Identification and characterization of a new enoyl coenzyme A hydratase involved in biosynthesis of medium-chain-length polyhydroxyalkanoates in recombinant Escherichia coli.
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.
Park
Si Jae
SJ
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.
Lee
Sang Yup
SY
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
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