From Biomass to Biofuels. Joint BioEnergy Institute Emeryville, CA

Transcription

1 From Biomass to Biofuels Joint BioEnergy Institute Emeryville, CA

2 JBEI at a glance Six Partners Lawrence Berkeley Nat l Lab Sandia Nat l Lab Lawrence Livermore Nat l Lab UC Berkeley UC Davis Carnegie Institute Four Science and Technology Divisions Feedstocks Deconstruction Fuels Synthesis Technologies Single location Emeryville, CA

3 Lignocellulosic Biomass to Fuels Plants Enzymes Microbes Lignocellulose Monomers

4 Some Key Challenges in Converting Lignocellulosic Biomass to Fuels Plants Enzymes Microbes Lignocellulose Monomers Challenges Cellulose and hemicellulose are occluded by lignin Lignin is recalcitrant to depolymerization Inhibitors released from biomass

5 Some Key Challenges in Converting Lignocellulosic Biomass to Fuels Plants Enzymes Microbes Lignocellulose Monomers Challenges Lignocellulose is difficult to depolymerize Pretreatment methods form inhibitory by-products

6 Some Key Challenges in Converting Lignocellulosic Biomass to Fuels Plants Enzymes Microbes Lignocellulose Monomers Challenges Natural microorganisms produce few biofuels Organisms can only utilize a fraction of the monomers Inhibitors released from biomass limit fuels production

7 JBEI: an interlocking approach with three scientific divisions

8 JBEI: an interlocking approach supported by a Technologies Division

9 JBEI: an interlocking approach underpinned by Genomics:GTL

10 Challenges Cellulose and hemicellulose are occluded by lignin, making deconstruction difficult Lignin is recalcitrant to depolymerization Functional groups on hemicellulose can inhibit fermentation & are not efficiently converted to fuels Feedstocks: Developing Bioenergy Crops Somerville et al. 2004, Science

11 Approach Understand & modify polysaccharide biosynthesis Focus on hemicellulose Reduce feruloylation by engineering alternative pathway Modify lignin to aid deconstruction Introduction of cleavable linkages Switchgrass, rice and Arabidopsis as model plants Feedstocks: Developing Bioenergy Crops

12 Engineering lignin composition Systematic analysis of (lignin) cell wall oxidases Develop replacement strategies for lignin Apply advanced imaging technology to determine structure of plant cell walls Objective: change the monomer composition of lignin

13 Engineering lignin composition phenylalanine cinnamate p-coumarate p-coumaroyl- CoA Rosmarinic acid synthase tyrosine Tyrosine aminotransferase 4-hydroxyphenylpyruvic acid 4-hydroxyphenyllactic acid Hydroxyphenylpyruvate reductase Cytochrome P450 hydroxylase Rosmarinic acid Export to Cell wall and integration into lignin

14 Impact Feedstocks: Developing Bioenergy Crops Improved understanding of all cell wall synthesizing and modifying enzymes in rice and Arabidopsis Transgenic plants with modified cell wall composition for optimal deconstruction Translate genetic developments from model plant systems to potential bioenergy crops

15 Challenges Lignocellulose is difficult to process due to: low accessibility of crystalline cellulose fibers presence of lignin seal & hemicellulose cross-links small pore sizes in lignocellulose Deconstruction: providing a source of fermentable sugars Acid pretreatment methods result in the formation of byproducts that are inhibitory to subsequent biofuels fermentation and result in a loss of sugars

16 Approach Deconstruction: providing a source of fermentable sugars Understand the chemical and structural changes resulting from current and new biomass pretreatment approaches Understand the fundamental interactions that govern lignocellulolytic enzymes Explore new microbial environments and employ directed evolution to produce more active and stable lignocellulolytic enzymes Ionic liquids as cellulose solvents Rain forest floor

17 Enzyme-substrate, enzyme complexes, and enzyme-product interactions are key components of enzyme performance Delineate the enzymatic mechanisms in lignocellulose depolymerization Utilize directed evolution to optimize enzymes to improve performance characteristics and lower cost Example: Improving enzyme performance Objective: optimize enzyme structure and function

