Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli

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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 maoC Ec gene in E. coli WB108. Also, E. coli W3110 possessing fully functional β-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoC Ec gene and the MCL-PHA synthase gene. For the enzymatic analysis, MaoC fused with His 6 -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 β-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.

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