过表达EHT1基因对酿酒酵母己酸乙酯生产能力的影响

Effects of Overexpressed EHT1 on Ester Production of Saccharomyces cerevisiae

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。

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. The results showed that after liquid fermentation by corn, the differences in fermentation performances (growth rate, fermentation rate and the yield of alcohol) between AY-PEK and AY15were negligible. However, the production of ethyl caproate produced by AY-PEK was markedly increased to 2.21-fold as high as that of parental strain AY15. Ethyl octanoate and ethyl decanoate were improved by 31.4% and 49.1%, respectively. The same amount of caproic acid was added to fermentation medium to imitate the actual fermentation, the production of ethyl caproate produced by AY-PEK was increased to nearly 2-fold higher than that of AY15, and the amount of total esters were also increased by 32.2%. Overexpression of EHT1 gene can significantly enhance the production of ethyl esters, especially ethyl caproate.