. These help plot the biosynthetic routes of natural

product compounds.

The various genetic studies on tenellin 87 and DMB 88 PKS-NRPS gene

clusters discussed in section 2.1 encouraged further transformation experiments in A.

oryzae. The objective of this Chapter is to analyse various strains prepared from

heterologous expression of tenellin 87 PKS genes and hybrid co-expression of tenellin-

DMB genes in A. oryzae. The hybrid tenellin and DMB genes co-expression was carried

out in quest to know the difference in programming of both tenellin and DMB gene

clusters. We also intended to analyse tenC-aRNA silenced clones of B. bassiana carried

out using amyB promoters.

All these strains were grown under standard fermentation conditions and their

organic extracts were examined using LC-MS which displays UV diode array and mass

spectrometry data of the compounds in the crude extract. Any interesting compounds

detected in these extracts were purified using preparative HPLC and the isolated pure

compounds were subjected to various structure techniques including IR, NMR, HRMS

and X-ray diffraction.

2.3.0 Results

2.3.1 Heterelogous Expression of tenS in A. oryzae M-2-3

The effective tenS gene knock out experiment in B. bassiana reported by Eley et

al.98

resulted in loss of production of tenellin 87. This generated interest to explore the

function of TENS PKS-NRPS protein. Halo et al. carried out heterologous expression

of tenS in A. oryzae M-2-3 using the pTAex3 expression system.104

Three new

compounds were produced from this experiment which were prototenellin A 111

(C21H23NO4, m/z 353), prototenellin B 112 (C18H19NO4, m/z 313) and prototenellin C

113 (C21H25NO6, m/z 387). Prototenellin C 113 was not fully characterized previously,

due to lack of material and instability during purification.104

The experiment was repeated to characterize prototenellin C 113. Spores of A.

oryzae pTAex3-tenS were first grown on plates (DPY media) for 7-10 days. A spore

solution was made using sterile deionized water and 1 ml of spore solution was added in

42

100 ml liquid culture in 500 ml Erlenmeyer flasks (10 × 100 ml) for 7 days with 200

rpm, at 25 °C. After completion of fermentation the cultures were homogenized and

extracted with ethyl acetate (section 4.11). The ethyl acetate extracts were dried and

evaporated to yield a crude extract of 70 mg, which was dissolved in HPLC methanol

(10 mg/ml). This extract was analyzed by LCMS (Method 1, section 4.4). Analytical

LCMS showed peaks of prototenellin C at Rt 29.4 mins (Figure 2.4). ES+ and ES-

showed masses of 388 and 386 respectively indicating a mass of 387 corresponding to

prototenellin C 113 (Figure 2.5). This peak was purified by mass-directed preparative

LCMS. 26 Injections were made using a 20 minute program (Method 1, section 4.5).

The purified fractions of prototenellin C were collected and dried under nitrogen gas

into pale yellow solid (2.4 mg/L).

Figure 2.4: Diodearray chromatogram of tenS expression in A. oryzae producing prototenellin C.

43

Figure 2.5: ES-, ES+ and UV spectrum of prototenellin C 113.

2.3.1(a) Characterization of Prototenellin C 113

The pure prototenellin C 113 was dissolved in deutrated methanol and 1D and

2D NMR experiments were carried out using a 500 MHz spectrometer. The 1H NMR

showed two prominent sets of doublets (Figure 2.6) which are characteristic of the para-

substituted hydroxy phenol found in tenellin and related compounds. In the 1H-

1H

COSY spectrum, the two diastereotopic protons H-16a and H-16b showed correlation

with H-5 which is a typical pattern of a tetramic acid (Figure 2.7). This was further

confirmed in the HMBC spectrum where the benzylic protons at H-16 showed two and

three bonds correlation to the phenolic protons and also to the H-5 proton of the

tetramic acid ring (Figure 2.7). Three methyl signals corresponding to H-13, H-14 and

H-15 were present in the alkane region. The HMBC correlation of H-15 with C-7 and

quaternary carbon C-6 confirmed methyl H-15 to be attached to C-7 indicating that the

polyketide chain of prototenellin C possess the same methylation pattern as

prototenellin A 111. HMBC signals at H-10 showed the occurrence of two hydroxyl

groups at H-11 and H-12. The signals at H-10 and H-14 are two overlapping doublets

and singlets respectively, providing evidence that the compound is a mixture of two

diastereomers. Collective NMR data confirmed prototenellin C structure to be 113. The

molecular formula and molecular mass was confirmed by HRMS to be C21H25NO6.

44

Fig 2.6: 1H NMR spectrum of prototenellin C 113 in methanol-d4.

Figure 2.7: 1H-

1H COSY (solid lines) and

1H-

13C HMBC correlations (arrows) in prototenellin C 113.

2.3.2 Heterelogous Expression of tenSPKS-dmbNRPS in A. oryzae M-2-3

Prototenellin C 113 was also produced from another strain tenSPKS-dmbNRPS

cloned in A. oryzae (This transformation was done by Katherine Williams in the School

of Biological Sciences). A. oryzae clones were grown in liquid culture (10 × 100 ml)

for 7 days, 200 rpm, at 25 °C. After fermentation the cultures were homogenized and

extracted with ethyl acetate (section 4.11). The ethyl acetate extracts were dried,

evaporated (yield was 137 mg) and dissolved in HPLC methanol (to a final

45

concentration of 10 mg/ml). This extract was analyzed by LCMS. The LCMS analysis

showed peak of prototenellin C. The ES+ and ES- showed obvious peaks of 386 and

388 respectively corresponding to the 387 mass of prototenellin C. This peak was

purified by mass- directed preparative LCMS. The purified fraction of prototenellin C

were combined and dried under nitrogen gas to give a pale solid mass of 9.6 mg.

1H NMR was carried out from this purified fraction on a 500 MHz spectrometer.

The 1H NMR showed the same proton signals as observed from the Prototenellin C 113

obtained from tenS expression clone (Figure 2.8).

Figure 2.8: Overlay of 1H NMR of prototenellin C 113 from A, A. oryzae pTAex3-tenS expression clone; B, tenSPKS-

dmbNRPS clone.

2.3.3 Analysis of A. oryzae dmbS–tenC Expression Clone

Different hybrid combinations of DMB 88 genes and tenellin 87 genes were

expressed in A. oryzae (This transformation was done by Mary N. Heneghan in the

School of Biological Sciences, University of Bristol) in order to investigate their mode

of programming in terms of chain length and methylation. Many different transformants

were produced. This section is an explanation of analysis of the LCMS of different

transformants and isolation of new compounds.

46

An A. oryzae dmbS–tenC transformant was grown in CMP liquid culture (10

flasks × 100 mL) at 25 °C at 200 rpm. The fermentation cultures were homogenized and

extracted as described in section 4.11. A crude extract of 111 mg was obtained and was

made into a solution of 10 mg/ml in HPLC methanol. This extract was analyzed by

analytical LCMS. A prominent peak at Rt 19 mins was observed (Figure 2.9). Mass

analysis indicated ES+ and ES- of 368 and 366 respectively (Figure 2.10). This peak

was purified with 25 preparative runs on LCMS (Method 1, section 4.5). The pure

compound was collected in a number of different tubes. They were all collected and

dried under nitrogen gas in the form of bright yellow powder (23 mg).

Figure 2.9: Diode array chromatogram of A, dmbS-tenC expression in A. oryzae; B, Aspergillus oryzae Wild type.

Figure 2.10: ES+, ES- and UV spectrum of the 19min peak.

47

2.3.3(a) Characterization of preDMB A 118

The high resolution mass spectrum (HRMS) of this fraction gave a molecular

formula of C22H26NO4 (observed 368.1851; calculated 368.1856 for M[H]+). The pure

compound was dissolved in deuterated methanol and 1D and 2D NMR experiments

were done on the purified fraction. The 1H NMR showed two separate doublets in the

aromatic region for H-19/23 and H-20/22 and diasteorotopic protons H-17a and H-17b

characteristic of benzylic protons of a tetramic acid (Figure 2.11). The diastereotopic

protons showed COSY connection with methine proton H-5 and HMBC correlation to

the C-19/23 of the phenol ring which confirms the tetramic acid structure (Figure 2.12).

There were two methyl signals, one for terminal H-15 and second for H-16. The HMBC

and COSY correlations confirmed that the pendant methyl at position 16 is attached to

C-13 (Figure 2.12). Six olefinic protons H-7, H-8, H-9, H-10, H-11 and H-12 in the

alkene region of the spectrum confirmed three double bonds typical for the DMB 88

polyketide chain. In the COSY spectrum the olefinic protons showed couplings of H-7

to H-8, H-9 to H-10 and H-11 to H-12. The alkene proton H-7 displayed a two bond

correlation to the quaternary carbon C-6 in HMBC. All 1D and 2D NMR data

confirmed the structure to be 118 which was named preDMB A.

48

Figure 2.11: Spectrum of 1H NMR of preDMB A 118 run in methanol-d4.

Figure 2.12: 1H-

1H COSY (solid lines) and

1H-

13C HMBC correlations (arrows) in preDMB A 118.

2.3.4 Heterologous expression of dmbS-dmbC in A. oryzae

The clone dmbS + dmbC gene (This transformation was done by Mary N.

Heneghan in the School of Biological sciences, University of Bristol) was analyzed

having both dmbS synthases and dmb enoyl reductase encoding genes cloned in A.

oryzae. It was grown in liquid culture (10 flasks × 100 ml) at 25 °C at 200 rpm. After

completion of fermentation the cultures were homogenized and extracted with ethyl

acetate (section 4.11). The ethyl acetate extracts were dried, evaporated (yield was 363

mg) and dissolved in HPLC methanol (10 mg/ml). The LCMS analysis showed peak of

49

predmbA at Rt 19 mins. The ES+ and ES- showed masses 368 and 366 respectively

which corresponds a mass of 367 of preDMB A 118. The peak was purified by mass-

directed preparative LCMS. This purification was accomplished by 18 injections on 20

minutes program with Method 1 described in section 4.5. The tubes containing purified

fractions were all collected and dried. The dried preDMB A was bright yellow with

yield of 17.6 mg.

The 1H NMR showed the same proton signals as the preDMB A obtained from

dmbS+ tenC clone as illustrated in Figure 2.13.

Figure 2.13: Overlay of 1H NMR of preDMB A 118 from A, dmbS-dmbC clone; B, dmbS-tenC expression in A. oryzae.

2.3.5 Heterologous expression of tenSPKS – dmbC in A. oryzae

In the course of investigating the biosynthetic potential of the tenellin 87

polyketide synthase-nonribosomal synthatase encoding genes, we now know that TENS

in the absence of tailoring enzymes is capable of producing tetramic acids prototenellin

A 111, protenellin B 112 and protenellin C 113 possessing different methylation and

reduction pattern than tenellin 87. When tenC is expressed with tenS, the ‘correct’

precursor pretenellin A 114 is produced. These results encouraged us to further expand

50

the gene expression experiments using the tenellin gene cluster. We now aimed to

express the HRPKS of TENS without its counterpart NRPS in A. oryzae. This

expression also included the enoyl reductase encoding gene dmbC from DMB cluster.

As the NRPS module affords a peptide or an amino acid, in its absence we

expected that the ACP bound pentaketide 106 synthesized by TENSPKS can either get

hydrolysed and released in the form of an open chain pentaketide 137 (Scheme 2.12) or

it can enter another chain extension cycle by addition of a malonyl CoA 25 by the KS

forming hexaketide 138. The hexaketide 138 can offload from the TENS by cyclisation

to form a pyrone 139 (Scheme 2.12).

Scheme 2.12: Possible compounds from expression of tenSPKS+ dmbC in A. oryzae.

Similar pyrone structures are also reported from other polyketide synthase

enzymes from other fungi. Tang et al. investigated the programming role of HRPKS-

NRPS encoding gene apdA involved in aspyridone A 84 biosynthesis in A. nidulans.107

He manipulated the megasynthase ApdA in different in vitro assays and reported

production of a number of α-pyrones. The in vitro assay of ApdA without the enoyl

reductase ApdC produced pentaketide and hexaketide pyrone 142 and 139 respectively

(Scheme 2.13, A). The NRPS module of ApdA is very specific to recognize only fully

reduced tetraketide synthesized from the ApdA PKS. This might be the reason that

unsaturated tetraketide in the absence of ApdC enters further chain extension cycles and

methylations and get offloaded by the ACP in the form of pyrones without being

processed by the NRPS module. Another tetraketide 140 and pentaketide 141 pyrones

51

without any methylations is observed in in vitro assay where methyltransferase domain

is removed from ApdA. The in vitro assay of ApdA-PKS with ApdC independent of the

NRPS module also produced a pyrone 143 with saturated linear chain similar to

aspyridone A 87.

Kennedy et al. reported two pyrones 144 and 145 when they over expressed

lovastatin polyketide synthase LNKS in Aspergillus nidulans without the enoyl

reductase LovC (Scheme 2.13, B).71, 105

A

B

Scheme 2.13: A, α-pyrones reported from different in vitro assays with PKS-NRPS megasynthase ApdA of aspyridone

84 by Tang et al.107

; B, pyrones reported from LNKS, LovB from lovastatin gene cluster.

2.3.6 Analysis of tensPKS–dmbC transformants

A total of 11 transformants were analysed (this transformation was carried out

by Dr. Walid Bakeer). They were grown in the same fermentation conditions for 7 days

and extracted with ethyl acetate (see section 4.11). The crude extracts were all in range

of 5 – 10 mg per 100 ml of liquid media. Each crude extract was made 10 mg/ml in

HPLC methanol and analysed by LCMS. In seven transformants we observed two

prominent peaks at 7.5 and 8.1 minutes (Figure 2.14) in a 15 minute program (Method

52

3, section 4.4). These were not observed in the A. oryzae wild type (M-2-3) strain. The

mass spectra of these two peaks were similar to the open chain polyketide 137, we

expected from this experiment (Scheme 2.12). The peak at 7.5 minutes showed mass of

213 in ES+ and 211 in ES- and was named compound A (Figure 2.15). The peak at 8.1

minutes gave a mass of 211 in ES+ and 209 in ES- and was named compound B (Figure

2.16). Neither compound showed a strong uv absorption.

Figure 2.14: A, LCMS chromatogram of tenSPKS-dmbC producing two new compounds at 7.5 mintues and 8.1

minutes; B, Wild type A. oryzae (M-2-3).

We thought there might be new polyketide compounds and thus planned to

purify these two peaks. These compounds were produced in low titre from each 100 ml

liquid culture. We chose one transformant producing this compound and grew large

scale fermentation of three litres liquid medium. The total crude extract was 239 mg. A

50 mg/ml sample was made in HPLC methanol for purification. About 24 runs of a 20

min program as stated in Method 2 (section 4.5) were carried out. The purified

compound A with mass (MH+) 213 was 9.5 mg and the second compound B with mass

(MH+) 211 was 9 mg.

53

Figure 2.15: A, ES- of compound A; B, ES+ of compound A; C, wavelength.

Figure 2.16: A, ES- of compound B; B, ES+ of compound B; C, wavelength.

2.3.6(a) Identification of Compound A

Compound A had a chemical formula of C11H17O4 (observed 213.1130;

calculated 213.1121 for M[H]+) on High Resolution Mass Spectrometry. The 9.5 mg of

pure compound was dissolved in 0.65 ml of deuterated chloroform. The structure of the

54

compound was elucidated using 1D and 2D 1H and

13C NMR spectroscopic analysis. A

number of 1D and 2D NMR experiments were carried out on 500 MHz NMR

Spectroscopy. The structure of the compound A 146 is given in Figure 2.17.

The carbon spectra revealed the presence of eleven carbons, which included two

carbonyl groups at δC 176.4 (C-11) and δC 170.1 (C-9), four methylene groups in the

alkane region of the spectra and one terminal methyl carbon at δC 14.4 (C-1) (Figure

2.18). The 1H-

13C HSQC gave eight protonated carbons. The

1H NMR showed two

distinct doublets in the alkene region at δH 5.95 and δH 6.54 which were assigned to two

geminal methylene protons of H-10. One of the H-10 methylene protons shows 1H-

13C

HMBC correlation to an alkene carbon at δC 136.1 which was assigned C-8. Both

geminal methylene protons (H-10) show HMBC connections to carbonyl carbon at δC

170.1 (C-9) and sp3 carbon at δC 45.2 (C-7). The triplet at δH 3.65 was assigned to

methine proton (H-7) (Figure 2.17) which show a 1H-

13C HMBC connection to terminal

carbonyl of a carboxylic acid at δC 176.4 (C-11), carbonyl carbon at δC 170.1 (C-9) and

an alkene carbon at δC 136.1 (C-8) (Figure 2.19).

Figure 2.17: 1H NMR of compound A run in chloroform-d.

55

Figure 2.18: 13

C NMR of compound A run in chloroform-d.

The methine proton at δH 3.65 (H-7) show 1H-

1H COSY connections to two

geminal methylene protons at δH 2.01 and 2.55 (H-6). Both geminal methylene protons

of H-6 show 1H-

1H COSY and

1H-

13C HMBC connections to a methine proton at δH

4.43 (C-5) linked to an oxygen atom (Figure 2.19). The chemical shifts of δC 79.6 for C-

5 support its link to an oxygen atom. The HMBC correlations of this compound

provided valid evidence for a pyran structure attached to a carboxylic acid at C-7. The

methine protons at δH 4.43 (H-5) and methylene protons at δH 2.01 and 2.55 (H-6) show

1H-

1H COSY connections to two other geminal protons at δH 1.65 and 1.81 (H-4). Two

multiplets at δH 1.37 and δH 1.46 were assigned to two geminal methylene protons H-3

which display 1H-

1H COSY link to one of the methylene at δH 1.8 (H-4). The broad

signal at δH 1.35-1.39 was assigned to methylene protons H-2 showing connections to δC

27.8 (C-3) in HMBC spectrum and to terminal methyl at δH 0.92 (H-1). Both HMBC

and COSY correlations show an aliphatic chain (C1-C4) attached to the pyran at

methine proton H-5.

56

Figure 2.19: Important 1H-

1H COSY and

1H-

13C HMBC connections in compound A 146.

Figure 2.20: Arrangement of protons in the pyran ring of 146 around the stereo centre at C-7 and C-5.

In the pyran ring of compound 146, the spatial orientation of the protons at the

two stereocentres C-5 and C-7 was established with the help of calculating the coupling

constant of H-5 and H-7 with their adjacent methylene protons at δH 2.01 (H-6a) and

δH 2.55 (H-6b). The coupling constant of 6 Hz between H-5 and H-6b indicated that H-

6b is equatorial. The J value of 10 Hz between H-5 and H-6a was 10 Hz, placing H-6a

in axial position. The coupling constant between the two geminal methylene protons H-

6a (2.00) and H-6b (2.55) was 12 Hz, which confirmed the conformation of H-6a to be

axial and H-6b as equatorial. The methine proton H-7 displayed J value of 12 Hz

between H-6a and 9 Hz with H-6b. These values depicts that H-7 orientation is axial.

The predicted conformation for the pyran of compound 146 from J values is given in

Figure 2.20 showing H-6a, H-7 and H-5 in axial direction and H-6b to be aligned in

equatorial position. Further confirmation of these spatial arrangements of atoms came

from 1D NOE, where both H-7 and H-5 show NOE correlations to each other which can

occur when both protons are in axial position (Figure 2.21 and 2.22).

57

Figure 2.21: In 1D-NOE, on irradiating H-5, signals of H-7 is observed in close proximity.

Figure 2.22: Irradiation of H-7 in 1D NOE, signals of H-5 are observed.

2.3.6(b) Identification of compound B

The structure of compound B 147 was interpreted with the help of HRMS and

NMR analysis and by comparison to compound A 146. The chemical formula given by

HRMS was C11H14O4 (observed 233.0799; calculated 233.0784 for [M]Na+). The 9 mg

of the pure compound was dissolved in 0.65 ml of deuterated chloroform. The ESI gave

58

idea that this compound may have the same structure with two protons less than

compound A 146. It was further confirmed by different NMR experiments.

The 13

C NMR displayed the presence of 11 carbons including two carbonyls at

δC 169.5 (C-9) and 171.7 (C-11), a methine carbon at δC 80.8 (C-5) attached to an

oxygen atom, a methyl group at δC 14.0 (C-1), three methylene groups and four olefinic

carbons at 125.0 (C-7), δC 128.5 (C-8), δC 133.6 (C-10) and δC 153.5 (C-6) (Figure

2.23).

Figure 2.23: 13

C NMR of compound B 147 run in chloroform-d.

The 2D HSQC showed 7 protonated carbons. In the 1H NMR, the broad doublet

signal downfield at δH 7.96 was assigned to the methine (H-6) (Figure 2.24) attached to

the olefinic carbon at δC 153.5 (C-6) in the 1H-

13C HSQC. In

1H-

13C HMBC spectra, H-

6 shows linkage to methine carbon at δC 80.8 (C-5) and to the olefinic carbon (C-7) at

δC 125.0. This confirmed the presence of a double bond between C-6 and C-7, which is

the difference in structure between compound 146 and 147. H-6 also shows HMBC

correlations with the olefin carbons, C-8 and C-11 (Figure 2.25). The two broad signals

at δH 6.79 and δH 7.19 were identified as geminal methylene protons H-10 showing

correlations to the methine protons H-5 and H-6 in 1H-

1H COSY and

1H-

13C HMBC

connections to the olefin carbon at δC 125.0 (C-7) and to the carbonyl group at δC 169.5

(C-9) (Figure 2.25).

59

Figure 2.24:

1H NMR of compound B run in chloroform-d.

The triplet signal at δH 4.99 was assigned to the methine H-5, displaying HMBC

correlations with the methylene groups at δC 27.3 (C-3) and δC 33.1 (C-4) and to olefin

carbons at δC 125.0 (C-7) and δC 153.5 (C-6). The two multiplet signals at δH 1.71 and

δH 1.79 were assigned to geminal methylene protons (H-4) linked to C-2 and C-3 at δC

22.6 δC 27.3 respectively in HMBC. The terminal methyl signal at δH 0.92 (H-1)

showed HMBC connections to the methylene groups at δC 22.6 (C-2) and δC 27.3 (C-3).

The 1H-

13C HMBC and

1H-

1H COSY shows an aliphatic chain comprising of C-1 to

C-4 attached to the methine proton H-5. From NMR analysis, this compound was

elucidated to be a carboxylic acid pyran ring attached to an aliphatic chain and possess a

double bond between C-6/C-7.

Figure 2.25: Important 1H-

1H COSY and

1H-

13C HMBC connections in compound B 147.

60

2.3.6(c) Discussion

The TENS-PKS synthesizes a polyketide chain from condensation of five

acetate units in four cycles, carrying out methylation and reduction in the first cycle and

another methylation in second cycle producing an open chain pentaketide 106. The

structures of compounds 146 and 147 were different than the compounds 137 and 139,

we proposed to be produced from this transformation. We concluded that they are not

products of TENS-PKS pathway.

