Cloning and Identification of Methionine Synthase Gene from Pichia pastoris

Methionine synthase(MS)is grouped into two classes.Class One MS(MetH)and Class TwoMS(MetE)share no homology and differ in their catalytic model.Based on the conserved sequences ofmetE genes from different organisms,a segment of the metE gene was first cloned from Pichia pastorisgenomic DNA by PCR,and its 5’and 3’regions were further cloned by 5’-and 3’-rapid amplification ofcDNA ends(RACE),respectively.The assembled sequence reveals an open reading frame encoding apolypeptide of 768 residues,and the deduced product shares 76% identity with MetE of Saccharomycescerevisiae.P.pastoris methionine synthase(PpMetE)consists of two domains common to MetEs.Theactive site is located in the C-terminal domain,in which the residues involved in the interaction of zinc withsubstrates are conserved.Homologous expression of PpMetE in P.pastoris was achieved,and the heterolo-gous expression of PpMetE in the S.cerevisiae strain XJB3-1D that is MetE-defective restored the growthof the mutant on methionine-free minimal media.The gene sequence has been submitted to GenBank/EMBL/DDBJ under accession No.AY601648.