Fundamental, Systems-Level Understanding of Microbes Relevant to Advanced Biofuels and Bioproducts Production
Biochemical pathways encoded in microbial genomes [Jonathan Remis, Joint Bioenergy Institute, Lawrence Berkeley National Laboratory]
The U.S. Department of Energy’s (DOE) Genomic Science program, managed within the Office of Biological and Environmental Research (BER), supports basic research aimed at identifying the foundational principles that drive biological systems. These principles govern the translation of the genetic code into integrated networks of proteins, enzymes, regulatory elements, and metabolite pools that underlie the functional processes of organisms including microbes and multispecies communities relevant to DOE missions in energy and the environment. To address the DOE mission in sustainable bioenergy development, BER’s Genomic Science program applies the “omics”-driven tools of modern systems biology to the challenges associated with microbial production of bioproducts and advanced biofuels (i.e., biologically synthesized compounds with the potential to serve as energy-dense transportation fuels such as gasoline, diesel, and aviation fuel).
Developing an increased understanding of how biological systems function and translating that knowledge to enhance the production of microbial and plant capabilities form the basis of DOE’s mission in sustainable bioenergy. Harnessing the biosynthetic processing power of the microbial world for producing advanced biofuels and bioproducts will require developing both an expanded set of platform organisms that have appropriate metabolic capabilities and stress tolerance characteristics, as well as a suite of modification tools. To foster this development, the Genomic Science program supports research aimed at understanding the principles that govern the functional properties of bioenergy-relevant organisms at the genomic scale. This endeavor is highly interdisciplinary, spanning multiple fields in biology, systems biology, chemical and metabolic engineering, and computational biology.
Recent progress in understanding biological systems and the ability to manipulate them is largely due to tremendous technological advances in the development of multiomics tools, high-throughput phenotypic screening approaches, and computational modeling methods used to analyze, modify, and select specific functional properties of biological systems. Continued research to understand the physiology and metabolism of unique microbes and advance them toward experimentally tractable organisms or systems presents an opportunity to potentially produce sustainable biofuels and bioproducts derived from lignocellulosic plant biomass or from photosynthetic capture of carbon dioxide (CO2).
In 2018, BER funded several new projects specifically targeting the production of advanced biofuels compatible with existing engines and fuel-distribution infrastructure and useful in producing bioproducts. The biological syntheses of advanced biofuels and bioproducts require significant advances in the basic understanding of microbial physiology and metabolism. Also needed are greater insights into the conversion of photosynthetically derived carbon compounds and how products can be shunted efficiently from central metabolism into complex products while rebalancing organismal carbon allocations and reduction-oxidation (redox) potential.
BER solicited applications for fundamental systems biology-driven basic research to enable production of advanced biofuels and bioproducts in two areas:
Project Goal and Summary: Use systems biology-guided approaches to develop a nonmodel, microbial metabolic engineering platform based on the most thermophilic lignocellulose-degrading organism known, Caldicellulosiruptor bescii, which grows optimally at 78°C. The latest metabolic reconstruction and modeling approaches will be applied to optimize biomass to product conversion using switchgrass as the model plant and acetone and 3-hydroxypropionate as products. Bioprocessing above 70°C can have important advantages over near-ambient operations. Highly genetically modified microorganisms usually have a fitness disadvantage and can be overtaken easily in culture when contaminating microbes are present. The high growth temperature of extreme thermophiles precludes growth or survival of virtually any contaminating organism or phage, thus reducing operating costs associated with reactor sterilization and maintenance of a sterile facility. In addition, at industrial scales, heat production from microbial metabolic activity vastly outweighs heat loss through bioreactor walls that would require cooling. Extreme thermophiles have other advantages—nonrefrigerated cooling water can be used if needed and heating requirements can be met with low-grade steam typically in excess capacity on plant sites. The overarching goal is to demonstrate that a nonmodel microorganism, specifically an extreme thermophile, can be a strategic metabolic engineering platform for industrial biotechnology.
