SbbR/SbbA, an Important ArpA/AfsA-Like System, Regulates Milbemycin Production in Streptomyces bingchenggensis

Abstract

Milbemycins, a group of 16-membered macrolide antibiotics, are used widely as insecticides and anthelmintics. Previously, a limited understanding of the transcriptional regulation of milbemycin biosynthesis has hampered efforts to enhance antibiotic production by engineering of regulatory genes. Here, a novel ArpA/AfsA-type system, SbbR/SbbA (SBI_08928/SBI_08929), has been identified to be involved in regulating milbemycin biosynthesis in the industrial strain S. bingchenggensis BC04. Inactivation of sbbR in BC04 resulted in markedly decreased production of milbemycin, while deletion of sbbA enhanced milbemycin production. Electrophoresis mobility shift assays (EMSAs) and DNase I footprinting studies showed that SbbR has a specific DNA-binding activity for the promoters of milR (the cluster-situated activator gene for milbemycin production) and the bidirectionally organized genes sbbR and sbbA. Transcriptional analysis suggested that SbbR directly activates the transcription of milR, while represses its own transcription and that of sbbA. Moreover, 11 novel targets of SbbR were additionally found, including seven regulatory genes located in secondary metabolite biosynthetic gene clusters (e.g., sbi_08420, sbi_08432, sbi_09158, sbi_00827, sbi_01376, sbi_09325, and sig24sbh) and four well-known global regulatory genes (e.g., glnRsbh, wblAsbh, atrAsbh, and mtrA/Bsbh). These data suggest that SbbR is not only a direct activator of milbemycin production, but also a pleiotropic regulator that controls the expression of other cluster-situated regulatory genes and global regulatory genes. Overall, this study reveals the upper-layer regulatory system that controls milbemycin biosynthesis, which will not only expand our understanding of the complex regulation in milbemycin biosynthesis, but also provide a basis for an approach to improve milbemycin production via genetic manipulation of SbbR/SbbA system.

Developing an endogenous quorum-sensing based CRISPRi circuit for autonomous and tunable dynamic regulation of multiple targets in industrialStreptomyces

“…Furthermore, we found that the promoter region of srbA showed high DNA sequence identity with that of sbbA from S. bingchengensis ( Fig. S1B), whose transcription is directly regulated by SbbR (31). This clearly suggests that the promoter region of srbA (srbAp) might be under the control of SrbR and srbAp is possible a GBL-responsive promoter in S. rapamycinicus.…”

“…Upon binding to GBL (at a high cell density), the receptor dissociates from target promoters, resulting in transcription derepression. The QS systems have been intensively investigated in several 7 / 39 streptomycetes, such as ArpA/AfsA from Streptomyces griseus (28), ScbR/ScbA from Streptomyces coelicolor (29,30) and SbbR/SbbA from Streptomyces bingchengensis (31). Among them, AfsA, ScbA and SbbA are the GBL synthases responsible for GBL biosynthesis, while ArpA, ScbR and SbbR function as the GBL receptors.…”

Elucidation of the Activation Pathways of ScyA1/ScyR1, an Aco/ArpA-Like System That Regulates the Expression of Nemadectin and Other Secondary Metabolic Biosynthetic Genes

“…All the expression plasmids were verified by nucleotide sequencing and then introduced into E. coli BL21 (DE3), respectively. Purification and protein concentrations were performed as described previously (He et al, 2018;Li C. et al, 2019).…”

“…noncyanogenus NMWT1 genomic DNA with primer pairs pscyA1GFPF/R and ScyR1GFPF/R, respectively. The gfp and the strong constitutive promoter SF14 were amplified from pSET152:P sbbA gfp:SF14sbbR with primer pairs GFPF/R and pSF14F/R, respectively (He et al, 2018). First, P A1 and gfp coding region were ligated into pSET152 using the ClonExpress TM MultiS One Step Cloning Kit (Vazyme) to obtain pSET152:P A1 gfp ( Supplementary Table 1).…”

Получение Авермектинов: Биотехнологии И Органический Синтез

“…Регуляция образования авермектинов в целом происходит в соответствии с общими закономерностями биосинтеза поликетидов (69) у стрептомицетов (81,82). Факторы регуляции биосинтеза авермектинов делятся на общие и специфические (83)(84)(85)(86)(87).…”