DOE Bioenergy Research Center (BRC) - jbei.org€¦ · DOE Bioenergy Research Center (BRC) - Joint BioEnergy Institute ... JBEI contacts Jay Keasling, Chief Executive Officer, [email protected],

DOE Bioenergy Research Center (BRC) - Joint BioEnergy Institute

Monthly Progress Report – May 2015

Fiscal Year: 2015

Year / Period: Year 8 / Month 8 / May 1 - 31, 2015

Department of Energy’s Joint BioEnergy Institute is supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through Contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy.

JBEI contacts Jay Keasling, Chief Executive Officer, [email protected], 510-495-2620 Nick Everson, Chief Operating Officer, [email protected], 510-486-6268

Executive Summary This Monthly Progress Report provides a summary of JBEI’s activities for the month of May 2015, eight month of fiscal year 2015:

Item Count Highlights

Publications in Print 9 "Acute limonene toxicity in Escherichia coli is caused by limonene-hydroperoxide and alleviated by a point mutation in alkyl hydroperoxidase (AhpC)"

Invention Disclosures 2 “PKS producing adipic acid”

Patent Applications 2 “Engineered Microorganisms Having Resistance to Ionic Liquids”

Media Citations 1 Women Getting Science Ph.D.s Still Face Gender Barriers, KQED

Outreach Interactions 29 Education: 10, Public: 1 and Scientific Community: 18.

On-Site Seminars 10 Professor Greg Challis from the Department of Chemistry, University of Warwick gave a seminar on “Discovery and Biosynthesis of Novel Polyketide Antibiotics”.

Industry Interactions 22 Interactions with Cummins and Genomatica.

FY15 Milestones Milestones completed: - Deconstruction: “Predict and develop task specific ILs for selective lignin or cellulose dissolution and low

temperature pretreatment”

- Deconstruction: “Expand our database of ionic liquid tolerant enzymes, particularly to include enzymes from the GH3, GH5, GH6, GH9, GH10, AA9 and AA10 families”

Milestones delayed: - Deconstruction: “Improve the overall sugar yields of our existing thermo and ionic liquid tolerant multi-

component enzyme mixture by including enzymes for hydrolysis of both cellulose and hemicellulose and by including LPMOs”

- Fuels Synthesis: “E. coli membrane editing: With JGI, generate 10-20 single and double mutants and test improvement in expression of beneficial tolerance pump and corresponding tolerance to toxic fuels”

- Technology: “Structural characterization of Fatty Acid Synthase and other large complexes by single particle cryo electron microscopy”

mailto:[email protected]

mailto:[email protected]

Dashboard - May 2015 - DOE

FY 2015 Dashboard For Period: May 1 - 31, 2015

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Publications

In Print

Chubukov, V., Mingardon, F., Schackwitz, W., Baidoo, E. E., Alonso-Gutierrez, J., Hu, Q., Lee, T. S., Keasling, J. D., & Mukhopadhyay, A. (2015). "Acute limonene toxicity in Escherichia coli is caused by limonene-hydroperoxide and alleviated by a point mutation in alkyl hydroperoxidase (AhpC)". Appl Environ Microbiol. doi, 10.1128/aem.01102-15 http://aem.asm.org/content/early/2015/04/27/AEM.01102-15.full.pdf

George, K. W., Thompson, M. G., Kang, A., Baidoo, E., Wang, G., Chan, L. J., Adams, P. D., Petzold, C. J., Keasling, J. D., & Soon Lee, T. (2015). "Metabolic engineering for the high-yield production of isoprenoid-based C alcohols in E. coli". Sci Rep, 5, 11128. doi, 10.1038/srep11128 http://www.ncbi.nlm.nih.gov/pubmed/26052683

Loque, D., Scheller, H. V., & Pauly, M. (2015). "Engineering of plant cell walls for enhanced biofuel production". Curr Opin Plant Biol, 25, 151-161. doi, 10.1016/j.pbi.2015.05.018

Mingardon, F., Clement, C., Hirano, K., Nhan, M., Luning, E. G., Chanal, A., & Mukhopadhyay, A. (2015). "Improving olefin tolerance and production in E. coli using native and evolved AcrB". Biotechnol Bioeng, 112(5), 879-888. doi, 10.1002/bit.25511 http://www.ncbi.nlm.nih.gov/pubmed/25450012

Mortimer, J. C., Faria-Blanc, N., Yu, X., Tryfona, T., Sorieul, M., Ng, Y. Z., Zhang, Z., Stott, K., Anders, N., & Dupree, P. (2015). "An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14". Plant J. doi, 10.1111/tpj.12898 http://onlinelibrary.wiley.com/store/10.1111/tpj.12898/asset/tpj12898.pdf?v=1&t=iapho8v0&s=ba7c1d428ade53affab67c35b6e2ed6dfdec0194

