Metabolic engineering of Chinese rice wine Saccharomyces cerevisiae with reduced urea production by CRISPR/Cas9 system
XIE Wenjuan1,2,3, WU Dianhui1,2,3, LI Xiaomin1,2,3, CAI Guolin1,2,3, XIE Guangfa3,4, LU Jian1,2,3*
1The Key Laboratory of Industrial BiotechnologyJiangnan University,Wuxi 214122, China 2National Engineering Laboratory for Cereal Fermentation TechnologyJiangnan University,Wuxi 214122, China 3School of Biotechnology,Jiangnan University,Wuxi 214122,China 4School of Biological and Enviromental Engineering,Zhejiang Shuren University,Hangzhou 310015,China
Abstract: The low urea-producing Saccharomyces cerevisiae was constructed through metabolic engineering to reduce the content of urea and ethyl carbamate (EC) formation in Chinese rice wine. This study constructed the DUR3 gene expression cassette HOL-PGK1p-DUR3-PGK1t-HOR using fusion PCR and electro-transformed into S. cerevisiae NaDUR1,2-Δcar1 by CRISPR/Cas9 system to obtain the modified strain S. cerevisiae NaDUR1,2/DUR3-Δcar1 with overexpressed DUR3 gene. It showed that the content of urea in Chinese rice wine samples fermented by S. cerevisiae NaDUR1,2/DUR3-Δcar1 reduced by 92.1% and the concentration of EC decreased by 58.6% compared to those of S. cerevisiae Na. Moreover, the content of urea in Chinese rice wine samples fermented by S. cerevisiae NaDUR1,2/DUR3-Δcar1 reduced by 43.4% and the concentration of EC decreased by 16.2% compared to those of S. cerevisiae NaDUR1,2-Δcar1. These results indicated that overexpressed DUR3 could transfer urea from fermentation liquor into yeast cells, which was beneficial to reduce the urea content and EC formation in Chinese rice wine. Furthermore, using CRISPR/Cas9 system introduced no foreign resistant genes to the yeast. In conclusion, S. cerevisiae NaDUR1,2/DUR3-Δcar1 can potentially be applied in industrial production to eliminate the contents of urea and EC in Chinese rice wine.
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