过表达谷氧还蛋白基因GRX5提高酿酒酵母乙酸耐性
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摘要
利用可再生的纤维素原料生产燃料乙醇是国内外研究的热点。但纤维素原料一些预处理过程产生的乙酸对酿酒酵母细胞生长和乙醇发酵产生强烈抑制,因此,提高酿酒酵母细胞的乙酸耐受性是提高纤维素乙醇发酵效率的重要手段。本文研究了谷氧还蛋白家族中GRX5p的编码基因的过表达对酿酒酵母在乙酸胁迫条件下细胞生长和发酵性能的影响。结果表明,过表达GRX5的重组菌株在含有5 g·L1乙酸的平板中生长优于对照菌株;在含有5 g·L1乙酸的培养基中进行乙醇发酵,过表达GRX5的重组菌株可在48 h基本消耗培养基中所有的葡萄糖,发酵周期比对照菌株缩短了12 h。过表达GRX5菌株的乙醇生产强度为0.897 g·L1·h1,比对照提高了28.5%。代谢物分析结果表明,过表达GRX5的重组菌株可产生更多的保护性物质海藻糖和甘油,有利于增强菌株胁迫耐受性。
Fuel ethanol production using renewable cellulosic materials has attracted widespread attention by researchers. However, acetic acid released from the pretreatment process exerts severe inhibition on yeast cell growth and ethanol fermentation. Therefore, improvement of acid tolerance of industrial Saccharomyces cerevisiae benefits efficient cellulosic ethanol production. In this work, glutaredoxin encoding gene GRX5 was overexpressed in S. cerevisiae, and it was found that the GRX5 overexpression strain grew better than that of the control strain in the plates containing 5 g·L1 acetic acid. When ethanol fermentation in the presence of 5 g·L1acetic acid was evaluated, it was found that the GRX5 overexpression strain consumed all glucose in the broth within 48 h, 12 h shorter than that of the control strain. Ethanol productivity of the GRX5 overexpressed strain was determined to be 0.897 g·L1·h1, with a 28.5% increase than that of the control strain. Analysis of the key metabolites showed that the GRX5 overexpression strain produced higher concentration of trehalose and glycerol, which might explain its improved cell viability in the presence of acetic acid stress.
Fuel ethanol production using renewable cellulosic materials has attracted widespread attention by researchers. However, acetic acid released from the pretreatment process exerts severe inhibition on yeast cell growth and ethanol fermentation. Therefore, improvement of acid tolerance of industrial Saccharomyces cerevisiae benefits efficient cellulosic ethanol production. In this work, glutaredoxin encoding gene GRX5 was overexpressed in S. cerevisiae, and it was found that the GRX5 overexpression strain grew better than that of the control strain in the plates containing 5 g·L1 acetic acid. When ethanol fermentation in the presence of 5 g·L1acetic acid was evaluated, it was found that the GRX5 overexpression strain consumed all glucose in the broth within 48 h, 12 h shorter than that of the control strain. Ethanol productivity of the GRX5 overexpressed strain was determined to be 0.897 g·L1·h1, with a 28.5% increase than that of the control strain. Analysis of the key metabolites showed that the GRX5 overexpression strain produced higher concentration of trehalose and glycerol, which might explain its improved cell viability in the presence of acetic acid stress.
引文
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[12]Kim I,Kim Y,Yoon H.Glutathione reductase from Oryza sativa increases acquired tolerance to abiotic stresses in a genetically modified Saccharomyces cerevisiae strain[J].Journal of Microbiology and Biotechnology,2012,22(11):1557-1567
[13]Rodríguez-Manzaneque M T,Ros J,Cabiscol E,Sorribas A,Herrero E.Grx5 Glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae[J].Molecular and Cellular Biology,1999,19(12):8180-8190
[14]Oh Y,Hong S,Yeon J,Cha M,Kim I.Interaction between Saccharomyces cerevisiae glutaredoxin 5 and SPT10 and their in vivo functions[J].Free Radical Biology and Medicine,2012,52(9):1519-1530
[15]He L Y,Zhao X Q,Bai F W.Engineering industrial Saccharomyces cerevisiae strain with the FLO1-derivative gene isolated from the flocculating yeast SPSC01 for constitutive flocculation and fuel ethanol production[J].Applied Energy,2012,100:33-40
[16]Nedjoud G,Fadila K,Mouna A,Zohra G,Sana G.Effect of zinc on growth,metabolism and activity of antioxidant enzymes in the yeast[J].Global Journal of Biodiversity Science and Management,2013,3(2):243-248
[17]Zhang Jina(张吉娜),He Xiuping(何秀萍),Guo Xuena(郭雪娜),Liu Nan(刘楠),Zhang Borun(张博润).Genetically modified industrial breing yeast with high-glutathione and low-diacetyl production[J].Chinese Journal of Biotechnology(生物工程学报),2006,21(6):942-946
[18]Wiemken A.Trehalose in yeast,stress protectant rather than reserve carbohydrate[J].Antonie van Leeuwenhoek,1990,58(3):209-217
