Biosystems Design to Enable Next-Generation Biofuels and Bioproducts Summary of projects awarded in 2017 under Funding Opportunity Announcement DE-FOA-0001650

Genomic Science Program genomicscience.energy.gov

Overview -omics datasets to solve grand challenges in biological engineering. he Genomic Science program within the U.S. Department of Energy’s In fiscal year 2017, BER solicited integra- T (DOE) Office of Biological and tive, multidisciplinary applications to con- Environmental Research (BER) supports duct fundamental genomics and systems basic interdisciplinary research aimed at biology research and technology develop- achieving a predictive, systems-level under- ment to design novel biosystems within the standing of plants, microbes, and microbial following research areas: communities to advance BER energy and • Integrating large-scale systems biol- environmental missions. Building on an ogy data to model, design, and ever-growing body of genomic sequence engineer microbial systems for the information, the program integrates large- production of biofuels and bioprod- scale systems biology (transcriptomics, pro- ucts. Projects should focus on the devel- teomics, metabolomics, and other “-omics” opment of innovative modeling and approaches) with computational modeling high-throughput genome-wide design, to comprehensively understand the foun- engineering, and testing of new strains dational principles that rule biological using in vivo or cell-free approaches for systems. Achieving this knowledge enables new or emerging eukaryotic and pro- the design and engineering of DOE- karyotic microbial systems that produce relevant plants and microbes for defined high levels of biofuels and bioproducts purposes and reveals insights into their from lignocellulosic biomass or by responses to environmental inputs. Within photosynthesis. the Genomic Science program, Biosys- tems Design research has contributed to • Designing plant systems for bio- the advancement of bioengineering, often energy. Projects should use multi- referred to as synthetic biology. disciplinary approaches to engineer New genome-scale engineering and syn- potential bioenergy crops for sustain- thetic biology tools are opening new ave- able production of biofuels and bio- nues for systems biology research. These products from lignocellulose or oils, tools not only enable the design of tailored while growing in marginal environ- plants and microbes by genome editing or ments. Project goals include increased introduction of megabase-size synthetic abiotic stress tolerance, higher water constructs with orthogonal pathways, they and nutrient use efficiency, improved also advance fundamental understanding photosynthetic capacity, and high pro- of biological function and inspire novel ductivity, as well as the development of approaches for its manipulation on a large technologies for the introduction and scale. Critical components of these tools are expression of large, stable, multigene computational modeling and computer- DNA constructs, aided by computa- aided design methods that integrate large tional modeling and design.

