基因组复制工程辅助的连续进化方法(GREACE)降低工业啤酒酵母乙醛产量的机理研究

The improving of stress resistance characters in yeast strains have been realized through the evolution engineering mrthod. Generally, high acetaldehyde concentration is believed to cause disgusting flavors, shorten the flavor fresh-keep period. Because of the back mutation of traditional genetic engineering strains, the complex genetic background of beer industrial yeast and food security reasons, the progress of evolution engineering to improve the yeast acetaldehyde metabolism is slow, which limits the improvement of beer flavor and quality. The evolutionary adaptation problems on the synthesis of acetaldehyde in beer industrial yeast strain and its corresponding haploid will be researched in this study. By the method of genome replication engineering assisted continuous evolution, the gene pol3-01 will be introduced in the beer yeast strains as an exogenous disturbance factor. As a result, the acetaldehyde synthetic pathway will be weakened, and the acetic acid synthesis pathway will be strengthened. The yeast strains with low yield of acetaldehyde will be used as the model strains, the mechanism of precursor supply, synthesis and tolerance in acetaldehyde metabolism will be studied using genomic sequencing, transcriptome, molecular biology and metabolic engineering method. The strategy and related mechanism will contribute to develop the follow-up industrial beer yeast strains with excellent properties, and provide the methodological and theoretical reference for other industrial strains in metabolic engineering improvement.

进化工程改造酵母菌株已实现实验室酵母菌抗逆性状的改良。乙醛含量过高会给啤酒带来生青味、缩短啤酒的风味保鲜期。由于传统基因工程菌株易回复突变、啤酒工业酵母菌遗传背景复杂及食品安全性等原因，进化工程改善酵母乙醛代谢方面的研究进展缓慢，限制了啤酒风味的改良和啤酒品质的提升。本课题针对乙醛在啤酒酵母工业菌株及相应单倍体内合成中的进化适应性问题进行研究。通过基因组复制工程辅助连续进化方法，在啤酒酵母菌株中引入基因pol3-01作为外源扰动因素，弱化乙醛合成途径、强化乙酸合成途径，削弱乙醛在啤酒酵母中的合成代谢。以研究中得到的低产乙醛啤酒酵母菌株为模型，利用基因组测序、转录组、分子生物学和代谢工程手段，研究在乙醛合成中，前体供给，产物合成和耐受机制。本研究中采用的策略和阐明的相关机制，将为后续开发啤酒酵母工业菌株的优良性能奠定坚实的基础，并为其它工业菌株的代谢工程改造提供方法和理论上的参考。

乙醛是啤酒中使饮酒者“上头”的物质之一，含量过高对啤酒风味具有不良影响。所以降低乙醛含量是目前啤酒行业需要解决的问题之一。啤酒酵母(Saccharomyces pastorianus, syn. Saccharomyces carlsbergensis)是啤酒酿造的灵魂，乙醛在啤酒中主要来自酵母代谢，因此，获得低产乙醛酵母菌株才是降低乙醛含量的根本途径。本课题采用常压室温等离子体(ARTP)诱变技术结合双硫仑-甲吡唑双抑制剂进行定向筛选，最终获得一种高效的低产乙醛酵母筛选培养基配方；进而构建了啤酒酵母表达系统，通过基因组复制工程辅助连续进化方法，获得低产乙醛啤酒酵母菌株；基于对出发菌株M14的全基因组测序，以及出发菌株和优选菌株MGA的转录组学分析结果，对酵母菌株低产乙醛的机制进行了多系统性层次分析；并通过强化TCA循环途径，使得发酵液中乙醛含量显著降低，进而考察了醇脱氢酶及NADH对啤酒发酵的影响，提出了利用反向代谢工程和辅因子工程手段增加啤酒酵母细胞内NADH/NAD+比率，降低啤酒产品中乙醛含量的新策略，为啤酒酵母菌株的乙醛代谢调控提供了有力的数据参考。

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