title: Metabolic engineering of Clostridium autoethanogenum for selective alcohol production.
abstract: Gas fermentation using acetogenic bacteria such as Clostridium autoethanogenum offers an attractive route for production of fuel ethanol from industrial waste gases. Acetate reduction to acetaldehyde and further to ethanol via an aldehyde: ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase has been postulated alongside the classic pathway of ethanol formation via a bi-functional aldehyde/alcohol dehydrogenase (AdhE). Here we demonstrate that AOR is critical to ethanol formation in acetogens and inactivation of AdhE led to consistently enhanced autotrophic ethanol production (up to 180%). Using ClosTron and allelic exchange mutagenesis, which was demonstrated for the first time in an acetogen, we generated single mutants as well as double mutants for both aor and adhE isoforms to confirm the role of each gene. The aor1+2 double knockout strain lost the ability to convert exogenous acetate, propionate and butyrate into the corresponding alcohols, further highlighting the role of these enzymes in catalyzing the thermodynamically unfavourable reduction of carboxylic acids into alcohols.
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, University Park, The University of Nottingham, Nottingham NG7 2RD, UK; LanzaTech Inc., 8045 Lamon Avenue, Suite 400, Skokie, IL, USA.
authors.forename: Fungmin
authors.initials: F
authors.lastname: Liew
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, University Park, The University of Nottingham, Nottingham NG7 2RD, UK.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Henstra
authors.affiliation: LanzaTech Inc., 8045 Lamon Avenue, Suite 400, Skokie, IL, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Kӧpke
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, University Park, The University of Nottingham, Nottingham NG7 2RD, UK.
authors.forename: Klaus
authors.initials: K
authors.lastname: Winzer
authors.affiliation: LanzaTech Inc., 8045 Lamon Avenue, Suite 400, Skokie, IL, USA.
authors.forename: Sean D
authors.initials: SD
authors.lastname: Simpson
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, University Park, The University of Nottingham, Nottingham NG7 2RD, UK. Electronic address: nigel.minton@nottingham.ac.uk.
authors.forename: Nigel P
authors.initials: NP
authors.lastname: Minton
mesh_terms: D000447:Aldehydes / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000502:physiology*; D003013:Clostridium / Q000145:classification / Q000502:physiology*; D000431:Ethanol / Q000302:isolation & purification; D024861:Genetic Enhancement / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000502:physiology
chemical_list: D000447:Aldehydes; D000431:Ethanol
keywords: Aldehyde:ferredoxin oxidoreductase (AOR); Bi-functional aldehyde/alcohol dehydrogenase (AdhE); Clostridium autoethanogenum; Gas fermentation; Metabolic engineering