Optimization Of Spinosad Heterologous Biosynthesis System

Spinosad is a class of macrolide compounds produced by S.spinosa.It is an ideal biopesticide with efficient and harmless insecticidal activity.However,there are some shortcomings when produce Spinosad with S.spinosa.Such as long fermentation cycle,with many by-products,difficultly to genetic manipulation,Previously,Song constructed an artificial gene cluster in which the 23 spinosad biosynthetic genes which were grouped into 7 operons under control of Streptomyces constitutive strong promoters.When the artificial gene cluster was expressed in Streptomyces albus J1074,the yield of spinosad was improved by 328 folds compared with the Streptomyces albus J1074 containing native spinosad gene cluster.Besides spinosad,the less active byproduct N-monodesmethyl spinosad was also detected at a considerable content in the fermentation broth of S.albus J1074 containing the artificial gene cluster.In this research we tuned expressions of the forosamine methyltransferase and the forosaminyl transferase to improving spinosad production in Streptomyces albus J1074:(1)Using an inducible promoter to regulate the expression of spnS gene,the result showed that N-monodesmethyl spinosad would be produced when the expression of spnS was low.(2)With overexpreesion of spnS gene N-monodesmethyl spinosad decreased by 90%,and the production of spinosad was only increased by 38%.(3)When the SpnP was further overexpressed,the production of spinosad was increased 5.3 folds(5.8±0.4 mg/L)and its N-monodesmethyl derivative was decreased by 90%.In this study,the omics analysis of Streptomyces albus J1074(pBACspnNEW)with high spinosad production was also carried out.The optimization of the biosynthesis system guided by multi-omics is an effective method to improve the biosynthesis efficiency.Fatty acid beta oxidation was proved that can provide sufficient precursors and energy during the secondary metabolites stage in Streptomyces.In order to further optimize the heterologous spinosad production system,(1)the genes that may be involved in β-oxidation was predicted by genome alignment,including 7 genes of TAG hydrolysis,17 genes encoding acyl-CoA synthetase,13 genes encoding acyl-CoA dehydrogenase,16 genes encoding FadB,and 7 genes encoding FadA.(2)The differential genes of beta-oxidation(xnr＿rs25305,xnr＿rs11285,xnr＿rs25440,xnr＿rs20830,xnr＿rs03170,xnr＿rs03175 and xnr＿rs06335-xnr＿rs06340)analysized with transcriptome in Streptomyces albus J1074(pBACspnNEW)were figured out.(3)Expression of these genes shown that expression of xnr＿rs25305,xnr＿rs11285,and xnr＿rs03175 genes could improve the titers of spinosyn A by 83%,43%and 26%,respectively.Futher,XNR＿RS06335-XNR＿RS06340 can increase the yield of spinosyn A by about 1 flod under the optimal conditions.In this study,genes invoved in FA uptaking and TAG anabolism were also expressed in Streptomyces albus J1074:(1)Combination with expressing of sco6196 gene,lipase from Streptomyces albus ZD11 and diacylglycerol acyltransferase(DGAT)from Streptomyces coelicolor was expressed,respectively.The results showed that expressing of DGAT with 72 h induced expression of sco6196 gene could increase the production of spinosyn A by 116%.(2)Using the Cumate-induced promoter to control the expression of lipid transport protein LTP from Rhodococcus jostii RHA1 showed that LTP can increase the production of spinosyn A at low expression level,and the production of spinosyn A decreased with the increasing of expression of LTP.The above results indicated that improving the synthesis and catabolism of TAG without competing with the precursors for polyketide synthesis in Streptomyces albus J1074 can promote production of spinosyn A.In sum,fine tuning expression of genes and multi-omics analysis guided the expression of related genes were used to effectively optimize the heterologous production system of spinosad in Streptomyces albus J1074.In the future,we will balance the expression of other synthetic genes of spinosad,such as PKS genes,to further improve the yield of spinosad.At the same time,we will enhance the TAG biosynthesis pathway in the primary metabolic stage and the beta oxidation of fatty acids in the secondary metabolic stage of Streptomyces albus J1074 by developing metabolic switch,which take advantage of TAG as a pool of carbon source when producing spinosad in Streptomyces albus J1074.