基于蛋白质组学分析尿嘧啶对出芽短梗霉产普鲁兰多糖的影响

Proteomics Analysis of the Effect of Uracil on Pullulan Polysaccharide Production by Aureobasidium pullulans

为了解尿嘧啶对出芽短梗霉生长及合成普鲁兰多糖的内在机制,研究了尿嘧啶在普鲁兰多糖发酵过程中的最适添加量与最适添加时间。利用非标记(Label-free)定量技术和液相色谱-串联质谱技术比较出芽短梗霉发酵后期(88 h)的蛋白质组分,并对其差异蛋白质进行生物信息学分析。结果表明:在48 h添加0.5 g/L的尿嘧啶对普鲁兰多糖的产量提高最为显著,在5 L发酵罐上验证,产量由70.13 g/L提高到86.27 g/L,提高了23%。进行蛋白质组分分析鉴定出80个差异性蛋白质,其中包括40个上调蛋白和40个下调蛋白(差异倍数>2,P<0.05),对这些差异蛋白进行聚类分析、GO功能富集分析、KEGG通路分析显示上述差异蛋白广泛涉及细胞过程、代谢过程等重要生物过程。差异蛋白质主要参与糖酵解、果糖和甘露糖代谢、乙醛酸和二羧酸代谢、丙酮酸代谢和TCA循环等代谢过程,最终引起普鲁兰多糖产量的变化。为进一步了解出芽短梗霉产普鲁兰多糖的代谢机理提供了分子基础。

In order to understand the mechanism of uracil on the growth by Aureobasidium pullulans and synthesis of pullulan, the optimal dosage and time of uracil in pullulan polysaccharide fermentation were studied. The label-free quantitative method and liquid chromatography-tandem mass spectrometry were used to compare the protein components in the late fermentation stage(88 h) by Aureobasidium pullulans and the differential proteins were analyzed by bioinformatics.The results showed that adding 0.5g/L uracil at 48 h had the most significant increasing in pullulan polysaccharide yield,which was verified on 5 L fermenter from 70.13 to 86.27 g/L, increasing by 23%. Protein component analysis identified 80different proteins, including 40 increases protein and 40 lower protein(difference>2, P<0.05), the protein of the differences of clustering analysis, GO function enrichment, KEGG pathway analysis showed that the differences in protein involved in cellular processes, metabolic processes, and other important biological processes. The differential proteins were mainly involved in glycolysis, metabolism of fructose and mannose, metabolism of glyoxylic acid and dicarboxylic acid, pyruvate metabolism and TCA cycle, which ultimately led to the change of pullulan polysaccharide yield. These results would provide a molecular basis for further understanding the metabolic mechanism of pullulan polysaccharides produced by Aureobasidium pullulans.