Construction Of Selenomethionine-resistant Pichia Pastoris On The Basis Of Sulfur Amino Acids Synthetic Pathway

Selenomethionyl protein,which contains selen formed by using selenomethionine to substitute methionine residues,is a noval tool in structural biology and can effectively solve phase problem in protein crystallography.Selenomethionyl protein is usually produced by Escherichia coli in selenomethionine-containing medium,however,E.coli often fail to express eukaryotic proteins that receive post-translational modification,such as glycosylation.Despite the fact that yeasts are ideal hosts for production of proteins from eukaryotes,incorporation of selenomethionine into proteins is limited because of inability of yeasts to grow in medium supplemented with selenomethionine.Although mechanisms underlying selenomethionine toxicity are still controversial,selenocystein generated from selenomethionine by endogenous enzymes is most likely to be a primary toxic compound.Thus,this thesis aimed at acquiring of selenomethionine-resistant yeast strain based on the study of sulfur amino acids synthetic pathway.Pichia pastoris is a very important expression host in industrial fermentation,while its sulfur amino acids biosynthetic pathway is still unclear.Our bioinformatics approach predicted that P.pastoris synthesizes sulfur amino acids from inorganic sulfate via O-acetyl-L-homoserine and O-acetyl-L-serine pathways.We also found that methionine and cysteine are interconvertible by trans-sulfuration pathway in this organism.These findings were confirmed by phenotypic analyses of a series of single or double knockout mutants generated by standard genetic engineering technology.The ability of P.pastoris to synthesize sulfur amino acids via O-acetyl-L-homoserine and O-acetyl-L-serine pathways.Combined analyses of genome sequence and disruptant phenotypes successfully identified genes involved in conversion of methionine to cysteine.These pathways is obviously different from other yeast species.In addition,we using genetic engineering technology disrupted CYS3 gene showing selenomethionine resistance by blocking selenocysteine biosynthesis from selenomethionine.The Ppcys3? resistant strain was cultured with different concentrations of selenomethionine.The results showed that when the concentration of selenomethionine reached 8mM,the growth rate of wild type cells was zero,while the Ppcys3?was close to 60%.In addition,by suppling 1mM cysteine to minus cysteine and methionine medium can effectively inhibit the cytotoxicity of selenomethionine.