synthetic biology for fuel biologistsbbq.iq.usp.br/biofuel/presentations/liao.pdfsynthetic biology...

Synthetic Biology for Fuel Biologist

James C. Liao University of California, Los

Angeles

1

Outline

• Alcohol synthesis (C2-C8)– CoA-dependent pathways

– Ketoacid pathways

– Non-natural alcohols

• Fatty acid sysnthesis (C12-C18)– Biodiesel

– Hydrocarbons

• Isoprenoid synthesis

2

CO2

CO2

Biomass to Ethanol

3

CO2

CO2

Microalgae to Diesel

Biodiesel

4

Lipid

CO2

CO2

A short-cut of biofuel cycle

Biofules

5

Designing bugs

6

Ideal organisms

Natural

Synthetic

Why non-native hosts?

ProsPros ConsCons

Grows fast

Easy to manipulate Tolerance?

High potential for homo-fermentative

production

Has not been shown to produce

higher alcohols

Oxygen indifferent

Why is it difficult?Efficiency, efficiency, efficiency

• Which pathway/gene to use?

• Gene expression, Protein activity?

• Metabolite toxicity?

• Oxygen sensitivity?

• Cofactor limitation?

• Theoretical yield? Titer? Productivity?

• Product recovery?

8

Yield, Titer, Productivity

9

Time (hr) Time (hr)

Eth

anol

(g/

L)

0 24 48 0 24 48

Glu

cose

(g

/L)100

50

0

200

100

0

Final Titer (concentration) = 75 g/L

Yield = product produces/ substrate consumed = 75 g / (200-20) g = 75/180 = 0.41 g/g

Productivity (g/L/hr) = 75/48 = 1.56 g/L/hr

Determines the ease of separation

Determines the profit limit

Determines the reactor size

Pathway overviews for fuels

Fatty acids

Isoprenoids

Sugars, CO2

Pyruvate

Acetyl-CoA

Keto acid pathwaysIso

prenoids

pathway

Fatty ac

id

pathw

ay

CoA-dependent

pathways Ethanol1-Butanol

Pathways for Ethanol Synthesis

Pyruvate

Acetyl-CoA Acetaldehyde

Ethanol

PDH

ADH

NADH

NADH NADH

11

CO2PDC

Pathways competing with ethanol synthesis

Pyruvate


Ethanol

PDH

ADH

NADH

NADH NADH

12

CO2

PDC

ADH

Formate

PFL

Acetyl- P TCA

Acetate

Lactate

Higher alcohols1-propanol

1-butanol

1-pentanol

1-hexanol

1-heptanol

Iso-propanol

Iso-butanol

2-methyl-1-butanol

3-methyl-1-butanol

No

Yes

No

No

No

Yes

No

No

No

Native producers?

OH

OH

Pros and Cons of Various Alcohols

Ethanol Long chain alcohols/alkanes

Energy content

Low High

Hygroscopicity

High Low

Flex Vehicle? Yes No

Production yield

High Zero/Low

14

Glucose

2 NAD

2 NADH

Acetoacetyl-CoA

Acetoacetate

2 Pyruvate2CoA, 2 NAD

2 CO2, 2 NADH

Acetone

adh (C. beijer.)adh (T. brock.)

NADPH

NADP

adc (C. aceto.)

atoD, atoActfA, ctfB (C. aceto.)

Acetate

2 Acetyl-CoA

CO2

CoA

CoA, ATP

Isopropanol

Isopropanol pathway

Acetyl-CoA

atoB (E. coli)thl (C. aceto.)

