关键基因过表达提高酿酒酵母抑制剂耐受性及乙醇发酵性能
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摘要
木质纤维素预处理过程中会产生多种抑制物,抑制酿酒酵母细胞生长及乙醇发酵性能,为挖掘耐性基因、构建新的菌株,进一步提高酿酒酵母对这些抑制物的胁迫耐受性,研究在硫酸锌添加条件下转录组学分析过程中筛选到的可能关键基因ADE17、SSZ1、SET5、PPR1、OGG1和YKL222C过表达对酿酒酵母环境胁迫耐受性的影响.结果显示,不同基因过表达对酿酒酵母在多种抑制物胁迫条件下生长性能的影响不同,其中ADE17过表达对菌株在乙酸、糠醛、苯酚、丙酸和氯化钠胁迫条件下的生长提升最显著,而OGG1和SSZ1过表达对菌株生长的影响相对较弱.进一步对菌株进行驯化,在混合抑制物条件下驯化得到的BADE17-2和BADE17-4菌株延滞期比对照菌株BADE17缩短23 h.上述研究表明,硫酸锌添加从多方面影响了酿酒酵母耐受性,且关键基因过表达对不同环境胁迫条件具有多样性的影响,并且通过基因过表达和驯化方式结合可进一步提高酿酒酵母环境胁迫耐受性,提高纤维素乙醇发酵效率.
Various toxic inhibitors are released during the pretreatment of lignocellulosic biomass, and inhibit cell growth and ethanol production of Saccharomyces cerevisiae. Therefore, development of yeast strains with improved stress tolerance is of importance to enhance ethanol production efficiency from cellulosic biomass. Multiple genes are involved in stress tolerance, but novel genes remain to be explored from omics analysis. In our previous studies, zinc sulfate has been shown to improve the acetic acid tolerance of S. cerevisiae, but key genes involved in stress tolerance by zinc sulfate addition are still not clear.Six key genes, including ADE17, SSZ1, SET5, PPR1, OGG1, and YKL222 C, were identified from the transcriptomic analysis of zinc sulfate addition, and these genes were overexpressed in S. cerevisiae. Stress tolerance of the engineered strains was compared, and adaptive evolution was performed to further improve ethanol production. Overexpression of the six genes improved growth ability under acetic acid conditions as well as tolerance to other inhibitory conditions. The six selected genes showed various effects under different stress conditions, and ADE17 showed the strongest stimulation of cell growth under acetic acid, furfural, phenol, propionic acid, and Na Cl stresses, whereas OGG1 and SSZ1 showed the weakest effects. Furthermore, evolutionary engineering was performed with the BADE17 strain overexpressing ADE17, and the results showed that the adapted strains BADE17-2 and BADE17-4 exhibited a significantly shortened lag phase, which was 23 h shorter under multiple stress conditions than that of the control strain carrying the empty plasmid. The results demonstrated that zinc sulfate exerts various influences on yeast stress tolerance, and different key genes are impacted differently. A combination of gene overexpression and evolutionary engineering is an efficient method to improve stress tolerance and cellulosic ethanol production in S. cerevisiae.
