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Background: Butenyl-spinosyn, produced by Saccharopolyspora pogona, is a promising biopesticide due to excellent insecticidal activity and broad pesticidal spectrum. Bacterioferritin (Bfr, encoded by bfr) regulates the storage and utilization of iron, which is essential for the growth and metabolism of microorganisms. However, the effect of Bfr on the growth and butenyl-spinosyn biosynthesis in S. pogona has not been explored. Results: Here, we found that the storage of intracellular iron influenced butenyl-spinosyn biosynthesis and the stress resistance of S. pogona, which was regulated by Bfr. The overexpression of bfr increased the production of butenyl-spinosyn by 3.14-fold and enhanced the tolerance of S. pogona to iron toxicity and oxidative damage, while the knockout of bfr had the opposite effects. Based on the quantitative proteomics analysis and experimental verification, the inner mechanism of these phenomena was explored. Overexpression of bfr enhanced the iron storage capacity of the strain, which activated polyketide synthase genes and enhanced the supply of acyl-CoA precursors to improve butenyl-spinosyn biosynthesis. In addition, it induced the oxidative stress response to improve the stress resistance of S. pogona. Conclusion: Our work reveals the role of Bfr in increasing the yield of butenyl-spinosyn and enhancing the stress resistance of S. pogona, and provides insights into its enhancement on secondary metabolism, which provides a reference for optimizing the production of secondary metabolites in actinomycetes. [ABSTRACT FROM AUTHOR]

Background: Acetoin utilization protein (acuC) is a type I histone deacetylase which is highly conserved in bacteria. The acuC gene is related to the acetylation/deacetylation posttranslational modification (PTM) system in S. spinosa. Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. However, the specific functions and influences of acuC protein in S. spinosa are yet to be characterized. Results: The knockout strain and overexpression strain were constructed separately with the shuttle vector pOJ260. The production of spinosyns A and D from S. spinosa-acuC were 105.02 mg/L and 20.63 mg/L, which were 1.82-fold and 1.63-fold higher than those of the wild-type strain (57.76 mg/L and 12.64 mg/L), respectively. The production of spinosyns A and D from S. spinosa-ΔacuC were 32.78 mg/L and 10.89 mg/L, respectively. The qRT-PCR results of three selected genes (bldD, ssgA and whiA) confirmed that the overexpression of acuC affected the capacities of mycelial differentiation and sporulation. Comparative proteomics analysis was performed on these strains to investigate the underlying mechanism leading to the enhancement of spinosad yield. Conclusions: This study first systematically analysed the effects of overexpression acuC on the growth of S. spinosa and the production of spinosad. The results identify the differentially expressed proteins and provide evidences to understand the acetylation metabolic mechanisms which can lead to the increase of secondary metabolites. [ABSTRACT FROM AUTHOR]

Butenyl-spinosyn, a secondary metabolite produced by Saccharopolyspora pogona , exhibits strong insecticidal activity than spinosyn. However, the low synthesis capacity and unknown metabolic characteristics of butenyl-spinosyn in wild-type S. pogona limit its broad application and metabolic engineering. Here, we showed that S. pogona exhibited increased glucose consumption ability and growth rate compared with S. spinosa , but the production of butenyl-spinosyn was much lower than that of spinosyn. To further elucidate the metabolic mechanism of these different phenotypes, we performed a comparative proteomic and metabolomic study on S. pogona and S. spinosa to identify the change in the abundance levels of proteins and metabolites. We found that the abundance of most proteins and metabolites associated with glucose transport, fatty acid metabolism, tricarboxylic acid cycle, amino acid metabolism, energy metabolism, purine and pyrimidine metabolism, and target product biosynthesis in S. pogona was higher than that in S. spinosa. However, the overall abundance of proteins involved in butenyl-spinosyn biosynthesis was much lower than that of the high-abundance protein chaperonin GroEL, such as the enzymes related to rhamnose synthesis. We speculated that these protein and metabolite abundance changes may be directly responsible for the above phenotypic changes in S. pogona and S. spinosa , especially affecting butenyl-spinosyn biosynthesis. Further studies revealed that the over-expression of the rhamnose synthetic genes and methionine adenosyltransferase gene could effectively improve the production of butenyl-spinosyn by 2.69- and 3.03-fold, respectively, confirming the reliability of this conjecture. This work presents the first comparative proteomics and metabolomics study of S. pogona and S. spinosa , providing new insights into the novel links of phenotypic change and metabolic difference between two strains. The result will be valuable in designing strategies to promote the biosynthesis of butenyl-spinosyn by metabolic engineering. [ABSTRACT FROM AUTHOR]

Polynucleotide phosphorylase is a highly conserved protein found in bacteria and fungi that can regulate the transcription of related enzymes involved in amino acid metabolism, organic acid metabolism, and cell biosynthesis. We studied the effect of polynucleotide phosphorylase on Saccharopolyspora pogona (S. pogona) growth and the synthesis of secondary metabolites. First, we generated the overexpression vector pOJ260-PermE-pnp via overlap extension PCR. The vector pOJ260-PermE-pnp was then introduced into S. pogona by conjugal transfer, thereby generating the recombination strain S. pogona-Pnp. Results showed that engineering strains possessed higher biomass than those of the wild-type strains. Moreover, the ability of these strains to produce spores on solid medium was stronger than that of the wild-type strains. HPLC results revealed that the butenyl-spinosyn yield in S. pogona-Pnp increased by 1.92-fold compared with that of S. pogona alone. These findings revealed that overexpression of polynucleotide phosphorylase effectively promoted butenyl-spinosyn biosynthesis in S. pogona. This result may be extended to other Streptomyces for strain improvement. [ABSTRACT FROM AUTHOR]

• The sensor kinase LytS-L influence the virulence against Helicoverpa armigera. • The expression of LytS-L influence growth and butenyl-spinosyn biosynthesis. • The LytS-L regulates the energy, carbon metabolism, and secondary metabolism. The LytTR family two-component system widely exists in bacterial cells and plays an important role in metabolic regulation. The lytS-L gene that encodes for a LytTR family sensor kinase was knocked out to study its influence on the growth, phenotype, and the biosynthesis of the insecticidal polyketide butenyl-spinosyn in Saccharopolyspora pogona NRRL 30141 (S. pogona). High performance liquid chromatography (HPLC) results showed that the butenyl-spinosyn yield of the lytS-L knockout mutant decreased by 58.9% compared with that of the parental strain. This is manifested by a weak toxicity of the mutant against the insect Helicoverpa assulta (H. armigera). Comparative proteomic analysis revealed the expression characteristics of the proteins in S. pogona and S. pogona- Δ lytS-L : a total of 14 proteins involved in energy metabolism were down-regulated, 9 proteins related to carbon metabolism such as glycolysis, and tricarboxylic acid cycle (TCA) were up-regulated, while 13 proteins involved in the biosynthesis of butenyl-spinosyn were down-regulated (fold change >1.2 or< 0.83). The qRT-PCR (Quantitative Real-time PCR) analysis illustrated that the changes in the expression levels of transcription and translation of the identified genes were consistent. This study explores the function of the two-component system of the LytTR family in S. pogona and shows that the lytS-L gene has an important influence on regulating primary metabolism and butenyl-spinosyn biosynthesis of S. pogona. [ABSTRACT FROM AUTHOR]