Mutagenic Screening And Mechanism Analysis Of Spinosad High-yield Strains And JL Producing Strains

Spinosyns is a secondary metabolite produced by aerobic fermentation of Saccharopolyspora spinosa.As one of the representatives of natural green pesticides,it is widely used in the prevention and control of pests in agriculture and especially in organic farm.Because of its unique insecticidal mechanism and very low-toxic to non-target animals and the environment,it has attracted the interest of many researchers.However,due to the difficulty of genetic engineering manipulation of the production strain of Saccharopolyspora spinosa,the immature secondary metabolic regulation mechanism and the existence of relevant patent barriers,the domestic research on spinosyn high-yield strain and its second-generation products development are still in its infancy and the large-scale industrial production has not been successed yet.At present,the methods of increasing yield and obtaining precursor production strains of second-generation products are mostly focusing on large-scale screening after random mutagenesis,but there are problems in this approach such as large-scale workload,strong randomness and poor stability,so it is still necessary to conduct a mechanism study to consider from the perspective of genetic engineering to achieve their goals.In this study,the original strain NRRL 18395 was mutagenized by NTG and1047 mutantstrains were screened,19 high-yield spinosyn and 1 spinosyn JL-producing strain(MFR JL)were obtained by initial screening in 96-well plates.A stable high-yield strain(MFR 3)was obtained after shake flask evaluation.The spinosyn yield was 374 μg/m L,which was 11 times higher than that of the original strain.The DNA microarray was used to analyze the difference in gene expression between the low-yield strains(NRRL 18395)and the high-yield strains(MFR 3).we found that 12 genes were significantly up-regulated and 4 genes were significantly down-regulated.There are 3 genes(LIP,ECH and HBD)related to substrate metabolic pathways in the up-regulated genes.Experiments show that LIP and ECH strengthen the fatty acid β oxidation process to increase the flow of acetyl-CoA and ultimately increase the production of spinosyn.While HBD is not a related to spinosyn yield.These findings provide us some hints for improving spinosyn production.This study also carried out a preliminary mechanism analysis of the induced spinosyn JL-producing strain(MFR JL).Through the analysis of the mutation site of the key gene spn K,it was found that,the amino acid 299 of spn K,(D)is mutated to asparagine(N),most of the spn K enzyme activities are lost,resulting in the accumulation of spinosyn JL.The mutation site has never been reported before,providing a basis for the study of the precursor of spinosyn second generation-Spinetoram.