Saccharomyces cerevisiae metabolizes arginine to ornithine and urea during wine fermentations. In the fermenta-
tion of Chinese rice wine, yeast strains of S. cerevisiae do not fully metabolize urea, which will be secreted into the
spirits and spontaneously reacts with ethanol to form ethyl carbamate, a potential carcinogenic agent for
humans. To block the pathway of urea production, we genetically engineered two haploid strains to reduce the
arginase (encoded by CAR1) activity, which were isolated from a diploid industrial Chinese rice wine strain. Final-
ly the engineered haploids with opposite mating type were mated back to diploid strains, obtaining a heterozy-
gous deletion strain (CAR1/car1) and a homozygous defect strain (car1/car1). These strains were compared to the
parental industrial yeast strain in Chinese rice wine fermentations and spirit production. The strain with the
homozygous CAR1 deletion showed significant reductions of urea and EC in the final spirits in comparison to
the parental strain, with the concentration reductions by 86.9% and 50.5% respectively. In addition, EC accumu-
lation was in a much lower tempo during rice wine storage. Moreover, the growth behavior and fermentation
characteristics of the engineered diploid strain were similar to the parental strain.