Genetic Improvement Of Saccharopolyspora Spinosa

Spinosad, a new microbial insecticide, is an intracellular secondary metabolites produced by actinomycetes Saccharopolyspora spinosa after aerobic fermentation.With its excellent insecticidal effect on agricultural pests and a very high security boundary on mammals, fish, birds and most of the beneficial insects, both the efficiency of chemical pesticides and the security of biological ones, it is becoming the most promising new generation of insecticides. Because of its low production and very long fermentation period up to two to three weeks, it has high production costs, which seriously hampered its production and practical application. Therefore, making strain improvement to increase the production of spinosad is becoming one of most urgent problem of development of the pesticides. Although traditional physical and chemical mutagenesis and screening is effective, it has the disadvantages such as arduous workload and unclear direction. In this study we attempted to increase the production of spinosad in2ways:the protoplast fusion and replacement of promoters.In this paper, using PCR we amplified a1.1kb fragment homologous with the eryAI gene internal sequence from Saccharopolyspora erythraea NRRL2338and also amplified the7.8kb spnA gene and the2.7kb tspnA gene fragment, a part of the starting module of the spnA gene, from Saccharopolyspora spinosa SP06081.After separate double digestion we ligated the1.1kb homologous fragment with the plasmid pOJ260, constructing a vector, pOJ260D, which was used to block the production of erythromycins in Saccharopolyspora erythraea.Vector pOJ260D was transformed into E. coli ET12567(pUZ8002) and then the resultant E. coli transformants were conjugated with Saccharopolyspora erythraea NRRL2338. After several days of culture, picked transformants and made them grow on BHI plates containing the appropriate concentration of Apramycin for2times. Finally we got a strain, Saccharopolyspora erythraea D, which didn’t produce erythromycins.The molecular nucleic acid characteristics of Saccharopolyspora erythraea D has been verified by PCR using primers designed according to the Apramycin resistant gene. We found it could produce more pigments than the original strain when it was grown on solid plates.There was no production of erythromycins after shake flasks fermentation by HPLC detection.We cultured separately the2strains, Saccharopolyspora erythraea D and Saccharopolyspora spinosa SP06081, and then made the protoplast fusion between them. Randomly picked80colonies from regeneration plates were grown on solid plates containing the appropriate concentration of Apramycin for2times. After shake flasks fermentation we used HPLC to detect the fused strains which could produce spinosyns and acquired4ones. Taking HPLC collected peaks from fermentation broth of these fused strains for mass spectrometry analysis, we got further confirmation for spinosyns.Because all the4fused strains had lower yield of spinosad than the original strains, we then direct screening without antibiotics, successfully acquiring a number of fused strains with high-yield spinosad, one of which, strain B-2, had a highest production of that with a increase of331%compared with the original ones. The yield of spinosad of fused strain B-2was stable by successive subculture, showing it had a good genetic stability.After separate double digestion we ligated the2.7kb tspnA fragment with the plasmid pLSB2, constructing a vector, pLSB2-tspnA, which was used to replace the original promoters of spinosyns synthetic gene clusters in Saccharopolyspora spinosa.Vector pLSB2-tspnA was transformed into E. coli S17-1and then the resultant transformants E. coli S17-1(pLSB2-tspnA) were acquired. We are ready to do the conjugation between E. coli S17-1(pLSB2-tspnA) and Saccharopolyspora spinosa. This research is a report on enhanced production of spinosad by protoplast fusion between Saccharopolyspora spinosa and Saccharopolyspora erythraea and will provide the important basis for constructing the fused strains with excellent features from Saccharopolyspora spinosa and Saccharopolyspora erythraea; It will also bring a new revelation for increasing the production of spinosad to replace the original promoter of Saccharopolyspora spinosa for spinosyns production with the act Ⅱ-ORF4/Pact Ⅰ activating/starting element and simultaneously increase the copy number of the spnA gene.