title: Engineering Escherichia coli for methanol conversion.
abstract: 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.
authors.affiliation: Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
authors.forename: Jonas E N
authors.initials: JEN
authors.lastname: Müller
authors.affiliation: Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
authors.forename: Fabian
authors.initials: F
authors.lastname: Meyer
authors.affiliation: Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
authors.forename: Boris
authors.initials: B
authors.lastname: Litsanov
authors.affiliation: Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
authors.forename: Patrick
authors.initials: P
authors.lastname: Kiefer
authors.affiliation: Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
authors.forename: Eva
authors.initials: E
authors.lastname: Potthoff
authors.affiliation: 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.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Heux
authors.affiliation: Department of Pharmaceutical Biology, University of Groningen, Groningen, The Netherlands.
authors.forename: Wim J
authors.initials: WJ
authors.lastname: Quax
authors.affiliation: Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Universitätsstr. 25, D-33615 Bielefeld, Germany.
authors.forename: Volker F
authors.initials: VF
authors.lastname: Wendisch
authors.affiliation: Department of Molecular Biology, SINTEF Materials and Chemistry, Trondheim, Norway; Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Trygve
authors.initials: T
authors.lastname: Brautaset
authors.affiliation: 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.
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Portais
authors.affiliation: Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland. Electronic address: vorholt@micro.biol.ethz.ch.
authors.forename: Julia A
authors.initials: JA
authors.lastname: Vorholt
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D000432:Methanol / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); D000432:Methanol
keywords: Escherichia coli; Methanol; Methylotrophy; One-carbon metabolism; Synthetic biology