DOE GTL Vertically Integrated BioEnergy Research Center(special thanks to Harvard Inst. for Biologically Inspired Engineering)

Time Agenda item (77 PIs 75 pages 390 minutes today)

8:30-8:45 Introduction and enabling technologies (GC)

8:45-9:15 Plan for integrated response to RFA (JA)

9:15-9:45 Plants and Agriculture

9:45-10:15 Saccharification + CBP

10:15-10:30 Break

10:30-11:00 Fermentation

11:00-11:15 Process modeling and improvement

11:15-11:30 Alternative biofuels and paradigms

11:30-12:00 Pretreatment, extraction, use

12:00-1:00 Working lunch: IP, other BRCs

1:00-2:00 Open Discussion

2:00-2:30 Budgets

2:30-3:00 Next steps

Brazil’s Bioethanol(& GtL Economic modeling needs)

Land use:45,000 km²Sugarcane: 344 million tons (76 tons/ha)Sugar: 23 million tonsEthanol:14 million m³ $0.26/L (feedstock 70%)

yield increase 3.5%/yrDry bagasse: 50 million tonsElectricity: 1350 MWBagasse ash 2.5% (vs 40% for coal), nearly no sulfur. Burns at low temperatures, so low nitrogen oxides.

Saccharum officinarum

The ‘G’ in GtL '77 Gilbert & Sanger papers '77 developed 1st auto-sequence-reading software'77 1st full plasmid sequence (recombinant DNA)'84 ‘Genomic Sequencing’ PNAS paper & DOE Alta meeting'87 1st Genome grant (DOE) '90 co-PI on 1st NIH Genome Centers, Stanford,GTC,MIT (now Broad)

'94 1st genome sequence H.pylori 1.7 Mbp (commercial) '02 Only DOE Center to address all 5 GtL goals'06 SynBERC grant with LBL, MIT, etc.

#1: Protein complexes & Mass Spec #2: Regulatory Networks & RNA#3: Microbial Communities #4: Computational models #5: Synthetic Biology

Plants Zea,Miscanthus,Poplar, Crambe, Algae

Pretreat acid, NH4 fiber explosion (AFEX), silage

Saccharify Trichoderma, Clostridia, AspergillusSoluble, cellulosome, consolidated

Ferment Saccharomyces, Escherichia, Zymomonas, Pichia, Klebsiella, communitiesExtract distill, gas strip, zeolite, phase

Process crack, blend ethanol, butanol, ethylesters, alkanes, H2 Uses transportation, chemicals, power

Vertical Integration (horizontal options)

AGRICULTURE

Enabling Tech: Modeling, Synthesis, Evolution, Sequencing

Our GTL network

Church-Harvard Silver Shih

Curtis-PSU

Chisholm-MIT

Leschine-UMA

Codon DevicesCollins-BU

LS9

Agrivida

Hamilton-INL

Ausubel-MGH

Liu-BNL

SynBERC-iGEM

Ingram-UFL

Chappell-UKY

Laible-ANL

Richard-PSU

Mendel

GreenFuel

Celunol

Chromatin

SunEthanol

DuPontShell

Vance-WashU

BioEnergy International

Endy

Polz

Pendse-UME

Cerrina-UW

Enabling Technologies #1: Computational & Systems Modeling

Enabling Technologies #2: Lab evolution (building on systems design)

#3: Functional metagenomics (interspecies DNA transfer)

Radiation resistance (Edwards & Battista)Tyr/Trp production & transport (Lin & Reppas)Citrate utilization (Lenski)Lactate production (Ingram)Temperature/acid tolerance (Marliere)Glycerol utilization (Palsson)Aldehyde resistance (Sommer & Dantas)

Position

TypeGen

eLocation Function Mechanism

986,334T >

Gomp

FPromoter-

10

Promoter of Non-specific transport channel

Makes promoter more consensus-like

985,797 T >

Gomp

FGlu > Ala Non-specific

transport channel

Makes pore bigger and more hydrophobic

931,9608

bp

lrp frameshiftGeneral

Transcriptional Regulator

?

Whole-Genome resequencing of evolved Trp

Shendure, et al. (2005) Science 309:1728

ompF – non specific transport channel• Glu-117 → Ala (in the pore)• Charged residue known to affect pore size and

selectivity• Can increase import & export capability

simultaneously

Tech #4: ‘Next Generation’ Sequencing

Multi-molecule Reaction Volume AB/APG Ligase beads 1 fL 454/Roche Pol beads 100,000 fL Solexa Pol term 1 fLCGI Ligase 1 fLAffymetrix Hybr array 100 fLIBS Pol beads 10,000 fLSingle molecules Helicos Biosci Pol <1fLVisigen Biotech Pol FRET <1fLPacific Biosci Pol <1fLAgilent Nanopores <1fL

fL =1E-15 liters(femto)

Our lab has been involved in 8/10

#5: Sequencing genomes from single cells

1) Environmental samples (poor or no lab growth)2) Candidate chromosome region sequencing3) Prioritizing or pooling (rare) species based on an initial DNA screen (metagenomics)4) Multiple chromosomes in a cell or virus5) RNA 5’, 3’ ends & splicing6) Cell-cell interactions (predator-prey, symbionts, commensals, parasites)

Phi-29 Polymerase Stand-displacement amplification

(single chromosome, cell , RNA or particle)Zhang, et al. (2006) . Nature Biotech. June ’06

#6: 10 Mbp of DNA / $1000 chip

8K Atactic/Xeotron/Invitrogen

Photo-Generated Acid

12K Combimatrix/Codon Electrolytic

44K Agilent Ink-jet standard reagents

380K Nimblegen/GA Photolabile 5'protection

Tian et al. Nature. 432:1050; Carr & Jacobson 2004 NAR; Smith & Modrich 1997

PNAS

#7: Recombinational Genome Engineering

Amplify pools of 50mers using flanking universal PCR primers &

3 paths to 10X error correction

Digital Micromirror Array

#8: 3D Structural Biology – especially membrane and fibrous structures

Hanson & Laible Shih, Douglas, iGEM

#9, 10: Proteomics, Metabolomics

DOE GTL Vertically Integrated Bioenergy and Novel Technologies(VIBRANT)

Time Agenda item (77 PIs 75 pages 390 minutes today)

8:30-8:45 Introduction and enabling technologies (GC)

8:45-9:15 Plan for integrated response to RFA (JA)

9:15-9:45 Plants and Agriculture

9:45-10:15 Saccharification + CBP

10:15-10:30 Break

10:30-11:00 Fermentation

11:00-11:15 Process modeling and improvement

11:15-11:30 Alternative biofuels and paradigms

11:30-12:00 Pretreatment, extraction, use

12:00-1:00 Working lunch: IP, other BRCs

1:00-2:00 Open Discussion

2:00-2:30 Budgets

2:30-3:00 Next steps

How do we distinguish ourselves from other GTL proposals?

The DOE-GTL-BRC competition (2 – 3 will be funded) 1. LBL-LLNL-Sandia-Stanford-U.IL 2. ORNL-NREL-Dartmouth-Noble-OK 3. Harvard-MIT-UMA-UME-BU-UFL-PSU-UWI-ANL-INL-BNL- 11 Companies4. San Diego-Iowa-JCVI5. Purdue-MIT26. UWI-MSU7. LANL

BP competition (1 will be funded)1. Imperial College, UK 2. Cambridge, UK 3. LBL-LLNL-Sandia-Stanford 4. San Diego-Iowa-JCVI 5. MIT-Purdue

Snapshots from the 1st DOE-VIBRANT meeting Boston 21-Nov-2006