过表达EHT1基因对酿酒酵母己酸乙酯生产能力影响.pdf

现代食品科技 Modern Food Science and Technology 2014, Vol.30, No.3 过表达 EHT1 基因对酿酒酵母己酸乙酯 生产能力的影响 李锋，陈叶福，郭建，郭学武，肖冬光 （天津科技大学生物工程学院，工业发酵微生物教育部重点实验室，天津 300457） 摘要：为了提高酿酒酵母己酸乙酯的产量，缩短浓香型白酒的发酵周期，降低生产粮耗。对酿酒酵母醇己酰基转移酶(ethanol hexanoyl transferase I)基因EHT1 进行了过表达，研究其对酿酒酵母己酸乙酯生产性能的影响。以大肠杆菌-酿酒酵母穿梭质粒YEp352 为载体，磷酸甘油酸激酶基因PGK1 启动子为上游调控元件， KanMX 抗性基因为筛选标记构建了酵母菌表达质粒 Yep-PEK ，经醋酸 锂转化和 G418 抗性筛选鉴定获得过表达 EHT1 基因的突变株 AY-PEK。经玉米原料液态白酒发酵后，实验结果显示突变株的生长速 度、发酵速度、酒精度等基本发酵性能没有明显变化，但己酸乙酯的产量提高为原菌种的 2.21 倍，辛酸乙酯和癸酸乙酯也分别提高 了31.4%和 49.1%。当加入等量的己酸模仿实际发酵过程中己酸菌提供己酸时，己酸乙酯的量提高为原菌种的近3 倍，总酯也相应的 提高了 32.2%。EHT1 基因的过表达对提高酿酒酵母产酯性能，特别是产己酸乙酯有显著作用。 关键词：中国白酒；酿酒酵母；己酸乙酯；醇己酰基转移酶 文章篇号：1673-9078(2014)3-93-98 Effects of Overexpressed EHT1 on Ester Production of Saccharomyces cerevisiae LI Feng, CHEN Ye-fu, GUO Jian, GUO Xue-wu, XIAO Dong-guang (College of Biotechnology,Tianjin University of Science and Technology, Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin 300457, China) Abstract: To increase the production of ethyl caproate by Chinese liquor yeast (S. cerevisiae AY15), shorten the fermentation period, and reduce the grainconsumption, a recombinant strain AY-PEK was constructed by overexpressing EHT1 (encoding ethanol hexanoyl transferase) to investigate the effect of EHT1-overexpression on the performance of ester-producing. The EHT1 gene was amplified from genomic DNA of AY15, the PGK1 promoter and resistance gene KanMX were orderly inserted into vector Yep352 to construct the expression plasmid Yep-PEK. Then EHT1-overexpression mutants AY-PEK were selected by YEPD agar plates containing 1400 μg/mL G418 after the plasmid Yep-PEK was transformed into AY15