genP基因在依纽小单孢菌中表达的分析

Exploration of the expression of genP in Micromonospora inyoens

庆大霉素生物合成过程中的genP基因和西索米星生物合成过程中的sisI基因属同工异源酶基因,均能催化绛红糖胺3′,4′-双脱羟基反应。本研究借助组合生物合成,探索绛红小单孢菌中的genP基因能否在依纽小单孢菌中表达。首先构建genP基因替换sisI基因的同源重组质粒pKPI4,并经接合转移将其导入依纽小单孢菌TS388中,最后再通过影印筛选及PCR鉴定获得genP基因替换sisI基因的工程菌PTI886。将野生型菌株TS388、sisI基因缺失突变株TI1102(TS388△sisI)及工程菌PTI886在同等条件下进行发酵,提取其代谢产物并经薄层层析及质谱分析。结果表明,突变株TI1102不能合成西索米星,而工程菌PTI886仍能继续合成西索米星,且代谢产物组分与野生型菌株TS388相比无明显变化,说明genP基因能在依纽小单孢菌中表达。本研究揭示了同工异源酶基因异源表达的可行性,可为今后不同微生物异源基因组合的研究提供借鉴。

The genP in gentamicin biosynthesis and sisI in sisomicin biosynthesis belong to heterologous genes encoding isozyme,and both can catalyze double dehydroxylation of purpurosamine at the C-3' and C-4' position.The express characterization of the genP of M.purpurea G1008 in M.inyoens TS388 was explored by combinatorial biosynthesis in this study.The recombinant plasmid pKPI4 used for the replacement of sisi with genP was constructed and was then introduced into M.inyoensis TS388 by conjugation.Finally,the mutant M.inyoensis PTI886 containing genP instead of sisi was screened out by replica plating and PCR validation method.Wild-type strain TS388,sisi deletion mutant TI1102(TS388 △sisI),and engineered bacteria PTI886 were fermented under the same conditions.Subsequently,their metabolites was isolated from the fermentation broth and then analyzed by thin-layer chromatography and mass spectrometry.The result indicates that TI1102 can't synthesize any sisomicin,while PTI886 continually can,and the metabolites components were roughly the same as that of the wild-type strain TS388.The results showed that genP can express in M.inyoens successfully.This research reveals that the heterologous expression of the heterologous genes encoding isozyme is feasible,which can provide a reference for the future study of heterologous gene combination of different microbial.