异柠檬酸脱氢酶基因调控对拉格酵母抗自溶性能的影响

Manipulation of isocitrate dehydrogenase genes affects the anti-autolytic ability of lager yeast

摘要:

啤酒酵母的自溶会影响啤酒的风味和品质，对酵母自溶的调控是工业啤酒生产的迫切需求。前期在啤酒酵母自溶的研究中发现柠檬酸循环相关基因对酵母自溶有较大影响。为探究柠檬酸循环中异柠檬酸脱氢酶基因调控对自溶的影响，在典型拉格啤酒酵母(Saccharomyces pastorianus H.) Pilsner中对IDP1和IDP2基因进行了破坏和过表达。结果发现IDP1基因的破坏能提高酵母的抗自溶能力，自溶96 h抗自溶指数为8.40，比原始菌提高了1.5倍。IDP1基因的破坏提高还原型辅酶Ⅱ(nicotinamide adenine dinucleotide phosphate,NADPH)的供应，NADPH/NADP⁺的比值为1.94，发酵结束时胞内ATP水平比原始菌提高1.8倍，活性氧(reactive oxygen species,ROS)相比原始菌减少10%。IDP2基因的缺失造成酵母快速自溶和NADPH供应的减少，自溶96 h抗自溶指数为4.03，NADPH/NADP⁺的比值为0.89。发酵结束时胞内ATP水平相比原始菌减少8%，ROS是原始菌的1.3倍。本研究进一步阐释了柠檬酸循环相关基因对酵母自溶的调控机理，为选育自溶性能可控的优良酵母提供了理论基础。

关键词:拉格酵母;抗自溶性能;柠檬酸循环;NADPH

Abstract:

Yeast autolysis affects the flavor and quality of beer. The regulation of yeast autolysis is a need for industrial beer production. Previous studies on brewer's yeast autolysis showed that the citric acid cycle-related genes had a great influence on yeast autolysis. To explore the contribution of isocitrate dehydrogenase genes in autolysis, the IDP1 and IDP2 genes were destroyed or overexpressed in typical lager yeast Pilsner. The destruction of IDP1 gene improved the anti-autolytic ability of yeast, and the anti-autolytic index after 96 h autolysis was 8.40, 1.5 times higher than that of the original strain. The destruction of IDP1 gene increased the supply of nicotinamide adenine dinucleotide phosphate (NADPH) and the NADPH/NADP⁺ ratio was 1.94. After fermentation, intracellular ATP level was 1.8 times higher than that of the original strain, while reactive oxygen species (ROS) was reduced by 10%. The destruction of IDP2 gene resulted in rapid autolysis and a decrease in the supply of NADPH. Anti-autolytic index after 96 h autolysis was 4.03 and the NADPH/NADP⁺ ratio was 0.89. After fermentation, intracellular ATP level was reduced by 8% compared with original strain, ROS was 1.3 times higher than that of the original strain. The results may help understand the regulation mechanism of citric acid cycle-related genes on yeast autolysis and provide a basis for the selection of excellent yeast with controllable anti-autolytic performance.

Key words:lager yeast;anti-autolytic ability;citric acid cycle;NADPH