Office of Biological and Environmental Research 2017 Awards Microbial Research Awards metabolites and other stresses, and produce valuable mol- ecules. Building on technologies and knowledge developed Systems engineering of Rhodococcus opacus with prior DOE funding from the Biosystems Design pro- to enable production of drop-in fuels from gram, the project will further develop the necessary techni- lignocellulose cal and computational infrastructure to achieve its goals, initially working with Escherichia coli and Saccharomyces Principal Investigator: •• Gautam Dantas (Washington cerevisiae and later transferring the technologies to other University in St. Louis) non-model bacteria and yeast species. •• Co-Investigators: Tae Seok Moon, Marcus Foston, Yinjie Tang, Fuzhong Zhang (Washington University in St. Louis); Hector Garcia Martin (Lawrence Berkeley Design, synthesis, and validation: National Laboratory) Genome-scale optimization of energy flux Building on prior funding through compartmentalized metabolic from BER for systems biology networks in a model photosynthetic work on Rhodococcus opacus, eukaryotic microbe this project will apply innova- •• Principal Investigator: Andrew Allen (J. Craig Venter tive combinations of -omics Institute) and mass spectrometry data •• Co-Investigators: Chris Dupont, Phil Weyman (J. Craig analysis, flux analysis, and Venter Institute); Graham Peers, Wen Zhou (Colorado machine learning to develop State University); Jamey Young (Vanderbilt University); improved genome-scale mod- Karsten Zengler, Bernhard Palsson (University of Califor- Fluorescent micrograph els that will guide multiplex nia, San Diego) of Rhodococcus opacus cells designed to convert genome engineering of R. The goal of this research is to integrate genome‐ phenolic compounds into opacus strains with branched- scale modeling efforts of the diatom Phaeodactylum lipids. Cells were stained chain fatty acid esters tricornutum to with Nile Red to observe (BCFAE) biosynthetic capac- inform experimen- lipid bodies as bright ity and improved tolerance to tal research for red spots. lignin degradation products. the production of The project will take advan- high-value and fuel‐ tage of this organism’s capability to use both polysaccharides related metabolites. and lignin as carbon sources as well as its natural tolerance Leveraging prior work on this organ- to phenolic compounds such as those produced by lignin ism under DOE breakdown. Biosystems Design funding, this project Design and engineering of synthetic will apply genome- control architectures scale engineering to optimize carbon, •• Principal Investigator: Ryan Gill (University of Colo- energy, photosyn- rado Boulder) thesis, and metabo- •• Co-Investigators: Christopher Voigt (Massachusetts lite flux to shift the Institute of Technology); Adam Arkin (Lawrence Berkeley native P. tricornutum Physical association between E. coli and diatoms during conju- National Laboratory); Carrie Eckert (National Renewable metabolism from gative delivery of designer dia- Energy Laboratory) carbohydrate stor- age into lipid and/ tom episomes by bacteria. The strength of this project is that it will advance biological or branched chain engineering to a new level of throughput and complexity. amino acid (BCAA) Guided by computer-aided design platforms, this research accumulation. The team will focus on compartmen- will develop new finely tuned microbial strains containing talizing relevant biosynthetic pathways on artificial orthogonal regulatory networks. The redesigned strains will chromosomes to obtain modified diatom strains with be tailored to grow on a variety of feedstocks, tolerate toxic high productivity of triacylglycerols or BCAAs.

2 U.S. Department of Energy Office of Science • Office of Biological and Environmental Research Genome-scale design and engineering of efforts to sequence the genomes of multiple Clostridium non-model yeast organisms for production strains and use a computer-aided design tool called Bio- of biofuels and bioproducts chemical Network Integrated Computational Explorer (BNICE) developed by team members to identify relevant •• Principal Investigator: Huimin Zhao (University of metabolic genes and pathways that will be subsequently Illinois at Urbana-Champaign) tested in cell-free systems. New CRISPR-based genome- •• Co-Investigators: Christopher Rao (University of Illinois editing techniques will be developed for Clostridium, and at Urbana-Champaign); Costas Maranas (Pennsylvania State metabolic ensemble modeling will be used to predict in University); Joshua Rabinowitz, Martin Wuhr (Princeton vivo pathway performance from cell-free assays. University); Yasuo Yoshikuni (Lawrence Berkeley National Laboratory) This project will focus on Rhodosporidium toruloides and Systems analysis and engineering of Issatchenkia orientalis, two industrial yeast species, to biofuel production in Chromochloris develop the tools needed to turn these organisms into zofingiensis, an emerging model green alga model systems for biological design. R. toruloides will be •• Principal Investigator: Krishna Niyogi (University of engineered to produce fatty alcohols from lignocellulose- California, Berkeley) derived sugars, and the target products for I. orientalis Co-Investigators: will be organic acids such as muconic acid and methyl •• Crysten Blaby (Brookhaven National Laboratory); Nanette Boyle (Colorado School of Mines); methacrylate. The project will leverage genome-scale Mary Lipton (Pacific Northwest National Laboratory); engineering tools previously developed for Saccharomyces Sabeeha Merchant (University of California, Los Angeles); cerevisiae by the PI’s laboratory that apply CRISPR-Cas Trent Northen (Lawrence Berkeley National Laboratory) methods for multi-gene disruptions (homology inte- grated CRISPR-Cas or HI-CRISPR) and CRISPR-based The model system on which this project will focus is the gene activation, interference, and deletion (CRISPR- oleaginous green alga Chromochloris zofingiensisbecause AID). A highly automated robotic pipeline already it is fast-growing, can be cultured at high densities, and operational at the University of Illinois will be used for accumulates triac- high-throughput genetic manipulation and engineering ylglcyerols (TAG) of the two yeast species. at the highest levels observed Establishing a Clostridia foundry for among microalgae. biosystems design by integrating C. zofingiensis met- abolic regulation computational modeling, systems- is unique because level analyses, and cell-free engineering in the presence of technologies glucose, photosyn- •• Principal Investigator: Michael Jewett (Northwestern thesis is switched University) Cryo-soft X-ray tomography of off by degrading •• Co-Investigators: Keith Tyo, Linda Broadbelt (Northwest- a reconstructed Chromochloris the thylakoid ern University); Michael Köpke, Sean Simpson (LanzaTech); zofingiensis cell with segmented membranes and Steven Brown, Timothy Tschaplinski, Robert Hettich, nucleus (purple), chloroplast photosynthetic Richard Gionnone (Oak Ridge National Laboratory) (green), mitochondria (red), lipids machinery, while This project will develop an integrated set of technolo- (yellow), and starch granules within lipid production the chloroplast (blue). gies for high-throughput metabolic pathway design and is substantially engineering of Clostridia, called the Clostridia Foundry increased; the for Biosystems Design (cBioFAB), to produce fuels and process is reversed when the glucose supply is exhausted. chemicals from lignocellulose-derived syngas. Using The project will leverage a high-quality genome assembly multi-omics, computational modeling, genome engineer- of the C. zofingiensis genome recently generated by the ing, and cell-free and in vivo technologies, the Foundry research team and will enhance it with multi-omics and will focus on the production of acetone, butanol, butane- physiological analyses to build a model to guide meta- diol, and mono-ethylene glycol as proof of concept. The bolic engineering of this alga for the production of bio- project will leverage DOE Joint Genome Institute (JGI) fuel precursors.

