Mining of crucial enzyme genes in the synthesis of spinosad in Saccharopolyspora spinosabased on transcriptomics

Abstract:

[Objective] To mine the key enzyme genes associated with spinosad synthesis and the biosynthetic gene clusters (BGCs) in Saccharopolyspora spinosa at different developmental stages by transcriptomics,thus laying the groundwork for the construction of high-yield strains.[Methods]The transcriptomes of S.spinosa during the logarithmic phase (T2-48 h) and the stationary phase (T6-144 h) were compared.The results from qRT-PCR and transcriptome sequencing were mutually validated.Gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) enrichment were performed for the differentially expressed genes (DEGs).Central carbon metabolism analysis was performed.[Results] The transcriptome sequencing of S.spinosa revealed 2 542 DEGs,including 1 188 genes with significantly up-regulated expression and 1 354 genes with significantly down-regulated expression.GO annotation indicated that the DEGs were primarily involved in carboxylic acid metabolic process,oxoacid metabolic process,organic acid metabolic process,and amino acid metabolic process.KEGG enrichment analysis demonstrated DEGs were mainly involved in pathways such as glycine,serine,and threonine metabolism,oxidative phosphorylation,and arginine biosynthesis.Further analysis identified seven genes related to spinosad biosynthesis.Among them,accB,Pfk,G6PD,and dsdA showed significantly up-regulated expression,while GAPDH,aceE,DLAT involved in the consumption of spinosad precursors,as well as genes in the TCA cycle and arginine biosynthesis,exhibited significantly down-regulated expression.The results of qRT-PCR were consistent with the trends observed in transcriptome sequencing,which revealed 12 upregulated BGCs:BGC2(43 846 bp),BGC4(18 330 bp),BGC9(20 501 bp),BGC18(62 621 bp),BGC22(19 626 bp),BGC25(42 896 bp),BGC26(40 086 bp),BGC28(39 392 bp),BGC30(20 282 bp),BGC31(53 657 bp),BGC34(20 787 bp),and BGC35(40 232 bp).[Conclusion]This study elucidated DEGs in S.spinosa at different developmental stages through transcriptome analysis,and analyzed the biosynthetic pathways and BGCs of spinosad.These findings pave the way for optimizing the spinosad biosynthetic pathways and genetically modifying S.spinosa to enhance the spinosad production in subsequent studies.