Genetic engineering to alter carbon flux for various higher alcohol productions by Saccharomyces cerevisiae for Chinese Baijiu fermentation

Abstract

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.

GAT1 Gene, the GATA Transcription Activator, Regulates the Production of Higher Alcohol during Wheat Beer Fermentation by Saccharomyces cerevisiae

“…S. cerevisiae synthesizes higher alcohols mainly through amino acid catabolism (Ehrlich pathway) and pyruvate anabolism pathways; the expression levels of genes in these metabolic pathways have a decisive effect on higher alcohol synthesis [ 32 , 33 , 34 ]. When the contents of fermentable sugar and FAN in the culture system are unusually sufficient, all higher alcohol anabolic pathways are in an active state consistently [ 23 , 35 , 36 ]. Genetic modification of genes in the higher alcohol metabolism pathway may not yield the desired results, and the metabolic regulation genes of carbon and nitrogen sources should be considered.…”

“…The kits were purchased from Takara Biotechnol (Dalian, China). The PCR procedure was set as reported by Li et al [ 23 ]. The primers (gene-F/gene-R) synthesized by GENEWIZ (Suzhou, China) are listed in the Supplementary Material .…”

A Seamless Gene Deletion Method and Its Application for Regulation of Higher Alcohols and Ester in Baijiu Saccharomyces cerevisiae

“…Corn hydrolysate medium was used in Baijiu fermentation of the parental strain and mutants. The details of the experiment procedures, including preparation of corn hydrolysate and control of the fermentation process, were based on those used in a previous study [31]. All fermentations were performed in triplicate.…”

Increasing Yield of 2,3,5,6-Tetramethylpyrazine in Baijiu Through Saccharomyces cerevisiae Metabolic Engineering

“…The method used to calculate the growth curve was obtained from Li W. et al (2018). Briefly, the yeast strain was grown in YPD medium for 12 h up until the stationary phase.…”