Strain Improvement Acremonium

8/10/2019 Strain Improvement Acremonium

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Strain improvement for cephalosporin production by Acremoniumchrysogenum using geneticin as a suitable transformation marker

Marta Rodrguez-Saiz a, Marianna Lembo b, Luca Bertetti b, Roberto Muraca b,Javier Velasco a, Antonella Malcangi b, Juan Luis de la Fuente a, Jose Luis Barredo a,*

a R+D Biology, Antibioticos S.A., Avenida de Antibioticos 59-61, 24009 Leon, Spainb R+D Biology, Antibioticos S.pA., Via Schiapparelli, 2, Settimo Torinese, 10036 Torino, Italy

Received 29 February 2004; received in revised form 5 April 2004; accepted 5 April 2004

First published online 17 April 2004

Abstract

An Acremonium chrysogenum strain improvement program based on the transformation with cephalosporin biosynthetic genes was

carried outto enhance cephalosporin C production. Best results were obtained with cefEFand cefGgenes, selecting transformants with

increased cephalosporin C production and lower accumulation of biosynthetic intermediates. Phleomycin resistant transformants,

designated B1 and C1, showed a single copy random integration event, higher levels ofcefEFtranscript and, according to immuno-

blotting analyses, higher amounts of deacetylcephalosporin C acetyltransferase (DAC-AT) protein than their parental strains.

Moreover, DAC-AT activity was higher in the transformants. Plasmids carrying geneticin resistance markers based on the nptIIgene

from Tn5 and theaphIgene from Tn903 were constructed to transform again B1 and C1, showing that the cassette PgdhnptIItrpC

was able to confer geneticin resistance to A. chrysogenumand demonstrating that geneticin is a helpful selection marker.

2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Acremonium; Cephalosporin; Improvement; Geneticin; Marker; Transformation; Resistance

1. Introduction

Although few species of filamentous fungi have been

used for the industrial production of antibiotics, Acre-

monium chrysogenumis the microorganism of choice for

cephalosporin production by fermentation in stirred

submerged cultures. For many years genetic manipula-

tion of industrial microorganisms was limited to im-

provement programs based on random mutation and

selection, and even today these techniques are indis-

pensable tools for the development of complex processes

in which there is little background molecular knowledge.

The development of recombinant DNA techniques over

the last 20 years for this filamentous fungus has allowed

yield increments and the design of new biosynthetic

pathways [1].

Cephalosporins are chemically characterized by a

cephem nucleus composed of a b-lactam ring fused to a

dihydrothiazine ring. Genes directly involved in the

biosynthesis of cephalosporin in A. chrysogenum have

been identified: pcbAB encoding a-aminoadipyl-cys-

teinyl-valine synthetase (ACVS) [2], pcbC coding for

isopenicillin N synthase (IPNS) [3], cefEFencoding de-

acetoxycephalosporin C synthase (DAOCS) and deace-

tylcephalosporin C synthase (DACS) activities [4], cefG

for deacetylcephalosporin C acetyltransferase (DAC-

AT) [5], and, more recently,cefD1and cefD2encoding a

two-step epimerase activity [6].

The major objective of our strain improvement pro-

gram was the selection of new strains able to produce

higher levels of cephalosporin C with reduced accumu-

lation of deacetoxycephalosporin C (DAOC) and deace-

tylcephalosporin C (DAC) biosynthetic intermediates.

There are few selection markers for transformation

in A. chrysogenum. They include phleomycin [7,8],

hygromycin B [810], and benomyl [11], whereas addi-

* Corresponding author. Tel.: +34-987-895826; fax: +34-987-895986.

E-mail address: [email protected](J.L. Barredo).

0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

doi:10.1016/j.femsle.2004.04.010

FEMS Microbiology Letters 235 (2004) 4349

www.fems-microbiology.org
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tional selection markers such as sulfonamide [12], oli-

gomycin [13,14], acetamide [15], G418 [16], and auxo-

trophic complementation [1723] have been described in

the filamentous fungi Penicillium chrysogenum, Asper-

gillus nidulansand Neurospora crassa. Although the use

of the aminoglycoside antibiotic G418 was described as a

selection marker in Cephalosporium acremonium [24]using the APH30I gene from Tn903, the transformationefficiency was very poor compared to the method de-

scribed here involving the expression of the nptIIgenet-

icin resistance gene from Tn5 under a strong fungal

promoter.

Transformation of A. chrysogenum is a common

technique used in our strain improvement programs.

