Construction Of Candida Glycerinogenes Genomic Library And Its Application

During the fermentation process,industrial yeast is subjected to various stresses from the substrate,product,and environment,which can have an impact on fermentation yield,conversion efficiency,and production cost.Candida glycerinogenes is a multi-stress resistant industrial yeast that can be found in many important industrial products,and is an excellent candidate for studying resistance genes.However,genome sequencing results indicate that there are very few similar and referenceable strains,making gene mining challenging.In this study,we constructed a C.glycerinogenes genomic library,screened for tolerance genes,and performed functional analysis and application study on resistance genes.This provides new genetic information and biomaterials for the development of stress tolerance mechanisms and unvirus-free cellulose fermentation in yeast.The main contents include:（1）In the pursuit of constructing a gene library for C.glycerinogenes,the methodological investigation was conducted,revealing that the attachment efficiency of the A-tail segment to the commercial T vector exceeded 90%.Conversely,the attachment rate to the self-constructed expression T vector was notably low.Consequently,it became apparent that the TA cloning method was not suitable for constructing the C.glycerinogenes genomic expression library.The enzyme digestion and linkage method was utilized,resulting in the successful construction of a C.glycerinogenes genomic library.Approximately 9×10⁴ transgeners were produced,with an average inserted DNA segment size of roughly 1500 bp,positive rate of approximately 75%,and storage capacity that was about 2.2 times that of the C.glycerinogenes genome.The probability of obtaining any DNA fragment was 99%,making it possible to screen for resistance genes.（2）The genomic library acquired was screened to identify resistance genes.Twenty resistant strains were obtained from the plates containing furfural,acetic acid,H₂O₂,Na Cl,and other compounds,out of which three genes were identified with observable phenotypes.Sequencing confirmed the presence of r RNA methyltransferase gene Cg BMT5,which can enhance the acetic acid tolerance of yeast;Rrna maturation and ribosome assembly related gene Cg EBP2,and cell wall synthesis related gene Cg ECM3,which can enhance the salt tolerance of yeast.To verify their functions,the three genes were expressed in S.cerevisiae,yielding the following results:under acetic acid stress,the biomass of S.cerevisiae/Cg BMT5 increased by20.1%;under Na Cl stress,the biomass of S.cerevisiae/Cg EBP2 and S.cerevisiae/Cg ECM3increased by 19.4%and 15.2%,respectively;and the ethanol fermentation yield of recombinant strains was increased by 9.9%-17.7%.Additionally,overexpression of Cg BMT5 in C.glycerinogenes led to a 17.6%increase in ethanol yield under acetic acid stress.Furthermore,when Cg EBP2 and Cg ECM3 were expressed separately,ethanol production increased by 12.7%and 21.3%,respectively under Na Cl stress.These findings indicate that Cg BMT5,Cg EBP2,and Cg ECM3 are indeed resistance genes.（3）Stress response analysis of strains overexpressing resistance genes and their application in undetoxified cellulose hydrolysate fermentation.Overexpression of Cg BMT5,Cg EBP2 and Cg ECM3 under acetic acid and Na Cl stress resulted in decreased lipid peroxidation levels and intracellular reactive oxygen species,while catalase（CAT）and superoxide dismutase（SOD）enzyme activities increased.Additionally,cell membrane permeability and intracellular levels of tryptophan,glutamic acid,and lysine were altered.q RT-PCR analysis revealed that Cg BMT5overexpression under acetic acid stress enhanced lipid metabolism,arginine synthesis,and ethanol synthesis pathways.Under Na Cl stress,overexpression of Cg EBP2 and Cg ECM3increased phospholipid synthesis,glycolysis,Na⁺transport,and ethanol synthesis.Furthermore,when the strains were utilized in cellulose hydrolysate ethanol fermentation,ethanol yield increased by 58.0%,18.1%,and 47.1%,and production intensity increased by 95.7%,73.9%,and 82.6%,respectively.These findings provide important insights into the resistance mechanisms of industrial yeast and their potential applications.