Reduction Of The Accumulation Of Urea In Rice Wine Fermentation By High-throughput Screening Strategy

Ethyl carbamate is a common potential hazard in different fermented foods.Urea and ethanol are the major precursors of ethyl carbamate in rice wine.Presently,the main methods to reduce accumulation of urea are metabolic engineering and genetic engineering,while it remains potential safety concerns in rule regulations and consumer acceptance.In this study,a high-throughput screening strategy was established to reduce the accumulation of urea,and therefore decrease the accumulation of ethyl carbamate in rice wine.An industrial strain Saccharomces cerevisiae XZ-11 was used in this study.Combined with high-throughput screening strategy,library of lower urea accumulating strains were achieved by using adaptive evolution,atmospheric and room temperature plasma(ARTP)mutagenesis,UV mutagenesis and chemical mutagenesis.Then,RT-qPCR analysis was applied to detect the transcription change of genes GLN3,GAT1,DUR1,2 and DUR3,which are related to urea metabolism or transportation.The main results are presented as follows:(1)Under the condition of adding different concentration of urea,continuous adaptive evolution were conducted on the strain S.cerevisiae XZ-11 for many generations,and finally the strains accumulating lower urea were obtained.Under the conditions of adding different concentration of urea(30,300,1500 and 3000 mg·L-1),continuous adaptive evolution were conducted for 360 generations on strain S.cerevisiae XZ-11.S.cerevisiae 4B with lower urea accumulation and genetic stability were obtained by using the established high-throughput urea detecting method.Compared with orginal strain,the accumulation of urea decreased by 48.0%.(2)Employing the successively mutagenesis with ARTP mutagenesis,UV mutagenesis and chemical mutagenesis on orginal strain S.cerevisiae XZ-11(the later treatment based on the fromer one).The lower urea accumulation strain 6-10 E was obtained by high-throughput urea detecting method based on MoFlo cytometry.Compared with the original strain S.cerevisiae XZ-11,the urea accumulation of 6-10 E was reduced by 51.0%.Besides,strain S.cerevisiae 6-10 E showed genetic stability after six generations of continuous subculture.(3)S.cerevisiae 4B and 6-10 E which were obtained from adaptive evolution,ARTP mutagenesis,UV mutagenesis and chemical mutagenesis were analyed the transcriptional change of genes GLN3,GAT1,DUR1,2 and DUR3.Light Cycler480 II system was used to detect the changes of gene transcription level in strains 4B and 6-10 E.The transcriptional changes of regulatory factors(GLN3,GAT1,DUR1,2 and DUR3)associated with urea metabolism were analyzed.Results showed that the transcriptional level of genes GLN3,GAT1,DUR1,2 and DUR3 in the strain 4B were upregulated 7.4-,6.2-,81.2-and 30.1-fold,respectively.In addition,the transcriptional level of genes GLN3,GAT1,DUR1,2 and DUR3 in the strain 6-10 E were upregulated 3.6-,5.3-,3.3-and 2.2-fold,respectively.