Therefore, the availability of a new selection marker will

permit the re-transformation of recombinant strains

previously transformed with resistance markers such as

phleomycin or hygromycin. In this paper we report the

characterization of cephalosporin C overproducing

transformants ofA. chrysogenum, and the use of shuttle

vectors containing nptIIas a resistance marker for the

transformation of phleomycin resistant strains of A.

chrysogenum. Differences among parental strains and

transformants are discussed.

2. Materials and methods

2.1. Microbial strains, plasmids and microbiological

procedures

A. chrysogenum strains B and C, two cephalosporinoverproducing strains belonging to the Antibioticos

S.pA. series [25], were cultured as previously described

[8]. Escherichia coli DH5a [26] was the recipient for

high-frequency plasmid transformation. pBluescript I

KS (+) and pBC KS (+) (both from Stratagene) were the

plasmids used for subcloning and sequencing. pAN52.1

was the source of Pgpd and TtrpC [27], Pgdh was ob-

tained from pALP30 [28], the aphI gene was from

pPIC3.5K (Invitrogen) andnptIIgene from pBI121 [29].

General procedures for plasmid purification, cloning

and transformation of E. coli were according to de-

scribed techniques [26]. Protoplast transformation ofA.

chrysogenum was performed according to previously

described protocols [710]. A. chrysogenum transfor-

mants were selected on TSA sucrose plates supple-

mented with 3 lg ml1 phleomycin (Cayla) or 7 lg ml1

G418 (Geneticin, Life Technologies) after incubation at

28 C for 10 days.

2.2. DNA manipulations, PCR, Southern and Northern

analysis

The glyceraldehyde-3-phosphate dehydrogenase pro-

moter (Pgpd) ofA. nidulans[27] was chosen to drive aphI

transcription in A. chrysogenum. The aphI gene was

purified as a 1.24 kb PstI fragment from pPIC3.5K, and

subcloned into pBC KS (+) to obtain pBCKan1. Primers

#82 (50-GTAATACAAGGGGTGCCATGGGCCAT-

ATTCAACGG-30) and M13 (50-GTAAAACGACG-

GCCAGT-30) were used to amplify the aphIgene from

pBCKan1 creating an NcoI restriction site (underlined)in the 50 end. A 1.1 kb NcoI-BamHI fragment from the

PCR product including the aphI gene was subcloned

into pAN52.1 to obtain pALG418Ap, which includes

the PgpdaphItrpC expression cassette. To avoid the

presence of the b-lactamase encoded by the ampicillin

resistance gene, the expression cassette was subcloned as

a 4.1 kb BglIIXbaI fragment into pBC KS (+) to give

pALG418, which includes the chloramphenicol resis-

tance gene as a marker for E. coli.

The glutamate dehydrogenase promoter (Pgdh) ofP.

chrysogenum[28] was chosen to drive nptIItranscription

in A. chrysogenum. An NdeI site was created in the 3 0

end of Pgdh by directed-mutagenesis using the Quik-

ChangeTM site-directed mutagenesis kit (Stratagene)

and the primers #113 (50-GAGTTAACAGTACC-

GGCCCATATGATGCAAAACCTTCCC-3 0) and

#114 (50-GGGAAGGTTTTGCATCATATGGGCC-

GGTACTGTTAACTC-3 0) (NdeI site underlined). After

amplification, the reaction mixture was digested with

DpnI (specific for methylated and hemimethylated

DNA) to cut the parental DNA template and facilitate

the selection of mutation-containing synthesized DNA.

The nicked plasmid incorporating the mutations was

then transformed into E. coli DH5a. Primers #94

(50-GGATCGTTTCATATGATTGAACAAGATGG-ATTGC-30; Nde I site underlined) and #95 (50-G-

CGGTGGATCCGAAATCTCGTGATGGCAGG-30;

BamHI site underlined), were used to amplify the nptII

gene from pBI121. After digestion of the PCR product,

the nptII gene was obtained as a 0.9 kb NdeIBamHI

fragment which was ligated to Pgdhto give pALGEN2.

Subsequently, the trpC terminator (TtrpC) of A. nidu-

lans [27] was placed downstream as a 0.7 kb BamHI

XbaI fragment to generate pALGEN3, which includes

the Pgdh nptIITtrpC expression cassette. The expres-

sion cassette was subcloned as a 2.2 kb EcoRIXbaI

fragment into pBC KS (+) to give pASG418, which

includes the chloramphenicol resistance gene as a mar-

ker for E. coli.

