发酵毕赤酵母柠檬酸转运蛋白基因功能分析及果酒降酸机制研究

The high content of citric acid will cause fruit wines taste tart and microbial instability. Deacidification of wines using yeasts capable of degrading citric acid has great practical relevance. In a previous work, we selected a yeast strain JT-1-3 with high citric acid degradation ability and great fruit wine making potential and was identified as P. fermentans. In this work, citric acid transporter genes of P. fermentans JT-1-3 will be identified by transcriptome analysis and investigated in a citrate-transport-deficient S. cerevisiae BY4741. The expression of citrate transporter genes will be further surveyed in a model fruit wine fermentation by real-time PCR method. More over, the biological pathways of P. fermentans JT-1-3 with over-expressed citrate transporter genes will be studied using untargeted metabolomic analysis platform. The results of this research will be a guideline to reduce citric acid concentration and improve quality in fruit wines.

柠檬酸含量过高会导致果酒口感尖酸且微生物稳定性差，应用酵母菌降低柠檬酸含量对于提高果酒质量具有重要意义。本项目拟以前期筛选的一株可利用柠檬酸且酿酒性能良好的发酵毕赤酵母（P. fermentans JT-1-3）为材料，基于转录组测序挖掘该菌跨膜转运柠檬酸蛋白基因，并转化酿酒酵母BY4741进行功能验证。利用实时荧光定量PCR技术分析柠檬酸转运蛋白基因在果酒酿造过程中的定量表达，进一步通过代谢组学分析柠檬酸转运蛋白基因过表达菌株在果酒酿造中参与柠檬酸降解的代谢通路，为果酒生物降酸提供理论依据。

本项目以发酵毕赤酵母（P. fermentans JT-1-3）为材料，研究其在果酒发酵过程中柠檬酸代谢特征，利用转录组学分析该酵母代谢柠檬酸的关键调控基因，基于代谢组学分析挖掘关键差异代谢物。结果表明，该酵母发酵所得果酒中柠檬酸、苹果酸和酒石酸含量均显著降低，并且发酵过程中总黄酮较为稳定，而总酚显著降低。将JT-1-3分别接入以葡萄糖和柠檬酸为单一碳源的基质中进行转录组学分析，共得到2416个差异基因，其中因代谢柠檬酸上调的基因数目为1292，进一步筛选得到12个潜在的关键调控基因（Gene1~12），qRT-PCR验证表明Gene1、Gene3、Gene10和Gene12与JT-1-3代谢柠檬酸密切相关。通过预测上述4个基因所翻译蛋白的跨膜区与三级结构，推断它们主要参与转运蛋白的合成。结合代谢组学分析JT-1-3于猕猴桃汁中代谢柠檬酸的差异代谢物，有四条通路活性明显上调。柠檬酸胁迫增加时提高花生四烯酸代谢与Serotonergic synapse两条通路的活性，JT-1-3利用柠檬酸过程中代谢产物会随着时间发生明显的变化。