一次敲除BAT2同时过表达Lg-ATF1对啤酒酵母产醇酯的影响

Effect of BAT2 Deletion with Lg-ATF1 Overexpression on the Production of Higher Alcohols and Ester in Brewer’s Yeast

摘要:

乙酸乙酯和高级醇是啤酒中的重要风味物质，为了探究BAT2基因和Lg-ATF1基因对啤酒酵母产醇酯能力的影响，进而解决啤酒中存在的醇高酯低的问题。本研究通过酶切连接法构建重组质粒pUC-PLABBK，采用醋酸锂转化法和胞内同源重组技术，以多倍体啤酒酵母菌株S5为出发菌株，KanMX基因作为筛选标记，最终获得过量表达Lg-ATF1基因同时敲除BAT2基因的重组菌株S5-Lg。通过啤酒发酵实验和数字PCR探究重组酵母菌株S5-Lg与出发菌株S5醇酯含量与相关基因表达量的变化。结果显示，与出发菌株相比，S5-Lg的总高级醇生成量降低9.12%，其中异丁醇和异戊醇的生成量分别降低了10.63%和9.55%，乙酸乙酯生成量提升了26.81%，BAT2基因表达量降低45.72%，Lg-ATF1基因表达量大幅提升。BAT2基因和Lg-ATF1基因可以影响啤酒酵母产生高级醇和乙酸乙酯的含量，对改善啤酒风味有重要参考意义。

Abstract:

Ethyl acetate and higher alcohols are important flavor substances in beer. The effect of BAT2 gene and Lg-ATF1 gene in beer yeast on producing alcohol esters was explored to solve the problem of more higher alcohols and less ethyl acetate in beer. The expression vector pUC-PLABBK was constructed by using the method of enzyme digestion and connection. Saccharomyces cerevisiae multiploid S5 was used as the original strain and the KanMX gene was used as a selection marker in this study. Lithium acetate transformation and homologous recombination were employed to screen for a mutant strain S5-Lg that overexpressing Lg-ATF1 and at the same time deleting BAT2. The variation between the content of higher alcohols, ethyl acetate and expression of related genes in the engineered yeast strain S5-Lg was investigated by fermentation experiments and digital PCR. The results showed that compared with the original strain S5, the ethyl acetate production of the S5-Lg strain was increased by 26.81%, total higher alcohol production decreased by 45.72%, of which the contents of isobutanol and isoamyl alcohol were reduced by 10.63% and 9.55%, respectively. The expression of Lg-ATF1 gene was greatly increased, and the expression level of BAT2 gene was decreased by 45.72%. It effectively changed the ability of brewer's yeast to produce ethyl acetate and higher alcohols and has important significance for improving beer flavor.