Research On Breeding Of Glutathione Producing Saccharomyces Cerevisiae Strains

Glutathione (GSH) constituting of γ-amide linkage and thiol is an active tripeptide thatwidely distributed in animal and plant cells and microorganisms. It consists of glutamic acid,cysteine and glycine and has lots of critical physiological functions. GSH can clear away freeradicals, detoxification, delaying senility and anti-fatigue and etc. Other than that, GSH can alsoprotect liver, cure tumors and treat endocrine disorders if used in clinical practice. There aremany different ways to produce glutathione. So far, fermentation is the most commonly used ofall.The basis of the fermentation is the stable and high yield breeding of a good strain. Genomeshuffling which was brought up in the90s which has combined the traditional mutation breedingwith protoplast fusion technology, therefore has greatly accelerated the process of strain forwardmutation. The study of this technology is relatively less intensive use in domestic scholars, thusit’s not much related literature in the breeding of high-yield glutathione strains utilizing thistechnology.The purpose of this paper is to pick up high-yield, short growth cycle and stableGSH-producing strain utilizing genome shuffling breed technology. The specific results werelisted as follows:1、After activation to the preservation of the strains and ten times of subculture，screen outstrains which was high-yield and stable named Y1and Y2. As starting strains, theGSH-production respectively was261.47mg/L and600.13mg/L. The conditions of haploidpreparation from two glutathione producing Saccharomyces cerevisiae Y1、 Y2wereoptimized.The optimum conditions of haploid preparation were as follows: Y1、Y2wereseparately hydrolyzed with2%and1.5%snailase for75min and90min, heat treatment8minand10min at55℃after enzymolysis.Under the above conditions,the sporulation rate couldreach100%.2. The optimum conditions of formation and regeneration of protoplast were as follows:Y1-110、Y2-206were separately pretreatment20min and25min, osmolarity is0.45mol/L and0.50mol/L, hydrolyzed together with2%snailase+1%cellulase for3h and4h.Underthe above conditions,the formation rate of the protoplast was88.02%and90.00%respectively,regeneration rate reached up to51.70%and47.86%respectively.3. By using protoplast inactivating for purpose strain screening in the genome shuffling.The optimum conditions of protoplast inactivated were as follows: protoplast of Y1-110、Y2-206through heat inactivated were separately treatment3min and5min under65℃, UV-inactivatedwere separately treatment90s and120s. The optimum conditions of protoplast fusion were asfollows: PEG6000was35%, fusion temperature was28~30℃,fusion time was120min, thefusion rate reached3.23×10-4%.After three rounds of genome shuffling、resistance screening andgenetic stability screening we got a recombinant strain YR-1which yield GSH highly. Theproduction was679.95mg/L, the production increased160.05%than the original strain Y1,increased13.30%than the original strain Y2. The yield characteristics of the recombinant strainswere caused by genome shuffling, rather than the protoplasts mutagenesis occured in thepreparation and regeneration process by control experiments.4. The fermentation medium and fermentation conditions of recombinant strain YR-1wereoptimized by single factor experiment and orthogonal experiment. The optimum fermentationmedium compositions were determined as follows: glucose4.6%、yeast powder2.4%、K2HPO40.21%、KH2PO40.30%、MgSO40.05%, The GSH-production reached730.12mg/L, which was7.38%higher than before.The optimal fermentation conditions were as follows: temperature28℃, the initial pH value5.5, and inoculation quantity10%, volume shake flask30mL/250mL,reciprocating shaker120r/min. Under the optimal conditions, the fermentation cycle was36h,The GSH-production reached750.65mg/L, which was2.81%higher than before.