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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
• Long-chain alkanes
• Few branches
• Some aromatics
Linear hydrocarbons from the fatty acid biosynthetic
pathways
PEP
Pyruvate
Acetyl-CoA
CIT
ICT
OGA
SUC
FUM
OAA
MAL
CO2
Glyoxylate
SUC-CoA
Acetaldehyde
Ethanol
Propionyl-CoA Propanol
Acetoacetyl-CoA Butyryl-CoA Butanol
Even chain fatty acid
Aliphatic hydrocarbons
Odd chain fatty acid
G3P
IPP DMAPP
GPP
FPP
GGPP
Isopentenol Isopentanol
Geraniol
Farnesol
Geranylgeraniol
Monoterpene
Sesquiterpene
Diterpene
DHAP
FBP
F6P
G6P 6PG X5P
E4P
S7P
Valeryl-CoA Isopentanol
Glucose
Xylose
Mannose M6P
Galactose G’1P G1P
Xylulose
R5P
Ribulose
Arabinose
Various esters
Alcohols
Aromatic hydrocarbons
Short, highly-branched
hydrocarbons
Linear hydrocarbons make great diesels
and jet fuels
Gasolines
• High octane
• Short-chain alkanes
• Many branches
• Some aromatics
Jet fuels
• Long-chain alkanes
• Few branches
• Some aromatics
Diesels
• Appropriate cetane numbers
• Long-chain alkanes
• Few branches
• Some aromatics
Linear hydrocarbons from the fatty acid biosynthetic
pathways
PEP
Pyruvate
Acetyl-CoA
CIT
ICT
OGA
SUC
FUM
OAA
MAL
CO2
Glyoxylate
SUC-CoA
Acetaldehyde
Ethanol
Propionyl-CoA Propanol
Acetoacetyl-CoA Butyryl-CoA Butanol
Even chain fatty acid
Aliphatic hydrocarbons
Odd chain fatty acid
G3P
IPP DMAPP
GPP
FPP
GGPP
Isopentenol Isopentanol
Geraniol
Farnesol
Geranylgeraniol
Monoterpene
Sesquiterpene
Diterpene
DHAP
FBP
F6P
G6P 6PG X5P
E4P
S7P
Valeryl-CoA Isopentanol
Glucose
Xylose
Mannose M6P
Galactose G’1P G1P
Xylulose
R5P
Ribulose
Arabinose
Various esters
Alcohols
Aromatic hydrocarbons
Short, highly-branched
hydrocarbons Fatty acids
Wild type E. coli produces no free fatty acids
because of strict acyl-ACP inhibition PEP
PYR
AcCoA
CIT
Fatty-ACP Fatty
acid
ACC FAS
Lipid
FadD Fatty
acyl-CoA
FadA
Steen et al. 2010.
Nature 463:559
FadB
FadE
β-keto
acyl-CoA
WT
Production of fatty acid ethyl esters
PEP
PYR
AcCoA
CIT
Fatty-ACP Fatty
acid
ACC FAS
Lipid
FadD Fatty
acyl-CoA
FadA
AtfA
TesA
Acetaldehyde Ethanol
Pdc
AdhB
Fatty acid ethyl
esters
Steen et al. 2010.
