Construction And Fermentation Research Of Glutathione Producing Saccharomyces Cerevisiae Recombinant Strain

Glutathione (y-glutamyl-L-cysteinyl-glycine or GSH), one of the major non-protein thiol compounds, widely distributes in almost all the aerobic organisms. As an important physiological active peptides, GSH plays many crucial physiological roles in oxidation resistance, immunity and detoxication. At present, GSH has been widely used in medical treatment, healthcare, functional foods, and cosmetics. Its market demand is rising, and the economic and social benefits are becoming more apparent.Many methods have been used to produce GSH, such as chemical method, enzymatic method and fermentative method, and the microbial fermentative method has emerged as the main approach for GSH production in industry. Currently, the GSH production technology in our country falls behind the others and the supply of GSH is mainly dependent on the import from Japan and other developed countries. So, it is of great importance to lower the production cost and increase the yeld of GSH.It is well known that the biosynthesis of GSH is an oxygen and energy consumption process. In the GSH fermentation process, the yeast cell growth is affected by insufficiency of dissolved oxygen, and as a result the GSH production is also affected. In previous study, in order to solve the dissolved oxygen problem, improving oxygen delivery capacity of bioreactors, increasing ventilatory capacity or sterring speed, or adding cosolvent had been tried in GSH fermentation. However, the practical use of these methods was restricted for the high demand of equipment or high cost. Therefore, how to solve the dissolved oxygen limitation has become one of the most crucial problems in GSH fermentation. For this reason, this paper is focusing on solving this problem.First, a zinc chloride-resistant and ethionine-resistant mutant S. cerevisiae NE101was obtained by complex mutagenesis with UV and nitrosoguanidine. The GSH productivity of the mutant reached84.2mg/L by the flask culture, which was 21.9%higher than the parent strain. Studies were carried out on the fermentation condition through single factor design, and the optimum medium composition was defined as:30g/L sucrose,8g/L yeast extract,5g/L urea,4.5g/L KH2PO4, and0.75g/L MgSO4, and the optimum fermentation condition was:culture temperature30℃, initial pH6.5, medium volumes30ml/250ml, inoculum size10%. In this fermental condition the GSH production reached117.8mg/L, which was39.9%higher than that of the initial condition.Second, the Vitreoscilla hemoglobin (VHb) was used to overcome the dissolved oxygen limitation. The primary function of VHb is to bind oxygen at low extracellular oxygen concentration and able to facilitate oxygen transfer to the terminal oxidase, and therefore VHb can enhance respiration under conditions of limited oxygen. Thus, the application of VHb will reduce the consumption of energy and the fermentation cost. In this study, we integrated the vgb gene into the GSH producing Saccharomyces cerevisiae genome. First, the integrating expression vector pYM-vgb containing the vgb gene was successfully constructed. Second, recombinant vector pYM-vgb was transformed into Saccharomyces cerevisiae NE101. Then the positive tranformants were selected on YPD plates containing G418and by PCR method. At last, expression of VHb in transformants and the bioactivity of the expressed VHb were verified by CO-difference spectrum and the result showed a typical VHb spectrum with a peak at around420nm and proved that the VHb had fuctional activity. To examine the VHb effects on cell densities and GSH yields, fermentation experiments were performed in shake flask under three different culture volume (30ml,50ml, and100ml per250ml) conditions. The result showed that the biomass and the GSH yield of the recombinant strain were both higher than the host strain in the different culture volumes and the GSH yield of the VHb-expressing stain increased about18.4%,29.2%and43.1%in the30ml,50ml and100ml, respectively. This proved that expression of VHb had positive effect on cell growth and GSH synthesis, especially under the limiting dissolved oxygen conditions.Third, the fermentation conditions were optimized by using the response surface method (RSM) in order to maximize the GSH production of VHb-expressing strain. L-cysteine, MgSO4and yeast extract were found to be the main factors affecting GSH production by Plackett-Burman design, and optimum levels of these three factors were defined by Box-Behnken design. From the response surface analysis, the optimal concentrations of the three major factors were:14.76g/L yeast extract,1.13g/L L-cysteine,1.54g/L MgSO4, and the extreme yield of GSH reached170.9mg/L, which was22.5%higher than that of the control.Forth, the separation and purification process of GSH from Saccharomyces cerevisiae101-V were investigated. Firstly, hot water extraction and ethanol extraction to extract GSH from yeast cells were compared and ethanol extraction was found to be the suitable method because it is an inexpensive, simple, efficient and GSH could be extracted without disrupting yeast cells. Secondly, membrane separation system (molecular weight cut-off5kDa) was selected to separate GSH and large proteins, and cation-exchange chromatography was used to separate and purify GSH, with a recovery of70.7%. Finally, the GSH with high purify was vacuumly concentrated and freeze-dried.