摘要

林可霉素A（Lin-A）是广泛使用的由林肯链霉菌（Streptomyces lincolnensis）发酵的抗菌抗生素。然而，林可霉素生物合成的转录调节机制很少被研究。在这里，我们首先确定了一种TetR家族转录调节因子（TFR），SLCG_2919，负责调节林肯链球菌LCGL中的林可霉素生物合成。发现SLCG_2919特异性结合林可霉素生物合成基因簇（lin簇）的启动子区域，包括24个结构基因，3个抗性基因和1个调控基因，并抑制这些基因的转录，证明了其直接调节作用。林可霉素生物合成。此外，我们发现SLCG_2919不是自动调节的，而是直接抑制其编码ATP / GTP结合蛋白的相邻基因SLCG_2920，其过表达增加了对林可霉素的耐药性和林肯A的产量。精确的SLCG_2919结合位点SLCG_2920的启动子区域通过DNA酶I足迹测定法和基于碱基置换诱变的EMSAs测定，内部10-nt富含AT的序列（AAATTATTTA）显示对于SLCG_2919结合是必需的。我们的研究结果表明，SLCG_2919是控制林肯氏菌中林可霉素生物合成的负调节剂。本研究提高了我们对林可霉素生物合成的分子调控的理解。重要性TetR家族转录调节因子（TFRs）通常被发现可调节细菌中不同的细胞过程，尤其是链霉菌中的抗生素生物合成然而，对它们在林肯氏菌中林可霉素生物合成中的功能的了解仍然未知。本研究提供了通过TFR，SLCG_2919调节林可霉素生物合成的新见解，其直接调节林可霉素的产生和抗性。有趣的是，SLCG_2919及其相邻基因SLCG_2920编码ATP / GTP结合蛋白，广泛分布于不同的链霉菌属物种中。此外，我们揭示了一种新的TFR结合基序，其中SLCG_2919与SLCG_2920的启动子区域结合，取决于介入的富含AT的序列而不是在其他TFR的结合位点中发现的侧翼反向重复序列。通过SLCG_2919对林可霉素生物合成的转录调控的这些见解将为链霉菌中的调节元件的基因工程提供有价值的方式以改善抗生素的产生。


Lincomycin A (Lin-A) is a widely used anti-bacterial antibiotic fermented by Streptomyces lincolnensis However, the transcriptionally regulatory mechanisms underlying lincomycin biosynthesis have seldom been investigated. Here, we first identified a TetR family transcriptional regulator (TFR), SLCG_2919, negatively modulating lincomycin biosynthesis in S. lincolnensis LCGL. SLCG_2919 was found to specifically bind to promoter regions of the lincomycin biosynthetic gene cluster (lin cluster), including twenty four structural genes, three resistance genes, and one regulatory gene, and to inhibit the transcription of these genes, demonstrating the directly regulatory role in lincomycin biosynthesis. Further, we found that SLCG_2919 was not auto-regulated, but directly repressed its adjacent gene SLCG_2920 encoding an ATP/GTP binding protein, whose overexpression increased the resistance against lincomycin and Lin-A yields in S.lincolnensis The precise SLCG_2919-binding site within the promoter region of SLCG_2920 was determined by a DNase I footprinting assay and by EMSAs based on base substitution mutagenesis, with the internal 10-nt AT-rich sequence (AAATTATTTA) shown to be essential for SLCG_2919 binding. Our findings indicate that SLCG_2919 is a negative regulator for controlling lincomycin biosynthesis in S. lincolnensis The present study improves our understanding of molecular regulation for lincomycin biosynthesis.IMPORTANCE TetR family transcriptional regulator (TFRs) are generally found to regulate diverse cellular processes in bacteria, especially antibiotic biosynthesis in Streptomyces However, knowledge of their function in lincomycin biosynthesis in S. lincolnensis remains unknown. The present study provides a new insight into the regulation of lincomycin biosynthesis through a TFR, SLCG_2919, that directly modulates lincomycin production and resistance. Intriguingly SLCG_2919 and its adjoining gene SLCG_2920 encoding an ATP/GTP binding protein were extensively distributed in diverse Streptomyces species. In addition, we revealed a new TFR binding motif, in which SLCG_2919 binds to the promoter region of SLCG_2920, dependent on the intervening AT-rich sequence rather than the flanking inverted repeats found in other TFRs' binding sites. These insights into transcriptional regulation of lincomycin biosynthesis by SLCG_2919 will set a valuable way for genetic engineering of regulatory elements in Streptomyces to improve antibiotic production.

https://www.ncbi.nlm.nih.gov/pubmed/30341075