Metabolic engineering of arginine permeases to reduce the formation of urea in Saccharomyces cerevisiae.

Urea is an important precursor of the harmful carcinogenic product ethyl carbamate in fermented wines. To decipher more fully the contributions of three arginine permeases, Can1p, Gap1p and Alp1p in urea formation, various engineered strains were examined for their ability to form urea. This included seven mutants with different combinations of permease deficiency and grown in both simple and more complex media, and the wild-type strain modified to overexpress the three arginine permeases. A truncated GATA transcription factor, Gln3p1-653, was also overexpressed in the arginine permease deficient mutants to determine whether the permeases have a synergistic effect on urea formation with other urea reducing modules. Additionally, in this study, transcriptional changes of four genes related to arginine metabolism and urea formation were investigated. We found that the three amino acids permeases affect urea formation mainly through the utilization of arginine in YNB medium containing the 20 common amino acids. The deletion mutant Δgap1Δcan1 showed a significant reduction (68%) in extracellular urea compared to the wild-type strain grown in YPD medium. Overexpression of a truncated Gln3p in Δgap1Δcan1 reduced the extracellular urea concentration even further (by 67%) than that in the wild-type strain and showed a synergistic effect with Δgap1Δcan1 and Δalp1Δgap1Δcan1 for extracellular reduction. Moreover, the results of this study provide a promising way to reduce urea accumulation during wine fermentation using S. cerevisiae, and present an approach to control metabolism and product formation through the regulation of amino acid permeases.