Marinacarbolines甲基化基因mcbD的筛选及其功能

发布时间：2018-05-03 22:31

本文选题：marinacarboline + 甲基化　；参考：《微生物学报》2017年07期

【摘要】：【目的】定位Marinactinospora thermotolerans SCSIO 00652基因组中负责marinacarbolines甲基化修饰的基因mcbD并鉴定其功能。【方法】游离M.thermotolerans SCSIO 00652的基因组序列中功能注释为甲基转移酶的蛋白,使用MEGA 6自带的ClustalW与来自于marformycins生物合成途径中的Mfn G(D/L-酪氨酸甲基化酶)进行多序列比对,用邻接法(Neighbor-Joining)构建系统进化树,筛选到与MfnG进化关系最近的Orf03255(Mcb D);扩增mcbD后克隆至pET28a(+)并在E.coli BL21(DE3)中表达,结合Ni-NTA亲和层析法纯化获取较高纯度的McbD活性蛋白;以marinacarboline B为底物,利用高效液相色谱检测McbD的体外酶活;利用基于PCR-targeting的遗传操作体系构建mcb D插入失活突变株((35)mcbD)并分析其与野生型菌株的发酵产物差异。【结果】N-末端融合组氨酸标签的McbD蛋白在E.coli BL21(DE3)中可溶性表达,亲和层析纯化的McbD能够以marinacarboline B为底物催化形成marinacarboline C;mcbD基因阻断突变株ΔmcbD与野生型相比不产生化合物marinacarboline C,同时累积marinacarboline B。【结论】McbD为marinacarbolines生物合成中必需的O-甲基转移酶。
[Abstract]:[objective] to locate the mcbD gene responsible for marinacarbolines methylation modification in the genome of Marinactinospora thermotolerans SCSIO 00652 and to identify its function. [methods] the genome sequence of free M.thermotolerans SCSIO 00652 was annotated as a methyltransferase protein. Using MEGA 6's own ClustalW and Mfn Gon D / L tyrosine methylase from the marformycins biosynthesis pathway, a phylogenetic tree was constructed by using neighbor method (Neighbor-Jointing). Orf03255(Mcb DX, which had the closest relationship with MfnG evolution, was screened, cloned into pET28a () and expressed in E.coli BL21DE3, then purified by Ni-NTA affinity chromatography to obtain high purity McbD active protein. Marinacarboline B was used as substrate. High performance liquid chromatography (HPLC) was used to detect the enzyme activity of McbD in vitro. PCR-targeting based genetic manipulation system was used to construct a mcb D insertion inactivated mutant, CbD35, and to analyze its difference from the wild-type strain. [results] the N-terminal fusion histidine tagged McbD protein was soluble expressed in E.coli BL21D3. McbD purified by affinity chromatography could catalyze the formation of marinacarboline CmcbD gene blocking mutant 螖 mcbD with marinacarboline B as substrate, and not produce a compound marinacarboline C and accumulate marinacarboline B. [conclusion] McbD is the necessary Omethyltransferase in marinacarbolines biosynthesis.
【作者单位】：安徽医科大学生命科学学院;中国科学院南海海洋研究所中国科学院热带海洋生物资源与生态重点实验室广东省海洋药物重点实验室中国科学院海洋微生物研究中心;中国科学院大学;
【基金】：安徽医科大学博士科研资助基金(XJ201512) 国家自然科学基金(41406195)~~
【分类号】：Q78

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