单细胞水平的工业酵母耐受木质纤维素水解抑制物关键信号通路的微进化研究

As interest in alternative energy sources increases, lignocellulose materials have become attractive potential low-cost feedstock for bioethanol production. For economic reasons, dilute acid hydrolysis is commonly used to prepare lignocelluloses for enzymatic saccharification and fermentation. However, numerous side-products are generated during the pretreatment, many of which inhibit microbial fermentation significantly. Development of genetically manipulated industrial Saccharomyces cerevisiae strains with in situ detoxification capacity and greater inhibitor tolerance is one of the most promising solutions. However, it is hindered by a lack of understanding of the microevolution mechanisms underlying inhibitor tolerance in yeast. In previous study, we have identified MAPK and phosphatidylinositol (PI) signaling pathways as the key tolerant signal transduction systems to inhibitors. .A single cell is the origin and a basic unit of evolution for cellular lives, therefore, fundamental understanding of microevolution necessitates tracing trait origin and development at single-cell resolution. In this research, we will combine single-cell sorting and single-cell functional genomics techniques to trace the genetic mutations and phenotype variations and to screen the distribution, frequency and evolution pattern of mutations at single cell resolution in industrial Saccharomyces cerevisiae strain NRRL Y-12632 and its mutants. Furthermore, the mutations identified at the single cell resolution will be compared to that at the population resolution. These efforts will reveal the heterogeneity among individual cells to the functioning and evolution of cellular population. Crucial genes and mutations in MAPK and PI signal pathways will be identified and their functions will be verified. This study will help unravel the evolution mechanism of inhibitor adaption and guide the genetic modification of industrial yeast.

生物质降解过程中产生的抑制物是限制燃料乙醇生产效率的瓶颈问题，诱导和筛选强耐受性酵母菌株是最优解决方案。基因型及遗传基础对耐受性具有决定性作用，因此揭示抑制物耐受性的微进化机制是获得耐受性菌株的重要基础。研究表明，MAPK和磷脂酰肌醇（PI）信号通路是酵母耐受抑制物的关键信号通路。.单个细胞是生命的基本单位和进化的起点，在单细胞水平考察性状的发生与发展，能从根本上认识微进化。本项目以工业酿酒酵母菌株Y-12632及其突变株为研究材料，结合单细胞分选和功能基因组技术，检测单细胞尺度的基因组突变和表型变异，考察遗传变异的演化机制和规律，并通过与种群水平基因组变化的比较分析，深入探讨细胞个体间的异质性和基因型与表型的相互关系，鉴定MAPK和PI信号通路上决定耐受抑制物的关键基因及突变，并初步验证其功能。研究结果对于阐明抑制物耐受性的进化机制，指导生产菌株的定向改造具有重要意义。

生物质降解过程中产生的抑制物是限制燃料乙醇生产效率的瓶颈问题，诱导和筛选抢耐受性酵母菌株是最优解决方案，基因型及遗传基础对耐受性具有决定性作用，揭示抑制物耐受性的微进化机制是获得耐受性菌株的重要基础。本项目以工业酿酒酵母高耐受性菌株Y50049和Y12632为研究材料，开发了能够进行酵母单细胞高效分选的微流控芯片技术，应用该技术进行单细胞液滴分选的成功率达到90%，单细胞液滴用于细胞培养的成功率达到80%，以单细胞液滴直接进行实时荧光定量PCR，48.3%的单细胞液滴可获得有效扩增曲线，CT值为44.86±2.86。对于获得的101个单细胞进行基因组测序，单个细胞基因组平均序列比对率为65.2%，基因组覆盖率达到43.3%。对单细胞基因组数据进行序列比对和SNP鉴定，在Y50049和 Y-12632各获得了27508和26785个SNP，其中Y50049中含有外显子SNP 8710个，内含子SNP 3449个，编码区上下游区域SNP 2268个，基因间隔区SNP 13081个。将SNP相关基因在GO和KEGG数据库进行了功能分层和代谢通路分析，解析了各类突变与单细胞耐受性差异的关联，应用选择消除分析分析，计算遗传分化指数（Fst）和核苷酸多态性指数（π）等遗传分离指标，鉴定了68个受选择的SNP位点和15个相关基因。将这些位点和基因映射到MAPK、和磷脂酰肌醇（PI）信号转导通路中，鉴定了与耐受性微进化相关的蛋白激酶C 1基因（pkc1）等关键节点基因。研究结果对于阐明酵母抑制物耐受性的进化机制，指导具有优良性状的生产菌株的定向筛选和改造具有重要意义。.同时，本项目研发过程中构建的针对微生物细胞的“基于微液滴的单细胞分离—核酸扩增—文库构建—功能基因组测序”的完整技术流程，为微生物单细胞基因组相关研究提供了重要的技术平台和保障。