allylix presentation at bio
TRANSCRIPT
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Protein Engineering and Chemobiosynthesis to Produce Novel
Sesquiterpenoids
BIO World Congress on Industrial Biotechnology & Bioprocessing – Washington, DC
June 28, 2010
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Allylix Technology
Production of rare and chemically complex
compounds by fermentation using genetically-
engineered yeast
Advantages:
– Sustainable supply
– Consistent Quality
– Environmentally-friendly
– Cost-effective
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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
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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
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Sesquiterpene production in Nature
CH3
CH3
CH2
CH3
Glucose
Many enzymatic
steps(ubiquitous)
FPP
Sesquiterpene
synthase(plant specific)
Valencene
OPP
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Sesquiterpene
Synthase
Diversity
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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
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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
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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
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Greenhagen et al., 2006 PNAS 103: 9826
Enzymatic Determinants of Product Specificity
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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
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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
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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
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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
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Combinatorial Chemobiosynthetic Production of Novel
Terpenes
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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)
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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
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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
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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