Both compounds 146 and 147 have not been reported before. We searched for

similar compounds in the literature and came across a number of similar compounds

(Figure 2.26). Horhant et al. isolated three paraconic acids, protolichesterinic acid 148,

lichesterinic acid 149 and roccellaric acid 150 from lichen, Cetraria islandica (L.)

Ach.113

Dahiya and Tewari characterized three plant growth factors from the fungus

Alternaria brassica, one of them was 3-carboxy-2methylene-4-pentenyl-4-butenolide

153. They reported that 153 reduces plant growth by causing chlorosis.114

The same

structure 153 was reported by Park et al.115

They named it methylenolactocin 153 (α-

methylene-γ-lactone) and obtained it from a penecillium sp.24-4 (FERM P-9437). They

stated that methylenolactocin 153 possess antimicrobial activity against Gram positive

bacteria. Huneck and Höfle isolated and characterized δ-lactone structures acaranoic

acid 151 and acarenoic acid 152 from the lichen Acarospora chlorophana.116

Figure 2.26: Similar compounds to A 146 and B 147, reported in literature.

61

Seshime et al. over expressed a type III PKS gene csyB in A. oryzae (M-2-3)

under the effect α-amylase promoter and produced a novel metabolite csypyrone B1 154

which is a 3-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl) proponoic acid (Scheme

2.14).117

In a [1, 2- 13

C2] feeding, csypyrone B1 154 showed incorporation of five

acetate units which confirmed it to be a product of a PKS pathway. They presented a

proposed biosynthetic pathway for csypyrone B1 154 in which a succinyl CoA

condenses with three malonyl CoA and by pyrone ring cyclization form csypyrone B1

154.

Scheme 2.14: Proposed biosynthetic pathway for csypyrone B1 154.

It seemed highly unlikely that compound A 146 and compound B 147 could be

product of the tenellin PKS. We thus re-examined wild type A. oryzae. Although peaks

for 146 and 147 could not be observed by uv, their characteristic masses could be

detected at the correct retention times using a more sensitive 60 minutes programme

with Method 5 described in section 4.4 (Figure 2.27). We proposed a hypothetical

pathway for the biosynthesis of compound 146 and 147 (Scheme 2.15).

Figure 2.27: Section of Single ion monitoring chromatogram of compound A in ES+ and ES- mode observed at 29.1

minutes in A. oryzae tenSPKS-dmbC and Wild type A. oryzae.

62

Scheme 2.15: Proposed biosynthetic pathway for compound A 146 and compound B 147.

2.3.7 tenC RNAi Silencing in B. bassiana with amyB promoter

Messenger RNA is an important type of ribonucleic acid (RNA) and it possesses

an important role in gene expression. During gene expression, the DNA molecule

transcribes the genetic information required for a protein synthesis to a single strand of

messenger RNA. This is carried out by RNA polymerase enzymes in a process called

transcription. The messenger RNA travels outside the nucleus carrying the genetic code,

which is then translated into proteins with the support of ribosomes and transfer RNA

by an organised cell process called translation.

Figure 2.28: Process of gene expression from DNA to proteins.

63

Transcription is a process which can be interrupted by RNA interference

(RNAi). RNAi is an effective tool in eukaryotes often used in recent advances in

molecular genetic techniques.118

It is used to diminish the function of a specific gene by

destroying the messenger RNA translation stage of the target gene; this can reduce, or

completely prevent, protein production. In this technique a single stranded RNA

comprising of reverse sequence or complementary sequence to that of target gene is

introduced into the cell. This single strand binds to the native single strand RNA to form

double stranded RNA. The double stranded RNA is considered as abnormal condition

by the cell and is then degraded by enzymes called dicer (Figure 2.29). The number of

native RNAs expressed into proteins becomes less and hence eliminates the gene

product. This reverse sequence dependant genetic tool is also referred to as ‘gene

silencing’. RNA silencing does not diminish the target gene but it causes reduction of

the function of the gene so it is also named as a ‘gene knock down’ technique.119

This method helps to understand the product and function of the target gene and

aids in the investigation of the biosynthetic pathways of natural products and secondary

metabolites from their gene clusters. A lot of efforts are underway to apply this gene

silencing technique in curing many diseases by destroying function of genes which are

causal factor of viral or tumour diseases in humans.

The concept of RNA interference system came into limelight among researchers

when in a number of cases of introducing homologous RNA for high gene expression

actually diminished the outcome of the native gene unexpectedly.120

In 1990, Napoli

and Jorgensen in their attempt to investigate the enzyme responsible for colouration of

petunia petals, brought forward the hypothesis of RNA silencing for the first time.121

They overexpressed chalcone synthase in the native plant, which is the putative enzyme

for colouration in violet petunias. Instead of obtaining deep violet colour, the petals of

flowers produced were white. This led to the conclusion that the original gene of the

flower was suppressed by the transgene introduced. This suppressing factor was later

discovered to be interfering RNA in the cell which diminishes the expression of the

genes.

64

Figure 2.29: RNA silencing pathway.

In the course of transcription of genes, ‘promoters’ play a vital role in initiating

conversion of the structural gene to proteins. Promoters are regulatory regions

consisting of DNA sequences and are located upstream of the gene they transcribe. The

promoter provides a binding site for RNA polymerase (the enzyme responsible for

generation of mRNA) and for transcriptional factors (proteins that initiate RNA

65

polymerase). The transcription factors are responsible for activation or suppression of

transcription.

Figure 2.30: Promoters are responsible for initiating gene expression.

Similarly in designing gene silencing procedures, promoters are a key feature

which initiates the gene silencing pathway. There are different kinds of promoters used

in gene silencing depending on the goal of an experiment. Most commonly utilized are

constitutive and inducible promoters. Constitutive promoters always direct expression

of genes and are independent of environmental or endogenous factors in cells. The

activity of inducible promoters are dependent on external stimuli such as light,

temperature and different media sources like alcohol, different nutrients including

carbon or many herbicides and antibiotics. The use of inducible promoters makes it

possible to control the level and time of expression of target genes.

Fuji reported that the starch inducible α-amylase promoter (PamyB) is an

effective promoter for heterologous expression systems of Iterative fungal polyketide

synthases in Aspergillus oryzae host.122

Halo et al. successfully expressed hybrid

polyketide synthase – nonribosomal peptide synthatase (PKS-NRPS) encoded by tenS

in A. oryzae using PamyB.104

PamyB is induced (switched on) by starch and repressed

(switched off) by glucose. The activity of genes driven by PamyB can thus be controlled

by the addition of starch or glucose to growth media.

A good example of a strong constitutive promoter is A. nidulans PgpdA

(glyceraldehyde-3-phosphate dehydrogenase promoter). Halo et al. obtained productive

results by using PgpdA to carry out RNA silencing (iRNA) for knocking down the

function of two oxidase enzymes encoded by tenA and tenB in B. bassiana.108

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The enoyl reductase encoding gene tenC is known to be vital for correct

programming of tenellin 87 production.104

Yakasai and colleagues carried out RNAi

silencing of tenC in B. bassiana with the constitutive PgpdA promoter.111

The B. bassiana

tenC RNAi transformant not only produced WT compounds tenellin 87, pretenellin A

114 and prototenellin D 155 but also reprogrammed compounds obtained in tenS

expression in A. oryzae, prototenellin A 111, prototenellin B 112. This silencing

experiment produced a new reprogrammed compound prototenellin E 156. The

presence of WT compounds in the culture show small varied level of TenC proteins

present in the transformant.

Figure 2.31: B. bassiana WT and reprogrammed compounds produced from RNAi tenC transformants by gpdA

promoter.111

The aim of the present project is to examine tenC RNAi transformants produced

under the effect of the inducible promoter amyB in B. bassiana. Here we design an

experiment to know whether the ‘concentration’ of TenC affects programming and new

or reprogrammed compounds are produced or not. The idea was to use an ‘inducible’

promoter ‘amyB’ the expression of which is dependent on the carbon source used in

liquid media. The expression of amyB is stimulated in the presence of maltose or starch

and is repressed by glucose.

67

The construction of the PamyB/tenC silencing vector and its transformation into

B. bassiana was done by Dr. Walid Bakeer. My role was to grow the six selected

silenced clones in different carbon sources and analyse the produced compounds.

The silenced clones (A, B, C, D, E and F) were grown along with wild type B.

bassiana using three carbon sources, mannitol, maltose and glucose.

2.3.7(a) Growth of B. bassiana transformants in TPM (mannitol)

The spores of the six silenced clones (A, B, C, D, E and F) and wild type (WT)

B. bassiana were each grown in standard tenellin production medium (TPM) (see

section 4.8). In TPM, mannitol is used as the carbon source.

Figure 2.32: tenC silencing clones and WT B. bassiana grown in standard TPM media.

The cultures were grown in 100 ml of TPM in 500 ml Erlenmeyer flasks. They

were incubated at 25 °C in shakers at 150 rpm. After ten days the cultures were filtered.

All cultures had varying degrees of yellow colour, which is an indication of tenellin

compounds. The mycelia in each flask were extracted in acetone (200 ml). The acetone

extract was concentrated under vacuum to a brown aqueous extract. It was further

diluted with deionized water (200 ml) and then extracted into ethyl acetate (200 ml).

The ethyl acetate layer was separated and dried with MgSO4. The extracts were

concentrated in vacuum to give a brown solid. All crude extracts of silenced clones

were in range of 5-8 mg and WT was 10 mg. A solution of 10 mg/ml for all extracts

was made with HPLC grade methanol and analysed with LCMS with Method 3

(section 4.4). The chromatograms of all six silenced clones and wild type are given in

figure 2.34 and 2.35.

WT A B C D E F

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The wild type B. bassiana produces four major compounds which are 15-

hydroxy tenellin 157 at 9.3 minutes, prototenellin D 155 at 10.0 minutes, pretenellin A

114 at 10.7 minutes and tenellin 87 at 11 minutes (Figure 2.33). The silenced clones A,

B, C, E and F still produce prototenellin D 155, pretenellin A 114 and tenellin 87. Clone

D produced only compound tenellin 87 and 157 (Figure 2.34 and 2.35). These

chromatograms showed that all the silenced clones still produce more or less the wild

type compounds. We did not observe any newly programmed or reprogrammed

compounds. This suggests that under these conditions tenC is not silenced and the amyB

promoter is inactive.

Figure 2.33: Diode array chromatogram of WT B. bassiana grown in TPM (mannitol) media showing production of 15-

hydroxytenellin 157 at 9.3 minutes, prototenellin D 155 at 10.0 minutes, pretenellin A 114 at 10.7 minutes and tenellin

87 at 11.0 minutes.

69

Figure 2.34: Diode array chromatograms of WT B. bassiana, Clone A, Clone B, Clone C grown in mannitol.

Figure 2.35: Diode array chromatograms of WT B. bassiana, Clone D, Clone E and Clone F grown in mannitol.

2.3.7(b) Growth of B. bassiana transformants in maltose media

The next experiment was to grow the six silenced clones A, B, C, D, E, F and

wild type B. bassiana in TPM medium but using maltose (30 g/L) as carbon source

instead of mannitol. As amyB promoter is turned on when grown with maltose medium,

we expected strong silencing of tenC.

70

Fig 2.36: tenC silencing clones and WT B. bassiana grown in maltose.

The six clones and WT B. bassiana were grown in the same conditions for ten

days at 25 °C at 150 rpm. All cultures were pale white colour, but produced mycelia.

The cultures were then extracted in the same way first with acetone, and then

concentrated and later diluted with deionized water and in the last extracted with ethyl

acetate. All dried extracts weighed from 28-34 mg from 100 ml liquid medium. The

concentrated extracts (10 mg/ml) in HPLC methanol were analysed in LCMS. The

chromatograms of silenced clones with WT B. bassiana are given in Figure 2.37 and

2.38.

Figure 2.37: Diode array chromatograms of WT B. bassiana, Clone A, Clone B, Clone C grown in maltose.

WT A B C D E

71

Figure 2.38: Diode array chromatograms of WT B. bassiana and tenC silencing Clone D, Clone E and Clone F grown in

maltose.

In maltose media the B. bassiana WT and tenC silenced clones did not produce

tenellin related compounds strongly. Only wild type produced small peaks of 15-

hydroxy tenellin 157 and prototenellin D 155. Tenellin 87 at 11 minutes was not

observed, the ESI only showed mass MH+ 354 at 11.08 minutes which may be

pretenellin B 115 but the uv was not strong or convincing. All the silenced clones failed

to produce compounds, even the wild type compounds were not detected. So, this

indicates that although amyB promoter is believed to be activated when there is maltose,

if mannitol is not used as the basic carbon source B. bassiana fails to produce any

compounds.

2.3.7(c) Growth of B. bassiana transformants in glucose media

After growing B. bassiana clones in maltose, the next experiment was to grow

them in TPM medium using glucose as carbon source (20 g/L). As the amyB promoter

is turned off when grown with glucose medium, we expected to observe an absence of

or weak silencing of tenC.

72

Fig 2.39: tenC silencing clones and WT B. bassiana grown in glucose.

The six clones and WT B. bassiana were grown in the same conditions for ten

days at 25 °C at 150 rpm. They all showed very faint colour which show poor

production of tenellin compounds. The cultures were extracted first with acetone, then

concentrated and diluted with deionized water and in the last extracted with ethyl

acetate. The dried extracts weighed 9-10 mg from 100 ml liquid media. The

concentrated extracts were made 10 mg/ml in HPLC methanol and analysed in LCMS.

The chromatograms of silenced clones with WT B. bassiana is given in Figures 2.40

and 2.41. In glucose medium the WT and all six silenced clones failed to produce any

tenellin related compounds.

Figure 2.40: Diode array chromatograms of WT B. bassiana and tenC silencing Clone A, Clone B and Clone C grown in

glucose.

WT A B C D E F

73

Figure 2.41: Diode array chromatograms of WT B. bassiana and tenC silencing Clone D, Clone E and Clone F grown in

glucose.

2.3.7(d) Growth of transformants in mannitol in combination with 1%

maltose

In the previous three experiments we observed that B. bassiana produces

tenellin 87 and related compounds only when mannitol is used in TPM medium. In

maltose and glucose even the WT could not produce. Here we used mannitol with 1%

maltose to see if maltose makes silencing in the presence of mannitol. The WT B.

bassiana and silenced clones were grown in the same conditions as explained in section

4.10. The crude extracts were made 10 mg/ml in HPLC methanol and analysed by

LCMS. The diode arrays of all extracts are given in Figure 2.42 and 2.43. In all

chromatograms wild type compounds, 15-hydroxy tenellin 157, prototenellin D 155,

pretenellin A 114 and tenellin 87 were produced.

74

Figure 2.42: Diode array chromatograms of WT B. bassiana and tenC silencing Clone A, Clone B and Clone C grown in

mannitol with 1% maltose.

Figure 2.43: Diode array chromatograms of WT B. bassiana and tenC silencing Clone D, Clone E and Clone F grown in

mannitol with 1% maltose.

2.3.7(e) Growth of transformants in mannitol in combination with

1% glucose

The WT and silenced clones of B. bassiana were grown on mannitol with 1%

glucose to see the effect of glucose on production of compounds. The WT B. bassiana

and silenced clones were grown in the same conditions as explained in section 4.10. The

75

crude extracts were made up to 10 mg/ml in HPLC methanol and analysed by LCMS.

The diode arrays of all extracts are given in figure 2.44 and 2.45. Wild type compounds

157, 155, 114 and 87 are observed in silenced clone A, B and D. Clones C, E and F also

produced more and less WT compounds.

Figure 2.44: Diode array chromatograms of WT B. bassiana and tenC silencing Clone A, Clone B and Clone C grown in

mannitol with 1% glucose.

Figure 2.45: Diode array chromatograms of WT B. bassiana and Clone D, E and F grown in mannitol with 1% glucose.

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2.3.7(f) Growth of transformants in mannitol with combination of

5% maltose

In this experiment the WT and silenced clones of B. bassiana were grown in mannitol

with increased percentage of maltose, 5%. The crude extracts were made 10 mg/ml in

HPLC methanol and analysed by LCMS. The chromatograms of all extracts are given in

Figure 2.46 and 2.47. WT B. bassiana produces only compound protenellin D 155 and

tenellin 87 when percentage of maltose is increased. The silenced produce WT

compounds 157, 155 and 87 in different concentrations.

Figure 2.46: Diode array chromatograms of WT B. bassiana and Clone A, B and C grown in mannitol with 5 % maltose.

Figure 2.47: Diode array chromatograms of WT B. bassiana and Clone D, E and F grown in mannitol with 5 % maltose.

77

2.3.7(g) Growth of transformants in mannitol with combination of

5% glucose

In last experiment the WT and silenced clones of B. bassiana were grown on

mannitol with increased percentage of glucose 5%. The crude extracts were made 10

mg/ml in HPLC methanol and analysed by LCMS. The chromatograms of all extracts

are given in figure 2.48 and 2.49. Wild type compounds prototenellin D 155 and tenellin

87 are produced in some clones.

Figure 2.48: Diode array chromatograms of WT B. bassiana and Clone A, B and C grown in mannitol with 5 % glucose.

Figure 2.49: Diode array chromatograms of WT B. bassiana and Clone D, E and F grown in mannitol with 5 % glucose.

78

These experiments showed that silencing a gene with a promoter whose function

is dependent on the carbon source was not successful in this case. B. bassiana will

always require mannitol in TPM media for production of its primary and secondary

metabolites.

We suggest that if we want tenC silencing in varying degrees in B. bassiana, we

may use another kind of promoter, the expression of which is not dependent on carbon

source for example, alcohol dehydrogenase PalcDH which is stimulated by glycerol or

lactose in the media.

2.4 Conclusions

Heterelogous expression of tenellin genes alone and co-expression with DMB

genes was effectively accomplished in A. oryzae (M-2-3) and their chemical products

were isolated.

The structure of prototenellin C 113 was elucidated and fully characterized.

Prototenellin C along with prototenellin A 111 and prototenellin B 112 were produced

from two clones; A. oryzae tenSPKS-NRPS and A. oryzae tenSPKS-dmbNRPS. The

TENS PKS-NRPS in absence of enoyl reductase TenC produce re-programmed or

unusual tetramic acids. The pattern of reduction, methylation and in case of 112, even

chain length was deviated than tenellin 87. This shows that TENS PKS controls the

programming of the polyketide chain while NRPS proves to possess a broader substrate

specificity to accept altered PKS chain from the TENS in case of 111, 112 and 113. The

NRPS deliberately serves its role to select and combine tyrosine with the PKS chain and

provide offloading mechanism for the chemical product. The production of 111, 112

and 113 from A. oryzae tenSPKS-dmbNRPS verifies successful development of a

Hybrid PKS-NRPS system, with proteins obtained from two different fungal strains

working together admirably. This paves way for further manipulation of PKS-NRPS

systems and exploring their enzymes for obtaining desired compounds. The yield of

prototenellin C 113 from A. oryzae tenSPKS-dmbNRPS was more (9.6 mg/L) than 113

obtained from A. oryzae tenSPKS-NRPS (2.4 mg/L), which further adds to the efficacy

of the hybrid TENS-PKS: DMB-NRPS system.

PreDMB A 118 was produced from A.oryzae dmbS –dmbC and A.oryzae dmbS-

tenC clones. PreDMB A 118 has similar polyketide chain pattern as DMB 88. This

79

heterologous expression shows that like tenellin pathway, the DMB PKS-NRPS in the

presence of enoyl reductase DMBC, produced the correctly programmed precursor

compound preDMB A 118. The expression of DMBS with TENC again produced

preDMB A 118. This shows that the enoyl reductase does not play any role in

programming of the polyketide chain but the presence of enoyl reductase, either DMBC

or TENC, DMBS ‘does not’ loses the fidelity of programming and compounds with

correct methylation and chain length are formed.

Heterologous expression tenSPKS-dmbC in A. oryzae without the NRPS

produced two new compounds 146 and 147, but they were not the products of tenellin

pathway. We ascertained that they are native wild type A. oryzae compounds. This

demonstrates that expression of TENS PKS without NRPS is a challenging experiment

and requires a more efficient expression system.

The silencing of tenC with amyB promoter in B. bassiana was carried out with

the purpose to achieve different level of concentration of the enoyl reductase and obtain

new compounds. This experiment was unsuccessful as substituting the carbon source

(mannitol) with maltose and glucose to obtain induction and repression of silencing,

severely abrogated the production of tenellin or related compounds. We suggested that

alternative inducible promoters can be considered in future, the induction of which is

not dependant on carbon source.

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Chapter 3

Investigation of the Role of Genes from the Aspyridone Pathway using

Heterologous Expression and Structural Elucidation of new

Compounds

3.1 Introduction

Aspergillus is a large genus of moulds which acquired its name because of its

distinctive spore bearing structure similar to an aspergillum (a holy water sprinkler). All

aspergilli have characteristic morphology consisting of a foot cell, elongated hyphae

called a conidiophore and a round vesicle bearing the asexual spores, the conidiospores

(Figure 3.1). In many species, the colour of the spores serves as identification, for

example Aspergillus niger produces black spores, Aspergillus ochraceus have yellow or

brown spores and the colour of spores from A. nidulans, A. fumigatus and A. flavus are

green.123

Because of their asexual airborne spores and their ability to grow with minimal

nutrients, aspergilli are widespread in all ecosystems.124

They mostly occur in terrestrial

habitats, soil and mostly on plant and animal debris. They are saprophytes. After they

get in contact to their food they first breakdown complex ingredients by secreting

enzymes and acids and then absorb the nutrients. The aspergilli play an important part

in decay and decomposition of organic matter driving carbon and other important

minerals back into the environment by natural recycling.125

They also provide a means

for supply of nutrients and food for a large number of other soil dwelling

microorganisms. Their ability of bio deterioration and degradation is a major problem in

spoiling foods, textiles, paper and even historic paintings.126

There are about 250 species in the genus Aspergillus. Many of the species aid in

fermentation and are important in the food industry. Since the early 20th

century

Aspergillus niger has been used in the production of citric and gluconic acids and has

also been used in the pharmaceutical industry.127

Aspergillus terreus is used in the

production of synthetic polymer.128

Aspergillus oryzae is used in the production of rice

vinegars, soy sauce, alcohol beverages,130

kojic acid used in a range of Japanese food

81

and also in making koji and synthesis of flavour enhancers.131

Many aspergillus species

are important in providing commercial enzymes.132

Aspergillus species can be easily grown in the laboratory on simple organic

media and have been studied extensively by molecular biologists and in studies for

developing biotechnology tools. A. oryzae has been an effective host for the

heterologous expression of different biosynthetic gene clusters of many secondary

metabolites133

and A. niger is used as a host for construction of heterologous proteins.134

Figure 3.1: A, microscopic view of Aspergillus spores; B, A. oryzae; C, A. terreus.

