枯草芽胞杆菌168不对称转化产生磷霉素的蛋白质组学分析

Proteomic analysis of Bacillus subtilis 168 transforming cis-propenylphosphonic acid to fosfomycin

旨在用蛋白质组学方法揭示枯草芽胞杆菌Bacillus subtilis 168将顺丙烯磷酸转化成磷霉素的机理。B.subtilis 168能够将顺丙烯磷酸不对称转化成磷霉素。气相色谱分析发现在转化培养基发酵液中的磷霉素的含量达816.6μg/mL,转化率为36.05%。将分别培养在含有底物和不含底物的培养基中的B.subtilis 168的胞质蛋白进行双向凝胶电泳。对两种条件下的电泳图谱进行比较,发现有98个差异表达蛋白。其中在有底物存在时,表达量下调的点有20个,表达量上调的点52个,底物特异性表达的点有26个。对差异表达蛋白进行质谱鉴定,共鉴定到80个蛋白点,其中下调的点17个,上调的点45个,底物特异性表达的点18个。这些蛋白分别参与胁迫反应、氧化还原反应、物质转运、核苷酸代谢、糖代谢、氨基酸和蛋白质代谢等。根据上述对B.subtilis 168蛋白质组学分析结果,推测菌株是通过两步将顺丙烯磷酸转化成磷霉素的。第一步是水化反应,第二步是脱氢反应。

In this study, we investigated the mechanism of transformation by Bacillus subtilis strain 168 by proteomic analysis. B. subtilis strain 168 was able to stereoselectively transform cis-propenylphosphonic acid （cPPA） to fosfomycin. The maximal fosfomycin production was 816.6μg/mL after two days cultivation, with a conversion rate of 36.05%. We separated the whole cellular proteins by two-dimensional gel electrophoresis （2-DE） method, and 562 protein spots were detected in the presence of cPPA in the medium, while 527 protein spots were detected in the absence of cPPA. Of them, 98 differentially expressed protein spots were found. Among them, 52 proteins were up-regulated whereas 20 were down-regulated in the presence of cPPA in the medium, and 26 induced at the presence of cPPA. The differentially expressed proteins were analyzed by combined MS and MS/MS methods. Eighty protein spots, including 45 up-regulated proteins, 17 down-regulated proteins, and 18 induced by cPPA were identified. Based on the results of proteomic analysis, we postulated two steps of transformation： in the first step, cPPA was hydrated to 2-hydroxypropylphosphonic acid; in the second step, 2-bydroxypropylphosphonic acid was transformed to fosfomycin via a dehydrogenation reaction.