AUTHOR=He Hairong , Ye Lan , Li Chuang , Wang Haiyan , Guo Xiaowei , Wang Xiangjing , Zhang Yanyan , Xiang Wensheng 

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

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.01064

DOI=10.3389/fmicb.2018.01064

ISSN=1664-302X

ABSTRACT=<p>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 <italic>S. bingchenggensis</italic> BC04. Inactivation of <italic>sbbR</italic> in BC04 resulted in markedly decreased production of milbemycin, while deletion of <italic>sbbA</italic> 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 <italic>milR</italic> (the cluster-situated activator gene for milbemycin production) and the bidirectionally organized genes <italic>sbbR</italic> and <italic>sbbA</italic>. Transcriptional analysis suggested that SbbR directly activates the transcription of <italic>milR</italic>, while represses its own transcription and that of <italic>sbbA</italic>. Moreover, 11 novel targets of SbbR were additionally found, including seven regulatory genes located in secondary metabolite biosynthetic gene clusters (e.g., <italic>sbi_08420</italic>, <italic>sbi_08432</italic>, <italic>sbi_09158</italic>, <italic>sbi_00827</italic>, <italic>sbi_01376</italic>, <italic>sbi_09325</italic>, and <italic>sig24<sub>sbh</sub></italic>) and four well-known global regulatory genes (e.g., <italic>glnR<sub>sbh</sub></italic>, <italic>wblA<sub>sbh</sub></italic>, <italic>atrA<sub>sbh</sub></italic>, and <italic>mtrA/B<sub>sbh</sub></italic>). 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.</p>