Project Goal and Summary: Carry out a comprehensive systems biology study of branched-chain alcohol (BCA) production and tolerance in yeast. BCAs, including isobutanol, isopentanol, and 2-methyl-1-butanol, are some of the most promising advanced biofuels in development. These alcohols have better fuel properties than bioethanol. They have higher energy density, their refinement is less expensive and energy intensive, and they have much better compatibility with the nation’s fuel use and distribution infrastructure. The project will leverage a recently developed, genetically encoded biosensor of BCA production to screen various yeast genomic libraries to measure the effects of different genetic perturbations (i.e., gene deletion, overexpression, or mutation) on BCA production or tolerance and to screen those collections using different substrates, nutritional requirements, or BCA-induced stress. In addition, by combining the biosensor with optogenetic regulation of BCA production, the team will establish a closed-loop control system for measuring transcriptomic changes under well- controlled conditions. These new insights will be used to develop improved strains for producing BCAs and to help make this very promising class of biofuels more economically competitive.
Project Goal and Summary: Use genetic manipulation techniques to enhance the exchange of metabolites between autotrophs and heterotrophs, creating superior synthetic lichens able to generate useful products of interest to the energy and chemical industries. Lichens are communities of microbes that collect sunlight and carbon dioxide (CO2) and apply them to power the group’s activities, allowing autotrophic members to optimize photosynthesis and metabolite generation while their heterotrophic fungal partners produce biochemical compounds for the community. Additional members may provide key functions such as nitrogen fixation. While lichens can thrive in the harshest environments on Earth, they also represent a novel biotechnology platform that can transform CO2 and sunlight into valuable energy-related biochemicals. Unfortunately, natural lichens have exceedingly slow growth rates, making them impractical for most industrial applications. Key metabolite excretion bottlenecks identified in cyanobacteria will be engineered to share particular metabolic intermediates with their heterotrophic partners for channeling into natural or engineered metabolic pathways, thus generating energy-related precursors of biochemicals or biofuels with high commercial value.
Project Goal and Summary: Develop and use technology for directly measuring synthesis of biofuels in living cells by employing a high-throughput platform for chemical imaging of biofuel production to improve Escherichia coli fatty acid production. Recent advances in the fields of synthetic biology and metabolic engineering have resulted in an unprecedented ability to engineer microbial genomes and to design and build gene circuits for improving biofuel production. Stimulated Raman scattering (SRS) microscopy will be introduced as a new technology for directly measuring chemical signatures in in vivo samples for engineering and optimizing biofuel production strains. This technology can work on a broad range of cell types (e.g., yeast, algae, and other bacteria besides E. coli) and can detect in vivo levels of other biofuels and products (e.g., diesel and jet fuels). SRS imaging is expected to be especially valuable for assessing chemical signatures in strains where tools for genetic manipulation are limited or nonexistent.
Project Goal and Summary: Elucidate fundamental design principles for the division of labor (DOL) in microbial ecosystems in the context of a Saccharomyces cerevisiae and Lactococcus lactis consortium that produces 2,3-butanediol, 2-butanol, and lactic acid. Microbial metabolic engineering is an attractive strategy for clean and sustainable production of biofuels and chemicals. Over the decades, this canonical paradigm, which involves pathway construction in single strains, has led to many breakthroughs; however, this paradigm has several key limitations including inefficient and slow substrate conversion, heavy burdens in energetics and reduction-oxidation (redox) balance, and unexpected accumulation of byproducts. Synthetic microbial consortia have recently emerged as a promising solution to address these challenges by expanding the programmability and enhancing the robustness of desired functionality. The hypothesis is that the structure of the cellular interaction network in this consortium is essential to ecosystem robustness, promising to deliver a quantitative and systematic understanding of DOL in microbial ecosystems. Thus, it will advance the fundamental knowledge of microbial ecology concerning community structure and dynamics. Achieving project goals also will provide valuable insights into the design and construction of artificial microbial consortia for the synthesis of bioproducts from cellulosic biomass.
Project Goal and Summary: Develop Methylobacterium extorquens as a catalyst to convert methoxylated aromatics from lignin hydrolysate into a model bioproduct, 1-butanol, and develop a novel approach that combines the advantages of gene editing, deep-sequencing, and analysis of phenotypic heterogeneity for both growth and production. Lignin-derived compounds from plant biomass are among those most recalcitrant for microbial conversion. Hydrolysates contain a wide variety of aromatic molecules, and a particular issue with these molecules is that many of them are methoxylated; these methoxy groups are released as formaldehyde during degradation, possibly overloading the detoxification ability of standard heterotrophs. Methylotrophic bacteria, on the other hand, not only rapidly generate internal formaldehyde from oxidation of single-carbon compounds, like methanol, but also can oxidize it fast enough to prevent toxicity. In an earlier DOE project, the team also discovered that some Methylobacterium strains grow exceptionally well on aromatics and do not release formaldehyde into the medium from the methoxy groups present, unlike classic systems for aromatic degradation (e.g., Pseudomonas putida). The project has since demonstrated that the pathways for methoxylated aromatic use can be introduced into the emerging model organism, M. extorquens, and enable it to grow on aromatics. These conceptual advances could broadly revolutionize work in DOE-relevant biosystems design.