Most Aspergillus species produce a large number of secondary metabolites and

many of them are important natural product drugs. Lovastatin 47, a cholesterol lowering

drug was isolated from A. terreus,135

cholecystokinin antagonist asperlicin 157 from A.

alliaceus,136

ion channel ligands137

and anti-fungal compounds 158 are reported from

aspergillus species.138

Besides many beneficial species of Aspergillus, there are many fungi which are

causal agents of human and animal diseases. They produce a number of mycotoxins139

mainly aflatoxin 48, patulin 159 and ochratoxin 160 which deteriorate stored seeds and

also cause loss of poultry and loss of domestic animals.140

The air borne spores may

cause respiratory tract disease like asthma, hay fever and cause a number of allergies.141

Aspergillosis caused mainly by A. fumigatus may become fatal in immunosuppressed

individuals.142

82

3.2 Aspergillus nidulans

Aspergillus nidulans (also known as Emericella nidulans) propagates both by

asexual spores called conidia and sexual spores called ascospores. Conidia are produced

on specialized structures called coniodiophores and ascospores are grown inside round

sexual fruiting bodies called cleistothecia.143

A. nidulans has been used as a model

organism for over fifty years, on which various research areas of mycology, eukaryotic

cell biology and genetics were initiated. For example, the parasexual cycle was first

discovered and studied in A. nidulans by Pontecorvo and was used as a means to

produce strain of fungus with desired genetic traits before the advent of modern

biotechnology techniques.144

Ronald Morris analysed the genetics of mitosis by

studying this organism.145

A. nidulans has also been used as a model to study genetic

metabolic diseases, genetic recombination,146

explain intron splicing, chromatin, DNA

repair and regulatory pathways.123

It has a defined sexual cycle which is used as a guide

to investigate reproductive mechanism in other fungi where the sexual phase is not

clearly defined.

Figure 3.2: Aspergillus nidulans strain 2.2 grown on plate.

83

The genome of A. nidulans has been sequenced.147

It shows that the organism is

distantly related to A. oryzae and A. fumigatus. The genome sequence indicates the

presence of various secondary metabolite gene clusters, of which 27 are polyketide

synthases, 14 are nonribosomal peptide synthetases, 6 fatty acid synthases, one

sesquiterpene cyclase and two dimethylallyl tryptophan synthase genes.148

3.3 Aspergillus nidulans metabolites

The role of secondary metabolites in the life cycle of a fungus is considered

mostly ambiguous but they often exhibit important bioactivities; most importantly

antitumor, antibacterial, antifungal and other vital pharmaceutical properties.5

Aspergillus nidulans is a producer of a range of biologically active metabolites,

some of which are toxic. But the numbers of secondary metabolite genes predicted

from sequencing of A. nidulans genome are more than the reported metabolites from

this fungus.

There are a number of elements which govern the formation of these

metabolites. These include environmental factors for example temperature, light, pH

and more important are the availability of different nutrients, nitrogen and carbon

sources.143

Synthesis of some metabolites is also related to fungal vegetative growth and

morphology. Recently it has been reported that G-proteins regulate growth of asexual

spores as well as mycotoxin production.149

A main reason for the number of compounds

discovered being less than predicted from the genome sequencing is that many genes

encoding the biosynthesis of metabolites are silent under normal fermentation

conditions and they require signals or stimulants to be activated and expressed in the

form of natural products.

Many efforts over a long period of time have been made to isolate metabolites

from A. nidulans and identify genes involved in the biosynthetic pathway for the

characterized metabolites. Mostly this has been achieved by targeted gene deletion

assisted by sequence comparison of the genes with those from the known library of

metabolites.150

The genes involved in the biosynthesis of a particular metabolite are

usually clustered; this helped the natural product chemists to recognize boundaries for

84

genes encoding all essential enzymes and catalysing each step in the biosynthesis of the

natural product.

The data provided by the genome sequence of A. nidulans encouraged

researchers to devise methods to activate silent gene clusters by different genetic

engineering using modern molecular tools and advanced genomics. Considerable

success has been achieved in this regard with the discovery and over expression of

global regulators of secondary metabolism genes such as laeA151

and replacing pathway

specific transcription regulators with inducing promoters.152

Moreover, manipulating

chromatin modifying proteins and epigenetic modifiers has facilitated the up-regulation

of previously silent clusters of many bioactive compounds and has unveiled hidden

biosynthetic potential present in this fungus.153

A review of recognized metabolites of

A. nidulans is explained below.

The well-known β-lactam antibiotic compound penicillin 161 is produced from

A. nidulans. It is produced from three amino-acids: L-α-aminoadipic acid, L-cysteine

and L-valine. The biosynthesis of penicillin 161 is encoded by three genes namely,

acvA, ipnA and aat.154

Understanding the genetic and molecular regulation of penicillin

161 biosynthesis in A. nidulans can help devise ways to increase production of this

antibiotic.

Sterigmatocystin 164 is a carcinogen polyketide mycotoxin, produced by about

20 species of Aspergillus including Aspergillus nidulans.155, 156, 157

There is a great

concern of contamination caused by mycotoxins in food and feed products resulting in

health issues and huge economic loss. It also causes high level of genotoxicity in liver

samples. It was necessary to study the biosynthesis of sterigmatocystin 164 on

enzymatic level and control them by molecular studies. A lot of research had been made

to investigate the metabolic pathway of strigmatocystin 164 and determine its gene

cluster. Brown et al. identified a 60 kb cluster in A. nidulans comprising of 25 genes

reported to conduct all the steps essential for sterigmatocystin 164 biosynthesis.150

In

this cluster a NADPH dependent reductase gene stcU158

and a P450 monooxygenase

85

gene stcS converts the intermediate compound versicolorin A 162 to sterigmstocystin

164159

and stcP encoding a methyltransferase carry out methylation of the intermediate

demethylsterigmatocystin 163 to form sterigmatocystin 164.160

Scherland and Hertweck identified four unique prenylated quinolone alkaloids,

aspoquinolones A-D 165, 166, 167 and 168 from Aspergillus nidulans (HKI 0410) by

growing the fungus in rice medium.161

They predicted that like other alkaloids, these

quinolones might have produced from its precursor compound anthranilic acid. The

presence of anthranilate synthase (AS) like genes in A. nidulans sequence supports this

assumption.

The conidia spore pigmentation in A. nidulans was proposed to be encoded by a

polyketide synthase gene wA. Heterologous expression of this PKS in A. oryzae

produced a yellow coloured, novel heptaketide naphthopyrone compound known as

YWA1 169. This compound was regarded as the intermediate in pigmentation of mature

green spores.162, 163

Fernandez et al. isolated shamixanthone 170, emiricellin 171, dehydroaustinol

172 and austinol 173 from Aspergillus nidulans. They reported that an essential 4’-

phosphopantetheinyl transferase (PPTase) encoding gene cfwA is required for the

production of secondary metabolites in A. nidulans particularly polyketides and NRPS

86

compounds. Xanthones are phenolic compounds and exhibit important biological

activities including antimicrobial, antioxidant, cytotoxic and neuropharmacological

activities.164

In recent years after genome sequencing of A. nidulans revealed that there are

many more secondary metabolite clusters present as compared to the isolated

metabolites form this fungus, a lot of efforts has been served to uncover factors

responsible for the repression of metabolites expression. Bok et al. detected a gene

called cclA, similar to an orthologous gene in S. cerevisae which is used in chromatin

mediated gene silencing by DNA modifications involving methylation of lysine of

Histones. The cclA deletion mutants in A. nidulans gave production of six aromatic

compounds not observed before in A. nidulans. They were monodictyphenone 174,

emodin 57 and emodin analogs 175, 176, 177 and 178 and two anti-osteoporosis

polyketides F-9775A 179 and F-9775B 180.153

Sanchez et al. reported two more xanthone compounds from A. nidulans

variecoxanthone A 181 and epishamixanthone 182. The gene cluster of xanthose

comprise of a cluster of ten genes including a PKS gene mdpG which is also involved in

monodictophenone 174 biosynthesis. They stated that 174 and emodin 57 are precursors

87

of prenyl xanthones. They also identified three prenyl transferase genes necessary for

encoding prenyl transferase part of the xanthone structures.165

Schroek et al. reported the production of the aromatic tetraketide orsellinic acid

56, a lichen metabolite lecanoric acid 183 and the two antiosteoporosis polyketides F-

9775A 179 and F-9775B 180 by growing A. nidulans in conjunction with a collection of

soil dwelling bacteria actinomycetes.166

They revealed that a NRPKS encoding gene

orsA (AN7909) is required for the biosynthesis of orsellinic acid 56, lecanoric acid 183,

179 and 180. Sanchez et al. confirmed orsA to be involved in orsellinic acid production

and in AN7909 deletion mutant isolated two bioactive aromatic compounds in A.

nidulans, gerfelin 184 and diorcinol 185.167

Nielsen et al. grew A. nidulans on eight different media and observed not only a

number of known metabolites but also arugosin A 186, arugosin H 187, antibiotic

compounds violaceol I 188 and violaceol II 189 not known before from this fungus.168

They obtained 32 PKS deletion mutants and linked violaceol I 188 to the orsA gene and

biosynthesis of arugosin 186 to the monodictophenone 174 gene mdpG.

88

Szewczyk et al. reported a new metabolite, asperthecin 192 by deleting a gene

SumO in Aspergillus nidulans which encodes a regulatory protein.169

They also

identified the gene cluster for this compound by a following a number of gene deletion

strategies. The gene cluster consists of an NR-PKS gene aptA, a gene aptB which

encodes a hydrolase and a monooxygenase gene, aptC. They proposed that the aromatic

structure 190 of asperthecin 192 is synthesized from one acetyl-CoA and seven

malonyl-CoA assembled by a NR-PKS, encoded by aptA. The PKS chain 190 is

hydrolysed by AptB into 191 and AptC carries out later oxidation to form asperthecin

192 (Scheme 3.1).

Scheme 3.1: Proposed biosynthetic pathway of asperthecin 192.

Bok and Keller identified a nuclear methyltransferase protein LaeA which

globally regulates transcription of secondary metabolites in A. nidulans.151

In the laeA

over expressed mutants, Bok and collegues (during genetic profiling of the mutants)

recognized an antitumor compound terrequinone 198 not reported before in A.

nidulans.170

They related a five open reading frame gene cluster to the biosynthesis of

terrequinone 198 named tdi. The cluster consists of a mono-modular NRPS encoding

gene tdiA encoding a protein comprising an adenylation domain, a thiolation domain

and a thioesterase domain but a condensation domain typical of NRPS was lacking.

89

Bouhired et al. proposed that the gene tdiD, which encodes a putative aminotransferase

is responsible for the deamination of L-tryptophan 193 to indole pyruvic acid 194, tdiA

encodes a protein which then adenylates the pyruvic acid 194 and dimerises it to a

quinone structure 195. A presumed prenyl transferase encoded by tdiB catalyses a first

prenylation to form 196 and then a reductase (tdiC) accomplishes hydroquinone

reduction 197 before a second prenylation (tdiE) to form terriquinone 198.171

Scheme 3.2: Proposed pathway of terriquinone A.

Emericellamide, an antibiotic known previously from marine emericella species,

is formed by the fusion of a polyketide and a nonribosomal peptide. Chiang et al.

identified emericellamide A 207 and its analogues in gene deletion studies of series of

NRPS sequences during genome mining experiments in A. nidulans. They also deduced

that emericellamides are synthesized from a gene cluster comprising of four contigious

open reading frames. The HR-PKS EasB forms a carboxylic acid polyketide chain 199

and is converted to CoA thiolester 200 by EasD (CoA ligase) and loaded on to the

acyltransferase (AT) of EasC, 201 (Scheme 3.3). The polyketide is then loaded to the

EasA which is an NRPS consisting of five modules. Each module of the EasA delivers

90

an amino peptide group to the growing chain, glycine and valine being the amino acid

provided in the first two cycles of the NRPS subsequently forming 202, 203, 204 and

205. At the end of the biosynthetic cycle the linear chain 206 is released, cyclised and

assembles to form emericellamide A 207 and its analogues C 208, D 209, E 210 and F

211.17

Scheme 3.3: Proposed biosynthetic pathway of emericellamides.

Dohren reviewed the non-ribosomal peptide synthetase encoding genes in A.

nidulans and listed 27 NRPS and NRPS related genes. He reported a number of peptide

91

and aminoacid metabolites reported from A. nidulans including echinocandin 212,

emericellamide 207, triacetylfusigen 214, fusarinine 215, terriquinone 198, emerin 213

and aspyridone 84.173

Wang et al. recognized a silent gene cluster in Aspergillus nidulans which

consist of two adjacent PKS genes, one a NR-PKS (afoE) and other a HR-PKS (afoG)

in the same cluster.152

They triggered the cluster by replacing a putative transcription

activator gene (afoA) with the inducible alcohol dehydrogenase promoter alcA. This led

to the production of a new metabolite asperfuranone 219 with subsequent gene deletion

experiments they identified five genes involved in its biosynthesis (Scheme 3.4). They

proposed a biosynthetic pathway for asperfuranone 219 which shows that the HRPKS

that (afoG) synthesizes a 3, 5-dimethyloctadienone moiety 216 from acetyl-CoA, three

malonyl-CoA and two S-adenosyl methionine (SAM). The 3, 5-dimethyloctadienone

216 is loaded on to the next NR-PKS (afoE) by the SAT domain. The NR-PKS extends

216 by condensing with four malonyl CoA and a SAM to form the first intermediate

217. A gene (afoD) encoding hydroxylase enzyme carries out hydroxylation at C-3 to

92

form 218. The afoF, encoding an FAD dependant oxygenase hydroxylates C-7 and afoC

encoding a hydrolase is involved in furan ring formation. A gene AN1030.3 encoding

an oxidoreducatse catalyzes the last reduction step forming asperfuranone 219 (Scheme

3.4).174

Scheme 3.4: Proposed biosynthetic pathway of asperfuranone.

3.4 Aspyridone pathway in A. nidulans

Hertweck and co-workers reported an 11.9 kilobase (kb), putative hybrid

polyketide synthase- nonribosomal peptide synthetase encoding gene in A. nidulans

which was named apdA.80

ApdA is homologous to TenS discussed in chapter 2. They

described the PKS-NRPS to be comprised of a number of domains, which are

ketosynthase (KS), acyltransferase (AT), ketoreductase (KR), dehydratase (DH), enoyl

reductase (ER), C-methyltransferase (C-MeT), acylcarrier protein (ACP), adenylation

domain (A), condensation domain (C), peptidyl carrier protein (PCP) and a reducase

domain (R). The apdA gene is clustered with a number of putative oxidoreductase

encoding genes; two of the genes encode cytochrome P450 monooxygenases and are

93

named as apdB and apdE and one is an FAD-dependent monooxygenases called apdD.

The PKS-NRPS is bordered downstream by a putative exporter gene apdF, an activator

gene apdR and an acyl-CoA dehydrogenase encoding gene apdG. The gene also has an

additional trans acting ER domain, apdC which is homologous to tenC. Hertweck et al.

believed this cluster to be silent as A. nidulans extracts grown in different laboratory

mediums do not contain any PKS-NRPS metabolites.80

They perceived that the

sequence of the activator gene apdR was similar to a transcription factor found in

Aspergillus fumigatus. Over-expression of apdR under the influence of the inducible

alcohol dehydrogenase promoter alcAp was achieved by homologous recombination in

A. nidulans. The mutant A. nidulans strain produced two new pyridone compounds

Aspyridone A 84 and Aspyridone B 226.

Scheme 3.5: Proposed biosynthesis pathway of aspyridone by Bergmann et al.80

94

Hertweck and coworkers proposed that the PKS synthesizes a tetraketide 220

from three malonyl CoA, an acetyl CoA and two SAM (S-adenosyl-methionine). The

enoyl reductase enzyme in ApdA was believed to be inactive and this function is

catalysed by the stand alone reductase protein ApdC acting in trans. This characteristic

is similar to the gene clusters of lovastatin, tenellin and desmethyl bassianin.104,109,105

The tetraketide 220 fuses with the tyrosine 107 catalysed by the condensation domain to

form a hybrid polyketide-peptide 221. Based on knowledge at the time, it was assumed

that a reductive release was catalysed by the reductase domain to form an aldehyde

intermediate 222 which after Knoevenagel closure forms pyrrolinone 223. The

cytochrome P450 encoded by apdB was proposed to perform the first oxidation to form

the tetramic acid 224 and the second oxidase encoding gene apdE makes the

hydroxylation of the tetramic acid forming an intermediate 225. The oxidative ring

expansion from tetramic acid to 2-pyridone is also proposed to be catalysed by the P450

enzymes to form aspyridone A 84. The last step of phenol hydroxylation of aspyridone

to form aspyridone B 226 is performed by the FAD dependent monooxygenase apdD

(scheme 3.5).

Tang et al. confirmed the biosynthesis of the aspyridone pathway by

reconstructing the function of apdA and apdC in in vitro studies and S. cerevisiae was

used as the expression host. They reported the production of preaspyridone A 224 by

incubating purified apdA and apdC in the presence of co-factors and building blocks

(Figure 3.3, A). The production of 4-hydroxy preaspyridone proves that the hybrid L-

tyrosine-tetraketide thiolester 221 after its release undergo ring closing by a Dieckman

cyclisation catalysed by the last C-terminal domain of the NRPS (Figure 3.3, B) and

disproves the presence of a reductase domain and consequent aldehyde intermediate 222

(Scheme 3.5). They highlighted the flexibility of the adenylation domain towards

different aromatic amino-acids by showing incorporation of L-tryptohan, L-4-

fluorophenylalanine and L-phenylalanine by apdA to form 227, 228 and 229.107

95

A

B

C

Figure 3.3: A, in vivo synthesis of preaspyridone A 224; B, Dieckmann cyclisation in preaspyridone A 224; C,

incorporation of different amino acid analogues by ApdA.

3.5 Objectives of the Chapter

Heterologous expression of biosynthetic genes in a foreign host, particularly

fungi, has been an important biotechnology tool to investigate the various steps in

biosynthetic pathways of novel natural products. It allows the determination of the role

of each gene in the gene cluster and is of particular use in the case of silent gene

clusters.175

Among different fungi, Aspergillus oryzae is considered as an effective

heterologous host, mainly because of its established use in producing large amount of

proteins.176

The Bristol Polyketide Group successfully studied the activities of a number of

HRPKS-NRPS gene clusters in detail by heterologous expression of the specific gene

clusters in A. oryzae, for example fusarin C 77,77

squalestatin 65,72

tenellin 87,104

desmethyl bassianin 88.109

These heterologous expressions experiments resolved the

methylation and chain length factors and cryptic programming between two similar yet

different compounds tenellin 87 and desmethyl bassianin 88.110

This encouraged us to

revisit and study the iterative PKS-NRPS gene cluster of aspyridone 84. We planned to

96

study heterologous expression of aspyridone biosynthetic genes in A. oryzae by using a

recently reported multiple gene expression plasmid system for A. oryzae

transformation.176

We also aimed to investigate and determine the function of each tailoring

enzyme, particularly the role of the different oxidative-enzyme-encoding genes in the

aspyridone gene cluster by expressing the genes in different groups in A. oryzae. These

experiments would also produce intermediate compounds and the structure elucidation

of these will aid in understanding the order of biosynthetic steps in the aspyridone

pathway.

The A. oryzae transformants obtained will be analysed. The new metabolites will

be isolated by preparative mass directed LCMS and their structures will be elucidated

and characterized. The stereochemistry of any crystallised compound will be studied

under X-ray crystallography. The chemical structure of a compound in a particular gene

expression will help to deduce the function and biosynthetic potential of the genes in the

cluster.

3.6 Heterologous Expression system used in fungal transformation

The number of secondary metabolite gene clusters annotated in A. nidulans

genome is more than the natural products known from the fungus. One of the reasons is

the mass metabolic background in the native fungus due to which many gene clusters

fail to express their products. A suitable way to determine the product of a secondary

metabolite encoding gene cluster is to express it in an appropriate host by fungal

transformation.178

Heterologous expression also helps in engineering fungal genes and provides an

alternative method for high production of novel metabolites which are formed in low

titre in their native host fungus. The first successful DNA-mediated fungal

transformation was reported in 1979.179,180

Since then, fungal transformation protocols

are still evolving and have improved through years but also have their limitations.

Fungal PKS genes encode large megasynthases and appropriate hosts with an

effective expression system are required for their expression. Quite a number of

organisms have been used for fungal PKS transformation181

for example yeast, bacteria,

plants and also fungi like A. nidulans and A. oryzae. For many PKS genes A. oryzae has

97

been a favourable host. This is because it belongs from the same fungal genus and due

to similar cellular mechanism the expressed genes function properly.122

A. oryzae has

the ability to translate mRNA from eukaryotes, carry out post-translational

modifications, produce large amounts of enzymes and secretes secondary metabolites in

the medium in high amount. It is among those fungal species which are generally

regarded as safe (GRAS) and they are easily fermented under simple laboratory

conditions. The genomic analysis as well as analysing the LCMS data of A. oryzae

transformants is easy to study. A. oryzae can also splice introns present in the PKS

genes.182

The heterologous expression plasmid used in fungal transformations usually

consists of the target genes themselves, suitable promoters and appropriate terminator

sequences and gene for the selection markers. A number of selection markers are used

in transformation, such as nutritional markers argB, amdS, pyrG or niaA used in

auxotrophic condition or different antibiotic resistance markers against hygromycin B,

phleomycin or benomyl.183

In our fungal transformations we will use the arginine

auxotrophic A. oryzae strain (M-2-3) as the host organism which is unable to grow in

the absence of arginine – i.e. on minimal media. The plasmid contains the argB gene of

A. nidulans. Thus transformed cells will be able to grow on minimal medium, whereas

the un-transformed cells will be unable to survive. In a standard fungal transformation

procedure the required genomic DNA is extracted from the subject fungus, amplified

and cloned in the respective plasmid used for the heterologous expression. The spores

of the host fungus are subjected to enzymatic treatment to break down the cell wall and

liberate protoplasts. The protoplasts are incubated with transforming DNA in a medium

containing CaCl2 and other additives and then grown on medium containing selective

nutrients allowing only heterologous transformants to grow (Figure 3.4).175

98

Figure 3.4: Steps in fungal transformation.

The PKS-NRPS encoding genes, like other fungal secondary metabolite genes,

exist in clusters and a simple precursor compound is synthesized by encoding of a

megasynthase enzyme accompanied by tailoring enzymes. This provoked biologists to

devise a multiple gene expression vector which can express four genes in a single

transformation experiment. This has been recently accomplished in the Lazarus group in

the School of Biological Sciences, University of Bristol.176

The vector comprises of

three constitutive promoters, PgpdA from A. nidulans, Padh (alcohol dehydrogenase) and

Peno (enolase) to express maximum of three tailoring genes from a gene cluster. For

expression of megasynthases like PKS-NRPS it consists of inducible PamyB (promoter of

taka-amylase coding gene in A. oryzae) and TamyB, the amyB terminator. The vector

99

contains of the argB gene as a selectable marker for expression in A. oryzae auxotroph

(M-2-3). This vector is termed as pTAYAGSargPage.

