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

Abstract:

In this study, we report a novel system of gene knocking-out in C. utilis SZU 07-01 by successfully 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 sequences among several different yeasts. Then, the disrupting vector, pPICZalpha A-kan 3 was constructed on the basis of plasmid pPICZalpha A, whose original TEF promoter responding for kananmycin 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 mutant 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.