Various toxic inhibitors are released during the pretreatment of lignocellulosic biomass, and inhibit cell growth and ethanol production of Saccharomyces cerevisiae. Therefore, development of yeast strains with improved stress tolerance is of importance to enhance ethanol production efficiency from cellulosic biomass. Multiple genes are involved in stress tolerance, but novel genes remain to be explored from omics analysis. In our previous studies, zinc sulfate has been shown to improve the acetic acid tolerance of S. cerevisiae, but key genes involved in stress tolerance by zinc sulfate addition are still not clear.Six key genes, including ADE17, SSZ1, SET5, PPR1, OGG1, and YKL222 C, were identified from the transcriptomic analysis of zinc sulfate addition, and these genes were overexpressed in S. cerevisiae. Stress tolerance of the engineered strains was compared, and adaptive evolution was performed to further improve ethanol production. Overexpression of the six genes improved growth ability under acetic acid conditions as well as tolerance to other inhibitory conditions. The six selected genes showed various effects under different stress conditions, and ADE17 showed the strongest stimulation of cell growth under acetic acid, furfural, phenol, propionic acid, and Na Cl stresses, whereas OGG1 and SSZ1 showed the weakest effects. Furthermore, evolutionary engineering was performed with the BADE17 strain overexpressing ADE17, and the results showed that the adapted strains BADE17-2 and BADE17-4 exhibited a significantly shortened lag phase, which was 23 h shorter under multiple stress conditions than that of the control strain carrying the empty plasmid. The results demonstrated that zinc sulfate exerts various influences on yeast stress tolerance, and different key genes are impacted differently. A combination of gene overexpression and evolutionary engineering is an efficient method to improve stress tolerance and cellulosic ethanol production in S. cerevisiae.
引文
1 Jsson LJ,Martín C.Pretreatment of lignocellulose:Formation of inhibitory by-products and strategies for minimizing their effects[J].Bioresour Technol,2016,199:103-112
2 Palmqvist E,Hahn-H Gerdal BR.Fermentation of lignocellulosic hydrolysates II:inhibitors and mechanisms of inhibition[J].Bioresour Technol,2000,74(1):25-33
3 Zhao XQ,Xiong L,Zhang MM,Bai FW.Towards efficient bioethanol production from agricultural and forestry residues:exploration of unique natural microorganisms in combination with advanced strain engineering[J].Bioresour Technol,2016,215:84-91
4 Mira NP,Palma M,Guer reiro J F,Sa-Cor reia I.Genome-wide identification of Saccharomyces cerevisiae genes required for tolerance to acetic acid[J].Microb Cell Fact,2010,9(1):1-13
5 赵心清,张明明,徐桂红,许建韧,白凤武.酿酒酵母乙酸耐性分子机制的功能基因组进展[J].生物工程学报,2014,30(3):368-380[Zhao XQ,Zhang MM,Xu GH,Xu JR,Bai FW.Advances in functional genomics studies underlying acetic acid tolerance of Saccharomyces cerevisiae[J].Chin J Biotechnol,2014,30(3):368-380]
6 Wang X,Bai X,Chen DF,Chen FZ,Li BZ,Yuan YJ.Increasing proline and myo-inositol improves tolerance of Saccharomyces cerevisiae to the mixture of multiple lignocellulose-derived inhibitors[J].Biotechnol Biofuel,2015,8(1):1-13
