Overproduction And Extracellular Accumulation Of Glutathione With Candida Utilis

Glutathione (GSH), the most abundant non-protein thiol compound distributedwidely in living organisms, performs important physical functions in cells. Candidautilis, a commonly industrial-used yeast strain, is a kind of Crabtree-negative andKluyver positive yeast. Multiple bio-based chemicals, such as organic acids, aminoacids and polypeptide, can be produced by C. utilis with hexose and/or pentose assubstrates. Especially, C. utilis can produce GSH efficiently with little cataboliterepression effect. Recently, microbial fermentation is considered as the most promisingapproach in the industrial production of GSH. However, few researches on GSHoverproduction have been carried out by regulating the physiological state of cells. Inthis study, we regulated cell growth and metabolism of the yeast by acid stress andsurfactants to promote GSH synthesis and excretion, and the mechanism on GSHoverproduction and extracellular accumulation were also explored.（1）Based on the analysis and comparison of parameters within batch GSHfermentation by C. utilis SZU07-01under constant pH values and no pH control, pH3.5and pH1.5were determined as the mild acid stress and severe acid stress conditions,respectively. In addition,6h for mild acid stress of pH3.5and2h for severe acid stressof pH1.5were chosen the optimal acid treating times during continuous culture of C.utilis SZU07-01in a chemostat. Furthermore, C. utilis SZU07-01was cultured underbatch culture with a total glucose concentration of60g/L by glucose feeding and acidstress, it was found that mild acid stress for6h was more propitious to GSHoverproduction than severe acid stress for2h and no acid stress. In the end, the reasonof GSH overproduction under mild acid stress were quantitatively interpreted by thecomparison of parameters obtained from batch fermentation kinetics analysis,intracellular cofactors as NADH and ATP determination, together with the distribution of flux on key metabolites.（2）It is common that GSH plays a key role in protecting organisms fromoxidative stress. In order to study whether GSH can play a role in acid stress and itsphysiological functions of resistance to acid stress, batch cultures of C. utilis SZU07-01and C. utilis gsh1under constant pH were conducted. Compared with the originalstrain of C. utilis SZU07-01, the intracellular GSH content of C. utilis gsh1wasdecreased by50%. The decrease of GSH may be due to the reduction of intracellularγ-glutamate cysteine ligase, which is resulted from the disruption of gsh1gene.Moreover, the DCW showed no significant difference between the two strains, whichindicated that the decrease of GSH had no obvious effect on cell growth. Low externalpH can lead to the decrease of pHi, we also found that pHidecreased under severe acidstress within the two strains. However, C. utilis gsh1with deficient biosynthesis abilityof GSH had a lower pHithan C. utilis SZU07-01.The activities of HK, PFK and PK were determined under different acid stress. Itwas obvious that the activities of these enzymes in both cells altered little under mildacid stress. However, at least80%decreased activities of HK, PFK and PK wasobserved in C. utilis gsh1when it was grown at pH1.5for2h. Although these enzymeactivities also decreased in C. utilis SZU07-01under severe acid stress, the decreasedextent was much lower than that of C. utilis gsh1except PK activities. SOD plays animportant role in protecting cells against damages caused by ROS. The activities ofSOD in both strains increased under the mild acid stress. Whereas the extent of SODactivity increment in C. utilis gsh1was more significant. SOD activity increased by180%under the severe acid stress while it only increased by69.5%in C. utilis SZU07-01at the same condition, which indicated that GSH played an important role inscavenging ROS.（3）The effects of typical surfactants as polyoxyethylene lauryl ether (Brij35),polyoxyethylene sorbitan monooleate (Tween80), sodium dedecyl sulfate (SDS) andcetyltrimethyl ammonium bromide (CTAB) on the cell growth and GSH productionwere investigated. Brij35could inhibit cell growth and total GSH production, DCW wasdecreased by19.7%,5%and17.8%with C. utilis SZU07-01, C. utilis gsh1and C. utilis gsh2, and GSH synthesis of C. utilis SZU07-01and C. utilis gsh2were alsoinhibited, the total GSH concentration were decreased by64.8%and37.72%,respectively. However, Brij35showed no positive effect on extracellular GSHaccumulation. Tween80seemed to have less effect on cell growth, but total GSHconcentration of C. utilis SZU07-01were decreased, and extracellular GSH increased.SDS and CTAB had critical inhibitory concentrations to cell growth during cultivation.When the concentrations of SDS and CTAB are higher than the critical levels, cellgrowth will be greatly inhibited or even ceased. When SDS and CTAB were added at18h, GSH accumulated extracellularly instead of intracellularly, and the intracellular GSHcontent decreased sharply to a very low level, especially when the concentration ofCTAB reached to0.04g/L and SDS to0.4g/L. The permeability of cell membrane wasimproved by CTAB and SDS, which would result in more extracellular GSH.