Project Goal and Summary: Use an integrated systems biology approach to develop the filamentous cyanobacterium Anabaena sp. PCC 33047 as a model fast-growing, photosynthetic, diazotrophic production platform. Cyanobacteria are photosynthetic prokaryotes with significant potential as cell factories for sustainable production of biofuels and chemicals by directly using energy from sunlight and carbon dioxide (CO2). One key issue with the current cyanobacterial production strains concerns the growth rates of these microbes. Compared to other oxygenic photosynthetic organisms such as plants and eukaryotic algae, many cyanobacterial strains have superior growth rates. However, they grow significantly slower than heterotrophic microbes such as Eshcherichia coli and yeast, which are commonly used in biofuels research. The team’s recent discovery of the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 demonstrates that there are strains available whose production potential far exceeds that of current model systems. Notably, most cyanobacterial production systems require the input of fixed nitrogen, which has been reported as one of the highest operational costs for biofuel production. This taxing requirement can be largely eliminated through the use of (N2)-fixing cyanobacteria. The team has identified Anabaena 33047, which has a remarkable 3.8-hour doubling time under nitrogen N2-fixing conditions. Because nitrogen demand is a major cost for photosynthetic bioproduction, the use of this fast-growing diazotrophic strain should significantly improve the cost outlook of target bioproducts. The team will pursue a systems approach to develop Anabaena 33047 as a versatile photosynthetic CO2-fixing and N2-fixing production platform for use by the bioenergy research community during the coming era.
Project Goal and Summary: Advance the systems biology understanding and predictive modeling of synthetic and syntrophic Clostridium microbial consortia, focusing on elucidation of metabolic networks and environmental signals in the consortia. Microbial communities are ubiquitous in nature and have a wide range of applications, including production of biofuels and chemicals. It is now well appreciated that the capabilities of multimicroorganism systems cannot be predicted by the sum of their parts. Rather, synergistic interactions at different levels often result in better overall performance of these systems. The emerging field of co-culture synthetic biology promises the assembly of different metabolic capabilities into functional systems, where the diversity of metabolic pathways and the ability of microorganisms to exchange metabolites and larger molecules dramatically expand the possible metabolic space. Clostridium organisms are uniquely capable of using a large variety of biomass-derived carbohydrates, and some of them can also fix carbon dioxide (CO2) autotrophically, thus enabling maximal use of substrate carbon. These organisms possess diverse biosynthetic capabilities for producing a broad spectrum of metabolites, which, together with their derivatives, could serve as commodity chemicals, biofuels, and biofuel precursors. Significantly, syntrophic Clostridia consortia can fix extensive CO2 amounts, thus achieving product yields that cannot be achieved by monocultures. The ultimate goal then is to use the knowledge developed from these systems as a basis for future developments of syntrophic systems to produce a broad spectrum of metabolites via modular syntrophic co-cultures, involving engineered and nonengineered microorganisms from various genera in addition to the Clostridium organisms.
Project Goal and Summary: Develop the commercially scalable emerging model organism, Clostridium autoethanogenum, which converts a single-carbon (C1) feedstock (e.g., carbon dioxide and carbon monoxide from waste gas emissions) into a short-chain fatty acid, 3-hydroxyproprionic acid. This 3-hydroxypropionic acid is an ideal biorenewable precursor to industrially important polymers such as acrylates. The team will apply several systems and synthetic biology technologies, coupling together algorithmic design approaches, highly multiplexed genome-scale engineering techniques, and omics measurements, to exert complete control over the metabolism of C. autoethanogenum. First, the team will employ an integrated computational, experimental approach to engineer optimized biosynthesis pathways for 3-hydroxypropionic acid in C. autoethanogenum. Second, to redirect metabolic flows toward 3- hydroxypropionic acid production, the team will develop and demonstrate a very highly multiplexed version of CRISPR (i.e., clusters of regularly interspaced short palindromic repeats) that uses highly nonrepetitive genetic parts to up-regulate or down-regulate up to 20 targeted genes simultaneously. Third, the team will perform technoeconomic assessments of C1 bioconversion to 3-hydroxypropionic acid and couple those assessments to algorithm-designed genetic modifications, determining genotype-phenotype-cost relationships across several metrics. This project will result in a commercially scalable emerging model organism capable of producing 3-hydroxypropionic acid at economically competitive, high productivities from low-cost C1 feedstock.
