Department of Chemical and Pharmaceutical Engineering, Hefei Normal University, Hefei, China.
School of Life Sciences, Institute of Physical Science and Information Technology, Anhui University, Hefei, China.
J Basic Microbiol. 2024 Jan;64(1):119-127. doi: 10.1002/jobm.202300203. Epub 2023 Aug 10.
The transcription factor (TF)-mediated regulatory network controlling lincomycin production in Streptomyces lincolnensis is yet to be fully elucidated despite several types of associated TFs having been reported. SLCG_2919, a tetracycline repressor (TetR)-type regulator, was the first TF to be characterized outside the lincomycin biosynthetic cluster to directly suppress the lincomycin biosynthesis in S. lincolnensis. In this study, improved genomic systematic evolution of ligands by exponential enrichment (gSELEX), an in vitro technique, was adopted to capture additional SLCG_2919-targeted sequences harboring the promoter regions of SLCG_6675, SLCG_4123-4124, SLCG_6579, and SLCG_0139-0140. The four DNA fragments were confirmed by electrophoretic mobility shift assays (EMSAs). Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) showed that the corresponding target genes SLCG_6675 (anthranilate synthase), SLCG_0139 (LysR family transcriptional regulator), SLCG_0140 (beta-lactamase), SLCG_6579 (cytochrome P450), SLCG_4123 (bifunctional DNA primase/polymerase), and SLCG_4124 (magnesium or magnesium-dependent protein phosphatase) in ΔSLCGL_2919 were differentially increased by 3.3-, 4.2-, 3.2-, 2.5-, 4.6-, and 2.2-fold relative to those in the parental strain S. lincolnensis LCGL. Furthermore, the individual inactivation of these target genes in LCGL reduced the lincomycin yield to varying degrees. This investigation expands on the known DNA targets of SLCG_2919 to control lincomycin production and lays the foundation for improving industrial lincomycin yields via genetic engineering of this regulatory network.
尽管已经报道了几种类型的相关 TF，但调控林肯链霉菌林肯霉素产生的转录因子 (TF)-介导的调控网络尚未完全阐明。SLCG_2919 是一种四环素阻遏物 (TetR) 型调控因子，是第一个在林肯霉素生物合成簇之外被表征的 TF，它直接抑制林肯霉素在林肯链霉菌中的生物合成。在这项研究中，采用了体外技术改进的基因组系统进化配体指数富集 (gSELEX) 来捕获额外的 SLCG_2919 靶向序列，这些序列含有 SLCG_6675、SLCG_4123-4124、SLCG_6579 和 SLCG_0139-0140 的启动子区域。通过电泳迁移率变动分析 (EMSA) 验证了这四个 DNA 片段。逆转录定量聚合酶链反应 (RT-qPCR) 显示，相应的靶基因 SLCG_6675（邻氨基苯甲酸合酶）、SLCG_0139（LysR 家族转录调节因子）、SLCG_0140（β-内酰胺酶）、SLCG_6579（细胞色素 P450）、SLCG_4123（双功能 DNA 引物/聚合酶）和 SLCG_4124（镁或镁依赖的蛋白磷酸酶）在ΔSLCGL_2919 中的表达分别比亲本菌株 S. lincolnensis LCGL 中的表达增加了 3.3、4.2、3.2、2.5、4.6 和 2.2 倍。此外，LCGL 中这些靶基因的单独失活在不同程度上降低了林肯霉素的产量。这项研究扩展了 SLCG_2919 已知的 DNA 靶标，以控制林肯霉素的产生，并为通过遗传工程改造这个调控网络来提高工业林肯霉素的产量奠定了基础。
