Construction Of A Chinese Rice Wine Yeast Strain With Low Urea Production By Disrupting CAR1Gene

A Chinese rice wine yeast strain with low urea production was constructed by disruptingits CAR1gene in this study. Arginase of this strain, which was encoded by CAR1was thendeficient, resulting in low urea production from arginine. Therefore the content of urea whichwas excreted to the fermentation broth of rice wine was decreased and so as the ethylcarbamate (EC) content. With the objective of obtaining the least urea content, the brewingtechnique of this engineering strain was also studied. The main results were as follows:A genetic engineering strain of Chinese rice wine yeast with low urea production wasconstructed. The genome DNA of the parental strain85~#was successfully extracted and all thegenes required in this study were obtained by PCR. Then a gene knockout cassette with the shble gene as the selective marker which flanked by the loxp sites and the470bp homologousarm sequence was constructed by overlap PCR to disrupt the CAR1gene in the yeast. Afterthat, the selective marker was excised by Cre enzyme, and the plasmid that could express Creenzyme was successively lost by passage. A genetic engineering strain with a copy of CAR1gene knocked out was obtained and named85~#: Δcar1.The fermentability of the85~#and85~#: Δcar1strain was analysed. The arginase activitiesof the two strains were detected. Urea and EC contents as well as the routine indexes of ricewine that fermented by the two strains respectively were also analysed. Results showed that,compared to the85~#, the arginase activity of the85~#: Δcar1strain decreased by56.2%,contents of urea and EC in rice wine decreased by72.1%and36.9%, respectively. After astorage for150d, EC content in rice wine fermented by the85~#: Δcar1strain was only48.7%of that fermented by the85~#strain with the routine indexes barely changed. Throughcontinuous generation fermentation, the85~#: Δcar1strain showed good stability of producinglower urea content.The brewing technique of the85~#: Δcar1strain was optimized to obtain the least ureacontent in the rice wine. The factors such as fermentation temperature, steeping time, ratio ofrice to water and kind of rice were studied. Through orthogonal experiment, the optimalconditions for the85~#: Δcar1strain to produce least urea content were fermentationtemperature at28℃, steeping time of28h, ratio of rice to water of1:2.0. Under theseconditions, urea content was7.13mg/L in rice wine fermented by the85~#: Δcar1strain, whichwas13.5%less than that before optimization of brewing process, and75.9%less than that ofthe parental strain85~#.