Fig.2 Comparison of maximum isopropanol production by each combination of pathway genes

0

20

40

60

isop

ropa

nol,

ace t

one

or e

than

ol

conc

entr

atio

n (m

M)

AcetoneEthanol

Isopropanol

Enzyme Genename

plasmidpTA41/pTA36 pTA29/pTA36 pTA30/pTA36 pTA39/pTA36 pTA39/pTA18

ACoAAT thl + + +atoB + +

ACoAT ctfAB + +atoAD + + +

ADC adc + + + + +SADH adh (cb) + + + +

adh (tb) +

2.4 g/L

0

10

20

30

40

0 5 10 15 20 25 30 350

50

100

0 5 10 15 20 25 30 35

0

25

50

75

100

0

5

10

15

20

25G

luco

se (

mM

)Is

opro

p ano

l (m

M)

OD

600

Eth

anol

or

ace t

one

(mM

)Ethanol

Acetone

Induction by IPTG

Addition of glucose

Time (h) Time (h)

5 g/L

Preliminary optimization

0

2 0 0

4 0 0

6 0 0

T B T B

G l y+

T B

G l c+

M 9

G l c+

0M 9

G l c++

C a s a m i n oA c i d s 37 ํํC 24 hr

550 mg/L

Growth in rich media increased 1-butanol production ~5 fold over cultures grown in M9.

Summary of Co-A Dependent Pathways for Alcohol Synthesis

n-Butanol

Pyruvate


EthanolAcetoacetyl-CoA

3-Hydroxybutyryl-CoA

Crotonyl-CoA ButyraldehydeButyryl-CoA

PDH

ADH

CoA-dependent pathway

NADH

NADH NADH

20

Ketoacid pathways for alcohol synthesis

n-Butanol

Pyruvate


EthanolAcetoacetyl-CoA

3-Hydroxybutyryl-CoA

Crotonyl-CoA ButyraldehydeButyryl-CoA

PDH PDC

ADH

CoA-dependent pathway

?

Non-CoA pathway

NADH

NADH NADH

21

Generalization of keto acid decarboxylase chemistry

Pyruvate

Acetylaldehyde

Ethanol

PDC

ADH

R-aldehyde

R-OH

KDC

ADH

2-keto acid

A simple keto acidLonger chain keto acids

Ehrlich, F. Ber. Dtsch. Chem.

Ges.1907 22

Opening the active site

Zymomonas mobilis PDC Lactococcus lactis KdcA

23

Further generalization of alcohol-producing chemistry

Atsumi et al. Nature 200824

Alternative Pathways for Alcohol Synthesis

Pyruvate


Ethanol

PDH PDC

ADH

?NADH

NADH NADH


Long-chain keto acids

Long-chain alcohols

Pyruvate

25

Non-polymeric Iterative Chain Elongation

XnXn+1

A B C D E F

26

Pyruvate NADPHCO2 H2O

+2CAHAS

Aceto-Hydroxy Acid Synthase (AHAS) Chain Elongation

27

Novel Pathways for C4, C5 Alcohol Synthesis


A Novel Pathway for Isobutanol Synthesis

PyruvateGlucose

Acetolactate

2,3-dihydroxy-isovalerate

2-keto-isovalerate

L-Valine

Isobutyraldehyde

Isobutanol

KDC

ADH2-keto-isovalerate


Improvement of isobutanol production

2.2 g/LGlucose

Pyruvate

IlvIH

IlvC

IlvD

2-keto-isovalerate

Isobutanol

KDC

ADH30 ํํC

M9 + 3.6% glc


Effects of Gene Deletions

adhldh

frd

fnr

pta

pflB

∆∆∆

∆∆∆∆

∆∆∆∆

∆ ∆ ∆

∆∆∆

∆ ∆

∆∆∆∆

∆ ∆

∆∆∆

∆∆∆∆

kn

oc

ko

ut

ge

ne

s

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

1 4 0 0 0

1 6 0 0 0

0

4

8

12

16

isob

uta n

ol (

g/L )

30 ํC 64hr

Pyruvate

Acetyl-CoA

LactateLdhA

AdhEEthanol

PtaAcetate

PflB

Glucose

PEPTCA cycle

Succinate

Citrate

PDHc

E.coli metabolism

Isobutanol

FrdABCD

Fnr

31

High yield production of isobutanol:86% of Theoretical

21.9 g/L

Glucose

Pyruvate

AlsS

IlvC

IlvD

2-keto-isovalerate

Isobutanol

KDC

ADH

Clostridium (1-butanol)

19.6 g/L

83% of theor.


