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AfsR is an important regulatory factor for growth and butenyl-spinosyn biosynthesis of Saccharopolyspora pogona

2019; BioMed Central; Volume: 69; Issue: 8 Linguagem: Inglês

10.1007/s13213-019-01473-8

1869-2044

Li Li, Liang Gong, Haocheng He, Zhudong Liu, Jie Rang, Jianli Tang, Shengnan Peng, Shuangqin Yuan, Xuezhi Ding, Ziquan Yu, Liqiu Xia, Yunjun Sun,

Fungal and yeast genetics research

To generate a AfsR-like (AfsR-L) overexpression strain Saccharopolyspora pogona-AfsR-L and investigate its effects on the morphology and metabolism of S. pogona. Firstly, we generated the overexpression vector pOJ260-PermE-afsR-L via overlap extension PCR. Then, the recombination strain S. pogona-AfsR-L was constructed via conjugal transfer. To monitor the growth and morphology, mycelia and sporulation were observed. The distinctive proteins and butenyl-spinosyn biosynthesis were investigated by SDS-PAGE, HPLC, and mass spectrometry. And the transcriptional level of afsR-L and other relative functional genes in S. pogona-AfsR-L was analyzed by qRT-PCR. Western blot verified the increased amount of AfsR-L protein in the overexpression strain. Growth curve and mycelia observation showed that afsR-L overexpression make the stationary phase of S. pogona-AfsR-L longer than that of wild S. pogona by approximate 3 days. Moreover, S. pogona-AfsR-L exhibited a more obvious white phenotype on the solid medium, which means afsR-L overexpression affects the sporulation ability of S. pogona. HPLC analysis revealed that the peak area of the butenyl-spinosyn yield of S. pogona-AfsR-L was 293.6, while that of S. pogona was 250.9. SDS-PAGE analysis showed that the two strains had different whole protein expression profiles, and the distinctive proteins were further identified by LC-MS/MS identification, which showed the possible control mechanism of afsR-L gene in S. pogona. We concluded that AfsR could directly or indirectly positively regulate the biosynthesis of butenyl-spinosyn and affect the growth features of S. pogona. We envisioned that this result can be expanded to other Streptomyces for strain improvement.