Producing Fuels the Old-Fashioned Way: Using Biology

Jim Swartz, Dept. of Chemical EngineeringDept. of Bioengineering

Stanford University

H2

Direct Solar BioHydrogen: Part II

With: Marcus Boyer, Jim Stapleton, Jon Keuchenreuther, and Phil Smith

Dept. of Chemical Engineering

Energy (Fossil fuels)

H2O O2 BiomassCO2

Biological Ecosystems

Solar Energy

Microorganisms, Plants

Consumption

CO2

Energy History: Planet Earth

Solar Energy

Sustainable Energy Future: Planet Earth

H2O O2

Fuel Cells, Power Plants Automobiles

Engineered Photosynthetic Microorganisms

Energy (H2 Economy)

H2

OurOverall

Proposal

The solar resources for generating power from concentrating solar power systems is plentiful. For instance, enough electric power for the entire country could be generated by covering about 9 percent of Nevada – a plot of land 100 miles on a side* – with parabolic trough systems.

Solar Energy Could Supply ALL of our Energy Needs

http://www.energylan.sandia.gov/sunlab/overview.htm#solres

* Equivalent to 1.7% of Current Cropland

The Direct Conversion Concept

TransparentGas PermeableMembrane

Cooling Fluid

Transparent Cover

OrganismSuspension

VacuumOne Candidate

Collector/ReactorCross Section

Incident Sunlight

Large Surface AreaCollector/Reactor

Engineered Organisms Rejuvenate

Culture

Low PressureH2 & O2Harvest

Temp. Control Fluid In

Temp. Control Fluid Out

2 H2O

O2+ 4 H+PS II PS I

hνReduced

Ferredoxin

SynechocystisCatabolism

Sunlight

Growth

hν

Glucose Pyruvate ReducedFerredoxin

Hydrogenase4 H+

2H2

Clostridium pasteurianum Catabolism

New Pathway

Aerobic Process

AnaerobicProcess

Direct PhotoBiological Hydrogen Production: Building a New Electron Pathway

Solar Energy

PhotolysisCenter

H+

O2

e-

H2O e-

Hydrogenase(Clostridial)

H2

Reduced Ferredoxin

Oxidized Ferredoxin

Goal: Engineered Synechocystis Bacterium

Direct PhotoBiological Hydrogen Production: Building a New Electron Pathway

Solar Energy

PhotolysisCenter

H+

O2

e-

H2O e-

Hydrogenase(Clostridial)

H2

Reduced Ferredoxin

Oxidized Ferredoxin

Goal: Engineered Synechocystis Bacterium

BUT, O2 Kills Hydrogenase Activity

Direct PhotoBiological Hydrogen Production: Building a New Electron Pathway

Clostridiumpasteurianum

Desulfovibriodesulfuricans

Scenedesmusobliquus

Chlamydomonasreinhardtii

Comparison of Different Fe-Fe Hydrogenases

The 3-D Structure is Known for theHydrogenase from Clostridium pasteurianum

2H+ + 2Fdred. H2 + 2Fdoxid.

Active Site

Ferredoxin Binding

Site

ElectronTransferPathway

Hydrogenase CpIfrom Clostridiumpasteurianum

SCys

O [FeFe] HydrogenaseActive Site Cluster

S

C

Cys

N

S

C

S

SS Fe

Fe

Fe

Fe

S Cys

SCysFe

S

SFe

C

H

CC C

CN O

NO

HH

HH

The Oxygen-Sensitive Active Site is Very Complex

1Peters JW, Lanzilotta WN, Lemon BJ, SeefeldtLC. 1998. Science 282(5395):1853-1858.

Hydrogenase CpI from Clostridium pasteurianum1

2H+ + 2Fdred H2 + 2Fdoxid

Active Iron-Sulfur Site

Electron transfer metal centers

Ferredoxin Binding Site

2Cohen J, Kim K, King P, Seibert M, SchultenK. 2005. Structure 13(9):1321.

Proposed model for O2 diffusion in CpI2

A Recent Molecular Dynamic Model Suggests Two Oxygen Channels

Create Collection of Hydrogenase Genes with

Genetic Diversity

Express Genes inCell-Free System

Identify CandidatesWith Increased Oxygen

Tolerance

Mutate Genes of Oxygen Tolerant

Hydrogenases

Evaluate InSynechocystis

Process ofDirectedEvolution

We Will Use Directed Evolution to Produce Oxygen Tolerant Hydrogenases

2H+ + 2Fdred. H2 + 2Fdoxid.