Nature 463:559
FadB
FadE
β-keto
acyl-CoA
Methyl ketones produced from fatty acyl-CoA
by overexpressing FadEBM PEP
PYR
AcCoA Fatty-ACP Fatty
acid
ACC FAS
Lipid
FadD Fatty
acyl-CoA
FadA
TesA
β-keto
acyl-CoA
FadM FadB
FadE
CIT
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
0.12
0.62
10
120
8.6
36
390
570
1350 1400
0.1
1
10
100
1000
10000
Me
thy
l ke
ton
es
(m
g/L
)
TSB
M9-MOPS (1% Gluc)
12020
Goh et al. 2012
‘tesA, fadE + + + + + + + + + +
fadM + + + + + + + +
aco, fadB, fadA + + + + + + + +
fadR, fadD + + + + +
poxB + + +
1-plasmid, co-aco
+ + +
ackA-pta + +
EG
S8
95
EG
S1
710
EG
S1
895
EG
S8
95
EG
S5
60
EG
S1
32
0
EG
S1
37
0
EG
S18
90
EGS860
EGS084
TesA-FadD combination wastes ATP
ATP is consumed
when free fatty acids
are
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
AcCoA
ACC
FAS1 FAS2
Fatty-CoA
TesA DGAT
FAR
AtfA
Tef1 FAS1
Chromosomal DNA
Tef1 FAS2
Tef1 ACC
b-oxidation
Engineered yeast strain WR1
! "
#! "
$! ! "
$#! "
%! ! " O
nOH
! "# $%"&'( )%
wt
mg/L
wt
TesA
WR1
TesA
WR1
! ACSs
TesA ! "
#"
$! "
$#"
%! "
O
O
O
O
O
O
n
n
n
! " #$%
wt wt
DGAT
WR1
DGAT
WR1
! ²-oxidation
DGAT
Lipid Content (%)
! "
#! "
$! "
%&! "
nOH
! "# $%%"&' ( ) ( &*%
mg/L
wt wt
FAR
WR1
FAR
WR1
! ²-oxidation
FAR
! "
#"
$"
%"O
nOEt
! " ##$%
mg/L
wt wt
AtfA
WR1
! ²-oxidation
AtfA
X
Fatty acid-derived fuels in S. cerevisiae
Unbalanced FAEE pathway caused strain instability
PEP
PYR
AcCoA
CIT
Fatty-ACP Fatty
acid
ACC FAS
Lipid
FadD Fatty
acyl-CoA
FadA
AtfA
TesA
Acetaldehyde Ethanol
Pdc
AdhB
Fatty acid ethyl
esters
Steen et al. 2010.
Nature 463:559
FadB
FadE
β-keto
acyl-CoA
Can FadR regulate FAEE production?
Peth
pdc adhB
PYR
Fatty-ACP Fatty
acid
ACC FAS FadD Fatty
acyl-CoA
TesA
Acetaldehyde Ethanol Pdc AdhB Fatty
esters
AtfA
AcCoA
PatfA
atfA PfadD
fadD
PBAD
fadR
PlacUV5
tesA
Zhang et al. 2012 Nat. Biotechnol. 30:354
FadR regulation improves FAEE production
PlacUV5
PAR
PFL1
PFL2
PFL3
0
200
400
600
800
1000
1200
1400
1600
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
• Short-chain alkanes
• Many branches
• Some aromatics
Jet fuels
• Long-chain alkanes
• Few branches
• Some aromatics
Diesels
• Appropriate cetane numbers
• Long-chain alkanes
• Few branches
• Some aromatics
Production of branched-chain fatty acids
AcCoA
Fatty Acyl-ACP
ACC
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
• Short-chain alkanes
• Many branches
• Some aromatics
Jet fuels
• Long-chain alkanes
• Few branches
• Some aromatics
Diesels
• Appropriate cetane numbers
• Long-chain alkanes
• Few branches
• Some aromatics
Branched-chain hydrocarbons
from isoprenoids
PEP
Pyruvate
Acetyl-CoA
CIT
ICT
OGA
SUC
FUM
OAA
MAL
CO2
Glyoxylate
SUC-CoA
Acetaldehyde
Ethanol
Propionyl-CoA Propanol
Acetoacetyl-CoA Butyryl-CoA Butanol
Even chain fatty acid
Aliphatic hydrocarbons
