title: Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes.
abstract: n-Butanol is generated as a natural product of metabolism by several microorganisms, but almost all grow at mesophilic temperatures. A synthetic pathway for n-butanol production from acetyl coenzyme A (acetyl-CoA) that functioned at 70°C was assembled in vitro from enzymes recruited from thermophilic bacteria to inform efforts for engineering butanol production into thermophilic hosts. Recombinant versions of eight thermophilic enzymes (β-ketothiolase [Thl], 3-hydroxybutyryl-CoA dehydrogenase [Hbd], and 3-hydroxybutyryl-CoA dehydratase [Crt] from Caldanaerobacter subterraneus subsp. tengcongensis; trans-2-enoyl-CoA reductase [Ter] from Spirochaeta thermophila; bifunctional acetaldehyde dehydrogenase/alcohol dehydrogenase [AdhE] from Clostridium thermocellum; and AdhE, aldehyde dehydrogenase [Bad], and butanol dehydrogenase [Bdh] from Thermoanaerobacter sp. strain X514) were utilized to examine three possible pathways for n-butanol. These pathways differed in the two steps required to convert butyryl-CoA to n-butanol: Thl-Hbd-Crt-Ter-AdhE (C. thermocellum), Thl-Hbd-Crt-Ter-AdhE (Thermoanaerobacter X514), and Thl-Hbd-Crt-Ter-Bad-Bdh. n-Butanol was produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to in vitro experiments, was used to determine relative enzyme ratios needed to maximize n-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to n-butanol was observed in vitro, but with a 60% decrease in the predicted pathway flux. With more-selective hypothetical versions of Bad and Bdh, >70% conversion to n-butanol is predicted, with a 19% increase in pathway flux. Thus, more-selective thermophilic versions of Bad, Bdh, and AdhE are needed to fully exploit biocatalytic n-butanol production at elevated temperatures.
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Loder
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Benjamin M
authors.initials: BM
authors.lastname: Zeldes
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: G Dale
authors.initials: GD
authors.lastname: Garrison
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA.
authors.forename: Gina L
authors.initials: GL
authors.lastname: Lipscomb
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA.
authors.forename: Michael W W
authors.initials: MW
authors.lastname: Adams
authors.affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, USA rmkelly@ncsu.edu.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Kelly
mesh_terms: D020001:1-Butanol / Q000378:metabolism*; D000102:Acetyl-CoA C-Acyltransferase / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000378:metabolism; D055162:Biocatalysis; D002849:Chromatography, Gas; D046969:Clostridium acetobutylicum / Q000378:metabolism; D048013:Acetivibrio thermocellus / Q000378:metabolism; D046968:Thermoanaerobacter / Q000378:metabolism
chemical_list: D000214:Acyl Coenzyme A; C030372:3-hydroxybutyryl-coenzyme A; D020001:1-Butanol; D000429:Alcohol Oxidoreductases; C060833:butanol dehydrogenase; D000426:Alcohol Dehydrogenase; D000445:Aldehyde Oxidoreductases; C018699:aldehyde dehydrogenase (NAD(P)+); D000102:Acetyl-CoA C-Acyltransferase