Biofuels Production projects
Metabolic Engineering of Methanogenic Archaea
This project taps methane, a clean-burning, renewable and underexploited energy source, by developing strains of methanogenic archaea with the ability to produce methane from the residual biomass present in the waste streams of biofuel production processes. Growing these engineered strains will capture energy that is lost in existing biofuel production strategies. In addition to their immediate impact on biofuel production, these studies will lend new insight into interspecies cooperation in methanogenesis, which may prove useful in efforts to optimize biological methane production in a variety of settings.
The goal of this project is to create engineered Methanosarcina strains with the ability to rapidly produce methane from residual biomass present in the waste streams of bioethanol production platforms. To this end, we have (1) constructed strains that efficiently produce methane from ethanol, (2) deciphered the mechanism of methanogenesis from pyruvate, and (3) selected strains with improved growth in thin stillage. Current efforts are focused on characterization and improvement of ethanol-dependent methanogenesis via stable isotope labeling, coupled with genetic manipulation of the engineered pathway and host genome. We are also benchmarking the engineered strains against currently used methanogenic waste treatment processes.
The goal of our research is to engineer Methanosarcina strains with the ability to produce methane from substrates present in the waste streams of biofuel production processes. Our efforts to date have focused on methanogenesis from ethanol and pyruvate. We have shown that pyruvate-utilizing mutants of Methanosarcina lack the enzyme pyruvate carboxylase and have up-regulated the enzyme pyruvate ferredoxin oxidoreductase. Unfortunately, these mutants require highly specialized growth conditions for utilization of pyruvate, which limits their utility in large-scale processes. Methanosarcina strains engineered to express the ethanol oxidation pathway from Clostridium kluyveri and the transhydrogenase from Escherichia coli acquire the ability to convert ethanol directly to methane, although they lack the ability to grow on this substrate. Preliminary evidence suggests that these strains stoichiometrically convert ethanol to acetate, which is subsequently converted to methane.
The year 2012 has seen significant progress in both the computational and engineering aspects of the project. First, we have completed an updated and highly accurate metabolic model of Methanosarcina barkeri, which complements the recently completed model of the closely related, but metabolically distinct, Methanosarcina acetivorans. These computer models accurately simulate growth rates and yields, as well as substrate uptake and product release rates and yields. Both models have subsequently been improved by modifying the cofactor specificity of selected redox reactions and by incorporating thermodynamic constraints on the substrates and products involved in the growth reaction. Second, we were successful, for the first time, in constructing engineered strains that completely convert ethanol to methane. Although these strains cannot grow on ethanol, they do convert a substantial fraction of this substrate to methane when growing on other substrates. Finally, we have completed a genome sequence of two methanogens that convert ethanol to a mixture of methane and acetate. Examination of these genome sequences revealed a novel mechanism for coupling ethanol oxidation to methane production. It is hoped that this alternate pathway for ethanol oxidation will improve the capacity of engineered Methanosarcina strains to completely convert ethanol to methane.
Researchers developed a quantitative, predictive computer model that describes the metabolism of the methane-producing organism Methanosarcina barkeri and will help design engineered strains that can produce methane from biomass substrates. Metcalf’s group also identified genetic mutations that enable Methanosarcina barkeri to metabolize pyruvic acid and constructed strains with the genes needed for conversion of lactate to pyruvate. In addition, researchers sequenced the genome of Methanogenium organophillum, a methanogen that can convert ethanol and CO2 to methane, which allowed identification of putative genes for a novel methane-generating pathway that will be useful in future genetic engineering efforts.
Published in 2013
Genomically and Biochemically Accurate Metabolic Reconstruction of Methanosarcina Barkeri Fusaro, iMG746, M. C. Gonnerman, M. N. Benedict, A. M. Feist, W. W. Metcalf, N. D. Price, Biotechnology Journal, 8, pp. 1070-1079, doi: 10.1002/biot.201200266, February 19, 2013.
Published in 2012
Updated Genome-Scale Metabolic Reconstruction of Methanosarcina barkeri, M. C. Gonnerman, M. N. Benedict, A. M. Feist, W. W. Metcalf and N. D. Price, Fusaro iMG746. Biotechnology Journal (in press).
Genome-Scale Metabolic Reconstruction and Hypothesis Testing in the Methanogenic Archeaon Methanosarcina acetivorans C2A, M. N. Benedict, M. C. Gonnerman, W. W. Metcalf and N. D. Price, Journal of Bacteriology 194:855-65. PMID:22139506.