Project Goal and Summary: Identify and characterize brown rot-specific gene regulation patterns to address key knowledge gaps, enabling in vivo manipulations such as CRISPR/Cas9 and metabolomics to map metabolite expression feedback over time to produce an integrated regulatory model for brown rot fungi. However, understanding of fungal brown rot metabolism is limited. Fungi dominate the biological decomposition of wood and other lignocellulosic plant tissues in nature through a range of pathways for unlocking the sugars embedded in lignin, offering a proven model for the sustainable production of energy from biomass. Modern approaches to bioenergy production aim to depolymerize polysaccharides to release fermentable sugars (saccharification), saving lignin as a coproduct that is a good fit for the carbohydrate-selective pathways of brown rot fungi. This project will enable omics-driven tools for organisms highly relevant to bioenergy, with broader scientific impacts in the fields of ecology, evolution, and biogeochemistry.
Project Goal and Summary: Maximize the potential of microorganisms to convert lignocellulose-derived compounds and carbon dioxide (CO2) to important synthetic precursor compounds such as ethylene and propylene, the most widely employed organic compounds in industry. With these compounds being used for the synthesis of several multibillion-dollar products, there is an increasing demand for bioproducts and biofuels from plentiful starting materials such as lignocellulose and CO2 feedstocks. In this project, a combination of systems biology and bioinformatics approaches, along with a unique toolbox of analytical, omics, molecular, and biochemical approaches, will be applied to meet project goals. Current chemical processes for precursor synthesis require huge amounts of energy derived from fossil fuels, but recently discovered, efficient anaerobic ethylene synthetic processes offer the potential to significantly impact biological ethylene (and propylene) formation, tenable with the plentiful starting materials of lignocellulose and/or CO2 feedstocks. Overall, this project aims to develop an industrially compatible process to synthesize ethylene in high yields using microbial systems.
Project Goal and Summary: Harness the potential of robust undomesticated Yarrowia lipolytica isolates to produce designer bioesters from undetoxified biomass hydrolysates. These isolates will be derived from genetic and phenotypic screening approaches using a rigorous microbe selection platform. Genomic and molecular characterization will be leveraged to elucidate and characterize the underlying mechanisms of how these new strains yield desirable bioesters and other bioproducts. Specifically, the team will detail how these Y. lipolytica (1) tolerate and effectively assimilate inhibitory biomass hydrolysates for superior lipid accumulation under hypoxic compared with oxygen-sufficient conditions; (2) tolerate organic solvents that are required to produce biofuels and bioproducts in a twophase fermentation system; and (3) endogenously degrade lipids to produce targeted esters with potential use as fuels, solvents, flavors, and fragrances. Project results will provide the needed tools to allow in situ production and integrated recovery of custom esters, as well as the insight necessary for engineering Yarrowia strains to produce a wide variety of biofuels and bioproducts from lignocellulosic biomass.
Project Goal and Summary: Deploy compartmentalization as a strategy to overcome a critical roadblock in biosynthesis— the requirement for reduction-oxidation (redox) cofactor recycling. Metabolic engineering holds great promise for creating efficient, competitive routes for the production of biofuels and biochemicals without harsh chemicals and hazardous byproducts. Successes in biochemical production include the use of bacteria for producing Dupont’s Sorona fibers from 1,3-propanediol from glucose and the use of yeast for the manufacture of the antimalarial drug artemisinin. However, roadblocks to biosynthesis prevent many biochemicals from being produced biologically, given current technology. Nature uses compartmentalization (e.g., organelles in eukaryotes and bacterial microcompartments in prokaryotes) to solve issues such as intermediate leakage, toxicity, and byproduct formation. In traditional systems, redox cofactors are lost to cellular growth and maintenance needs. Compartmentalizing redox cofactors with the biochemical synthesis enzymes is anticipated to increase thermodynamic efficiency and prevent the loss of valuable intermediates and cofactors. If successful, this strategy would be the first direct demonstration of this feature of a bacterial microcompartment and would provide a tool for improving metabolic pathway performance for all enzymes with redox or other cofactors. In addition, this work would reveal insights into the native function of these structures, while also providing a detailed method for selecting and improving biochemical pathway performance. Ultimately, research results will lead to the cost-efficient production of chemicals currently derived from petroleum.
