title: Escherichia coli enoyl-acyl carrier protein reductase (FabI) supports efficient operation of a functional reversal of β-oxidation cycle.
abstract: We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the -oxidation cycle for fuel and chemical production in Escherichia coli (J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541–554, 2012, http://dx.doi.org/10.1021/sb3000782).While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the Euglena gracilis trans-enoyl-CoA reductase [EgTER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native E. coli enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a -oxidation reversal. Overexpression of FabI proved as effective as EgTER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of fabI, we investigated whether bacterial ENRs from other families were able to complement a fabI deletion without promiscuous reduction of crotonyl-CoA. These characteristics from Bacillus subtilis FabL enabled deltaffabI complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the β-oxidation reversal in E. coli.
authors.forename: Jacob E
authors.initials: JE
authors.lastname: Vick
authors.forename: James M
authors.initials: JM
authors.lastname: Clomburg
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Blankschien
authors.forename: Alexander
authors.initials: A
authors.lastname: Chou
authors.forename: Seohyoung
authors.initials: S
authors.lastname: Kim
authors.forename: Ramon
authors.initials: R
authors.lastname: Gonzalez
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D002384:Catalysis; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054889:Fatty Acid Synthase, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction
chemical_list: D000214:Acyl Coenzyme A; D029968:Escherichia coli Proteins; D005227:Fatty Acids; D009243:NAD; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); C493924:fabI protein, E coli; D054889:Fatty Acid Synthase, Type II