The Studies On Breeding Yeast With High Selenium And Yeast With High Glutathione

This paper not only focused on the breeding of a high-biomass, selenium-enriched yeast strain and optimization of cultivation conditions, but also focused on increasing glutathione (GSH) formation by functional expression of the γ-glutamylcysteine synthetase gene GSH-I in Saccharomyces cerevisiae.Strains exhibiting comparatively high Se resistance were selected out of 300 industrial strains. Among them, S.cerevisiae ZY-67 with high biomass and S. kluyveri ZY-198 with high Se content were chosen as initial parental strains. Haploids ZY-67-18 (a) with high biomass and ZY-198-21 (a) with high Se content were mutagenized by NTG. The auxotrophic mutant ZY-67-18-34 (a, leu") with high biomass and ZY-198-21-6 (a, trp-) with high Se content were chosen as parental strains for fusion. Fusant ZFF-28 was selected with higher biomass and Se content than the parental strains. Its biomass and Se content are 2.8 times and 2.0 times of the initial parental strains ZY-67 and ZY-198. Fusant ZFF-28 is stable in genetics.The cultivation conditions were optimized through single factor tests and a L16 (43 26) orthogonal design. Under the optimized cultivation conditions, the biomass (dry weight) reached 8.2 g/L and the Se content of the cells reached 2050 g/g. Organic Se content of the cells is 91%.A 4.2kb DNA amplified by PCR was inserted into the SmaI site of YEp352 to construct plasmid pGF-2. The pGF-2 was introduced into S.cerevisiae YS58. The GSH content of the recombinant strain was 1.5 fold of that in the host.A recombinant plasmid pGMF, containing GSH-I gene from pGF-2, was constructed with a copper resistance gene MTI as the selection marker and was first introduced into S.cerevisiae YSF-31 from Tsingtao Beer Brewery. The GSH content of the recombinant strain was 1.5 fold of that in the host strain. In addition, the biomass of the transformant was not affected by introduction of the plasmid pGMF. After growing the transformants under non-selective conditions (in YEPD) for 144h, 85% of cells still contained plasmid.