Aldehydes and phenols in the undetoxified hydrolysate inhibited growth and fermentation of the strain.Candida glycerinogenes have multiple resistance properties, and recombinant strains were constructed by overexpressing relevant resistance genes and screened.Compared with the control strain, the recombinant strain overexpressing CgGCN4 increased the biomass of C.glycerinogenes by 25.2%, 14.6%, and 5.2%, and the glucose consumption rate by 9.9%, 5.5%, and 13.1%, respectively, in a high-glucose medium containing 3.0 g/L furfural, 2.0 g/L vanillin and 3.5 g/L acetic acid, and increased glycerol production by 23.4% under the stress of 3.0 g/L furfural.Under the stress of furfural, vanillin or acetic acid, the increase of intracellular reactive oxygen species level of the recombinant strains was reduced by 35.2%, 28.0%, and 15.3%, respectively, and the binding level of propidium iodide was reduced to different degrees, suggesting that the recombinant strains could maintain the excellent growth and fermentation performance of the strains by reducing the accumulation of intracellular reactive oxygen species and maintaining the integrity of the cell membrane.Application of the resistant recombinant strain to open fermentation of undetoxified bagasse hydrolysate for glycerol production resulted in a 20.0% increase in sugar consumption rate, a 12.3% increase in biomass and a 21.7% increase in glycerol yield.This study provides new ideas and genetic resources for the application of undetoxified cellulose hydrolysate.
[1] AMORIM H V, LOPES M L, DE CASTRO OLIVEIRA J V, et al.Scientific challenges of bioethanol production in Brazil[J].Applied Microbiology and Biotechnology, 2011, 91(5):1267-1275.
[2] LIU Z L.Molecular mechanisms of yeast tolerance and in situ detoxification of lignocellulose hydrolysates[J].Applied Microbiology and Biotechnology, 2011, 90(3):809-825.
[3] HADI S M, SHAHABUDDIN, REHMAN A.Specificity of the interaction of furfural with DNA[J].Mutation Research Letters, 1989, 225(3):101-106.
[4] KLINKE H B, THOMSEN A B, AHRING B K.Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass[J].Applied Microbiology and Biotechnology, 2004, 66(1):10-26.
[5] WANG X, LI B Z, DING M Z, et al.Metabolomic analysis reveals key metabolites related to the rapid adaptation of Saccharomyce cerevisiae to multiple inhibitors of furfural, acetic acid, and phenol[J].Omics, 2013, 17(3):150-159.
[6] JÖNSSON L J, ALRIKSSON B, NILVEBRANT N O.Bioconversion of lignocellulose:Inhibitors and detoxification[J].Biotechnology for Biofuels, 2013, 6(1):16.
[7] TAHERZADEH M J, ADLER L, LIDÉN G.Strategies for enhancing fermentative production of glycerol—a review[J].Enzyme and Microbial Technology, 2002, 31(1-2):53-66.
[8] ANITHA M, KAMARUDIN S K, KOFLI N T.The potential of glycerol as a value-added commodity[J].Chemical Engineering Journal, 2016, 295:119-130.
[9] WANG Z X, ZHUGE J, FANG H Y, et al.Glycerol production by microbial fermentation: A review[J].Biotechnology Advances, 2001, 19(3):201-223.
[10] JIANG D Q, WANG M Y, ZHAO X H, et al.Glycerol production from undetoxified lignocellulose hydrolysate by a multiresistant engineered Candida glycerinogenes[J].Journal of Agricultural and Food Chemistry, 2024, 72(3):1630-1639.
[11] ZHAO M L, SHI D C, LU X Y, et al.Ethanol fermentation from non-detoxified lignocellulose hydrolysate by a multi-stress tolerant yeast Candida glycerinogenes mutant[J].Bioresource Technology, 2019, 273:634-640.
[12] WANG Y Q, ZHANG Z Y, LU X Y, et al.Transcription factor Hap5 induces gsh2 expression to enhance 2-phenylethanol tolerance and production in an industrial yeast Candida glycerinogenes[J].Applied Microbiology and Biotechnology, 2020, 104(9):4093-4107.
[13] WU C D, ZHANG J, WANG M, et al.Lactobacillus casei combats acid stress by maintaining cell membrane functionality[J].Journal of Industrial Microbiology & Biotechnology, 2012, 39(7):1031-1039.
[14] 张国友, 唐玲, 陈玮, 等.分光光度法测定蒙古栎叶中多酚的含量[J].中国科学院研究生院学报, 2009, 26(3):319-322.
ZHANG G Y, TANG L, CHEN W, et al.Spectrophotometric determination of polyphenols in Quercus mongolicus Fisch leaves by means of Folin-ciocalteu reagent[J].Journal of University of Chinese Academy of Sciences, 2009, 26(3):319-322.
[15] ZHANG M M, ZHANG K Y, MEHMOOD M A, et al.Deletion of acetate transporter gene ADY2 improved tolerance of Saccharomyces cerevisiae against multiple stresses and enhanced ethanol production in the presence of acetic acid[J].Bioresource Technology, 2017, 245:1461-1468.
[16] UJOR V C, OKONKWO C C.Microbial detoxification of lignocellulosic biomass hydrolysates:Biochemical and molecular aspects, challenges, exploits and future perspectives[J].Frontiers in Bioengineering and Biotechnology, 2022, 10:1061667.
[17] LIMA C S, NEITZEL T, PIROLLA R, et al.Metabolomic profiling of Spathaspora passalidarum fermentations reveals mechanisms that overcome hemicellulose hydrolysate inhibitors[J].Applied Microbiology and Biotechnology, 2022, 106(11):4075-4089.
[18] NATARAJAN K, MEYER M R, JACKSON B M, et al.Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast[J].Molecular and Cellular Biology, 2001, 21(13):4347-4368.
[19] QI S H, HE L, ZHANG Q, et al.Cross-pathway control gene CPC1/GCN4 coordinates with histone acetyltransferase GCN5 to regulate catalase-3 expression under oxidative stress in Neurospora crassa[J].Free Radical Biology and Medicine, 2018, 117:218-227.
[20] JI H, LU X Y, WANG C Y, et al.Identification of a novel HOG1 homologue from an industrial glycerol producer Candida glycerinogenes[J].Current Microbiology, 2014, 69(6):909-914.
[21] ENJALBERT B, SMITH D A, CORNELL M J, et al.Role of the Hog1 stress-activated protein kinase in the global transcriptional response to stress in the fungal pathogen Candida albicans[J].Molecular Biology of the Cell, 2006, 17(2):1018-1032.
[22] 何艳艳, 孜力汗, 张宝会, 等.过表达长链鞘氨醇激酶基因LCB4提高酿酒酵母抑制物耐受性[J].生物工程学报, 2018, 34(6):906-915.
HE Y Y, ZI L H, ZHANG B H, et al.Improvement of inhibitors tolerance of Saccharomyces cerevisiae by overexpressing of long chain sphingoid kinases encoding gene LCB4[J].Chinese Journal of Biotechnology, 2018, 34(6):906-915.
[23] PELICANO H, CARNEY D, HUANG P.ROS stress in cancer cells and therapeutic implications[J].Drug Resistance Updates, 2004, 7(2):97-110.
[24] WANG X H, CUI S Q, HU J J, et al.Saccharides in straw hydrolysate decrease cell membrane damage by phenolics by inducing the formation of extracellular matrix in yeast[J].Carbohydrate Polymers, 2019, 219:414-422.
