Breeding Of Saccharopolyspora Spinosa Producing Spinosad

Spinosyns is a class of macrolide antibiotics,produced by Saccharopolyspora spinosa.The mechanism of this action is to paralyze the target phytophagous pests by continuously activating the acetylcholine receptor and γ-aminobutyric acid receptor,and ultimately leads to the death of pests.The novel mechanism makes spinosyns have good biological safety and highly effective insecticidal properties.However,due to the low fermentation potency of wild-type S.spinosa,it is difficult to apply to industrial production.In this study,a mutant SS-168 with high yield of spinosad was screened by a series of physical and chemical modifications performed on wild-type SS-WT.The high yield mechanism of the mutant was analyzed by transcriptomics method.The expression of genes in some key metabolic pathways during fermentation was verified,and amino acids were added to the fermentation medium to further improve the spinosad fermentation titer of the high-yield mutant.The research results are as follows:1.The strain SS-WT was mutated by EMS(ethyl methane sulfonate)in the first round,followd by streptomycin resistance breeding in the second round.In the third round ARTP(atmospheric pressure room temperature plasma)mutation breeding was conducted to further consolidate the breeding results.The fermentation level of the mutant strain was investigated by methionine-added fermentation medium.The mutant SS-168 was obtained by compound mutagenesis,and the fermentation level was higher than 1000 mg/L.In the fermentation medium containing methionine,the fermentation level was further improved to about 1600mg/L.In addition,another mutant strain ESA-598 produced a higher level of spinosad J,while spinosad A decreased significantly.2.Transcriptome analysis showed that compared to the wild-type SS-WT,255 genes were up-regulated and 1086 genes were down-regulated in S.spinosa SS-168 after 7 days of fermentation.Functional clustering analysis of differentially expressed genes showed that the significantly down-regulated genes were mainly enriched in biological processes and molecular functions.The prediction results of KEGG(Kyoto Encyclopedia of Genes and Genomes)metabolic pathway showed that during the fermentation process of SS-168,about40 % of the genes in alanine/aspartate/glutamate metabolic pathway were significantly down-regulated,about 34 % of the genes involved in fatty acid degradation pathway were significantly up-regulated,and about 33 % of the genes involved in arginine/proline metabolic pathway were significantly down-regulated.In addition,about 32 % of the genes involved in glycine/serine/threonine metabolic pathway were significantly down-regulated.3.The glycine/serine/threonine metabolic pathway was selected as the research object according to the difference in gene expression abundance.Three kinds of amino acids were added to the medium to investigate their effects on the production of spinosad.The results showed that the addition of glycine at 1 mmol/L promoted the synthesis of spinosad most significantly.The optimal concentration for serine to promote the increase of spinosad yield was 15 mmol/L;threonine had a significant promoting effect on the yield of multiple killing strains when the concentration was greater than a certain threshold(5 mmol/L).In this study,a rational chemical structure-oriented molecular design breeding method was used to select high-yielding Saccharopolyspora spinosa mutants,which had better mutagenesis effect and mutation frequency compared with traditional mutagenesis methods.The high-yield mechanism of the mutant strain provides a theoretical basis for the subsequent in-depth development,and great significance to promote the large-scale production of spinosyn in domestic.