Metabolic Engineering of the Regulators in Nitrogen Catabolite Repression To Reduce the Production of Ethyl Carbamate in a Model Rice Wine System

Abstract

Rice wine has been one of the most popular traditional alcoholic drinks in China. However, the presence of potentially carcinogenic ethyl carbamate (EC) in rice wine has raised a series of food safety issues. During rice wine production, the key reason for EC formation is urea accumulation, which occurs because of nitrogen catabolite repression (NCR) in Saccharomyces cerevisiae. NCR represses urea utilization by retaining Gln3p in the cytoplasm when preferred nitrogen sources are present. In order to increase the nuclear localization of Gln3p, some possible phosphorylation sites on the nuclear localization signal were mutated and the nuclear localization regulation signal was truncated, and the disruption of URE2 provided an additional method of reducing urea accumulation. By combining these strategies, the genes involved in urea utilization (DUR1,2 and DUR3) could be significantly activated in the presence of glutamine. During shake flask fermentations of the genetically modified strains, very little urea accumulated in the medium. Furthermore, the concentrations of urea and EC were reduced by 63% and 72%, respectively, in a model rice wine system. Examination of the normal nutrients in rice wine indicated that there were few differences in fermentation characteristics between the wild-type strain and the genetically modified strain. These results show that metabolic engineering of the NCR regulators has great potential as a method for eliminating EC during rice wine production.

Regulation of Sensing, Transportation, and Catabolism of Nitrogen Sources in Saccharomyces cerevisiae

“…In contrast, Gln3 can activate the transcription of NCR genes independently (162). Gln3 and Gat1 will be translocated into the nucleus under nitrogen starvation conditions, while under nitrogen repletion conditions, they are sequestered in the cytoplasm by TOR-mediated phosphorylation (14,163). In addition, the transcriptional regulator Ure2 works as another anchor to prevent the translocation of Gln3 into the nucleus.…”

“…increasing the nuclear localization of Gln3 by mutating potential phosphorylation sites on its nuclear localization signal, truncating its nuclear localization regulation signal, and disrupting URE2 significantly activated the expression of genes in the urea metabolism pathway (DUR1,2 and DUR3), which ultimately reduced the levels of urea and EC by 63% and 72%, respectively, during rice wine fermentation (14).…”

“…In industrial biotechnology processes, nitrogen sources play an essential role in the production of various products, e.g., antibiotics (6)(7)(8)(9)(10), amino acids (11), and enzymes (12). In addition, some hazardous nitrogen compounds that accumulate during the production of fermented food, such as ethyl carbamate (EC) during alcoholic beverage production (13)(14)(15) and soy sauce production (16)(17)(18), are also related to the preferential utilization of nitrogen sources. Therefore, nitrogen catabolism could also seriously impact the quality of different fermented foods, such as Chinese rice wine (19) and cheese (20).…”

Effects of nitrogen catabolite repression-related amino acids on the flavour of rice wine

“…A model system for rice wine production was designed according to the manufacturing process of Shaoxing rice wine in China. The process of fermentation has been described in detail . After fermentation for 25 days, samples were taken to measure the concentrations of the flavour compounds in the model system.…”

Effect of citrulline metabolism inSaccharomyces cerevisiaeon the formation of ethyl carbamate during Chinese rice wine fermentation

“…A logical solution is to decrease the EC level by reducing the content of its precursors. The dominant precursor of EC in alcoholic beverages is considered to be urea, which is mainly generated by the arginine metabolism of Saccharomyces cerevisiae; thus, the approach of metabolically engineering this yeast strain has been applied to eliminate EC in wine, sake, stone fruit spirits, Chinese rice wine and other alcoholic beverages (7)(8)(9)(10)(11)(12)(13)(14)(15). One method is to knock out the CAR1 gene, which encodes arginase, the enzyme that cleaves arginine into ornithine and urea (8,9,11,13,15).…”