Constructing Low Methanol Saccharomycetes By Knocking Out Gene Of Glycine Glycocoll Lyase System And Its Application

Fermented alcoholic drink is often used in people’s daily life, that is produced on a variety of grain crops which are fermented by microorganism’s metabolism on its own. However, some of the other harmful substances will be produced in the process of producing wine. Methanol is one of them. Methanol is mainly produced through two methods when people brew wine. A method is that starting crude have methanol by itself, another way is that saccharomyces cerevisiae through its own metabolism produce methanol. In the process of brewing wine, glycine glycocoll in the fermentation liquor produces methanol through the catalysis of yeast glycine decarboxylase. Therefore, it increases the harm to people’s body. In conclusion,this paper mainly uses the method of gene knockout to change the saccharomyces cerevisiae metabolic pathways to realize the lower content of methanol in the brewing wine, which is more suitable for human to drink.This experiment uses a strain of slow methanol yeasts for original strain which is preserved in the laboratory of Institutes Of Technology Of South China. It is successful of using Cre-Lox P recombination system and lithium acetate conversion method to knockout the low methanol yeast’s gene GCV2, and as well it gains a strain of gene deletion strains which is named for S2. It uses S2 strain and the original strain for ferment in the preparation of Liquid Malt juice. It uses fermented wine through static headspace gas chromatography to determine relative methanol ’s content and at the same time to construct the standard curve equation for the determination of methanol: y=0.0003x-0.0064. Fermentation results: the relative content of methanol in the low methanol yeast is 411.13 mg/L, 20 degrees of alcohol is 8.8vol%, the relative content of methanol of S2 yeast is 329.88mg/L, 20 degrees of alcohol is 9.7vol%, compared with strain, the latter’s relative content of methanol is reduced by 19.76% and the content of alcohol is increases by 10.23%.It continues to use Cre-lox P recombination system to knockout glycine cleavage enzyme gene GCV1. And this experiment selects 7 positive strains in the first experiment to get one strain which have relatively good fermentation performance, the strain in the fermentation liquid of low relative content of methanol is named for 4, the result for its alcohol fermentation is 12%vol and its relative content of methanol is 223.33mg/l. with the strain to continue the following experiment, it is successful to construct the deleted saccharomycetes of gene GCV1, which is named for S21, using strain S2 and strain S21 separately in the malt extract(12°P) and 28 ℃ temperature ferments 5 days, fermentation results of using headspace gas chromatograph for the relative content of methanol with strain S2 and strain S21 are 323.33mg/l,223.33mg/l, and both of their alcoholic strength of 20℃ measured by alcohol meter are 12%vol, therefore, the relative content of methanol of strain S21 in the products of fermentation is lower by 30.93% than strain S2.This paper using three single factors including temperature, time and sugar degree, have more effects for this ferment experiment, through response surface analysis software, it chooses Factors=3, Runs=18, Box-Behnken devise to conduct combined experiment. According to the experimental results. Through the analysis of regression equation, the optimal fermentation conditions of the transformation were determined: the temperature is 24 ℃、sugar degree is 150g/L、the time is 8 days, the relative content of methanol is 75.92 mg/L. Experimental verification which found that relative content of methanol under the conditions in the fermentation production is closer to this result of response surface experiment.