Abstract:[Objective] Sulfite, an intermediate metabolite in yeast sulfur-containing amino acid biosynthesis pathway, plays an important role in beer flavor stabilizing because of its antioxidant activity. In Saccharomyces cerevisiae, excretion of sulfite is regulated by sulfite transporter protein Ssu1p which is encoded by SSU1. We constructed a high sulfite excreting brewing yeast strain to solve the beer staling problem by SSU1 gene over-expressing. [Methods] Sulfite transporter gene SSU1-1 and SSU1-2 that contain different length of 5′ untranslated region were cloned by PCR, with the genomic DNA of an industrial mutant strain M8 as template. The amplified DNA was digested with BamHⅠand SalⅠ, and then inserted into plasmid YEp352 digested by BamHⅠ-SalⅠand constructed the expression vector pSU1 and pSU2. pSU1 and pSU2 were transformed into laboratory strain YS58 and tested the effect of SSU1 multi-copy expression on Saccharomyces cerevisiae sulfite production. Furthermore, pSU2 was transformed into industrial brewing yeast mutant strain M8, and SO2, H2S production and beer antioxidant abilities of transformant Y2, screened out by sulfite resistance plate, were tested. [Result] The SO2 production of laboratory yeast transformants pSU1-4 and pSU2-3 enhanced remarkably, but H2S production remained unchanged. Compared with the S. cerevisiae M8, SO2 production of transformant Y2 increased by 74.4%, TBA value decreased by 14.9%, DPPH radical scavenging ratio enhanced by 38.2%, but H2S production had little change. [Conclusion] Over-expression of SSU1 in both laboratory S. cerevisiae strain and industrial brewing yeast strain increased their sulphite excretion, enhanced beer antioxidant abilities and had no negative effect on sulfur-containing amino acids biosynthesis.