Odd chain fatty acid
G3P
IPP DMAPP
GPP
FPP
GGPP
Isopentenol Isopentanol
Geraniol
Farnesol
Geranylgeraniol
Monoterpene
Sesquiterpene
Diterpene
DHAP
FBP
F6P
G6P 6PG X5P
E4P
S7P
Valeryl-CoA Isopentanol
Glucose
Xylose
Mannose M6P
Galactose G’1P G1P
Xylulose
R5P
Ribulose
Arabinose
Various esters
Alcohols
Aromatic hydrocarbons
Short, highly-branched
hydrocarbons
Isoprenoids
Branched-chain hydrocarbons
from isoprenoid biosynthetic pathway FDP
G3P DHAP
PEP
PYR
AcCoA IPP DMAPP
OAA
MAL
CIT
MEV
DXP
FPP quinones
GPP
Sesquiterpenes
Monoterpenes
Hemiterpenes
DMAPP Monoterpenes
FDP
G3P DHAP
PEP
PYR
AcCoA IPP
OAA
MAL
CIT
MEV
DXP
FPP quinones
GPP
Sesquiterpenes
IPP 3-methyl-3-butenol
phosphatase
DMAPP 3-methyl-2-butenol
3-methyl-butanolreductase
0
2
4
6
8
10
12
14
16
18
20
C5
Alc
oh
ols
(m
g/L/
OD
)
HAD-likePhosphataseLibrary
(-)control
0
100
200
300
400
500
Iso
pen
tan
ol(
mg/
L)
(-)control
OYELibrary
OHOPP
OPP OH
OHBiofuel
Water solubility
(g/L)
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
(116
MJ/gal)
Hemiterpenes
Branched-chain hydrocarbons
from isoprenoid biosynthetic pathway
DMAPP
FDP
G3P DHAP
PEP
PYR
AcCoA IPP
OAA
MAL
CIT
MEV
DXP
FPP quinones
GPP
Sesquiterpenes
Hemiterpenes IPP 3-methyl-3-butenol
phosphatase
DMAPP 3-methyl-2-butenol
3-methyl-butanolreductase
0
2
4
6
8
10
12
14
16
18
20
C5
Alc
oh
ols
(m
g/L/
OD
)
HAD-likePhosphataseLibrary
(-)control
0
100
200
300
400
500
Iso
pen
tan
ol(
mg/
L)
(-)control
OYELibrary
OHOPP
OPP OH
OHBiofuel
Water solubility
(g/L)
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
(116
MJ/gal)
Monoterpenes Limonene
synthase
Pinene
synthase
Pd/C, H2
Chemical
catalyst
Branched-chain hydrocarbons
from isoprenoid biosynthetic pathway
DMAPP
FDP
G3P DHAP
PEP
PYR
AcCoA IPP
OAA
MAL
CIT
MEV
DXP
FPP quinones
GPP
Hemiterpenes
Monoterpenes Limonene
synthase
Pinene
synthase
Pd/C, H2
Chemic
al
catalyst
IPP 3-methyl-3-butenol
phosphatase
DMAPP 3-methyl-2-butenol
3-methyl-butanolreductase
0
2
4
6
8
10
12
14
16
18
20
C5
Alc
oh
ols
(m
g/L/
OD
)
HAD-likePhosphataseLibrary
(-)control
0
100
200
300
400
500
Iso
pen
tan
ol(
mg/
L)
(-)control
OYELibrary
OHOPP
OPP OH
OHBiofuel
Water solubility
(g/L)
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
(116
MJ/gal)
Sesquiterpenes
Peralta-Yahya et al. 2011. Nature Comm. 2:483
McAndrews et al. 2011. Structure 19:1876
Branched-chain hydrocarbons
from isoprenoid biosynthetic pathway
DMAPP Monoterpenes
Branched-chain hydrocarbons
from isoprenoid biosynthetic pathway
FDP
G3P DHAP
PEP
PYR
AcCoA IPP
OAA
MAL
CIT
MEV
DXP
FPP quinones
GPP
Sesquiterpenes
IPP 3-methyl-3-butenol
phosphatase
DMAPP 3-methyl-2-butenol
3-methyl-butanolreductase
0
2
4
6
8
10
12
14
16
18
20
C5
Alc
oh
ols
(m
g/L/
OD
)
HAD-likePhosphataseLibrary
(-)control
0
100
200
300
400
500
Iso
pen
tan