7 Chen YY,Sheng JY,Jiang T,Stevens J,Feng XY,Wei N.Transcriptional profiling reveals molecular basis and novel genetic targets for improved resistance to multiple fermentation inhibitors in Saccharomyces cerevisiae[J].Biotechnol Biofuel,2016,9(9):1-18
8 Tanaka K,Ishii Y,Ogawa J,Shima J.Enhancement of acetic acid tolerance in Saccharomyces cerevisiae by overexpression of the HAA1gene,encoding a transcriptional activator[J].Appl Environ Microb,2012,78(22):8161-8163
9 徐桂红,赵心清,李宁,白凤武.锌离子提高絮凝酵母乙酸胁迫耐受性[J].化工学报,2012,63(6):1823-1829[Xu GH,Zhao XQ,Li N,Bai FW.Improvement of acetic acid tolerance of self-floculating yeast by zinc supplementation[J].CIESC J,2012,63(6):1823-1829]
10 Wan C,Zhang MM,Fang Q,Xiong L,Hasunuma T,Bai FW,Kondo A.The impact of zinc sulfate addition on the dynamic metabolic profiling of Saccharomyces cerevisiae subjected to long term acetic acid stress treatment and identification of key metabolites involved in the antioxidant effect of zinc[J].Metallomics,2015,7(2):322-332
11 Zhang MM,Zhang KY,Mehmood MA,Zhao ZB,Bai FW,Zhao XQ.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].Bioresour Technol,2017,Doi:10.1016/j.biortech.2017.05.191
12 张明明,万青青,张克俞,熊亮,赵心清,白凤武.过表达分支酸歧化酶编码基因ARO7对酿酒酵母抑制物耐受性的影响[J].应用与环境生物学报,2016,22(2):201-205[Zhang MM,Wan QQ,Zhang KY,Xiong L,Zhao XQ,Bai FW.Effect of overexpression of chorismate mutase encoding gene ARO7 on the inhibitor tolerance of Saccharomyces cerevisiae[J].Chin J Appl Environ Biol,2016,22(2):201-205]
13 方青,张明明,陈洪奇,熊亮,赵心清,白凤武.过表达谷氧还蛋白基因GRX5提高酿酒酵母乙酸耐性[J].化工学报,2015,4:1434-1439[Fang Q,Zhang MM,Chen HQ,Xiong L,Zhao XQ,Bai FW.Improvement of acetic acid tolerance of Saccharomyces cerevisiae byoverexpressing glutaredoxin encoding gene GRX5[J].CIESC J,2015,4:1434-1439]
14 He LY,Zhao XQ,Bai FW,Engineering industrial Saccharomyces cerevisiae strain with the FLO1-derivative gene isolated from the flocculating yeast SPSC01 for constitutive flocculation and fuel ethanol production[J].Appl Energy,2012,100:33-40
15 Zhang MM,Zhao XQ,Cheng C,Bai FW.Improved growth and ethanol fermentation of Saccharomyces cerevisiae in the presence of acetic acid by overexpression of SET5 and PPR1[J].Biotechnol J,2015,10(12):1903-1911
16 Zhao XQ,Li Q,He LY,Bai FW.Exploration of a natural reservoir offlocculating genes from various Saccharomyces cerevisiae strains and improved ethanol fermentation using stable genetically engineered flocculating yeast strains[J].Process Biochem,2012,47(11):1612-1619
17 陈洪奇,于欣水,张明明,白凤武,赵心清.硫酸锌添加对酿酒酵母乙酸胁迫条件下全局基因转录的影响[J].微生物学通报,2017,44(6):1312-1321[Cheng HQ,Yu XS,Zhang MM,Bai FW,Zhao XQ.Impact of zinc sulfate supplementation on global gene expressionprofiling of Saccharomyces cerevisiae in response to acetic acid stress[J].Microbiol Chin,2017,44(6):1312-1321]
18 徐桂红.锌对自絮凝酵母乙酸胁迫的保护作用及分子机理研究[D].大连:大连理工大学,2012[Xu GH.Protective effect of zinc on the selfflocculating yeast against acetic acid stress and studies of underlying molecular mechanisms[D].Dalian:Dalian Technology University,2012]
19 Lee Y,Nasution O,Choi E,Kim W.,Choi W.Transcriptome analysis of acetic-acid-treated yeast cells identifies a large set of genes whose overexpression or deletion enhances acetic acid tolerance[J].Appl Microbiol Biotechnol,2015,99(15):6391-6403
