Glutathione Biosynthesis By Engineered Saccharomyces Cerevisiae

Glutathione (GSH) is a non-protein thiol compound in living cells. Under the supply of ATP, the GSH could be synthesized from L-glutamic acid, L-cysteine and glycine in two consecutive steps catalysed by y-glutamylcysteine synthase (GSH1) and y-glutamylcysteine (GSH2). With its important physiological functions such as antioxidant. detoxification of xenobiotics. and immune booster and so on, GSH has been widely used in medicine, food additive and cosmetic products. In recent years the demand for GSH has tended to increase. The main contents and results were as follows:(1) The GSH1and GSH2genes were amplified from the extracted total DNA of Saccharomyces cerevisiae S288c by PCR and expressed under the control of alcohol dehydrogenase (ADH1) promoter and their respective native terminators. With Kanr gene from pFA6a-kanMX6or Hygr gene from pYM20as the selective markers, two expression vectors YEplace181AK-GSH1and YEplace181AH-GSH2were constructed. The recombinant plasmids were transformed into an industry strain Saccharomyces cerevisiae TS013respectively and simultaneously by electroporation, and three recombinant strains S.TS013/GSH1, S.TS013/GSH2and S.TS013/GSH1+GSH2were generated by screening on YPED plates supplemented with G418or (and) Hygromycin. The protein expression of GSH1, GSH2and genetic stability of recombinant S. cerevisiae were evaluated and investigated. Strong protein bands could be observed both in host and recombinant strains. No significant differences of protein bands could be observed from the gel electrophoresis. After the transformants were cultured under non-selective conditions (in YEPD) for10generations,92%of cells still contained plasmid.(2) Comparison of GSH synthesis in different engineered strains. The intracellular GSH content of S.TS013, S.TS013/GSH1, S.TS013/GSH2, S.TS013/GSH1+GSH2were8.39mg/g dry cells,10.46mg/g dry cells,9.56mg/g dry cells, llmg/g dry cells; the total GSH content were103.4mg/L,118.86mg/L,110.51mg/L,121mg/L. Compared with the content of control strain, the intracellular GSH content of recombinant strains increased by25%,14%and31%; the total GSH content increased by16%,7%and17%. The experiment was used to optimize fermentation parameters of the medium and cultivation condition of the recombinant strain S.TS013/GSH1+GSH2. The maximal yield of GSH and the biomass could be obtained (9.91g/L,150.02mg/L) when the concent of glucose, tryptone, KH2PO4, MgSO4-7H2O, inositol were set at40g/L,40gL,2g/L,0.5g/L,0.1g/L, respectively at the optimal cultivation pH6.0, inoculation size10%, rotate200r/min, flask fluid volume50mL/250mL.(3) There was great correlation in the OD values and cell dry weights both in the logarithmic growth and stable phase. According to the linear functions, the yeast biomass could be easily detected and converted, which would be helpful for the measurement and comparison of the yeast’s biomass under different conditions. The addition of precursor amino acids by two steps and hydrogen peroxide were used to improve GSH in recombinant strain S.TS013/GSH1+GSH2. The results showed that adding1Ommol/L cys at the beginning of fermentation6h, the GSH content reached224.7mg/L, increased by40.53%compared to control (160.02mg/L). Mixed amino acids of30mmol/L were added at middle logarithmic growth phase, the GSH content reached267.9mg/L, increased by67.42%compared to control. lmol/L H2O2were added at the stationary phase24h, the GSH content reached286.7mg/L, increased by79.17%compared to control.(4) Enzymatic GSH production using metabolically engineered Saccharomyces cerevisiae as a whole-cell biocatalyst. The cells were treatment with0.4%Triton X-100to cell permeation. The rate of the release of GSH from permeated yeast cells was saturated at87%within36h. The GSH contents of S.TS013, S.TS013/GSH1, S.TS013/GSH2, S.TS013/GSH1+GSH2were349.06mg/L,488.13mg/L,388.97mg/L and405.24mg/L. The GSH contents of recombinant strains increased by40%,11%and16%compared to control. By cell dosage proportions of two engineered yeast cells S.TS013/GSH1and S.TS013/GSH2to GSH synthesis, GSH content reached537.25mg/L at the ratio of4:1at48h, increased by10%and38%compared to S.TS013/GSH1or S.TS013/GSH2used only. In a two-stage reaction by two engineered strains, in the start stage, glutamic acid, cysteine and S.TS013/GSH1strain were added into the reaction mixture. Glycine, S.TS013/GSH2strain were then added to the reaction mixture at24h. GSH productivity synthesized reached 644.7mg/L at48h, was20%higher than that of the GSH synthesized by two engineered strains in one-stage reaction, and84.7%higher than that of host strain.