Cell-Free Protein Synthesis (CFPS) –Can Easily Conduct Multiple Parallel Reactions

Combined Transcription/Translation: E.coli1. Grow and

Lyse E.coli2. Prepare

Extract3. Add

Substrates,Salts, andFolding Aids

4. AddTemplate

5. Incubate

Provides Direct Access and Control and Rapid Analysis

NewPolypeptide

Ribo-some

RNAPolym-erase

mRNAHalflife

- 3-5 min

TranslationFactors

MessengerRNA

N’sorNDP’s

TranscriptionTranslation

Phosphoenol Pyruvate Pyruvate

PEP Pyruvate

(PEP)

Phosphate

Pi

ATP Regeneration System

ATP Regeneration System

ADP ATPNTP’s

t-RNA’s+

Amino Acids

GTP GDP

ADP ATP

Screening Strategy

Anaerobic Chamber, 3% H 2

Expose to Oxygen by Addition of Air-Saturated Buffer

Cell-Free Protein Synthesis:Gene Library

Protein Library

Error-Prone PCR,Family Shuffling,Rational Design

Mutant Library

Isolate Mutants by Dilution

PCR PCR

Amplify MutantsGenerate Diversity

Oxidized Methyl Viologen Hydrogen Consumption Assay

2MV ox+ H2 2MVred+ 2H +

Jim Stapleton

• Dilution to single molecule level is crucial

• Poisson distribution describes single molecule statistics

• For Leu66 (107 dilution of library):

( )!k

emkPmk −

=P is the probability of getting k DNA molecules in a specified well, when m = average # DNA molecules per well

( ) mm

eemP −−

==!0

00 ( )[ ]0ln Pm −=

21/29

The 107 dilution contains 1.23 molecules/µLMethodology developed by Jim StapletonData from Phil Smith

Single Molecule PCR (smPCR) Allows Clonal Separation

For K = 0

Cell-Free Produced Ferredoxins are Fully Active

Activity of Ferredoxins on per Iron basis

020406080

100120140160

In-Vivo KC1 pRKISC

Fd Sample

Activ

ity (M

Cyt

/ M

Iron

/ m

in)

Cell-Free Cell-Free

Synechocystis Ferredoxin

We Began with Ferredoxin. It also Needs an Fe-S Center

Marcus Boyer

Cys

Cys

CysCys

The Active Site of Fe-Fe Hydrogenases is Complicated:Stabilized by Cysteines, Carbon Monoxide, and Cyanide

CN

CN

COCO

CO

FIG. 5. Hydrogen production rates from purified HydA1 heterologously expressed in E. coli either alone or co-expressed with the indicated Hyd proteins.

Hydrogen production was measured using the methyl viologen-based assay. The data shown represent the average of four independent experiments; average deviations from the mean are shown.

*Genes Taken from Chlamydomonas reinhardtii

In-vivo Co-expression of Maturation Enzymes and Hydrogenases

0

50

100

150

200

250

300

Chlamydomonas HydA1

Chlamydomonas HydA1 +

ChlamydomonasHydG,EF

Chlamydomonas HydA1 +

Shewanella HydGxEF

Clostrium CpI(H2ase) +

ShewanellaHydGxEF

ChlamydomonasHydA1 (synthetic

gene) +ShewanellaHydGxEF

Hyd

roge

nase

Act

ivity

[mm

olH

2/(m

in*m

L*O

D)]

Marcus Boyer, Chia-Wei Wang. Jackie Ng

Hydrogenase Expression in E.coliIdentified Much Better Helper Proteins

Marcus Boyer, Jim Stapleton, Jonny Kuchenreuther, Chia Wei Wang

Producing Active Cell Extract Is a Complicated Procedure

Anaerobic Production of Cell Extract for CellAnaerobic Production of Cell Extract for Cell--Free Synthesis of Active [Free Synthesis of Active [FeFeFeFe] Hydrogenase] Hydrogenase

Aerobic Growth PhaseE. Coli BL21(DE3) w/pACYC HydGXEF;

8 L fermentation at 37 °C

O2

O2

Argon

Argon

O2 to Argon at OD595 = 5.0;Addition of IPTG, fumarate, ferrous iron, and cysteine;

Temp change: 37 ° C to 16 °C

Cells thawed,resuspended, homogenized,

centrifuged

Cells frozen with liquid N2

CentrifugedAnaerobic Cell Anaerobic Cell Extract PreparationExtract Preparation

Cell extract decanted

AnaerobicallyAnaerobically--produced cell extract produced cell extract

with with HydEHydE, , HydFHydF, and , and HydGHydG maturation maturation

enzymesenzymes Cell extract frozen with liquid N2

Cell HarvestCells

collected after 16 – 20

hr of anaerobic

induction at 16 °C

Anaerobic Growth Phase

0.0

2.0

4.0

6.0

8.0

AnaerobicCell Extract

ReconstitutedAnaerobicCell Extract

Oxygen-Exposed

AnaerobicCell Extract

Oxygen-Exposed

ReconstitutedAnaerobic

Cell Extract

Activ

e H

ydA1

(mg/

ml)

