卡西霉素生物合成基因簇中调控基因calR1的功能

Functional analysis of the regulatory gene calR1 in calcimycin biosynthetic gene cluster

摘要:

【背景】卡西霉素(calcimycin)是重要的离子载体抗生素，其生物合成基因簇已从教酒链霉菌NRRL3882的基因组DNA中成功克隆，但基因簇内的部分生物合成基因及调控基因的功能有待研究。【目的】研究卡西霉素产生菌教酒链霉菌NRRL3882中编码TylR家族同源转录调控蛋白的calR1基因的功能。【方法】通过PCR-targeting的方法，构建calR1基因敲除突变株及回补菌株，对突变菌株及回补菌株进行发酵，通过HPLC分析其代谢产物。利用荧光定量PCR检测ΔcalR1突变菌株和野生菌株的生物合成基因转录水平。【结果】calR1基因敲除突变株丧失产生卡西霉素的能力，但仍有中间产物噻唑霉素的积累，回补菌株中卡西霉素的产量有一定程度的恢复。RT-qPCR结果表明，卡西霉素合成相关的一些重要基因calC、calG、calU3等基因的表达量明显改变。【结论】TylR家族转录调控基因calR1是卡西霉素生物合成的调控基因。

Abstract:

[Background] Calcimycin is an essential ionophoric antibiotic. The gene cluster for calcimycin biosynthesis has been cloned from the genome of Streptomyces chartreusis NRRL3882, but functions of some biosynthetic genes and regulatory genes in calcimycin biosynthesis remain unclear. [Objective] To study the functions of the putative regulatory gene calR1 encoding TylR-family transcriptional regulator in the biosynthetic gene cluster of calcimycin. [Methods] ThecalR1-knockout mutant and complementary strain were constructed by PCR-targeting and fermented, and their metabolites were analyzed by HPLC. The transcriptions of biosynthetic genes in ΔcalR1 mutant and wild-type strain were analyzed by RT-qPCR. [Results] ΔcalR1 mutant lost the ability to produce calcimycin, while the accumulation of cezomycin was observed. The complementary strain restored the production of calcimyc into a certain degree. The transcriptions of some essential genes, including calC, calG, and calU3, were substantially changed in ΔcalR1 mutant compared with those in the wild-type strain. [Conclusion] The putative TylR-family transcription regulatory gene calR1 is involved in calcimycin biosynthesis.