86% of theoretical yield

32

Direct CO2 to Isobutanol UsingPhotosynthetic Bacteria

33

Isobutanol production from CO2

34

Direct-Solar Fuel from CO2

Direct CO2 to isobutyraldehyde/isobutanol in S. elongatus

Atsumi et al. Nature Biotech, 2009 35

Acetyl-CoA NADH CO2

Pyruvate NADPHCO2 H2O

+1CIPMS

+2CAHAS

Isopropylmalate Synthase (IPMS) Chain Elongation

36

Novel Pathways for C5 alcohol synthesis

Atsumi et al. Nature 2008Connor and Liao, 2008 AEM37

Novel Pathway for C3 alcohol synthesis

Atsumi et al. Nature 2008Shen and Liao 2008, Metabolic Engineering38

Novel Pathway for C4 alcohol synthesis

Atsumi et al. Nature 2008Shen and Liao 2008, Metabolic Engineering39

Pyruvate

2-ketobutyrate

2-keto-3-methylvalerate (C6)

2-ketovalerate 2-ketoisovalerate

2-keto-4-methylvalerate

1-butanol

(C4)

1-propanol

(C3)

2-methyl-1-butanol

(C5)

3-methyl-1-butanol

(C5)

Isobutanol

(C4)

(B)

(B)

(A)

(A)

(B)

(A)Leucine biosynthesis (leuABCD)

Isoleucine biosynthesis (ilvGM(ST)-ilvCD)

Synthetic Network for Higher Alcohols

40

Chain ElongationCOOHR

O

COOHR

NH2

Natural amino acids

OHR

Natural keto acids

Natural alcohols (Cn<6)

COOHR'

O

Nonnatural keto acids

OHR'

Nonnatural alcohols (Cn>=6)

41

Glucose

Ketoisoleucine (C6)

COOH

O

3-methyl-1-pentanol (C6)

Ketohomoisoleucine (C7)

Nonnaturalmetabolic pathway

IPMS chain elongation

COOH

O

OH

Design a Nonnatural Metabolic Pathway to Biosynthesize C6 Alcohol

Isoleucine

OH

2-methyl-1-butanol (C5)

Ehrlich pathway

IlvE

COOH

O

Ketovaline (C5)LeuA

Ketoleucine

LeuC,LeuDLeuB

COOH

O

L-leucine

IlvE/TyrB

Leucine biosynthesis

LeuABCD may be promiscuous on ketoisoleucine?

42

Optimizing LeuA for Ketoisoleucine

43

Ketoisoleucine (C6)

Ser139

Natural substrate

COOH

OTarget substrate

COOH

O

Ketovaline (C5)

Engineering LeuA to relieve steric hindrance

Glycine

Asn167

Ser139

His97

Combinatorial mutagenesis of H97, S139 and N167 to leucine, alanine and glycine

Engineering KIVD for Ketohomoisoleucine

44

Leucine Alanine

Enlarge the Binding Pocket

M538

S286

F381V461

ThDP cofactor

Binding pocket

Natural substrate

COOH

O

Target substrate

Ketovaline (C5)

Ketohomoisoleucine (C7)

COOH

O

Decarboxylation

Milestones

45

E. coli LeuA: wt wt wt wt S139GE. coli LeuA: wt wt wt wt S139G

L. lactis KIVD: wt V461A V461A V461A V461A /M538A /F381L /F381L L. lactis KIVD: wt V461A V461A V461A V461A /M538A /F381L /F381L

3-methyl-1-pentanol (C6)

OH

Zhang et al. PNAS 2009 105(52): 20653–8.

