engineering microbial metabolism for production of fuels

Engineering Microbial Metabolism for

Production of Fuels and Chemicals

Jay Keasling

Joint BioEnergy Institute

University of California, Berkeley

Lawrence Berkeley National Laboratory

Hydrocarbon fuels

Gasolines

• High octane

• Short-chain alkanes

• Many branches

• Some aromatics

Jet fuels

• Long-chain alkanes

• Few branches

• Some aromatics

Diesels

• Appropriate cetane numbers

• Few branches

• Some aromatics

Linear hydrocarbons from the fatty acid biosynthetic

pathways

Pyruvate

Acetyl-CoA

Glyoxylate

SUC-CoA

Acetaldehyde

Ethanol

Propionyl-CoA Propanol

Acetoacetyl-CoA Butyryl-CoA Butanol

Even chain fatty acid

Aliphatic hydrocarbons

Odd chain fatty acid

IPP DMAPP

Isopentenol Isopentanol

Geraniol

Farnesol

Geranylgeraniol

Monoterpene

Sesquiterpene

Diterpene

G6P 6PG X5P

Valeryl-CoA Isopentanol

Glucose

Xylose

Mannose M6P

Galactose G’1P G1P

Xylulose

Ribulose

Arabinose

Various esters

Alcohols

Aromatic hydrocarbons

Short, highly-branched

hydrocarbons

Linear hydrocarbons make great diesels

and jet fuels

Gasolines

• High octane

• Many branches

• Some aromatics

Jet fuels

• Few branches

• Some aromatics

Diesels

• Few branches

• Some aromatics

Linear hydrocarbons from the fatty acid biosynthetic

pathways

Pyruvate

Acetyl-CoA

Glyoxylate

SUC-CoA

Acetaldehyde

Ethanol

IPP DMAPP

Geraniol

Farnesol

Geranylgeraniol

Monoterpene

Sesquiterpene

Diterpene

G6P 6PG X5P

Glucose

Xylose

Mannose M6P

Xylulose

Ribulose

Arabinose

Various esters

Alcohols

hydrocarbons Fatty acids

Wild type E. coli produces no free fatty acids

because of strict acyl-ACP inhibition PEP

Fatty-ACP Fatty

ACC FAS

FadD Fatty

acyl-CoA

Steen et al. 2010.

Nature 463:559

β-keto

acyl-CoA

Production of fatty acid ethyl esters

Fatty-ACP Fatty

ACC FAS

FadD Fatty

acyl-CoA

Acetaldehyde Ethanol

Fatty acid ethyl

esters

Steen et al. 2010.

Nature 463:559

β-keto

acyl-CoA

Methyl ketones produced from fatty acyl-CoA

by overexpressing FadEBM PEP

AcCoA Fatty-ACP Fatty

ACC FAS

FadD Fatty

acyl-CoA

β-keto

acyl-CoA

FadM FadB

Methyl ketones

Goh et al. 2012.

AEM 78:70

Diesel-range methyl ketones in E. coli

Goh et al. 2012 AEM 78:70

Goh et al. 2014 Metab. Eng. 26:67

Re-engineering β-oxidation Titers exceeding 1.4 g/L (1% Glu, M9)

40% of maximum theoretical yield

1350 1400

M9-MOPS (1% Gluc)

Goh et al. 2012

‘tesA, fadE + + + + + + + + + +

fadM + + + + + + + +

aco, fadB, fadA + + + + + + + +

fadR, fadD + + + + +

poxB + + +

1-plasmid, co-aco

ackA-pta + +

EGS860

EGS084

TesA-FadD combination wastes ATP

ATP is consumed

when free fatty acids

esterified to CoA Haushalter et al. 2015. Metab. Eng. 30:1-6

Acyl-ACP inhibits fatty

acid biosynthesis

Type I FAS nearly doubles fatty alcohol titers

compared to native FAS

• Improvement in

production

(2.9 g/L versus

1.6 g/L)