U.S. Department of Energy Office of Science • Office of Biological and Environmental Research 3 Plant Research Awards SyPro poplar: Improving poplar biomass production under abiotic stress conditions: Phylogenomic discovery and engineering an integrated omics, bioinformatics, of nitrogen fixation in the bioenergy synthetic biology, and genetic engineering woody crop poplar approach •• Principal Investigator: Matias Kirst (University of •• Principal Investigator: Eduardo Blumwald (University Florida) of California, Davis) •• Co-Investigators: Sushmita Roy, Jean-Michel Ané •• Co-Investigators: Amir Ahkami (Pacific Northwest (University of Wisconsin, Madison); Robert Guralnick, National Laboratory); C. Neal Stewart (University of Pamela Soltis, Douglas Soltis (University of Florida) Tennessee); Stephen DiFazio (West Virginia University) To identify the plant genes involved in the develop- To engineer bioenergy crops that can grow in dry, marginal ment of symbiotic nodules containing nitrogen-fixing lands, this project will introduce into poplar trees tolerance microbes (diazotrophs), this project will analyze the to different environmental stresses such as water deficit, evolution of those symbioses. Prior studies by members high soil salinity, and heat. As plants are often exposed of the research team showed that microbe-mediated to combinations of these different types of abiotic stress, nitrogen fixation was originated and lost multiple times poplar lines will be engineered to express multiple stress- during the evolution of the nodulating plant clade. related genes, each one controlled by a synthetic promoter Therefore, investigators intend to identify the genetic that is only induced under specific stress conditions and machinery used by nodulating plants and introduce it within the tissues where its function is needed, improving into non-nodulating bioenergy crops such as poplar, biomass production under unfavorable conditions while thereby reducing this crop’s need of nitrogen fertiliza- avoiding negative effects of unwanted transgene expression. tion. To achieve this ambitious goal, the team will The project will focus on a poplar hybrid clone for which conduct extensive phylogenetic analysis of over 20,000 a genome sequence is available and will design tissue- and species using available genomic sequence data as well stress-specific synthetic promoters using plant promoter as samples from multiple plant species, including pre- databases and computational tools to identify cis-regulatory served museum specimens from around the world. elements. Selected engineered poplar lines will be tested for their ability to establish symbiotic relationships with diazo- trophs, as well as for their biomass productivity and Using systems approaches to improve composition. photosynthesis and water use efficiency in sorghum •• Principal Investigator: Thomas Brutnell (Donald Danforth Plant Science Center) •• Co-Investigators: Ivan Baxter (U.S. Department of Agriculture and Donald Danforth Plant Science Center); Todd Mockler, Hector Quemada (Donald Danforth Plant Science Center); Asaph Cousins (Washington State University); Jose Dinneny, Sue Rhee (Carnegie Institu- tion for Science); Albert Kausch (University of Rhode Island); Andrew Leakey (University of Illinois, Urbana- Champaign); Daniel Voytas (University of Minnesota) Building on prior work on the model grass Setaria, funded by DOE as part of the Biosystems Design pro- gram, this project aims at increasing water use and pho- tosynthetic efficiencies in the bioenergy crop sorghum. Transgenic roots of the model legume Medicago truncatu- Leveraging the resources previously developed by the la expressing DsRed, with nodules formed by its symbiont (Sinorhizobium meliloti). research team for Setaria, investigators will accelerate the generation of engineered high-biomass sorghum lines

