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

Abstract

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, Gln3p, 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.

Metabolic Engineering of Four GATA Factors to Reduce Urea and Ethyl Carbamate Formation in a Model Rice Wine System

“…10,11 As shown in previous works, the reduction of extracellular urea formation is closely related to high expression levels of both DUR1,2 and DUR3 genes; however, the high expression of individual DUR1,2 or DUR3 genes is not directly related to urea reduction. 13,17,31 Furthermore, the transcription level changes of these two genes differed in GATA transcriptional factor mutants (Figures 1B, 3, and 5), indicating that the influence of four GATA factors on the individual expression of DUR1,2 and DUR3 is different. Apart from the inhibition of phosphorylation modification on the nucleus import of Gln3p and Gat1p, 29,32 the transcription level changes of DUR1,2 gene were consistent with extracellular urea formation (Figure 5).…”

“…Four GATA factors were involved in the transcription regulation of multiple NCR-sensitive genes; however, their responses to different environment conditions and preferences to bind to target genes are diverse. , In previous studies, two truncated GATA factors, Gln3p 1–653 and Gat1p 1–375 , exhibit enormous enhancement in urea or nonpreferred nitrogen source utilization , and show a synergistic effect with arginine permease disruption in urea reduction . However, a comprehensive influence of these four GATA factors and their truncated variants on the urea formation remains unclear.…”

“…29,30 In previous studies, two truncated GATA factors, Gln3p 1−653 and Gat1p 1−375 , exhibit enormous enhancement in urea or nonpreferred nitrogen source utilization 16,17 and show a synergistic effect with arginine permease disruption in urea reduction. 13 However, a comprehensive influence of these four GATA factors and their truncated variants on the urea formation remains unclear. Herein, several S. cerevisiae mutants were used for the analysis of the comprehensive effects of four GATA transcriptional factors (Gln3p, Gat1p, Dal80p, and Gzf3p) on extracellular urea and EC formation in YPD medium and a model rice wine system.…”

“…Furthermore, the effects of arginine transporters and some upstream transcriptional regulators on extracellular urea and EC formation have also been studied. Deficiency or mutation in two arginine permease genes, CAN1 and GAP1 , − or the overexpression of truncated NCR regulators, Gln3p and Gat1p, , showed an effective alternative to reduce the formation of urea and EC. Recently, the CRISPR/Cas9 system and adaptive evolution methods were applied to obtain low-urea production industrial yeast strains, which could avoid some potential safety concerns. , …”

Molecular Engineering of Bacillus paralicheniformis Acid Urease To Degrade Urea and Ethyl Carbamate in Model Chinese Rice Wine

“…Ethyl carbamate (EC or urethane), a carcinogenic compound that has been classified as a Group 2A carcinogen by the International Agency for Research on Cancer (IARC), is widely present in fermented foods and alcoholic beverages. − In alcoholic beverages, EC is mainly synthesized by condensation of urea and alcohol. − Increasing food safety concerns call for the elimination or decrease in the concentration of EC, especially using enzymatic methods. , Considerable efforts have been made to hydrolyze EC directly [CH 3 CH 2 OC­(O)­NH 2 + H 2 O → C 2 H 6 O + CO 2 + NH 3 ] or degrade urea to prevent the formation of EC. ,,− To hydrolyze EC, some urethanases from Bacillus licheniformis, Citrobacter sp., Rhodococcus equi strain TB-60, and Penicillium variabile have been purified and investigated. − Unfortunately, they are not suitable for industrial applications considering the unsatisfactory enzymatic properties, such as low ethanol tolerance and instability under acid conditions. , Many acid ureases from lactic acid bacteria (LAB) and Enterobacter species have been isolated and evaluated for the ability to decompose urea to prevent EC formation in rice wine. − Alternatively, metabolic engineering strategies have been developed to engineer Saccharomyces cerevisiae to reduce urea while simultaneously producing the desired aroma. − Although the concentration of urea was significantly reduced in rice wine, particularly by the enzymatic strategies, the residual urea (1–10 mg/L) could still react with alcohol during long-term storage to form 50–750 μg/L EC, which is still a hazardous level. ,,,− …”