0

100

200

300

400

500

600

700

800

900

Alc

ohol

tite

r

(mg/L

)

Protein engineering enables a 122-fold increase in yield

0.04 g/ g glucose yield

10% of theoretical maximum

PEP, Pyruvate (C3)

2-Ketobutyrate (C4)

2-Keto-3-methylvalerate (C6)

2-Keto-4-methylhexanoate (C7)

2-Keto-5-methylheptanoate (C8)

2-Ketovalerate (C5)

2-Ketohexanoate (C6)

2-Ketoheptanoate (C7)

2-Ketoisovalerate (C5)

2-Keto-4-methylvalerate (C6)

2-Keto-5-methylhexanoate (C7)

1-Butanol (C4)

2-Keto-6-methyloctanoate (C9)

(A)

(K)

(I)

(K)

(A)(I)

(I)

(A)

(K)

AHAS Elongation

IPMS Elongation

KDC + ADH Reaction

Glucose, CO2

1-Pentanol (C5)

1-Hexanol (C6)

1-Propanol (C3)

Isobutanol (C4)

3-Methyl-

1-butanol

(C5)

4-Methyl-1-

pentanol (C6)

2-Methyl-

1-butanol

(C5)3-Methyl-

1-pentanol

(C6)

4-Methyl-1-hexanol

(C7)

5-Methyl-1-

heptanol (C8)

(I)

(I)

(I)

(I)

(K)

(K)

(K)

(K)

(K)

(K)

(K)

(K)(I)

(I)

(K)

46

Outline





– Hydrocarbons


47

What is Biodiesel?• Alternative fuel for diesel engines• Currently made from plant oil or animal fat • Meets health effect testing (CAA)• Lower emissions, High flash point (>300F),

Safer• Biodegradable, Essentially non-toxic.• Chemically, biodiesel molecules are mono-

alkyl esters produced usually from triglyceride esters

Fatty AcidAlcoholGlycerin

Vegetable Oil

BiodieselFA

FAFA

FA

NADPH NADHH2OACP,CO2Malonyl-ACP+2C

FattyAcid

Fatty acid chain elongation

49

Acetyl-CoA

CO2

Fatty acid production by E. coli

50

JBC 1995TesA (E. coli thioesterase I), a periplasmic enzyme

Trapping TesA in cyctol inreases FFA synthesis

51

Plant thioesteraseincreased FA

Block degradation

releasing feedback inhibition caused by long-chain fatty acyl-ACPs through over- expression of an endogenous thioesterase.

53

JOURNAL OF BACTERIOLOGY, 1997, 2969–2975

FAR

Unspecific acyl transferase fromAcinetobacter baylyi strain ADP1

55

Kalscheuer et al. 2006

Lu et al., 2008

Adelsberger et al,2004

57

Xyn10B : endoxylanase catalytic domain from Clostridium stercorarium20 Xsa: xylanase from Bacteroides ovatus21

0.2%glucose

xynB

xyn10Bxsa

Production of Hydrocarbons

58

Enzymes and biochemical steps are identified

59Schirmer et al. Science 2010

Genome comparision between cyanobacterai that produce hydrocarbons and cyanobacteria that do not.

60Schirmer et al. Science 2010

PCC7942_orf1593

PCC7942_orf1594

S. elongatus

S. elongatus

61

Expression in E. coli

E. Coli MG 1655

62

Synechocystis sp. PCC6803

Deletion of the two adjacent Synechocystis sp. PCC6803 orfs sll0208 and sll0209

63

Aldehyde decarbonylase Ribonucleotide reductase R2

Outline





– Hydrocarbons


64

Isoprenoid pathway reengineered to produce alcoholsYan, Y, Liao JC. J Ind Microbiol Biotechnol 2009, 36 471-479

IPP PPDMAPP

66

Isoprene productionfrom CO2

in Synechocystis

Lingerg et al. Metabolic Engineering2010

synthetic biology for fuel biologistsbbq.iq.usp.br/biofuel/presentations/liao.pdfsynthetic biology...

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