• Changes in

distribution of

fatty acids

FAS1 FAS2

Fatty-CoA

TesA DGAT

Tef1 FAS1

Chromosomal DNA

Tef1 FAS2

Tef1 ACC

b-oxidation

Engineered yeast strain WR1

$! ! "

%! ! " O

! "# $%"&'( )%

! ACSs

TesA ! "

! " #$%

! ²-oxidation

Lipid Content (%)

! "# $%%"&' ( ) ( &*%

! ²-oxidation

! " ##$%

! ²-oxidation

Fatty acid-derived fuels in S. cerevisiae

Unbalanced FAEE pathway caused strain instability

Fatty-ACP Fatty

ACC FAS

FadD Fatty

acyl-CoA

Acetaldehyde Ethanol

Fatty acid ethyl

esters

Steen et al. 2010.

Nature 463:559

β-keto

acyl-CoA

Can FadR regulate FAEE production?

pdc adhB

Fatty-ACP Fatty

ACC FAS FadD Fatty

acyl-CoA

Acetaldehyde Ethanol Pdc AdhB Fatty

esters

atfA PfadD

PlacUV5

Zhang et al. 2012 Nat. Biotechnol. 30:354

FadR regulation improves FAEE production

PlacUV5

PlacUV5PAR

PFL1PFL2

PFL3Promoters for

ethanol production

Promoters

for atfA

FAEE titer (mg/L)

• Four-fold improvement

in FAEE production

• Significantly improved

stability of host

Zhang et al. 2012 Nat. Biotechnol. 30:354

Melting points of various biofuels

12-methyl tetradecanoic acid methyl ester

Melting point = -5.3°C

pentadecanoic acid methyl ester

Melting point = 18°C

Farnesane

Melting point = <-78°C

Bisabolane

Melting point = <-78°C

13-methyl tetradecanoic acid methyl ester

Melting point = 6.4°C

A few branches are important!

Gasolines

• High octane

• Many branches

• Some aromatics

Jet fuels

• Few branches

• Some aromatics

Diesels

• Few branches

• Some aromatics

Production of branched-chain fatty acids

Fatty Acyl-ACP

FAS (E. coli FabH)

Mal-CoA Ketoacyl-CoA

Branched

Fatty Acyl-ACP

FAS (B. subtilis FabH)

Alkanes Esters Ketones Alcohols

Leu, Val, Ile BCKA

Decarboxylase

Production of branched-chain fatty acids

• Branched-chain amino acid biosynthetic pathway produces precursors to branched-chain fatty acids

• B. subtilis fabH2 initiates fatty acid synthesis with branched acyl-CoA precursors

Melting point = -5C (methyl ester)*

Melting point = 18C (methyl ester)*

All genes in blue

were overexpressed

Haushalter et al. 2014. Met. Eng. 26:111-118

Anteiso-branched fatty acid production

• E. coli MG1655ΔfadE

expressing ‘tesA and fadR

• only straight-chain saturated

and unsaturated fatty acids

Straight-chain, even carbon

Straight-chain, odd carbon

Iso-branched

Anteiso-branched

• Branched acyl-CoA production,

coupled with B. subtilis fabH2

• 22% anteiso branched species

Haushalter et al. 2014. Met. Eng. 26:111-118

A few branches are important!

Gasolines

• High octane

• Many branches

• Some aromatics

Jet fuels

• Few branches

• Some aromatics

Diesels

• Few branches

• Some aromatics

Branched-chain hydrocarbons

from isoprenoids

Pyruvate

Acetyl-CoA

Glyoxylate

SUC-CoA

Acetaldehyde

Ethanol

IPP DMAPP

Geraniol

Farnesol

Geranylgeraniol

Monoterpene

Sesquiterpene

Diterpene

G6P 6PG X5P

Glucose

Xylose

Mannose M6P

Xylulose

Ribulose

Arabinose

Various esters

Alcohols

hydrocarbons

Isoprenoids

from isoprenoid biosynthetic pathway FDP

G3P DHAP

AcCoA IPP DMAPP

FPP quinones

Sesquiterpenes

Monoterpenes

Hemiterpenes

DMAPP Monoterpenes

G3P DHAP

AcCoA IPP

FPP quinones

Sesquiterpenes

IPP 3-methyl-3-butenol

phosphatase

DMAPP 3-methyl-2-butenol

3-methyl-butanolreductase

HAD-likePhosphataseLibrary

(-)control

OYELibrary

OPP OH

OHBiofuel

Water solubility

Research Octane

Numbera

Energy

Density

3-methyl-2-butenol

6 + 0.34 90

3-methyl-3-butenol

9 + 0.15 102

3-methyl-butanol

15 + 3.2 102

96% of gasoline

MJ/gal)

