Jim Swartz Jim Stapleton Jon Kuchenreuther Phil Smith Alyssa Bingham and Monica Ortiz

Dept of Chemical Engineering and Dept of Bioengineering Stanford U

Direct Solar BioHydrogenFive Years and Beyond

Amino Acids+

Information (DNA)

+Energy

Ribosomes (Catalysts) Product

Cell-Free Protein Synthesis

H2

Photo-Synthetic

GCEP Research Symposium ndash

October 2 2008

Solar Energy

Sustainable Energy Future Planet Earth

H2 O O2

Power Plants FertilizerCement Automobiles

Engineered Photosynthetic Microorganisms

Energy (H2 Economy)

H2

OurOverall

Proposal

What is the Potential for Solar EnergyCurrent US Consumption

Of Liquid Fuels plus Nat Gascong

60 x 1015 Btu yr

Assume Solar Incidence of7 kWhm2-day

and 15 Energy Efficiency(Overall)

Need 17000 Square Miles

= 25 of Current Cropland

(But Do Not Need to Convert Current Crop Land)

The Direct BioConversion

ConceptIncident

Sunlight

Large Surface AreaCollectorReactor

Engineered Organisms Rejuvenate

Culture

Low Partial PressureH2

amp O2Harvest

Temp Control Fluid In

Temp Control Fluid Out

or Purge Gas (N2

)

EstimatedEconomicsAllow About$20-30ft2

TransparentGas PermeableMembrane

Cooling Fluid

Transparent Cover

OrganismSuspension

Low PressureOne Candidate

CollectorReactorCross Section

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-

H2 O e-

Hydrogenase(Clostridial)

H2

Reduced Ferredoxin

Oxidized Ferredoxin

Goal Engineered Synechocystis Bacterium

Direct PhotoBiological

Hydrogen Production Building a New Electron Pathway

H+

O2

e-

H2 O e-

Hydrogenase(Clostridial)

H2

Reduced Ferredoxin

Oxidized Ferredoxin

BUT O2 Kills Hydrogenase Activity

Direct PhotoBiological

Hydrogen Production Building a New Electron Pathway

Challenges1

Need O2

toleranthydrogenase

2

Need to expressand activate foreign H2

ase

3

Need to remodelantennae

4

Need to controlelectron andprotonflux rates

1Peters JW Lanzilotta

WN Lemon BJ Seefeldt

LC 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 Schulten

K 2005 Structure 13(9)1321

Proposed model for O2

diffusion in CpI2

Can We Evolve an Oxygen Tolerant HydrogenaseA Molecular Dynamic Model Suggests at Least Two Oxygen Channels

We Will Undoubtedly Need Multiple Cooperative MutationsWe Will Need Precise Measurements to Detect Incremental Improvements

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 Evolve Oxygen Tolerant Hydrogenases

2H+ + 2Fdred H2 + 2Fdoxid

Cell-Free Protein Synthesis (CFPS) ndash

Combined TranscriptionTranslation Ecoli1 Grow and

Lyse Ecoli2 Prepare

Extract3 Add

SubstratesSalts 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

NrsquosorNDPrsquos

TranscriptionTranslation

Phosphoenol Pyruvate Pyruvate

PEP Pyruvate

(PEP)

Phosphate

Pi

ATP Regeneration System

ATP Regeneration System

ADP ATPNTPrsquos

t-RNArsquos+

Amino Acids

GTP GDP

ADP ATP

Why Use Cell-Free Protein Synthesis (CFPS)

1

We need to make a very complex protein we can control theconcentrations of helper proteins and co-factors

2

We avoid the need to make plasmids transform organisms withthe plasmids select single colonies grow the organisms and purify the protein

3

We can more easily measure specific activity (using radioactivelabeling to measure protein yields)

4

We can more easily control background activities

5

We can use automated equipment to precisely evaluate10rsquos of thousands of candidates

6 The technique may also enable evaluation of millions of candidates

Screening Strategy

Anaerobic Chamber 2 H 2

Expose to Oxygen by Addition of Air-Saturated Buffer

Cell-Free Protein SynthesisGene Library

Protein Library

Mutant Library

Isolate Mutants by Dilution

PCR PCR

Amplify MutantsGenerate Diversity

Oxidized Methyl Viologen Hydrogen Consumption Assay

2MV ox+ H2 2MVred+ 2H +

Error Prone PCRDNA ShufflingRational Design

bull

Dilution to single molecule level is crucial

bull

Poisson distribution describes single molecule statistics

( )k

emkPmk minus

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

2129

The 107

dilution contains 123 moleculesμL

Single Molecule PCR (smPCR) Allows Clonal

Separation

The Active Site of Fe-Fe Hydrogenases is ComplicatedStabilized by Cysteines Carbon Monoxide and Cyanide

Cys

Cys

CysCys

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 +

ChlamydomonasHydGEF

Chlamydomonas HydA1 +

Shewanella HydGxEF

Clostrium CpI(H2ase) +

ShewanellaHydGxEF

ChlamydomonasHydA1 (synthetic

gene) +ShewanellaHydGxEF

Hyd

roge

nase

Act

ivity

[mm

olH

2(m

inm

LO

D)]