ol(
mg/
L)
(-)control
OYELibrary
OHOPP
OPP OH
OHBiofuel
Water solubility
(g/L)
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
(116
MJ/gal)
Hemiterpenes
Branched-C5 alcohols
are promising gasoline replacements
• High energy content
– 96% of gasoline
• Low solubility in
water
• High blending
octane number
OH
OH
OH
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
OPP
OPP
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
0
2
4
6
8
10
12
14
16
18
20
C5 A
lco
ho
ls (
mg
/L/O
D)
HAD-like Phosphatase Library
(-) control
Nudix Hydrolase Library
0
2
4
6
8
10
12
14
16
18
20
C5 A
lco
ho
ls (
mg
/L/O
D)
(-) control
2X
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
OPP
OPP
Howard Chou
Novel reductases for synthesizing isopentanol
0
50
100
150
200
250
300
350
400
450
Iso
pen
tan
ol (m
g/L
)
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
AraC binding sites GFP
X
Mev IPP
IPP IPP
Mev added to culture
Mev absent from culture
DMAPP
Chou et al 2013 Nat Commun 4:2595
A Hybrid Sensor for IPP
AraC binding sites GFP
X
Mev IPP
IPP IPP
Mev added to culture
DMAPP
Chou et al 2013 Nat Commun 4:2595
Using the Hybrid Sensor to Select for Mutations that
Increase [IPP]
AraC binding sites MutD5
AraC binding sites MutD5
X
Mev IPP
IPP IPP
High [IPP] in cells Low mutation rate
Low [IPP] in cells High mutation rate
DMAPP
AcCoA
x
x
x
x
x
x
Chou et al 2013 Nat Commun 4:2595
Mutated Cells with Increased [IPP] Produce more
Lycopene
0
1000
2000
3000
4000
5000
6000
7000
8000
0 72 144 216 288 360 432
Lyco
pen
e (
p.p
.m.)
Time (hrs)
AraC IA32 IA44
Mev IPP DMAPP
AcCoA
GGPP Lycopene
E. coli chromosome
Non-synonymous
SNPs
All SNPs
Chou et al 2013 Nat Commun 4:2595
DMAPP
Branched-chain hydrocarbons
from isoprenoid biosynthetic pathway
FDP
G3P DHAP
PEP
PYR
AcCoA IPP
OAA
MAL
CIT
MEV
DXP
FPP quinones
GPP
Sesquiterpenes
Hemiterpenes IPP 3-methyl-3-butenol
phosphatase
DMAPP 3-methyl-2-butenol
3-methyl-butanolreductase
0
2
4
6
8
10
12
14
16
18
20
C5
Alc
oh
ols
(m
g/L/
OD
)
HAD-likePhosphataseLibrary
(-)control
0
100
200
300
400
500
Iso
pen
tan
ol(
mg/
L)
(-)control
OYELibrary
OHOPP
OPP OH
OHBiofuel
Water solubility
(g/L)
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
(116
MJ/gal)
Monoterpenes Limonene
synthase
Pinene
synthase
Pd/C, H2
Chemical
catalyst
Engineering pinene production
IPP
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
Sarria et al 2014 ACS Syn Bio In press
Pinene synthase competes for GPP
IPP
DMAPP GPP Pinene
GPP Synthase Pinene Synthase
FPP Growth
Sarria et al 2014 ACS Syn Bio In press
Linking enzymes to improve GPP access
IPP
DMAPP
GPP
Pinene
GPP Synthase Pinene Synthase
Sarria et al 2014 ACS Syn Bio In press
GPPS at the N-terminus with a sufficient linker length is
best
Sarria et al 2014 ACS Syn Bio In press