We planned to use the pTAYAGSargPage vector to transform the silent gene

cluster from the A. nidulans aspyridone pathway. The strong constitutive promoters

provided in this vector will allow the PKS-NRPS and the tailoring genes to trigger and

express into proteins.

Figure 3.5: Multiple gene expression vector pTAYAGSargPage used in fungal transformation.

3.7.0 Results

3.7.1 Heterologous expression of apdA and apdC in A. oryzae (M-2-3)

From in vitro studies by Tang et al.107

and the aspyridone pathway proposed by

Bergmann et al.,80

we knew that the megasynthase HRPKS-NRPS encoded by apdA

and the enoyl reductase apdC synthesize preaspyridone A 224, the first compound in

the aspyridone pathway. This step is similar to the biosynthesis of pretenelllin A 114104

and preDMB A 118109

in B. bassiana. So, to confirm this hypothesis in vivo we carried

out heterologous expression of apdA and apdC in A. oryzae. The cloning and fungal

transformation was done by Dr. Khomaizon Pahirulzaman from the School of

Biological Sciences, University of Bristol.

The pTAYAGSargPage vector was used to combine the iterative HRPKS-NRPS

apdA and apdC genes to form the vector pTAYAargAC. Transformation of this vector

into A. oryzae produced a number of transformants (denoted A. oryzae apdAC) and the

100

incorporation and expression of the genes apdA and apdC was confirmed by qRT-PCR.

The best producing transformants were then selected for chemical analysis and

identification and purification of novel compounds by LCMS and NMR. Wild-type A.

oryzae M-2-3 was grown in parallel in all experiments as a control.

Figure 3.6: Expression vector pTAYAargAC containing apdA and apdC.

Figure 3.7: A, A. oryzae mycelia in liquid media in a flask; B, A. oryzae apdAC expression clone on CDA plate.

The A. oryzae transformant was grown first on Czapek Dox agar (CDA) (Figure

3.7, B) and later on DPY solid media for maximum sporulation. The transformant was

grown on plates for 7-10 days. The mature spores were scratched with a sterile loop and

spores were collected in deionized water. 1 ml of the spore solution was added to 100

A B

101

ml of CMP liquid media (see section 4.9) in a 250 ml baffled Erlenmeyer flask. The

cultures were grown at 28 °C in a shaker at a speed of 200-250 rpm for 7 days. The A.

oryzae transformants mycelia grow in a form of small balls (Figure 3.7, A). After 7 days

the cultures were removed from the shaker, and the entire fermentation mixture

(mycelia with the liquid) was acidified (pH= 3) and homogenized with ethyl acetate

(section 4.11). The organic extract was separated, then concentrated under vacuum on a

rotary evaporator and then the residue was defatted. The mass of the dried crude extract

was 40 mg (from 100 ml fermentation). A solution of 10 mg/ml of the crude extract was

made with HPLC grade methanol and 20 µl was injected and analysed by a Waters

2795HT HPLC system. It measures wavelength between 200 and 400 nm with a Waters

998 diode array detector and provides an electrospray (ES) mass spectrum with Waters

ZQ spectrometer sensing masses between the ranges of 150 to 600 m/z units.

The LCMS chromatogram of A. oryzae apdAC displayed two new peaks, one

minor peak at 13.2 minutes and the second major peak at 14.0 minutes which were not

present in the A. oryzae wild type used as a control (Figure 3.8). Both the peaks showed

m/z 332 [M]H+

and a λmax of 279 nm which is the same as described for preaspyridone

224107

by Tang et al. although they reported that only a single compound was produced

in their experiments. The ESI chromatogram showed two peaks of identical masses

indicating that they are probably isomers. To confirm the production of preaspyridone

224 and determine the two isomers we purified each compound for NMR structural

determination.

The purified minor (0.61 mg) and major (60.7 mg) components were dissolved

in deuterated chloroform (CDCl3) and 1D, 2D 1H NMR and

13C NMR spectroscopic

studies were carried out. Both compounds were identified as preaspyridone A 224 and

the chemical shifts of the NMR spectra matched with those reported by Tang et al.107

The 1D 1H and

13C chemical shifts of both minor and major components in CDCl3 were

same with only negligible differences of 0.01ppm. But NMR spectra run in dimethyl

sulfoxide-d6 discovered key differences between the structures of the minor and major

compounds. Reinjection of the pure compounds showed that they did not interconvert.

102

Figure 3.8: A, Diode array chromatogram of A. oryzae apdAC expression clone showing two new peaks at 13.2 and

14.0 minutes; B, Diode array chromatogram of Wild type A. oryzae used as a control.

Figure 3.9: A, ES+, ES- and UV spectrum of minor isomer at 13.2 minutes; B, major isomer at 14.0 minutes observed

in A. oryzae apdAC expression clone

3.7.1(a) Identification of minor isomer of preaspyridone A 224

The minor compound eluting at 13.2 minutes was purified in the form of pale

crystalline solid. HRESIMS gave a molecular formula of C19H26NO4 (observed

332.1852; calculated 332.1856 for M[H]+).

AU

0.0

1.0e+1

2.0e+1

3.0e+1

4.0e+1

5.0e+1

6.0e+1

-0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

AU

0.0

2.5e+1

5.0e+1

7.5e+1

1.0e+2

1.25e+2

1.5e+2

ZW-II-92H 14.0

3.45

2.656.23

28.7726.93

ZW-II-92M

3.22

2.60

3.45

5.80

4.25

27.43

13.2

Wildtype A. oryzae

A

B

%

100

%

0

100 ZW-II-92H 262 (14.078)

330

325317297 309302 308 315 320

331

366332 363355348344337368

ZW-II-92H 263 (14.105) 332

333

334

[M]H+

[M]H-

%

100

%

0

100

ZW-II-92H 246 (13.218)

330

325316313305300 321

331

363332352346341 362 366 371376

332

357

341333

336 342 358373370365

[M]H-

[M]H+

AU

1.0e-2

2.0e-2

3.0e-2

4.0e-2

5.0e-2

6.0e-2

7.0e-2

8.0e-2

ZW-II-92H 793

(13.200) 222

280

apdA,C-repeat

AU

0.0

2.0e-1

4.0e-1

6.0e-1

8.0e-1

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

ZW-II-92H 841 (14.000)279

223

A

B

103

The 13

C NMR indicated the presence of 19 carbons which included two carbonyl

groups at δC 175.4 (C-2) and δC 191.9 (C-6), an enol group at δC 194.4 (C-4), a

quaternary carbon at δC 99.9 (C-3), a methine carbon at δC 62.3 (C-5), two sp3 carbons

at δC 33.4 (C-7) and δC 31.5 (C-9), two methylene groups at δC 39.6 (C-8) and δC 28.7

(C-10) and three methyl carbons at δC 16.7 (C-13), δC 18.9 (C-12) and δC 10.8 (C-11).

Figure 3.10: 1H NMR of minor component of preaspyridone A 224 run in DMSO-d6.

The 1D 1H NMR contains two distinct doublets in the aromatic region at δH 6.88

(2H, H-16, H-20) and δH 6.59 (2H, H-17, H-19) (Figure 3.10) which is characteristic of

a para- substituted phenol ring and is further corroborated by 1H-

13C HMBC correlation

of both the aromatic protons with the C-OH at δc 155.7 (C-18) (Figure 3.12). The two

singlets at δH 9.17 and δH 8.93 were assigned to the para- substituted hydroxyl group at

H-18 and to H-1 attached to a nitrogen atom respectively by HMBC correlations. The

multiplet peaks at δH 2.82 were assigned to the diastereotopic protons (H-14a, H-14b)

linked to the methine proton at δH 4.08 (H-5) in 1H-

1H COSY (Figure 3.12) and to a

quaternary carbon at δC 125.3 (C-15), aromatic carbon at δC 130.6 (C-16/20) and

104

carbonyl group at δC 194.4 (C-4) in 1H-

13C HMBC and confirms benzylic protons

linked to the pyrrolidine ring at C-5 (Figure 3.11).

Figure 3.11: Segment of 1H-

13C HMBC NMR spectrum of minor isomer of preaspyridone A 224 showing correlations of

benzylic protons H-14 with C-5, C-16, C-15 and C-4.

The diastereotopic protons (H-14a, H-14b) and the methine proton H-5 are three

different nuclei (A, B, X) coupled to each other and have separate chemical shifts. Each

of these proton signals are doublets of doublets but the signals of H-14a and H-14b have

merged into each other creating distorted peaks typical pattern of an ABX system and

the signals at H-5 have combined to a broad peak (Figure 3.10). The quartet at δH 3.53

was assigned to H-7 linked to the carbonyl carbon at δC 191.9 (C-6), the methyl carbon

at δC 17.1 (C-13) and methylene group at δC 39.6 (C-8) in HMBC. The broad multiplet

between δH 1.24-1.27 was assigned to the methylene protons H-8 and to the methine

proton H-9. The multiplet at δH 1.09 and δH 1.26 corresponds to the geminal protons of

H-10. The doublet at δH 1.03 was assigned to the methyl group at C-13 exhibiting a

HMBC connection to C-6, C-7 and C-8. The terminal multiplet at δH 0.80-0.82 was

consigned to the last methyl H-11 and methyl group H-12 showing HMBC connections

to C-10, C-9 and C-8.

105

Figure 3.12: 1H-

1H COSY (solid lines) and

1H-

13C HMBC correlations (arrows) in preaspyridone minor isomer 224.

Crystals of the minor component 224 were formed in chloroform and were

analyzed on a Microstar X-ray instrument. The crystal data (ccdc code 941137) gave

the relative stereochemistry of the three chiral centres in minor preaspyridone as (5R,

7S, and 9R). The methyl groups at C-7 and C-9 possess anti-configuration in the crystal

data. Bergmann et al. reported syn configuration for both methyls in aspyridone A 84 2,

4-dimethylhexanoyl side chain.80

Figure 3.13: Crystal structure of the minor isomer of preaspyridone A 224.

3.7.1(b) Identification of major isomer of preaspyridone A 230

The major compound eluting at 14.0 minutes was purified in the form of pale

brown solid. HRESIMS gave a molecular formula of C19H25NO4Na (observed

354.1677; calculated 354.1676).

The 13

C NMR revealed the presence of 19 Carbons (Figure 3.15) having similar

chemical shifts as the 13

C NMR spectra of minor preaspyridone 224. The 1H NMR of

the major component was similar to minor preaspyridone A 224 in having similar

aromatic doublets at δH 6.59 (H-17, H-19), δH 6.90 (H-16, H-20) and the singlet at δH

9.17 (H-18) which is distinctive of para- substituted phenol in preaspyridone A. The 1H

NMR showed difference in the signals of three protons H-14, H-8 and H-13 (Figure

3.16). The splitting pattern of the diastereotopic protons H-14a and H-14b in major

106

preaspyridone 230 was different because both protons appeared at the same chemical

shift δH 2.82 as a doublet coupled to methine proton H-5 with J constant of 4.5 Hz,

unlike in minor preaspyridone 224 where both H-14 protons appeared at separate ppm

(Figure 3.10). The methylene group at H-8 in major preaspyridone 230 appeared as

geminal protons resonating at two separate chemical shifts δH 1.29 and δH 1.38. The

chemical shift of the methyl H-13 was also different as it produced a doublet at δH 0.95

(Figure 3.16) as compared to δH 1.03 in the minor isomer. From the above variations in

the 1H NMR and the crystal structure of the minor preaspyridone 224, we deduced that

both the minor and major compound observed in the apdAC expression clone, are

diastereomers of preaspyridone A being epimeric at C-5 (Figure 3.13). The 1H-

13C

HMBC showed similar correlations as observed in minor preaspyridone 224 (Figure

3.14).

Figure 3.14: 1H-

1H COSY (solid lines) and

1H-

13C HMBC correlations (arrows) in preaspyridone major isomer 230.

Figure 3.15: 13

C NMR of major isomer of preaspyridone A 230 run in DMSO-d6.

107

Figure 3.16: 1H NMR of major isomer of preaspyridone A 230 run in DMSO-d6.

3.7.1(c) Discussion on the biosynthesis of preaspyridone A 224 and 230

Heterologous expression of apdA and apdC in A. oryzae (M-2-3) produced two

diastereomers of preaspyridone A, 224 and 230, which confirms the in vitro

reconstitution of apdA and apdC function by Tang and colleagues.107

There are a

number of 3-acyl tetramic acids reported from analogous iterative HRPKS-NRPS gene

clusters from other fungi. For example, fusarin C 77, pramanicin 83, militarinone C 82,

equisetin 81, pseurotin A 78, chaetoglobosin A 80, 2-oxo-cyclopiazonic acid 85 (Figure

3.17), pretenellin A 114 and predesmethylbassianin A 118 (see chapter 1). The

biosynthetic pathway of preaspyridones is more similar to pretenellin A 114.

The minor 224 and major 230 preaspyridone A are epimers at methine proton H-

5. This illustrates that the adenylation domain of the NRPS is able to select D-tyrosine

231 during biosynthesis of minor preaspyridone 224 and L-tyrosine 232 to form the

major preaspyridone 230 (Scheme 3.5). This further confirms the in vitro studies by

Tang and coworkers where they reported the flexibility of the adenylation domain of

apdA to incorporate different aromatic amino acids.107

We also proposed that minor

preaspyridone 224 might have formed by reduction of 267.

108

Scheme 3.5: Incorporation of amino acids in isomers of preaspyridone A 224 and 230.

The structures of the diastereomers of preaspyridone A 224 and 230 also reflects

the exclusive potential of the enoyl reductase domain (ApdC) of aspyridone gene

cluster, particularly setting the opposite R and S stereo centres at the pendant methyls in

the 2, 4-dimethyl hexanoyl side-chain of preaspyridone A 224 and 230. In the initial

cycle of the polyketide chain biosynthesis the AT and KS domains form a diketide,

followed by methyl group transfer by CMeT domain and ketoreduction by the KR. The

dehydratase (DH) further reduces the diketide to an enol group. The enoyl reductase is

defective in the ApdA protein and the reduction is carried out by the stand alone ER

protein encoded by apdC. The ER reduces the enoyl group to a saturated bond and at

the same time sets the stereochemistry of the methyl chain, and in pre aspyridone A the

first methyl group is arranged in R configuration 233. The same steps repeat in the

second cycle by the iterative domains and the second methyl group is settled in S

configuration 234 by the ER domain (Scheme 3.6). In the last cycle a β-keto tetraketide

220 is formed.

109

Scheme 3.6: Proposed biosynthetic steps of polyketide chain in pre aspyridone A 224 and 230.

A similar stereoselective programming is observed by KR domain in (6’S,

10’S)-7, 8’-dehydrozearalenol (DHZ 236) biosynthesis, which is an intermediate of

hypothemycin 237. The HRPKS Hpm8 synthesizes a hexaketide 235 where KR

catalyzes opposite stereo reduction at C-6’ and C-10’. The hexaketide 235 is transferred

to a NRPKS which combines with three malonyl coenzyme A to form DHZ 236 and

later steps eventually form hypothemycin 237 (Scheme 3.7).184

Scheme 3.7: Stereoselective reduction during biosynthesis of hypothemycin 237.

110

Figure 3.17: 3-acyl tetramic acids reported from hybrid PKS-NRPS pathway, amino acids are highlighted in red.

3.7.2 Heterologous expression of apdACE in A. oryzae (M-2-3)

After successful expression of apdA and apdC, pTAYAGSargPage was

modified to develop another plasmid pTAYAargACE, containing apdA, apdC and

apdE, which was then transformed into A. oryzae M-2-3. The apdE gene encodes a

cytochrome P450 monooxygenase and it shares 48% protein identity to the ring

expandase P450 enzyme TenA present in B. bassiana 110.2.108

This expression was

aimed to delineate the role of P450 enzymes in aspyridone A 84 pathway. Selected

transformants were screened for production of new metabolites by LCMS.

The A. oryzae ACE expression clone was grown on DPY plates and mature

spores were inoculated in CMP liquid media for 7 days at 28 °C (see section 4.10). The

A. oryzae culture was homogenised, acidified and extracted with ethyl acetate. The

organic extract was concentrated under vacuum and defatted. The dried extract (112.6

mg from 100 ml of culture) was made to a solution of 10 mg/ml in HPLC grade

methanol and analysed by LCMS.

111

Figure 3.18: A, Diode array chromatogram of A. oryzae apdACE expression clone showing four new peaks at 8.8, 14.5,

14.9 and 24.8 minutes; B, Diode arrat chromatogram ofvWild type A. oryzae used as a control.

The diode array chromatogram of A. oryzae apdACE expression clone indicated

four new peaks which were not present in the A. oryzae wild type (WT) extract. The

first peak at 8.8 minutes possessed a λmax (222, 281) which is similar to uv absorption of

the 3-acyl tetramic acid preaspyridone A 224. The ESI spectrum of this peak showed

m/z 348 [M]H+

which is 16 mass units more than that of preaspyridone A 224. The

second peak at 14.5 minutes showed mass ion of 238 [M]H+ and a longer λmax (229, 325

nm). The peak eluting at 14.9 minutes showed a uv absorption (λmax 246, 344) and ESI

(m/z 330 [M]H+), spectra corresponding to aspyridone A 84. The last peak at 24.8

minutes had a mass ion of m/z 316 [M]H+

, which is 16 mass units less than

preaspyridone A 224 (m/z 332 [M]H+) and a λmax 281 nm.

The transformant was grown on large scale (100 ml × 10 flasks) and mass-

directed purification of the above peaks was performed on a Waters LCMS

autopurification system (see section 4.5). The dried crude extract (1303 mg/L) was used

to prepare a solution of 50 mg/ml and about 200 µl was injected in each preparative run.

The gradient use on a 30 duration programme was acetonitrile Method 5 (section 4.5)

on a C18 Phenomenex LUNA column. The purified fractions of each compound were

collected and dried under nitrogen gas. Structural elucidation was achieved using 1D

and 2D NMR spectroscopy and High Resolution Mass Spectrometry.

0.0

1.0e+1

2.0e+1

3.0e+1

4.0e+1

5.0e+1

6.0e+1

-0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.000.0

1.0e+2

2.0e+2

3.0e+2

ZW-II-92K-50-70ch3cn 14.5

8.8

3.28

2.58

3.726.4

14.9

24.8

ZW-II-92M

3.22

2.60

3.45

5.80

4.25

27.43

Wildtype A. oryzae

A

B

112

3.7.2(a) Identification of 14-Hydroxypreaspyridone A 238

The compound eluting at 8.8 minutes was obtained in the form of waxy light

brown solid (67 mg/L). HRESIMS gave a molecular formula of C19H25NO5 (observed

370.1624; calculated 370.1625 for M[Na] +

). The 13

C NMR in methanol-d4 showed

many signals coming at similar chemical shifts as preaspyridone A 224. These included

two methylene carbons at δC 41.2 (C-8) and δC 30.2 (C-10), a methine carbon at δC 68.5

(C-5), two sp3 carbons at δC 33.5 (C-9) and δC 35.5 (C-7), three methyl groups at δC 17.5

(C-13), δC 19.6 (C-12) and δC 11.4 (C-11), two aromatic carbon at δC 115.5 (C-17, C-19)

and δC 129.6 (C-16, C-20) and carbonyl group at δC 195.2 (C-6). A new signal at δC 74.8

indicated carbon attached to a hydroxyl (OH) group. The 1H NMR presented a similar

spectrum to preaspyridone A 224 spectra. A doublet at δH 4.99 attached to δC 74.8 (C-

14) in the 1H-

13C HSQC was assigned to H-14 (Figure 3.20). The aromatic doublets at

δH 7.11 (H-16, H-20) and δH 6.65 (H-17, H-19) exhibited HMBC correlations to para-

substitited phenol at δC 158.6 (C-18), quaternary carbon at δC 130.3 (C-15) and benzylic

carbon δC 74.8 (C-14) (Figure 3.19). The doublet at δH 4.22 was assigned to methine H-

5 showing a 1H-

1H COSY to H-14 and HMBC correlation to benzylic carbon at C-14

which confirmed para-substituted phenol attached at hydroxy-benzyl to a pyrrolidine

nucleus at methine H-5 (Figure 3.21). The quartet at δH 3.55 (H-7) was linked to C-8

and C-13 in HMBC. The multiplet spread between δH 1.29-1.40 was assigned to

methylene protons (H-8) and δH 1.09-1.33 to methylene groups (C-10) and methine at

C-9. The terminal methyl at δH 0.81 (H-11) and methyl at 0.82 (H-12) showed HMBC

connection to C-13, C-9 and C-8 confirming a similar 2,4-dimethylhexanoyl side chain

possessed by preaspyridone A 224 (Figure 3.19). All correlations confirmed the

structure to be 14-hydroxypreaspyridone A 238.

Figure 3.19: 1H-

1H COSY (solid lines) and

1H-

13C HMBC correlations (arrows) in 14-hydroxypreaspyridone 238.

113

Figure 3.20: 1H NMR of 14-hydroxy preaspyridone A 238 run in methanol-d4.

Figure 3.21: Segment of 1H-

13C HMBC NMR spectrum of 14-hydroxy preaspyridone A 238 showing key correlations of

hydroxyl benzyl at C-14 with a methine at H-5 and aromatic proton H-16.

114

Figure 3.22: ES+ and UV spectrum of 14-hydroxy preaspyridone A 238 eluted at 8.8 mins.

3.7.2(b) Identification of 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 239

The compound eluting at 14.5 minutes was obtained as pale white solid (63

mg/L). The HRESIMS gave a molecular formula of C13H20NO3 (observed 238.1428;

calculated 238.1437 for M[H] +

). The 13

C NMR in DMSO-d6 indicated the presence of

all 13 carbons consisting of two carbonyl groups at δC 177.6 (C-4) and δC 211.9 (C-7),

two methylene groups at δC 40.0 (C-9) and δC 29.7 (C-11) and three methyl groups at δC

11.1 (C-12), δC 16.7 (C-14) and δC 18.8 (C-13). The 1H NMR showed a triplet

downfield at δH 7.60 and a doublet at δH 5.92 which were assigned to H-6 and H-5

respectively (Figure 3.24), linked to an amide proton at δH 11.49 (H-1) in 1H-

1H COSY

(Figure 3.23). The aromatic protons H-5 and H-6 showed 1H-

13C HMBC correlations to

quaternary carbon C-3 (δC 105.9), a hydroxyl group at C-4 (δC 177.6) and carbonyl

group at δC 161.8 (C-2) verifying the structure of 2-pyridone (Figure 3.25). The quartet

at δH 4.25 was assigned to a methine proton H-8 linked to the carbonyl group at δC 211.9

(C-7), methyl group at C-14 and to a methylene group at C-9 (Figure 3.23). The methyl

group at δH 1.02 (H-14) showed HMBC connection to carbonyl at C-7 and methine at C-

8 which established that it is attached to C-8. The multiplets at δH 1.21 and δH 1.49 were

assigned to geminal protons at H-9 and multiplets at δH 1.09 and δH 1.25 were allocated

to geminal protons at H-11. A number of COSY and HMBC showed the structure to be

a 2, 4-dimethylhexanoyl side chain attached to 2-pyridone at C-3 and was named as 4-

hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 239.

