allylix presentation at bio
TRANSCRIPT
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