微嗜酸寡养单胞菌中磷酸吡哆胺氧化酶基因pnpox生物学功能研究

Role of phosphopyridoxamine oxidase gene pnpox in Stenotrophomonas acidaminiphila

摘要:

【目的】研究微嗜酸寡养单胞菌(Stenotrophomonas acidaminiphila) CW117中磷酸吡哆胺氧化酶基因pnpox (phosphopyridoxamine oxidase, pnpox)在维生素B₆ (VB₆)合成中的贡献及对黄曲霉毒素B1 (aflatoxin B1, AFB1)的降解活性。【方法】采用基因插入突变方式，对菌株CW117中磷酸吡哆胺氧化酶基因pnpox进行突变，得到突变菌株。通过高效液相色谱法(high performance liquid chromatography, HPLC)检测突变株对AFB1的降解活性，以及突变株中吡哆醇和吡哆醛的合成情况，确定基因pnpox在寡养单胞菌体内VB₆合成中的贡献和黄曲霉毒素降解代谢作用。【结果】成功构建了磷酸吡哆胺氧化酶基因突变子pnpox::pK19mobΩ2HMB，突变子吡哆醛的合成量较野生型菌株显著减少，吡哆醇合成量与野生型菌株无显著性差异；同时，突变子与野生型株CW117对AFB1的降解活性未发现显著性差异。【结论】菌株CW117中磷酸吡哆胺氧化酶在吡哆醛合成的过程中起着重要作用，该基因突变会导致VB₆的严重缺乏，影响寡养单胞菌正常生长，但该基因对CW117降解黄曲霉毒素无显著性贡献。

Abstract:

[Objective] To reveal the role of the phosphopyridoxamine oxidase gene pnpox in vitamin B₆ (VB₆) synthesis and aflatoxin B1 (AFB1) degradation in Stenotrophomonas acidaminiphila CW117. [Methods] The phosphopyridoxamine oxidase gene was mutated in strain Stenotrophomonas acidaminiphila CW117 by the recombinant plasmid pK19mobΩ2HMB/P-pnpox, and thus the mutant pnpox::pK19mobΩ2HMB was constructed. The vitamin B₆ (i.e., pyridoxine and pyridoxal) synthesis and AFB1 degradation activities of the mutant and the wild-type strain CW117 were detected by high performance liquid chromatography (HPLC). The detemination results were compared to reveal the role of pnpox in VB₆ synthesis and AFB1 degradation. [Results] The mutant pnpox::pK19mobΩ2HMB was successfully constructed. It had significantly lower yield of pyridoxal than the wild-type strain CW117, while no significant difference was observed in the yield of pyridoxine between the mutant and CW117. Meanwhile, the mutant showed no significant difference in AFB1 degradation from CW117.[Conclusion] Phosphopyridoxamine oxidase plays an important role in the pyridoxal synthesis of CW117. The mutation of pnpox reduced the VB₆ production and thus seriously delayed the growth of the strain, while the gene showed no significant contribution to AFB1 degradation in strain CW117.