Abstract
PII signal transduction proteins are widely found in bacteria and plant chloroplast, and play a central role in nitrogen metabolism regulation, which interact with many key proteins in metabolic pathways to regulate carbon/nitrogen balance by sensing changes in concentrations of cell-mediated indicators such as α-ketoglutarate. In this study, the knockout strain Saccharopolyspora pogona-ΔpII and overexpression strain S. pogona-pII were constructed using CRISPR/Cas9 technology and the shuttle vector POJ260, respectively, to investigate the effects on the growth and secondary metabolite biosynthesis of S. pogona. Growth curve, electron microscopy, and spore germination experiments were performed, and it was found that the deletion of the pII gene inhibited the growth to a certain extent in the mutant. HPLC analysis showed that the yield of butenyl-spinosyn in the S. pogona-pII strain increased to 245% than that in the wild-type strain while that in S. pogona-ΔpII decreased by approximately 51%. This result showed that the pII gene can promote the growth and butenyl-spinosyn biosynthesis of S. pogona. This research first investigated PII nitrogen metabolism regulators in S. pogona, providing significant scientific evidence and a research basis for elucidating the mechanism by which these factors regulate the growth of S. pogona, optimizing the synthesis network of butenyl-spinosyn and constructing a strain with a high butenyl-spinosyn yield.
Key points
• pII key nitrogen regulatory gene deletion can inhibit the growth and development of S. pogona.
• Overexpressed pII gene can significantly promote the butenyl-spinosyn biosynthesis.
• pII gene can affect the amino acid circulation and the accumulation of butenyl-spinosyn precursors in S. pogona.
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Data availability
The datasets generated during in the current study are available from the corresponding author on reasonable request.
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Funding
This work was supported by funding from the National Natural Science Foundation of China (31770106, 3107006), the National Basic Research Program (973) of China (2012CB722301), and the National High Technology Research and Development program (863) of China (2011AA10A203).
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LX conceived the project. JH and ZX generated the concepts and designed the research. JH and LS performed mutant construction. JC, ZZ, LC, and JX performed HPLC analysis. JH, ZD, YH, WH, SH, YS, and HSB performed LC–MS/MS and data analysis. JH, ZX, LX, LS, and JC wrote the manuscript. All authors discussed the results and approved the final manuscript.
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Hu, J., Xia, Z., Shuai, L. et al. Effect of pII key nitrogen regulatory gene on strain growth and butenyl-spinosyn biosynthesis in Saccharopolyspora pogona. Appl Microbiol Biotechnol 106, 3081–3091 (2022). https://doi.org/10.1007/s00253-022-11902-5
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DOI: https://doi.org/10.1007/s00253-022-11902-5