Solaiman Daniel K Y
Eastern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Wyndmoor, PA 19038, USA.
J Ind Microbiol Biotechnol. 2003 May;30(5):322-6. doi: 10.1007/s10295-003-0059-8. Epub 2003 May 22.
Pseudomonas resinovorans harbors two isogenic poly(hydroxyalkanoates) (PHAs) synthase genes ( phaC1(Pre), phaC2(Pre)) responsible for the production of intracellular medium-chain-length (mcl-)PHAs. Sequence analysis showed that the putative gene-products of these genes contain a conserved alpha/beta-hydrolase fold in the carboxy-terminal half of the proteins. Hybrid genes pha7 and pha8 were constructed by exchanging the alpha/beta-hydrolase-fold coding portions of phaC1(Pre) and phaC2(Pre) at the 3' terminal. When grown with decanoate as carbon source, the pha7- or pha8-transformed Escherichia coli LS1298 produced PHAs containing 73-75% beta-hydroxydecanoate (beta-HD) and 25-27% beta-hydroxyoctanoate (beta-HO). Deletion mutants, Delta pha7 and Delta pha8, were isolated during the PCR-based construction of pha7 and pha8, respectively. Cells harboring these mutants produced PHAs containing 55-60 mol% beta-HD and 40-45 mol% beta-HO. These results demonstrate the feasibility of generating active hybrid mcl-PHA synthase genes and their mutants with the potential of producing polymers having a varied repeat-unit composition.
树脂糖假单胞菌含有两个同基因的聚羟基脂肪酸酯（PHA）合酶基因（phaC1(Pre)、phaC2(Pre)），负责细胞内中链长度（mcl-）PHA的合成。序列分析表明，这些基因的推定基因产物在蛋白质的羧基末端含有保守的α/β-水解酶折叠结构。通过交换phaC1(Pre)和phaC2(Pre) 3'末端的α/β-水解酶折叠编码部分构建了杂合基因pha7和pha8。当以癸酸作为碳源生长时，pha7或pha8转化的大肠杆菌LS1298产生的PHA含有73-75%的β-羟基癸酸（β-HD）和25-27%的β-羟基辛酸（β-HO）。在基于PCR构建pha7和pha8的过程中，分别分离出缺失突变体Delta pha7和Delta pha8。携带这些突变体的细胞产生的PHA含有55-60 mol%的β-HD和40-45 mol%的β-HO。这些结果证明了产生具有活性的杂合mcl-PHA合酶基因及其突变体的可行性，这些基因和突变体有潜力生产具有不同重复单元组成的聚合物。
