Ethyl carbamate (EC) is a naturally occurring substance in alcoholic beverages from the reaction of ethanol with urea during fermentation and storage. EC can cause dizziness and vomiting when consumed in small quantities and develop kidney cancer when consumed in excess. Thus, the reduction of EC formation in alcoholic beverages is important for food safety and human health. One of the strategies for reducing EC contents in alcoholic beverages is developing a new yeast starter strain to enable less formation of EC during fermentation. In this study, we isolated a polyploid wild-type yeast Saccharomyces cerevisiae strain from the Nuruk (Korean traditional grain-based inoculum of wild yeast and mold) and developed a starter culture by genome engineering to reduce EC contents in alcoholic beverages. We deleted multiple copies of the target genes involved in the EC formation in the S. cerevisiae by a CRISPR/Cas9-based genome editing tool. First, the CAR1 gene encoding for the arginase enzyme responsible for the formation of urea was completely deleted in the genome of S. cerevisiae. Additionally, the GZF3 gene encoding the transcription factor controlling expression levels of several genes (DUR1, 2, and DUR3) related to urea absorption and degradation was deleted in S. cerevisiae to further reduce the EC formation. The effects of gene deletion were validated by RT-qPCR to confirm changes in transcriptional levels of the EC-related genes. The resulting strain of S. cerevisiae carrying a double deletion of CAR1 and GZF3 genes successfully reduced the EC contents in the fermentation medium without significant changes in alcohol contents and fermentation profiles when compared to the wild-type strain. Finally, we brewed the Korean traditional rice wine Makgeolli using the double deletion strain of S. cerevisiae dCAR1&GZF3, resulting in a significant reduction of the EC content in Makgeolli up to 41.6% when compared to the wild-type strain. This study successfully demonstrated the development of a starter culture to reduce the EC formation in an alcoholic beverage by CRISPR/Cas9 genome editing of the wild yeast.
Keywords: CRISPR/Cas9; Saccharomyces cerevisiae; alcoholic beverages; ethyl carbamate; genome editing.