RS24090, a TetR family transcriptional repressor, negatively affects the rimocidin biosynthesis in Streptomyces rimosus M527

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.