| The physiological processes of microbial secondary metabolites biosynthesis are controlled by a series of elaborative and orderly complex metabolic regulation in cells, to explore the key regulatory pathways or protein factors in biosynthesis route is the immediate areas of research focus. In recent years, the technology of proteomic analysis has been used in the research of the regulatory networks of microbial secondary metabolites biosynthesis, to identify and evaluate the proteins and the difference of their expression abundance in the proteome, and then excavate the potential regulatory factors and obtain the metabolic regulatory networks during the secondary metabolites process.Spinosad, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. S. spinosa is a Gram-positive actinomycete and an important producer of antibiotic spinosad with clarified biosynthesis pathway, but its complex regulation networks associated with primary metabolism and secondary metabolites production have been little concerned or studied before. The proteomic analysis of S. spinosa strain CCTCC M206084 was performed and aimed to provide a global profile of regulatory proteins. Two-dimensional liquid chromatography tandem mass spectrometry(2D LC-MS/MS) identified 1090, 1166, 701, and 509 proteins from four phases respectively, i.e., the logarithmic growth phase(T1), early stationary phase(T2), late stationary phase(T3), and decline phase(T4). Among the identified proteins, 1579 were unique to the S. spinosa proteome, including almost all the enzymes for spinosad biosynthesis. Trends in protein expression over the various time phases were deduced from using the modified protein abundance index(PAI), revealed the importance of stress pathway proteins and other global regulatory network proteins during spinosad biosynthesis. qRT-PCR analysis of genes glnA, metE, spnK, psda and cndp showed a positive correlation between changes at translational and transcriptional expression level. This study is the first systematic analysis of the S. spinosa proteome during fermentation and its valuable proteomic data of regulatory proteins may have profound significance to the further application of the metabolic regulatory networks research of spinosad biosynthesis.One of the most important measures to study the biological function of a selected gene is to block this gene and followed by the observation of phenotypic difference between the mutant strain and the parental strain. Cobalamin-independent methionine synthase(MetE) is a key enzyme which catalyzes the last step in methione biosynthesis. In the earlier functional proteomic analysis of S. spinosa, the expression abundance of MetE was found to be significantly upregulated with the spinosad accumulation in fermentation broth. Therefore, we inferred that MetE not only elicits response to methionine biosynthesis but also affects secondary metabolism in S. spinosa. Its corresponding gene metE was inactivated by single homologous recombination, and then a ΔmetE mutant strain was successfully obtained. Compared to the wild-type strain of S. spinosa, the ΔmetE mutant strain had a more rapid growth rate both in the liquid fermentation medium and on the TSB solid medium; In the liquid fermentation medium, the wild-type S. spinosa strain undergoes a reproducible biphasic growth and growth arrest before the long stationary phase, however, the ΔmetE mutant strain only undergoes one stationary phase; The inactivation of gene metE resulted in almost loss of spinosad production, the decrease range of spinosad yield in the ΔmetE mutant strain was more than 95% while compared to the wild-type strain. In addition, the ΔmetE mutant strain could not produce white spores on the TSB solid solid medium. So, we hypothesized that the blocking of gene metE resulted in a metabolic disorder in S. spinosa, which affected the spinosad biosynthesis and the spores development.Comparative proteomic analysis is an efficient tool to investigate differences in global protein abundance profiling between mutant strain and wild-type strain, and then providing novel insights into regulatory pathways of secondary metabolite biosynthesis and bacterial development or differentiation to clarify the function or regulatory effect of the specific gene. A label-free quantitative proteomics-based approach was employed to obtain insights into the mechanism by which the metabolic network adapts to the absence of MetE in this study. A total of 1440 proteins were detected from wild-type and ΔmetE mutant strains at three time points: stationary phase of Δmet E mutant strain(S1ΔmetE, 67 h), first stationary phase of wild-type strain(S1WT, 67 h) and second stationary phase of wild-type strain(S2WT, 100 h). Protein expression patterns were determined using an exponentially modified protein abundance index(emPAI) and analysed by comparing S1ΔmetE/S1 WT and S1Δmet E/S2 WT. Results showed that differentially expressed enzymes were mainly involved in primary metabolism and genetic information processing. Then the complex metabolic adaptation mechanism of enzymes participated in glucose and amino acid metabolism(mainly were methione, aspartate and glutamate) were analyzed in detail. This study demonstrated that the role of MetE is not restricted to methionine biosynthesis but rather is involved in global metabolic regulation in S. spinosa.Overall, we have investigated the differences of S. spinosa proteome profile from different growth phases during fermentation by comparative proteomic analysis, discussed the influence on growth and phenotype of S. spinosa with the disruption of gene metE, and provided proteomic insights into metabolic adaptation to deletion of gene metE in S. spinosa. These results were the basis of further study to explore the metabolic regulatory networks of spinosad biosynthesis in detail, and have also demonstrated the powerful role of proteomic research technique in studying the regulation mechanism of microbial secondary metabolites synthesis. |
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