Optimization Of Key Regulatory Genes And Metabolic Networks Of Butenyl-spinosyn Biosynthesis

Butenyl-spinosyn is a secondary metabolite with insecticidal activity produced by aerobic fermentation of Saccharopolyspora pogona.The anti SMASH analysis showed that the genome of S.pogona contains 32 different gene clusters,suggesting a large number of potentially competing metabolisms during butenyl-spinosyn biosynthesis.Therefore,reducing metabolic competition by deleting other non-essential gene clusters to improve butenyl-spinosyn production is a feasible research strategy.However,identifying target gene clusters from the genome that can be reduced and optimized is a great challenge due to the complex genetic background and metabolic networks of Saccharopolyspora pogona.In this study,three mutants S.pogona-?clu4,S.pogona-?clu13 and S.pogona-?clu22 were successfully constructed by knocking out the corresponding polyketide gene cluster in S.pogona using CRISPR/Cas9 technology.The HPLC analysis showed that S.pogona-?clu13 exhibited a greater ability to produce butenyl-spinosyn,with a 4.06-fold increase in production compared to the wild-type.To investigate how S.pogona-?clu13 promotes butenyl-spinosyn biosynthesis,we analyzed the 52 open reading frames in clu13 and found a gene coding for polyketide synthase RppA-L,which we hypothesized could competitively utilize coenzyme A precursors and thus interfere with butenyl-spinosyn biosynthesis.With the construction of S.pogona-?rppA-L,we found that the deletion of rppA-L promoted the production of butenyl-spinosyn,but the increase was not significant,indicating that rppA-L was not the main cause affecting the biosynthesis of butenyl-spinosyn.After analyzing the transcriptional abundance of the genes in clu13,we found that it contained a potential regulator,Sp1764,which had a high transcriptional abundance.After suppressing the gene expression of sp1764 in S.pogona,the production of butenyl-spinosyn increased 3.15-fold compared that in the wild-type strain,indicating that the absense of sp1764 is the main cause of increased production of butenyl-spinosyn in S.pogona-?clu13.The differential proteomic analysis of the wild-type strain and S.pogona-?clu13 was performed,and a series of genes that regulated the butenyl-spinosyn biosynthesis and growth were altered in expression.To optimize the metabolic network of butenyl-spinosyn biosynthesis,we performed an intensive analysis of differentially expressed proteins,and found that more proteins affecting butenyl-spinosyn biosynthesis were associated with amino acid metabolism,in addition to a series of potential regulatory factors that also underwent expression changes.In proteomics,we first investigated the potential regulators Reg and the down-regulated biotin carboxyl carrier protein BccA,which were subsequently overexpressed in the wild-type.The results demonstrated that the production of butenyl-spinosyn in S.pogona-Reg and S.pogona-BccA was increased by 2.16-fold and 0.77-fold,respectively,compared that in the wild-type strain.The above results showed that proteomics could effectively identify the factors regulating the biosynthesis of butenyl-spinosyn,and also confirmed that amino acid metabolism was closely related to the biosynthesis of butenyl-spinosyn.KEGG analysis showed that amino acid degradation could synthesize the precursor of coenzyme A required for the synthesis of butenyl-spinosyn.In addition to BccA,we also screened two proteins,MmsA and PaaF,those could potentially regulate the precursor biosynthesis for butenyl-spinosyn according to proteomics,and constructed two overexpression muatnts S.pogona-MmsA and S.pogona-PaaF,respectively.The results showed that the yield of butenyl-spinosyn in S.pogona-MmsA was 0.84 times higher than that in the wild-type strain.However,S.pogona-PaaF produced an unknown metabolite,but the production of butenyl-spinosyn decreased by 81.1 %.Branched-chain amino acid degradation can produce a large number of coenzyme A precursors.To strengthen this metabolic route,we added the corresponding amino acids to fermentation broth and performed measurements of butenyl-spinosyn production and different precursor concentrations.The results showed that methylmalonyl-Co A and propionyl-Co A promoted the biosynthesis of butenyl-spinosyn in a certain concentration range,while beyond the limits,these two precursors generated feedback inhibition,leading to a decrease in the yield of butenyl-spinosyn.In the differential proteomics of S.pogona-?clu13,the expression of BccA protein was down-regulated,while the expression of MmsA was not significantly changed.To further optimize the metabolic network of butenyl-spinosyn biosynthesis,we used S.pogona-?clu13 as the dominant chassis strain in which the bcc A and mmsA genes were overexpressed,respectively.The results showed that the butenyl-spinosyn production in the two mutants increased by 6.4-fold and 10.1-fold,respectively,compared that in the wild-type strain.By combining genome reduction with multi-omics analysis,the butenyl-spinosyn biosynthesis was found to be regulated by several key genes,and closely related to the amino acid degradation pathway,whose metabolic network was improved through a combination optimization strategy,resulting in the construction of two mutants with high production potential.This study provides a research basis for constructing dominant chassis strains and optimizing the metabolic network of natural products,expands the research ideas of amino acid degradation and precursor supply in secondary metabolite biosynthesis,and also provides a reference and guidance for screening key factors and efficient construction of production strains.