淀粉质燃料乙醇发酵胁迫及菌株耐受性改造
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摘要
在淀粉质燃料乙醇生产过程中,酿酒酵母面临的多重胁迫严重制约了燃料乙醇的生产效率。针对这一现状,该文结合国内淀粉质燃料乙醇主要生产工艺,综合分析了该工艺下燃料乙醇发酵过程中影响酿酒酵母生长和生产的各种胁迫因素;阐述了微生物对不同胁迫因子的抗逆分子机制;介绍了菌株耐受性改造的方法;提出实现菌株全局的耐受性和鲁棒性,并且开发基于全基因组的快速进化方法是未来工业微生物耐受性改造的方向。
In the industrial production of starchy fuel ethanol, Saccharomyces cerevisiae faces multiple stresses during fermentation, which substantially limits the efficiency of ethanol production. Aiming at this phenomenon, this article analyzes the current domestic fuel ethanol production status and production process and the stress factors in fermentation process, illustrates the molecular mechanisms of S. cerevisiae tolerance to various stresses, and introduces the modification methods about yeast tolerance. We suggest that realizing global tolerance and robustness of S. cerevisiae and developing genome-wide discretionary multipoint genome modification approach are the probable direction of industrial microorganism modification in the future.
In the industrial production of starchy fuel ethanol, Saccharomyces cerevisiae faces multiple stresses during fermentation, which substantially limits the efficiency of ethanol production. Aiming at this phenomenon, this article analyzes the current domestic fuel ethanol production status and production process and the stress factors in fermentation process, illustrates the molecular mechanisms of S. cerevisiae tolerance to various stresses, and introduces the modification methods about yeast tolerance. We suggest that realizing global tolerance and robustness of S. cerevisiae and developing genome-wide discretionary multipoint genome modification approach are the probable direction of industrial microorganism modification in the future.
引文
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[27]Zheng P,Liu M,Liu XD,et al.Genome shuffling improves thermotolerance and glutamic acid production of Corynebacteria glutamicum[J].World Journal of Microbiology&Biotechnology,2012,28(3):1035-1043.
[28]Wang Hao(王灏),Wang Hang(王航),Meng Chun(孟春),et al.Study of breeding Saccharomyces cerevisiae with improved temperature and ethanol tolerance by genome shuffling[J].Microbiology China(微生物学通报),2007,34(4):705-708.
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[30]Chen Y,Sheng J,Jiang T,et al.Transcriptional profiling reveals molecular basis and novel genetic targets for improved resistance to multiple fermentation inhibitors in Saccharomyces cerevisiae[J].Biotechnology for Biofuels,2016,9:9.
[31]Yang W,Zhang J,Wang H,et al.AngiotensinⅡdownregulates catalase expression and activity in vascular adventitial fibroblasts through an AT1R/ERK1/2-dependent pathway[J].Mol Cell Biochem,2011,358(1/2):21-29.
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[34]Shahsavarani H,Sugiyamaa M,Kanekoa Y,et al.Superior thermotolerance of Saccharomyces cerevisiae for efficient bioethanol fermentation can be achieved by overexpression of RSP5ubiquitin ligase[J].Biotechnol Adv,2012,30:1289-1300.
[35]Qiu Z,Deng Z,Tan H,et al.Engineering the robustness of Saccharomyces cerevisiae by introducing bifunctional glutathione synthase gene[J].J Ind Microbiol Biotechnol,2015,42:537-542.
[36]Luhe A L,Tan L,Wu J,et al.Increase of ethanol tolerance of Saccharomyces cerevisiae by error-prone whole genome amplification[J].Biotechnology Letters,2011,33(5):1007-1011.
[37]Kitichantaropas Y,Boonchird C,Sugiyama M,et al.Cellular mechanisms contributing to multiple stress tolerance in Saccharomyces cerevisiae strains with potential use in high-temperature ethanol fermentation[J].AMB Express,2016,6(1):107.
[38]Thompson O A,Hawkins G M,Gorsich S W,et al.Phenotypic characterization and comparative transcriptomics of evolved Saccharomyces cerevisiae strains with improved tolerance to lignocellulosic derived inhibitors[J].Biotechnology for Biofuels,2016,9:200.
[39]Doudna J A,Charpentier E.Genome editing.The new frontier of genome engineering with CRISPR-Cas9[J].Science,2014,346(6213):1258096.
[40]Rauscher B,Heigwer F,Breinig M,et al.Genome CRISPR a database for high-throughput CRISPR/Cas9 screens[J].Nucleic Acids Research,2017,45(Database issue):D679-D686.
[41]Wang H,Yang H,Shivalila C S,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[J].Cell,2013,153(4):910-918.
[42]Ryan O W,Skerker J M,Maurer M J,et al.Selection of chromosomal DNA libraries using a multiplex CRISPR system[J].ELife,2014,3:e03703.
[43]Zehua B,Mohammad H R,Pu X,et al.Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision[J].Nature Biotechnology,2018.DOI:10.1038/nbt.4132.