Mukhopadhyay, A. (2015). "Tolerance engineering in bacteria for the production of advanced biofuels and chemicals". Trends Microbiol. doi, 10.1016/j.tim.2015.04.008

Parsons, H. T., & Heazlewood, J. L. (2015). "Beyond the Western front: targeted proteomics and organelle abundance profiling". Front Plant Sci, 6, 301. doi, 10.3389/fpls.2015.00301 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4419601/pdf/fpls-06-00301.pdf

Scullin, C., Stavila, V., Skarstad, A., Keasling, J. D., Simmons, B. A., & Singh, S. (2015). "Optimization of renewable pinene production from the conversion of macroalgae Saccharina latissima". Bioresour Technol, 184, 415-420. doi, 10.1016/j.biortech.2014.09.105 http://www.ncbi.nlm.nih.gov/pubmed/2531890

Velasquez, S. M., Ricardi, M. M., Poulsen, C. P., Oikawa, A., Dilokpimol, A., Halim, A., Mangano, S., Denita Juarez, S. P., Marzol, E., Salgado Salter, J. D., Dorosz, J. G., Borassi, C., Moller, S. R., Buono, R., Ohsawa, Y., Matsuoka, K., Otegui, M. S., Scheller, H. V., Geshi, N., Petersen, B. L., Iusem, N. D., & Estevez, J. M. (2015). "Complex regulation of prolyl-4-hydroxylases impacts root hair expansion". Mol Plant, 8(5), 734-746. doi, 10.1016/j.molp.2014.11.017 http://www.ncbi.nlm.nih.gov/pubmed/25655826



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Media Citations

Date Article Source

May 18 Women Getting Science Ph.D.s Still Face Gender Barriers KQED

Outreach – Education

Date Presenters Audience or Presentation Title Activity Location

May 6 Peter Matlock and Blake Simmons

University of Hawaii, Hilo Meeting Off-site

May 7 Sarah Richardson Rock Canyon High School - Biotech Club Meeting Off-site

May 8 Ee-Been Goh and Belle Smith

Bioscience Career Institute Tour On-site

May 8 Peter Matlock Lisa Margonelli, writer for The Atlantic Meeting On-site

May 14 Jeff Kimbrel, Aram Kang, Tristan de Rond and Irina Silva

Wageningen University Tour On-site

Invention Disclosures Inventor(s)

2015-106 - “PKS producing adipic acid” Andrew R. Hagen, Leonard Katz, Jay Keasling and Sean Poust

2015-114 - “Droplet-based single molecule detection” Anup Singh

Patent Applications Inventor(s)

2013-120-02, US 14/717,962 “Engineered Microorganisms Having Resistance to Ionic Liquids” Thomas L. Ruegg, Michael Thelen and Douglas Higgins

2013-188-02, PCT/US2015/031444 “Compositions and methods for single-molecule construction of DNA”

Jay Keasling, Lane Weaver, William Holtz and Daniel Arlow.



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May 15 Sarah Richardson Tennyson High School Seminar Off-site

May 18 Irina Silva iCLEM Alumni Meeting Off-site

May 21 Irina Silva, Dominique Loque, Patrick Shih and Virginia Tartaglio

Agroecology Program at Florida International University

Tour On-site

May 26 Sarah Richardson Oakland Unified School District Dinner with a Scientist

Event Off-site

May 28 Sarah Richardson Castro Valley High School - Biotechnology Course Project

Interview Off-site

Outreach – Public


May 11 Diane Pierotti and Irina Silva Lawrence Hall of Science Meeting Off-site

Outreach – Scientific Community


May 4 - 6 Steven Singer The Promise and Perils of Automating Metagenomic Binning

Presentation Genome Standards Consortium-17, Joint Genome Institute, Walnut Creek, CA

May 6 Taek Soon Lee Microbial production of advanced biofuels Presentation National University of Singapore, Biochemistry Department Seminar, Singapore

May 6 Dominique Loque Plant Engineering to Synthetic Biology and Back

Presentation UC Berkeley, CA

May 8 Jay Keasling Engineering Microbes to Solve Global Challenges

Presentation Abbott Lecture, Rensselaer Polytechnic Institute, Troy, New York



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May 11 Taek Soon Lee Microbial production of advanced biofuels Presentation Kunkuk University, Bioscience and Biotechnology Department Seminar, Seoul, South Korea

May 12 Dominique Loque Plant Synthetic Biology and Engineering Presentation UC Berkeley, CA

