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 target, and the kanamycin resistance gene (neo) under the control of PrimA was chosen as 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. Seventy-nine mutants were obtained in total, of which sixty-seven mutants showed 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 to M527-pAN. Notably, three 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 to 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 to M527-pAN. These results confirmed that the RGMS approach was successful in improving the rimocidin production in S. rimosus M527.

中文翻译：

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通过报告基因突变选择提高 Streptomyces rimosus M527 中利莫西丁的产量

在这项研究中，我们采用报告基因引导的突变选择 (RGMS) 策略来提高 Streptomyces rimosus M527 的利莫西丁产量，该策略基于单一报告基因质粒 pAN 和大气和室温等离子体 (ARTP)。在质粒 pAN 中，选择利莫西丁生物合成加载模块 (RimA) 的天然启动子 PrimA 作为靶标，并选择 PrimA 控制下的卡那霉素抗性基因 (neo) 作为报告基因。通过接合将整合质粒 pAN 引入到 S. rimosus M527 的染色体中，以产生初始菌株 S. rimosus M527-pAN。随后，M527-pAN 的突变体由 ARTP 生成。共获得79个突变体，其中67个突变体表现出比初始菌株M527-pAN更高水平的卡那霉素抗性(Kanr)。与 M527-pAN 相比，大多数突变体的利莫西丁产量略有增加。值得注意的是，三种突变体 M527-pAN-S34、S38 和 S52 在所有 Kanr 突变体中表现出最高的卡那霉素抗性，与 M527-pAN 相比，利莫西丁产量分别增加了 34%、52% 和 45%。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 基因的转录水平增加。这些结果证实，RGMS 方法成功地提高了 S. rimosus M527 中利莫西丁的产量。与 M527-pAN 相比，利莫西丁产量分别增加了 52% 和 45%。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 基因的转录水平增加。这些结果证实，RGMS 方法成功地提高了 S. rimosus M527 中利莫西丁的产量。与 M527-pAN 相比，利莫西丁产量分别增加了 52% 和 45%。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 基因的转录水平增加。这些结果证实，RGMS 方法成功地提高了 S. rimosus M527 中利莫西丁的产量。