Project Goal and Summary: Characterize and engineer carbon-carbon (C-C) bond-forming enzymes to enable novel biosynthetic pathways to a variety of long-chain and di-functional fatty acids. More broadly, this project will harness enzyme substrate promiscuity to access a broad range of products not found in nature, while mitigating toxic or undesirable side reactions, potentially unlocking facile synthesis of new classes of molecules to enable biomanufacturing. Its significance lies in two areas—enabling synthesis of new molecules difficult to obtain by petrochemical routes and modeling the genome-scale consequences of enzyme promiscuity. First, many useful fuel and chemical molecules, like heptanoic and suberic acid, are difficult to produce by oil refining and petrochemistry. Functionalizing the ends of alkanes is particularly difficult because interior carbons are more reactive. Biosynthesis of terminally functionalized molecules mediated by enzymatic coupling reactions would allow production of valuable biochemicals, making scale-up more feasible and lower risk. Secondly, the team will develop and use tools to predict enzyme promiscuity and mitigate the negative consequences associated with unwanted side reactions. This research and accompanying tool development will help identify deleterious promiscuous reactions and pave the way for other metabolic engineers to readily avoid toxicity and productivity loss due to unwanted side reactions.
Project Goal and Summary: Use a model co-culture of photoautotroph-methanotroph Synechococcus sp. PCC 7002 and Methylomicrobium alcaliphilum 20ZR for developing experimental and computational tools to gain a qualitative and quantitative understanding of the interactions and dynamics of the co-culture at both systems and molecular levels. Biogas from conversion of organic waste streams has immense potential for use as a feedstock to produce high-density fuels and commodity chemicals. However, the use of biogas represents a significant challenge due to its low pressure and the presence of contaminants such as hydrogen sulfide, ammonia, and volatile organic carbon compounds. Tapping into this immense potential requires effective biotechnologies that co-utilize both carbon dioxide and methane. Fundamental understanding of the photoautotroph-methanotroph interaction will lay the foundation for designing and optimizing synthetic binary consortia to produce fuels and chemicals from biogas. Knowledge gained from this project may be generally applicable to other cross-feeding binary consortia, and the tools developed can be adapted to study the interactions and dynamics of other multiorganism platforms.
Project Goal and Summary: Develop Megasphaera elsdenii as a platform for the conversion of lignocellulosic biomass sugars and organic acids into hexanol and other valuable chemicals. The native ability to condense acetyl-coenzyme A (CoA) groups to efficiently generate C4 to C8 compounds makes M. elsdenii a compelling platform for producing fuels and chemicals from lactate and plant carbohydrates. Engineering M. elsdenii to efficiently produce next-generation, drop-in lignocellulosic fuels such as hexanol at high yield and titer could provide an efficient bioengineering platform. Initially, lignocellulosic sugars will be converted to hexanol and related products; however, because M. elsdenii ferments lactate to organic acids, this project also will lay the foundation for more advanced processing options such as a co-culture or sequential fermentation in which one organism converts sugars to lactate and an engineered M. elsdenii converts the lactate to a higher-value product.
Project Goal and Summary: Develop the thermotolerant yeast Kluyveromyces marxianus as a platform host for industrial bioprocessing. A critical area of the U.S. industrial biotechnology sector is the conversion of biomass and other renewable feedstocks to fuels and chemicals. Robust microorganisms that are genetically accessible can grow rapidly at high temperature and low pH, and those that can effectively assimilate a wide range of different sugars, such as K. marxianus, are needed to sustain technological and economic growth. New genome-wide CRISPR-based tools for genome editing, genetic screening, and rapid strain development will be developed and applied in systems biology studies to understand industrially desirable phenotypes and for metabolic engineering. A key aim will be to enhance acetyl-coenzyme A (CoA) production, a central precursor in the synthesis of many fuels and chemicals. Anticipated outcomes include rapid engineering of K. marxianus strains that produce biofuels and chemicals at high titer, rate, and yield, leading to a new, robust platform for low-cost bioprocessing.