Hemiterpenes

from isoprenoid biosynthetic pathway

G3P DHAP

AcCoA IPP

FPP quinones

Sesquiterpenes

Hemiterpenes IPP 3-methyl-3-butenol

phosphatase

(-)control

OYELibrary

OPP OH

OHBiofuel

Water solubility

Research Octane

Numbera

Energy

Density

3-methyl-2-butenol

6 + 0.34 90

3-methyl-3-butenol

9 + 0.15 102

3-methyl-butanol

15 + 3.2 102

96% of gasoline

MJ/gal)

Monoterpenes Limonene

synthase

Pinene

synthase

Pd/C, H2

Chemical

catalyst

G3P DHAP

AcCoA IPP

FPP quinones

Hemiterpenes

synthase

Pinene

synthase

Pd/C, H2

Chemic

catalyst

phosphatase

(-)control

OYELibrary

OPP OH

OHBiofuel

Water solubility

Research Octane

Numbera

Energy

Density

3-methyl-2-butenol

6 + 0.34 90

3-methyl-3-butenol

9 + 0.15 102

3-methyl-butanol

15 + 3.2 102

96% of gasoline

MJ/gal)

Sesquiterpenes

Peralta-Yahya et al. 2011. Nature Comm. 2:483

McAndrews et al. 2011. Structure 19:1876

DMAPP Monoterpenes

G3P DHAP

AcCoA IPP

FPP quinones

Sesquiterpenes

phosphatase

(-)control

OYELibrary

OPP OH

OHBiofuel

Water solubility

Research Octane

Numbera

Energy

Density

3-methyl-2-butenol

6 + 0.34 90

3-methyl-3-butenol

9 + 0.15 102

3-methyl-butanol

15 + 3.2 102

96% of gasoline

MJ/gal)

Hemiterpenes

Branched-C5 alcohols

are promising gasoline replacements

• High energy content

– 96% of gasoline

• Low solubility in

• High blending

octane number

3-methyl-2-butenol

3-methyl-3-butenol

3-methyl-butanol

Constructing de novo synthetic pathways

for biofuel targets

OH IPP 3-methyl-3-butenol

phosphatase

OH DMAPP 3-methyl-2-butenol

OH 3-methyl-butanol

reductase

Novel phosphatases for synthesizing isopentenols

Superfamily Haloacid dehalogenase-like

(HAD) Phosphatase

Nudix Hydrolase

# sequenced >3000 >4000

# in E. coli 29 13

Mechanism Covalent Enzyme-

Substrate Intermediate

Metal-assisted hydrolysis

HAD-like Phosphatase Library

(-) control

Nudix Hydrolase Library

(-) control

Howard Chou

Constructing de novo synthetic pathways

for biofuel targets

OH IPP 3-methyl-3-butenol

phosphatase

OH DMAPP 3-methyl-2-butenol

OH 3-methyl-butanol

reductase

Howard Chou

Novel reductases for synthesizing isopentanol

Cultures fed 1g/L 3-methyl-2-butenol for 24 hours.

