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Metabolic Network Plasticity in Ethylene-producing Cyanobacteria

Algae Biomass Summit

Jianping Yu

October 2, 2014

2

Metabolic network plasticity vs rigidity

A rigid or “hard-wired” metabolic network has limited flux

A flexible metabolic network has

expandable flux

A

B

C

E

D

1

2

3

45

7

68

9

10

F

A

B

C

E

D

1

2

3

45

7

68

9

10

F

3

Cyanobacteria can produce ethylene

CO2

in airEthylene

Measured by GC

Ethylene-forming enzyme

Ungerer et al 2012, Energy and Environmental Science

4

Ethylene production in Synechocystis

5

Up to 10% of fixed carbons go to Ethylene

0

2

4

6

8

10

12

0

100

200

300

400

500

600

700

800

900

C C

2H4

(mo

l)/C

CO

2(m

ol)

(%

)

C2H

4µ

L/L/

hr/

OD

730

Specific ethylene productivity

Carbon partition

Growth rates are the same for all these strains!

6

Kinetic 13C-Metabolic flux analysis

M0 M1 M2 M3 m/zMeasuredSimulated

A

B

C

E

D

Isotopic substrate

Cell culture

Flux-dependent metabolite isotope patterns

Metabolic modeling

Fitting

Feeding

MS measurement over time

Comparison

Simulation

1

2

3

45

7

68

9

10

7

Strains: Synechocystis OD730 ~1.5

Medium: liquid BG11 20ml

Light Intensity: 80 µE

Culture conditions: shaking at 200rpm at 30oC in 5% CO2 growth chamber

Cell Harvest: filtering with 0.8µM nylon membrane

Quenching: -80oC methanol

Extraction: 50% cold methanol

Post processing: evaporated with speed vacuum and re-dissolve in 200 µl 50% cold methanol; the supernatant is ready for LC-MS analysis

Kinetic labeling

8

Metabolic flux analysis using 13C labeling

Ribulose-bp

0.25 0.5 1 2 4 8 16 32 640.0

0.2

0.4

0.6

0.8

1.0

Ribulose-5p

0.25 0.5 1 2 4 8 16 32 640.0

0.2

0.4

0.6

0.8

1.0

Phosphoglutarate

0.25 0.5 1 2 4 8 16 32 640.0

0.2

0.4

0.6

0.8

1.0

Glucose-6p

0.25 0.5 1 2 4 8 16 32 640.0

0.2

0.4

0.6

0.8

1.0

2-oxoglutarate

0.25 0.5 1 2 4 8 16 32 640.0

0.2

0.4

0.6

0.8

1.0

Fructose-bp

0.25 0.5 1 2 4 8 16 32 640.0

0.2

0.4

0.6

0.8

1.0

Malate

0.25 0.5 1 2 4 8 16 32 640.0

0.5

1.0

Fructose-6p

0.25 0.5 1 2 4 8 16 32 640.0

0.2

0.4

0.6

0.8

1.0

Sedoheptulose-7p

0.25 0.5 1 2 4 8 16 32 640.0

0.2

0.4

0.6

0.8

1.0

Phosphoglycerate

0.25 0.5 1 2 4 8 16 32 640.0

0.2

0.4

0.6

0.8

1.0

Sedoheptulose-bp

0.25 0.5 1 2 4 8 16 32 640.0

0.2

0.4

0.6

0.8

1.0

Succinate

0.25 0.5 1 2 4 8 16 32 640.0

0.2

0.4

0.6

0.8

1.0

Aspartate

0.25 0.5 1 2 4 8 16 32 640.0

0.2

0.4

0.6

0.8

1.0

Phosphoenolpyruvate

0.25 0.5 1 2 4 8 16 32 640.0

0.5

1.0

CO2

CO2

Acetyl CoA

Citrate

Isocitrate

Malate

FumarateSuccinyl semialdehyde

2-oxoglutarate

Fructose-6p

Sedoheptulose-7p

Pentose-5p

Glucose

Glucose-6p

Fructose-

bp

Glyceraldehyde-3p

(Dihydroxyacetone-p)

Phosphoglycerate

Ribulose-bp

Erythrose-4p

Sedoheptulose-bp

CO2

CO2

Succinate

Oxaloacetate

phosphoenolpyruvate

pyruvate

CO2

Glutamate

Aspartate CO2

CO2

Glutamate

0.25 0.5 1 2 4 8 16 32 640.0

0.2

0.4

0.6

0.8

1.0

Time (min) Time (min)