4 U.S. Department of Energy Office of Science • Office of Biological and Environmental Research that grow in marginal lands using innovative genome- relative to oilseed crops. Computational modeling will editing tools, genome-scale modeling, -omics approaches, be used to design improved photosynthetic capacity genome-wide association studies, and high-throughput that they expect to reach 50%, along with increased phenotyping in both laboratory and field settings. The nitrogen and water use efficiency, to account for the work will exploit the efficient transformation and screen- required redirection of carbon flow from lignocellu- ing pipelines available for Setaria to facilitate gene dis- losic biomass to oil in the redesigned plant lines. covery and pathway design that can be transferred to sorghum. Because biomass productivity strongly depends on photosynthesis and water availability, the engineering Biological design of Lemnaceae aquatic work will focus on three main topics: enhanced carbon plants for biodiesel production assimilation, increased root water uptake, and reduced •• Principal Investigator: Robert Martienssen (Cold Spring water loss through stomata. Harbor Laboratory) •• Co-Investigators: James Birchler (University of Missouri); Building on success in systems design of Eric Lam (Rutgers University); Jorg Schwender, John high-yielding, low-input energy canes for Shanklin (Brookhaven National Laboratory) marginal lands The goal of this project is to divert carbon flow from •• Principal Investigator: Stephen Long (University of starch accumulation to oil production in resting fronds Illinois, Urbana-Champaign) of Lemnaceae (duckweed) species, thereby redesigning •• Co-Investigators: Don Ort, Vijay Singh, Li-Qing Chen existing (University of Illinois, Urbana-Champaign); John Shanklin commercial (Brookhaven National Laboratory); Fredy Altpeter (Univer- sity of Florida); Brian Baldwin (Mississippi State University) strains towards This project will pursue an innovative approach to biofuel engineer oil accumulation in vegetative tissues of produc- bioenergy crops. Leveraging previous efforts toward tion. producing Lemnaceae’s oil in sugar cane leaf rapid tissue, the growth, researchers high pro- will attempt portion Duckweed fronds (photosynthetic part of to redirect of photo- the plant body) and flowers (white protru- and increase synthetic sions). TAG accu- tissue mulation in due to its the stems of lack of lignified supportive tissue, and its ability to grow energy cane in marginal wastewater environments are among the Section of a Miscanthus stem under a con- and Mis- advantages of these small aquatic plants. An available focal microscope showing oil accumula- canthus. As genome sequence and other -omics resources will be these species tion within parenchymatic cells. leveraged, along with other products from prior DOE are close funding (ARPA‐E) to significantly increase oil content relatives of Lemnaceae sugar cane, CRISPR/Cas9-based genome-editing tech- in . Metabolic pathways will be re‐engi- nologies previously developed in sugar cane will likely neered to maximize TAG yields while preserving rapid be transferable to these new plant systems along with biomass accumulation, and tools for genetic manipula- novel genome-engineering methods. The ultimate goal tion, metabolic flux models, and a catalog of key tran- of the project is to reach a TAG accumulation of up to scription factors and promoters will be developed to 20% of dry stem weight, which represents a produc- elevate this emerging plant model system to a potential tivity increase of more than one order of magnitude bioenergy crop.