115

Figure 3.23: 1H-

1H COSY (solid lines) and

1H-

13C HMBC correlations (arrows) in 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-

pyridone 239 and crystal structure of 239.

Figure 3.24: 1H NMR of 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 239 run in DMSO-d6.

Figure 3.25: Section of 1H-

13C HMBC NMR spectrum of 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 239 showing

important correlations of pyridone protons H-5 and H-6 with quaternary carbons C-2, C-4 and C-3.

116

Figure 3.26: ES+ and UV spectrum of 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 239.

3.7.2(c) Identification of aspyridone A 84

The compound separating at 14.9 minutes (Figure 3.18) was obtained as light

brown solid. The HRESIMS gave a molecular formula of C19H24NO4 (observed

238.1692; calculated 330.1699 for M[H]+). From 1D

1H and

13C NMR and 2D NMR the

compound was identified as aspyridone A 84.80

The two aromatic doublets at δH 7.26

(H-16, H-20) and δH 6.80 (H-17, H-19) (Figure 3.28) exhibited HMBC correlations to

para-substituted phenol carbon at δC 158.3 (C-18) and quaternary carbon at δC 125.2 (C-

15). The singlet at δH 7.47 was assigned to methine proton H-6 which displayed 1H-

13C

HMBC connections to a hydroxyl group at δC 177.6 (C-4) and carbonyl group at C-2

(δC 163.9) (Figure 3.27). The H-6 singlet also showed HMBC connection to quaternary

carbon C-15 which confirmed 2-pyridone linked to para-substituted phenol at C-15.

The quartet at δH 4.39 was allotted to methine proton H-8 linked to methylene group at

δC 41.2 (C-9) and to methyl group at δC 17.6 (C-14). The last methyl at δH 0.87 (H-12)

showed correlations to C-10 (δC 33.7) and methylene group at δC 33.7 (C-11). The

methyl group at δH 0.90 (H-13) showed connections to C-9, C-10 and C-11 giving

117

evidence that a similar polyketide chain of preaspyridone A 224 has combined intact to

2-pyridone at C-7 (δC 214.4) to give a structure of aspyridone A 84.

Crystals of aspyridone A 84 were formed. The methyl groups at C-8 and C-10

possess anti-configuration in the crystal data, similar to preaspyridone A 224 minor

isomer and 239.

Figure 3.27: 1H-

1H COSY (solid lines) and

1H-

13C HMBC correlations (arrows) in aspyridone A 84 and crystal structure

of aspyridone A 84.

Figure 3.28: 1H NMR of aspyridone A 84 run in methanol-d4.

118

Figure 3.29: ES+ and UV spectrum of aspyridone A 84.

3.7.2 (d) Identification of 18-deshydroxypreaspyridone A 240

The final compound from A. oryzae apdACE expression clone separated at 24.8

minutes (Figure 3.18). It was obtained as light brown solid (12 mg/L) and HRESIMS

showed molecular formula of C19H25NO3 (observed 338.1735; calculated 338.1726 for

M[Na]+).

The 13

C NMR revealed the presence of 15 carbons and quaternary carbons were

observed in the 2D NMR. A significant difference in the 1H NMR of compound 240

was observed due to the presence of distinct multiplets in the aromatic region (Figure

3.31) in contrast to the distinct pair of doublets which are characteristic of the para-

substituted phenol present in the spectra of preaspyridone 224 and aspyridone A 84

(Figure 3.10 and 3.28). The multiplets from δH 7.16-7.22 were assigned to three

aromatic protons (H-17/19), (H-18) and (H-16/20) (Figure 3.31). The aromatic protons

H-16/20 and H-17/19 displayed HMBC correlations to quaternary carbon at δC 136.9

(C-15) and benzylic carbon at δC 38.3 (C-14) (Figure 3.32). Two set of doublets of

doublets at δH 2.98 and δH 3.07 were assigned to two diastereotopic protons at the

benzylic position, H-14 and showed connection to δC 130.7 (C-17/19), C-15 and to

methine carbon at δC 63.5 (C-5) confirming the structure to be benzene ring attached to

pyrrolidine ring at C-5 connected through benzylic carbon, C-14. The multiplet at δH

3.65 was allotted to methine proton H-7 linked to a carbonyl group at δC 196.0 (C-6),

methylene group at C-8 (δC 41.3), a methine at δC 33.5 (C-9) and a methyl group at δC

119

17.6 (C-13) (Figure 3.30). The multiplets at δH 1.33 and 1.44 were assigned to geminal

protons at H-8 correlated to C-6, C-13, C-12 (δC 19.5), C-10 (δC 30.3), C-9 (δC 33.5) and

C-7 in 1H-

13C HMBC. The multiplets at δH 1.09 and δH 1.34 were assigned to a second

methylene group at H-10 linked to C-11 (δC 11.5), C-12 and C-9 in the HMBC

correlations. All the 1D and 2D NMR elucidated the structure to be 18-

deshydroxypreaspyridone A 240.

Figure 3.30: 1H-

1H COSY (solid lines) and

1H-

13C HMBC correlations (arrows) in 18-deshydroxypreaspyridone A 240.

Figure 3.31: 1H NMR of 18-deshydroxypreaspyridone A 240 run in methanol-d4.

120

Figure 3.32: Segment of 1H-

13C HMBC NMR of 18-deshydroxypreaspyridone A 240.

Figure 3.33: ES+ and UV spectrum of 18-deshydroxypreaspyridone A 240.

3.7.2 (e) Role of apdE in aspyridone pathway

Heterologous expression of hrPKS-NRPS ApdA, enoyl reductase ApdC and

cytochrome P450 enzyme ApdE in A. oryzae was accomplished using the

121

pTAYargACE expression vector. The transformant produced three new products; 14-

hydroxy preaspyridone 238, 4-hydroxy-3-(2,4-dimethylhexanoyl)2-pyridone 239,

aspyridone A 84 and 18-deshydroxypreaspyridone A 240. This experiment exemplified

the role of ApdE in the aspyridone pathway as the new compounds 238, 239 and 240

were not observed in the A. oryzae apdAC expression clone.

The apdE gene encodes a monooxygenase enzyme which belongs to a large

family of cytochrome P450 oxidases. Cytochrome P450 enzyme in fungi are known to

perform important bioconversions and reactions in the biosynthesis of many natural

products, for example selective olefin epoxidation and oxidation of methyl groups of

natural product compounds. They also execute hydroxylation of complex polyaromatic

hydrocarbons, steroids such as progesterone. Some plant pathogenic fungi are reported

to encode P450 enzymes.185

Cytochrome P450 contain a heme cofactor and are known

as hemo proteins. Scheme 3.8 illustrates the reaction by which Ferrous (II) ions in heme

uses molecular oxygen as the oxidant and carries out oxidation of organic substrates by

introduction of oxygen into C-H bond.

Scheme 3.8: The heme in Cytochrome P450 uses molecular oxygen for oxidation of organic substrates.

In aspyridone biosynthesis, ApdE catalyses three important reactions which are:

conversion of preaspyridone A 224 and 230 to aspyridone A 84 by oxidative ring

expansion; hydroxylation of preaspyridone A to form 238; and oxidative dephenylation

of preaspyridone A to from 239. A similar oxidative ring expansion reaction takes place

in tenellin 87 biosynthesis in B. bassiana 110.0 by a cytochrome P450 enzyme known

as TenA. It drives the formation of pretenellin B 115 from pretenellin A 114 by

oxidative ring expansion of 3-acyl tetramic acid to 2-pyridone (see chapter 2).

Prototenellin D 155 is another compound similar to 14-hydroxy preaspyridone A 238 in

tenellin pathway. It is formed by hydroxylation of pretenellin A by direct rebound

122

mechanism by an unknown oxidase enzyme in B. bassiana as it is only produced in the

native organism and not observed during heterologous expression of tenellin 87 genes

in A. oryzae. Halo and colleagues have comprehensively studied and presented a

hypothesis for the mechanism of oxidative ring expansion and benzylic hydroxylation

of tetramic acid in tenellin 87 pathway.104,108

Scheme 3.9: Biosynthesis of pretenellin B 115 and protonellin D 155 in B.bassiana.

Scheme 3.10: A, Proposed oxidative mechanisms for biosynthesis of hydroxyl tetramic acid 238; B, Proposed

mechanism for oxidative ring expansion during biosynthesis of 2-pyridone 84.

123

We proposed similar biosynthetic routes for compounds 238, 84 and 239

(Scheme 3.10, 3.11). Cytochrome P450 initiates by hydrogen atom abstraction from the

benzylic position and forms carbon-centred radical 241. The C-centred radical reacts

with an iron bound hydroxyl radical and gets hydroxylated directly by the oxygen

rebound mechanism186

to form 14-hydroxy preaspyridone 238 (Scheme 3.10, A). The

carbon centred radical 241 can seemingly also follow another route of single electron

transfer and form cyclopropyl oxy-radical 242,187

followed by another short lived

intermediate 243, which consequently leads to ring expansion and form 2-pyridone

compound aspyridone A 84 (Scheme 3.10, B).

Scheme 3.11: Proposed oxidative dephenylation for biosynthesis of 239.

A third mechanism was proposed for the biosynthesis of 4-hydroxy-3-(2,4-

dimethylhexanoyl) 2-pyridone 239. A peroxo-iron intermediate 244 can make

hydroxylation at the phenyl-bridge head carbon of preaspyridone A 224 which results in

formation of assumed hydroxyquinone 245 (Scheme 3.11). Further electron transfer

leads to ring expansion of the pyrrolidone ring to 2-pyridone 246 and the hydroquinone

separates causing dephenylation. The 2-pyridone rearranges and forms 239 (Scheme

3.11). A number of related natural products are known. For example Torrubiellone A

247 and Torrubiellone B 248 from the spider-pathogenic fungi Torrubiella sp. BCC

2165,188

(+)-N-deoxymilitarinone A 249 and militarinone A 250 from

entomopathogenic fungi Paecilomyces farinosus,189

all feature structures which have

been hydroxylated at the carbon corresponding to the oxidation target in the proposed

mechanism, while jacaglabroside B 251190,191

features a more highly oxidised version of

the same structural feature.

124

TenA in the tenellin 87 gene cluster has a similar catalytic activity to ApdE, yet

the latter displays a broader chemical diversity as no dephenylated or similar

compounds were reported from the tenellin 87 pathway. There are a number of reported

structures which are similar to dephenylated compound 239. Piericidins A 252 and B

253 are natural insecticides isolated from Streptomyces mobaraensis.192

They exhibit

specific inhibitory action for electron transport system in mitochondria.

Sapinopyridione 254 and 255 were purified from a fungal pathogen of conifers,

Sphaeropsis sapinea.193

Atpenins 256, 257, 258 are antifungal metabolites of molds

Penicillium sp.FO-125. They are known to inhibit the succinate-ubiquinone reductase

activity of mitochondrial complex II.194

The atpenins also possess pendant methyls

arranged in anti-configuration.

125

3.7.3 Heterologous Expression of apdABC in A. oryzae

The apdB gene encodes a second cytochrome P450 monooxygenase in the

aspyridone cluster.80

The pTAYAargABC plasmid was constructed to reveal the role of

apdB. Selected transformants were grown to be analysed for production of new

metabolites. The transformants were grown on DPY medium on plates and spores were

afterwards inoculated in CMP (section 4.9.b) growth media for seven days according to

methods dercribed in section 4.10. The cultures of A. oryzae apdABC transformants

were extracted in the usual way and the crude extract was defatted with wet methanol

and hexane and then evaporated (80.5 mg from 100 ml).

Figure 3.34: A, Diode array chromatogram of A. oryzae apdABC expression clone showing peaks of preasyridone A

minor isomer 224 at 13.2 and major preaspyridone A 230 at 14.0 minutes; B, Wild type A. oryzae used as a control.

A solution of 10 mg/ml in HPLC grade MeOH of the crude extract was injected

for analysis on LCMS. The diode array chromatogram showed two peaks at 13.2 and

14.0 minutes. They were identified as the minor and major isomers of preaspyridone A

224 and 230 which were characterised from the apdAC expression clone. We observed

an increase in the titres of both compounds in the apdABC expression clone to be 2.2

mg/L and 141.0 mg/L for the minor and major components of preaspyridone A,

respectively. This was double the amount of preaspyridone A in comparison to apdAC

expression clone where the yields of minor and major component were 0.6 mg/L and 60

apdA,B,C

Time0.0

1.0e+1

2.0e+1

3.0e+1

4.0e+1

5.0e+1

6.0e+1

-0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.000.0

5.0e+1

1.0e+2

1.5e+2

ZW-II-92D

14.0

3.32

2.67 6.104.52 29.25

ZW-II-92M

3.22

2.60

3.45

5.80

4.25

27.43

13.2

Wildtype A. oryzae

A

B

126

mg/L, respectively. This expression experiment showed that the ApdB cytochrome

P450 cannot chemically act on the tetramic acid preaspyridone A, but it may act later in

the pathway.

3.7.4 Heterologous Expression of apdACEB in A. oryzae

The A. oryzae apdABC expression clone did not produce any new compounds,

compared to A. oryzae apdAC. We next planned to express the apdB gene in the

presence of the megasynthase ApdA, enoyl reductase ApdC and monooxygenase ApdE

in A. oryzae. The pTAYAGSargPage vector has a capacity to express maximum of four

genes owing to the presence of four promoters.176

Thus pTAYAGSargPage was

modified to construct a plasmid pTAYAargASP inserting all four genes apdA, apdB,

apdC and apdE (Figure 3.35). The expressions of all the genes were confirmed by qRT-

PCR.

Figure 3.35: (left) pTAYAargASP expression plasmid, (right) A. oryzae apdACEB expression clone on Czapek Dox

Agar.

The A. oryzae clones were initially grown on selection media and later on DPY

media (section 4.7.b) for maximum production of spores. The one week old spores were

inoculated in liquid media (CMP) at 28 °C at 200 rpm for 7 days (section 4.10). The

fermentations were extracted in the usual way and the organic layer was concentrated

under vacuum and then defatted with wet methanol and hexane. The resulting crude

127

extract was dried to a thick mass weighing 30 mg/100ml. 10 mg/ml of the crude extract

was made with HPLC methanol and 20 µl was injected in LCMS.

Figure 3.36: A, Diode array chromatogram of A. oryzae apdACEB expression clone showing production of 14-

hydroxypreaspyridone A 238, minor 224 and major isomer of preaspyridone A 230, aspyridone A 84 and three new

compounds at 16.5, 20.0 and 20.3 minutes; B, A. oryzae Wild type.

The LCMS chromatogram displayed a number of peaks which were not present

in the diode array chromatogram of the Wild type (WT) A. oryzae. The wavelength and

mass spectrum of first peak at 9.2 min. was identified as 14-hydroxy preaspyridone 238,

the second peak at 13.6 min. was minor isomer of preaspyridone A 224, third peak at

14.2 min. was major isomer of preaspyridone A 230 and the compound eluting at 14.5

min was aspyridone A 84 (Figure 3.36). These compounds were later confirmed by 1H

NMR.

Three new peaks were detected at 16.5, 20.0 and 20.3 min. (Figure 3.36). The

mass spectrum for the compound eluting at 16.5 min. was m/z 254 [M]H+, 16 mass units

more than the dephenylated aspyridone 239 and observed λmax of 278, 335 nm. The

peak at 20.0 minutes showed a uv absorption 228, 330 nm and ESI spectrum presented

m/z 268 [M]H+. The last peak at 20.3 min. showed a higher uv absorption (294, 376 nm)

and m/z 330 [M]H+.

For purification of all the peaks 1 litre (100 ml x 10 flasks) of the cultures of the

transformant was grown according standard fermentation conditions (section 4.10). The

Time2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00

14.2

13.53.43

2.25

9.14.18

16.5 20.320.0

3.22

2.60

3.45

5.8

4.25

Wildtype A. oryzae B

A

128

crude extract obtained after extraction and concentration was 1.13 g per liter. A

concentration of 50 mg/ml was made with HPLC grade methanol. 200 µl of the solution

was injected in Waters LCMS mass directed purification in each preparative run. The

purification was achieved with an acetonitrile and water solvent system with Method 3

(section 4.5) in a 30 min. duration programme. The purified fraction of each peak was

collected and dried under nitrogen. The last peak at 20.3 min. was not collected due to

lower titres (it was later purified in another experiment explained in section 3.7.5). The

structures of pure compounds were each studied with the help of 1D and 2D NMR

spectrometer and high resolution mass spectrum.

3.7.4 (a) Identification of 1, 4-dihydroxy-3-(2, 4-dimethylhexanoyl) 2-

pyridone 259

The compound eluting at 16.5 min. was obtained as light brown solid 6.2 mg/L.

The HRESIMS provided the molecular formula to be C13H19NO4 (observed 276.1218;

calculated 276.1206 for [M]Na+). The

13C NMR showed the presence of all 13 carbons,

with many chemical shifts similar to dephenylated aspyridone 239. The 13

C NMR

composed of two carbonyls groups at δC 213.6 (C-7) and δC 160.0 (C-2), a quaternary

carbon at δC 107.4 (C-3), a hydroxyl group at C-4 (δC 176.0), two methylene groups at

δC 31.0 (C-11) and δC 41.1 (C-9) and three methyl groups at δC 19.3 (C-13), δC 17.3 (C-

14) and δC 11.7 (C-12). A quartetet at δH 4.31 allocated to H-8 displayed 1H-

1H COSY

correlation to H-14 (δH 1.11) and to the methylene carbon C-9 in the 2D 1H-

13C HMBC.

The multiplets at δH 1.33 and δH 1.62 were assigned to geminal protons at H-9 linked to

C-7, C-14, C-13, C-11, C-10 and C-8 in the HMBC correlations (Figure 3.37 and 3.39).

The methine proton H-10 signals at δH 1.41 and multiplets at δH 1.18 and δH 1.36 were

consigned to the two geminal protons at H-11. These correlations verified that the

aliphatic chain is the same as in dephenylated preaspyridone 239 and no hydroxylation

has occurred on the polyketide chain. The pair of aromatic douplets at δH 5.97 and δH

7.94 were assigned to protons H-5 and H-6 respectively, of the 2-pyridone (Figure

3.38). The distance between the doublets was more than observed in the 1H NMR of

dephenylated aspyridone 239 and this was ascribed to the presence of a hydroxyl group

attached to the pyridone nitrogen. From the NMR correlations and HRMS the structure

was established to be 1, 4-dihydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 259.

129

Figure 3.37: 1H-

1H COSY (solid lines) and

1H-

13C HMBC correlations (arrows) in 1, 4-dihydroxy-3-(2, 4-

dimethylhexanoyl) 2-pyridone 259.

Figure 3.38: 1H NMR of 1, 4-dihydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 259 run in methanol-d4.

Figure 3.39: Key 1H-

13C HMBC correlations in 1, 4-dihydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 259.

130

Figure 3.40: ES+ and UV spectrum of 1, 4-dihydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 259.

3.7.4 (b) Identification of 1-methoxy, 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-

pyridone 260

The compound 260 separating at 20.0 minutes was purified in the form of light

brown solid (2.7 mg/L) and HRESIMS analysis presented a molecular formula of

C14H21NO4 (observed 290.1368; calculated 290.1362 for [M]Na+). The

13C NMR

displayed presence of 14 Carbons. Many chemical shift values were similar to

dephenylated aspyridone A 239 as it consists of three methyl groups at δC 11.7 (C-12),

δC 17.3 (C-14) and δC 19.3 (C-13), two methylene groups at δC 31.0 (C-11) and δC 41.1

(C-9), two methine at δC 42.2 (C-8) and δC 33.5 (C-10), two carbonyls at δC 213.6 (C-7)

and δC 159.7 (C-2) and aromatic carbons at δC 100.7 (C-5) and δC 143.0 (C-6). A new

carbon signal was observed at δC 65.6 which indicated that it is attached to an oxygen

atom.

The 1H NMR consists of two sets of doublets at δH 6.02 (H-5) and δH 8.03 (H-6),

assigned to aromatic protons of 2-pyridone (Figure 3.43). The 1H NMR presented a

singlet at δH 4.01 integrating to three protons which was not observed in dephenylated

aspyridone A 239. It was attached to δC 65.6 in 1H-

13C HSQC. A characteristic feature

which aided in elucidation of the structure was revealed in 1D NOESY when δH 8.03

131

(H-6) showed correlation to δH 4.01, which determined that an O-methyl is attached to

nitrogen atom at position 1 in 2-pyridone (Figure 3.41).

Figure 3.41: 1D NOESY showing connection of H-6 with methoxy group at N-1.

In 1H-

13C HMBC spectrum, H-5 presented two bond and three bond correlations

with hydroxyl group at C-4 (δC 177.3) and quaternary carbon (δC 108.2) respectively

(Figure 3.44). The aromatic proton H-6 displayed connections to carbonyl group at C-2,

aromatic carbon C-5 and hydroxyl group at C-4. We didn’t observe any HMBC

correlations from H-15 to C-2 or C-6; this might be because four bond correlations are

rarely seen. The terminal methyl at δH 0.87 (H-12) was linked to methylene group at δC

31.0 (C-11) and methine at C-10 (δC 33.5) and adjacent methyl signal at δH 0.92 (H-13)

diplayed HMBC correlations to C-10, C-11 and C-9 (Figure 3.42). The geminal protons

at δH 1.31 and δH 1.61 at H-9 shows connections to carbonyl group at C-7 (δC 213.6),

methine carbon at δC 42.2 (C-8), C-10, C-11 and C-13. This confirms a 2, 4-

dimethylhexanoyl side chain identical to aspyridone A 84. These correlations establish

the structure to be 1-methoxy, 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 260.

132

Figure 3.42: 1H-

1H COSY (solid lines) and

1H-

13C HMBC correlations (arrows) in 1-methoxy, 4-hydroxy-3-(2, 4-

dimethylhexanoyl) 2-pyridone 260.

Figure 3.43: 1H NMR of 1-methoxy, 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 260 run in methanol-d4.

Figure 3.44: Key 1H-

13C HMBC correlations in 1-methoxy, 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 260.

133

Figure 3.45: ES+ and UV spectrum of 1-methoxy, 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 260.

3.6.4 (c) Discussion on role of apdB

The apdABCE expression clone produced a range of compounds including the

two diastereomers 224 and 230 observed when megasynthase ApdA was expressed with

enoyl reducatse ApdC and 14-hydroxy preaspyridone A 238, dephenylated aspyridone

239 and aspyridone A 84 which are products of the monooxygenase ApdE. This

illustrates that as more genes from a cluster are expressed in a heterologous experiment,

we observe novel biosynthetic potential of the specific gene cluster.

