The Breeding Of High Glutathione Producing Strains By Mutagenesis Coupled Protoplast Fusion

Glutathione was widely used in various fields because of its special function. The glutathione of our country depend on importation, so it is urgent to study on glutathione production. The breeding of high glutathione-producing yeast strain and optimization of cultivation conditions were studied.The biomass and GSH content of 75 yeast strains from different species and genera were tested. Diploid strain Saccharomyces cerevisiae Y003 with high GSH content and Saccharomyces cerevisiae 2B48 with high biomass were chosen as parental strains. Haploids Sl(a) with high cell GSH content and SB1 (a) with high biomass were selected through determination of the mating- type, the biomass and the GSH content of the haploids.The two haploid strains were mutagenesised by DES, UV and UV-DES, selecting ZnCl₂ resistance and L-Ethionine resistance mutants, respectively. The mutant F4 (a,Arg^-,ZnCl₂^r,Eth^r) with high cell GSH content and the mutant with high biomass were selected, finally. The intracellular content of glutathione and the accumulation of glutathione ofthe mutant F4 (a,Arg^-,ZnCl₂^r,Eth^r)increased 73.5 % and 118.1% in comparison with the original strain S1 (a), respectively. The biomass and the accumulation of glutathione of the mutant 3-8(a,ZnCl₂^r,Eth^r) increased 25.6 % and 39.0% in comparison with the original strain SBl(a), respectively.Fusant R1 that was stable and excellent in biomass and glutathione yield was constructed by inactived protoplast fusion, selection and identification. The condition of production, regeneration of protoplast and protoplast fusion were studied. The biomass of Rl was 9.8g/L, the intracellular content of glutathione was 21.2mg/g and the accumulation of glutathione was 208.5mg/L. The accumulation of glutathione of the mutant Rl increased 129.4% and 155.5% in comparison with the two original strain F4 (a, Arg",ZnCl2r,Ethr) and 3-8(a, ZnCl2r,Ethr) , respectively.The effect of medium composition and culture condition on the production of glutathione by fusant Rl were studied in shaking-flask culture. The optimum ferment condition was culture under 32°C for 33h, the original PH of the medium was 7.0, the inoculating Concentration was 10%, the liquid volume of 30 ml in 250 ml.The effect of factors such as carbon source, nitrogen source, inorganic salt, that affected the GSH productivity of Rl were tested. The optimal fermentation conditions for Rl which were determined by directcross experiment was:10g/L yeast extract,30g/L glucose, NH4C1 3g/L,...