Yu Dan, Lin Hengyi, Bechthold Andreas, Yu Xiaoping, Ma Zheng
Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou, Zhejiang Province 310018, China.
University of Freiburg, Institute for Pharmaceutical Sciences, Pharmaceutical Biology and Biotechnology, 79104 Freiburg, Germany.
Int J Biol Macromol. 2025 Jan;285:138043. doi: 10.1016/j.ijbiomac.2024.138043. Epub 2024 Nov 26.
The TetR family of regulators (TFRs), commonly reported as repressors, plays a role in regulating secondary metabolite production in Streptomyces. In this study, we sought to elucidate the relationship between TFRs and rimocidin production of Streptomyces rimosus M527. Through transcriptomic analysis, we identified the protein RS24090, which exhibited significant differential expression. Phylogenetic analysis of its amino acid sequence and structural alignment predicted it to be a TetR family regulator. Thus, RS24090 was named TetR24. The role of TetR24 in biosynthesis of rimocidin was verified through gene-deletion, -complementation, and -overexpression experiments. The TetR24 gene-deletion mutant (ΔTetR24), which was generated using CRISPR/Cas9 technology, produced 38.08 % more rimocidin than the wild-type (WT) strain M527. Complementary expression of the TetR24 gene in the mutant ΔTetR24 restored rimocidin production to levels comparable to the WT strain. In contrast, the recombinant strain M527-TetR24, which harbored an overexpression of the TetR24 gene, exhibited a 40.31 % decrease in rimocidin production compared to the WT strain. A similar trend in the transcription levels of the rim genes (rimA, rimC, rimG, rimR1, and rimR2), all located in the rimocidin biosynthetic gene cluster, was revealed by quantitative RT-PCR analysis in M527-ΔTetR24, M527-ΔTetR24::TetR24, and M527-TetR24. EMSA and DNase I footprinting assays confirmed that TetR24 regulates the transcription of rim genes by binding to promoter regions of rimA and rimR2.
调控因子的TetR家族（TFRs）通常被报道为阻遏物，在调节链霉菌次生代谢产物的产生中发挥作用。在本研究中，我们试图阐明TFRs与龟裂链霉菌M527产生龟裂杀菌素之间的关系。通过转录组分析，我们鉴定出了表现出显著差异表达的蛋白质RS24090。对其氨基酸序列进行系统发育分析和结构比对预测它是一个TetR家族调控因子。因此，RS24090被命名为TetR24。通过基因缺失、互补和过表达实验验证了TetR24在龟裂杀菌素生物合成中的作用。使用CRISPR/Cas9技术构建的TetR24基因缺失突变体（ΔTetR24）产生的龟裂杀菌素比野生型（WT）菌株M527多38.08%。在突变体ΔTetR24中对TetR24基因进行互补表达使龟裂杀菌素产量恢复到与WT菌株相当的水平。相反，携带TetR24基因过表达的重组菌株M527-TetR24与WT菌株相比，龟裂杀菌素产量降低了40.31%。在M527-ΔTetR24、M527-ΔTetR24::TetR24和M527-TetR24中进行的定量RT-PCR分析揭示了所有位于龟裂杀菌素生物合成基因簇中的rim基因（rimA、rimC、rimG、rimR1和rimR2）转录水平的类似趋势。电泳迁移率变动分析（EMSA）和DNase I足迹分析证实TetR24通过结合rimA和rimR2的启动子区域来调控rim基因的转录。
