Construction Of Self-cloning Industrial Brewing Yeast With High-glutathione Production And Low-ADH Ⅱ Enzyme Activity

Acetaldehyde is one of the most important flavor compounds in beer.Excess acetaldehyde produces a green,grassy,or apple-like off-flavor.The content of acetaldehyde is usually 5-12mg/L in domestic beer,and below 3mg/L in oversea high-quality beer.At present,the means,which was used to diminish the acetaldehyde content in beer,was improvement of producing technology.The method,however,was not so effective for diminishing the acetaldehyde.Acetaldehyde content had been a major factor hard to overcome in improvement of beer flavor.Because of the development of DNA recombination technology,it had been possible to reduce the acetaldehyde content in beer by modifying the genes of Saccharomyces cerevisiae.In this research,a new self-cloning industrial brewers' yeast was constructed.The content of important production and fermentation profiles were investigated in a flask fermentation test.The major content and results of this research were listed as follow:A fragment of DNA consist of ORF and its up-stream 700bp fragment ofADH2 gene, which encoded alcohol dehydrogenaseâ…¡,was amplified by PCR from YSF31.The primers were designed from the reported sequences of ADH2 in Saccharomyces cerevisiae.The amplified ADH2 gene DNA fragment was inserted into YEp352 to construct a recombinant plasmid pYA.Then,the screening marker gene CUP1 and GSH1 encodingγ-glutamylcysteine synthetase were inserted into the ADH2 gene in plasmid pYA to construct a recombinant plasmid pACG.A fragment of DNA,which consisted of CUP1 gene,GSH1 gene,and flanged with ADH2 gene sequences,was retrieved by digested with restriction enzyme of Pvuâ…¡.The industrial yeast strain YSF31 was transformed with the recombinant DNA fragment and the fragment recombined into the locus of adh2 on the chromosome of YSF31.The transformant was selected by the resistance to Cu²⁺ of the cells and verified by PCR.Because the DNA referred to genetic manipulation was all from S.cerevisiae itself,the engineering strain was called self-cloning strain.The Cu²⁺ resistance test indicated that the host yeasts and self-cloning could grow on YEPD with 5mmol/L CuSO₄.But the host could not grow on YEPD with 6mmol/L CuSO₄. And the self-cloning strain could grow even on YEPD with 6mmol/L,7mmol/L,8mmol/L CuSO₄.The Cu²⁺ resistance of self-cloning strain with one more copy of CUP1 gene was 8.5mmol/L CuSO₄.The GSH content test showed that intracellular GSH of self-cloning strain was 1.15 fold compared with that of host strain,and GSH of self-cloning strain was 1.42 fold compared with that of host in fermenting liquor.The results of Cu²⁺ resistance test and GSH content test suggested that CUP1 gene and GSH1 gene were successfully recombined into the chromosome of host,and successfully co-expressing in self-cloning strain.The flask fermentation test manifested that GSH content of self-cloning strain was 1.34 fold compared with that of host strain in fermenting liquor.Resulting with the knockout of ADH2 gene,ADHâ…¡activity of self-cloning strain decreased to 65%compared with that of host strain.The other fermentation profiles,such asα-N-amino acid assimilation,real degree of fermentation,were almost identical between these of self-cloning and host strain.According to the results of CO2 weight reducing and real degree of fermentation test,the fermentation capability of self-cloning strain was intact under the genetic modification.It is first time to construct a self-cloning strain with low ADHâ…¡activity and high GSH content by inserting CUP1 and GSHlgene into locus of ADH2 on chromosome to disrupt ADH2 gene.It made a foundation for the work Wing to construct the industrial brewing yeast with low-acetaldehyde-content and anti-aging capability.