Tianjin Industrial Microbiology Key Laboratory, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, People's Republic of China.
Appl Microbiol Biotechnol. 2018 Feb;102(4):1783-1795. doi: 10.1007/s00253-017-8715-5. Epub 2018 Jan 5.
Higher alcohols significantly influence the quality and flavor profiles of Chinese Baijiu. ILV1-encoded threonine deaminase, LEU1-encoded α-isopropylmalate dehydrogenase, and LEU2-encoded β-isopropylmalate dehydrogenase are involved in the production of higher alcohols. In this work, ILV1, LEU1, and LEU2 deletions in α-type haploid, a-type haploid, and diploid Saccharomyces cerevisiae strains and ILV1, LEU1, and LEU2 single-allele deletions in diploid strains were constructed to examine the effects of these alterations on the metabolism of higher alcohols. Results showed that different genetic engineering strategies influence carbon flux and higher alcohol metabolism in different manners. Compared with the parental diploid strain, the ILV1 double-allele-deletion diploid mutant produced lower concentrations of n-propanol, active amyl alcohol, and 2-phenylethanol by 30.33, 35.58, and 11.71%, respectively. Moreover, the production of isobutanol and isoamyl alcohol increased by 326.39 and 57.6%, respectively. The LEU1 double-allele-deletion diploid mutant exhibited 14.09% increased n-propanol, 33.74% decreased isoamyl alcohol, and 13.21% decreased 2-phenylethanol production, which were similar to those of the LEU2 mutant. Furthermore, the LEU1 and LEU2 double-allele-deletion diploid mutants exhibited 41.72 and 52.18% increased isobutanol production, respectively. The effects of ILV1, LEU1, and LEU2 deletions on the production of higher alcohols by α-type and a-type haploid strains were similar to those of double-allele deletion in diploid strains. Moreover, the isobutanol production of the ILV1 single-allele-deletion diploid strain increased by 27.76%. Variations in higher alcohol production by the mutants are due to the carbon flux changes in yeast metabolism. This study could provide a valuable reference for further research on higher alcohol metabolism and future optimization of yeast strains for alcoholic beverages.
高级醇显著影响中国白酒的质量和风味特征。ILV1 编码的苏氨酸脱氨酶、LEU1 编码的α-异丙基苹果酸脱氢酶和 LEU2 编码的β-异丙基苹果酸脱氢酶参与高级醇的生成。在这项工作中，构建了α-型单倍体、a-型单倍体和二倍体酿酒酵母菌株中 ILV1、LEU1 和 LEU2 的缺失以及二倍体菌株中 ILV1、LEU1 和 LEU2 的单等位基因缺失，以检验这些改变对高级醇代谢的影响。结果表明，不同的遗传工程策略以不同的方式影响碳通量和高级醇代谢。与亲本二倍体菌株相比，ILV1 双等位基因缺失的二倍体突变体产生的正丙醇、活性戊醇和 2-苯乙醇的浓度分别降低了 30.33%、35.58%和 11.71%。此外，异丁醇和异戊醇的产量分别增加了 326.39%和 57.6%。LEU1 双等位基因缺失的二倍体突变体表现出 14.09%的正丙醇增加、33.74%的异戊醇减少和 13.21%的 2-苯乙醇减少，与 LEU2 突变体相似。此外，LEU1 和 LEU2 双等位基因缺失的二倍体突变体的异丁醇产量分别增加了 41.72%和 52.18%。ILV1、LEU1 和 LEU2 的缺失对α-型和 a-型单倍体菌株中高级醇生成的影响与二倍体中双等位基因缺失相似。此外，ILV1 单等位基因缺失的二倍体菌株的异丁醇产量增加了 27.76%。突变体中高级醇产量的变化是由于酵母代谢中碳通量的变化。本研究可为进一步研究高级醇代谢和未来优化酒精饮料酵母菌株提供有价值的参考。
