[关键词]
[摘要]
酿酒酵母BAT基因编码支链氨基酸转氨酶,其中BAT1和BAT2基因分别编码线粒体和细胞质氨基酸转氨酶,位于细胞不同的位置导致二者的生理功能有所差异,BAT1基因在线粒体中倾向催化α-酮酸合成氨基酸,细胞质中的BAT2基因将氨基酸转化为α-酮酸,通过敲除BAT2以减少α-酮酸合成,过表达BAT1以增加α-酮酸消耗达到降低酿酒酵母高级醇的合成的目的。本研究以酿酒酵母AY15单倍体α5为出发菌株,结合融合PCR技术构建重组质粒pUC-BABPB1K,获得BA-PGK-BAT1-BB重组盒,并利用醋酸锂转化法和同源重组技术筛选出缺失BAT2基因同时过表达BAT1基因的突变株B-8,将其和亲本菌株α5、BAT2基因缺失菌株α5ΔBAT2进行酒精发酵实验,发酵结束后进行发酵性能和高级醇的测定。实验结果表明,与亲本菌株相比,异丁醇降低了25%,异戊醇降低了15%,活性戊醇降低了30%;与α5ΔBAT2菌株相比,异丁醇提高了0.5倍,异戊醇增加了0.1倍,活性戊醇增加了0.3倍。
[Key word]
[Abstract]
Branched-chain amino acid transaminases are encoded by BAT genes in Saccharomyces cerevisiae, and BAT1 and BAT2 encode mitochondrial and cytosolic branched-chain amino acid transaminases, respectively. Owing to their different locations in cells, BAT1 and BAT2 have different physiological functions. BAT1 in the mitochondria has a tendency to carry out the catalytic synthesis of amino acids from α-keto acids, and BAT2 in the cytoplasm is responsible for converting amino acids into α-keto acids. This study attempted to reduce the synthesis of α-keto acids and increase the consumption of α-keto acids in S. cerevisiae by knocking out BAT2 and overexpressing BAT1, in order to decrease the level of higher alcohols in wine. Saccharomyces cerevisiae AY15 haploid α5 was used as the parental strain in this study, and fusion polymerase chain reaction (PCR) was adopted to construct the recombinant plasmid pUC-BABPB1K, and the recombinant gene cassette BA-PGK-BAT1-BB was obtained. Lithium acetate transformation and homologous recombination were employed to screen for a mutant strain B-8 overexpressing BAT1 and lacking BAT2. B-8, parental strain α5, and strain α5ΔBAT2 lacking BAT2 were used to conduct ethanol fermentation, and the fermentation performance and yield of higher alcohols were determined at the end of fermentation. The yields of isobutanol, isopentanol, and 2-methyl-1-butanol decreased by 25%, 15%, and 30%, respectively, compared with those of parental haploid α5, while the production of these higher alcohols were 0.7, 0.1, and 0.3 fold higher than those from the α5ΔBAT2 mutant, respectively.
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[基金项目]
天津市科委自然基金重点项目 (14JCZDJC32900);国家自然科学基金项目(31471724)