| The soil actinomycete Saccharopolyspora spinosa produces secondary metabolites called spinosyns, polyketide-derived macrolide active ingredients in a family of insect control agents. The most active and abundant components of the spinosyn family of compounds are spinosyn A and D (spinosad). Spinosad is widely used in agriculture as a potent insect control agent and animal health product because of its high efficiency against target insects and environment-friendly characteristics. However, the biosynthesis of spinosad in wild-type S. spinosa is very low, therefore considerable interest is in the researches of the strain improvement and its physiological properties. The major strategies for enhancing spiosad productivity include improving flux through spinosad biosynthetic pathway, increasing precursor supply, reducing by-product formation and improving othe properties of S. spinosa. In this study, the traditional mutagenesis and genetic engineering were applied to improve the wild-type S. spinosa SP06081. While the differential proteomic researches were also performed to investigate the molecular mechanism leading to the increased spinosad production.The preparation and regeneration conditions of protoplasts of S. spinosa SP06081were optimized according to the effects of glycin concentration and the operational time, temperature and lysozyme concentration on the protoplasts preparation in order to improve the spinosad-producing S. spinosa. The yield of S. spinosa SP06081protoplast was the highest under these conditions:the collected mycelial from SP06081cultured in tryptic soy broth (TSB) medium with0.2%glycin for48h was treated by0.1mg/mL lysozyme at28℃for20min, then plated on the R2YE medium, the number of regeneration protoplast was up to10/mL. Further investigation of the physiological characteristics of the protoplast-regenerated strains showed that they exhibited changes in morphology and spinosad production, and there is significant correlation between morphological differentiation and spinosad yield. On these bases, UV induced mutation combining natural isolation of the SP06081protoplasts were performed. A rapid method for hierarchy screening was established by comprehensive analysis of the physiological characteristics of the mutants, and a high spinosad-producing strain named PR2was obtained. Further Cobalt mutagensis on the PR2protoplast resulted in a high-yeild mutant Coy129with stable heredity, its spinosad yield increased128.5%compared with the original strain SP06081.Then a comparative proteomic analysis was performed on the S. spinosa SP06081and PR2during the first rapid growth phase (RG1) in seed medium (SM) by using label-free quantitative shotgun proteomics to investigate the underlying mechanism leading to the enhancement of spinosad yield. In total,224proteins from the SP06081strain and204proteins from the PR2strain were unambiguously identified by liquid chromatography-tandem mass spectrometry analysis, in which,140proteins were shared by the two strains, a total of12proteins directly related to spinosad biosynthesis were identified. Further comparative analysis of the shared proteins revealed that approximately31%of them changed their abundance significantly and fell in all of the functional groups, such as tricarboxylic acid cycles, glycolysis, biosynthetic processes, catabolic processes, transcription, translation, oxidation and reduction. Several key enzymes involved in the synthesis of primary metabolic intermediates used as precursors for spinosad production, energy supply, deoxysugar methylation, and antioxidative stress were differentially expressed in the same pattern of facilitating spinosad production by the PR2strain. Real-time reverse transcriptase polymerase chain reaction analysis revealed that four of five selected genes showed a positive correlation between changes at the translational and transcriptional expression level, which further confirmed the proteomic analysis. Moreover, in order to improve the oxygen supply limitation during high cell density submerged cultivations of S. spinosa, the open reading frame of Vitreoscilla hemoglobin gene (vgb) was placed under the control of the promoter PermE by overlap PCR, and cloned into the integration vector pSET152, then introduced into S. spinosa SP06081by conjugal transfer and integrated into its chromosome. The resultant conjugant was named S. spinosa SP-VHb which has excellent stability. The integration was further confirmed by PCR and southern blotting detection; Carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin (VHb) was successfully expressed in S. spinosa SP-VHb; Fermentation results revealed that the expression of vgb gene could significantly promote the biosynthesis of spinosad under both normal oxygen and moderate limited oxygen supply conditions (p<0.01).In conclusion, our results provide valuable technologic information and data for further improving spinosad-producing S. spinosa by rational application of genetic and metabolic engineering. |
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