基于转录组学分析刺糖多孢菌多杀菌素合成代谢途径关键酶基因挖掘

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

摘要:

【目的】通过对不同时期的刺糖多孢菌进行转录组分析，探究多杀菌素生物合成的相关代谢通路，挖掘代谢途径关键酶基因，探究多杀菌素竞争基因簇，为高产工程菌的构建奠定基础。【方法】选取刺糖多孢菌株对数生长期(T2-48 h)和稳定期(T6-144 h)进行比较转录组分析，并通过实时荧光定量PCR (real-time fluorescence quantitative PCR,qRT-PCR)与转录组测序进行相互验证。采用基因本体论(gene ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)对差异表达基因进行功能和代谢通路注释并进行中心碳代谢分析。【结果】刺糖多孢菌通过转录组测序发现有2 542个差异表达基因，其中具有显著上调基因1 188个，显著下调基因1 354个。GO注释表明，差异表达基因主要参与羧酸代谢过程、含氧酸代谢过程、有机酸代谢过程和氨基酸代谢过程。KEGG富集结果表明，差异表达基因主要参与甘氨酸、丝氨酸和苏氨酸代谢，以及氧化磷酸化和精氨酸生物合成等通路。进一步分析得到7个与多杀菌素生物合成相关的基因，其中accB、Pfk、G6PD、dsdA表达量显著上调，而涉及多杀菌素前体消耗的GAPDH、aceE、DLAT以及TCA循环和精氨酸生物合成途径中的基因表达量都呈现显著性下调趋势。qRT-PCR与转录组测序结果发现双方同时上调的基因有12个，分别为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)和BGC35(40 232 bp)。【结论】本研究通过转录组学分析获得了不同时期刺糖多孢菌的差异基因以及多杀菌素生物合成通路，并分析了刺糖多孢菌中多杀菌素的竞争基因簇，为后期开展多杀菌素生物合成途径的优化和对刺糖多孢菌进行遗传改造从而达到提高多杀菌素产量的目的奠定了基础。

关键词:多杀菌素;刺糖多孢菌;转录组分析;代谢途径;实时荧光定量PCR

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.

Key words:spinosad;Saccharopolyspora spinosa;transcriptomics;metabolic pathways;qRT-PCR