(-) control OYE Library

Williams & Bruce. Microbiol. 148, 1607-14 (2002).c

Old Yellow Enzyme Family

Howard Chou

A Hybrid Sensor for IPP

AraC binding sites GFP

IPP isomerase

linker

AraC DNA

binding domain

Mev IPP

IPP IPP

Mev added to culture

Mev absent from culture

Chou et al 2013 Nat Commun 4:2595

A Hybrid Sensor for IPP

Mev IPP

IPP IPP

Mev added to culture

Using the Hybrid Sensor to Select for Mutations that

Increase [IPP]

AraC binding sites MutD5

Mev IPP

IPP IPP

High [IPP] in cells Low mutation rate

Low [IPP] in cells High mutation rate

Mutated Cells with Increased [IPP] Produce more

Lycopene

0 72 144 216 288 360 432

Time (hrs)

AraC IA32 IA44

Mev IPP DMAPP

GGPP Lycopene

E. coli chromosome

Non-synonymous

All SNPs

G3P DHAP

AcCoA IPP

FPP quinones

Sesquiterpenes

Hemiterpenes IPP 3-methyl-3-butenol

phosphatase

(-)control

OYELibrary

OPP OH

OHBiofuel

Water solubility

Research Octane

Numbera

Energy

Density

3-methyl-2-butenol

6 + 0.34 90

3-methyl-3-butenol

9 + 0.15 102

3-methyl-butanol

15 + 3.2 102

96% of gasoline

MJ/gal)

synthase

Pinene

synthase

Pd/C, H2

Chemical

catalyst

Engineering pinene production

DMAPP GPP Pinene

GPP Synthase Pinene Synthase

Sarria et al 2014 ACS Syn Bio In press

GPP and pinene synthases from Grand Fir produce the

highest pinene titers

Pinene synthase competes for GPP

DMAPP GPP Pinene

FPP Growth

Linking enzymes to improve GPP access

Pinene

GPPS at the N-terminus with a sufficient linker length is

G3P DHAP

AcCoA IPP

FPP quinones

Hemiterpenes

synthase

Pinene

synthase

Pd/C, H2

Chemic

catalyst

phosphatase

(-)control

OYELibrary

OPP OH

OHBiofuel

Water solubility

Research Octane

Numbera

Energy

Density

3-methyl-2-butenol

6 + 0.34 90

3-methyl-3-butenol

9 + 0.15 102

3-methyl-butanol

15 + 3.2 102

96% of gasoline

MJ/gal)

Sesquiterpenes

Peralta-Yahya et al. 2011. Nature Comm. 2:483

McAndrews et al. 2011. Structure 19:1876

IPP DMAPP

Branched-chain hydrocarbons from the isoprenoid

biosynthetic pathway

G3P DHAP

AcCoA IPP

FPP quinones

IPP DMAPP

Branched-chain hydrocarbons from the isoprenoid

biosynthetic pathway

G3P DHAP

AcCoA IPP

FPP quinones

Bisabolane as a renewable diesel

No 2 Diesel Bisabolane

Cetane No. 41.6 41.9

Freeze pt. N/a <-81C

Cloud pt. -21C <-78C

Density 864.6 g/L 819 g/L

Bisabolene Bisabolane

Peralta-Yahya et al. 2010 Nature Comm. 2:483

Finding the best bisabolene synthase

Peralta-Yahya et al. 2010 Nature Comm. 2:483

Finding the best bisabolene synthase

McAndrew et al. 2011 Structure 19:1876

Bisabolene (C15) Synthase Aristolochene (C15) Synthase

Squalene Hopene (C30) Cyclase Trichodiene (C15) Synthase

Structural comparison

Pathway balance is essential to prevent

accumulation of toxic intermediates

G3P DHAP

IPP DMAPP

[Mevalonate]

0 2 4 6 8 10 12

Time (hours)

Balancing expression is difficult with inducible

promoters

• Typically, we use inducible promoters to

control expression of genes in a metabolic

pathway

• Inducers are expensive

• Tuning expression can be challenging

PMK MK PMD Idi IspA FPP ADS Mev Terpene

IPTG Arabinose

Sensing toxic intermediates to balance metabolic

pathway fluxes

• Can we use the toxicity of FPP to regulate the

metabolic pathways that produce and consume

• Can we reduce accumulation of toxic

intermediates?

• How do we regulate the pathway dynamically …

in the face of changing environmental

conditions?