[19]Wang X,Li B Z,Ding M Z,Zhang W W,Yuan Y J.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
[20]Zancan P,Sola-Penna M.Trehalose and glycerol stabilize and renature yeast inorganic pyrophosphatase inactivated by very high temperatures[J].Archives of Biochemistry and Biophysics,2005,444:52-60
[2]Xu Guihong(徐桂红),Zhao Xinqing(赵心清),Li Ning(李宁),Bai Fengwu(白凤武).Improvement of acetic acid tolerance of self-flocculating yeast by zinc supplementation[J].CIESC Journal,2012,63(6):1823-1829
[3]Ding M Z,Wang X,Yang Y,Yuan Y J.Metabolomic study of interactive effects of phenol,furfural,and acetic acid on Saccharomyces cerevisiae[J].Omics:a Journal of Integrative Biology,2011,15(10):647-653
[4]Li Hongxing(李洪兴),Zhang Xiaoran(张笑然),Shen Yu(沈煜),Dong Yongsheng(董永胜),Bao Xiaoming(鲍晓明).Inhibitors and their effects on Saccharomyces cerevisiae and relevant countermeasures in bioprocess of ethanol production from lignocellulose–a review[J].Chinese Journal of Biotechnology(生物工程学报),2009,25(9):1321-1328
[5]Zheng D Q,Wu X C,Wang P M,Chi X Q,Tao X L,Li P,Jiang X H,Zhao Y H.Drug resistance marker-aided genome shuffling to improve acetic acid tolerance in Saccharomyces cerevisiae[J].Journal of Industrial Microbiology&Biotechnology,2011,38(3):415-422
[6]Zhao Xinqing(赵心清),Zhang Mingming(张明明),Xu Guihong(徐桂红),Xu Jianren(许建韧),Bai Fengwu(白凤武).Advances in functional genomics studies underlying acetic acid tolerance of Saccharomyces cerevisiae[J].Chinese Journal of Biotechnology(生物工程学报),2014,30(3):368-380
[7]Gaida S M,Al-Hinai M A,Indurthi D C,Nicolaou1 S A,Papoutsakis E T.Synthetic tolerance:three noncoding small RNAs,Dsr A,Arc Z and Rpr A,acting supra-additively against acid stress[J].Nucleic Acids Research,2013,41(18):8726-8737
[8]Graves T,Narendranath N V,Dawson K,Power R.Effect of p H and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash[J].Journal of Industrial Microbiology and Biotechnology,2006,33(6):469-474
[9]Hasunuma T,Sanda T,Yamada R,Yoshimura K,Ishii J,Kondo A.Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae[J].Microbial Cell Factories,2011,10(1):2-13
[10]Rodríguez-Manzaneque M T,Tamarit J,BellG,Ros J,Herrero E.Grx5 is a mitochondrial glutaredoxin required for the activity of iron/sulfur enzymes[J].Molecular Biology of the Cell,2002,13(4):1109-1121
[11]Pérez Gallardo R V,Briones L S,Díaz Pérez A L,Gutiérrez S,Rodríguez Zavala J S,Campos García J.Reactive oxygen species production induced by ethanol in Saccharomyces cerevisiae increases because of a dysfunctional mitochondrial iron-sulfur cluster assembly system[J].FEMS Yeast Research,2013,13(8):804-819
[12]Kim I,Kim Y,Yoon H.Glutathione reductase from Oryza sativa increases acquired tolerance to abiotic stresses in a genetically modified Saccharomyces cerevisiae strain[J].Journal of Microbiology and Biotechnology,2012,22(11):1557-1567
[13]Rodríguez-Manzaneque M T,Ros J,Cabiscol E,Sorribas A,Herrero E.Grx5 Glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae[J].Molecular and Cellular Biology,1999,19(12):8180-8190
[14]Oh Y,Hong S,Yeon J,Cha M,Kim I.Interaction between Saccharomyces cerevisiae glutaredoxin 5 and SPT10 and their in vivo functions[J].Free Radical Biology and Medicine,2012,52(9):1519-1530
[15]He L Y,Zhao X Q,Bai F W.Engineering industrial Saccharomyces cerevisiae strain with the FLO1-derivative gene isolated from the flocculating yeast SPSC01 for constitutive flocculation and fuel ethanol production[J].Applied Energy,2012,100:33-40
[16]Nedjoud G,Fadila K,Mouna A,Zohra G,Sana G.Effect of zinc on growth,metabolism and activity of antioxidant enzymes in the yeast[J].Global Journal of Biodiversity Science and Management,2013,3(2):243-248
[17]Zhang Jina(张吉娜),He Xiuping(何秀萍),Guo Xuena(郭雪娜),Liu Nan(刘楠),Zhang Borun(张博润).Genetically modified industrial breing yeast with high-glutathione and low-diacetyl production[J].Chinese Journal of Biotechnology(生物工程学报),2006,21(6):942-946
[18]Wiemken A.Trehalose in yeast,stress protectant rather than reserve carbohydrate[J].Antonie van Leeuwenhoek,1990,58(3):209-217
[19]Wang X,Li B Z,Ding M Z,Zhang W W,Yuan Y J.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
[20]Zancan P,Sola-Penna M.Trehalose and glycerol stabilize and renature yeast inorganic pyrophosphatase inactivated by very high temperatures[J].Archives of Biochemistry and Biophysics,2005,444:52-60