Study On The Resistance Mechanism Of Candida Tropicalis To Inhibitors In Corn Stalk Hydrolysate

A wide range of corn straw is adopted to produce bioethanol with fine prospect,which is identified as the ideal alternative for non-renewable fossil fuels（such as coal,natural gas and oil）.When corn straw is used to produce bioethanol by microorganism,Candida tropicalis no only produce ethanol similar to Saccharomyces cerevisiae,but also utilize xylose to generate xylitol.In this process,corn straw must be convert into monosaccharides for fermentation.However,these pretreatments will generate massive inhibitors which can significantly depress the cell growth and fermentation.However,the studies on the tolerance mechanism of C.tropicalis to the complex of inhibitors derived from corn straw hydrolysate was currently not conducted.In this paper,Candida tropicalis strain SHC-03 is selected as experimental material to explore its biochemical and molecular tolerance mechanism in corn straw hydrolysate at different growth stages.In this paper,we observed the morphological changes of subcellular structures by fluorescence confocal microscopy and used the second generation of high-throughput sequencing technology to find out the key genes related to tolerance to inhibitors in hydrolysate.1.Morphological observations on subcellular structures of samples at 3 h demonstrated that complex of inhibitors will induce accumulation of reactive oxygen species（ROS）and the morphological change of the mitochondria.With an increase in the time of culture,we found the decreased proportion of cell with ROS and the decrease of effect on the mitochondria induced by inhibitors.Based on the RNA-seq data,we found that the 21genes related to these processes are up-regulated.Among these genes,CTRG＿04311（GRX2）and CTRG＿04173（GRX5）encoding glutaredoxin-2 and glutaredoxin-5respectively were up-regulated by inhibitors,which indicated that glutaredoxin system was motivated to eliminate the ROS and reduce the toxic damage on cells.In addition,in this process,CTRG＿00152,CTRG＿05019 and CTRG＿02226（TSA1）encoding peroxidase and CTRG＿01087（SOD6）encoding superoxide dismutase exhibit up-regulated expression,which imply that peroxidase catalytic reaction system plays an important role in elimination of free radical.Furthermore,due to Ycp4p and Pst2p are responsible for the remodeling of vitamin K1 and K2,the up-regulation of CTRG＿00571（YCP4）,CTRG＿00572（PST2）and CTRG＿00589（PST2）induced by inhibitors in hydrolysate can accelerate the synthesis of massive vitamin K1 and K2 to protect the cells suffering peroxidation damage.2.When C.tropicalis strain SHC-03 was incubated into hydrolysate,we found that the morphological structure of endoplasmic reticulum（ER）of cells are not significantly altered.However,the date from RNA-seq show that 44 genes related to ER stress and protective unfolded protein response are significantly up-regulated.The protection mechanism of ER in which these genes are involved retains the homeostasis of ER.This protection mechanism includes the degradation of unfolded protein for which CTRG＿01299（KAR2）and CTRG＿01440（MNL1）are responsible,molecular chaperone-dependent protein protection for which CTRG＿00613（HSP60）,CTRG＿04728（SSA2）,CTRG＿05008（HSP82）,CTRG＿05840（AHA1）,CTRG＿02733（WOS2）,CTRG＿02883（AHA1）,CTRG＿03067（HSP104）are responsible,and homeostasis of Zn²⁺in ER retained by CTRG＿01049（ZRC1）and CTRG＿03775（MSC2）.3.The MIPS enrichment analysis of up-regulated expression genes from RNA-seq demonstrated that 36 genes related to detoxification are up-regulated.Among these genes,CTRG＿00385（QDR1）,CTRG＿01434（ROD1）,CTRG＿02846（YCR023C）,CTRG＿03729（SPBC409.08）related to multi-drug resistance,CTRG＿04983（YM9711.02C）related to detoxification and CTRG＿01697（SSU1）and CTRG＿05905（SSU1）related to efflux pump system are up-regulated by inhibitors in hydrolysate,which contribute to the detoxification and exhaust of endogenous toxic compounds induced by inhibitors in hydrolysate.Meanwhile,we found that a range of genes related to the reduction of inhibitors in hydrolysate are significantly up-regulated,such as CTRG＿00771（YNL134C）,CTGR＿01653（YML131W）,CTRG＿05072（YDR541C）encoding aldehyde reductase,CTRG＿04683（YPL088W）encoding aromatic alcohol dehydrogenase,CTRG＿02797（ADH7）,CTRG＿05127（ADH2）,CTRG＿05197（ADH2）encoding ethanol dehydrogenase,GTRG＿05318（FDH2）,CTRG＿00534（FDH1）,CTRG＿00547（FDH1）encoding formate dehydrogenase,CTRG＿03917,CTRG＿03729 encoding salicylic acid dehydrogenase,CTRG＿01751（ZTA1）encoding quinone oxidoreductase,CTRG＿03131（YALI0B16192g）encoding mannitol dehydrogenase,CTRG＿03026（ARD）encoding arabinose dehydrogenase,CTRG＿00569（BDH2）encoding hypothetical diacetyl reductase,CTRG＿04713（YIR035C）encoding hypothetical cytoplasmic short chain dehydrogenase,which contribute to the in situ detoxification of inhibitors by C.tropicalis strain SHC-03.In this paper,the oxidative stress mechanism and key genes related to reactive oxygen species induced by inhibitor of yeast C.tropicalis are preliminarily analyzed.At the same time,we explored the mechanism and key genes of endoplasmic reticulum stress,and analyzed genes related to situ and multidrug resistance of yeast C.tropicalis.Meanwhile,we constructed the recombinant expression plasmid related to situ.This paper preliminarily describes the tolerance mechanism of yeast C.tropicalis response to inhibitor complex in hydrolysate,and also provides a reference for the study of yeast C.tropicalis in lignocellulosic fermentation of bioethanol and construction of new engineering yeast strains.