The production of 1-methoxy dephenylated pyridone 260 and N-hydroxy

dephenylated pyridone 259 ascribes a role for the cytochrome P450 enzyme ApdB, that

it is involved in N-hydroxylation of 5-dephenylated pyridone 239 to form 259 and 260.

It exhibits substrate specificity for dephenyalted pyridines only, because we didn’t

observe any N-hydroxylation in tetramic acids 224 and 230 and neither in aspyridone A

84.

In tenellin, the cytochrome P450 enzyme TenB also displays N-hydroxylation

for only pretenellinB to form tenellin 87 and TenB does not N-hydroxylates tetramic

acids pretenellin A 114 (see chapter 2). We assumed that the O-methylation in 260 is

carried out by a native enzyme present within A. oryzae as there is no enzyme for

putative methyl transferase characteristic in aspyridone cluster.80

There are a number of

reported compounds which possess N-methoxy group, for example Cordypyridone C

134

261 and Cordypyridone D 262 from the insect pathogenic fungus Cordyceps

nipponica.195

Kumarihamy et al. isolated four new N-methoxy-2-pyridinone compounds

from the plant pathogen Septoria pistaciarum, which are 17-hydroxy-N-(O-methyl)

septoriamycin A 263, 17-acetoxy-N-(O-methyl) septoriamycin A 264, 13-(S)-hydroxy-

N-(O-methyl) septoriamycin A 265 and 13-(R)-hydroxy-N-(O-methyl) septoriamycin A

266.196’’

3.7.5 Heterologous expression of apdACED in A. oryzae

ApdD encodes a FAD dependent monooxygenase. We planned to coexpress the

apdD gene in the presence of the megasynthase ApdA, enoyl reductase ApdC and

monooxygenase ApdE in A. oryzae to illustrate the role of ApdD in aspyridone

biosynthesis. The pTAYAargASP plasmid was used to construct pTAYAargACED

consisting of apdA, apdE, apdC and apdD and this was transformed into A. oryzae. The

transcription levels of all the genes were confirmed by qRTPCR (This transformation

was done by Dr. Khomaizon Pahirulzaman).

The selected transformant was grown on DPY plates and when mature spores

were visible after seven days, a spore solution was made in sterilised deionized water

and was inoculated in each 100 ml liquid media kept in 250 ml Erlenmeyer flask. 1

Litre of the media (10 x 100 ml flasks) was grown with constant shaking at 200 rpm at

28 °C. After seven days the fungal cultures were extracted with ethyl acetate (section

4.11). The organic layer was concentrated and defatted with hexane. Drying the crude

extract under nitrogen, gave a brown mass (1.128 g / 1000 ml). A 10 mg/ml solution

was made with HPLC grade methanol and analysed on LCMS using CH3CN/H2O

analytical programme with a gradient (50-65%) with 30 min. duration (Figure 3.46).

135

Figure 3.46: A, Diode array chromatogram of apdACED consisting of 14-hydroxy preaspyridone A 238 at 10.2 minutes,

dephenylated pyridone 239 at 16.6 minutes, aspyridone A 84 at 17.3 minutes and a new compound at 24.4 minutes

(green); B, Diode array chromatogram of wild type A. oryzae.

The LCMS chromatogram displayed four noticeable peaks which were not

present in the wild type A. oryzae. The peak at 10.2 min. was identified as 14-hydroxy

preaspyridone 238 possessing a mass of m/z 348 in [M]H+ and λmax of (221, 283 nm);

the second peak at 16.6 min. was recognized as dephenylated pyridone 238 with a λmax

of (221, 283 nm) and a m/z of 238 in the ES+. The third peak at 17.3 min. had a uv

absorption of (246, 345 nm), characteristic of aspyridone A 84, exhibiting a molecular

ion of m/z 330 [M]H+. The last peak at 24.4 min. showed a m/z of 330 as [M]H

+ and a

λmax of (294, 376 nm). This peak was observed in the apdABCE expression clone but

was not purified and characterised previously because of low titre (section 3.7.4).

The crude extract (1.13g) was prepared to a concentration of 100 mg/ml and 200

µl was injected in each run in mass directed preparative LCMS. The gradient used was

(50-65%) in acetonitrile/water (Method 6, section 4.5). Each of the above peaks were

purified, collected and dried under nitrogen. The first three compounds at 10.2, 16.6 and

17.3 min. after purification were analysed by 1D 1H NMR and confirmed to be 14-

hydroxy preaspyridone 238, dephenylated pyridone 239 and aspyridone A 84. The last

peak which was a new compound was studied with 1D 1H and

13C and 2D NMR

experiments.

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

16.6

3.55

4.0210.2

17.3

24.4

3.22

3.45

5.80

4.25

Wildtype A. oryzae

A

B

136

From the above experiment we couldn’t observe any precise role for ApdD.

Bergmann et al. guessed that apdD performs hydroxylation of aspyridone A 84 to form

aspyridone B 226.80

But we did not observe any peak confirming the production of

aspyridone B 226 in the expression clone. However, we observed that the presence of

ApdD in the expression clone with ApdE reduced the production of metabolites to 71.5

mg as compared to A. oryzae apdACE where total titres of metabolites were 390.6 mg.

(section 3.7.10).

3.7.5(a) Identification of Z-5, 14-anhydropreaspyridone A 267

The compound eluting at 24.4 min. was obtained as a bright yellow solid (4 mg)

after repurification. The molecular formula in HRESIMS analysis was given as

C19H23NO4 (observed 352.1528; calculated 352.1519 for [M]Na+). The

13C NMR

spectra consisted of three methyl groups at δC 11.6 (C-11), δC 17.5 (C-13), δC 19.5 (C-

12), two methylene groups at δC 30.6 (C-10) and δC 41.5 (C-8) and a methine group at

δC 33.6 (C-9), two aromatic signals at 116.9 (C-17, C-19) and 132.3 (C-16, C-20), a

quaternary signal at 126.5 (C-15) and a hydroxyl signal at C-18 (δC 159.4). A

quaternary carbon at δC 184.0 observed in 2D HMBC was assigned for C-4 and an

alkene carbon at δC 110.4 in 2D HSQC was assigned for C-14.

A structural feature of this compound was revealed in the 1H NMR by the

presence of a singlet at δH 6.52 (Figure 3.47), joined to δC 110.4 in the 1H-

13C HSQC.

The signal at δH 6.52 displayed 1H-

13C HMBC connection to 4-hydroxy of the

pyrrolidine nucleus at δC 184.0 (C-4) and to aromatic carbons at C-16/20 (Figure 3.49

and 3.50). From the above correlations the signal at δH 6.52 was assigned to H-14

attached at the benzylidene position of a tetramic acid. The aromatic doublet at δH 7.40

assigned to H-16/H-20 displayed HMBC connections to benzylidene carbon at δC 110.4

(C-14) and hydroxyl group at C-18. The second aromatic doublet at δH 6.83 assigned to

H-17 and H-19 show couplings to quaternary carbon C-15 and para hydroxyl group at

C-18 (Figure 3.49 and 3.50).

137

Figure 3.47: 1H NMR of Z-5, 14-anhydropreaspyridone A 267 run in methanol-d4.

In order to determine the orientation of the olefin between C-5 and C-14, a three

bond long range J coupling between H-14 and quaternary carbon C-4 was measured

from a 2D selective EXSIDE NMR experiment.191

The EXSIDE NMR is a 2D spectrum

and displays cross peak similar to a HMBC spectrum. The cross peak is a split in the

Carbon dimensions and this splitting gives the value of J (C-H) constant. Figure 3.48

shows cross peaks between H-14 and C-4, labelled in Hertz. The second number in each

peak is the 13

C frequency in Hertz and the difference between both peaks (22974.16 Hz

-23019.82 Hz) is 45 Hz. A 15-fold J-scaling factor is used which give a value of 3 Hz

coupling constant between H-14 and C-4. The value of 3JHC for two possible E/Z

isomers of 267 by DFT calculations gave a value of 7.5 and 4.5 Hz respectively, which

further supported the EXSIDE experiment and the double bond between C-5 and C-14,

was confirmed to be a cis configuration.191

(DFT calculations were performed by Dr.

Craig Butts, School of Chemistry, University of Bristol). We failed to determine the

geometry of this compound from 1D NOESY and 1D ROESY because of exchangeable

protons between N-H and hydroxyl group at C-18.

138

Figure 3.48: Selective EXSIDE NMR spectrum showing cross peaks coupling between H-14 and C-4 to be 3 Hz in 267.

The multiplet at δH 3.85, assigned to methine at H-7 showed couplings to

pendant methyl group at C-13 and to methylene group at C-8. The geminal protons at δH

1.40 and δH 1.59 were assigned to H-8, linked to methyls at C-13, C-12 and to

methylene at C-10 and to methine at C-9. The multiplets at 0.87 and 0.90 were allocated

to terminal methyl H-11 and pendant methyl H-12 respectively displaying HMBC

correlations to C-9, C-10 and methylene C-8 (Figure 3.49). These connections showed

that this compound also possess a similar dimethylated tetraketide chain as observed in

preaspyridone 224 and 230. The above correlations decided the structure to be Z-5, 14-

anhydropreaspyridone A 267.

Figure 3.49: 1H-

1H COSY (solid lines) and

1H-

13C HMBC correlations (arrows) in Z-5, 14-anhydropreaspyridone A 267.

139

Figure 3.50: Key 1H-

13C HMBC NMR correlations in Z-5, 14-anhydropreaspyridone A 267.

Figure 3.51: ES+ and UV spectrum of Z-5, 14-anhydropreaspyridone A 267.

3.7.6 Coexpression of apdG with apdACEB in A. oryzae

The apdG gene, located downstream of megasynthase apdA and exporter gene

apdR in the gene cluster, encodes an acyl dehydrogenase. Bergmann et al.80

predicted

that it assists in tetramic acid ring closing and reductive release of preaspyridone A 224

from the PKS-NRPS domains (section 3.4). However, the production of preaspyridone

140

A 224 and 230 from A. oryzae apdAC expression clone and from in vitro analysis by

Tang et al. (section 3.4), contradicts Bergmann et al. hypothesis. In order to explore the

function of ApdG, we planned to co-express apdG with apdA, apdB, apdC and apdE in

A. oryzae.

The vector pTAYAargASP carried apdABCE and can express only four genes.

To express apdG Dr. Khomaizon Pahirulzaman designed another plasmid which

contained a different selection marker, the bleomycin resistance gene, ble. The argB

gene in the original vector pTAYAGSargPage was replaced by bleomycin resistance

gene ble and the insertion of this gene was driven by PtrpC. This plasmid was used to

enclose apdG inserted next to PgpdA, this plasmid was named pTAYAGSbleG (Figure

3.52). A. oryzae (M-2-3) was co-expressed with pTAYAargASP (carrying apdABCE)

and pTAYAGSbleG (carrying apdG). The minimal media was supplemented with

antibiotic for selection of transformants.

Figure 3.52: Vector pTAYAGSbleG carrying apdG used in A.oryzae apdABCEG expression clone.

The selected transformant was grown on DPY plates for a week, spores were

prepared in sterile deionized water and then transferred to liquid media (CMP media,

section 4.9.b). One flask (100 ml media in 250 ml Erlenmeyer flask) was grown for

141

seven days at 28 °C with constant shaking at 200 rpm. The mycelia and liquid media

were homogenized and acidified before extraction and defatting in the usual way to give

crude extract weighing 62.2 mg/100 ml. A 10 mg/ml solution was made and analysed

with LCMS on a 30 minutes analytical programme using CH3CN/H2O (50-70%

gradient) (Figure 3.53).

Figure 3.53: A, Diode array chromatogram of apdABCEG expression clone showing the production of 14-hydroxy

preaspyridone 238 at 8.9 minutes, diastereomers of preaspyridone A 224 and 230 at 13.9 and 14.4 minutes, aspyridone

A 84 at 14.9 minutes, N-hydroxy dephenylated pyridone 259 at 17 minutes and O methoxy dephenylated pyridone 260

at 21.2 minutes; B, Diode array chromatogram of wild type A. oryzae.

The yield of these compounds from 1 litre fungal culture was calculated by

quantification experiment (see section 3.6.10). The quantity of 14-hydroxy

preaspyridone 238 was 42.6 mg/L, major diasteomer of preaspyridone A 230 was 98

mg/L, aspyridone A 84 was 12.6 mg/L, N-hydroxy dephenylated pyridone 259 was 15.8

mg/L and N-O methoxy dephenylated pyridone 260 was 8.2 mg/L. The above

mentioned compounds are the same as observed in the apdABCE expression clone only

they are produced in higher titres in apdABCEG expression clone (see section 3.7.4).

From the above heterologous expression we couldn’t settle a precise role for apdG only

that when it was expressed with other four genes from the cluster it displayed increase

in the production of metabolites.

Time6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00

6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00

14.4

8.9

6.48

14.921.217.0

8.28 10.02 13.27

Wildtype A. oryzae

A

B

142

3.7.7 Heterologous expression of apdAC and tenA in A. oryzae

The tenA gene encodes a cytochrome P450 enzyme during tenellin 87

biosynthesis in B. bassiana 110.25 and the protein encoded by apdE in the aspyridone

gene cluster displays 48 % sequence similarity to TenA. TenA catalyses the oxidative

ring expansion of the pyrrolidone in pretenellin A 114 to a 2-pyridone compound,

pretenellin B 115.108

We planned to express megasynthase apdA and enoyl reductase

encoding gene apdC with tenA in A. oryzae to determine if TenA displays broader

substrate selectivity and turnover of preaspyridone A 224 and 230 produced by apdAC

to aspyridone A 84. Earlier the Cox group achieved effective gene swaps between

tenellin 87 and DMB 88 when enoyl reductase dmbC from DMB 88 cluster and PKS-

NRPS tenS were co-expressed in A. oryzae, they produced a precursor compound of

tenellin 87 pathway, pretenellin A 114.109

Dr. Khomaizon Pahirulzaman constructed a vector pTAYAargACtenA carrying

PKS-NRPS coding gene apdA, enoyl reductase encoding gene apdC and ring expandase

gene tenA and expressed it in A. oryzae. One producing transformant was grown on

DPY solid media and later mature spores were inoculated in aspyridone growth media

(CMP media) after growing for 7-10 days. The fungal culture was grown in 100 ml

liquid media in 250 ml flask at 28 °C on shakers at a speed of 200 rpm for seven days.

Mature mycelia were homogenised and extracted with ethyl acetate (see section 4.11).

The organic layer was concentrated and defatted. A crude extract of 65.3 mg/100 ml

was obtained after drying. The crude extract prepared in HPLC grade methanol (10

mg/ml) was analysed on analytical LCMS programme with CH3CN/H2O having a

gradient of 55-65% in 30 minutes (Figure 3.54).

143

Figure 3.54: A, Diode array chromatogram of apdACtenA expression clone showing production of minor preaspyridone

A diastereomer 224 at 13.3 min. and major preaspyridone A diastereomer 230 at 14.3; B, Diode array chromatogram

of wild type A. oryzae.

The LCMS chromatogram displayed production of major and minor diasteomers

of preaspyridone A 224 and 230 presenting a molecular ion of 332 [M]H+

and a uv

spectrum of 222, 278 nm. This experiment indicated that TenA has restricted substrate

specificity and didn’t convert preaspyridone A 224 to aspyridone A 84. But the titres of

minor component of preaspyridone A 224 (2.2 mg/L) and major preaspyridone A 230

(121 mg/L) in apdACtenA expression clone was higher than produced in apdAC

expression (see section 3.7.1). A similar effect in increase in preaspyridone A 224 and

230 was observed in apdABC expression. With presence of ApdB and TenA we didn’t

observe any new metabolites but the production of preaspyridone A 224 and 230 was

increased.

3.7.8 Heterologous expression of apdACE and tenB in A. oryzae

TenB, a cytochrome P450 protein in tenellin 87 gene cluster catalyses the N-

hydroxylation of the 2-pyridone in pretenellin B 115 and froms tenellin 87.108

In an A.

oryzae transformation we planned to express tenB with apdACE to explore the catalytic

activity of TenB and whether it N-hydroxylates the 2-pyridone compound, aspyridone A

Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

14.3

3.38

2.62 6.074.55 13.3 28.52

3.22

2.60

3.45

5.80

4.25

27.43

Wildtype A. oryzae

A

B

144

84. Dr.Khomaizon Pahirulzaman modified the vector pTAYAargASP to form

pTAYAargACEtenB carrying genes apdACE and tenB.

A number of A. oryzae transformants were obtained and one was selected for

screening by LCMS. The transformant was grown on DPY media and after 7-10 days

when spores were well grown, spore solution was inoculated in CMP liquid media (1 x

100 ml) in a 250 ml flask for seven days at 28 °C (see section 4.10). The fungus culture

was extracted and defatted in the usual way. A solid mass weighing 27.4 mg/100ml was

obtained. 10 mg/ml in HPLC methanol was made from the crude extract and analysed

by LCMS with CH3CN/H2O with gradient 55-65% in 30 minutes.

Figure 3.55: A, Diode array chromatogram of A. oryzae apdACEtenB expression clone displaying production of 14-

hydroxy preaspyridone A 238, preaspyrdione A 230, dephenylated aspyridone 239 and aspyridone A 84; B, Diode array

chromatogram of wild type A. oryzae.

The LCMS chromatogram revealed the production of 14-hydroxy preaspyridone

A 238 (m/z 348 [M]H+, λmax 223, 281 nm), preaspyridone A 230 (m/z 332 [M]H

+,

λmax223, 280 nm), dephenylated aspyridone 239 (m/z 238 [M]H+, λmax229, 325 nm ) and

aspyridone A 84 (m/z 330 [M]H+, λmax247, 344 nm). One litre (10 x 100 ml) of

apdACEtenB clone was grown for purification of compounds. The crude extract

achieved after extraction and concentration was 764 mg/L. The extract was made to a

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

15.4

4.42

2.78

4.78

14.55.02 9.5

3.22

2.60

3.45

5.80

4.25

15.9

Wildtype A. oryzae

B

A

145

concentration of 50 mg/ml and 100 µl was injected in each run of auto purification on

mass directed preparative LCMS. The solvents used were acetonitrile/water with a

gradient of 50-75% carried out in 30 min (Method 3) programme. The purified

fractions of each compound was collected and dried. All above stated compounds were

verified with 1H NMR spectroscopy. The yield of 14-hydroxy preaspyridone A 238 was

5.2 mg, preaspyridone A 230 was 11 mg, dephenylated aspyridone 239 was 28 mg and

aspyridone A 84 was 2.3 mg.

We didn’t observe any new or N-hydroxylated 2-pyridone compounds which

confirm that tenB is highly selective and didn’t take any 2-pyridone compounds from

aspyridone pathway as its substrate.

3.7.9 [1-13

C] L-Tyrosine feeding

Earlier aspyridone A 84 and preaspyridone A 224 were reported from hybrid

PKS-NRPS gene cluster by Bergmann et al.80

and Tang107

and co-workers. They are

synthesized from the fusion of a tetraketide 220 and an amino acid tyrosine 107. With

hetererologous expression of apdACE genes and consequential later experiments we

achieved novel dephenylated aspyridone compound, 4-hydroxy-3-(2, 4-

dimethylhexanoyl) 2-pyridone 239. We carried out [1-13

C] L-tyrosine feeding with 5-

dephenylated pyridone 239 producing clone apdACEtenB, firstly to determine if 5-

dephenylated pyridone 239 utilize L-tyrosine as its amino acid precursor and secondly

to ascertain that dephenylated 2-pyridone 239 is an oxidative breakdown product of

aspyridone pathway.

25 mg of [1-13

C] L-tyrosine was dissolved in 3 ml of deionized water with 5µl of

1 molar NaOH. 1 ml of this solution was added in three flasks each containing 100 ml

of 3 days old fungal culture of A. oryzae apdACEtenB in 250 ml Erlenmeyer flask. This

was repeated on 4th

and 5th

day as well. One 250 ml flask containing 100 ml of media

was grown as a control with no [1-13

C] feeding. The fungal cultures were grown

according to the standard fermentation conditions (see section 4.10). On the seventh day

the fungal mycelia was homogenized, acidified and extracted with ethyl acetate. The

organic layer was semi concentrated and defatted. After defatting the extract was dried

to a brown mass weighing 71.7 mg/300 ml. A 50 mg/ml solution was made with HPLC

146

grade methanol and 50µl was injected in each run of mass directed auto-purfication on

LCMS instrument. The gradient used on a CH3CN/H2O solvent was 55-60 % in 30 min.

duration. The purified fractions were collected and fully dried under nitrogen gas. The

yield of 14-hydroxy preaspyridone 238 was 2.8 mg, preaspyridone A 230 was 6.8 mg,

5-dephenylated pyridone 239 was 9.8 mg and aspyridone A 84 was 1.4 mg. Each of

these compounds was studied by 1D 13

C NMR spectroscopy and compared parallel with

un-labelled pure compounds.

Figure 3.56: A, 13

C NMR showing incorporation of [1-13

C] L-tyrosine at C-4 in preaspyridone A 230; B, 13

C NMR of

preaspyridone A 230 without isotope feeding.

The products of A. oryzae apdACEtenB expression clone are 14- hydroxy

preaspyridone A 238, preaspyridone A 230, dephenylated 2- pyridone 239 and

aspyridone A 84. In [1-13

C] L-tyrosine feeding preaspyridone A 230 exhibited 30%

incorporation of L-tyrosine at C-4 (Figure 3.56) and dephenylated 2-pyridone 239

displayed 13% incorporation of [1-13

C] L-tyrosine at C-4 (Figure 3.57). We didn’t

observe any incorporation of isotope labelled tyrosine in 14- hydroxy preaspyridone A

238 and aspyridone A 84. The feeding experiment confirmed that dephenylated

13C feeding

No feeding

A

B

147

pyridone 239 derives the same PKS-NRPS biosynthetic pathway as preaspyridone A

230 and aspyridone A 84. It also confirms that the adenylation domain of the NRPS

selects the amino acid, L-tyrosine.

Figure 3.57: A, 13

C NMR showing incorporation of [1-13

C] L-tyrosine at C-4 in dephenylated 2-pyridone 239; B, 13

C

NMR of dephenylated 2-pyridone 239 without isotope feeding.

3.7.10 Quantification of metabolites

The titres of different compounds produced in various heterologous expression

clones can vary owing to differences in metabolic and environmental conditions. For

this reason, we carried out quantification of all aspyridone metabolites by measuring

standard calibration curves. Serial dilutions of pure compounds from 30 µg/ml-1000

µg/ml were made with HPLC grade methanol and diode array chromatograms were

obtained on LCMS with acetonitrile/water using gradient 50-70% in 20 min. Integration

values of absorption peaks were recorded at the λmax value for each compound. Graphs

were plotted between integration values versus different concentration used in serial

dilution. This gave a standard calibration curve and linear equation for each compound.

