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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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 beta-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 beta-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.
Publisher
AMER SOC MICROBIOLOGY
Issue Date
2003-09
Language
English
Article Type
Article
Keywords

ACID BETA-OXIDATION; PSEUDOMONAS-AERUGINOSA; AEROMONAS-CAVIAE; COA HYDRATASE; BACTERIAL POLYHYDROXYALKANOATES; MOLECULAR CHARACTERIZATION; HYDROXYBUTYRIC ACID; FATTY-ACIDS; PHAG GENE; CLONING

Citation

JOURNAL OF BACTERIOLOGY, v.185, pp.5391 - 5397

ISSN
0021-9193
URI
http://hdl.handle.net/10203/21887
Appears in Collection
CBE-Journal Papers(저널논문)
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