Rel

ativ

e Fr

acti

on

Rel

ativ

e Fr

acti

on

9

TCA cycle is bifurcated in WT

Citrate

Isocitrate

2-oxoglutarate

Succinate

Fumarate

Malate

Oxaloacetate

Phosphoenolpyruvate

Pyruvate

Acetyl CoA

EthyleneCitrate

Isocitrate

2-oxoglutarate

Succinate

Fumarate

Malate

Oxaloacetate

Phosphoenolpyruvate

Pyruvate

Acetyl CoA

10

Mapping TCA cycle flux using labeled glutamate

NH4+

Glutamate Glutamine

2-oxoglutarate

NH4+

GDH

GOGAT

GS

U-13

C-15

N-g

luta

mat

e

GT

Succinate

Ethylene

EfeMalate

TCA cycle

GS/GOGAT cycle

11

TCA cycle topology is changedWT JU547

• Succinate is derived from Glu by Efe

• Malate is also derived from Glu

12

Central carbon metabolism in WT

G6P Ru5P

FBP

DHAP GAP

PGA

RuBP

E4P

SBP

S7P

CO2

F6P

CIT

ICT

2OG

SUC

FUM

MAL

OAA

SSA

PEP

Pyr

AcCoA

GlycogenCO2

X5P

R5P

CO2

CO2

CO2

CO2

CO2

1005020≤5

RBC

135.2 ±17.2

PGI

27.1±5.5

G6PD

24.2±5.5

PFK

65.5±29.3

PK

13.2 ±4.1

ENO

23.4 ±2.1

GAPDH

244.0 ±10.9

FBA

65.5±29.3

PPE

75.5 ± 0.5

PPI

35.4 ±0.2

PRK

135.2 ±5.5

TKT

75.5 ±0.5

TKT

38.4 ±0.2

TKT

37.2 ±0.2

TAL

0.0 ± 25.5

TAL

0.0 ± 25.5

SBA

37.2 ±26.8

PEPC

7.8 ± 0.3

ME

1.5 ± 0.3

PDH

11.8 ±0.3

CS

3.0 ± 0.3ACO

3.0 ± 0.3

ICTDH

3.0 ± 0.3

SDH

0.0 ± 0.3

FUS

1.7 ± 0.3

MDH

0.2 ± 4.6

SSADH

-0.4 ± 0.2

SBPS

37.2 ±26.8

TPI

102.6 ±11.3

A B

FBP

S7P RuBP

SUC

G6P

PGA PEP 2OG

OAA*

F6P

SBP

2OGDH

0.0 ± 1.5

INCA

13

Central carbon metabolism in JU547

G6P Ru5P

FBP

DHAP GAP

PGA

RuBP

E4P

SBP

S7P

CO2

F6P

CIT

ICT

2OG

SUC

FUM

MAL

OAA

SSA

PEP

Pyr

AcCoA

GlycogenCO2

X5P

R5P

CO2

CO2

CO2

CO2

CO2

Ethylene

CO2

F6P FBP

S7P SBP RuBP

SUC

G6P

PGA PEP 2OG

MAL

1005020≤5

RBC

137.6±0.6

PGI

4.1±0.7

G6PD

1.5±0.7

PFK

50.0±1.2

PK

9.1 ±1.3

ENO

33.2 ±0.2

GAPDH

239.4 ±1.4

FBA

50.0±1.2

PPE

92.2 ±0.2

PPI

44.0 ±0.1

PRK

137.6 ±0.7

TKT

92.2 ±0.2

TKT

46.6 ±0.1

TKT

45.5 ±0.1

TAL

0.6

TAL

0.6

SBA

46.1 ±0.2

PEPC

21.8 ±0.7

ME

7.6 ± 1.3

PDH

17.7 ±0.2

CS

9.8 ± 0.2 ACO

9.8 ± 0.2

ICTDH

9.8 ± 0.2

Efe

2.5 ± 0.0

SDH

2.1 ± 0.2

FUS

3.6 ± 0.2

MDH

7.6 ± 1.3

SSADH

-0.4 ± 0.2

SBPS

46.1 ±0.2TPI

96.2 ±0.6

A B

14

Flux rates in TCA cycle

-400

-200

0

200

400

600

800

Ne

t fl

ux

(µm

ol/

g-D

W/h

)