20 Kim S K,Jin Y S,Choi I G,Park YC,Seo JH.Enhanced tolerance of Saccharomyces cerevisiae to multiple lignocellulose-derived inhibitors through modulation of spermidine contents[J].Metab Eng,2015,29:46-55
21 Conz C,Otto H,Peisker K,Gautschi M,Wfle T,Mayer MP,Rospert S.Functional characterization of the atypical Hsp70 subunit of yeast ribosome-associated complex[J].J Biol Chem,2007,282(47):33977-33984
22 Hohmann S.Control of high osmolarity signalling in the yeast Saccharomyces cerevisiae[J].FEBS Lett,2009,583(24):4025-4029
23 Pahlman AK,Granath K,Ansell R,Hohmann S,Adler L.The yeast glycerol 3-phosphatases GPP1P and GPP2P are required for glycerol biosynthesis and differentially involved in the cellular responses to osmotic,anaerobic,and oxidative stress[J].J Biol Chem,2001,276(5):3555-3563
24 Almario MP,Reyes LH,Kao KC.Evolutionary engineering of Saccharomyces cerevisiae for enhanced tolerance to hydrolysates of lignocellulosic biomass[J].Biotechnol Bioeng,2013,110(10):2616-2623
2 Palmqvist E,Hahn-H Gerdal BR.Fermentation of lignocellulosic hydrolysates II:inhibitors and mechanisms of inhibition[J].Bioresour Technol,2000,74(1):25-33
3 Zhao XQ,Xiong L,Zhang MM,Bai FW.Towards efficient bioethanol production from agricultural and forestry residues:exploration of unique natural microorganisms in combination with advanced strain engineering[J].Bioresour Technol,2016,215:84-91
4 Mira NP,Palma M,Guer reiro J F,Sa-Cor reia I.Genome-wide identification of Saccharomyces cerevisiae genes required for tolerance to acetic acid[J].Microb Cell Fact,2010,9(1):1-13
5 赵心清,张明明,徐桂红,许建韧,白凤武.酿酒酵母乙酸耐性分子机制的功能基因组进展[J].生物工程学报,2014,30(3):368-380[Zhao XQ,Zhang MM,Xu GH,Xu JR,Bai FW.Advances in functional genomics studies underlying acetic acid tolerance of Saccharomyces cerevisiae[J].Chin J Biotechnol,2014,30(3):368-380]
6 Wang X,Bai X,Chen DF,Chen FZ,Li BZ,Yuan YJ.Increasing proline and myo-inositol improves tolerance of Saccharomyces cerevisiae to the mixture of multiple lignocellulose-derived inhibitors[J].Biotechnol Biofuel,2015,8(1):1-13
7 Chen YY,Sheng JY,Jiang T,Stevens J,Feng XY,Wei N.Transcriptional profiling reveals molecular basis and novel genetic targets for improved resistance to multiple fermentation inhibitors in Saccharomyces cerevisiae[J].Biotechnol Biofuel,2016,9(9):1-18
8 Tanaka K,Ishii Y,Ogawa J,Shima J.Enhancement of acetic acid tolerance in Saccharomyces cerevisiae by overexpression of the HAA1gene,encoding a transcriptional activator[J].Appl Environ Microb,2012,78(22):8161-8163
9 徐桂红,赵心清,李宁,白凤武.锌离子提高絮凝酵母乙酸胁迫耐受性[J].化工学报,2012,63(6):1823-1829[Xu GH,Zhao XQ,Li N,Bai FW.Improvement of acetic acid tolerance of self-floculating yeast by zinc supplementation[J].CIESC J,2012,63(6):1823-1829]
10 Wan C,Zhang MM,Fang Q,Xiong L,Hasunuma T,Bai FW,Kondo A.The impact of zinc sulfate addition on the dynamic metabolic profiling of Saccharomyces cerevisiae subjected to long term acetic acid stress treatment and identification of key metabolites involved in the antioxidant effect of zinc[J].Metallomics,2015,7(2):322-332
