Ethyl carbamate (EC), a water-soluble and semi-volatile polar compound, exists widely in fermented foods and alcoholic beverages and is classified as a Group 2 A carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization (Ma et al., 2022, Gowd et al., 2018; Luiset et al., 2022). At present, the content of EC in wine is limited in some countries. For example, the content of EC in table wine is limited legally at 30 µg/L in Canada (Conacher, & Page, 1986), while in the United States, the content of EC is limited to 15 µg/L for wines with alcohol content less than 14% (Fu et al., 2021). There is no limit standard for the content of EC in wine in China, but it has attracted more and more attention with the improvement in the requirements for food safety and quality.

In wine production, EC is formed in the fermentation and storage processes by a spontaneous chemical reaction of ethanol and carbamyl compounds (Zhou et al., 2021; Ough et al., 1988). During the alcoholic fermentation phase, the spontaneous reaction of urea and ethanol is the main pathway for EC formation in wine (Guo et al., 2016). Grapes contain abundant arginine, which is transported into yeast cells and decomposed into urea and ornithine by arginase. Regulated by nitrogen catabolite repression (NCR), the expression of urea transporters and hydrolytic enzymes involved in urea utilization is strongly inhibited in the presence of preferred nitrogen sources. As the accumulation of a large amount of urea within cells is harmful, urea is transported out of the cell through urea permease and accumulates outside of the cell, and then spontaneously reacts with ethanol to form EC (Hofman-Bang., 1999). Therefore, reducing the content of intracellular arginine is the main way to reduce the production of urea and EC in wine.

In order to reduce the EC content in alcoholic beverages, metabolic-engineered yeast starter breeding, fermentation process optimization, enzymatic removal and physical adsorption were used and reported ( Zhao et al., 2013). For example, Dong et al. obtained a specific ECH from Acinetobacter calcoaceticus (A. calcoaceticus) through protein sequence analysis using NCBI, and found that this enzyme is a highly specific esterase that can directly degrade EC without degrading urea (Dong et al., 2022). Among them, breeding the metabolic engineered Saccharomyces cerevisiae with low EC production is an effective measure aimed at EC production, while the other measures are aimed at treating EC that has already been generated. Zhang et al. knocked out the genes CAN1 and GAP1 encoding arginine permease and amino acid permease in S. cerevisiae BY4741 resulted in the significant decrease of extracellular urea content (68%) of the mutant compared with that of the wild-type strain (Zhang, & Hu, 2018). Zhou et al. reported that the expression of the arginine uptake gene (VBA2 gene) in vacuoles was significantly upregulated in the fermentation of rice wine by adding bamboo leaf extract, and thus inhibited the arginine metabolism and the formation of EC (Zhou et al., 2020). Dahabieh et al. constructed the recombinant yeast strain K9 (DUR1,2) by overexpressing the gene DUR1,2 encoding urea amidase, which resulted in a decrease of 68% in EC production compared with the original strain (Dahabieh et al., 2010). In our previous study, CAR1 coding arginase, which was responsible for urea formation, was deleted in industrial diploid S. cerevisiae WY1, and this resulted in a 77.89% and a 73.78% reduction in urea and EC content in wine (Guo et al., 2016).

In addition to affecting the synthesis of urea and EC, arginine in yeast cells is also closely related to the synthesis of higher alcohols and esters. For example, Alicia Gutierrez et al. reported that wines produced using arginine as an additive had lower alcohol content and higher ester concentration, respectively (Gutierrez et al., 2013). Wang et al. found that knocking out the gene CAN1 encoding arginine transaminase could reduce the n-propanol production of the recombinant strain and simultaneously increase the production of isobutanol, isoamyl alcohol and 2-phenylethanol (Wang et al., 2020). Therefore, in addition to affecting the synthesis of urea and EC, the presence of arginine in yeast cells is closely related to the synthesis of higher alcohols and esters. As is well known, higher alcohols and esters play a key role in the flavor and quality of wine. However, the flavor and overall quality of the alcoholic beverages were rarely investigated in the previous studies on EC control by regulating the genes involved in the metabolism of the precursors of EC synthesis in S. cerevisiae, which greatly limited the effective role of these strategies for regulating EC content in the actual brewing of alcoholic beverages.

In this study, genetic modification of the transport and synthesis pathways of arginine in S. cerevisiae were investigated to reduce the accumulation of arginine in yeast cells (Fig. 1). S. cerevisiae mutants were obtained by (1) deleting genes encoding arginine permease (Can1p) and amino acid permease (Gap1p) on the cell membrane as well as argininosuccinate synthase (Arg1) respectively, (2) overexpressing the gene encoding amino acid transporter (Vba2) and (3) co-modifying the genes related to the transport and synthesis pathways of arginine. The growth performance, fermentation performance of the mutants as well as the content of urea, EC and the flavor substance of the wine in must fermentation of Cabernet Sauvignon were systematically researched.