[Construction of high sulphite-producing industrial strain of Saccharomyces cerevisiae]

Abstract

In the process of beer storage and transportation, off-flavor can be produced for oxidation of beer. Sulphite is important for stabilizing the beer flavor because of its antioxidant activity. However, the low level of sulphite synthesized by the brewing yeast is not enough to stabilize beer flavor. Three enzymes involve sulphite biosynthesis in yeast. One of them, APS kinase (encoded by MET14) plays important role in the process of sulphite formation. In order to construct high sulphite-producing brewing yeast strain for beer production, MET14 gene was cloned and overexpressed in industrial strain of Saccharomyces cerevisiae. Primer 1 (5'-TGTGAATTCCTGTACACCAATGGCTACT-3', EcoR I) and primer 2 (5'-TATAAGCTTGATGA GGTGGATGAAGACG-3', HindIII) were designed according to the MET14 sequence in GenBank. A 1.1kb DNA fragment containing the open reading frame and terminator of MET14 gene was amplified from Saccharomyces cerevisiae YSF-5 by PCR, and inserted into YEp352 to generate recombinant plasmid pMET14. To express MET14 gene properly in S. cerevisiae, the recombinant expression plasmids pPM with URA3 gene as the selection marker and pCPM with URA3 gene and copper resistance gene as the selection marker for yeast transformation were constructed. In plasmid pPM, the PGK1 promoter from plasmid pVC727 was fused with the MET14 gene from pMET14, and the expression cassette was inserted into the plasmid YEp352. The dominant selection marker, copper-resistance gene expression cassette CUP1-MTI was inserted in plasmid pPM to result in pCPM. Restriction enzyme analysis showed that plasmids pPM and pCPM were constructed correctly. The laboratory strain of S. cerevisiae YS58 with ura3, trp1, leu2, his4 auxotroph was transformed with plasmid pPM. Yeast transformants were screened on synthetic minimal medium (SD) containing leucine, histidine and tryptophan. The sulphite production of the transformants carrying pPM was 2 fold of that in the control strain YS58, which showed that the MET14 gene on plasmid pPM was expressed functionally in YS58. The industrial brewing yeast strain YSF-38 was transformed with the plasmid pCPM and yeast transformants were selected on YEPD medium containing 4mmol/L copper sulphate. The recombinant strain carrying pCPM showed a 3.2-fold increase in sulphite production when compared to the host strain YSF-38 under laboratory culture conditions. Flask fermentation under brewing-like conditions was performed in Tsingtao Beer Brewery. The sulphite production of the recombinant strain began to be higher than that of the host strain YSF-38 at the fourth day and reached the maximum at the eighth day. At the end of fermentation, the sulphite produced by recombinant strain is 1.4 fold of that in the host strain. The overexpression of MET14 gene in both laboratory and industrial strains of S. cerevisiae increases the sulphite formation. It is the first time to construct high sulphite-producing industrial strain by functional expression of MET14 in S. cerevisiae. Such study provides the foundation for construction of an excellent brewing yeast strain that can produce proper sulphite and can be used in commercial beer production.