DMAPP
Branched-chain hydrocarbons
from isoprenoid biosynthetic pathway
FDP
G3P DHAP
PEP
PYR
AcCoA IPP
OAA
MAL
CIT
MEV
DXP
FPP quinones
GPP
Hemiterpenes
Monoterpenes Limonene
synthase
Pinene
synthase
Pd/C, H2
Chemic
al
catalyst
IPP 3-methyl-3-butenol
phosphatase
DMAPP 3-methyl-2-butenol
3-methyl-butanolreductase
0
2
4
6
8
10
12
14
16
18
20
C5
Alc
oh
ols
(m
g/L/
OD
)
HAD-likePhosphataseLibrary
(-)control
0
100
200
300
400
500
Iso
pen
tan
ol(
mg/
L)
(-)control
OYELibrary
OHOPP
OPP OH
OHBiofuel
Water solubility
(g/L)
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
(116
MJ/gal)
Sesquiterpenes
Peralta-Yahya et al. 2011. Nature Comm. 2:483
McAndrews et al. 2011. Structure 19:1876
IPP DMAPP
FPP
GPP
Branched-chain hydrocarbons from the isoprenoid
biosynthetic pathway
FDP
G3P DHAP
PEP
PYR
AcCoA IPP
OAA
MAL
CIT
MEV
DXP
FPP quinones
IPP DMAPP
FPP
GPP
Branched-chain hydrocarbons from the isoprenoid
biosynthetic pathway
FDP
G3P DHAP
PEP
PYR
AcCoA IPP
OAA
MAL
CIT
MEV
DXP
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
FDP
G3P DHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
DXP
FPP
MEV
5 mM
10 mM
20 mM
40 mM
[Mevalonate]
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12
Time (hours)
Cell
Gro
wth
(O
D 6
00 )
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
it?
• 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
FPP
Dahl et al. 2013 Nat. Biotechnol. 31:1039.
Two FPP-responsive promoters
HMGS atoB tHMGR PMK MK PMD Idi IspA FPP ADS
IPTG
Plac Plac Plac
HMGS atoB tHMGR PMK MK PMD Idi IspA FPP ADS
PrstA PgadE PgadE
Inducible system
Dynamically-controlled system
Dahl et al. 2013 Nat. Biotechnol. 31:1039.
0
300
600
900
1200
1500
1800
pADS prstA-ADS pADS prstA-ADS
lacUV5-MevT-MBIS pgadE-MevT-MBIS
Am
orp
ha
die
ne
Pro
du
cti
on
(m
g/L
)
-IPTG
+IPTG
FPP-responsive promoters
improve terpene production
HMGS atoB tHMGR PMK MK PMD Idi IspA
IPTG
Plac Plac
HMGS atoB tHMGR PMK MK PMD Idi IspA FPP
PgadE PgadE
FPP ADS
PrstA
FPP ADS
PrstA
ADS
IPTG
Plac
ADS
IPTG
Plac
High product titers with
inducer-free control
Dahl et al. 2013 Nat. Biotechnol. 31:1039.
Fuel toxicity
a-pinene limonene
E. coli M9 Minimal Media (vol/vol)
No
rmal
ize
d C
ell
De
nsi
ty
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
Dunlop et al. 2011. Mol. Sys. Biol. 7:487.
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
Pool
strains in
equal
proportion
Pool is grown in M9 medium (control)
OR M9 medium with biofuel (test)
Arrays used
to identify
pumps
remaining in
the pool at
every
dilution
Plasmid isolated
from pools at
every dilution and
identify winner
Dunlop et al. 2011. Mol. Sys. Biol. 7:487.
Biofuel competitions
Dunlop et al. 2011. Mol. Sys. Biol. 7:487.
Engineering pinene tolerance into E. coli
Dunlop et al. 2011. Mol. Sys. Biol. 7:487.