11 Zhang MM,Zhang KY,Mehmood MA,Zhao ZB,Bai FW,Zhao XQ.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].Bioresour Technol,2017,Doi:10.1016/j.biortech.2017.05.191
12 张明明,万青青,张克俞,熊亮,赵心清,白凤武.过表达分支酸歧化酶编码基因ARO7对酿酒酵母抑制物耐受性的影响[J].应用与环境生物学报,2016,22(2):201-205[Zhang MM,Wan QQ,Zhang KY,Xiong L,Zhao XQ,Bai FW.Effect of overexpression of chorismate mutase encoding gene ARO7 on the inhibitor tolerance of Saccharomyces cerevisiae[J].Chin J Appl Environ Biol,2016,22(2):201-205]
13 方青,张明明,陈洪奇,熊亮,赵心清,白凤武.过表达谷氧还蛋白基因GRX5提高酿酒酵母乙酸耐性[J].化工学报,2015,4:1434-1439[Fang Q,Zhang MM,Chen HQ,Xiong L,Zhao XQ,Bai FW.Improvement of acetic acid tolerance of Saccharomyces cerevisiae byoverexpressing glutaredoxin encoding gene GRX5[J].CIESC J,2015,4:1434-1439]
14 He LY,Zhao XQ,Bai FW,Engineering industrial Saccharomyces cerevisiae strain with the FLO1-derivative gene isolated from the flocculating yeast SPSC01 for constitutive flocculation and fuel ethanol production[J].Appl Energy,2012,100:33-40
15 Zhang MM,Zhao XQ,Cheng C,Bai FW.Improved growth and ethanol fermentation of Saccharomyces cerevisiae in the presence of acetic acid by overexpression of SET5 and PPR1[J].Biotechnol J,2015,10(12):1903-1911
16 Zhao XQ,Li Q,He LY,Bai FW.Exploration of a natural reservoir offlocculating genes from various Saccharomyces cerevisiae strains and improved ethanol fermentation using stable genetically engineered flocculating yeast strains[J].Process Biochem,2012,47(11):1612-1619
17 陈洪奇,于欣水,张明明,白凤武,赵心清.硫酸锌添加对酿酒酵母乙酸胁迫条件下全局基因转录的影响[J].微生物学通报,2017,44(6):1312-1321[Cheng HQ,Yu XS,Zhang MM,Bai FW,Zhao XQ.Impact of zinc sulfate supplementation on global gene expressionprofiling of Saccharomyces cerevisiae in response to acetic acid stress[J].Microbiol Chin,2017,44(6):1312-1321]
18 徐桂红.锌对自絮凝酵母乙酸胁迫的保护作用及分子机理研究[D].大连:大连理工大学,2012[Xu GH.Protective effect of zinc on the selfflocculating yeast against acetic acid stress and studies of underlying molecular mechanisms[D].Dalian:Dalian Technology University,2012]
19 Lee Y,Nasution O,Choi E,Kim W.,Choi W.Transcriptome analysis of acetic-acid-treated yeast cells identifies a large set of genes whose overexpression or deletion enhances acetic acid tolerance[J].Appl Microbiol Biotechnol,2015,99(15):6391-6403
20 Kim S K,Jin Y S,Choi I G,Park YC,Seo JH.Enhanced tolerance of Saccharomyces cerevisiae to multiple lignocellulose-derived inhibitors through modulation of spermidine contents[J].Metab Eng,2015,29:46-55
21 Conz C,Otto H,Peisker K,Gautschi M,Wfle T,Mayer MP,Rospert S.Functional characterization of the atypical Hsp70 subunit of yeast ribosome-associated complex[J].J Biol Chem,2007,282(47):33977-33984
22 Hohmann S.Control of high osmolarity signalling in the yeast Saccharomyces cerevisiae[J].FEBS Lett,2009,583(24):4025-4029
23 Pahlman AK,Granath K,Ansell R,Hohmann S,Adler L.The yeast glycerol 3-phosphatases GPP1P and GPP2P are required for glycerol biosynthesis and differentially involved in the cellular responses to osmotic,anaerobic,and oxidative stress[J].J Biol Chem,2001,276(5):3555-3563
24 Almario MP,Reyes LH,Kao KC.Evolutionary engineering of Saccharomyces cerevisiae for enhanced tolerance to hydrolysates of lignocellulosic biomass[J].Biotechnol Bioeng,2013,110(10):2616-2623