title: Metabolomics-driven approach to solving a CoA imbalance for improved 1-butanol production in Escherichia coli.
abstract: High titer 1-butanol production in Escherichia coli has previously been achieved by overexpression of a modified clostridial 1-butanol production pathway and subsequent deletion of native fermentation pathways. This strategy couples growth with production as 1-butanol pathway offers the only available terminal electron acceptors required for growth in anaerobic conditions. With further inclusion of other well-established metabolic engineering principles, a titer of 15g/L has been obtained. In achieving this titer, many currently existing strategies have been exhausted, and 1-butanol toxicity level has been surpassed. Therefore, continued engineering of the host strain for increased production requires implementation of alternative strategies that seek to identify non-obvious targets for improvement. In this study, a metabolomics-driven approach was used to reveal a CoA imbalance resulting from a pta deletion that caused undesirable accumulation of pyruvate, butanoate, and other CoA-derived compounds. Using metabolomics, the reduction of butanoyl-CoA to butanal catalyzed by alcohol dehydrogenase AdhE2 was determined as a rate-limiting step. Fine-tuning of this activity and subsequent release of free CoA restored the CoA balance that resulted in a titer of 18.3g/L upon improvement of total free CoA levels using cysteine supplementation. By enhancing AdhE2 activity, carbon flux was directed towards 1-butanol production and undesirable accumulation of pyruvate and butanoate was diminished. This study represents the initial report describing the improvement of 1-butanol production in E. coli by resolving CoA imbalance, which was based on metabolome analysis and rational metabolic engineering strategies.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Ohtake
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA 90095, USA.
authors.forename: Sammy
authors.initials: S
authors.lastname: Pontrelli
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Walter A
authors.initials: WA
authors.lastname: Laviña
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA 90095, USA.
authors.forename: James C
authors.initials: JC
authors.lastname: Liao
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: sastia_putri@bio.eng.osaka-u.ac.jp.
authors.forename: Sastia P
authors.initials: SP
authors.lastname: Putri
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: fukusaki@bio.eng.osaka-u.ac.jp.
authors.forename: Eiichiro
authors.initials: E
authors.lastname: Fukusaki
mesh_terms: D020001:1-Butanol / Q000378:metabolism*; D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism; D003065:Coenzyme A / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D055442:Metabolome; D055432:Metabolomics
chemical_list: D029968:Escherichia coli Proteins; D020001:1-Butanol; D000426:Alcohol Dehydrogenase; C068383:adhE protein, E coli; D000445:Aldehyde Oxidoreductases; D003545:Cysteine; D003065:Coenzyme A
keywords: 1-Butanol; CoA balance; Escherichia coli; Metabolomics; Strain improvement