基于耐热酵母人工菌群实现共利用葡萄糖和木糖高产3-HP及其阻遏机制

目前酵母等真核微生物葡萄糖阻遏效应的分子机制尚未完全明确，极大的限制了混合碳源(例如木质纤维素的水解产物葡萄糖和木糖)的同步共利用。为了克服这一问题，本项目以GRAS耐热性马克斯克鲁维酵母(Kluyveromyces marxianus)为研究对象，基于瞬时CRISPR/Cas9技术构建选择性利用葡萄糖、木糖以及甘油为底物合成3-羟基丙酸的模块化功能菌株，采用串联、并联和混联的模块化组装策略建立共利用混合碳源的酵母人工菌群共培养体系，比较不同的模块化组装方式对合成效率的影响，明确细胞间的信息互作和调控，对细胞间的物质流和能量流进行表征。比较功能菌株的转录组和SNP差异，鉴定马克斯克鲁维酵母中葡萄糖阻遏木糖代谢的关键基因，阐明其作用机制。单物种多菌株的人工菌群相比多物种菌群更加稳定，又可以完成单一菌株培养体系难以实现的多底物共利用和复杂化合物合成过程，本项目将拓展其在食品合成生物学中的应用。

The key mechanisms of carbon catabolite repression have not been fully clarified in yeast, thus the simultaneous co-utilization of mixed carbon sources (such as the glucose and xylose mixture from lignocellulose hydrolysis) have been limited a lot. This project proposes to develop co-culture systems with engineered Kluyveromyces marxianus strains that selectively use glucose, xylose or glycerol to produce 3-hydroxypropionic acid. By co-culturing these strains with different assembly strategies, we will obtain the relationship between product synthesis efficiency and assembly strategies, clarify the information interaction and regulation between cells, and characterize the material and energy flows. By comparing the transcriptomes differences between strains using different carbon sources, and the SNPs between strains, we will reveal the molecular mechanism of carbon catabolite repression of glucose on xylose in K. marxianus. This single-species and multi-strains co-culture system is more stable than a multi-species co-culture system and suitable for complex biochemical synthesis which is difficult for a monoculture system. These efforts should significantly expand the application of single-species and multi-strains co-culture system in food synthetic biology.