Marinacarbolines甲基化基因mcbD的筛选及其功能

Screening and characterization of methylation tailoring gene-mcbD in marinacarboline biosynthesis

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MfnG（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（McbD）；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mcbD插入失活突变株（ΔmcbD）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.coliBL21（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] The aim of this study was to screen the methylation tailoring gene-mcbD in marinacarboline biosynthesis pathway from the genomic sequence of Marinactinospora thermotolerans SCSIO 00652, and to characterize McbD both in vitro and in vivo.[Methods] Orf03255 (McbD) was selected by phylogenetic analysis of the methyltransferases from M. thermotolerans SCSIO 00652 with MfnG (D/L-Tyr methyltransferase) using Mega6. The mcbD gene was amplified from the M. thermotolerans SCSIO 00652 genomic DNA, cloned into pET28a(+) vector, and expressed in E. coli BL21 (DE3); the McbD protein was purified with Ni-NTA affinity chromatography. The McbD-mediated enzymatic reaction was performed with marinacarboline B as substrate and detected with HPLC. The ΔmcbD mutant was constructed with PCR-targeting genetic manipulation system. Difference in fermentation extracts between M. thermotolerans SCSIO 00652 wild-type and ΔmcbD was analyzed with HPLC.[Results] The N-His tagged McbD was successfully expressed in E. coli BL21 (DE3) in soluble form, and purified by Ni-NTA affinity chromatography; further biochemical reaction showed that McbD can catalyze the transformation of marinacarboline B to marinacarboline C with SAM. The ΔmcbD inactivated mutant completely abolished the production of marinacarboline C, and accumulated marinacarboline B.[Conclusion] McbD, responsible for the methylation tailoring of marinacarboline, is indispensable in marinacarboline biosynthetic pathway.