Protein Engineering and Chemobiosynthesis to Produce Novel

Sesquiterpenoids

BIO World Congress on Industrial Biotechnology & Bioprocessing – Washington, DC

June 28, 2010

Allylix Technology

Production of rare and chemically complex

compounds by fermentation using genetically-

engineered yeast

Advantages:

– Sustainable supply

– Consistent Quality

– Environmentally-friendly

– Cost-effective

Plant natural products– Aromas

– Insect repellent and attractants

– Antimicrobial and antiherbivorial compounds

– Antioxidants

• Hydrocarbons, alcohols, ketones– Multicyclic

– Multiple chiral centers

– Over 300 known carbon skeletons

Sesquiterpenoids

CH3

CH3

CH2

CH3

O CH3

CH3

O

CH3

CH2

CH3

CH3

CH2

CH3

Sesquiterpenes

Of significant commercial interest– Flavors & Fragrances

– Urban Pesticide & Crop Protection Agents

– Functional Ingredients

– Pharmaceutical Intermediates

Historically expensive to produce– Multicyclic, multichiral compounds difficult and expensive to

synthesize

– Low natural abundance makes them expensive to extract

Allylix technology offers:– Step change in cost of production

– Ability to create novel products

Sesquiterpene production in Nature

CH3

CH3

CH2

CH3

Glucose

Many enzymatic

steps(ubiquitous)

FPP

Sesquiterpene

synthase(plant specific)

Valencene

OPP

Sesquiterpene

Synthase

Diversity

Allylix Technology for Terpene Production

• Gene Isolation– Synthesis, informatics, cloning

• Protein engineering – Generating improved synthases

• Metabolic engineering– Production of high levels of FPP for conversion to terpenes

• Fermentation – Economical production of terpenes

• Combinatorial chemobiosynthesis– Chemical modification of biosynthetic terpenes to produce

novel or commercially-inaccessible products

Protein Engineering – What and Why?

Altering specific amino acids in a protein to generate one

with improved characteristics

Improved synthases:

• Specificity – what product it makes

• Selectivity – Some enzymes generate product mixtures

– Proportions can be changed

• Catalytic efficiency – more active, robust enzymes

Altering Specificity:

Structural Studies to Elucidate Specificity Determinants

Henbane

Premnaspirodiene

Synthase

Tobacco

5-epi-Aristolochene

Synthase•Mechanistically similar

•72% amino acid identity

Greenhagen et al., 2006 PNAS 103: 9826

Greenhagen et al., 2006 PNAS 103: 9826

Enzymatic Determinants of Product Specificity

Protein Engineering for Improved Catalytic Efficiency

• For valencene production, terpene cyclase catalytic

efficiency appears to be limiting factor

• Error-prone PCR with high-throughput screening in

microvials

• Improved mutants isolated, sequenced, recombined

2x

4x

wt

2A2

2H66A7

9-41

9-70

D1

E8

epPCR

epPCR

Recomb DNA

Valencene Production Improved by Protein Engineering

Mutations are generally:

•Additive

•Conservative

•Unpredicted

Improved mutants have greater stability, expression, and/or catalytic efficiency

Protein Engineering for Improved Terpene Synthases

• Altered specificity and selectivity have been

demonstrated for several enzymes

• Productivity increases for valencene synthase translate

from vials to shake flasks to fermentors

• Improved valencene synthase variants allow production

at commercially-viable levels

• Systematic mutant generation, screening, and

recombination has identified many mutations and

combinations of mutations that have beneficial effects

on valencene production

Random vs. Rational Approach to Protein Engineering

• In general, the rational approach works better for

specificity and selectivity changes

• Random mutagenesis and screening, followed by

recombination is better approach for activity

improvement – rules are less well known

However,

• A combined approach will lead to highest probability of

success

Combinatorial Chemobiosynthetic Production of Novel

Terpenes

Discovery of and Screening of Novel Terpenes

Chemical modification of biosynthetic terpene scaffolds to

produce novel or commercially-inaccessible products

“Smart” libraries built on chiral scaffolds more likely to produce

compounds with desirable properties – “hits”– Natural products produce higher proportion of hits than randomly

generated products of combinatorial chemistry

– Natural products more closely resemble bioactive compounds

– Renewed emphasis on natural products screening and screening of

semisynthetic natural product libraries

CH3

CH3

CH2

CH3

CH3

CH3

CH2

CH3

CH3

CH3

CH2

CH3

X XY

Scaffolds

(Several)

Primary Derivatives

(Dozens per scaffold)

Secondary Derivatives

(Hundreds per scaffold)

Premnaspirodiene LibrariesStructure CAS # Common Name Name Stereoisomers

82189-85-3 Premna-

spirodiene

(2R,5S,10R)-6,10-dimethyl-2-(prop-1-en-2-yl)spiro[4.5]dec-6-ene

54878-25-0 Solavetivone

(2R,5S,10R)-6,10-dimethyl-2-(prop-1-en-2-yl)spiro[4.5]dec-6-en-8-one

1

42483-52-3 Epi- -vetivone

(5S,10R)-6,10-dimethyl-2-(propan-2-ylidene)spiro[4.5]dec-6-en-8-one

1

54878-30-7 Tetrahydro- -

vetivone

(5R,6R)-2-isopropyl-6,10-dimethylspiro[4.5]decan-8-one

1 set of 2 or 4

901767-03-1 Solavetivol

(2R,5S,10R)-6,10-dimethyl-2-(prop-1-en-2-yl)spiro[4.5]dec-6-en-8-ol

2

39850-92-5 Epi- -vetivol

(5S,10R)-6,10-dimethyl-2-(propan-2-ylidene)spiro[4.5]dec-6-en-8-ol

2

54878-29-4 Tetrahydro- -

vetivol

(5R,6R)-2-isopropyl-6,10-dimethylspiro[4.5]decan-8-ol

2 sets of 2 or 4

O

O

O

HO

HO

HO

One scaffold leads to 14-16 primary derivatives

Terpene Product Discovery

Each scaffold can lead to hundreds of derivatives

These derivatives can be screened as:– Flavors or fragrances

– Insect repellants, insect attractants, pesticides

– Antimicrobials

– Pharmaceutical intermediates

Summary

• Production by fermentation of metabolically-

engineered yeast allows economic production of

sesquiterpene hydrocarbons

• This production platform allows us to “improve on

Nature”– Protein engineering for modified product profile or improved product

yields

– Chemobiosynthetic library generation and screening for commercially

inaccessible or novel products

• High titer production technology ensures sustainable

cost-effective production at scale