Early Transcriptional Response In Corn Straw Hydrolysate And Functional Identification Of Aldehyde Reductase Genes Of Saccharomyces Cerevisiae

Lignocellulose,represented by corn straw,is regarded as"a neglected resource",it is an important biomass energy,but which is treated in an unscientific way and causes both the environmental and economic losses.Its carbohydrates can be used for fermentation by microorganisms such as Saccharomyces cerevisiae to produce ethanol,and ethanol fuels are considered as the ideal alternatives to liquid fuels because of the clean and renewable characteristics.In order to destroy the crystalline structure of cellulose and the protective barrier of lignin,some pretreatment is needed in the process of straw saccharification.However,these pretreatment processes will produce a series of inhibitors that effect cell growth and ethanol yield.Therefore,a strain,YA-B-6-1,with high tolerance to acetic acid,furfural and other hydrolysates was selected in this study.The early transcriptional response and subcellular structure of Saccharomyces cerevisiae in corn straw hydrolysis were studied by transcriptome sequencing and fluorescence microscopy.The following experimental results are obtained:1.The lag phase of Saccharomyces cerevisiae was prolonged by corn straw hydrolysate.The length of the lag phase was negatively correlated with the inoculum size,and the cells were in the lag phase before 6 h in the culture medium with 100%inoculum size.The results of transcriptome analysis at 3 h and 6 h showed that the cells at 3 h were responsing the stress.Compared with the control group,the differentially expressed genes were enriched in the antioxidant reaction,and the cells at 6 h had begun to synthesize macromolecules to prepare for the later growth,such as receptor proteins and so on.2.By analyzing the differentially expressed genes in the transcriptome data at 3 h,we found that genes TRX3 and TSA2 encoding the thioredoxin could effectively eliminate reactive oxygen species produced in mitochondria in time.And the expression of peroxidase genes GPX1,SYM1,CTT1 were up-regulated,they can clear reactive oxygen species in the cytoplasm,ASK10 and RCK1 were also up-regulated,which are genes involved in HOG-MAPK signal transduction pathway,which can regulate the transcription of YAP2 and participate in oxidative stress response.The observation of fluorescence microscope shows that a large amount of reactive oxygen species was accumulated in the cells at 3 h,but the proportion of positive signal cells decreased significantly at 6 h.The results showed that corn straw hydrolysate could induce a large number of ROS in cells,but up-regulation of peroxidase genes and geness encoding glutaredoxin/thioredoxin could effectively scavenge reactive oxygen species,activate the response mechanism and reduce the damage of reactive oxygen species.3.The functional catalogue of differentially expressed genes at 3 h showed that 17up-regulated genes were enriched in protein folding and stability.SSA1,SSA2,SSA3,SSE2and ECM10 are the HSP70 family members,they can bind with newly translated proteins to help them to fold correctly and prevent them from aggregating or misfolding,and they are also involved in the disassembly of misfolded protein aggregates,both APJ1 and SIS1are the members of HSP40 family that promote HSP70's ATPase activity,and they are also up-reglulated.In addition,HSP82 can specifically repair some important transcription factors and protein receptors,and PCH2,which maintain the structure of chromosome axon protein,were also up-regulated.Most of these above genes'promoters upstream contain stress response elements,and can be regulated by Msn2p/Msn4p,whose fold changes reach 1.2 and 3.0 respectively in this study,and SFL1 which is negatively regulated by protein kinase A also up-regulated,these suggest the inhibition of c AMP-PKA signal transduction pathway may activate the mechanism of protein-protective response.Because the structure of endoplasmic reticulum would be damaged by the accumulation of unfolded proteins or misfolded proteins,and then the structure of endoplasmic reticulum was destroyed.The endoplasmic reticulum membrane structure was observed by fluorescence microscope at different time points.The endoplasmic reticulum was slightly damaged at 3 h,but it was alleviated at 6 h.This is the function of the protecting and correctly folding of the new proteins,and renaturation and clearance of the misfolded proteins.These functions released the stress of endoplasmic reticulum and maintained the stability of endoplasmic reticulum structure.4.During the process of functional catalogue and gene annotation,many genes related to detoxification were also found to be up-regulated.For example,the aldehydes reductase gene YDR541C,GRE2,ADH6,GOR1,GRE3,GRE2 and the ketoreductase genes DAK1,DAK2 and YDL124W can reduce different aldehydes and ketones as nontoxic or less toxic alcohols,and the genes that regulate H+ATPase on the cell membrane,HSP30,PMP2,PMP1 were also up-regulated,they can pump out the intracellular H~+to maintain the inner neutral environment of cell.The up-regulation of these above genes lead to the harmfulness of the toxic inhibitor was reduced.And three possible aldehyde reductase genes,NRE1,BDH1,BDH2 were found in this study.5.Cloning and expression of these three possible aldehydes reductase genes and detection of their related enzyme activities showed that they all had aldehyde reductase activities,and they all preferred NADH as co-factor for reducing,NRE1 can reduce at least three substrate,including acetaldehyde,cyclopentanone and 2-pentanone,BDH1 showed activity to six substrates under the condition of NADH as co-factor,and the reduce activity of acetaldehyde was as high as 79 U/mg.BDH2 could reduce at least 7 substrates,and the enzyme activity for acetaldehyde was the highest,reaching 92 U/mg.In this paper,it was preliminarily found that Saccharomyces cerevisiae can scavenge the active oxygen species in various subcellular organelle,protect proteins to promote proper folding and maintain the structure of the endoplasmic reticulum,detoxify aldehydes,ketone compounds and pump intracellular proton to maintain the cellular neutral environment.These results could be used as a reference for the construction of the engineering strain of Saccharomyces cerevisiae with high tolerance to corn straw hydrolysate.