Construction Of Chinese Rice Wine Yeast With Low Urea Production

Ethyl carbamate （EC） is a potential carcinogen for human, which has been found inmany fermented alcoholic beverages. The main precursor for EC is urea produced by yeast.To reduce EC content in Chinese rice wine, a study concerning construction and applicationof a Chinese rice wine yeast strain with low urea production was conducted.This paper consists of three parts:（1） genetic engineering of a Chinese rice wine yeastwith low urea production by constitutive overexpression of the DUR1,2gene,（2） comparisonof urea and EC contents in Chinese rice wine fermented by the engineering and parentalstrains respectively in laboratory brewing tests,（3） analysis of key factors during Chinese ricewine brewing on urea production by the engineering strain. Main results were as follows:The DUR1,2gene which encoded urea amidolyase from Chinese rice wine yeast85^#（Sacchromyces cerevisiae） was cloned into plasmid pYX212between the TPI1promoter andterminator sites to form the DUR1,2expression cassette. The fact that urea degradation washighly accelerated by overexpression of urea amidolyase was demonstrated. The expressioncassette was then integrated into the genome of the Chinese rice wine yeast85^#by linearizingthe vector. Thus a Chinese rice wine yeast85^DUR1,2with low urea production was obtained.According to rice wine brewing tests at laboratory scale, urea and EC contents inChinese rice wine fermented by yeast85^DUR1,2were8.34mg/L and58.84μg/L,69.88%and41.26%less than those of85^#, respectively. EC content in rice wine produced by85^DUR1,2wasbarely changed after storage for150d at room temperature, while that of the control groupwas increased by39.07%. Meanwhile,85^DUR1,2was substantially equivalent to85^#in terms offermentation ability, content of ethanol, total acid and amino nitrogen. Through continuousgeneration fermentation, the engineering strain85^DUR1,2showed good stability of producinglow urea content.Effects of key factors during Chinese rice wine brewing, including fermentationtemperature, pitching rate of yeast, use of wheat Qu and ratio of rice to water, on ureaproduction by85^DUR1,2were studied. The optimal conditions for85^DUR1,2to produce least ureacontent were fermentation temperature at28℃, pitching rate of1×10⁷cell/mL, total wheatQu amount of13%, ratio of raw wheat Qu to cooked wheat Qu at4:1, and ratio of rice towater at1:2. Under these conditions, urea content was7.22mg/L in rice wine fermented by85^DUR1,2, which was13.43%less than that before brewing process optimization, and73.93%less than that of the parental strain85^#.