Effect Of Glutathione On Resisting To Oxidative Stress In Candida Utilis

Candida utilis is a very important industrial strain. It has been approved as a food additive by U.S. Food and Drug Administration. However, when Candida utilis is fermented to produce products, it will be subjected to oxygen stress by (O2-), (OH·) and (H₂O₂). Excessive reactive oxygen can damage proteins, fetty acids and nucleic acids to make cell death. This study aimed to analyze the regulatory mechanism and response to oxygen stress, so as to reduce the adverse impact by oxygen stress, improve the capabilities of resisting oxygen stress, and finally improve the production of target products.Glutathione (GSH) is the most abundant non-protein thiol compound widely distributed in living organisms. Glutathione can be produced using enzymatic methods in the presence of ATP and its three precursor amino acids (L-glutamic acid, L-cysteine and glucine). GSH performs the important physical function, especially in maintaining the oxidative-reduction environment in cells. In order to uncover the physical functions of GSH, we first cloned the gsh1 gene (encodingγ-GCS protein) from C. utilis SZU 07-01 by genome walking method. Secondly, we constructed the gene knocking-out systems in C. utilis and obtained the corresponding gsh1 and gsh2 gene disrupting heterozygotic mutants GSH-3 and GSH2(2). Finally, the two gene disrupting mutants were subjected to H₂O₂ stress and their responses were analyzed.(1) First of all, the sequences ofγ-GCS protein among several different yeasts were aligned to aquire the conserved sequence. The gsh1 gene (encodingγ-GCS protein) in C. utilis was firstly cloned by genome walking method, using the primers designed by CODEHOP on line. Its gene bank accession number was assigned to be HQ171204.(2) In this study, we reported a novel system of gene knocking-out in C. utilis SZU 07-01 and successfully disrupting the gene of gsh1. The disrupting vector, pPICZalphaA-kan3 was constructed on the basis of plasmid pPICZalpha A, whose original TEF promoter for kananmycin resistance gene (kan) was replaced by GAP promoter (pGAP) isolated from C. utilis SZU 07-01. pPICZalphaA-kan3 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 which was deficient in glutathione biosynthesis showed 17.5%, 61% and 18.5% decreases inγ-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 functions of GSH in C. utilis at the molecular level.The plasmid pPICZalphaA-zeocin, pPICZalphaA-CYH, pPICZalphaA-HPH were constructed on the basis of plasmid pPICZalphaA-kan, and then transformed into C. utilis to obtain gsh1 gene disrupting mutants of GSH-6(gsh1△), GSH-3(gsh1△²)and GSH-9(gsh1△³). Moreover, in order to knockout the the gsh2 gene, plasmids pPICZ-gsh2-kan and pPICZ-gsh2-zeocin were constructed and transformed into C. utilis to obtain gsh2 gene disrupting mutants of GSH2(1)( gsh2△) and GSH2(2)(gsh2△2). These part of work will play a important role in the molecular research of C. utilis.(3) Glutathione plays a key role in protecting organisms from oxidative stress. In order to study its physiological functions of resisting to oxidative stress, we first tested the fermentation profiles of strains SZU07-01, GSH-3, and GSH2(2) without any oxidative stress. Compared with the wild type strain SZU07-01, the intracellular GSH of strains GSH-3 and GSH2(2) were decreased by 89% and 53%, respectively, and their total GSH were decreased by 64% and 29%. The resulted GSH decrease may be due to the reduction of intracellular gama-glutamate cysteine ligase and glutamate synthetase expression, which are the consequence of the low gsh1 and gsh2 copy numbers. Moreover, the DCW of strains GSH-3 and GSH2(2) were increased by 11% and 10% as compared with the wild type strain, which indicates that the decreasing of GSH have no obvious effect on cell growth in the case of no H₂O₂ stress. Next, we determined the fermentation profiles of strains SZU07-01, GSH-3, and GSH2(2) with H₂O₂ stress. We found that addtion of 10 mmol/L H₂O₂ in SZU07-01 wide control strain at 18 h can increased the GSH synthetic, which indicated that the cells may resisted to H₂O₂ stress by increasing its GSH synthsis. Addition of H₂O₂ to strains GSH-3 and GSH2(2) at the same concentration and time point as above increased the total GSH concentration of these two strains by 22% and 1%, respectively, which indicated that the cells may resisted to H₂O₂ stress by increasing its GSH synthsis. However, the Addition of H₂O₂ decreased the DCW by 6% and 9%, respectively, which indicated that the two strains may be sensitive to H₂O₂ because of the low gsh1 and gsh2 copy numbers and then the low GSH contents. Moreover, the CAT activitiy of strains GSH-3 and GSH2(2) were increased by 7% and 12%, which means that CAT may be the important defensive factor as C. utilis was subjected to H₂O₂-induced oxidative stress. In conclusion, GSH and intracellular CAT may also play a important role upon the H₂O₂ stress.