18 Raw switchgrass samples were processed to isolate different parts of the plant Intact bulk samples were then exposed to 1-ethyl- 3-methylimidazolium (acetate salts) at 120 o C Biomass deconstruction tracked as a function of temperature and time Confocal images taken with dual wavelength excitation (405 and 543 nm) Ionic Liquid Pretreatment of Switchgrass Initial T = 120 o C, t = 20 mins T = 120 o C, t = 50 mins

19 Impact Deconstruction: providing a source of fermentable sugars Optimal pretreatment methods for target biomass feedstocks Improved lignocellulolytic enzymes with enhanced activity and stability Understanding of how microbial communities degrade lignocellulose Cost-effective pretreatment & enzymatic depolymerization methods with minimal byproducts and inhibitor formation

20 Challenges Advanced biofuels are needed to replace petroleum-based: - Jet fuel - Diesel - Gasoline Microorganisms convert only a limited number of precursors to fuels Inhibitors resulting from the pretreatment process prevent growth and biofuel production Fuels Synthesis: ethanol and advanced biofuels

21 Approach Engineer microorganisms to consume lignocellulose monomers Develop pathways for production of future biofuels Engineer organisms to produce & withstand high concentrations of biofuels Fuels Synthesis: ethanol and advanced biofuels

22 Production of isoprenoid-based fuels Isoprenoid (mg/l) Glucose DHAP Construct G6P FDP G3P PYR AcCoA Pathways previously constructed for artemisinin biosynthesis used for production of advanced biofuels. DXP MEV DMAPP Cyclic alkanes and alkenes Cyclic alkanes and alkenes IPP GPP FPP Isopentenol AcCoA Geraniol Genes for isopentenol production isolated from Bacillus subtilis Future fuels will include potential diesel and jet-fuel replacements Geraniol Acetate

23 Impact Fuels Synthesis: ethanol and advanced biofuels Organisms engineered to produce and withstand high concentrations of biofuels Organisms resistant to by-products formed during deconstruction Sequence and regulatory information for metabolic pathways producing biofuels Models of metabolic pathways for fuel synthesis and their mode of regulation

24 Challenges Few tools available for bioenergy/biomass research New high throughput biochemical and `omics approaches needed for all aspects of bioenergy research Advanced imaging techniques can be leveraged to characterize biomass and biomass deconstruction processes Technology: new tools for biofuels research

25 Approach Provide technologies for scientific discovery Implement highthroughput off-the-shelf systems Automate, parallelize and miniaturize throughputlimiting procedures Develop new technologies for enzyme characterization Technology: new tools for biofuels research

26 Challenges: Screening for enzyme activities Bioprospecting and directed evolution will generate glycosyl enzymes with improved or modified function Rapid screening of hydrolase and transferase functions will be required to screen for activity (e.g. endo-, exo-, betaglucosidase) We are developing array-based devices that present glucan moieties for enzymatic modification and subsequent readout. Anup Singh, SNL This system will also be tailored for identification of lignase and other lignin-degrading enzymes

27 Impact Technology: new tools for biofuels research Plant cell wall characterization pipeline High-throughput technologies for large scale analysis of plant and microbial enzyme activities Chips for rapid screening of lignolytic and cellulolytic enzyme function `Omics pipelines for biofuels systems biology Computational tools for biofuels development

28 JBEI Facility: EmeryStation East

29 JBEI leverages key capabilities of partner Systems & Synthetic Biology institutions Plant Research Energy Research Synthetic Biology Engin. Res. Center (SynBERC) DOE: Genomics VIMSS ESPP Joint Genome Institute UC Davis Genome Center & Plant Genomic Program Helios National Combustion Research Facility JBEI Imaging Carnegie Institute of Stanford Nanoscience Computation UC Berkeley Imaging National Center for Electron Microscopy Molecular Foundry Center for Integrated Nanoscience National Energy Res. Supercomputing Center Red Storm Supercomputer Center for Accelerator Mass Spectrometry Advanced Light Source

30 Interactions with industry Equipment makers/suppliers Bioinformatics companies Crop genetics companies Biofuels companies Automobile companies Biomass suppliers

31 Impacts Elucidate & modify plant cell wall structure and synthesis Efficient, cost-effective routes for deconstruction of lignocellulose Engineered organisms for scalable production of ethanol and next generation biofuels Enabling and integrating technologies for bioenergy research Integrated science & technology to transform the U.S. biofuels industry

