LEU2基因敲除对工业啤酒酵母高级醇生成量的影响  

Effect of LEU2 gene knockout on higher alcohols production in industrial Saccharomyces cerevisiae.

作　　者：佐一含[1] 朱旭东[2] 陈叶福[1] 吕鸿雁[1] 肖冬光[1] 

机构地区：[1]工业微生物教育部重点实验室,天津市工业微生物重点实验室,天津科技大学生物工程学院,天津300457 [2]南开大学生命科学学院,天津300071

出　　处：《中国酿造》2011年第3期27-30,共4页China Brewing

基　　金：“十一五”国家科技支撑计划子课题资金资助(2008BAI63B06)

摘　　要：通过敲除啤酒酵母中β-丙基苹果酸脱氢酶基因(LEU2),研究该基因对工业啤酒酵母高级醇特别是异戊醇生成量的影响。通过醋酸锂一步转化法,将一段两端具有LEU2基因序列同源区,中间为遗传霉素(G418)抗性基因的DNA片段转入酵母细胞中,与LEU2基因的ORF(open reading flames)进行同源重组,利用遗传霉素抗性进行筛选。将突变株与出发菌株进行发酵实验,测定其发酵性能和高级醇的生成量。筛选获得了LEU2基因突变的工业啤酒酵母。在支链氨基酸含量较低的培养基中进行发酵测定,突变株发酵液中高级醇含量比出发菌株降低了9.97%,其中异戊醇的含量降低了11.82%,而酒精度、发酵速度等发酵性能没有明显变化。LEU2基因敲除可降低工业啤酒酵母在支链氨基酸含量较低的培养基中高级醇特别是异戊醇的生成量。Effect of β-isopropylmalate dehydrogenase gene （LEU2） on production of higher alcohols, especially isoamyl alcohol, in industrial brewing yeast was studied with the method ofgene knockout. The recombinant cassette Po3-KanMX-Po4 with LEU2 gene homology region at both ends and with KanMX gene in the middle was amplified by PCR. The obtained Po3-KanMX-Po4 fragment was transformed into Saccharomyces cerevisiae using lithium acetate method. The mutant with LEU2 knocked out was selected out by the resistance to geneticin （G418） and further confirmed by PCR. Fermentation performance and production of higher alcohols by the mutant was compared with parental strain S6 in different fermentation medium. Mutant S6-1 with at least one copy ofLEU2 gene knockout was obtained. The fermentation test results showed that the production of higher alcohols from this mutant was reduced by 9.97%, of which the content of isoamyl alcohol was reduced by 11.82% when using a low branched-chain amino acids containing medium, while alcohol content, fermentation rate and other fermentation performance did not change significantly. Production of higher alcohols, especially isoamyl alcohol in the industrial S. cerevisiae S6 with LEU2 gene knockout was decreased when the branched-chain amino acids in the medium were insufficient.

关 键 词：LEU2基因 高级醇 酿酒酵母 基因敲除 

分 类 号：TS261.1[轻工技术与工程—发酵工程]