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

Abstract

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.