Improvement of rimocidin production in Streptomyces rimosus M527 by reporter-guided mutation selection.
Improvement of rimocidin production in Streptomyces rimosus M527 by reporter-guided mutation selection.
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DOI：
10.1093/jimb/kuac030
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发表时间：
2023-02-13
影响因子：
3.4
通讯作者：
中科院分区：
文献类型：
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作者：
在这项研究中，我们采用了一个细菌导向突变选择（RGMS）的策略，以提高rimocidin生产的龟裂链霉菌M527，这是基于一个单报告质粒pAN和大气和室温等离子体（ARTP）。在质粒pAN中，选择了裂解霉素生物合成装载模块（RimA）的天然启动子PrimA作为靶基因，并选择PrimA控制下的卡那霉素抗性基因（neo）作为报告基因。将整合质粒pAN导入S. rimosus M527进行接合以产生初始菌株S.裂膜菌M527-pAN.随后，通过ARTP产生M527-pAN的突变体。共获得79株突变株，其中67株突变株的卡那霉素抗性（Kanr）高于出发菌株M527-pAN。与M527-pAN相比，大多数突变体显示出杀rimocidin产量的轻微增加。值得注意的是，在所有Kanr突变体中表现出最高卡那霉素抗性的3个突变体，M527-pAN-S34、S38和S52，与M527-pAN相比，分别显示出34%、52%和45%的杀菌素产量增加。定量RT-PCR分析表明，与M527-pAN相比，突变体M527-pAN-S34、S38和S52中neo和rim基因的转录水平均有所提高。这些结果证实了RGMS方法在提高S.裂膜菌M527。
In this study, we employed a reporter-guided mutation selection (RGMS) strategy to improve the rimocidin production of Streptomyces rimosus M527, which is based on a single-reporter plasmid pAN and atmospheric and room temperature plasma (ARTP). In plasmid pAN, PrimA, a native promoter of the loading module of rimocidin biosynthesis (RimA) was chosen as a target, and the kanamycin resistance gene (neo) under the control of PrimA was chosen as the reporter gene. The integrative plasmid pAN was introduced into the chromosome of S. rimosus M527 by conjugation to yield the initial strain S. rimosus M527-pAN. Subsequently, mutants of M527-pAN were generated by ARTP. 79 mutants were obtained in total, of which 67 mutants showed a higher level of kanamycin resistance (Kanr) than that of the initial strain M527-pAN. The majority of mutants exhibited a slight increase in rimocidin production compared with M527-pAN. Notably, 3 mutants, M527-pAN-S34, S38, and S52, which exhibited highest kanamycin resistance among all Kanr mutants, showed 34%, 52%, and 45% increase in rimocidin production compared with M527-pAN, respectively. Quantitative RT-PCR analysis revealed that the transcriptional levels of neo and rim genes were increased in mutants M527-pAN-S34, S38, and S52 compared with M527-pAN. These results confirmed that the RGMS approach was successful in improving the rimocidin production in S. rimosus M527.
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