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Cyanobacterial ethylene production

Jianping Yu

October 25, 2016

Algal Biomass Summit2015 R&D 100 AwardEditor’s Choice Award

Outline

• Ethyleneformingenzyme

• Cyanobacterialethyleneproduction

• Stimulationofphotosynthesis

BiologicalEthyleneProduction• Regulatesmanyprocessesinplantgrowth• Plantpathogensuseethyleneasaweaponto

weakenplantdefense.• Ethyleneformingenzyme(EFE);presentin

Pseudomonassyringae.Stillpoorlyunderstood.

A

B

C

D

Eckert et al. Biotechnology for Biofuels 2014

Arginine

EFE enables cyanobacterial ethylene production

Enhanced EFE synthesis

Re-design efe gene

Stronger promoter

Multiple efe copy

Stronger ribosome binding site (RBS)

gene stability

transcription

gene dosage

translation

Ungerer et al. Energy and Environmental Science 2012

Justin Ungerer

5

EFEexpressionisnolongerrate-limitingstepinethyleneproduction

0

100

200

300

400

500

600

700

WT 1xefe 2xefe 3xefe

Ethylene

(µL/L/hr/O

D 730)

0510152025303540

547R 646 641

Ethylene

(nmol/m

L/h/OD 7

30)

Expected

Observed

(1xefe*)(2xefe*)(3xefe*)

Current efforts aim to improve supply of AKG and arginine, the substrates of EFE.

Bo Wang

Onlineethyleneproductionanddetection

Gas-MixingController

5%CO2

PBRfromPSIcompany F-900EthyleneAnalyzerColdtrap

- Ethyleneproductionshowsdiurnalcyclingundercontinuouslight.- Isitregulatedbybiologicalclock,andhow?

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Ethylene

(ppm

)

Time(day)

