多质粒共转化组合筛选方法构建木糖利用酿酒酵母的研究

快速得到目标代谢路径相关基因的大量组合以及实现组合库的高效筛选，是合成生物学领域中一个重要的研究内容。建立了三质粒共转化酵母菌株组合筛选方法并以XR-XDH木糖代谢路径在酿酒酵母中的应用为例进行阐释。首先利用Yeast Golden Gate连接法在三种不同表达载体上构建不同启动子控制下的XR、XDH、XK单个基因的表达盒，然后直接用三质粒共转化系统构建100种不同组合的重组酵母。经过木糖平板初筛筛选出16个能利用木糖的组合，将这16个组合对应的三基因表达模块组装至同一表达载体后转化底盘菌株，再通过限氧发酵进行复筛，最终筛选出木糖代谢能力、木糖醇和乙醇生成速率最优菌株Sc-LQH35（TDH3p-XR-ACS2t-FBA1p-XDH-ENO2t-PDC1p-XK-ASC1t），在培养基中含有20 g/L木糖的条件下，其木糖醇产量为7.14 g/L，乙醇产量为5.92 g/L，而菌株Sc-LQH39（TDH3p-XR-ACS2t-FBA1p-XDH-ENO2t-ZEO1p-XK-ASC1t）则表现出较强的木糖醇生产能力，特别在限氧发酵时，其木糖醇得率可高达0.71 g/g。三质粒共转化组合筛选方法实现了木糖利用菌株的灵活构建和快速筛选，并成功得到具有优良木糖利用性能的酿酒酵母菌株，表明其在重组菌株的构建和筛选工作中有一定的应用价值。

Construction of Recombinant Xylose-utilizing Saccharomyces cerevisiae by Three-plasmid Co-transformation Combinatorial Screening Method

In the field of synthetic biology, quick construction of target metabolic pathways and rapid screening of combinatorial libraries is of great significance. A combinatorial screening method by co-transforming Saccharomyces cerevisiae with three plasmids was established and XR-XDH pathway was constructed in Saccharomyces cerevisiae as an application of the method. The gene expression cassettes of XR,XDH and XK were constructed efficiently using the Yeast Golden Gate (yGG) method. 100 recombinant strains with different promoter combinations were obtained through the three plasmids co-transformation system. Then the colonies were screened through spot assay on 2% SX plate and 16 colonies were chosen. In order to make the property more stable, the separate three gene modules of the corresponding colonies were assembled together to the expression vector pRS426, and then transformed to BY4741 to obtain the new recombinant strains. Oxygen-limited fermentation was carried out to test these strains. Among the 16 strains, Sc-LQH35(TDH3p-XR-ACS2t-FBA1p-XDH-ENO2t-PDC1p-XK-ASC1t) showed the highest products yield and fastest xylose utilization speed. Under oxygen-limited condition, xylitol and ethanol could accumulate to 7.14 g/L and 5.92 g/L separately when the medium contained 20 g/L xylose. The strain Sc-LQH39(TDH3p-XR-ACS2t-FBA1p-XDH-ENO2t-ZEO1p-XK-ASC1t) showed strong ability of producing xylitol on the oxygen-limited fermentation and xylitol yield could reach as high as 0.71 g/g. Three-plasmid co-transformation combinatorial screening method realized the flexible construction and rapid screening of the xylose-utilizing strains. Strains with high fermenting performance were obtained, and it showed that the method has great potential application in construction and screening of the recombinant strains.