May 13 Sacha Escamez Umeå Plant Science Centre Tour Umeå Plant Science Centre

May 14 Taek Soon Lee Microbial production of advanced biofuels Presentation Seoul National University, Chemistry Department Seminar, Seoul, Korea

May 18 Hector Garcia Martin Quantitative metabolic modeling for biofuel synthesis

Presentation Cell factories and sustainability, Hillerod, Denmark

May 18 Blake Simmons, Jenny Mortimer, Seema Singh, Chris Petzold

Exxon Tour Exxon

May 19 Irina Silva, Nathan Hillson LBL Govt Affairs Tour LBL Govt Affairs

May 20 - 22

Blake Simmons Advanced Biofuel Development at JBEI Presentation GLBRC Annual Retreat, South Bend, IN

May 20 Erika Yoshida Ajinomoto Co., Inc. Tour Ajinomoto Co., Inc.

May 20 Steve Singer PNNL Tour PNNL

May 22 Ryan Phelan, Susan Gardner

UC Berkeley Tour UC Berkeley

May 22 Nathan Hillson, Henrik Scheller, Dominique Loque, Aindrila Mukhopadhyay, Chris Petzold

House Energy and Water Development Appropriations Subcommittee

Tour House Energy and Water Development Appropriations Subcommittee



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May 30 - June 2

Ee-Been Goh Substantial Methyl Ketone Production in E. coli using Rational Metabolic Engineering and Random Mutagenesis Approaches

Presentation 115th ASM General Meeting, New Orleans, LA

May 30 - June 2

Blake Simmons Comparative 'Omics for Understanding and Harnessing Lignocellulose Deconstruction in Microbial Communities

Presentation 115th ASM General Meeting, New Orleans, LA



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Onsite Seminars

Date Type Presenter Title Affiliation

May 5 General Seminar Deconstruction Guest

Junko Yaegashi Application of Genome-wide Strategies for the Mining of Secondary Metabolite Biosynthetic Pathways in Filamentous Fungi

Decon Interview Candidate

May 6 JBEI Seminar/Training

Sarah Richardson Cas9 Working Group Fuels Synthesis Division

May 6 Group Seminar Rachel Li Identification of enzymes involved in the biosynthesis of the diterpenoid Taxol

Fuels Synthesis Division

May 11 General Seminar SynBerc Guest

Professor Greg Challis Discovery and Biosynthesis of Novel Polyketide Antibiotics

Department of Chemistry, University of Warwick

May 13 Group Seminar Sarah Rodriguez Biotransformation of methane into n-butanol by a methanotrophic methane monooxygenase


May 13 Group Seminar Edward Baidoo Developing CE-MS methods for metabolite analyses.


May 13 General Seminar SynBerc Guest

Jessica Walter, Ph.D Efficient Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas

Amyris

May 14 General Seminar

Deconstruction Guest

Amanda Kohler Pathogenesis at an Atomic Scale Decon Interview Candidate

May 18 Group Seminar Ee-Been Goh Recent advancements in the production of methyl ketones in E. coli




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May 18 Group Seminar Maren Wehrs Developing a CRISPR/Cas platform for metabolic engineering of Saccharomyces cerevisiae




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Industry Interactions

Date Company / Contact

May 1 Afingen / Ai Oikawa

May 7 California Technology Council / Matt Gardner

May 7 Evolva

May 7 Arzeda / Alexandre Zanghellini

May 7 ZymoChem / Harshal Chaokhawala

May 7 Illium / Aaron Socha

May 12 MMI / Pat Smith

May 14 Monsanto / John Hamer

May 14 Monsanto / Ginni Ursin

May 14 Renault SA / Federic Maire

May 14 Mass Insight / Tom Gilman

May 14 PolyOne / Chris Murphy

May 18 Arbiom / John Evans

May 18 PolyOne / Roger Avakian

May 19 Ajinomoto / Dr. Yoshio Kawahara

May 26 Total / Florence Mingardon



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May 26 Virdia / Manja Sachet (Wilson Sonsini)

May 27 Cummins, Inc. / John Wall

May 28 Total / Vincent Schachter

May 28 Genomatica / Mark Burk

May 28 Dupont / Luis Cascao-Pereira



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Milestones

Feedstocks Division

Area Initiatives Milestone/Deliverables Due % Complete

Plant Cell Wall Biosynthesis and Its Regulation

Novel acetyl and feruloyltransferase activities characterized (CWB) Sept. 2015 72

Genetic characterization of mutant candidates from rice stem saccharification screen (GG)

Sept. 2015 66

Correlation between cell wall modification and environmental interactions investigated (GG)

