强化酿酒酵母固定二氧化碳生产二元羧酸的"生物分子阀门"研究

Dicarboxylic acids are widely used in food, pharmaceutical and chemical industries. In recent years, they have attracted increasing attention since they can be used as precursors of therapeutic drugs and starting materials for polymerization and esterification reactions. The yeast Saccharomyces cerevisiae was regarded as the first choice for dicarboxylic acids production in biotechnological industries. Moreover, the production process involves biological CO2 fixation instead of release, which increases concerns of climate change. Pyruvate carboxylase plays a key role in the dicarboxylic acid production in S. cerevisiae. Therefore, more in-depth knowledge about its functional rule is needed. In this project, production of fumaric acid, a typical dicarboxylic acid, is selected as a model for bio-molecular valve analysis. The pyruvate pool and fumaric acid biosynthetic pathway will be constructed for the optimal pyruvate carboxylase selecting. After that, effects of the pyruvate carboxylase expression level on carbon metabolic flux will be investigated. With the optimized expression level of pyruvate carboxylase, the bio-molecular valve will finally be fixed. In order to understand the regulating rule of bio-molecular valve, the effects of cofactor of pyruvate carboxylase on the carbon metabolic flux will also be investigated. The research presented here will provide improved sight on regulating the carbon metabolic flux by pyruvate carboxylase, and novel strategy to achieve the high efficient-production of dicarboxylic acids.

二元羧酸广泛地用于食品、医药和化学等产业，近年来，由于它可用于药物前体和聚合反应或酯化反应材料而备受关注。酿酒酵母是生产二元羧酸的潜在最适微生物，利用酿酒酵母生产二元羧酸还可以固定二氧化碳，具有重要的的环保意义。丙酮酸羧化酶在强化酿酒酵母固定二氧化碳高效生产二元羧酸中起着关键的作用，但对其作用规律还缺乏系统的研究和深入的认识。本研究以酿酒酵母生产延胡索酸为研究模型，在构建"丙酮酸库"及延胡索酸的合成途径的基础上，选择最佳的丙酮酸羧化酶，研究其表达水平对碳代谢流分布的影响，从而确定合适的表达水平，实现"生物分子阀门"的构建。此外，通过研究丙酮酸羧化酶的辅因子对碳代谢流分布的影响，阐明"生物分子阀门"的控制规律。本研究将对丙酮酸羧化反应在调控碳代谢流方面的作用提供理论依据，为实现二元羧酸的高效生产提供重要的指导作用。

二元羧酸（延胡索酸、琥珀酸和苹果酸等）在食品、医药、化工以及畜牧等领域有着十分重要的用途。微生物发酵法生产二元羧酸备受关注，酿酒酵母菌因具有诸多优势而成为生产二元羧酸潜在最适生产菌株。丙酮酸羧化酶充当着“生物分子阀门”的作用—将碳流由丙酮酸引入到目标羧酸的合成途径。. 本项目以酿酒酵母CEN.PK2-1C为底盘微生物，通过敲除PDC1和ADH1基因来减少乙醇生成，并对ACE2等基因调控乙醇发酵的规律展开研究。在pdc1adh1缺失株中通过敲除FUM1基因来构建延胡索酸的合成途径，发酵96 h时积累1944.0 mg/L延胡索酸。本项目将米根霉的丙酮酸羧化酶基因RoPYC在酿酒酵母中进行表达后，延胡索酸产量达222.0 ± 2.2 mg/L。随后，重点研究了利用饱和突变技术改变丙酮酸羧化酶RoPYC的特性来提高延胡索酸产量。考虑到丙酮酸羧化酶的突变体P458S能够促进谷氨酸棒杆菌生产氨基酸，本项目对RoPYC的同源位点P474进行饱和突变，获得了酶活性提高的突变体P474N，它表达后，发酵96 h时延胡索酸产量为2855.7 mg/L，较对照提高13.3%。之后，又筛选出正突变体RoPYC N315F、R485P和N1078F，发酵96 h，延胡索酸产量分别为290±11.2、312±14.0和309±12.0 mg/L，较对照分别提高了22.0%、31.1%和29.9%。将丙酮酸羧化酶基因密码子优化得到的基因RoPYC*进行表达，延胡索酸产量达426.4±8.9 mg/L，较对照提高32.2%。对RoPYC*进行定点突变，仅有突变体RoPYC* R485P的表达促进了延胡索酸的积累，进而研究生物素调控延胡索酸的规律，发酵120 h可积累延胡索酸465.5±6.5 mg/L，较对照提高了24.6%。最后，在7 L发酵罐条件下，探究了菌株pdc1adh1fum1↑RoPYC生产延胡索酸的条件，36 h时通入CO2，发酵120 h可积累延胡索酸453.6 mg/L，较对照提高了20%。此外，本项目还测试了丙酮酸羧化酶在琥珀酸等生产中的作用。. 本项目回答了“如何更好地将碳代谢流从丙酮酸引流到目的产物的合成途径？如何调节碳代谢流由丙酮酸通往目的产物的合成途径？”的科学问题。为在酵母中有效地将碳流由丙酮酸引入到二元羧酸的合成途径，从而实现高效生产提供了重要的理论指导