阿拉伯糖快速发酵酿酒酵母的反向代谢工程

Arabinose is the second most abundant pentose, less than glucose and xylose, present in the cellulosic ethanol production. The Saccharomyces cerevisiae will acquire the arabinose utilization ability through the introduction of the arabinose metabolic pathway from other species, and the fermentation performance of the strain further improved during serial transfer in the arabinose as the sole carbon medium, while the underlying mechanism is unclear. Combining the genetic engineering and adaptive laboratory evolution , we constructed a fast arabinose fermentation strain based on a xylose fermentation yeast developed by our laboratory previously, the arabinose metabolic rate of which is superior to those reported in literature. Choosing before and after evolution strains as materials, this project aims to identify mutations resulting in efficient arabinose fermentation and to reveal their molecular mechanisms by genome sequencing，comparative genomic analysis, gene disruption, retro-complementation and biochemical analysis .Then, through the strains rational design to further confirm the mechanisms uncovered. The achievements of this project will contribute to developing better performance cellulosic ethanol producing yeast.

阿拉伯糖是纤维素乙醇生产中仅次于葡萄糖和木糖的可发酵糖原料。酿酒酵母阿拉伯糖发酵能力可通过导入异源阿拉伯糖发酵途径获得，并在阿拉伯糖培养基中连续传代进一步提升，但传代过程中发生的突变及机制不明。申请人在一株木糖发酵酵母中进行上述操作使其发酵阿拉伯糖快于文献报道的速率。本项目拟以连续传代前后的酿酒酵母为材料，通过比较基因组学、基因中断、遗传回补和生化分析，鉴定并解析阿拉伯糖快速发酵表型的突变及机制，并通过理性重构进一步证实。本项目成果将有助于选育更高效的纤维素乙醇酵母。

赋予酿酒酵母阿拉伯糖利用能力是提高纤维素乙醇经济性的重要手段之一，酿酒酵母阿拉伯糖发酵能力可通过导入异源阿拉伯糖发酵途径获得，并在阿拉伯糖培养基连续传代（驯化）进一步提升，但是传代过程中发生的有利突变及机制不明，使得不依赖驯化理性构建阿拉伯糖酵母难以实现。.以自行构建的可快速发酵木糖的工业二倍体酿酒酵母CIBTS0735为出发菌株，整合阿拉伯糖代谢基因表达盒（AUC），经驯化后获得快速发酵阿拉伯糖的菌株CIBTS1974。比较基因组学及real time PCR分析发现AUC拷贝数由1个扩增到12个，推测机制为AUC两端重复序列有丝分裂时发生非等位交换，引起AUC串联复制。将CIBTS1974所有AUC敲除，所得菌株CIBTS1975无法利用阿拉伯糖，证明高拷贝AUC对菌株发酵阿拉伯糖是必要的。在CIBTS1975染色体回补AUC拷贝数至12，菌株阿拉伯糖代谢表型完全回补。此外，直接在出发菌CIBTS0735迭代整合AUC拷贝至12，所得菌株完全回补驯化株CIBTS1974表型且具有剂量效应，表明高拷贝AUC是菌株获得阿拉伯糖发酵能力的充分条件。菌株AUC拷贝数进一步增加到14，阿拉伯糖发酵能力略优于驯化株，表明可通过理性设计获得表型更好的阿拉伯糖酵母。另外我们还发现AUC包含的两个转运蛋白Gal2和Stp2，Gal2起主导作用而Stp2基本无用。以上研究首次阐明了酿酒酵母经驯化获得快速发酵阿拉伯糖能力的遗传机制，为理性设计构建工业阿拉伯糖酵母菌株提供指导。