Sept. 2015 66

Characterization of novel nucleotide sugar transporters (SB) Sept. 2015 70

Characterization of novel GT activities (SB) Sept. 2015 75

Lignin and Hemicellulose Modification for Fuels Production

Evaluation of engineered plants with new lignin traits (including analysis with the Deconstruction and Technology Divisions) (CWE)

Sept. 2015 78

Transgenic plants with dominant repressors of hemicellulose biosynthesis characterized (CWB)

Sept. 2015 65

Nucleotide sugar transporters used for cell wall engineering (CWB) Sept. 2015 71

Optimization of translation control technologies to improve tissue-specific expression (CWE)

Sept. 2015 65

Analysis of stacked traits in Arabidopsis (CWE) Sept. 2015 68

Heterologous screening of transferases to promote in planta design of polysaccharides (SB).

Sept. 2015 60



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Milestones

Feedstocks Division (continued)


Engineering of Modifications in Different Plant Species

Cell wall related genes in switchgrass functionally annotated, integrated into phylogenomics databases, and integrated with Kbase (GG)

Sept. 2015 66

Resequencing of Kitaake mutants (with JGI) and a database set up for hi throughput in silico screening (GG).

Sept. 2015 68

Translation of best technologies developed in Arabidopsis to other plant species (CWE)

Sept. 2015 68



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Milestones

Deconstruction Division


Biomass Pretreatment

Predict and develop task specific ILs for selective lignin or cellulose dissolution and low temperature pretreatment

May 2015 100

Complete characterization of lignin streams from various IL pretreatment process configurations

July 2015 85

Design, synthesis and assessment of lignin and hemicellulose derived IL mixtures for mixed and engineered feedstock pretreatment

Aug. 2015 75

Use multi-scale simulations strategies (from first principle quantum mechanics to force field based) to understand interactions between biomass derived ILs and lignocellulosic biomass

Sept. 2015 75

Enzyme Optimization

Develop an assay for screening lignin degrading enzymes on insoluble lignin and begin populating a lignin degrading enzyme database (LigDB).

Feb. 2015 100

Expand our database of ionic liquid tolerant enzymes, particularly to include enzymes from the GH3, GH5, GH6, GH9, GH10, AA9 and AA10 families.

May 2015 100

Improve the overall sugar yields of our existing thermo and ionic liquid tolerant multi-component enzyme mixture by including enzymes for hydrolysis of both cellulose and hemicellulose and by including LPMOs.

July 2015 60

Demonstrate using directed evolution to engineer enhanced stability -glucosidase in up to 20% [C2mim][OAc].

Sept. 2015 72



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Milestones

Deconstruction Division (continued)


Microbial Communities

Complete characterization of active component of JTherm March 2015 100

Establish complementary thermophilic cellulase cocktail focusing on crystalline substrates

July 2015 95

Identify five bacterial strains capable of metabolizing mixtures of aromatics from pretreatment-derived lignin streams

Sept. 2015 95

Fungal Biotechnology

Design new expression systems based on recent “omics” discoveries in regard to promoters and assess heterologous GH expression

Feb. 2015 100

Initiate ChIP-Seq analysis of histone modifications in A. niger aimed at identifying epigenetic determinants of high productivity

April 2015 100

Optimize cultivation conditions for heterologous enzyme production, including temperature and pH, and initiate ‘omic analysis of optimal vs. baseline conditions

July 2015 83

Validate candidate genes for hyperproduction phenotypes through gene deletion and/or overexpression

Sept. 2015 70



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Milestones



Discovery of Novel Hydrocarbon Biochemistries

In vitro, anaerobic testing of toluene biosynthesis candidates Sept. 2015 67

In vitro, anaerobic testing of ladderane biosynthesis candidates Sept. 2015 67

Optimization of Hydrocarbon Biosynthetic Pathways

Improve methyl ketone production in E. coli with NIMS-aided HT screening and metabolic modeling

Sept. 2015 67

E. coli pathway engineering for isopentenol production both in aerobic and anaerobic conditions

Sept. 2015 75

Genes encoding isoprenoid pathway integrated into E. coli chromosome

Sept. 2015 67

Three enzymes in S. cerevisiae glycolytic pathway replaced and impact on fatty acid-derived fuel tested

Sept. 2015 100

Type 1 fatty acid synthases from several organisms expressed in E. coli and production of fatty acid-based fuels tested

March 2015 100

Develop analytical method for LC-MS of MEV and DXP intermediates April 2015 100

Host Engineering Improve overall xylose utilization capability in S. cerevisiae strains and examine performance in industrially relevant yeast strains.

