金霉素生物合成基因簇中调控基因ctcB的功能

Function of Streptomyces antibiotic regulatory proteins family transcriptional regulator ctcB in the biosynthetic cluster of chlortetracycline

摘要:

[目的]研究金霉素产生菌中SARP家族转录调控基因ctcB的作用。[方法]利用大肠杆菌、链霉菌的属间接合转移和同源重组双交换的方法，构建ctcB基因缺失突变株。通过cDNA在相邻同转录方向的基因间隔进行PCR验证，确定金霉素生物合成基因簇中的转录单元。利用荧光定量RT-PCR方法进行突变株金霉素生物合成基因簇的转录水平检测。随后，生物信息学预测分析了金霉素生物合成基因簇内CtcB与DNA的结合位点。[结果]获得了ctcB基因缺失的双交换突变株。发酵结果显示，该突变株失去产生金霉素与四环素的能力。金霉素生物合成基因簇内有6个共转录单元，其中4个共转录单元在ctcB基因缺失突变株中转录水平明显下降。软件分析预测到一致性较高的CtcB结合重复序列。[结论]ctcB正调控金霉素生物合成结构基因ctcG-D、ctcH-K、ctcN-P、ctcW-T 4个转录单元和ctcQ，为进一步研究ctcB调控机制奠定了基础。

关键词:金霉素;SARP家族转录调控基因;基因敲除;生物合成

Abstract:

[Objective] In order to understand the regulatory mechanisms of chlortetracycline biosynthesis in an industrial strain, function of an Streptomyces antibiotic regulatory proteins (SARP) family transcriptional regulator ctcB in the biosynthetic gene cluster of chlortetracycline was studied.[Methods] By double crossover recombination, we constructed Streptomyces aureofaciens F3 with disrupted SARP family transcriptional regulator ctcB gene. The amplicons of RT-PCR were designed to cover the adjacent genes for verification of the operons in chlortetracycline biosynthetic cluster. Transcriptional level was analyzed in the chlortetracycline biosynthetic gene cluster in the wild type strain and the ctcB gene disrupted mutant LJIA02 by quantitative real-time RT-PCR.[Results] The disruption mutant LJIA02 abolished tetracycline and chlortetracycline production. In RT-PCR six operons were confirmed in chlortetracycline biosynthetic cluster. Quantitative real-time RT-PCR indicated that ctcB directly activated five promoters from ctcG-D, ctcH-K, ctcN-P, ctcW-T and ctcQ.[Conclusion] CtcB is an essential activator as an SARP family transcriptional regulator in the chlortetracycline biosynthetic gene cluster.

Key words:chlortetracycline;Streptomyces antibiotic regulatory proteins (SARP) family transcriptional regulator;gene disruption;biosynthesis