title: Identification and characterization of a new enoyl coenzyme A hydratase involved in biosynthesis of medium-chain-length polyhydroxyalkanoates in recombinant Escherichia coli.
abstract: The biosynthetic pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) from fatty acids has been established in fadB mutant Escherichia coli strain by expressing the MCL-PHA synthase gene. However, the enzymes that are responsible for the generation of (R)-3-hydroxyacyl coenzyme A (R3HA-CoAs), the substrates for PHA synthase, have not been thoroughly elucidated. Escherichia coli MaoC, which is homologous to Pseudomonas aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1), was identified and found to be important for PHA biosynthesis in a fadB mutant E. coli strain. When the MCL-PHA synthase gene was introduced, the fadB maoC double-mutant E. coli WB108, which is a derivative of E. coli W3110, accumulated 43% less amount of MCL-PHA from fatty acid compared with the fadB mutant E. coli WB101. The PHA biosynthetic capacity could be restored by plasmid-based expression of the maoCEc gene in E. coli WB108. Also, E. coli W3110 possessing fully functional beta-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoCEc gene and the MCL-PHA synthase gene. For the enzymatic analysis, MaoC fused with His6-Tag at its C-terminal was expressed in E. coli and purified. Enzymatic analysis of tagged MaoC showed that MaoC has enoyl-CoA hydratase activity toward crotonyl-CoA. These results suggest that MaoC is a new enoyl-CoA hydratase involved in supplying (R)-3-hydroxyacyl-CoA from the beta-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.
authors.affiliation: Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering, BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon, Republic of Korea.
authors.forename: Si Jae
authors.initials: SJ
authors.lastname: Park
authors.forename: Sang Yup
authors.initials: SY
authors.lastname: Lee
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D004274:DNA, Recombinant; D004746:Enoyl-CoA Hydratase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D020868:Gene Silencing; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D011091:Polyesters / Q000737:chemistry / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D005227:Fatty Acids; D011091:Polyesters; D006639:Histidine; C010701:crotonyl-coenzyme A; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; C406162:R-specific trans-2,3-enoylacyl-CoA hydratase; D004746:Enoyl-CoA Hydratase; C477835:enoyl coenzyme A hydratase MaoC, E coli