PMK MK PMD Idi IspA FPP ADS Mev Terpene

Identification of FPP-responsive promoters

• No terpene synthase to

consume FPP

• Examine differences in gene

expression in the presence and

absence of a terpene synthase

PMK MK PMD Idi IspA Mev FPP

PMK MK PMD Idi IspA FPP ADS Mev Amorp

Dahl et al. 2013 Nat. Biotechnol. 31:1039.

Expression of about 40 promoters significantly changed

in response to FPP

Without sesquiterpene

synthase

With sesquiterpene

synthase

Two FPP-responsive promoters

HMGS atoB tHMGR PMK MK PMD Idi IspA FPP ADS

Plac Plac Plac

HMGS atoB tHMGR PMK MK PMD Idi IspA FPP ADS

PrstA PgadE PgadE

Inducible system

Dynamically-controlled system

pADS prstA-ADS pADS prstA-ADS

lacUV5-MevT-MBIS pgadE-MevT-MBIS

FPP-responsive promoters

improve terpene production

HMGS atoB tHMGR PMK MK PMD Idi IspA

Plac Plac

HMGS atoB tHMGR PMK MK PMD Idi IspA FPP

PgadE PgadE

FPP ADS

High product titers with

inducer-free control

Fuel toxicity

a-pinene limonene

E. coli M9 Minimal Media (vol/vol)

Bioprospecting for solvent resistance pumps (SRPs)

Many different pumps in many

microbes

How do we find the right ones?

Dunlop et al. 2011. Mol. Sys. Biol. 7:487.

Bioprospecting for solvent resistance pumps (SRPs)

• Search genomes of all

sequenced gram-negative

bacteria for efflux pumps

• Final library contained

pumps distributed across

the homology profile

• Used SLIC-based strategy

to create efflux pump strain

library

Pumps screened by competition

For this we designed custom microarrays which contain

probes for all known efflux pumps from sequenced bacteria.

Expression

plasmids and

individual

strains

strains in

proportion

Pool is grown in M9 medium (control)

OR M9 medium with biofuel (test)

Arrays used

to identify

remaining in

the pool at

dilution

Plasmid isolated

from pools at

every dilution and

identify winner

Biofuel competitions

Engineering pinene tolerance into E. coli

a-pinene

Microorganisms engineered

for consolidated bioprocessing

Problem: Enzymes

add significant

cost to the biofuel

Solution: Engineer fuel-producing

microbes to secrete enzymes

Biofuel

Pretreatment Enzymes

E. coli growth on Beechwood Xlyan

Beechwood Xylan Xylodextrins

Xylose/

Arabinose

Endoxylanase β-xylosidase

M9 minimal medium with

0.2% ionic liquid-

extracted switchgrass

xyn10B OsmY xsa

Endoxylanase β-xylosidase

Beechwood xylan

Xylose

Bokinsky et al. 2011. Proc. Natl. Acad. Sci. USA. 108:19949.

Endocellulase: OsmY-Cel Betaglucosidase: Cel3A

E. coli growth on phosphoric

acid-swollen cellulose

cel OsmY cel3A

Endocellulase β-glucosidase

Switchgrass Eucalyptus

E. coli growth on

ionic liquid-treated plant biomass

Growth on IL-treated garden waste

Bokinsky et al. 2011.

Proc. Natl. Acad. Sci. USA. 108:19949.

Fatty acid ethyl esters (biodiesel)

Butanol (gasoline substitute)

Pinene (jet fuel precursor)

Jet fuel, diesel fuel, and gasoline

from switchgrass

Polyketide-based fuels

Pyruvate

Acetyl-CoA

Glyoxylate

SUC-CoA

Acetaldehyde

Ethanol

IPP DMAPP

Geraniol

Farnesol

Geranylgeraniol

Monoterpene

Sesquiterpene

Diterpene

G6P 6PG X5P

Glucose

Xylose

Mannose M6P

Xylulose

Ribulose

Arabinose

Various esters

Alcohols

hydrocarbons

Isoprenoids

Erythromycin Nystatin

Lipomycin

Polyketides

Erythromycin

AT ACP KS AT ACP

TE KS AT ACP

KS AT ACP

AT ACP

KS AT ACP

KS ACP

Donadio 1991 Science

Erythromycin synthase

DEBS1 DEBS2

Module 1 Module 2 Module 3 Module 4

Module 5 Module 6

DEBS1 DEBS2

Module 5 Module 6

AT ACP KS AT ACP

TE KS AT ACP

KS AT ACP

AT ACP

KS AT ACP

KS ACP

Polyketide synthases (PKSs)

