AfsR is an important regulatory factor for growth and butenyl-spinosyn biosynthesis of Saccharopolyspora pogona
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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.
KeywordsSaccharopolyspora pogona AfsR Regulatory factor Butenyl-spinosyn biosynthesis Strain morphology
L. L. and S. Y. developed the bacterial recombinant and performed bacterial strain isolation. Y. S., L. G., and J. T. performed HPLC. J. R., X. D., and Z. L. performed LC-MS/MS and data analysis. L. G., H. H., Z. Y., and S. P. performed Total RNA isolation and qRT-PCR analysis. L. G. and H. H. performed Western blot. L. L., H. H., Y. S., and L. X. designed the study and wrote the draft of manuscript. L. G. and Y. S. modified and corrected this manuscript. All authors discussed the results and approved the final manuscript.
This work was supported by funding from the National Natural Science Foundation of China (31770106), the National Basic Research Program (973) of China (2012CB722301), the Major Research Projects in Hunan Province (2017NK1030), and the Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Research involving human participants and/or animals
Ethical approval “All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.” All procedures performed in studies involving animals were in accordance with the ethical standards of the Animal Care Committee of Hunan Normal University at which the studies were conducted.
Informed consent was obtained from all individual participants included in the study.
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