32 Forward screen of rice mutants to identify cell wall alterations Fast Neutron mutagenesis Seed bulk (M 0 ) M 0 (+/-) Screen (M 1 ) (+/+, +/-, -/-) 2000 M1/month Confirm phenotypes: cell wall composition, saccharification, imaging Identify genes in deleted region and complement mutant phenotype (transgenic or transient assays) Carry out whole genome comparative hybridization on rice tiling array Extract DNA from wild-type and mutant 100,000 M1 seed harvested February, 2008 Pam Ronald, UCD

33 Mutations generated by fast neutrons localized with tiling arrays Whole Genome Comparative Hybridization with NimbleGen 2.1M tiling array Mutant 11-2 The deleted region contains 12 genes Pam Ronald, UCD

34 Identification of glycosyl transferases Objectives: Determine enzyme function & alter composition of cell walls Future studies: Effect on cell wall properties in plants with altered expression of the enzymes are being investigated Crystallization and structural determination of selected enzymes Prediction of enzyme function Henrick Scheller, LBNL

35 Cellulose crystallinity as a function of pretreatment X-ray diffraction Initial Biomass Recovered Product Loss of crystallinity and structural order in pretreated biomass : greater surface area and accessibility for enzyme saccharification S. Singh, M. Auer, M. Clift, P. Adams, B. Simmons

36 Enzymatic cellulose depolymerization kinetics Studied crystalline cellulose microfibrils Utilized in situ atomic force microscopy 500 X 500 nm 200 X 200 nm 100 X 100 nm Introduced cellulase cocktail and tracked depolymerization kinetics t=0 Final D. Dibble, B. Simmons

37 Challenges: Cell Wall Characterization High throughput methods are required to rapidly assess the effects on biomass of mutations in cell wall synthesis or pretreatment methods Many plant cell wall synthesis mutations showed no changed phenotype using currently applied techniques

38 Example: Automation of Limiting Processes Cloning, protein expression and enzyme assays will be rate limiting without high-throughput technologies. JBEI will implement HT cloning and expression technologies and develop new microfluidic tools for HT enzyme assays Current Cloning Protein Expression Enzyme Assays JBEI

39 Functional genomics of biofuel toxicity Butanol toxicity studied by proteomics I-Traq labeling, 2D-LC-MS/MS Stress responses identified Transcriptomics Proteomics Analysis 0% 0.80% 1.20% 1.60% 2% Metabolomics Leu Lys Ile Glu Arg Val Phe His Pro MetAsp Time [min]

40 Engineering Sulfolobus solfataricus metabolism S. solfataricus is being engineered to produce ethanol May allow extractive fermentations with real feedstocks S. solfataricus can grow on cellobiose S. solfataricus can withstand high T, low ph

41 Computational analysis Comparative genomics analysis of fully sequenced genomes Metabolic inference Regulation inference Protein function prediction Metagenomic data Species distribution analysis Comparative species/function enrichment Data Management Transcriptomics Metabolite data Proteomics Strain/phenotype data in the works 16S RNA library/chip production Comparative functional genomics Technology: Functional genomics pipeline

42 Commercializing JBEI s Inventions JBEI interacts with industry in order to: Accelerate biofuels innovation and adoption Provide collaboration opportunities Create a climate for successful spin off companies Promote dialogue among researchers, industry, and VCs

43 Industry Advisory Committee Companies from key sectors: feedstocks, enzymes, biotechnology, genetics, chemicals, fuels production, transportation Provide feedback on JBEI research from industry perspective Arborgen Boeing BP America Chevron DuPont GM Pacific Ethanol Plum Creek POET StatoilHydro

44 JBEI Industry Partnership Program Opportunities: Sponsor research complementary to JBEI mission Sponsor post-doctoral fellowships Place a visiting scholar from your company at JBEI Meet up and coming biofuels researchers Receive advance notice of JBEI inventions 30 days in advance of public promotion Attend JBEI scientific summit Recruiting assistance Receive copy of JBEI publications

45 Technology: Functional genomics pipeline Transcripts Proteins Analysis Microbial Biomass Production Metabolites