are very large, multi-activity proteins

Protein 1 Protein 2 Protein 3

DEBS1 DEBS2

Module 5 Module 6

AT ACP KS AT ACP

TE KS AT ACP

KS AT ACP

AT ACP

KS AT ACP

KS ACP

Each extension module adds 2 carbons (plus pendant

groups) to the polyketide backbone

Extension

Module

Loadin

Module

DEBS1 DEBS2

Module 5 Module 6

AT ACP KS AT ACP

TE KS AT ACP

KS AT ACP

AT ACP

KS AT ACP

KS ACP

Polyketide synthases (PKSs)

are very large, multi-activity proteins

Extension

Module

Loadin

Module

Termination

Module

DEBS1 DEBS2

Module 5 Module 6

AT ACP KS AT ACP

TE KS AT ACP

KS AT ACP

AT ACP

KS AT ACP

KS ACP

Each domain in a PKS has an individual enzyme activity

Domains

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

Basic PKS

AT KS AT ACP ACP TE

HSCoA SCoA

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

R1 SCoA

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

Basic PKS

AT KS AT ACP ACP TE

Polyketide synthase structure

Whicher et al. 2014. Nature. 510(7506): 560–564

ACP moves from active site to active site

like an arm on a robot

Whicher et al. 2014. Nature. 510(7506): 560–564

PKSs function like

molecular assembly lines

Lots of naturally occurring Load, Extension, and

Termination modules

AT KS AT ACP ACP TE

S1 mAT ACPKSQ

ACPKSQS2

ACPCoLS3 ERR

E1mAT ACPKS

E2 mAT

E3 mAT

E6 mmAT(

S)ACPKS

E7ACPKS

KR(S)mmAT(

E8ACPKS

KR(R)mmAT(

KR(S)mmAT(

KR(R)mmAT(

E11mmAT

E12ACPKS

E13ACPKS

cMTmmAT

ACPKS eAT (S)

E16ACPKS

eAT (S)

ACPKS pAT (S)

Red-TE

Red-TEOH

Polyesters

• Used for

– Cushioning, clothing, bottles, films, liquid crystal

displays, holograms, filters, dielectric films, insulation for

– Finishes on wood products, paints, etc.

Product Type 2002 (M tons/yr) 2008 (M tons/yr)

Textile-PET 20 39

Resin, bottle PET 9 16

Film-PET 1.2 1.5

Special polyester 1 2.5

Total 31.2 49

Hydroxyacids are needed for polyesters

3-hydroxy acids

Erythromycin

AT ACP KS AT ACP

TE KS AT ACP

KS AT ACP

AT ACP

KS AT ACP

KS ACP

DEBS1 DEBS2

Module 5 Module 6

Erythromycin

AT ACP KS AT ACP

TE KS AT ACP

KS AT ACP

AT ACP

KS AT ACP

KS ACP

DEBS1 DEBS2

Module 5 Module 6

Cortes et al. 1995. Science

AT ACP KS AT ACP

TE KS AT ACP

Protein Engineering

Module 1 Module 2

Cortes et al. 1995. Science

AT ACP KS AT ACP

TE KS AT ACP

Protein Engineering

Module 1 Module 2

AT ACP KS AT ACP

Production of 3-hydroxyacids

Module 1

Satoshi Yuzawa, Clara Eng

Potential polymers that can be produced with various 3-

hydroxyacids

Interesting polyesters with novel properties

LipPks1 1 LipPks2, LipPks3, LipPks4, LipNrps

b-Lipomycin

AT ACP KS AT ACP

Lipomycin PKS

Yuzawa et al. 2013. Biochemistry 52:3791-3793.

