Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Xueyuan Street, Xiasha Higher Education District, Hangzhou, 310018, Zhejiang, People's Republic of China.
Institute for Pharmaceutical Sciences, Pharmaceutical Biology and Biotechnology, University of Freiburg, 79104, Freiburg, Germany.
Microb Cell Fact. 2023 Feb 21;22(1):32. doi: 10.1186/s12934-023-02039-9.
Streoptomyces rimosus M527 is a producer of the polyene macrolide rimocidin which shows activity against various plant pathogenic fungi. Notably, the regulatory mechanisms underlying rimocidin biosynthesis are yet to be elucidated.
In this study, using domain structure and amino acid alignment and phylogenetic tree construction, rimR2, which located in the rimocidin biosynthetic gene cluster, was first found and identified as a larger ATP-binding regulators of the LuxR family (LAL) subfamily regulator. The rimR2 deletion and complementation assays were conducted to explore its role. Mutant M527-ΔrimR2 lost its ability to produce rimocidin. Complementation of M527-ΔrimR2 restored rimocidin production. The five recombinant strains, M527-ER, M527-KR, M527-21R, M527-57R, and M527-NR, were constructed by overexpressing rimR2 gene using the promoters permE, kasOp, SPL21, SPL57, and its native promoter, respectively, to improve rimocidin production. M527-KR, M527-NR, and M527-ER exhibited 81.8%, 68.1%, and 54.5% more rimocidin production, respectively, than the wild-type (WT) strain, while recombinant strains M527-21R and M527-57R exhibited no obvious differences in rimocidin production compared with the WT strain. RT-PCR assays revealed that the transcriptional levels of the rim genes were consistent with the changes in rimocidin production in the recombinant strains. Using electrophoretic mobility shift assays, we confirmed that RimR2 can bind to the promoter regions of rimA and rimC.
A LAL regulator RimR2 was identified as a positive specific-pathway regulator of rimocidin biosynthesis in M527. RimR2 regulates the rimocidin biosynthesis by influencing the transcriptional levels of rim genes and binding to the promoter regions of rimA and rimC.
嗜热链霉菌 M527 是聚烯大环内酯类利莫菌素的产生菌，对各种植物病原真菌具有活性。值得注意的是，利莫菌素生物合成的调控机制尚未阐明。
在这项研究中，通过结构域、氨基酸比对和系统发育树构建，首次发现并鉴定了位于利莫菌素生物合成基因簇中的 rimR2，它是更大的 ATP 结合调节因子 LuxR 家族（LAL）亚家族调节因子。进行了 rimR2 缺失和互补实验，以探讨其作用。突变株 M527-ΔrimR2 丧失了产生利莫菌素的能力。M527-ΔrimR2 的互补恢复了利莫菌素的产生。通过使用 permE、kasOp、SPL21、SPL57 和其天然启动子分别过表达 rimR2 基因，构建了五个重组菌株 M527-ER、M527-KR、M527-21R、M527-57R 和 M527-NR，以提高利莫菌素的产量。与野生型（WT）菌株相比，M527-KR、M527-NR 和 M527-ER 分别表现出 81.8%、68.1%和 54.5%的利莫菌素产量增加，而重组菌株 M527-21R 和 M527-57R 的利莫菌素产量与 WT 菌株相比没有明显差异。RT-PCR 分析表明，rim 基因的转录水平与重组菌株中利莫菌素产量的变化一致。通过电泳迁移率变动分析，我们证实 RimR2 可以结合 rimA 和 rimC 的启动子区域。
鉴定出 LAL 调节因子 RimR2 是 M527 中 rimocidin 生物合成的正特异途径调节因子。RimR2 通过影响 rim 基因的转录水平和结合 rimA 和 rimC 的启动子区域来调节 rimocidin 生物合成。