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
Bokinsky et al. 2011. Proc. Natl. Acad. Sci. USA. 108:19949.
Switchgrass Eucalyptus
E. coli growth on
ionic liquid-treated plant biomass
Bokinsky et al. 2011. Proc. Natl. Acad. Sci. USA. 108:19949.
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
Bokinsky et al. 2011. Proc. Natl. Acad. Sci. USA. 108:19949.
Polyketide-based fuels
PEP
Pyruvate
Acetyl-CoA
CIT
ICT
OGA
SUC
FUM
OAA
MAL
CO2
Glyoxylate
SUC-CoA
Acetaldehyde
Ethanol
Propionyl-CoA Propanol
Acetoacetyl-CoA Butyryl-CoA Butanol
Even chain fatty acid
Aliphatic hydrocarbons
Odd chain fatty acid
G3P
IPP DMAPP
GPP
FPP
GGPP
Isopentenol Isopentanol
Geraniol
Farnesol
Geranylgeraniol
Monoterpene
Sesquiterpene
Diterpene
DHAP
FBP
F6P
G6P 6PG X5P
E4P
S7P
Valeryl-CoA Isopentanol
Glucose
Xylose
Mannose M6P
Galactose G’1P G1P
Xylulose
R5P
Ribulose
Arabinose
Various esters
Alcohols
Aromatic hydrocarbons
Short, highly-branched
hydrocarbons
Isoprenoids
Erythromycin Nystatin
Lipomycin
Polyketides
Erythromycin
AT ACP KS AT ACP
KR
DH
ER
TE KS AT ACP
KR
KS AT ACP
KRx
AT ACP
KR
KS AT
KR
KS AT ACP
KR
KS ACP
Donadio 1991 Science
Erythromycin synthase
DEBS1 DEBS2
Module 1 Module 2 Module 3 Module 4
DEBS3
Module 5 Module 6
DEBS1 DEBS2
Module 1 Module 2 Module 3 Module 4
DEBS3
Module 5 Module 6
AT ACP KS AT ACP
KR
DH
ER
TE KS AT ACP
KR
KS AT ACP
KRx
AT ACP
KR
KS AT
KR
KS AT ACP
KR
KS ACP
Polyketide synthases (PKSs)
are very large, multi-activity proteins
Protein 1 Protein 2 Protein 3
DEBS1 DEBS2
Module 1 Module 2 Module 3 Module 4
DEBS3
Module 5 Module 6
AT ACP KS AT ACP
KR
DH
ER
TE KS AT ACP
KR
KS AT ACP
KRx
AT ACP
KR
KS AT
KR
KS AT ACP
KR
KS ACP
Each extension module adds 2 carbons (plus pendant
groups) to the polyketide backbone
Extension
Module
Loadin
g
Module
DEBS1 DEBS2
Module 1 Module 2 Module 3 Module 4
DEBS3
Module 5 Module 6
AT ACP KS AT ACP
KR
DH
ER
TE KS AT ACP
KR
KS AT ACP
KRx
AT ACP
KR
KS AT
KR
KS AT ACP
KR
KS ACP
Polyketide synthases (PKSs)
are very large, multi-activity proteins
Extension
Module
Loadin
g
Module
a
Termination
Module
DEBS1 DEBS2
Module 1 Module 2 Module 3 Module 4
DEBS3
Module 5 Module 6
AT ACP KS AT ACP
KR
DH
ER
TE KS AT ACP
KR
KS AT ACP
KRx
AT ACP
KR
KS AT
KR
KS AT ACP
KR
KS ACP
Each domain in a PKS has an individual enzyme activity
Domains
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH SH
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH SH
HSCoA SCoA
O
R1
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SHS
O
R1
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH
HSCoA
S
O
R1 SCoA
O
OH
O
R2
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
S
O
R1
S
O
OH
O
R2
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
CO2
S
O
R1
S
O
R2
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH S
O
R1
O
R2
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH S
O
R1
O
R2
OH
O
R1
O
R2
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH SH
OH
O
R1
O
R2
