解脂亚洛酵母酮酸转运蛋白的生理功能与调控机制

During the production of central metabolism pathway related carboxylic acids by microorganisms, there are always kinds of non-target carboxylic acids byproducts. The regulation on the central metabolism pathway and cofactor balances had little effects on the elimination of these non-target carboxylic acids. Therefore, based on the demonstration of the dynamics and regulation mechanisms of carboxylic transporters, the regulation of these transporters should effectively solve this issue. During the production of alpha-ketoglutaric acid by Yarrowia lipolytica, the pyruvic acid is always existed at a high level. However, there is no related reports about which transporters are responsible for these two kinds of typical keto acids. In this project, we plan to discover the transporters responsible for the two keto acids based on the combination of next-generation high-throughput sequencing and molecular biology routes. Besides, the regulation mechanisms of these transporters on the central metabolism pathway, cofactor balance, and pH homeostasis, would also be investigated with pH-sensitive fluorescent probes, real-time PCR, RNA sequencing (RNASeq). Furthermore, the carboxylic acids transportation process would be regulated by metabolism and extracellular culture environment optimization, results in a Y. lipolytica strain with significant decrease in the pyruvic acid production and enhanced alpha-ketoglutaric acid production. By using this strain, the role of these transporters in the response of environmental factors and cell survival should also be well deomonstrated.

发酵法生产中心代谢途径相关羧酸过程中，通常存在非目标羧酸干扰。针对中心代谢途径和辅因子平衡的调控，对于非目标羧酸的消除效果有限。在阐明特定羧酸转运蛋白的动力学特性及其调控机制的基础上，对其进行调控可以有效解决上述问题。在解脂亚洛酵母合成alpha-酮戊二酸的过程中，存在严重的丙酮酸积累。目前alpha-酮戊二酸和丙酮酸的特异转运蛋白还缺乏报道。本项目拟采用基于二代测序和分子生物学技术相结合的方法，发现负责两种典型酮酸跨膜运输的转运蛋白。采用基于pH敏感的荧光染料、定量PCR、转录组测序等技术，分析酮酸转运蛋白在中心代谢途径、辅因子平衡和胞内pH稳定过程中的作用及其调控机制。在此基础上，采用代谢工程和胞外环境优化策略，调控羧酸转运过程，得到一株胞外丙酮酸积累显著消除、alpha-酮戊二酸积累显著增强的菌株，并初步阐明酮酸特异转运蛋白对于环境因素的响应及其存在对于解脂亚洛酵母生存的生理学意义。

(1) 采用Illumina MiSeq高通量测序技术和PacBio RS高通量测序技术对α-KG高产菌株Y. lipolytica WSH-Z06进行了全基因组测序，获得了大小为20.14 Mbp包括6条染色体和1条环状线粒体的基因组序列。通过对比分析Y. lipolytica WSH-Z06菌株与Y. lipolytica CLIB122菌株的α-KG合成相关途径，发现了10条对α-KG合成有帮助的基因发生了拷贝数的变化。相对于Y. lipolytica CLIB122菌株，Y. lipolytica WSH-Z06菌株具有缺失甘油三脂合成途径中酶编码基因和增加异柠檬酸脱氢酶拷贝数的α-KG合成基因组特异性。.(2) 基于Y. lipolytica WSH-Z06全基因组测序结果，从其基因组范围内筛选出YALI0B19470p、YALI0C15488p、YALI0C21406p、YALI0D24607p、YALI0D20108p和YALI0E32901p可能的酮酸转运蛋白，并在酮酸转运功能缺失的Saccharomyces. cerevisiae细胞中验证其多功能的转运功能。过量表达YALI0B19470p的重组菌株细胞外积累的α-KG从36.6 g L-1上升至46.7 g·L-1，而胞外的丙酮酸含量从17.8 g·L-1降低至12.3 g·L-1，强化了α-KG在细胞外的积累。从酮酸转运的机制角度表明，细胞同时编码6条能以多种羧酸为底物的酮酸转运蛋白是Y. lipolytica WSH-Z06菌株能同时积累多种有机酸的原因之一。.(3) 通过基于双向电泳的比较蛋白组学研究揭示了在发酵生产α-KG过程中降低培养环境中的pH值能促进α-KG的过量积累。在低环境pH条件下，细胞为维持细胞内环境的相对恒定，通过提高两个H+-转运ATPases的表达强化将胞浆中H+转运出细胞。同时，细胞提高电子传递链上4个组分的表达量将线粒体内的H+通过电子链传递给活性氧形成H2O并加快ATP的生成。为回补细胞内加速消耗的ATP和NADH/NADPH等高能化合物，细胞通过提高糖异生途径中的7个蛋白和三羧酸循环中7个蛋白的表达量，强化对底物甘油和丙酮酸分解利用为细胞提供能量。

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