Isopropanol production with engineered Cupriavidus necator as bioproduction platform
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Alleviating our society’s dependence on petroleum-based chemicals has been highly emphasized due to fossil fuel shortages and increasing greenhouse gas emissions. Isopropanol is a molecule of high potential to replace some petroleum-based chemicals, which can be produced through biological platforms from renewable waste carbon streams such as carbohydrates, fatty acids, or CO2. In this study, for the first time, the heterologous expression of engineered isopropanol pathways were evaluated in a Cupriavidus necator strain Re2133, which was incapable of producing poly-3-hydroxybutyrate [P(3HB)]. These synthetic production pathways were rationally designed through codon optimization, gene placement, and gene dosage in order to efficiently divert carbon flow from P(3HB) precursors toward isopropanol. Among the constructed pathways, Re2133/pEG7c overexpressing native C. necator genes encoding a β-ketothiolase, a CoA-transferase, and codon-optimized Clostridium genes encoding an acetoacetate decarboxylase and an alcohol dehydrogenase produced up to 3.44 g l-1 isopropanol in batch culture, from fructose as a sole carbon source, with only 0.82 g l-1 of biomass. The intrinsic performance of this strain (maximum specific production rate 0.093 g g-1 h-1, yield 0.32 Cmole Cmole-1) corresponded to more than 60 % of the respective theoretical performance. Moreover, the overall isopropanol production yield (0.24 Cmole Cmole-1) and the overall specific productivity (0.044 g g-1 h-1) were higher than the values reported in the literature to date for heterologously engineered isopropanol production strains in batch culture. Strain Re2133/pEG7c presents good potential for scale-up production of isopropanol from various substrates in high cell density cultures.
KeywordsCupriavidus necator Ralstonia eutropha Isopropanol Branched-chain alcohols Biofuel Metabolic engineering
This work was supported by grants from the MIT-France Seed Fund and U.S. Department of Energy, Advanced Research Projects Agency-Energy (ARPA-E). Dr. Estelle Grousseau was funded by a Post-Doctoral grant from the French National Center for Scientific Research (CNRS) and the French Ministry of Higher Education and Research following the France-MIT Energy Forum in June 29, 2011. We thank Dr. Jens K. Plassmeier and Dr. Christopher J. Brigham for the helpful discussions. We thank Mr. Stephan Grunwald for his assistance, and Mr. John W. Quimby for the review of this manuscript. We also thank Dr. Toshiaki Fukui from the Tokyo Institute of Technology for the generous gift of the pBBad plasmid.
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