Regulation of Arginine Metabolism by Transcription Factor Btn2p in a Mixed Culture of Saccharomyces cerevisiae and Pediococcus pentosaceus and Subsequent Ethanol Tolerance

Ethyl carbamate (EC) is a natural carcinogen widely found in fermented alcoholic beverages. The compound is mainly generated through the reaction of urea and citrulline with ethanol. The transcription factor Btn2p may affect arginine metabolism, and thus regulate EC in different fermentation systems. Therefore, in this study, we investigated the effects of Btn2p on arginine metabolism in Saccharomyces cerevisiae in different culture systems and analyzed the potential regulatory mechanisms of EC formation. In addition, we studied the ethanol tolerance of BTN2-modified yeast to determine its applicability in huangjiu fermentation and to provide a theoretical basis for subsequent studies. We found that BTN2 knockout inhibited two major EC precursors, and the inhibitory effect was better in mixed cultures with Pediococcus pentosaceus. In addition, BTN2 knockout promoted the activities of urease and ornithine transcarbamoylase, but reduced the activity of arginine deiminase, which led to the reduction of urea and citrulline concentrations. The growth conditions of BTN2-modified strains under different ethanol concentrations were also studied for future applications in huangjiu fermentation. The results showed that BTN2 overexpression promoted cell growth and increased ethanol tolerance, whereas BTN2 knockout reduced the ethanol tolerance of cell. The findings indicated that Btn2p was involved in arginine metabolism, possibly through the regulation of urea and citrulline metabolism, and BTN2-knockout strains can be used as a potential target for EC reduction.