Optimized amplification reactions (20 ll in a DNA

Thermal Cycler 480, PerkinElmer Cetus) contained

about 50 ng of genomic DNA, 20 mM TrisHCl pH 8.8,

2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1%

Triton X-100, 0.1 mg ml1 nuclease-free BSA, 0.25 lM

of each primer, 1.0 U of Turbo Pfu DNA polymerase

(Stratagene), and dNTPs 200 lM each. The reaction

mixtures were overlaid with mineral oil and subjected to

different programs for each pair of primers: (I) #82-

M13. 94 C, 60 s (2 min for the first cycle); 55 C, 60 s;

44 M. Rodrguez-Saiz et al. / FEMS Microbiology Letters 235 (2004) 4349


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72 C, 90 s (5 min for the last cycle) 25 cycles. (II) #94-

#95. 94 C, 60 s (2 min for the first cycle); 55 C, 60 s; 72

C, 60 s (5 min for the last cycle) 25 cycles. (III) #113-

#114. 95 C, 30 s (60 s for the first cycle); 55 C, 60 s; 68

C, 8 min (5 min for the last cycle) 16 cycles. Amplified

fragments from #82-M13 and #94-#95 were purified

from agarose gel.DNAs ofA. chrysogenum were purified as previously

described [30] and used as templates for PCR reactions

with (i) primers AS36 (50-CCCTGAATGAACTG-

CAGGACG-30) and AS37 (50-AAGGCGATA-

GAAGGCGATGC-30) to amplify an internal 611 bp

fragment of the nptII gene, and (ii) primers AS28 (5 0-

CGCGAGGGTGCATCGCAACG-3 0) and AS29 (50-

GTCCAGGACGATACCGGTCG-3 0) to amplify an

internal 875 bp fragment of the actA gene ofA. chrys-

ogenum [31]. Amplification reactions were as above for

30 cycles with the following program: 95 C, 60 s (5 min

for the first cycle); 60 C, 45 s; 72 C, 60 s (10 min for the

last cycle). Amplified fragments were analyzed in aga-

rose gel.

Southern analyses were according to described tech-

niques [26]. DNAs were digested with BamHI, HindIII

and SalI, blotted to a nylon filter and hybridized

with the following probes ofA. chrysogenum: 2.95 kb

BamHI internal to pcbAB gene encoding ACVS [2],

2.25 kb EcoRI/XmnI including a portion of pcbC

gene encoding IPNS [3], 0.5 kb SalI internal to cefEF

gene encoding DAOCS/DACS [4] and 2.0 kb HindIII

including a portion of cefG gene coding for DAC-AT

[5].

RNA was extracted from A. chrysogenum myceliumgrown in flask according to described protocols [32] and

Northern analyses were as previously described [26]. A

0.8 kb SacI probe including a portion of the actA gene

encoding c-actin [31] was used as a control of the RNA

quantity.

2.3. Immunodetection of IPNS and DAC-AT proteins

Cell extracts prepared by mycelium sonication were

loaded onto a 15% SDSPAGE (40 lg of protein per

lane) and, after electrophoresis, the proteins were

transferred to a polyvinylidene difluoride membrane

(Immobilon-P, Millipore) using a Minitransblot elec-

troblotting system (BioRad). Membranes were treated

with polyclonal antibodies generated by immunization

of rabbits with recombinant IPNS or DAC-AT proteins

expressed in E. coli. Membranes were then washed and

treated with a commercial secondary Ab-HRP conju-

gate. Immunoreactive bands were detected with the

ECLTM Western blotting analysis system (Amersham

Biosciences) and the intensity of the chemiluminescence

signals was quantified with a Shimadzu spectrodensi-

tometer.

2.4. Measurement of IPNS and DAC-AT enzymatic

activities

IPNS activity was measured by monitoring the for-

mation of isopenicillin N (IPN) from Bis-ACV as pre-

viously described [33] and IPN was quantified by

bioassay against Micrococcus luteus. DAC-AT activitywas measured monitoring by HPLC in vitro conversion

of DAC and acetyl-CoA into cephalosporin C as de-

scribed [5]. Total protein in cell extracts was determined

by Bradford using ovalbumin as standard.

3. Results and discussion

3.1. Transformation of A. chrysogenum with cephalo-

sporin biosynthetic genes to improve cephalosporin C

production

The strains B and C ofA. chrysogenum were trans-

formed with the cephalosporin biosynthetic genes

pcbAB, pcbC, cefEF and cefG using phleomycin resis-

tance as selection marker. As a result, cephalosporin C

production was improved and the accumulation of the

biosynthetic intermediates DAC, DAOC, IPN and

penicillin N decreased in transformants B1 and C1,

which have an extra copy of the cefEFand cefGgenes

(Table 1). This is a logical result if parental strains B and

C have a bottleneck in the biosynthetic steps catalyzed

by DAOCS/DACS or DAC-AT, the enzymes encoded

by cefEFand cefG, respectively. Reduction of interme-

diates, especially DAOC and DAC, is a very importanttask to improve the quality of commercial preparations.