Hydrogenase Expression in Ecoli Identified Much Better Helper Proteins

Collaboration with Professor Alfred Spormann

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 HydGxEF8 L fermentation at 37 degC

O2

O2

Argon

Argon

O2 to Argon at OD595 = 50Addition of IPTG fumarate ferrous iron and cysteine

Temp change 37 deg C to 16 degC

Cells thawedresuspended 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 HydE HydF and with HydE HydF and HydG maturation HydG maturation

enzymesenzymes Cell extract frozen with liquid N2

Cell HarvestCells

collected after 16 ndash 20

hr of anaerobic

induction at 16 degC

Anaerobic Growth Phase

Boyer et al 2008 Cell-free synthesis and maturation of [FeFe] hydrogenases BiotechampBioeng 9959-67

Screening Strategy

Anaerobic Chamber 2 H 2

Expose to Oxygen by Addition of Air-Saturated Buffer

Cell-Free Protein SynthesisGene Library

Protein Library

Mutant Library

Isolate Mutants by Dilution

PCR PCR

Amplify MutantsGenerate Diversity

Oxidized Methyl Viologen Hydrogen Consumption Assay

2MV ox+ H2 2MVred+ 2H +

Error Prone PCRDNA ShufflingRational Design

Specific activity expressed aspmol

H2

consumed per minute per ng

hydrogenase

Discovered a More Active H2

ase (2 mutations)Mutation 1 Confers Higher Specific Activity

Cell-Free Expression In vivo Expression

0

10

20

30

40

50

Wild-type Mutation 1

Mutation 2

Mutant E7(1 + 2)

HydA1 Hydrogenase

[FeF

e] H

ydro

gena

seC

ell-f

ree

Yie

ld

0

100

200

300

400

500

[FeF

e] H

ydro

gena

seSp

ecifi

c A

ctiv

ity

Grey cell-free yieldRed specific activity

Activity expressed aspmol

H2

consumed per minuteper mg wet cell mass

Red Expressed Activity

0

1000

2000

3000

HydA1 Wild-type

HydA1Mutant E7

[FeF

e] H

ydro

gena

se

Act

ivity

This is the CpI

structure but the mutations are in HydA1

Red Complex = Top Portion of H-clusterYellow Complexes = Other FeS clustersPink = mutations

Mutation 1G116D (HydA1 residue ) is close to the active site in an alpha helix on the rightThis is the one that causes the improved activity

Mutation 2N211S is on the left on an externalloop and has no significant effect onspecific activity

12

The Influential Mutation is Near the Active Site(Glycine

to Aspartic Acid)

N-terminal domain

Peptide loop

ClostridialCpI

ChlamydomonasHydA1

3-D Structures Suggest HydA1 May Never Become O2

TolerantBUT

H-cluster

Improvements In Cp1 Expression and Activation

CpI Hydrogenase Activity

003086

196

715

000100200300400500600700800

Jan 2007 WildType

Feb 2007 FrontEnd Optimized

Nov 2007Standardized

Reagents

June 2008 (noCysteineAddition)

CpI A

ctiv

ity

(nm

ol m

in-1

microL

CF R

xn p

rodu

ct-1

)

Improving the Oxygen Inactivation Procedure

0

20

40

60

80

100

120

140

0 14 28 42 56 69 83 97 111

O2 Concentration for Exposure (μM)

Pos

t-O2 E

xpos

ure

Act

ivity

()

Early ProcedureResults from Three Different Days

0

20

40

60

80

100

120

140

0 50 75 100 150 200 225

O2 Concentration for Exposure (μM)

Pos

t-O2 E

xpos

ure

Act

ivity

()

Optimized ProcedureResults from Two Different Days

Revised Plan for More Intense Hydrogenase

Evolution

Introduce Rational Mutations to Block or Immobilize Oxygen

ChannelsAND

Conduct Agressive

Mutagenesis to ldquoFillrdquo

Gene with Permissible

Mutations

2

Conduct DNA Shuffling to Combine Permissible Beneficial Mutations

3

Develop Ultra-High Throughput Screen (Emulsion System with FACS Sorting)to Evaluate Millions and Isolate Thousands of Promising Candidates

4

Use 96-well Plate Screening for Precise Comparison of Candidates

5

Sequence to Identify Beneficial Mutations

6

Randomize Influential Sequences and Search Again ANDOR

Conduct DNA Shuffling and Search Again

7 Continue until Done (May also need to improve other attributes)

1

Streptavidin-coated beads

Biotinylated mutant genes attachedlt1 gene per bead

Biotinylated anti-tag antibodies attached

Beads are added to cell-free protein synthesis reaction mixture

Emulsification in oil creates isolated femtoliter-scale reactors

Mutant genes are expressed as taggedmutant proteins which bind to antibodies

Emulsions are broken and CFPS mixture removed

Emulsion-Based H2 ase Screening

Griffiths amp Tawfik

Oil

H2 2H++2e-O2O2

O2

O2

O2

O2O2

O2

O2

O2

O2

O2

O2

Beads are exposed to oxygen levels sufficient to deactivate wild-type proteins

Beads are mixed with fluorogenic

substrate

and re-emulsified in oilMutants surviving O2

exposure consume dissolved H2

The resulting electrons reduce the fluorogenic

substrate

generating a fluorescent signal

The hydrophobic fluorophore sticks to the hydrophobic beads

when they are removed from the emulsion droplets

Fluorescence-Activated Cell Sorting (FACS)