[44]Snoek T,Picca Nicolino M,Van den Bremt S,et al.Large-scale robot-assisted genome shuffling yields industrial Saccharomyces cerevisiae yeasts with increased ethanol tolerance[J].Biotechnology for Biofuels,2015,8:32.
[2]Yue Guojun(岳国君),Wu Guoqing(武国庆),Hao Xiaoming(郝小明),et al.The status quo and prospects of fuel ethanol process technology in china[J].Progress in Chemistry(化学进展),2007,19(7/8):1084-1090.
[3]Zhang G,Lin Y,Qi X,et al.TALENs-assisted multiplex editing for accelerated genome evolution to improve yeast phenotypes[J].ACSSynthetic Biology,2015,4(10):1101-1111.
[4]Liu Y,Zhang G,Sun H,et al.Enhanced pathway efficiency of Saccharomyces cerevisiae by introducing thermo-tolerant devices[J].Bioresource Technology,2014,170(5):38-44.
[5]Abbott D A,Ingledew W M.Buffering capacity of whole corn mash alters concentrations of organic acids required to inhibit growth of Saccharomyces cerevisiae and ethanol production[J].Biotechnology Letters,2004,26(16):1313-1316.
[6]Zi Z,Liebermeister W,Klipp E.A quantitative study of the Hog1MAPK response to fluctuating osmotic stress in Saccharomyces cerevisiae[J].PloS One,2010,5(3):e9522.
[7]Gong Y,Kakihara Y,Krogan N,et al.An atlas of chaperone-protein interactions in Saccharomyces cerevisiae:implications to protein folding pathways in the cell[J].Molecular Systems Biology,2014,5(1):275-283.
[8]Li J R,Yu P.Expression of Cu,Zn-superoxide dismutase gene from Saccharomyces cerevisiae in Pichia pastoris and its resistance to oxidative stress[J].Applied Biochemistry and Biotechnology,2007,136(1):127-139.
[9]Choi K,Batke S,Szakal B,et al.Concerted and differential actions of two enzymatic domains underlie Rad5 contributions to DNA damage tolerance[J].Nucleic Acids Research,2015,43(5):2666-2677.
[10]Petitjean M,Teste M A,Francois J M,et al.Yeast tolerance to various stresses relies on the trehalose-6P synthase(Tps1)protein,not on trehalose[J].Journal of Biological Chemistry,2015,290(26):16177-16190.
[11]Busti S,Mapelli V,Tripodi F,et al.Respiratory metabolism and calorie restriction relieve persistent endoplasmic reticulum stress induced by calcium shortage in yeast[J].Scientific Reports,2016,6:27942.
[12]Teixeira M C,Monteiro P,Jain P,et al.The yeastract database:a tool for the analysis of transcription regulatory associations in Saccharomyces cerevisiae[J].Nucleic Acids Research,2006,34(Database issue):D446-D451.
[13]Lam F H,Ghaderi A,Fink G R,et al.Biofuels.Engineering alcohol tolerance in yeast[J].Science,2014,346(6205):71-75.
[14]Izawa S,Ikeda K,Miki T,et al.Vacuolar morphology of Saccharomyces cerevisiae during the process of wine making and Japanese sake brewing[J].Applied Microbiology and Biotechnology,2010,88(1):277-282.
[15]Caspeta L,Chen Y,Ghiaci P,et al.Biofuels.Altered sterol composition renders yeast thermotolerant[J].Science,2014,346(6205):75-78.
[16]Jia H Y,Sun X Y,Sun H,et al.Intelligent microbial heat-regulating engine(IMHeRE)for improved thermo-robustness and efficiency of bioconversion[J].ACS Synthetic Biology,2016,5(4):312-320.
[17]Zhang Y X,Perry K,Vinci V A,et al.Genome shuffling leads to rapid phenotypic improvement in bacteria[J].Nature,2002,415(6872):644-646.
[18]Alper H,Moxley J,Nevoigt E,et al.Engineering yeast transcription machinery for improved ethanol tolerance and production[J].Science,2006,314(5805):1565-1568.
[19]Isaacs F J,Carr P A,Wang H H,et al.Precise manipulation of chromosomes in vivo enables genome-wide codon replacement[J].Science,2011,333(6040):348-353.
[20]Auesukaree C,Koedrith P,Saenpayavai P,et al.Characterization and gene expression profiles of thermotolerant Saccharomyces cerevisiaeisolatesfrom Thai fruits[J].J Biosci Bioeng,2012,114:144-149.
[21]Sridhar M,Sree N K,Rao L V.Effect of UV radiation on thermotolerance,ethanol tolerance and osmotolerance of Saccharomyces cerevisiae VS1and VS3 strains[J].Bioresource Technology,2002,83(3):199-202.
[22]Cha Y L,Ana G H,Yang J,et al.Bioethanol production from Miscanthus using thermotolerant Saccharomyces cerevisiaembc 2 isolated from the respiration-deficient mutants[J].Renew Energ,2015,80:259-265.
[23]Shen Naikun(申乃坤),Wang Qingyan(王青艳),Qin Yan(秦艳),et al.Breeding of a thermotolerant and high ethanol-producing Saccharomyces cerevisiae strain by UV-NTG composite protoplast mutagenesis[J].Liquor-Making Science&Technology(酿造科技),2011,203(5):23-26.
