酿酒酵母基因组位置效应对外源基因表达的影响

Effect of integration loci of genome on heterologous gene expression in Saccharomyces cerevisiae

摘要:

外源基因元件和模块在底盘细胞中发挥特定功能是合成生物学研究的基本过程，而外源元件和模块在基因组中的位置对其功能的实现具有显著影响。为了系统、全面地表征酿酒酵母基因组位置效应对外源基因的表达影响，以绿色荧光蛋白为报告基因，通过双交换同源重组方法，对酿酒酵母单基因敲除库进行高通量转化，构建酿酒酵母基因组单位点荧光标记菌株库。结合流式细胞术和高通量测序技术对单位点荧光标记库菌株进行分析，构建高表达位点库和低表达位点库，共发现促进绿色荧光蛋白表达的位点428个，抑制绿色荧光蛋白表达的位点444个。通过分析高、低表达位点在酵母染色体上的分布，从全基因组尺度上对酿酒酵母基因组整合位置对基因表达的影响进行表征。本研究可为酿酒酵母基因组位置效应的分布规律和产生机理研究提供重要参考，对外源蛋白工业生产和合成生物学中的基因表达精细调控也具有重要的指导意义。

Abstract:

Chromosomal integration of heterologous genes or pathways is preferred over the use of episomal plasmids for its inherently stability and thus more desirable in the industrial setting. However, the position of integration of heterologous genes in the genome influences the expression levels. In combination of high throughput transformation of the Yeast Knock-out Collection (YKO) and FACS analysis, the position effect on heterologous reporter gene gfp was identified across the whole genome in yeast. In total 428 high-expressed sites and 444 low-expressed sites were spotted, providing massive data to analyze patterns and reasons for region dependency of gene expression on the genome-wide scale.