Engineering Escherichia coli for methanol conversion.

Müller JEN; Meyer F; Litsanov B; Kiefer P; Potthoff E; Heux S; Quax WJ; Wendisch VF; Brautaset T; Portais JC; Vorholt JA

doi:10.1016/j.ymben.2014.12.008

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Journal article

Engineering Escherichia coli for methanol conversion.

Müller JEN Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
Meyer F Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
Litsanov B Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
Kiefer P Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
Potthoff E Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
Heux S Université de Toulouse; INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
Quax WJ Department of Pharmaceutical Biology, University of Groningen, Groningen, The Netherlands.
Wendisch VF Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Universitätsstr. 25, D-33615 Bielefeld, Germany.
Brautaset T Department of Molecular Biology, SINTEF Materials and Chemistry, Trondheim, Norway; Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway.
Portais JC Université de Toulouse; INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
Vorholt JA Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland. Electronic address: vorholt@micro.biol.ethz.ch.

2015-01-18

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Metabolic engineering. - 2015

English Methylotrophic bacteria utilize methanol and other reduced one-carbon compounds as their sole source of carbon and energy. For this purpose, these bacteria evolved a number of specialized enzymes and pathways. Here, we used a synthetic biology approach to select and introduce a set of "methylotrophy genes" into Escherichia coli based on in silico considerations and flux balance analysis to enable methanol dissimilation and assimilation. We determined that the most promising approach allowing the utilization of methanol was the implementation of NAD-dependent methanol dehydrogenase and the establishment of the ribulose monophosphate cycle by expressing the genes for hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi). To test for the best-performing enzymes in the heterologous host, a number of enzyme candidates from different donor organisms were selected and systematically analyzed for their in vitro and in vivo activities in E. coli. Among these, Mdh2, Hps and Phi originating from Bacillus methanolicus were found to be the most effective. Labeling experiments using (13)C methanol with E. coli producing these enzymes showed up to 40% incorporation of methanol into central metabolites. The presence of the endogenous glutathione-dependent formaldehyde oxidation pathway of E. coli did not adversely affect the methanol conversion rate. Taken together, the results of this study represent a major advancement towards establishing synthetic methylotrophs by gene transfer.

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