The Helper Proteins HydE, HydG, and HydF are all 4Fe-4S Proteins.*

*Brazzolotto, …, Marc Fontecave, J. Biol. Chem. 281:769, 2006

Activity of Cell Extracts Can be Significantly Increased by “Reconstitution”

“Reconstitution” is Effected by Incubation of Cell Extract with:1mM Fe(NH4)2(SO4)2, 1mM Na2S, and 1 mM Dithiothreitol (DTT)

Jonny Kuchenreuther and Marcus Boyer

0

8

16

24

32

40

HydA1 CAT9-HydA1

HydA1 fromLinear

Templates

CAT9-HydA1 from

LinearTemplates

Act

ive

Cel

l-Fre

e Y

ield

(µg

/ml)

Active Protein

Improved Translational Initiation Gives Higher Active Yields

Jonny Kuchenreuther

New Insights: The Devil is in the Details

We must better understand:The Function and the Activation of the Helper Proteins as well as any other Requirements for Hydrogenase Activation:

A) Required for Effective Screening of Hydrogenase Mutants

B) Required for Activation of Hydrogenase when it is Expressed inPhotosynthetic Organism.

Fontecave’s work suggests requirements for:Guanosine Tri-Phosphate (GTP)

and S-Adenosyl Methionine (SAM)

What do GTP and SAM do?What is the Origin of the Active Site Atoms?

SAM

Post-Synthesis Activation (Maturation) of ApoHydrogenaseEnables Detailed Studies of Maturation Requirements

A. Hydrogenase polypeptide is produced without helper proteinsand under aerobic conditions. Small molecules are removed.

B. Cell Extract with Helper Proteins is Reconstituted under anaerobicconditions. Small molecules are removed.

C. Under anaerobic conditions, the preparations are mixed and candidate small molecules are added.

Marcus Boyer and Jonny Kuchenreuther

0%5%

10%15%20%25%30%

2 mM GTP+ 2 mM

SAM

2 mM GTP+ 0.33 mM

NAD

2 mM SAM+ 0.33 mM

NAD

2 mM GTP+ 2 mM

SAM + 0.33mM NAD

% A

pohy

drog

enas

eM

atur

ed

Screening Strategy

Anaerobic Chamber, 3% H 2

Expose to Oxygen by Addition of Air-Saturated Buffer

Cell-Free Protein Synthesis:Gene Library

Protein Library

Error-Prone PCR,Family Shuffling,Rational Design

Mutant Library

Isolate Mutants by Dilution

PCR PCR

Amplify MutantsGenerate Diversity

Oxidized Methyl Viologen Hydrogen Consumption Assay

2MV ox+ H2 2MVred+ 2H +

Jim Stapleton

Initial Screening Assays Were Highly Variable

Pre-exposure Reproducibility

0

0.5

1

1.5

2

2.5

A1 B2 C5 D4 E8 F7 G8 H11

Norm

aliz

ed S

lope

(AU/

s/uL

) Initial screenRetest

For Unmutated Hydrogenase

Pre-exposure

00.20.40.60.8

11.21.4

A3 A4 B3 B4 C3 C4 D3 D4 E3 E4 F3 F4 G3 G4 H3 H4

Wild-type Reaction

Nor

mal

ized

to

Med

ian

Post-exposure

00.20.40.60.8

11.21.4

A3 A4 B3 B4 C3 C4 D3 D4 E3 E4 F3 F4 G3 G4 H3 H4

Wild-type Reaction

Nor

mal

ized

to

Med

ian

We are Working to Make the Screen Performance More Reproducible

JimStapleton,

SeanKendall,

PhilSmith

Conclusions

Solar BioHydrogen Appears to be Technically and Economically Feasible

BUT, We must first Evolve an Oxygen Tolerant Hydrogenase

We can express Fe-Fe Hydrogenases With Cell-Free Technology Using Activated Cell Extracts

We can accomplish Single-Molecule PCR for Clonal Separation

GTP, SAM, and NAD are Required for H2ase Activation

We Have Gained Significant Knowledge To Help Us ExpressThe Hydrogenases in Photosynthetic Organisms

The Search for Oxygen Tolerant Hydrogenases is Underway

Acknowledgements

• Stanford School of Engineering For Seed Funding

• Stanford Global Climate and EnergyProgram (GCEP) for Major Funding