酿酒酵母内外源杂合半乳糖代谢途径构建和优化

Galactose, the important component of hemicellulose, which was just less than xylose and arabinose. Total-sugar utilization is being attracted much concerns, and tons of work focused on the xylose-glucose co-utilization had been done. However, the researches about the co-utilization of galactose and glucose were significant less than that of xylose-glucose co-utilization. Also, limited galactose metabolism studies mainly focused on the modification of yeast native Leloir pathway, in which galactose got metabolized through five enzymatic steps to generate glucose-6-phosphate before entering the glycolytic pathway, and which will cause competent with the glucose metabolism. Therefore, novel strategy is required to enhance galactose utilization rates and simultaneous utilized galactose and glucose. Interestingly, two alternative short galactose metabolism pathways (Entner-Doudoroff or De Ley-Doudoroff) were found in some microbes, in these two pathways only 4-5 enzymatic steps were needed to convert the galactose into pyruvate and glyceraldehyde-3-phosphate. In this study, combining with galactose transporters screening and modification, the short exogenous galactose metabolic pathway was reconstructed by synthetic biology and transported into the model yeast strain Saccharomyce cerevisiae. Then, the adaptive evolution and CCR modification will be conducted further to optimize the engineered stain.

半乳糖是组成半纤维素的重要单糖，含量仅次于木糖和阿拉伯糖。在生物质全糖利用研究中对木糖和葡萄糖共利用研究最多，而半乳糖和葡萄糖的共利用研究较少，且已有研究主要集中在对半乳糖代谢的经典途径Leloir的改造，以期减少葡萄糖抑制效应 (CCR)，半乳糖经过Leloir途径中5个酶的作用转化为葡萄糖-6-磷酸，然后进入糖酵解途径，会和葡萄糖代谢产生竞争。该方法不能真正实现半乳糖与葡萄糖的共利用。自然界的个别微生物中存在不依赖糖酵解的Entner-Doudoroff或De Ley-Doudoroff半乳糖代谢途径，只需要4-5步反应，就可将半乳糖降解为丙酮酸和3-磷酸甘油醛。本项目将在酿酒酵母中重构新半乳糖代谢短途径，结合半乳糖转运蛋白筛选改造构建出半乳糖代谢杂合酵母菌株，并通过适应进化和CCR途径关键靶点调控，实现葡萄糖和半乳糖的高效共利用。

本课题以酿酒酵母为研究对象，利用合成生物学思路，重点强化半乳糖转运模块，导入外源半乳糖代谢途径，形成酿酒酵母中半乳糖内外杂合途径，提升底物利用效率。课题按照研究计划进行，重点解析了嗜热毁丝霉在葡萄糖和半乳糖条件下的转录组数据，挖掘半乳糖转运及代谢关键基因；通过整合半乳糖新转运元件GAL-2，结合导入关键代谢元件：半乳糖脱氢酶基因galdh、D-半乳糖酸脱水酶基因gdh以及KDGal醛缩酶基因Ankdg，构建了完整的De Ley-Doudoroff途径，获得含有内外杂合半乳糖代谢途径的酿酒酵母工程菌株，重组菌株的半乳糖利用速率提升了32.1%，葡萄糖利用速率提升了82.3%。本课题在酿酒酵母中研究了内外源杂合半乳糖代谢途径的构建及优化，为解除CCR以及实现葡萄糖和半乳糖的共利用、高效利用提供新思路和技术支撑，对生物炼制生产生物基化学品和生物燃料成本的降低具有重要意义。课题执行期间，发表文章2篇，申请中国专利5项，联合培养研究生2名。较好的完成了课题研究目标。