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Streptomyces bingchenggensis is the main industrial producer of milbemycins, which are a group of 16-membered macrocylic lactones with excellent acaricidal, insecticidal and anthelmintic activities. In past decades, scientists tried a lot to solve its low productivity. But less understanding the regulatory network of milbemycin biosynthesis limited the generation of high producing strains with regulatory rewiring approach. SARPs（Streptomyces Antibiotic Regulatory Proteins）family regulators are widely distributed and play key roles in regulating antibiotics production in actinobacteria. In this paper, MilR3 (encoded by sbi_06842) has been screened out for significantly affecting milbemycin production from all the 19 putative SARP family regulators in S. bingchenggensis with the DNase-deactivated Cpf1-based integrative CRISPRi system. Interestingly, milR3 is about 7 Mb away from milbemycin biosynthetic gene cluster and adjacent to a putative type II PKS （the core minimal PKS encoding genes are sbi_06843, sbi_06844 and sbi_06845）gene cluster, which was proved to be responsible for producing a yellow pigment. The quantitative real-time PCR (qRT-PCR) analysis proved that MilR3 positively affected the transcription of milR, milA1, milA2, milA4, milF, milR3 and sbi_06844 in both clusters. Unlike previous “small” SARP family regulators played pathway specific roles, MilR3 was probably a unique SARP family regulator and played a pleotropic role. MilR3 was an upper-level regulator in MilR3-MilR cascade. This study firstly illustrated the co-regulatory role of this unique SARP regulator. This greatly enrich our understanding of SARPs and lay a solid foundation for milbemycin yield enhancement in the near future. |
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