Lipomycin load has a broad substrate range

Substrate kcat (min-

kcat/Km

(M-1/s-1)

isobutyryl-CoA 0.053 2.9 304.1

propionyl-CoA 0.056 13.4 70.3

n-butyryl-CoA 0.036 26.4 22.7

2-methylbutyryl-CoA 0.126 8.8 237.0

isovaleryl-CoA 0.290 128.1 38.0

pivaloyl-CoA 0.002 8.8 4.1

Constructing a 3-hydroxyacid synthase

AT ACP KS AT ACP

Thioesterase (TE) from DEBS PKS

Load and extension modules

from Lipomycin PKS

Load Extension

AT ACP KS AT ACP

Production of 3-hydroxy-2-methyl acids

with Lip PKS

Extension

Product

Native substrate

Potential polymers that can be produced with various 3-

hydroxyacids

Interesting polyesters with novel properties

The acyltransferase (AT) determines the acyl-CoA

loaded onto the ACP

AT KS AT ACP ACP TE

R1 SCoA

loaded onto the ACP

AT KS AT ACP ACP TE

R1 SCoA

Specifies that a methylmalonyl group

will be loaded onto the ACP.

Native DEBS1

Module 1 Module 2

AT ACP KS AT ACP

KS AT ACP

Acyl transferase (AT) specifies the acyl group that

gets loaded onto the ACP

Module 1 Module 2

AT ACP KS AT ACP

KS AT ACP

Modified DEBS1

Oliynyk et al 1996 Chem. Biol. 3:883

loaded onto the ACP

AT KS mAT ACP ACP TE

Specifies that a malonyl group will be

loaded onto the ACP.

AT ACP KS mAT ACP

Production of 3-hydroxyacids with Lip PKS

Extension

Product

Native substrate

Yuzawa et al 2015 unpublished

Ketones

• Solvents

– Methyl ethyl ketone (MEK)

– Ethyl ethyl ketone (EEK)

• Flavors and fragrances

– Long-chain alkyl groups

– Stereochemistry matters

• Fuels

– Diesels (C14-C16)

- NADPH

Δ AT ACP KS AT ACP

In vitro production of ethyl ketones

LipPK1 + DEBS TE

Δ AT ACP KS AT ACP

In vivo production of ethyl ketones

LipPK1 + DEBS TE

Knocking out activity of ketoreductase (KR)

disables reduction of ketone to alcohol

Δ AT ACP KS mAT ACP

In vivo production of methyl ketones

Exchanging the native methylmalonyl-

CoA—specific acyltransferase (AT) for a

malonyl-CoA—specific AT enables

production of methyl ketones

Acknowledgements

• US Department of Energy for funding

• All of the members of JBEI

#Jay Keasling has a financial interest in Amyris, LS9, and Lygos.

Artemisinic Acid

Glucose Artemisinin

A Novel Semi-synthetic Route

•US Department of Energy for

funding

•All of the members of JBEI

funding

Bill & Melinda Gates Foundation

Department of Energy

National Science Foundation

Funding:

Thanks to:

Eric Steen

Connie Kang

Greg Bokinsky

Robert Haushalter

Leonard Katz

Fuzhong Zhang

James Carothers

Andrew Hagen

Sean Poust

Ee-Been Goh

Harry Beller

Edward Baidoo

Chris Petzold

Tanveer Batth

Dan Groff

Sam Deutsch

Ted Chavkin

Simon Brunner

Satoshi Yuzawa

Clara Eng

Tristan de Rond

Clem Fortman

George Wang

Weerawat Runguphan

Funding:

US Department of Energy

National Science Foundation

University of California Discovery Grant Program

Institutes and Centers:

Joint BioEnergy Institute (JBEI)

Synthetic Biology Engineering Research Center (Synberc)

Disclosure:

Jay Keasling has a financial interest in Amyris & Lygos

Thanks to …

DOE, NSF for funding

The JBEI Team

Disclosure: Jay Keasling has a financial interest in Amyris & Lygos.

engineering microbial metabolism for production of fuels

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