酿酒酵母是构建细胞工厂的重要平台微生物，而甲酸结构简单、来源丰富，且易于储存和运输。本项目以酿酒酵母高效利用甲酸为目的，研究内容包括：借鉴真核生物的甲醇利用机制，设计新型甲酸同化途径，并结合木糖代谢途径优选甲酸还原反应；利用果糖-6-磷酸的积累推动非氧化戊糖磷酸途径再生关键底物-木酮糖-5-磷酸；通过弱化甲酸氧化途径，减少甲酸损耗；借助基于梯度混合碳源的适应进化，解决甲酸同化途径、甲酸氧化途径、磷酸戊糖再生途径和糖酵解途径之间的适配问题和细胞的甲酸耐受问题；利用组学分析，确定进化突变的关键靶基因信息，并阐明途径之间的互作机制、适配策略以及甲酸耐受的强化机理；采用间接反馈控制式流加培养提高细胞的甲酸利用效率，发挥甲酸代谢途径的应用潜力。本项目的实施有望为酿酒酵母提供甲酸高效利用的实现策略，研究成果有助于进一步拓展酿酒酵母在生物炼制中的应用前景。

Saccharomyces cerevisiae is an important platform microorganism for cell factory construction, while formic acid has a simple structure, abundant sources, and is easy to be stored and transported. This project aims to develop a metabolically engineered S. cerevisiae that is capable of efficient use of formic acid as carbon source. It includes the following researches: Design a new formic acid assimilation pathway based on the methanol utilization mechanism of eukaryotes. Optimize the reduction reaction for formic acid with use of the xylose metabolic pathway. Force the non-oxidative pentose phosphate pathway to regenerate the key substrate, xylulose-5-phosphate, via fructose-6-phosphate accumulation. Reduce the loss of formic acid by weakening the formic acid oxidation pathway. Balance the formic acid assimilation pathway, the formic acid oxidation pathway, the phosphate pentose regeneration pathway, and the glycolytic pathway by the adaptive evolution using a gradient mixed carbon source. Meanwhile, increase the cell tolerance to formic acid. Identify the target genes through omics analysis. Clarify the interaction mechanisms between different pathways and the mechanisms of strengthening the formic acid tolerance. Improve the formic acid utilization via indirect feedback batch operation. This work will provide a strategy for S. cerevisiae to efficiently utilize formic acid, and promote the application of S. cerevisiae in biorefinery.