| The growth and secondary metabolites biosynthesis of actinomycetes are usually under precise and intricate regulation to respond to the changes of external environment and the intracellular signal factors orderly.The study of regulatory factors is the key to revealing the regulation mechanism of secondary metabolites biosynthesis,and it is also the basis for obtaining high-yielding engineered strains via metabolic engineering and synthetic biology strategies.Butenyl-spinosyn is a secondary metabolite with insecticidal activity produced by Saccharopolyspora pogona via aerobic fermentation,and has broad application prospects in agricultural production.However,the extremely low yield and long production cycle of butenyl-spinosyn make it difficult to realize the large-scale industrial production and application.In order to increase the production of butenylspinosyn,it is necessary to conduct in-depth analysis of physiological characteristics such as the growth and secondary metabolism of S.pogona,and study its biosynthesis regulation mechanism,so as to propose a systematic transformation strategy.In this study,the quantitative proteomics technique was used to analyze the proteome of S.pogona at different growth stages,and a large number of proteins related to the metabolic pathways of butenyl-spinosyn biosynthesis precursors were obtained as well as their expression abundance.According to the growth trend of the strain and the biosynthesis characteristics of butenyl-spinosyn,some potential key functional proteins and regulatory factors were screened out.Quantitative proteomics analysis provides a new way to gain insight into the growth and secondary metabolic characteristics of S.pogona,and to carry out functional studies on these screened proteins via genetic manipulation combined with phenotypic analysis,which is an effective strategy to improve the yield of butenyl-spinosyn.In the study of the medium composition,it was found that S.pogona was very sensitive to the iron concentration,and the strain growth and butenyl-spinosyn biosynthesis could be significantly promoted when under appropriate concentration of iron.The ability of strains to store and utilize iron is regulated by bacterioferritin(Bfr).It was found that the overexpression of bfr increased the production of butenyl-spinosyn by3.14-fold,and enhanced the tolerance of S.pogona to iron toxicity and oxidative damage.However,knocking out bfr had significant side effects on the growth and secondary metabolism of the strain.The underlying mechanisms of these phenomena were investigated via quantitative proteomic analysis and phenotypic experimental validation.It was found that overexpression of bfr increased iron storage and utilization capacity of the strain,which indirectly activated polyketide synthase genes and enhanced the metabolic capacity of the central carbon metabolism-related pathways,thereby increasing the supply of acyl-Co A precursors to promote butenyl-spinosyn biosynthesis.In addition,it also activated the expression of related antioxidant enzymes by inducing oxidative stress,thereby improving the stress resistance of S.pogona.A TetR family transcription regulator Sp13016 was discovered in the proteomics data mining of S.pogona.The transcription level of its coding gene was closely related to the growth and butenyl-spinosyn biosynthesis of the strain.Overexpression of sp13016 increased the production of butenyl-spinosyn to 2.84-fold higher than that of the wild-type strain,while the deletion of sp13016 resulted in a sharp decline in production,the growth of the strain was inhibited and sporulation was earlier.Comparative proteomics revealed that these phenotypic changes were attributed to the influence of Sp13016 on the central carbon metabolism pathway,which regulated the supply of acyl-Co A precursors to affect butenyl-spinosyn biosynthesis,and also indirectly activated most of the genes in the biosynthesis gene cluster.The production potential of butenyl-spinosyn was further tapped via analyzing the potential utilization advantages of soluble starch in ::sp13016 and targeted optimization of the medium.The functions of two potential regulatory proteins AraC and GntR discovered by quantitative proteomics were studied.The results showed that the overexpression of the encoding genes araC and gntR both resulted in a significant decrease in the strain's ability to utilize glucose and growth was inhibited,and the yield of butenyl-spinosyn was only 41% and 56% of the wild-type strain,respectively.In contrast,after knocking out araC and gntR,the yields of butenyl-spinosyn were 2.05-fold and 1.87-fold that of the wild-type,respectively.LC-MS/MS identification of the differential proteins in these engineered strains implied that araC and gntR may have similar influence patterns,both regulating the supply of acetyl-Co A by affecting the glycolytic pathway,which in turn affected the primary and secondary metabolism of the strains.In summary,quantitative proteomic analyses of different phases for S.pogona were used to systematically characterize the growth and metabolism of the strain,and a large number of potential key proteins and transcriptional regulators were successfully uncovered.Four of the regulatory factors have been functionally verified via genetic manipulation and phenotypic experiments,revealing their regulatory mechanisms in strain growth and the biosynthesis of secondary metabolites.These results not only lay an important foundation for the study of metabolic regulatory network of S.pogona and the improvement of butenyl-spinosyn production,but also provide a basis for the subsequent systematic modification by metabolic engineering and synthetic biology strategies to obtain highyielding engineered strains. |
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