WT JU547

15

Fluxes in amphibolic reactions and TCA cycle are increased

Citrate

Isocitrate

2-oxoglutarate

Succinate

Fumarate

Malate

Oxaloacetate

Phosphoenolpyruvate

Pyruvate

Acetyl CoA

Ethylene

JU547

Citrate

Isocitrate

2-oxoglutarate

Succinate

Fumarate

Malate

Oxaloacetate

Phosphoenolpyruvate

Pyruvate

Acetyl CoA

WT

0.00

0.10

0.20

0.30

0.40

0.50

WT JU547

Intr

ace

llula

rle

vel o

f su

ccin

ate

(µm

ol/

gDW

)

16

Carbon is redistributed toward TCA metabolites

G6P Ru5P

FBP

DHAP GAP

PGA

RuBP

E4P

SBP

S7P

CO2

F6P

CIT

ICT

2OG

SUC

FUM

MAL

OAA

SSA

PEP

Pyr

AcCoA

GlycogenCO2

X5P

R5P

CO2

CO2

CO2

CO2

CO2

Ethylene

CO2

1005020≤5

RBC

137.6±0.6

PGI

4.1±0.7

G6PD

1.5±0.7

PFK

50.0±1.2

PK

9.1 ±1.3

ENO

33.2 ±0.2

GAPDH

239.4 ±1.4

FBA

50.0±1.2

PPE

92.2 ±0.2

PPI

44.0 ±0.1

PRK

137.6 ±0.7

TKT

92.2 ±0.2

TKT

46.6 ±0.1

TKT

45.5 ±0.1

TAL

0.6

TAL

0.6

SBA

46.1 ±0.2

PEPC

21.8 ±0.7

ME

7.6 ± 1.3

PDH

17.7 ±0.2

CS

9.8 ± 0.2 ACO

9.8 ± 0.2

ICTDH

9.8 ± 0.2

Efe

2.5 ± 0.0

SDH

2.1 ± 0.2

FUS

3.6 ± 0.2

MDH

7.6 ± 1.3

SSADH

-0.4 ± 0.2

SBPS

46.1 ±0.2TPI

96.2 ±0.6

Decreased pool sizes for sugar phosphates

Increased pool sizes for Suc, Fum, MalDecreased pool size for 2OG

17

Ethylene production increases energy demand

0

5

10

15

20

25

30

35

WT ATP cost JU547 ATPcost

WT NADPHcost

JU547NADPH cost

mm

ol/

gDW

/hMetabolic Fluxes Biomass Growth

18

Ethylene production stimulates photosynthesis

0

1

2

3

4

5

6

7

8

9

10

0

2

4

6

8

10

12

14

16

18

20

WT JU547

Ch

loro

ph

yll

(µg

/mL/

OD

730

µm

ol O

2/L/

min

/OD

730

O2 evolution

Chlorophyll content

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

WT JU547

mm

ol/

g-D

W/h

Tota

l fix

ed

car

bo

n %

into

th

e T

CA

cyc

le Total fixed carbon % into the TCA cycleCO2 fixation rate

Light Reaction Carbon Metabolism

19

Conclusions

Metabolic network in a cyanobacterium is very flexible• TCA cycle topology can change in response to an engineered

ethylene pathway• Amphibolic reactions and TCA cycle fluxes can be increased to

accommodate this engineered pathway• Carbon can be pulled from elsewhere to accommodate an

engineered pathway• Photosynthesis can run faster to meet the increased demand

20

Metabolic network in a cyanobacterium is very flexible• TCA cycle topology can change in response to an engineered

ethylene pathway• Amphibolic reactions and TCA cycle fluxes can be increased to

accommodate this engineered pathway• Carbon can be pulled from elsewhere to accommodate an

engineered pathway• Photosynthesis can run faster to meet the increased demand

Source-sink relationship in photosynthesis• Sink expansion can stimulate photosynthesis, so that loss of

carbon from an engineered pathway is fully compensated without slowing down cell growth.

Conclusions

21

Conclusions

Metabolic network in a cyanobacterium is very flexible• TCA cycle topology can change in response to an engineered

ethylene pathway• Amphibolic reactions and TCA cycle fluxes can be increased to

accommodate this engineered pathway• Carbon can be pulled from elsewhere to accommodate an

engineered pathway• Photosynthesis can run faster to meet the increased demand

Source-sink relationship in photosynthesis• Sink expansion can stimulate photosynthesis, so that loss of

carbon from an engineered pathway is fully compensated without slowing down cell growth.

• How much is the unrealized potential in photosynthesis?• Can we find the trigger to unleash that potential and increase

algal productivity?

22

Acknowledgements

Wei XiongJustin Ungerer

Pin-Ching ManessJohn Morgan

Funding fromDOE BETO

DOE BERNREL