A new knocking-out system in Candida utilis and its application on disrupting the gsh1 gene

In this study,we report a novel system of gene knocking-out in C.utilis SZU 07-01 by suc-cessfully disrupting the gene of gsh1.First of all,the gsh1(encoding γ-GCS protein) gene was cloned by genome walking method from C.utilis SZU 07-01 according to γ-GCS protein conservative se-quences among several different yeasts.Then,the disrupting vector,pPICZalpha A-kan 3 was con-structed on the basis of plasmid pPICZalpha A,whose original TEF promoter responding for kananmy-cin resistance gene(kan) transcription was replaced by GAP promoter(pGAP) isolated from C.utilis SZU 07-01.pPICZalpha A-kan 3 was linearized and then transformed into C.utilis,resulting in a gsh1 deleted heterozygotic mutant strain designated as GSH-6.After cultured in the same condition,the mu-tant deficient in glutathione biosynthesis showed decreases of 17.5%,61%,18.5% in γ-GCS activity,glutathione content and dry cell weight,respectively.The disruption element(pGAP:kan) used in this study supplies a new gene genetic manipulation approach to research the physiological function of GSH in C.utilis at a molecular level.