Ethylene production stimulates photosynthesis

Wei XiongMetabolic flux analysis

Xiong et al., Nature Plants 2015

9

CentralcarbonmetabolisminWTG6P Ru5P

FBP

DHAPGAP

PGA

RuBP

E4P

SBP

S7P

CO2

F6P

CIT

ICT

2OG

SUC

FUM

MAL

OAA

SSA

PEPPyr

AcCoA

GlycogenCO2

X5P

R5P

CO2

CO2

CO2

CO2

CO2

1005020≤5

RBC135.2 ±17.2

PGI27.1±5.5

G6PD24.2±5.5

PFK65.5±29.3

PK13.2 ±4.1

ENO23.4 ± 2.1

GAPDH244.0 ± 10.9

FBA65.5±29.3

PPE75.5 ± 0.5

PPI35.4 ± 0.2

PRK135.2 ± 5.5

TKT75.5 ± 0.5

TKT38.4 ± 0.2

TKT37.2 ± 0.2

TAL0.0 ± 25.5

TAL0.0 ± 25.5

SBA37.2 ± 26.8

PEPC7.8 ± 0.3

ME1.5 ± 0.3

PDH11.8 ± 0.3

CS3.0 ± 0.3 ACO

3.0 ± 0.3

ICTDH3.0 ± 0.3

SDH0.0 ± 0.3

FUS1.7 ± 0.3

MDH0.2 ± 4.6

SSADH-0.4 ± 0.2

SBPS37.2 ± 26.8

TPI102.6 ± 11.3

A B

FBP

S7P RuBP

SUC

G6P

PGA PEP 2OG

OAA*

F6P

SBP

2OGDH0.0 ± 1.5

INCA

*

10

CentralcarbonmetabolisminJU547G6P Ru5P

FBP

DHAPGAP

PGA

RuBP

E4P

SBP

S7P

CO2

F6P

CIT

ICT

2OG

SUC

FUM

MAL

OAA

SSA

PEPPyr

AcCoA

Glycogen CO2

X5P

R5P

CO2

CO2

CO2

CO2

CO2

Ethylene

CO2

F6P FBP

S7P SBP RuBP

SUC

G6P

PGA PEP 2OG

MAL

1005020≤5

RBC137.6±0.6

PGI4.1±0.7

G6PD1.5±0.7

PFK50.0±1.2

PK9.1 ±1.3

ENO33.2 ± 0.2

GAPDH239.4 ± 1.4

FBA50.0±1.2

PPE92.2 ± 0.2

PPI44.0 ± 0.1

PRK137.6 ± 0.7

TKT92.2 ± 0.2

TKT46.6 ± 0.1

TKT45.5 ± 0.1

TAL0.6

TAL0.6

SBA46.1 ± 0.2

PEPC21.8 ± 0.7

ME7.6 ± 1.3

PDH17.7 ± 0.2

CS9.8 ± 0.2 ACO

9.8 ± 0.2

ICTDH9.8 ± 0.2

Efe2.5 ± 0.0

SDH2.1 ± 0.2

FUS3.6 ± 0.2

MDH7.6 ± 1.3

SSADH-0.4 ± 0.2

SBPS46.1 ± 0.2

TPI96.2 ± 0.6

A B

*

11

Fluxesinamphibolic reactionsandTCAcycleareincreased

Citrate

Isocitrate

2-oxoglutarate

Succinate

Fumarate

Malate

Oxaloacetate

Phosphoenolpyruvate

Pyruvate

AcetylCoA

Ethylene

JU547

Citrate

Isocitrate

2-oxoglutarate

Succinate

Fumarate

Malate

Oxaloacetate

Phosphoenolpyruvate

Pyruvate

AcetylCoA

WT

PK

• TCAcycleoperationchangedfrombifurcatedtocyclic.

• Enhancedamphibolicreactionsfeed3XfluxintoTCAcycle.

• CarbonispulledfromupperglycolysistoTCAcycle.

12

Ethyleneproductionincreasesenergydemand

05

101520253035

WTATPcost JU547ATPcost

WTNADPHcost

JU547NADPHcost

mmol/gDW

/h

MetabolicFluxes BiomassGrowth

13

Ethyleneproductionstimulatesphotosynthesis

012345678910

02468101214161820

WT JU547

Chloroph

yll(µg

/mL/OD7

30

µmolO2/L/min/O

D730

O2evolution Chlorophyllcontent

00.511.522.533.544.55

0%5%10%15%20%25%30%35%40%45%50%

WT JU547

mmol/g-DW/h

Totalfixed

carbon

%intoth

eTCA

cycle

Totalfixedcarbon%intotheTCAcycleCO2fixationrate

LightReaction CarbonMetabolism

Rubiscoactivityalsoincreased

14

EnhancedelectrontransfercapacityandPSIIefficiency

Electrontransportcapacityishigher

PhotosystemIefficiencyis

comparabletoWT

PhotosystemIIefficiencymaybehigher

WTJU547

High Carbon Low Carbon

15

Enhancedcarbonuptakecapacity

SbtA:bicarbonatetransporter

• HollandetalAlgalResearch2016• Sinklimitationofphotosynthesis.• Whatisthemolecularmechanismthatregulatephotosynthesiscapacity?

16

ConclusionsPhotosynthesiscanbestimulatedtosupportanengineeredcarbonsink-ethylene

• CarbonfixationreactionsandTCAcyclefluxescanincrease

• Capacity/efficiencyoflightreactionsandcarbonuptakecanincreasetomeettheincreaseddemandforcarbonandenergy

• Togetherwithotherstudies,theseresultsshowthatmetaboliteexcretionisemergingasageneralstrategytoincreasephotosyntheticproductivity.

Arginine

Expandlightharvestingspectrumandtruncateantenna

Minimizenativesinkstoredirectcarbonflux

Developcarbon-efficientpathways

EnhanceEFEexpressionandsubstratesupply

UnderstandEFEmechanismforenzymeengineering

Improvereactordesigntoenhancelightpenetrationandgastransfer

CO2

FutureDirections Markham et al 2016 Green Chemistry

Acknowledgment: DOE BETO

Glycogen mutant; Carrieri et al 2012 Energy & Environmental Science

Phosphoketolase pathway; Xiong et al 2015 Nature Plants

E. coli model; Lynch et al 2016 Biotechnology for Biofuels