Genomic DNAs from the parental untransformed

strains (B and C) and from the transformants (B1 and

C1) were analyzed by Southern blotting using the probes

described in Section 2, showing that a single copy of

each gene was integrated into the genome without dis-

ruption of the endogenouspcbAB,pcbC,cefEFand cefG

genes (Fig. 1). Additionally, transcription levels of these

genes were analyzed in same strains after 48, 96 and 144

h of flask fermentation (Fig. 2). Bands of pcbAB

and cefG transcripts (not shown) were too weak to

Table 1

Cephalosporin (CPC), deacetylcephalosporin (DAC), deacetoxyceph-

alosporin (DAOC), and penicillin N and isopenicillin N (PenN + IPN)

productions expressed as percentages of totalb-lactams

Strain CPC

(%)

DAC

(%)

DAOC

(%)

PenN+ IPN

(%)

A. chrysogenumB 71.0 15.6 1.0 12.4

A. chrysogenumB1 86.7 7.4 0.2 5.7

A. chrysogenumC 81.6 8.6 0.0 9.8

A. chrysogenumC1 87.9 6.7 0.0 5.4

B1 and C1 include an extra copy of thecefEFand cefGgenes.

Results are the average of three different flask fermentations.

M. Rodrguez-Saiz et al. / FEMS Microbiology Letters 235 (2004) 4349 45


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distinguish any differences between the parental and

transformant strains and the variations detected inpcbC

transcript were equivalent to that of the actAgene used

as a control of RNA quantity. However, B1 and C1

transformants showed notably higher levels of cefEF

transcript than their parental strains throughout the

fermentation process.

IPNS (pcbC) and DAC-AT (cefG) proteins in the

same samples employed for RNA extraction were ana-

lyzed by immunoblotting using polyclonal antibodies

generated against the recombinant proteins expressed inE. coli. IPNS showed maximal accumulation after 48 h,

being almost undetectable at the end of the fermentation

(144 h). As expected, there were no significant differ-

ences between transformants and parental strains. In

contrast, highest concentrations of DAC-AT were de-

tected between 48 and 96 h, decreasing later on. Strains

B1 and C1 showed higher amounts of DAC-AT protein

than their parental strains throughout the fermentation

process (Fig. 3). Furthermore, IPNS and DAC-AT ac-

tivities were quantified to determine their potential effect

on cephalosporin production. DAC-AT was higher in

B1 and C1 strains, whereas IPNS did not show a clear

difference between strains (Fig. 3). While DAC-AT

Fig. 2. Transcription level ofpcbC, cefEF, and actAgenes after 48, 96

and 144 h of flask fermentation in parental strains (B and C) and

transformants (B1 and C1).

Fig. 1. Southern analyses of parental strains (B and C) and phleomycin resistant transformants (B1 and C1). DNAs were digested with BamHI,

HindIII and SalI, and hybridized with probes from pcbAB, pcbC, cefEFand cefGgenes fromA. chrysogenum.

Fig. 3. Western analysis of IPNS (pcbC) and DAC-AT (cefG) proteins using polyclonal antibodies, and IPNS and DAC-AT enzymatic activities in

parental strains (B and C) and transformants (B1 and C1).



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activity was present until the final stages of the fer-

mentation, IPNS was greatly decreased at the end of the

process.

All of these results indicate that the introduction of

an extra copy of the cefEFand cefGgenes leads to an

increase in DAC-AT activity (and probably in DAOCS/

DACS) causing the improvement of cephalosporin Cproduction and the decrease of penicillin N, DAOC and

DAC accumulation. The improvement of an industrial

strain ofA. chrysogenumby introduction of extra-copies

of the cefEFgene was previously described by Eli Lilly

researchers [10], and the occurrence of an inefficient

conversion of DAC into cephalosporin C mediated by a

limiting expression of the cefG gene was also known

[34].

3.2. Transformation of A. chrysogenum by geneticin

resistance

The minimal inhibitory concentration (MIC) of A.

chrysogenum was determined by seeding fresh colonies

and protoplasts of A. chrysogenum into TSA-sucrose

and checking a G418 range from 1 to 75 lg ml1. MIC

values described forA. chrysogenum(12.5lg ml1) [24]

were lower than the obtained in this work (5 lg ml1),

but a different culture medium and a cephalosporin

overproducing strain were used. Resistance differences

observed could be related to the transport of geneticin

into the cells.