No feeding

13C feeding

A

B

148

100 ml of each A. oryzae transformant in a 250 ml flask was grown and crude extracts

were obtained according to the method given in section 4.11. LCMS was run on these

samples and peaks were first identified by standard retention time, uv spectra and mass

spectra. The yields of each compound in the crude extracts were then calculated from

their respective standard linear equation at their respective retention time and λmax value.

The values are arranged in Table 3.1.

149

Table 3.1: Quantification of metabolites in mg/L.

Transformant

238 224 230 84

260 259

239

267 240 Total

yield

apdA,C

430 mg

0 0.61 60.70 0 0 0 0 0 trace 61.3

apdA,C,E

1126 mg

183.6 0 0 119.8 0 0 72.2 0 15 390.6

apdA,B,C

805 mg

0 2.2 141.0 0 0 0 0 0 trace 143.2

apdA,C,tenA

653 mg

0 2.2 121.0 0 0 0 0 0 trace 123.2

apdA,C,E,B

300 mg

2.6 5.7 34.6 1.8 2.7 6.2 trace 4 trace 53.6

apdA,C,E,tenB

(274mg from 100ml)

2.9 0 5.3 3.2 0 0 6.9 6 0 24.3

apdA,C,E,D

448 mg

8.2 0 0.3 5.1 0 0 49.9 8 0 71.5

apd A,B,C,E,G

(62.2 mg from

100ml)

42.6 0 98 12.6 8.2 15.8 0 trace trace 177.2

150

3.7.11 Discussion and Conclusions

Aspyridone A 84 and aspyridone B 226 were reported to be the exclusive

products of a silent hybrid PKS-NRPS gene cluster in A. nidulans.80

It was discovered

by the Hertweck group80

and they activated the cluster by overexpressing the

transcription regulator apdR. In this Chapter we studied the gene cluster of aspyridone

84 using a heterologous gene expression technique in A. oryzae via a lately devised

multi gene expression plasmid pTAYAGSargPage.176

It consists of strong constitutive

promoters for each subject gene and resulted in successful transcription of apd genes in

the heterologous host. The coexpression of apd genes in A. oryzae in an orderly scheme

disclosed advance oxidative modification and programming potential of respective

genes leading to a number of new compounds.

Heterologous expression of apdAC formed the previously known precursor

compound preaspyriodone A 224.107

In our project preaspyridone A was isolated as a

major and a minor diastereomer compounds 224 and 230, being epimers at C-5 (section

3.7.1). The formation of crystals of minor preaspyridone A isomer 224 presented anti-

arrangement of the pendent methyl groups of the alkane chain which is opposite to that

reported before.80

We established that aspyridone A 84 is the product of three genes

apdAC and apdE. Similar result is reported by Niehaus et al.,197

they studied the

biosynthesis of fusarin C in the fungus Fusarium fujikuroi. The fusarin gene cluster

consists of nine genes but they proved that only four genes are required for the

biosynthesis of fusarin C. So, we concluded that it is difficult to predict what a gene

actually does and how many genes are required for the biosynthesis of a single

compound.

The A. oryzae apdACE expression clone showed distinctive catalytic feature of

cytochrome P450 apdE that it performs oxidative ring expansion of preaspyridone A

224 to form aspyridone A 84, benzylic hydroxylation of 224 to form 14-hydroxy

preaspyridone A 238 and loses a phenoxide to form the novel compound, 5-

dephenylated pyridone 239. The apparent incorporation of [1-13

C]-L-tyrosine by 5-

dephenylated pyridone 239 and the precursor compound preaspyridone 230 confirms

that tyrosine is the precursor amino acid and biosynthesis of 239 is linked to

preaspyridone 230. The structure of 5-dephenylated pyridone 239 and its derivatives

259 and 260 are similar to known fungal metabolites, the Atpenins 256-258. The

151

biosynthetic pathway of Atpenins is not reported and we suggest that they must be

products of a similar PKS-NRPS cluster like the aspyridone genes. The polyketide chain

in Atpenins and in 84, 224, 239, possess pendant methyls arranged in anti configuration.

The methyls are derived form S-adenosyl methionine by the CMeT domain. The ER

domain in ApdA is inactive and the stand alone enoyl reductase ApdC acting in trans

sets the stereochemistry of the carbon bearing the methyls. The ApdC remarkably sets

an opposite stereochemistry of the two methyls during the first and second cycle of the

tetraketide producing anti dimethylation pattern. The characterization of 18-deshydroxy

preaspyridone A 240 in apdACE and in trace amounts in other transformants (Table 3.1)

shows a wider specificity of the adenylation domain to incorporate other amino acids

than L-tyrosine. This is also in agreement of the in vitro studies performed by Tang and

coworkers.107

The gene apdB encodes a cytochrome P450 enzyme which didn’t accomplish

any chemical step when expressed with apdAC alone but was observed to increase the

titres of the metabolites. However, when expressed with apdACE in A. oryzae apdACEB

expression it was resolved that it catalyses N- hydroxylation of dephenylated 2 pyridone

239 to form a new compound 259. The apdACEB was an exclusive clone to form nine

different compounds (Table 3.1) owing to the presence of two cytochrome P450s but

the overall yield of this transformant was less (53.6 mg/L) as compared to apdACE (390

mg/L).

We did not perceive a role for the FAD dependent mono oxygenase apdD. As

we didn’t observe the production of aspyriodone B 226, we suspect it might have been

catalysed by a gene outside the cluster which is possible in transcription mediated

recombination where simulataneously many genes are turned on. The presence of apdD

also lowered the yield (71.5 mg/L) of metabolites in apdACED expression than

achieved in apdACE transformant (Table 3.1). The gene apdG didn’t exhibit any

catalytic role in apdACEBG expression clone but increased the titres of metabolites

produced. This might due to the fact that biosynthetic proteins work in clusters and with

more proteins in a heterologous expression there is more protein-protein interaction and

more production. But it cannot be concluded decisively without further experimental

studies. The expression of tenellin genes with apd genes showed high substrate

specificity of tenellin biosynthetic genes as we didn’t observe their particular role in the

152

transformants. The presence of tenA with apdAC increased the titres of the compounds

with a similar effect as apdB in apdABC expression clone.

Thus heterologous expressions of apd genes in suitable host, A. oryzae produced

new compounds and help understand the role of specific genes in aspyridone cluster of

A. nidulans.

153

Chapter 4

Experimental

4.1 General Chemicals and Equipment

All chemicals and reagents used were of analytical grade and obtained from

Sigma Aldrich, Fischer, Fluka analytical and BDH laboratories. All solvents used in

HPLC and purification were HPLC grade. Deionized water was used in all experiments.

Weighing balances were of Sartorius AX224. Small centrifuge for obtaining clear

sample were from AG Hanburry 22331. Sterilizations were carried out using Astell

Autoclave at 121 ºC for 15 minutes. Optical rotations were measured with an ADP 220

polarimeter at 589 nm. Melting points were determined using Electrothermal apparatus.

IR data were obtained using a Perkin–Elmer FTIR instrument.

4.2 Mass Spectrometry

Electrospray ionization (ESI) mass spectra were recorded on a VG Quattro-Mass

spectrometer or Bruker microtof mass spectrometer.

4.3 Nuclear Magnetic resonance Spectroscopy

NMR experiments were conducted on Varian VNMRS-500 spectrometer, 1H

NMR at 500 MHz and 13

C NMR at 125 MHz. Chemical shifts were recorded in parts

per million (ppm) and coupling constant (J) in Hz.

4.4 Analytical LCMS

All crude extracts were prepared to a concentration of 10 mg/ml in HPLC grade

methanol, centrifuged for 60 seconds and supernatant was placed in LCMS vials. 20 µl

of the extracts were injected and analysed on a Waters 2795HT HPLC system.

Detection was achieved by uv between 200 and 400 nm using a Waters 998 diode array

detector, and by simultaneous electrospray (ES) mass spectrometry using a Waters ZQ

spectrometer detecting between 150 and 600 m/z units. Chromatography (flow rate 1

ml/min) was achieved using a Phenomenex LUNA column (5 μ, C18, 100 Å, 4.6 × 250

mm) equipped with a Phenomenex Security Guard precolumn (Luna C5 300 Å) or

Chromatography was achieved using a Phenomenex Kinetex column (2.6 µ, C18, 100

154

Å, 4.6 x 100 mm) equipped with a Phenomenex Security Guard precolumn (Luna C5

300 Å). Solvents used were: A, HPLC grade H2O containing 0.05 % formic acid; B,

HPLC grade MeOH containing 0.045 % formic acid; and C, HPLC grade CH3CN

containing 0.045 % formic acid. The following gradients were used:

Method 1. Luna/MeOH: 0 min, 25% B; 5 min, 25% B; 51 min, 95% B; 53 min, 95%

B; 55 min, 25% B; 59 min, 25% B; 60 min, 25% B.

Method 2. Luna/MeOH: 0 min, 25% B; 13 min, 95% B; 15min, 95% B; 17 min,

25% B; 20 min, 25% B.

Method 3. Kinetex/MeOH: 0 min, 10% B; 10 min, 90% B; 12 min, 90% B; 13 min,

10% B; 15 min, 10% B.

Method 4. Kinetex/CH3CN: 0 min, 10% C; 10 min, 90% C; 12 min, 90% C; 13 min,

10% C; 15 min, 10% C.

Method 5. Luna/CH3CN: 0 min, 5% C; 5 min, 5% C; 45 min, 75% C; 46 min, 95% C;

50 min, 95% C; 55 min, 5% C; 60 min, 5% C.

4.5 Preparative HPLC

Purification of compounds was generally achieved using a Waters mass-directed

autopurification system comprising of a Waters 2767 autosampler, Waters 2545 pump

system, a Phenomenex LUNA column (5µ, C18, 100 Å, 10 × 250 mm) equipped with a

Phenomenex Security Guard precolumn (Luna C5 300 Å) eluted at 4 ml/min. Solvents

used were: A, HPLC grade H2O + 0.05% formic acid; solvent B, HPLC grade MeOH +

0.045% formic acid; solvent C, HPLC grade CH3CN + 0.045% formic acid. The post-

column flow was split (100: 1) and the minority flow was made up with solvent A to 1

ml/min for simultaneous analysis by diode array detector (Waters 2998), evaporative

light scattering (Waters 2424) and ESI mass spectrometry in positive and negative

modes (Waters Quatro Micro).

Method 1: 0 min, 25% B; 13 min, 95% B; 15 min, 95% B; 17 min, 25% B; 20 min,

25% B.

Method 2: 0 min, 40% C; 15 min, 80% C; 15.50 min, 95% C; 16.50 min, 95% C;

17 min, 40% C; 20 min, 40% C.

Method 3: 0 min, 50% C; 22 min, 75% C; 24 min, 95% C; 26 min, 95% C; 27 min,

50% C; 30 min, 50% C.

155

Method 4: 0 min, 55% C; 22 min, 60% C; 24 min, 95% C; 26 min, 95% C; 27 min,

55% C; 30 min, 55% C.

Method 5: 0 min, 55% C; 22 min, 75% C; 24 min, 95% C; 26 min, 95% C; 27 min,

55% C; 30 min, 55% C.

Method 6: 0 min, 50% C; 22 min, 65% C; 24 min, 95% C; 26 min, 95% C; 27 min,

50% C; 30 min, 50% C.

4.6 X-ray Crystallography

X-ray diffraction data were analysed on a Bruker Microstar rotating anode

diffractometer using Cu-Kα radiation (λ = 1.54178 Å). Data collections were performed

using a CCD area detector from a single crystal mounted on a glass fibre. Absorption

corrections were based on equivalent reflections using TWINABS or SADABS. The

structures were solved using direct methods using SHELXS and refined against all Fo2

data with hydrogen atoms on carbon and oxygen atoms riding in calculated positions

using SHELXL.

4.7 Solid media for growth of fungal spores

The Aspergillus oryzae transformants and were first grown on Czapek Dox

Agar (minimal media) and for further production of spores, they were inoculated and

grown on DPY solid media for 7-10 days at 25 °C. Beauveria bassiana WT and RNAi

strains of B. bassiana were grown on Potato Dextrose Agar (PDA) for 10 days at 25 °C.

4.7(a) Czapek Dox Agar

50 grams of Czapek Dox agar was dissolved on 1 litre of deionized water and

sterilized in autoclave.

4.7(b) DPY media

The spores of A. oryzae transformants were grown on plates in DPY media

(dextrin-peptone-yeast extract). It is made by the following ingredients 2% (w/v)

dextrin, 1% (w/v) polypeptone, 0.5% (w/v) yeast extract, 0.5% (w/v) potassium

dihydrogen phosphate, 0.05% (w/v) magnesium sulphate and 2.5% (w/v) agar.

156

4.7(c) PDA media

B. bassiana was grown on potato dextrose agar for growth of spores. 39 gram of

PDA was dissolved in 1 litre of deionized water and sterilized by autoclave.

4.8 Preparation of Tenellin production Media

The liquid media used for growing B. bassiana spores was Tenellin production

media (TPM). The ingredients include D-mannnitol (50 g), KNO3 (5 g), KH2PO4 (1 g),

MgSO4·7H2O (0.5 g), NaCl (0.1 g), CaCl2 (0.2 g), FeSO4·7H2O (20 mg) and mineral

ion solution (10 ml, ZnSO4·7H2O (880 mg), CuSO4·5H2O (40 mg), MnSO4·4H2O (7.5

mg), boric acid (6 mg), and (NH4)6Mo7O24·4H2O (4 mg) made up to 1 L in deionized

water) made up to 1 L in deionized water. This medium solution was divided into 100

ml each in 500 ml Erlenmeyer flask, covered with foam bung covered with aluminium

foil and sterilized by autoclaving.

4.9 Czapek-Dox minimal medium (CD)

A. oryzae tenPKS-dmbC strain was grown in CD minimal media. It was

prepared by adding 10 g peptone, 20 g glucose, and 30 g sucrose, 50 ml of solution A

(40 g NaNO3, 40 g KCl, 10 g MgSO4.7H2O, 0.2 g FeSO4.7H2O in 1 litre deionized

water) and 50 ml of solution B (20 g K2HPO4 in 1 litre deionized water) in 1 litre

deionized water. 100 ml of the media was divided in 500 ml conical Erlenmeyer flasks,

covered with bung form and alumium foil and autoclaved. The A. oryzae spores were

inoculated in flasks and were incubated for 4 days at 28 °C and shaken at 200 rpm. This

was followed by changing the media (under sterile condition) with induction medium

for production of secondary metabolites. The inducing medium was made by dissolving

20 g starch, 10 g peptone, 50 ml solution A and 50 ml solution B in 1 litre deionized

water and incubated under the same previous conditions over 5-7 days.

4.9(a) Czapek-Maltose-Polypeptone (CMP) medium A

This medium was prepared by adding 20 g maltose, 10 g polypeptone, 50 ml

solution A and 50 ml solution B in 900 ml deionized water. The medium was divided

into 100 ml each in 500 ml Erlenmeyer flask, covered with foam bung and aluminium

foil and then sterilized by autoclaving.

157

4.9(b) Czapek-Maltose-Polypeptone (CMP) medium B

The liquid medium for A. oryzae transformants studied in aspyridone

biosynthesis (chapter 3) was made by adding 30 g sucrose, 20 g maltose, 10 g

polypetone, 50 ml solution B and 50 ml solution C (60 g NaNO3, 10 g KCl, 10 g

MgSO4.7H2O and 0.2 g FeSO4.7H2O in 1 litre deionized water) in 900 ml deionized

water. The medium (100 ml) was divided in 500 ml Erlenmeyer flasks as described

previously.

4.10 Culturing and inoculation of fungal spores in liquid media

10 mL of deionized water was added on plates having 10 days old growing

spores of the fungus. The spores were made to pass into the deionized water by careful

scratching the surface with sterilized loop. The spore suspension (1 ml) from the plate

was added in each 100 mL liquid medium contained in 500 ml Erlenmeyer flask. The A.

oryzae spores were allowed to grow in the liquid culture for 7 days on shakers at 200

rpm at 25 °C- 28 °C and B. bassiana spores were grown on shakers for at least 10 days

at 25 °C at 150-200 rpm.

4.11 Extraction of A. oryzae transformants

Cells and media (1 L) were homogenized using a hand-held electric blender and

then acidified to pH 4.0 using 37% aqueous HCl. An equal volume of ethyl acetate was

added and stirred for 10 min. The resulting mixture was vacuum filtered through

Whatman no. 1 filter paper. The filtrate was transferred into a separating funnel and

shaken vigorously. The mixture was allowed to stand to separate the layers. The organic

layer was washed once with concentrated brine solution and then with deionized water.

The organic phase was dried (MgSO4), filtered and evaporated to dryness. The crude

extract was dissolved in 10 % aqueous methanol and defatted by extraction with

hexane. The methanolic layer was evaporated to dryness and then made into a solution

of 10 mg/ml in HPLC grade methanol and analysed by LCMS. For purification of

compounds the crude extract was made into a solution of 50 mg/ml in HPLC grade

methanol and 200 µl aliquots were injected in each run of mass-directed HPLC

preparative purification.

158

4.12 Extraction of B. bassiana transformants

The 10 days old cultures of B. bassiana were vacuum filtered and the mycelia

was collected and kept in equal volume of acetone overnight. The acetone layer was

separated from the mycelia by filtration with Whatman filter paper and acetone was

concentrated by vacuum to form a brown aqueous extract. It was further diluted with

deionized water and then extracted with ethyl acetate (2 × 500 ml). The ethyl acetate

layer was separated from the water layer by a separating funnel. MgSO4 was added to

absorb any trapped water droplets. The ethyl acetate was filtered and concentrated under

vacuum to obtain a semi solid crude extract. The extract was dissolved in 10 % aqueous

methanol and defatted by extraction with hexane. The methanolic layer was obtained in

separated glass vial and dried. The crude extract was prepared to a concentration of 10

mg/ml in HPLC grade methanol and analysed by LCMS.

4.13 Purification of metabolites from A. oryzae pTAex3-tenS and A. oryzae

tenSPKS-dmbNRPS

The 1 litre culture of A. oryzae pTAex3-tenS was extracted by the procedure

explained above and a crude extract of 70 mg was achieved. The extract was dissolved

in HPLC grade methanol to a concentration of 50 mg/ml and used for mass-directed

HPLC preparative purification. 100 – 200 μl of the crude solution was injected during

successive rounds of a 20 minute HPLC program (Method 1). Fractions corresponding

to prototenellin C 113 were collected and evaporated to yield 2.4 mg of pure compound.

Similar protocols were used in purifying the prototenellin C 113 from 137 mg of a crude

extract of A. oryzae tenSPKS-dmbNRPS obtained from 1 L fermentation and produced

9.6 mg of the pure compound.

4.13(a) Characterization of prototenellin C 113109

Prototenellin C 113, pale yellow solid, 1H NMR (CD3OD, 500 MHz) δ = 1.15 (d, J =

6.4 Hz, 3H, H-13), 1.29 (s, 3H, H-14), 1.93 (s, 3H, H-15), 2.85 (m,1H, H-16a), 2.99 (m,

1H, H-16b), 3.62 (m, 1H, H-12), 3.94 (m, 1H, H-5), 6.15 (d, 1H, J = 15 Hz, H-10), 6.68

159

(d, J = 8.2 Hz, 2H, H-19, H-21), 6.71 (m, 1H, H-9), 6.98 (m, 1H, H-8), 7.03 (d, J = 8.2

Hz, 2H, H-18, H-22); 13

C NMR (CD3OD, 125 MHz), δ = 11.5 (C-15), 16.3 (C-13),

22.9 (C-14), 36.3 (C-16), 61.2 (C-5), 73.3 (C-12), 75.3 (C-11), 114.4 (C-19, C-21),

124.2 (C-9), 126.3 (C-17), 130.5 (C-18, C-22), 131.7 (C-7), 138.9 (C-8), 145.5 (C-10),

155.0 (C-20), 163.1 (C-2), 188.3 (C-6), 194.0 (C-4). The 13

C signal for C-3 was not

observed. HRMS calculated for C21H25NO6Na: 410.1580; found 410.1578 [M]Na+.

4.14 Purification of metabolites from A. oryzae dmbS –tenC and A. oryzae dmbS-

dmbC

A 1 litre fermentation media was grown for A. oryzae dmbS –tenC at 25 °C (10

flasks × 100 mL) for 7 days on shakers at 200 rpm. The fungal cultures were extracted

with protocol explained in section 4.14, which gave a dark brown crude extract of 111

mg. This was made to a solution of 50 mg/ml with HPLC grade methanol for

purification of compounds on mass- directed preparative HPLC. 100µl - 200µl of crude

extract was injected in each successful preparative run. Pure fractions having bright

yellow colour of predmB A were collected after 25 preparative runs and dried to

evaporation. 23 mg of pure compound was achieved. 363 mg of crude extract was

obtained from a 1 litre culture of A. oryzae dmbS-dmbC and 17.6 mg of pure predmb A

was produced by following a similar protocol as explained above.

4.14(a) Characterization of predmbA 118

PreDMB A 118, brownish yellow solid; IR (neat): νmax 3277, 2961, 2922, 2876,

1648, 1594, 1514 cm-1

; 1H NMR (CD3OD, 500 MHz) δ = 0.92 (t, J = 7.5 Hz, 3H, H-

15), 1.09 (d, J = 7.5 Hz, 3H, H-16), 1.41 (m, 2H, H-14), 2.20 (m, 1H , H-13), 2.90

(brd, 1H, H-17a), 3.00 (brd, 1H, H-17b), 4.08 (brs, 1H, H-5), 5.98 (dd, J = 7.8, 15.2 Hz,

1H, H-12), 6.27 (dd, J = 10.8, 15.2 Hz, 1H, H-11), 6.43 (dd, J = 11.9, 14.5 Hz, 1H, H-

9), 6.70 (d, J = 8.1 Hz, 2H, H-20, H-22), 6.83 (dd, J = 14.5, 10.8 Hz, 1H, H-10), 7.00

(d, J = 8.1 Hz, 2H, H-19, H-23), 7.21 (d, J = 15.4 Hz, 1H, H-7), 7.53 (dd, J = 11.9, 15.4,

160

Hz, 1H, H-8), 13

C NMR (CD3OD, 125 MHz) δ = 10.7 (C-15), 18.7 (C-16), 29.2 (C-14),

36.2 (C-17), 38.8 (C-13), 62.8 (C-5), 114.7 (C-20, C-22), 119.7 (C-7), 126.3 (C-18),

128.7 (C-11), 128.8 (C-9), 130.4 (C-19, C-23), 144.3 (C-10), 145.2 (C-8), 147.8 (C-12),

155.9 (C-21), 173.3 (C-6), 173.7 (C-2), 195.9 (C-4). The 13

C signal for C-3 was not

observed. HRMS calculated for C22H26NO4: 368.1856; found 368.1851 [M]H+

.