U.S. Department of Energy Office of Science • Office of Biological and Environmental Research 5 A systems approach to increasing and genomics tools and resources, and suitability of its carbon flux to seed oil for biofuels oil for transportation as liquid fuel blends are some of and bioproducts production in Camelina’s advantages over other oilseed crops. How- Camelina sativa ever, oil yield from Camelina seeds is relatively low. To improve oil production, an integrative approach includ- •• Principal Investigator: Danny Schnell (Michigan State ing metabolic flux modeling, proteomics, regulatory University) network analysis, and multi‐gene stacking and genome- •• Co-Investigators: Erich Grotewold, Yair Shachar-Hill editing techniques will be employed to optimize pho- (Michigan State University); Heike Sederoff (North Caro- tosynthetic carbon fixation in leaves, its transport to lina State University); Kristi Snell (Yield10 Bioscience) seeds, and the allocation of fixed carbon to TAG. With This project will establish Camelina sativa as a com- this approach, the team aims at increasing oil produc- mercially viable, dedicated biofuel and bioproduct tion to 300% per hectare, thus greatly enhancing the feedstock. Its low agronomic input requirements, natu- feasibility of developing Camelina as a cost‐competitive ral tolerance to different stresses, available molecular bioenergy crop.

Contact and Websites

BER Program Manager for Biosystems Design Pablo Rabinowicz 301.903.0379 [email protected]

Websites Biosystems Design report •• genomicscience.energy.gov/biosystemsdesign/

Biosystems Design Report BER Genomic Science program •• genomicscience.energy.gov Further information on BER objectives in this area of research can be found in Biosystems Design: Report DOE Office of Biological and Environmental Research from the July 2011 Workshop. The DOE report, along •• science.energy.gov/ber/ with this list of funded projects, is available: genomicscience.energy.gov/biosystemsdesign/. DOE Office of Science •• science.energy.gov

Image Credits Page 1: (1) Saccharomyces cerevisiae image adapted from Das Murtey, M. and P. Ramasamy. 2016. “Sample Preparations for Scanning Electron Micros- copy – Life Sciences.” In Modern Electron Microscopy in Physical and Life Science. Eds. M. Janecek and R. Kral. DOI: 10.5772/61720. Reprinted under a Creative Commons Attribution License (CC BY 3.0). (2) Poplar leaf image courtesy U.S. Department of Energy Joint Genome Institute. Page 2: (1) Image courtesy William Henson and Aki Yoneda, Washington University in St. Louis. (2) Reprinted under a Creative Commons Attribution License (CC BY 4.0) from Karas, B.J. et al. 2015. “Designer Diatom Episomes Delivered by Bacterial Conjugation,” Nature Communications 6, 6925. Page 3: Image courtesy National Center for X-ray Tomography. Reprinted from Roth, M. S. et al. 2017. “Chromosome-Level Genome Assembly and Tran- scriptome of the Green Alga Chromochloris zofingiensis Illuminates Astaxanthin Production,” Proceedings of the National Academy of Sciences of the USA 114(21), E4296–4305. Page 4: Image courtesy Jean-Michel Ané University of Wisconsin–Madison. Page 5: (1) Image courtesy Ashley Spence, University of Illinois. (2) Image courtesy Evan Ernst, Martienssen Lab, Cold Spring Harbor Laboratory.

November 2017 6 U.S. Department of Energy Office of Science • Office of Biological and Environmental Research