Construction Of Engineering Yeast And Study On Its Fermentative Production For Glutathione

Glutathione plays an important role in protein and DNA synthesis as well as amino acids transporting.It also has the health function of anti-oxidation,anti-aging,detoxification of heavy metal and improving immunity.The latest medical researches suggest that glutathione has a certain positive effect in treating liver disease,cancer and HIV.Up to now,there are three main methods to produce glutathione including chemical process,enzymatic method and fermentation.While,through improving the production technique and strain,the fermentation method is gaining more and more popularity and becoming the dominant method to produce glutathione industrially.This thesis presented a new method which enables yeast to synthesize cysteine-the precursor of glutathione by another pathway existing in Escherichia coli and eventually successfully obtained an engineering strain which could produce more glutathione than the original one.The main results are as follows:1.Two key genes named cysE and gsh I in cysteine and glutathione synthesis were cloned from DH5? Escherichia coli.Compared with the sequences reported in Genbank,cysE had two mutations at 143bp and 196bp,in which the variation at 196bp resulted in the change of amino acid;also,the mutation at 1343bp in gsh I caused the change of amino acid.2.Through a convenient site-directed mutagenesis method,the cloned gene was consistent with the sequences reported in Genbank.A double gene expression vector which carried two genes was constructed and verified by PCR.The constructed vector was transformed into saccharomyces cerevisiae.After 31 hours'fermentation,the maximum concentration of glutathione produced by engineered yeast reached 1.315mg/ml.While the original strain needed 54 hours to reach the maximum concentration which was 15.1%less than the engineered yeast.3.Comparing the intracellular content of glutathione by different methods of disrupting the cell wall,it was concluded that the ultrasonic method was the most efficient among all those methods,1.63mg/ml.The concentration of chemical method was 0.78mg/ml and 0.62mg/ml by boiling and freezing method.It showed that the ultrasonic method was respectively 2.1 times and 2.6 times more efficient than them.