Identification of a TetR family regulator and a polyketide synthase gene cluster involved in growth development and butenyl-spinosyn biosynthesis of Saccharopolyspora pogona

• Published in: Applied microbiology and biotechnology 2021-02, Vol.105 (4), p.1519-1533
• Main Authors: Rang, Jie, Zhu, Zirong, Li, Yunlong, Cao, Li, He, Haocheng, Tang, Jianli, Hu, Jinjuan, Chen, Jianming, Hu, Shengbiao, Huang, Weitao, Yu, Ziquan, Ding, Xuezhi, Sun, Yunjun, Xie, Qingji, Xia, Liqiu
• Format: Article
• Language: English
• Subjects: Actinomycetes; Analysis; Antibiotics; Applied Genetics and Molecular Biotechnology; Bacteria; Biocides; Biomedical and Life Sciences; Biosynthesis; Biotechnology; Cell growth; CRISPR; Cytology; Deletion; Editing; Genes; Genetic aspects; Genetic modification; Genome editing; Genomes; Growth; Identification and classification; Life Sciences; Macrolides; Microbial Genetics and Genomics; Microbiology; Morphology; Multigene Family; Pesticides; Polyketide synthase; Polyketide Synthases - genetics; Polyketides; Production processes; Proteins; Proteomics; Regulatory mechanisms (biology); Repressor proteins; Saccharopolyspora; Saccharopolyspora - genetics; Sporulation
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-021-11105-4

Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and broad pesticidal spectrum. However, its synthetic level was low in the wild-type strain. At present, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficiently editing its genome to improve the butenyl-spinosyn yield. To accelerate the genetic modification of S. pogona , we conducted comparative proteomics analysis to screen differentially expressed proteins related to butenyl-spinosyn biosynthesis. A TetR family regulatory protein was selected from the 289 differentially expressed proteins, and its encoding gene (SP_1288) was successfully deleted by CRISPR/Cas9 system. We further deleted a 32-kb polyketide synthase gene cluster (cluster 28) to reduce the competition for precursors. Phenotypic analysis revealed that the deletion of the SP_1288 and cluster 28 resulted in a 3.10-fold increase and a 35.4% decrease in the butenyl-spinosyn levels compared with the wild-type strain, respectively. The deletion of cluster 28 affected the cell growth, glucose consumption, mycelium morphology, and sporulation by controlling the expression of ptsH , ptsI , amfC , and other genes related to sporulation, whereas SP_1288 did not. These findings confirmed not only that the CRISPR/Cas9 system can be applied to the S. pogona genome editing but also that SP_1288 and cluster 28 are closely related to the butenyl-spinosyn biosynthesis and growth development of S. pogona . The strategy reported here will be useful to reveal the regulatory mechanism of butenyl-spinosyn and improve antibiotic production in other actinomycetes. Key points • SP_1288 deletion can significantly promote the butenyl-spinosyn biosynthesis. • Cluster 28 deletion showed pleiotropic effects on S. pogona. • SP_1288 and cluster 28 were deleted by CRISPR/Cas9 system in S. pogona.