4.15 Purication of metabolites from A. oryzae tensPKS –dmbC

3 litres of A. oryzae tenSPKS-dmbC strain (30 flasks × 100 ml) were grown at 28

°C at 200 rpm. After extraction of fungal cultures with ethyl acetate (see section 4.14), a

crude extract of 239 mg was obtained. HPLC grade methanol was added to make a 50

mg/ml solution for mass-directed purification of compounds on preparative HPLC. The

crude extract was subjected to 24 successful preperative runs with Method 2 and two

pure metabolites were obtained. The yield of compound 146 was 9.5 mg and compound

147 was 9 mg.

4.15(a) Characterization of Compound A 146

Light brown viscous oil, [α]22

D -16.9 (c = 0.23, MeOH); IR (neat): νmax 2932, 2872,

2342, 1761, 1631, 1355, 1190 1084 cm-1

; 1H NMR (CDCl3, 500 MHz) δ = 0.92 (t, J = 7

Hz, 3H, H-1), 1.36 (m, 2H, H-2), 1.37 (m, 1H, H-3a), 1.45 (m, 1H, H-3b), 1.65 (m, 1H,

H-4a), 1.81 (m, 1H, H-4b), 2.01 (ddd, J = 12, 10.5, 12 Hz 1H, H-6a), 2.55 (ddd, J = 9,

6, 12 Hz, 1H, H-6b), 3.65 (t, J = 9, 12 Hz, 1H, H-7), 4.43 (m, 1H, H-5), 5.95 (b, 1H, H-

10a), 6.54 (b, 1H, H-10b), 13

C NMR (CDCl3, 125 MHz) δ = 14.4 (C-1), 22.9 (C-2), 27.8

(C-3), 35.5 (C-4), 36.1 (C-6), 45.2 (C-7), 79.6 (C-5), 131.8 (C-10), 136.1 (C-8),

170.1(C-9), 176.4 (C-11). HRESIMS calculated for C11H17O4: 213.1121; observed

213.1130 [M]H+.

161

4.15(b) Characterization of compound B 147

light color viscous oil, IR (neat): νmax 2962, 2930, 2873, 1736, 1582, 1454, 1045, 878

cm-1

; 1H NMR (CDCl3, 500 MHz) δ = 0.92 (t, J = 7 Hz, 3H, H-1), 1.38 (m, 2H, H-2),

1.46 (m, 2H, H-3), 1.71 (m, 1H, H-4a), 1.79 (m, 1H, H-4b), 4.99 (t, J = 6.2 Hz, 1H, H-

5), 6.79 (b, 1H, H-10a), 7.19 (b, 1H, H-10b), 7.96 (b, 1H, H-6); 13

C NMR (CDCl3, 125

MHz) δ = 14.0 (C-1), 22.6 (C-2), 27.3 (C-3), 33.1 (C-4), 80.8 (C-5), 125.0 (C-7), 128.5

(C-8), 133.6 (C-10), 153.5 (C-6), 169.5 (C-9), 171.7 (C-11). HRESIMS calculated for

C11H14O4Na: 233.0784; observed 233.0799 [M]Na+.

4.16 Purication of metabolites from A. oryzae apdACE

A brown crude extract of 1303 mg was formed from 1 litre culture of A. oryzae

apdACE strain by growing the fungus according to standard conditions and extraction

with ethyl acetate. The crude extract was made to a solution of 50 mg/ml with HPLC

grade methanol for purification of metabolites on the preparative HPLC by following

the gradient given in Method 5. Pure fractions of corresponding metabolites were

collected and dried to evaporation.

Characterisation of metabolites from A. oryzae apdACE

4.16(a) 14- Hydroxy preaspyridone A 238

Brown viscous oil, [α]22

D -158.8 (c = 1.29, MeOH); IR (neat): νmax 3317, 3020, 2964,

1651, 1214 cm-1

; 1H NMR (CD3OD, 500 MHz) δ = 0.81 (m, 3H, H-11), 0.82 (m, 3H,

H-12), 0.95 (d, J = 6.5 Hz, 3H, H-13), 1.09 (m, 1H, H-10a), 1.23 (m, 1H, H-9), 1.29 (m,

1H, H-8a), 1.33 (m, 1H, H-10b), 1.40 (m, 1H, H-8b), 3.55 (q, J = 7 Hz, 1H, H-7), 4.22

(d, J = 3.5 Hz, 1H, H-5), 4.99 (d, J = 4 Hz, 1H, H-14), 6.65 (d, J = 8.5 Hz, 2H, H-17,

H-19), 7.11 (d, J = 8.5 Hz, 2H, H-16, H-20) ; 13

C NMR (CD3OD, 125 MHz) δ = 11.4

162

(C-11), 17.5 (C-13), 19.6 (C-12), 30.2 (C-10), 33.5 (C-9), 35.5 (C-7), 41.2 (C-8), 68.5

(C-5), 74.8 (C-14), 115.5 (C-17, C-19), 129.6 (C-16, C-20), 130.3 (C-15), 158.6 (C-

18), 195.2 (C-6). The 13

C signals for C-2, C-3 and C-4 were not observed. HRESIMS

calculated for C19H25NO5Na: 370.1624; observed 370.1624 [M]Na+.

4.16(b) 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 239

Light brown solid, mp 120 °C; [α]22

D -24.3 (c = 0.82, MeOH); IR (neat): νmax 3408,

3298, 2923, 2287, 1734, 1601, 1462, 1227 cm-1

; 1H NMR (DMSO, 500 MHz) δ = 0.79

(m, 3H, H-12), 0.81 (m, 3H, H-13), 1.02 (d, J = 7 Hz, 3H, H-14), 1.09 (m, 1H, H-11a),

1.21(m, 1H, H-9a), 1.25 (m, 1H, H-11b), 1.33 (m, 1H, H-10), 1.49 (m, 1H, H-9b), 4.25

(q, J = 7 Hz, 1H, H-8), 5.92 (d, J = 7.5 Hz, 1H, H-5), 7.60 (t, J = 6.5, 7 Hz, 1H, H-6),

11.49 (s, 1H, H-1); 13

C NMR (DMSO, 125 MHz) δ = 11.1 (C-12), 16.7 (C-14), 18.8 (C-

13), 29.7 (C-11), 31.7 (C-10), 39.9 (C-8), 40.0 (C-9), 99.1 (C-5), 105.9 (C-3), 142.8 (C-

6), 161.8 (C-2), 177.6 (C-4), 211.9 (C-7). HRESIMS calculated for C13H20NO3:

238.1437; observed 238.1428 [M]H+.

Crystals were formed by slow evaporation in methanol. Crystals size/mm3 =

0.29 × 0.25 × 0.06, formula C13H19NO3, (M =237.29): monoclinic, space group P21 (no.

4), a = 9.0939(16) Å, b = 30.695(5) Å, c = 9.1390(16) Å, β = 94.405(5)°, V =

2543.5(8) Å3, Z = 8, T = 100(2) K, μ/mm

-1 = 0.713, Dcalc = 1.239 g/mm

3, range for

data collection = 9.7 to 133.62°, reflections collected 60175, independent reflections

8598 (Rint = 0.0426), final R1 was 0.0294 and wR2 was 0.0759, Largest diff. peak/hole/e

Å-3

= 0.12/-0.19, flack parameter 0.03(8), ccdc code 941139.

163

4.16(c) Aspyridone A 8480

Light brown solid, mp 178 °C; [α]22

D -8.2 (c = 0.48, MeOH); IR (neat): νmax 2961,

2928, 1648, 1610, 1516, 1458, 1378, 1217, 1175, 992, 835, 588 cm-1

; 1H NMR

(CD3OD, 500 MHz) δ = 0.87 (t, J = 7.5 Hz, 3H, H-12), 0.90 (d, J = 6.5 Hz, 3H, H-13),

1.13 (d, J = 6.5 Hz, 3H, H-14), 1.16 (m, 1H, H-11a), 1.33 (m, 1H, H-9a), 1.34 (m, 1H,

H-11b), 1.41 (m, 1H, H-10), 1.64 (m, 1H, H-9b), 4.39 (q, J = 6.8 Hz, 1H, H-8), 6.80 (d,

J = 8.5 Hz , 1H, H-17, H-19), 7.26 (d, J = 8.5 Hz, 1H, H-16, H-20), 7.47 (s, 1H, H-6);

13C NMR (CD3OD, 125 MHz) δ = 11.7 (C-12), 17.6 (C-14), 19.4 (C-13), 31.0 (C-11),

33.7 (C -10), 41.2 (C-9), 41.9 (C-8), 107.0 (C-3), 116.0 (C-5), 116.15 (C-17, C-19),

125.2 (C-15), 131.5 (C-16, C-20) 140.6 (C-6), 158.3 (C-18), 163.9 (C-2), 177.6 (C-4),

214.4 (C-7). HRESIMS calculated for C19H24NO4: 330.1699; observed 330.1692

[M]Na+. The specific rotation and melting point of aspyridone A 84 are not reported

before in literature.

Crystals were formed by slow evaporation in methanol and diethyl ether. Brown

crystals, formula C19H23NO4, M =329.38, monoclinic, space group P21, a =

7.3372(7) Å, b = 22.647(3) Å, c = 20.618(2) Å, β = 90.112(6)°, V = 3426.0(6) Å3, Z =

8, T = 100(2) K, μ/ mm-1

= 0.727, Dcalc = 1.277 g/mm3, range for data collection = 3.9

to 132.38°, reflections collected 56007, independent reflections 5995 (Rint = 0.0792),

final R1 was 0.0452 and wR2 was 0.1139, Largest diff. peak/hole/e Å-3

= 0.28/-0.29,

flack parameter 0(10), ccdc code 941138.

4.16 (d) 18-deshydroxypreaspyridone A 240

Brown viscous oil, [α]22

D -142.3 (c = 0.15, MeOH); IR (neat): νmax 3019, 2962, 2875,

1709,1656, 1600, 1214 cm-1

; 1H NMR (CD3OD, 500 MHz) δ = 0.83 (m, 3H, H-11),

164

0.84 (m, 3H, H-12), 1.02 (d, J = 7 Hz, 3H, H-13), 1.09 (m, 1H, H-10a), 1.27 (m, 1H, H-

9), 1.34 (m, 1H, H-10b), 1.33 (m, 1H, H-8a), 1.44 (m, 1H, H-8b), 2.98 (dd, J = 5.5, 14

Hz, 1H, H-14a), 3.07 (dd, J = 4, 14 Hz, 1H, H-14b), 3.65 (q, J = 6.7 Hz, 1H, H-7), 4.11

(t, J = 4.5 Hz, 1H, H-5), 7.16 (m, 2H, H-17, H-19), 7.17 (m, 1H, H-18), 7.22 (m, 2H, H-

16, H-20); 13

C NMR (CD3OD, 125 MHz) δ = 11.5 (C-11), 17.6 (C-13), 19.5 (C-12),

30.3 (C-10), 33.5 (C-9), 36.0 (C-7), 38.3 (C-14), 41.3 (C-8), 63.5 (C-5), 128.1 (C-18),

129.5 (C-16, C-20), 130.7 (C-17, C-19), 136.9 (C-15), 196.0 (C-6), 197.7 (C-4). The

13C signals for C-2 and C-3 were not observed. HRESIMS calculated for C19H25NO3Na:

338.1726; observed 338.1735 [M]Na+.

4.17 Purification of metabolites from A. oryzae apdACEB

1 litre culture (100 ml x 10 flasks) of the fungal strain A. oryzae apdACEB was grown

in CMP medium B at 28 °C for 7 days at 200 rpm. The culture was then extracted with

ethyl acetate according to standard method given in section 4.11. A crude extract of

1.13 g was obtained after vacuum concentration of the ethyl acetate layer and 50 mg/ml

solution was made with HPLC grade methanol. For purification of metabolites 200 µl of

the crude extract was injected in each successive run on the preparative HPLC

following the gradient in Method 3. The purified fractions of respective metabolites

were collected and dried.

Characterization of new metabolites from A. oryzae apdACEB

4.17(a) Preaspyridone A (minor) 224

Pale white crystalline solid, mp 146-148 °C; [α]22

D 98.4 (c = 0.51, CHCl3); IR (neat):

νmax 3262, 2962, 1693, 1650, 1589 cm

-1;

1H NMR (DMSO, 500 MHz) δ = 0.80 (m, 3H,

H-12), 0.82 (m, 3H, H-11), 1.03 (d, J = 6 Hz, 3H, H-13), 1.09 (m, 1H, H-10a), 1.24 (m,

1H, H-9), 1.26 (m, 1H, H-10b), 1.27 (m, 2H, H-8), 2.82 (td, J = 4.5, 15.5 Hz, 2H, H-

14), 3.53 (q, J = 6.8 Hz, 1H, H-7), 4.08 (brs, 1H, H-5), 6.59 (d, J = 8 Hz, 2H, H-17, H-

165

19), 6.88 (d, J = 8.5 Hz, 2H, H-16, H-20), 8.93 (s, 1H, H-1), 9.17 (brs, 1H, H-18); 13

C

NMR (125 MHz, DMSO) δ = 10.8 (C-11), 16.7 (C-13), 18.9 (C-12), 28.7 (C-10), 31.5

(C-9), 33.4 (C-7), 35.4 (C-14), 39.6 (C-8), 62.3 (C-5), 99.9 (C-3), 114.7 (C-17, C-19),

125.3 (C-15), 130.6 (C-16, C-20), 155.7 (C-18), 175.4 (C-2), 191.9 (C-6), 194.4 (C-4).

HRESIMS calculated for C19H26NO4: 332.1856; observed 332.1852 [M]H+.

Crystals were formed by slow evaporation in methanol. Formula C19H25NO4,

M =331.40, monoclinic, space group P21, a = 8.3357(13) Å, b = 11.294(2) Å, c =

9.9646(17) Å, α = 90.00°, β = 103.551(8)°, V = 912.0(3) Å3, Z = 2, T = 100(2) K, m/

mm-1

= 0.683, Dcalc = 1.207 g/mm3, range for data collection = 9.12 to 132.5°,

reflections collected 10439, independent reflections 3021( Rint = 0.0533),

final R1 was 0.0467 and wR2 was 0.1226, Largest diff. peak/hole/e Å-3

= 0.28/-0.29,

flack parameter 0.2 (2), ccdc code 941137.

4.17(b) Preaspyridone A (major diastereomer) 230107

Brown viscous oil, [α]22

D -166.3 (c = 0.95, CHCl3) ; IR (neat): νmax 3020, 2964, 1653,

1602, 1214 cm-1.

1H NMR (500 MHz, DMSO) δ = 0.80 (m, 3H, H-12), 0.79 (m, 3H, H-

11), 0.95 (d, J = 7 Hz, 3H, H-13), 1.07 (m, 1H, H-10a), 1.28 (m, 1H, H-9), 1.29 (m, 1H,

H-10b), 1.29 (m, 1H, H-8a), 1.38 (m, 1H, H-8b), 2.82 (d, J = 4.5 Hz, 2H, H-14), 3.51

(q, J = 6.6 Hz, 1H, H-7), 4.08 (brs, 1H, H-5), 6.59 (d, J = 8.5 Hz, 2H, H-17, H-19),

6.90 (d, J = 8 Hz, 2H, H-16, H-20), 8.92 (s, 1H, H-1), 9.17 (brs, 1H, H-18); 13

C NMR

(125 MHz, DMSO), 10.9 (C-11), 17.1 (C-13), 18.9 (C-12), 28.6 (C-10), 31.7 (C-9),

33.5 (C-7), 35.7 (C-14), 39.1 (C-8), 62.4 (C-5), 99.8 (C-3), 114.7 (C-17, C-19), 125.4

(C-15), 130.6 (C-16, C-20), 155.9 (C-18), 175.6 (C-2), 191.9 (C-6), 194.3 (C-4).

HRESIMS calculated for C19H26NO4Na: 354.1676; observed 354.1677 [M]Na+.

166

4.17 (c) 1, 4-dihydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 259

Brown viscous oil, [α]22

D -14.3 (c = 0.20, MeOH) ; IR (neat): νmax 3104, 2928, 1731,

1635, 1611, 1453, 1200, 751 cm-1

; 1H NMR (CD3OD, 500 MHz) δ = 0.87 (t, J = 7.2 Hz,

3H, H-12), 0.92 (d, J = 7 Hz, 3H, H-13), 1.11 (d, J = 6.5 Hz, 3H, H-14), 1.18 (m, 1H,

H-11a), 1.33 (m, 1H, H-9a), 1.36 (m, 1H, H-11b), 1.41 (m, 1H, H-10), 1.62 (m, 1H, H-

9b), 4.31 (q, J = 7 Hz, 1H, H-8), 5.97 (d, J = 8 Hz , 1H, H-5), 7.94 (d, J = 7.5 Hz, 1H,

H-6); 13

C NMR (CD3OD, 125 MHz) δ = 11.7 (C-12), 17.3 (C-14), 19.3 (C-13), 31.0 (C-

11), 33.5 (C -10), 41.1 (C-9), 42.1 (C-8), 98.9 (C-5), 107.4 (C-3), 142.3 (C-6),160.2 (C-

2), 176.0 (C-4), 213.6 (C-7). HRESIMS calculated for C13H19NO4Na: 276.1206;

observed 276.1218 [M]Na+.

4.17(d) 1-methoxy, 4-hydroxy-3-(2, 4-dimethylhexanoyl) 2-pyridone 260

Brown viscous oil, [α]22

D -32.5 (c = 0.12, MeOH) ; IR (neat): νmax 2961, 2930, 1661,

1611, 1465, 1388, 975 cm-1

; 1H NMR (DMSO, 500 MHz) δ = 0.80 (t, J = 7.2 Hz, 3H,

H-12), 0.84 (d, J = 6.5 Hz, 3H, H-13), 1.03 (d, J = 7 Hz, 3H, H-14), 1.13 (m, 1H, H-

11a), 1.25 (m, 1H, H-9a), 1.25 (m, 1H, H-11b), 1.37 (m, 1H, H-10), 1.49 (m, 1H, H-9b),

3.93 (s, 3H, H-15), 4.15 (q, J = 7 Hz, 1H, H-8), 5.99 (d, J = 7.5 Hz , 1H, H-5), 8.24 (d,

J = 7.5 Hz, 1H, H-6); 13

C NMR (DMSO, 125 MHz) δ = 11.1 (C-12), 16.5 (C-14), 18.6

(C-13), 29.4 (C-11), 31.8 (C-10), 39.6 (C-9), 39.9 (C-8), 64.4 (C-15), 98.4 (C-5), 146.2

(C-6), 109.9 (C-3), 159.9 (C-2), 177.3 (C-4), 214.5 (C-7). HRESIMS calculated for

C14H21NO4Na: 290.1362; observed 290.1368 [M]Na+.

4.18 Purification of metabolites from A. oryzae apdACED

The A. oryzae apdACED was grown in 1 litre CMP medium B (10 flasks ×100 ml) at

28°C for 7 days with constant shaking at 200 rpm. The fungal cultures were then

167

extracted with ethyl acetate following the protocol explainedin section xx. A dried crude

extract of 1.128 g was obtained which was made to a concentration of 50 mg/ml with

HPLC grade methanol for purification of metabolites on preparative HPLC with

gradient given in Method 6. The pure fractions of metabolites were collected and dried.

Characterization of new metabolites from A. oryzae apdACED

4.18(a) Z-5, 14-anhydropreaspyridone A 267

Bright yellow solid, [α]22

D 15.1° (c = 0.13, MeOH); IR (neat): νmax 3667, 3190, 2961,

2876, 2366, 1691, 1584 cm-1

; 1H NMR (DMSO, 500 MHz) δ = 0.79 (m, 3H, H-11),

0.82 (m, 3H, H-12), 1.05 (d, J = 6.5 Hz, 3H, H-13), 1.08 (m, 1H, H-10a), 1.32 (m, 1H,

H-9), 1.32 (m, 1H, H-10b), 1.38 (m, 1H, H-8a), 1.47 (m, 1H, H-8b), 3.75 (q, J = 6.6 Hz,

1H, H-7), 6.37 (s, 1H, H-14), 6.78 (d, J = 8.5 Hz, 2H, H-17, H-19), 7.46 (d, J = 8.5 Hz,

2H, H-16, H-20); 13

C NMR (DMSO, 125 MHz) δ = 10.9 (C-11), 16.8 (C-13), 18.8 (C-

12), 28.7 (C-10), 31.5 (C-9), 40.4 (C-8), 110.4 (C-14), 115.6 (C-17, C-19), 124.8 (C-

15), 131.5 (C-16, C-20), 158.8 (C-18), 184.0 in CD3OD (C-4). The 13

C signals for C-2,

C-3, C-4, C-5, C-6 and C-7 were not observed. HRESIMS calculated for C19H23NO4Na:

352.1519; observed 352.1528 [M]Na+.

168

Chapter 5

Summary and Future Perspective

Iterative HRPKS-NRPS produce a wide array of diverse, bioactive and

biosynthetically intriguing compounds. The programming rules embedded in the PKS-

NRPS enzymes have been under investigation for many years and still provide

undiscovered horizons for researchers.

In this research we isolated and characterized new compounds produced from

different genetically modified transformants (particularly from heterologous expression

and gene silencing). The structures of the isolated compounds gave clues to the

biosynthetic potential of the PKS-NRPS enzymes and also the tailoring enzymes present

in the PKS-NRPS clusters.

We characterized two new putative A. oryzae wild type compounds 146 and 147

from A. oryzae tenSPKS-dmbC but couldn’t determine the chemical product of

TENSPKS acting without its NRPS moiety. The tenC silencing in B. bassiana by the

carbon inducible promoter PamyB failed to deliver any outcome because B. bassiana

didn’t produce tenellin compounds when we changed the carbon source in TPM media

from mannitol to maltose and glucose.

The investigation of the aspyridone pathway of A. nidulans and heterologous

expression of apd genes in A. oryzae gave insight into the unique catalytic capabilities

of the cytochrome P450 present in the apd cluster and the biosynthetic potential of the

PKS-NRPS. The cytochrome P450 ApdE has oxidative ring expanding and unusual

dephenylation activity. ApdB catalyses N-hydroxylation in dephenylated 2-pyridones.

The production of 18-deshydroxy preaspyridone 240 displays wider amino acid

selectivity besides tyrosine. The compounds 239, 259, 260 and 267 show more diverse

chemical activities for apd PKS-NRPS enzymes as compared to tenellin and desmethyl

bassianin PKS-NRPS gene clusters. The crystal structures of 224, 84 and 239 gave

knowledge about the relative stereochemistry of their structures. The enoyl reductase

ApdC showed a significant property for setting opposite stereochemistry for the

dimethyls in different cycles of the polyketide chain of aspyridone.

169

The ApdC, ApdE and ApdB enzymes should be further studied in vitro to

explore their catalytic potential and may contribute to be used as biocatalysts in

biosynthetic reactions.

170

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