Project Goal and Summary: Develop technologies to optimize cyanobacteria and other microbes for producing renewable chemicals at commercially feasible rates and yields by establishing a rapid flux phenotyping platform that can be applied to accelerate metabolic engineering of cyanobacterial hosts. The ability to quantify flux alterations in response to targeted genetic manipulations is a key requirement for rational metabolic engineering, but the time needed to complete a comprehensive 13C flux analysis can far exceed the time needed to introduce new genetic modifications to a recombinant host. Matching the throughput of 13C flux phenotyping to the rate of strain generation will provide the foundation for a rational “design-build-test-learn” metabolic engineering cycle. Project findings are expected to be generalizable to a diverse range of biochemical products derived from major metabolic hubs, enabling a systematic strategy for metabolic engineering of cyanobacteria and other microbes.
Project Goal and Summary: Construct symbiotic relationships between microbial phototrophs (cyanobacteria or algae) and heterotrophs (bacteria or yeast), enabling development of a coupled system for light-driven CO2 fixation and high-efficiency synthesis of oils suitable for use as biofuels. Drawing inspiration from the natural symbiotic pairing of algae and fungi that form lichens, the investigators will examine specific factors that facilitate beneficial pairings between organisms and increase overall process efficiency. These pairings will focus on a phototroph engineered to secrete carbohydrates (Synechococcus elongates), first with a model bacterium (Escherichia coli) and later with an oleaginous yeast (Yarrowia lipolytica) that naturally accumulates oils. Transcriptomic and metabolomic approaches will facilitate construction of a genomescale model to inform engineering efforts and enable fundamental insights into the development of consortial relationships among microbes. In the future, this type of relationship could enable effective division of labor in biofuels production, with specialization mitigating the metabolic overload observed in some engineered species that both photosynthesize and produce fuels.
Project Goal and Summary: Conduct large-scale mapping of genotype to phenotype in oleaginous yeast, focused on the genes underlying lipid production, plant feedstock hydrolysate tolerance, low-oxygen metabolism, and co-utilization of sugars in plant material. The yeast chosen for this study, Rhodosporidium toruloides, has several advantages over more traditional model yeasts, including its native ability to metabolize the sugars in plant hydrolysates (i.e., glucose, xylose, arabinose, and cellobiose) and high de novo lipid productivity. Techniques for R. toruloides genetics and genome engineering will be developed and used to map the determinants of complex growth and lipid productivity traits in wild isolates and engineered strains. Establishment of a robust, versatile model yeast that natively accumulates high lipid levels will enable greater flexibility in developing new biofuels that produce industrial strains and will provide fundamental insights into the origins of complex traits useful for biofuels production.
Project Goal and Summary: Develop Rhodococcus opacus, a soil bacterium capable of converting phenolic compounds to biofuel precursors, as a model microbe and potential platform for biofuels production. Phenolic compounds are released from lignin during biomass deconstruction and are problematic because of their potential toxicity to the microbes used for biofuels synthesis. By metabolizing these toxic compounds, R. opacus bypasses this problem and could increase biofuel production titers by utilizing lignin as a feedstock. Beginning with model phenolic substrates and eventually moving to thermochemically depolymerized lignin, the investigators will construct strains of R. opacus for increased tolerance to phenolics and increased biofuel precursor production. The evolved strains will then undergo a battery of omics testing to elucidate the mechanisms responsible for these desirable characteristics. Research results will advance systems biology understanding of genetic factors involved in resistance to lignin-derived phenolic toxins and could provide a promising new candidate platform organism for converting lignin compounds into biofuels.
Project Goal and Summary: Investigate systems biology properties of several nonmodel algal species in the laboratory and under simulated outdoor conditions. This approach will allow investigators to focus their efforts on understanding regulatory and metabolic networks that impact growth rates and lipid yields in realistic biofuels production scenarios and enable potential improvements via targeted genetic modification. Investigators will work with four species representing the two major algal subgroups—the green chlorophytes (Scenedesmus obliquus and a Coelastrum strain) and the stramenophile diatoms (Thalassiosira pseudonana and Cyclotella cryptica). This research will develop several algal species as model organisms and potential platforms for biofuels production, emphasizing the identification of specific genetic factors underlying performance in realistic biofuel production conditions.
