Effect of inactivation of poly(hydroxyalkanoates) depolymerase gene on the properties of poly(hydroxyalkanoates) in Pseudomonas resinovorans
Effect of inactivation of poly(hydroxyalkanoates) depolymerase gene on the properties of poly(hydroxyalkanoates) in Pseudomonas resinovorans
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DOI：
10.1007/s00253-003-1317-4
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发表时间：
2003-05
影响因子：
5
通讯作者：
D. Solaiman;R. Ashby;T. Foglia
中科院分区：
文献类型：
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作者：
D. Solaiman;R. Ashby;T. Foglia
噬树脂假单胞菌的phaZ基因是一种聚羟基烷酸酯（PHA）解聚酶。通过体外转座和同源重组的方法，构建了噬脂假单胞菌NRRL B-2649的两个突变体FOAC 001和FOAC 002。转座子插入位点的详细作图和所得序列的分析表明，推定的融合多肽PhaZFOAC 001（239个氨基酸残基）和PhaZFOAC 002（85个氨基酸残基）可以分别由FOAC 001和FOAC 002的muplanphaZ基因产生。体内PHA降解实验结果表明，PhaZFOAC 001可能仍保留部分PHA解聚活性，而PhaZFOAC 002则完全丧失了这一功能。当细胞在限制氮源（低氮）条件下生长长达5天或在过量氮源（高氮）条件下生长3天时，B-2649、FOAC 001和FOAC 002的细胞产量和PHA含量相似。在PhaZ活性B-2649和FOAC 001细胞中观察到PHA含量在高N培养基中的延长细胞生长期间（5天）或响应于氮源的上移而显著降低。当在高N条件下生长时，来自FOAC 001和FOAC 002的PHA的重复单元组成含有比野生型B-2649略低量的β-羟基辛酸酯和较高量的β-羟基十四烯酸酯。尽管来自FOAC 001和FOAC 002的PHA的分子量在本研究中使用的任何条件下都没有变化，但野生型B-2649的分子量在高氮条件下生长5天或经历氮源向上转移的细胞中显著增加。因此，这些突变体为研究PHA解聚酶对中链PHA的积累和性质的影响提供了一个有价值的系统。
ThephaZgene ofPseudomonas resinovoranscodes for a poly(hydroxyalkanoates) (PHA) depolymerase. TwophaZmutants ofPseudomonas resinovoransNRRL B-2649, FOAC001 and FOAC002, were constructed by an in vitro transposition procedure followed by chromosomal integration via homologous recombination. A detailed mapping of the transposon insertion sites and an analysis of the resultant sequences showed that putative fusion polypeptides PhaZFOAC001(239 amino-acid residues) and PhaZFOAC002(85 amino-acid residues) could result from the mutantphaZgenes of FOAC001 and FOAC002, respectively. In vivo PHA degradation data indicated that PhaZFOAC001might still retain a partial PHA depolymerization activity, while PhaZFOAC002is completely devoid of this function. The cell yields and PHA contents of B-2649, FOAC001, and FOAC002 were similar when the cells were grown either under a limiting nitrogen-source (low-N) condition for up to 5 days or in excess N-source (high-N) for 3 days. A dramatic decrease in PHA content was observed in the PhaZ-active B-2649 and FOAC001 cells during prolonged cell growth (5 days) in high-N medium or in response to a shift-up in nitrogen-source. The repeat-unit compositions of the PHAs from FOAC001 and FOAC002 contained slightly lower amounts of β-hydroxyoctanoate and higher β-hydroxytetradecenoate than that of the wild-type B-2649 when grown under a high-N condition. While the molecular masses of the PHAs from FOAC001 and FOAC002 did not vary under any conditions used in this study, those of the wild-type B-2649 were markedly increased in cells either grown for 5 days under a high-N condition or subjected to a nitrogen-source shift-up. ThesephaZmutants thus provide a valuable system to study the influence of PHA depolymerase on the accumulation and properties of medium-chain-length PHA.