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH
KR
S
O
R1
O
R2
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH
KR
S
O
R1
HO
R2
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH
KR
SH
OH
O
R1
HO
R2
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH S
O
R1
O
R2
KR DH
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH
KR DH
S
O
R1
HO
R2
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH
KR DH
S
O
R1
R2
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH SH
KR DH
OH
O
R1
R2
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH
KR ER
DH
S
O
R1
O
R2
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH
KR ER
DH
S
O
R1
HO
R2
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH
KR ER
DH
S
O
R1
R2
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH
KR ER
DH
S
O
R1
R2
Basic PKS
AT KS AT ACP ACP TE
Load Extension Termination
SH
KR ER
DH
SH
OH
O
R1
R2
Polyketide synthase structure
Whicher et al. 2014. Nature. 510(7506): 560–564
KS
AT
KR
ACP
ACP moves from active site to active site
like an arm on a robot
Whicher et al. 2014. Nature. 510(7506): 560–564
6-dEB
PKSs function like
molecular assembly lines
Lots of naturally occurring Load, Extension, and
Termination modules
AT KS AT ACP ACP TE
Load Extension Termination
S1 mAT ACPKSQ
mmAT(
S)
ACPKSQS2
ACPCoLS3 ERR
O
E1mAT ACPKS
OH
E2 mAT
ACPKS
KR(S)
OH
E3 mAT
ACPKS
KR(R)
E4mAT
ACPKS
KR
DH
E5mAT
ACPKS
KR
DH ER
O
E6 mmAT(
S)ACPKS
OH
E7ACPKS
KR(S)mmAT(
S)
OH
E8ACPKS
KR(R)mmAT(
S)
E9
OH
ACPKS
KR(S)mmAT(
R)
E10
OH
ACPKS
KR(R)mmAT(
R)
E11mmAT
ACPKS
KR
DH
E12ACPKS
KR
DH ER
mmAT(
S)
E13ACPKS
KR
DH ER
mmAT(
R)
E14
O
ACPKS
cMTmmAT
E15
O
ACPKS eAT (S)
E16ACPKS
KR
DH ER
eAT (S)
E17
O
ACPKS pAT (S)
TE
R O
O
TE
OH
O
Red-TE
H
O
Red-TEOH
ST TE
Polyesters
• Used for
– Cushioning, clothing, bottles, films, liquid crystal
displays, holograms, filters, dielectric films, insulation for
wires
– 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
HO O
O
O
O
O
O
O
O
O
O
O
O
OH
O
3-hydroxy acids
Erythromycin
AT ACP KS AT ACP
KR
DH
ER
TE KS AT ACP
KR
KS AT ACP
KRx
AT ACP
KR
KS AT
KR
KS AT ACP
KR
KS ACP
Erythromycin synthase
DEBS1 DEBS2
Module 1 Module 2 Module 3 Module 4
DEBS3
Module 5 Module 6
Erythromycin
AT ACP KS AT ACP
KR
DH
ER
TE KS AT ACP
KR
KS AT ACP
KRx
AT ACP
KR
KS AT
KR
KS AT ACP
KR
KS ACP
Erythromycin synthase
DEBS1 DEBS2
Module 1 Module 2 Module 3 Module 4
DEBS3
Module 5 Module 6
Cortes et al. 1995. Science
AT ACP KS AT ACP
KR
TE KS AT ACP
KR
Protein Engineering
DEBS1
Module 1 Module 2
Cortes et al. 1995. Science
AT ACP KS AT ACP
KR
TE KS AT ACP
KR
Protein Engineering
DEBS1
Module 1 Module 2
AT ACP KS AT ACP
KR
TE
Production of 3-hydroxyacids
DEBS1
Module 1
Satoshi Yuzawa, Clara Eng
Potential polymers that can be produced with various 3-
hydroxyacids
HO O
O
O
O
O
O
O
O
O
O
O
O
OH
O
Interesting polyesters with novel properties
LipPks1 1 LipPks2, LipPks3, LipPks4, LipNrps
b-Lipomycin
AT ACP KS AT ACP
KR
Lipomycin PKS
Yuzawa et al. 2013. Biochemistry 52:3791-3793.