Allows Us To Capture the Beads with

the Desirable Mutants

Cells flow single-file past a laserand detector and can be sorted based on fluorescence or size

httpwwwbiodavidsoneduCOURSESGENOMICSmethodFACShtml

100 101 102 103 104

FL4 TR

0

20

40

60

80

100

o

f Max

100

101

102

103

104

FL4 TRN

umbe

r of b

eads

Hydrogenase-coated beads

Negative control beads

Fluorescence

bull ~1 molecule DNA (wild type)attached per bead

bull Beads in Cell-free Reaction Mixture wereemulsified in oil

bull Cell-free protein synthesis conducted in emulsions

bull Emulsion broken and CFPS solution removed

bull Beads re-emulsified with fluorogenic

compound

bull 1-day incubationbull Second emulsion broken bull Beads analyzed by FACS

The Procedure is Working

Cell-Free Reaction MixtureWith Beads

OilPhase

OilPhase

We will Now Use MicroFluidic

Technology toProduce Emulsions With Uniform Reactor Sizes

httpraindancetechnologiescomtechnologypcr-genomics-researchasp

Also Need Ability to Activate [FeFe] Hydrogenasein Synechocystis sp --

First We Need to Know the Co-Factor Requirements

1

Purify ApoProtein(no active site

H-Cluster)

2

Remove small molecules from thecell extract thatcontains maturases

3

Determine which small moleculemetabolites are required for activation

PEP = phosphoenol

pyruvateSAM = S-adenosyl

methionineNAD = nicotinamide

adenine dinucleotide

0

10

20

30

40

20 AminoAcids

InorganicSulfur

Fe(II) PEP SAM NAD None (AllIncluded)Cofactor(s) Not Included

[FeF

e] H

ydro

gena

seSp

ecifi

c A

ctiv

ity

CoFactors

Omitted from Activation Reactions

Then We Need to Optimize in vivo H2

ase Activation (Initially we are working in Ecoli)

0

1000

2000

3000

4000

No Additions Iron(III) L-cysteine Iron(III) + L-cysteine

Addtions to Growth Medium

[FeF

e] H

ydro

gena

seA

ctiv

ity

(Activity expressed in nmol

H2

consumed mg cells (wet) ndash

min)

ConclusionsSolar BioHydrogen Appears to be Technically

and Economically Feasible

BUT We must first Evolve an Oxygen Tolerant Hydrogenase

[FeFe]Hydrogenases Can Be Produced In Cell-Free Rxns

Single-Molecule PCR can be used for Clonal Separation

More active HydA1 Mutants were isolated but not O2 Tolerance

An ultra-high throughput screen is being implemented for the more complicated CpI

Hydrogenase

SAM NAD PEP amp amino acids assist H2ase Activation

Heterologous expression and activation is feasible

The Quest Continues

CysCys

CysCys

CN

CN

COCO

CO

Pending Future Projects

Participant in a DOE Energy Frontiers Research CenterldquoCenter for Evolved NanoBioHybridsrdquo

Energy Biosciences ProposalHow is H-Cluster Assembled

AndFunction of Amino Acid ScaffoldSupporting and Activating H-Cluster

Acknowledgements

bull

Stanford School of Engineering For Seed Funding

bull

Stanford Global Climate and Energy Program (GCEP) for Major Funding

Thank You

Acknowledgements

bull

Stanford School of Engineering For Seed Funding

bull

Stanford Global Climate and Energy Program (GCEP) for Major Funding

Thank You

What Rates of Gas Permeation Are Required

TransparentGas PermeableMembrane

Cooling Fluid

Transparent Cover

OrganismSuspension

VacuumOne Candidate

CollectorReactorCross Section

Assume 20 Overall EfficiencyGenerates asymp

2 moles of gasm2-hr

Under standard conditions requires 004 mhr permeationDOE Objective is 30mhr

with hydrogen ΔP = 20 psi

for hydrogen separation membranes

• Slide Number 1
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• Fluorescence-Activated Cell Sorting (FACS) Allows UsTo Capture the Beads with the Desirable Mutants
• Slide Number 28
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• Acknowledgements
• Slide Number 35
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• Slide Number 21
• Slide Number 22
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• Slide Number 24
• Slide Number 25
• Slide Number 26
• Fluorescence-Activated Cell Sorting (FACS) Allows UsTo Capture the Beads with the Desirable Mutants
• Slide Number 28
• Slide Number 29
• Slide Number 30
• Slide Number 31
• Slide Number 32
• Slide Number 33
• Acknowledgements
• Slide Number 35
• Slide Number 36
• Slide Number 37