[24]Geng Xiaolin(耿笑林),Wang Qinhong(王钦宏),Song Andong(宋安东).Adaptive evolution for screening thermo-tolerant,xylitolproducing Candida maltosa[J].China Brewing(中国酿造),2013,1:36-39.
[25]Caspeta L,Nielsen J.Thermotolerant yeast strains adapted by laboratory evolution show trade-off at ancestral temperatures and preadaptation to other stresses[J].mBio,2015,6(4):e00431.
[26]Satomura A,Miura N,Kuroda K,et al.Reconstruction of thermotolerant yeast by one-point mutation identified through whole-genome analyses of adaptively-evolved strains[J].Scientific Reports,2016,6:23157.
[27]Zheng P,Liu M,Liu XD,et al.Genome shuffling improves thermotolerance and glutamic acid production of Corynebacteria glutamicum[J].World Journal of Microbiology&Biotechnology,2012,28(3):1035-1043.
[28]Wang Hao(王灏),Wang Hang(王航),Meng Chun(孟春),et al.Study of breeding Saccharomyces cerevisiae with improved temperature and ethanol tolerance by genome shuffling[J].Microbiology China(微生物学通报),2007,34(4):705-708.
[29]Lu Y,ChengY F,He X P,et al.Improvement of robustness and ethanol production of ethanologenic Saccharomyces cerevisiaeunder co-stress of heat and inhibitors[J].J Ind Microbiol Biotechnol,2012,39:73-80.
[30]Chen Y,Sheng J,Jiang T,et al.Transcriptional profiling reveals molecular basis and novel genetic targets for improved resistance to multiple fermentation inhibitors in Saccharomyces cerevisiae[J].Biotechnology for Biofuels,2016,9:9.
[31]Yang W,Zhang J,Wang H,et al.AngiotensinⅡdownregulates catalase expression and activity in vascular adventitial fibroblasts through an AT1R/ERK1/2-dependent pathway[J].Mol Cell Biochem,2011,358(1/2):21-29.
[32]Zhao Xinqing(赵心清),Jiang Rujiao(姜如娇),Li Ning(李宁),et al.Improving ethanol tolerance of Saccharomyces cerevisiae industrial strain by directed evolution of SPT3[J].Chin J Biotech(生物工程学报),2010,26(2):159-164.
[33]Hiroyuki H,Mai M,Hiroshi T.Enhancement of stress tolerance in Saccharomyces cerevisiaeby overexpression of ubiquitin ligase Rsp5and ubiquitin-conjugating enzymes[J].Biosci Biotechnol Biochem,2006,70:2762-2765.
[34]Shahsavarani H,Sugiyamaa M,Kanekoa Y,et al.Superior thermotolerance of Saccharomyces cerevisiae for efficient bioethanol fermentation can be achieved by overexpression of RSP5ubiquitin ligase[J].Biotechnol Adv,2012,30:1289-1300.
[35]Qiu Z,Deng Z,Tan H,et al.Engineering the robustness of Saccharomyces cerevisiae by introducing bifunctional glutathione synthase gene[J].J Ind Microbiol Biotechnol,2015,42:537-542.
[36]Luhe A L,Tan L,Wu J,et al.Increase of ethanol tolerance of Saccharomyces cerevisiae by error-prone whole genome amplification[J].Biotechnology Letters,2011,33(5):1007-1011.
[37]Kitichantaropas Y,Boonchird C,Sugiyama M,et al.Cellular mechanisms contributing to multiple stress tolerance in Saccharomyces cerevisiae strains with potential use in high-temperature ethanol fermentation[J].AMB Express,2016,6(1):107.
[38]Thompson O A,Hawkins G M,Gorsich S W,et al.Phenotypic characterization and comparative transcriptomics of evolved Saccharomyces cerevisiae strains with improved tolerance to lignocellulosic derived inhibitors[J].Biotechnology for Biofuels,2016,9:200.
[39]Doudna J A,Charpentier E.Genome editing.The new frontier of genome engineering with CRISPR-Cas9[J].Science,2014,346(6213):1258096.
[40]Rauscher B,Heigwer F,Breinig M,et al.Genome CRISPR a database for high-throughput CRISPR/Cas9 screens[J].Nucleic Acids Research,2017,45(Database issue):D679-D686.
[41]Wang H,Yang H,Shivalila C S,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[J].Cell,2013,153(4):910-918.
[42]Ryan O W,Skerker J M,Maurer M J,et al.Selection of chromosomal DNA libraries using a multiplex CRISPR system[J].ELife,2014,3:e03703.
[43]Zehua B,Mohammad H R,Pu X,et al.Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision[J].Nature Biotechnology,2018.DOI:10.1038/nbt.4132.
[44]Snoek T,Picca Nicolino M,Van den Bremt S,et al.Large-scale robot-assisted genome shuffling yields industrial Saccharomyces cerevisiae yeasts with increased ethanol tolerance[J].Biotechnology for Biofuels,2015,8:32.