Project Goal and Summary: Construct and evolve model strains of bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) to rely solely on an engineered pathway, non-oxidative glycolysis (NOG), for glucose utilization. The NOG pathway enables full retention of carbon from sugars when combined with reducing equivalents from formate or hydrogen, theoretically increasing carbon yield from 66% to 100%. Numerous genetic modifications are envisioned, including enzyme introduction in the NOG pathway and removal of essential glycolytic enzymes to force utilization of the engineered pathway. This development will be followed by selection for growth on glucose, as well as genomic and transcriptomic evaluation of the evolved strains to characterize their adaptions to NOG. The project also will explore direction of carbon flux into n-butanol, a potential advanced biofuel, in NOG-utilizing bacteria. Conversion of all carbon in sugar, aside from that required for cellular function, to next-generation biofuels could greatly improve the efficiency of microbial biofuels synthesis as well as illuminate the basic governing principles underlying fundamental rewiring of central carbon metabolism.
Project Goal and Summary: Develop a systematic procedure to construct cell-wide kinetic models of two biofuel-relevant microbes through an ensemble modeling approach. This innovative modeling approach uses experimental transcriptomic, metabolomic, and fluxomic data to limit possible metabolic pathways for carbon flux in model space to those most likely to represent actual physiology. This research focuses on two related microbes—Clostridium thermocellum and Moorella thermoacetica. Previously engineered for biofuels production, C. thermocellum is an efficient degrader of lignocellulosic biomass. M. thermoacetica is a homoacetogen being developed as a potential platform organism for converting synthesis gas ("syngas," a mixture of H2, CO2, and carbon monoxide produced during thermochemical conversion of biomass) to liquid biofuels. Research results will significantly advance the status of these two bioenergy-relevant microbes as model organisms and provide predictive modeling tools that will facilitate more sophisticated approaches to engineering microbes for improved biomass deconstruction and biofuels synthesis.
Project Goal and Summary: Examine the systems biology properties of the cyanobacterium Synechococcus elongatus strain UTEX 2973 and develop modeling and bioengineering capabilities for this organism. Researchers recently demonstrated that this strain possesses the fastest growth rate of any known cyanobacterium. Despite this pronounced difference in growth rate from other more developed model strains of Synechococcus, genome sequencing has revealed a less than 1% difference in sequence in the 2973 strain, suggesting that this capability could be leveraged for more efficient biofuels synthesis and potentially engineered into other organisms. As such, development of this strain as a model organism and potential platform for light-driven conversion of CO2 into biofuel compounds could have major impacts.
Project Goal and Summary: Use an omics-driven systems biology approach to identify key points of integration between regulatory and metabolic networks in two microorganisms—the biofuel-producing yeast Saccharomyces cerevisiae and the cellulosedegrading bacterium Clostridium celluloyticum. Tight regulation of important metabolic pathways that control carbon flux continues to be a challenge for engineered biofuels production, and systematic elucidation of these control strategies is the first step to overriding this regulation. By analyzing data from metabolomics, proteomics, and fluxomics experiments, investigators will determine where the observed metabolic flux variations of reactions cannot be accounted for under standard models, identifying possible points of regulation and enabling use of a computational modeling approach to predict mediating elements. These predictions will be experimentally validated (1) in vivo by tuned modulation of expression of enzymes of interest, (2) in vitro by biochemical analysis of reaction kinetics displayed by enzymes in the presence of putative regulators, and (3) through systems-level genetic modification aimed at "unwiring" regulation in select cases. The resulting data will be used to refine systemslevel metabolic and regulatory models for the two organisms, which will serve as valuable resources for both understanding their systems biology and guiding bioengineering strategies. This research will advance a sophisticated new technical approach for analyzing metabolic and regulatory networks and will generate new understanding of systems biology properties of two biofuel-relevant model organisms of broad value to the scientific community.
Project Goal and Summary: Determine how lysine acetylation alters production rates and yields in biofuel-producing strains of Escherichia coli. Multiple studies have shown that metabolic enzymes are highly acetylated and that growth on different carbon sources changes their acetylation profile. This finding leads to the hypothesis that bacteria employ lysine acetylation as a global mechanism to regulate metabolism in response to their energy and redox status. The investigators will test the hypothesis that lysine acetylation plays a role in central metabolic pathways involved in the synthesis of biofuel compounds, leveraging previously engineered E. coli strains capable of synthesizing a variety of potential biofuel precursors. This research will address a key knowledge gap in global regulatory mechanisms (specifically at the translational level) used by bacteria to modulate large-scale cellular processes, focusing on the integration to regulatory and metabolic networks involved in biofuels synthesis.
