库德里阿兹威毕赤酵母四碳二羧酸转运蛋白PkJEN2功能解析及其对耐酸性状的影响研究

The yeast species Pichia kudriavzevii possess excellent acid resistance capabilities and could grow in harsh low pH environment (pH1.5~2.2). Although it has been developed as a successful host to produces organic acids at industrial scale, the detailed mechanism underpinning acid-resistance still remain unclear. In nature, most of acid-resistance yeast species belong to Krebs-positive yeast, which can use carboxylic acids from the Krebs cycle, for example succinic acid and malic acid, as sole carbon and energy sources. For this reason, whether C4-dicarboxylate permease could contribute to acid-resistance phenotype deserves further discussion. In previous work, we isolate an excellent acid resistance P.kudriavzevii strain CY902 and assembled its draft genome. We cloned one potential C4 dicarboxylate permease gene and named PkJEN2, because sequence analysis showed it as the homologous gene of Kluyveromyces lactis KlJEN2 (47.2% amino acids sequence identity). Deletion of PkJEN2 in CY902 marked attenuation of cell transportation capability for succinic acid and malic acid. Meanwhile, growth of ΔPkJEN2 under low pH condition was significantly inhibited. In the following study, we plan to explore the detail physiologic function for PkJEN2, including identification of substrate, measuring transport rate, monitoring subcellular location, and determination of its transcriptional regulation pattern. In addition, RNA-seq profiling and monitoring dynamic change of intracellular key metabolite (such as ATP, NADH, reactive oxygen species) for PkJEN2 null or over-expression mutants also will be performed, the results will contribute to interprets how PkJEN2 permease exert an influence on the global metabolic pathway and organic acids resistance phenotype.

库德里阿兹威毕赤酵母能在严苛酸环境下生长，但内在耐酸机制还不清楚。自然界中耐酸较强的酵母多属Krebs-positive酵母，即能以TCA中间产物如丁二酸、苹果酸作为唯一碳源生长，因而羧酸转运是否与耐酸表型相关值得进一步研究。前期实验室分离出一株耐酸优秀的库德里阿兹威毕赤酵CY902，并完成了其基因组测序分析。序列分析发现了一个与乳酸克鲁维酵母四碳二羧酸转运蛋白KlJEN2 具有47.2%序列一致性的同源基因，命名为 PkJEN2。在CY902中敲除PkJEN2使得细胞对丁二酸和苹果酸的转运能力明显下降，且在低pH条件下生长显著变差。本研究拟进一步对PkJEN2转运底物谱、底物转运速率、亚细胞定位、转录调控模式等基础问题进行系统研究；并通过转录组和酸胁迫条件下胞内一些关键组分如ATP、NADH、活性氧自由基的浓度变化的测定，阐述PkJEN2转运蛋白对胞内整体代谢以及耐酸表型的影响。

非传统酵母库德里阿兹威毕赤酵母（Pichia kudriavzevii）以其对低pH的极强耐受能力而闻名，已被开发成细胞工厂用于各种有机酸的工业生产，特别是丁二酸。然而，库德里阿兹威毕赤酵母也可以利用Krebs循环的二羧酸和三羧酸中间产物作为唯一碳源供细胞生长，这可能对有机酸的细胞外积累产生不利影响。目前我们对库德里阿兹威毕赤酵母的有机酸转运相关知识了解较少，因此本项目从挖掘和表征新分离的库德里阿兹威毕赤酵母菌株CY902中二羧酸转运蛋白的角度，重点研究了其羧酸转运特征。通过基因组测序和转录组分析，发现两个JEN家族羧酸转运蛋白PkJEN2-1和PkJEN2-2参与了四碳二羧酸的转运。底物特异性分析表明，这两个转运蛋白均属于二羧酸转运蛋白，能有效地将丁二酸、富马酸和L-苹果酸借助质子梯度摄入细胞。此外，PkJEN2-1可以转运α-酮戊二酸，而PkJEN2-2却不能。由于PkJEN2-1的转录丰度高于PkJEN2-2，因此其在二羧酸转运中的作用比PkJEN2-2更重要。此外，PKJEN2-2还负责柠檬酸盐的摄取。据我们所知，这是首次发现酵母中的JEN2亚家族转运蛋白参与三羧酸转运。基于同源建模结构分析和蛋白理性设计进一步证明PkJEN2-2第十跨膜区（TMS-X）的392-403氨基酸残基在决定三羧酸底物的特异性方面起着重要作用。此外，通过构建丁二酸生产底盘菌株，我们发现这两种PkJEN转运蛋白仅对丁二酸表现出内向转运活性。同时失活两种PkJEN转运蛋白可减少丁二酸生产过程中有机酸的内流，有利于丁二酸在发酵后期细胞外积累。本项目实施为研究库德里阿兹威毕赤酵母利用二/三羧酸的机理提供了有益的信息，有助于提高该菌的产酸性能。

内容获取失败，请点击重试