Synthesis of poly(3-hydroxyalkanoates) in Escherichia coli expressing the PHA synthase gene phaC2 from Pseudomonas aeruginosa:: comparison of PhaC1 and PhaC2
Synthesis of poly(3-hydroxyalkanoates) in Escherichia coli expressing the PHA synthase gene phaC2 from Pseudomonas aeruginosa:: comparison of PhaC1 and PhaC2
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DOI：
10.1111/j.1574-6968.1997.tb12767.x
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发表时间：
1997-12-01
影响因子：
2.1
通讯作者：
Steinbüchel, A
中科院分区：
文献类型：
--
作者：
Qi, QS;Rehm, BHA;Steinbüchel, A
为了获得来自铜绿假单胞菌的PHA合酶基因phaC 2在大肠杆菌中的功能性表达，将phaC 2的编码区（包括核糖体结合位点）亚克隆到与lac启动子共线的pBluescriptSK中。该质粒pBHR 71-C2使phaC 2在E.在含有0.5%（w/v）各种脂肪酸的LB培养基中生长时，（C-8-C-14）当提供十二烷酸作为碳源时，观察到PHA的最强积累，并且PHA贡献细胞干重的15%，其由35mol%3-羟基十二烷酸组成，60摩尔%的3-羟基癸酸酯和5摩尔%的3-羟基辛酸酯。质粒pBHR 78含有铜绿假单胞菌的基因phaC 1和phaC 2，在pBluescriptSK中的Inc启动子控制下，在E. coli LS 1298细胞在癸酸盐培养基上生长时，PHA的积累量占细胞干重的13%。与PhaC 1或PhaC 2相比，PHA组合物中只有轻微的差异。从E.表达PhaC 1或PhaC 2或同时表达两种PHA酶的coliLS 1298中的PHA酶活分别为106 × 10 ~（3）、70 × 10 ~（3）和67 × 10 ~（3）。该研究清楚地表明，当在大肠杆菌中表达时，来自铜绿假单胞菌的两种PHA酶表现出非常相似的性质，导致相似的PHA积累程度、相似的组成和分子量。并且脂肪酸β-氧化为两种PHA酶提供底物。
In order to obtain functional expression of PHA synthase gene phaC2 from Pseudomonas aeruginosa in Escherichia coli, the coding region of phaC2 was subcloned, including the ribosomal binding site, into pBluescript SK- collinear to the lac promoter. This plasmid pBHR71-C2 enabled functional expression of phaC2 in E. coli LS1298 (fadB) under Inc promoter control, leading to PHA accumulation, when grown in LB medium containing 0.5% (w/v) of various fatty acids (C-8-C-14) The strongest accumulation of PHA was observed, when dodecanoate was provided as carbon source, and PHA contributed to 15% of cell dry weight, which was composed of 35 mol% 3-hydroxydodecanoate, 60 mol% 3-hydroxydecanoate and 5 mol% 3-hydroxyoctanoate. Plasmid pBHR78, which contained both genes phaC1 and phaC2 from P. aeruginosa under Inc promoter control in pBluescript SK- led in E. coli LS1298 to PHA accumulation, which contributed to 13% of cell dry weight, when cells were grown on decanoate. Only slight differences in PHA composition compared with either PhaC1 or PhaC2 were obtained. The weight average molecular masses of PHA purified from decanoate-grown cells of E. coli LS1298 expressing PhaC1 or PhaC2 alone or both PHA synthases, were 106 x 10(3), 70 x 10(3) or 67 x 10(3), respectively. This study clearly demonstrated that both PHA synthases from P. aeruginosa exhibit very similar properties resulting in similar extent of PHA accumulation, similar composition and molecular mass, when expressed in E. coli and that fatty acid beta-oxidation provides substrates for both PHA synthases.