Project Goal and Summary: Examine the genetic basis of wood deconstruction in brown rot fungi, a subclass of wood-degrading fungi evolved from the more common white rot fungi. As compared to white rot fungi, brown rot species use a distinct and potentially more efficient mechanism to circumvent the lignin barrier during biomass breakdown. Investigators will use coupled omics techniques to spatially and sequentially map the expression of genes involved in wood deconstruction in fungal hyphae at the microbelignocellulose interface. This approach will employ an analytical technique developed in a prior DOE Early Career Research award that enables high-resolution transcriptomics and proteomic analysis of fungal hyphae in the active reaction zones in wood samples. This strategy will facilitate a highly focused comparison of expressed genes and secreted cellulolytic enzymes, resulting in development of a "connectome" co-localizing expression of implicated genes and proteins with specific degradative reactions in the wood. This work will advance overall understanding of mechanisms used by filamentous fungi to deconstruct lignocellulose and facilitate their potential application to production of next-generation biofuels.
Project Goal and Summary: Examine interactions in a model microbial consortium relevant to consolidated bioprocessing and develop a computationally enabled pipeline for high-throughput omics analysis, predictive modeling, and targeted optimization of microbial consortia. The investigators have selected four model microorganisms, each with a different functional capability: (1) cellulose/hemicellulose degradation (Streptomyces reticuli), (2) lignin demethoxylation (Methylobacterium extorquens), (3) lignin degradation (Streptomyces viridosporus), and (4) biofuels synthesis (Yarrowia lipolytica). A stable, optimized consortium of these organisms theoretically would be capable of near-complete conversion of plant biomass into lipids suitable for biodiesel production. Research results will advance understanding and predictive modeling capabilities for community-scale microbial interactions and enable development of new consolidated bioprocessing strategies for biofuels synthesis.
Project Goal and Summary: Examine systems biology properties of Bacillus megaterium strain SR7, a bacterium isolated from a deep subsurface salt dome capable of growth in the presence of super critical CO2 (scCO2). By developing this microbe as a potential biofuel production platform organism, investigators hope to enable development of continuous-flow bioreactors that are highly resistant to contamination and utilize scCO2 chemistry for extraction of biofuel compounds. In addition to examining fundamental physiological properties of B. megaterium SR7, the team will engineer metabolic pathways for synthesizing medium-chain hydrocarbons and develop a bench-scale scCO2 bioreactor system.
Research results will advance fundamental understanding of a newly discovered set of stress-tolerance characteristics highly relevant to DOE missions in bioenergy and environmental process understanding and enable development of a novel platform organism suitable for use in an innovative new biofuel-production process.
Project Goal and Summary: Better understand lipid accumulation in the oleaginous yeast Rhodotorula glutinis through development of single-cell analysis techniques. R. glutinis natively produces high levels of lipids and can accumulate them to a large fraction of its dry cell weight, making it a very promising organism for biofuels production. This research will focus on analyzing transcriptomic data from single cells correlated with quantitative measurements of lipid production in vivo through development of a stimulated Raman scattering microscopy (SRS-M)-enabled microfluidic flow sorter. The genes and transcription factors found to be potentially responsible for lipid accumulation will be verified through metabolic engineering strategies. This work will develop a novel technology for imaging and analysis of shifts in gene expression at the single-cell level, helping to advance this particular model organism for biofuels production and for systems biology research in the broader scientific community.
Project Goal and Summary: Examine systems biology properties of the acetogenic bacterium Acetobacterium ljungdahlii and construct a genome-scale metabolic model. A. ljungdahlii is a potential chassis organism for biological conversion of synthesis gas ("syngas," a mixture of H2 , CO2 , and carbon monoxide produced during thermochemical conversion of biomass) to liquid biofuels. By advancing understanding of the nested genetic and metabolic networks of this organism and developing an in silico model of its biosystems, investigators aim to identify targets for metabolic engineering of strains capable of converting syngas to a variety of medium-chain alcohols. In addition to generating modified strains of A. ljungdhalii that potentially can be further developed as chassis organisms for syngas conversion, project results will facilitate new approaches to computationally driven design of biological systems and will significantly advance systems biology understanding of the homoacetogenesis, a form of chemolithotrophic metabolism with relevance to multiple DOE missions.
U.S. Department of Energy
Office of Biological and Environmental Research