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首页> 外文期刊>Journal of bacteriology >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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Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli

机译：新型大肠杆菌重组酶中中链长度多羟基链烷酸酯生物合成中的新酶辅酶A水合酶的鉴定与表征

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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 maoCEc 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 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 β-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.
机译：通过表达MCL-PHA合酶基因，在 fadB 突变大肠杆菌菌株中建立了脂肪酸中链长度（MCL）多羟基链烷酸酯（PHA）的生物合成途径。 。但是，尚未完全阐明负责（ R ）-3-羟酰基辅酶A（R3HA-CoAs）（PHA合酶底物）生成的酶。鉴定出与铜绿假单胞菌（ R ）特异性烯酰-CoA水合酶（PhaJ1）同源的大肠杆菌 MaoC对于 fadB 突变体 E中的PHA生物合成很重要。大肠杆菌菌株。当引入MCL-PHA合酶基因时， fadB maoC 双突变体 E。大肠杆菌WB108，它是 E的衍生物。大肠杆菌W3110，与 fadB 突变体 E相比，脂肪酸中MCL-PHA的累积量减少了43％。大肠杆菌WB101。 PHA的生物合成能力可以通过质粒表达 E中的 maoC Ec 基因来恢复。大肠杆菌WB108。另外， E。具有完全功能性β-氧化途径的大肠杆菌 W3110可通过 maoC Ec 基因和MCL-PHA合酶基因的共表达从脂肪酸中产生MCL-PHA 。为了进行酶分析，在 E中表达了在其C-末端融合有His 6 -Tag的MaoC。大肠杆菌并纯化。标记的MaoC的酶分析表明，MaoC对巴豆酰-CoA具有烯酰-CoA水合酶活性。这些结果表明，MaoC是一种新的烯酰-CoA水合酶，参与从 fadB R ）-3-羟酰基-CoA。 >突变体 E。大肠杆菌菌株。

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