Lipomycin load has a broad substrate range
Substrate kcat (min-
1)
Km
(uM)
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
Yuzawa et al. 2013. Biochemistry 52:3791-3793.
Constructing a 3-hydroxyacid synthase
AT ACP KS AT ACP
KR
TE
Thioesterase (TE) from DEBS PKS
Load and extension modules
from Lipomycin PKS
Load Extension
Yuzawa et al. 2013. Biochemistry 52:3791-3793.
AT ACP KS AT ACP
KR
TE
Production of 3-hydroxy-2-methyl acids
with Lip PKS
+
Load
Extension
Product
Native substrate
Yuzawa et al. 2013. Biochemistry 52:3791-3793.
Potential polymers that can be produced with various 3-
hydroxyacids
HO O
O
O
O
O
O
O
O
O
O
O
O
OH
O
Interesting polyesters with novel properties
The acyltransferase (AT) determines the acyl-CoA
loaded onto the ACP
AT KS AT ACP ACP TE
Load Extension Termination
SH
HSCoA
S
O
R1 SCoA
O
OH
O
R2
The acyltransferase (AT) determines the acyl-CoA
loaded onto the ACP
AT KS AT ACP ACP TE
Load Extension Termination
SH
HSCoA
S
O
R1 SCoA
O
OH
O
Specifies that a methylmalonyl group
will be loaded onto the ACP.
Native DEBS1
Module 1 Module 2
AT ACP KS AT ACP
KR
KS AT ACP
KR
Acyl transferase (AT) specifies the acyl group that
gets loaded onto the ACP
Module 1 Module 2
AT ACP KS AT ACP
KR
KS AT ACP
KR
Modified DEBS1
TE TE
Oliynyk et al 1996 Chem. Biol. 3:883
The acyltransferase (AT) determines the acyl-CoA
loaded onto the ACP
AT KS mAT ACP ACP TE
Load Extension Termination
SH
HSCoA
S
O
R1
Specifies that a malonyl group will be
loaded onto the ACP.
SCoA
O
OH
O
AT ACP KS mAT ACP
KR
TE
Production of 3-hydroxyacids with Lip PKS
+
Load
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)
O
O
- NADPH
Δ AT ACP KS AT ACP
KR
TE
In vitro production of ethyl ketones
LipPK1 + DEBS TE
Yuzawa et al 2015 unpublished
Δ AT ACP KS AT ACP
KR
TE
In vivo production of ethyl ketones
LipPK1 + DEBS TE
X
Knocking out activity of ketoreductase (KR)
disables reduction of ketone to alcohol
Yuzawa et al 2015 unpublished
Δ AT ACP KS mAT ACP
KR
TE
In vivo production of methyl ketones
X
Exchanging the native methylmalonyl-
CoA—specific acyltransferase (AT) for a
malonyl-CoA—specific AT enables
production of methyl ketones
Yuzawa et al 2015 unpublished
Acknowledgements
• US Department of Energy for funding
• All of the members of JBEI
#Jay Keasling has a financial interest in Amyris, LS9, and Lygos.
4
Artemisinic Acid
O
O
O
O
O
H
HH
Glucose Artemisinin
A Novel Semi-synthetic Route
•US Department of Energy for
funding
•All of the members of JBEI
•US Department of Energy for
funding
•All of the members of JBEI
•US Department of Energy for
funding
•All of the members of JBEI
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
LS9
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.