Sept. 2015 65

E. coli membrane editing: With JGI, generate 10-20 single and double mutants and test improvement in expression of beneficial tolerance pump and corresponding tolerance to toxic fuels.

Sept. 2015 45



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Milestones

Fuels Synthesis Division (continued)


Synthetic Bio Informatics

Codon optimization tool (GeneDesign) integrated into the DIVA platform.

Sept. 2015 67

Clonal sequence validation (Sanger and/or MiSeq) pipeline implemented and integrated with DIVA/ICE

Sept. 2015 67

Quantitative Metabolic Modeling for Host and Pathway Engineering

Usage of EDD, MvT and 2S-13C MFA to improve production of fatty acids in S. cerevisiae.

Sept. 2015 100

Perform flux analysis of S. cerevisiae sip1 strain June 2015 94

Implement interactive metabolic engineering on MvT Sept. 2015 85



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Milestones

Technology Division


High Throughput Screening

Support the optimization of T and IL tolerant cellulose/hemicellulase cocktail using HT-NIMS

Sept. 2015 75

Apply HT NIMS screen for fatty acids based fuel molecules (e.g. FAMK) in collaboration with the Fuels Division.

Sept. 2015 75

Implement software to automate unit operations (e.g., droplet dispensing, routing, merger, and sorting) on an integrated droplet chip

Sept. 2015 66

Demonstrate microfluidic droplet platform for integration of DNA assembly, transformation of cells, and culturing

July 2015 80

Proteomics Develop rapid untargeted proteomics methods for relative quantification of >800 E. coli and S. cerevisiae proteins

Sept. 2015 85

Develop absolute quantification targeted proteomic assays for 150 S. cerevisiae proteins

Sept. 2015 85

Implement membrane proteomics assays to enable E. coli membrane editing with Fuels Synthesis Division

Sept. 2015 80



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Milestones

Technology Division (continued)


Physical Characterization

SEM and TEM analysis of genetically altered feedstock biomass (including lignin and protein mapping) with Feedstocks and Deconstruction Divisions

Sept. 2015 75

Electron tomographic analysis of plant cell walls of engineered feedstocks, and CAD model generation for mechanical properties simulation.

Sept. 2015 75

Structural Biology Test expression of multiple GT clones in insect cell platform to produce quantities of protein for crystallization trials

Sept. 2015 75

Characterize lignin degrading enzymes in collaboration with GLBRC Sept. 2015 100

Structural characterization of Fatty Acid Synthase and other large complexes by single particle cryo electron microscopy

Sept. 2015 50

Synthetic Bio Informatics

EDD further developed to support enzyme characterization data and workflows, with Deconstruction Division

Sept. 2015 67

EDD further developed to support genetic expression tool data and workflows, with Fuels Synthesis

Sept. 2015 67

BioCad tools enabled to query ICE and EDD for enzymes and expression tools satisfying specified performance characteristics

Sept. 2015 67



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Milestones

Operations Management Division


Intellectual Property Management

Generate >10 inventions Sept. 2015 95

Educate researchers about the tech transfer process Sept. 2015 79

Licensing Increase number of patent applications or inventions licensed or optioned and/or licensing agreements executed relative to the average of years 1 - 7.

Sept. 2015 65

Start-up Company Creation

Identify any IP or portfolios of IP that could lead to a start-up company and create an internal commercialization team to develop an initial go-to-market plan around that IP.

Sept. 2015 73

Maintain regular JBEI on-site office hours for the Berkeley Lab entrepreneur adviser.

Sept. 2015 70

Business Development Outreach

Promote each JBEI invention to at least 700 contacts in the biofuels industry.

Sept. 2015 79

Conduct meetings with at least fifteen companies each year, at least one-third of which are new to JBEI

Sept. 2015 68

Industry Advisory Committee

Hold an annual meeting of the IAC to inform JBEI research of industry concerns and technology bottlenecks.

Sept. 2015 90

Industry Partnership Program

Strengthen relationships with current partners and pursue collaborations with new industry partners in areas of complementary research.

Sept. 2015 79



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Milestones

Operations Management Division (continued)


Annual Scientific Advisory Meeting

Hold the annual meeting and report to DOE on the findings of the committee

Sept. 2015 67

Annual JBEI Conference

Annual JBEI Retreat Sept. 2015 89

Human Resources Staffing for year 8 complete Sept. 2015 67

Documents

DOE Bioenergy Research Center (BRC) - jbei.org€¦ · DOE Bioenergy Research Center (BRC) - Joint BioEnergy Institute ... JBEI contacts Jay Keasling, Chief Executive Officer, [email protected],