To transform again the above described phleomycin

resistant strains B1 and C1, plasmids carrying geneticin

resistance markers based on the aphIgene from Tn903(pALG418) and the nptII gene from Tn5 (pASG418)

were constructed. These plasmids also include chlor-

amphenicol resistance as a marker forE. coli, and single

sites for routine subcloning (Fig. 4). Sequence analysis

of PgpdaphI transcriptional fusion of pALG418

showed a change in the second residue of the protein

(serine by glycine) caused by the introduction of the

NcoI site, whereas the PgdhnptII fusion did not showany mutation.

A. chrysogenum strains B, C, B1 and C1 were trans-

formed with pALG418 and pASG418, and transfor-

mants were selected in TSA-sucrose supplemented with

7 lg ml1 geneticin. To determine the stability of the

transformants, isolated colonies were streaked again

onto selective medium and tested for G418 resistance.

Only those colonies growing properly were counted as

transformants. Whereas stable transformants were ob-

tained using pASG418 with a frequency of around 510

transformants per lg of DNA, we were unable to select

any stable transformant using pALG418. Additionally,

segregational stability of transformants was demon-

strated after growing on geneticin-free medium. Trans-

formation frequencies obtained in A. chrysogenum were

similar to those described for phleomycin [7,8] or hy-

gromycin [810]. Therefore, the nptII gene from Tn5

expressed under the control of Pgdh can be used as a

transformation marker in A. chrysogenum. The failure

to obtain pALG418 transformants could be due to in-

efficient function of the protein encoded by aphIor to

the Ser2 !Gly2 mutation for PgdhaphI construction.Plasmids containing the aphIgene from Tn903 and the

nptII gene from Tn5 were also constructed for trans-

formation of Cryptococcus neoformans, but those con-taining theaphIgene never produced any transformants

SalIcmR

ColE1

f1(+)

nptII

NdeI

Pgdh

TtrpCNcoI

PstI

SacINotIXbaI

PvuII

PvuII

PvuII

XhoI

EcoRVEcoRI

KpnI

HindIII

SalI/AccI

ClaII

pASG418ASG 8

5.6 Kb

pALG418ALG 8

7.5 Kb

PvuII

BamHI

NcoI

ClaII

XhoI

cmR

ColE1

f1(+) aphI

Pgpd

TtrpC

HindIII

NcoI

SacI

NotIXbaI

PvuII

PvuII

PvuII

PvuII

BamHI

NcoI

XhoI

SalI/AccI

EcoRVEcoRI

KpnI

ClaII

HindIII

XhoI

SacI

SacI

ClaII

XhoI

Fig. 4. Combined physical and genetic maps of pASG418 and pALG418. Singles sites useful for subcloning are shown in bold. The genetic maps were

compiled from the data of the progenitors: pBluescript I KS (+) and pBC KS (+) (Stratagene), Pgpdand TtrpC[27], Pgdh[28], nptII[29], and aphI

(pPIC3.5K, Stratagene).



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[35]. However, there is a report that A. chrysogenum

transformed to G418 resistance using theAPH30Igenefrom Tn903 expressed under the control of the yeast

constitutive ADCI promoter gave a very low transfor-

mation frequency (0.3 transformants/lg of DNA) [24].

Genomic DNA was purified from 10 geneticin resis-

tant transformants and from the parental phleomycinresistant transformants B1 and C1. After PCR amplifi-

cation using primers for the actA gene ofA. chrysoge-

num [31] and the nptII gene of Tn5 [29], the expected

DNA fragments for geneticin transformants were ob-

tained: 611 bp for nptIIand 875 bp for actA(Fig. 5). In

contrast, only the actA fragment was amplified using

DNA from B1 and C1. Monocopy random integration

of pASG418 into the B1 and C1 genomes was confirmed

by Southern analysis of three randomly chosen trans-

formants (data not shown).

Geneticin presents some interesting characteristics as

low background and absence of spontaneous resistant

colonies that make it attractive as selection marker. Inthis way, geneticin resistance can be used as primary

marker for routine transformation experiments or as

secondary marker in recombinant species where other

resistances have been employed for primary selection.

Acknowledgements

The authors thank P. Merino, M. Sandoval, M.

Medici, B. Comoglio, and L. Cresto for their excellent

technical assistance.

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