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
|
摘要
木质纤维素预处理过程中产生的有毒副产物严重影响了纤维素乙醇发酵,提高酿酒酵母抑制物耐受性是提高纤维素乙醇发酵效率的有效方法。文中通过过表达LCB4基因,研究了重组菌株S288C-LCB4在乙酸、糠醛和香草醛胁迫下的细胞生长和乙醇发酵性能。结果表明,LCB4过表达菌株在分别含有10 g/L乙酸、1.5 g/L糠醛和1 g/L香草醛的平板中生长均优于对照菌株;在分别含有10 g/L乙酸、3 g/L糠醛和2 g/L香草醛的液体乙醇发酵过程中,重组菌株S288C-LCB4乙醇发酵产率分别为0.85 g/(L·h)、0.76 g/(L·h)和1.12 g/(L·h),比对照菌株提高了34.9%、85.4%和330.8%;且糠醛和香草醛胁迫下发酵时间分别缩短了30 h和44 h。根据发酵终点发酵液代谢物分析发现重组菌株比对照菌株产生了更多甘油、海藻糖和琥珀酸,这些物质有利于增强菌株的抑制物耐受性。综上所述,LCB4基因过表达可显著提高酿酒酵母S288C在乙酸、糠醛和香草醛胁迫下的乙醇发酵性能。
By-products released from pretreatment process of lignocellulose seriously hinder the development of cellulosic fuel ethanol. Therefore, the great way to increase the efficiency of cellulosic ethanol production is improvement of Saccharomyces cerevisiae tolerance to these inhibitors. In this work, the effects of LCB4 gene overexpression on cell growth and ethanol fermentation in S. cerevisiae S288 C under acetic acid, furfural and vanillin stresses were studied. Compared to the control strain S288 C-HO, the recombinant strain S288 C-LCB4 grew better on YPD solid medium containing 10 g/L acetic acid, 1.5 g/L furfural and 1 g/L vanillin. Ethanol yields of recombinant strain S288 C-LCB4 were 0.85 g/(L·h), 0.76 g/(L·h) and 1.12 g/(L·h) when 10 g/L acetic acid, 3 g/L furfural and 2 g/L vanillin were supplemented into the fermentation medium respectively, which increased by 34.9%, 85.4% and 330.8% than the control strain S288 C-HO. Meanwhile, ethanol fermentation time was reduced by 30 h and 44 h under furfural and vanillin stresses respectively. Further metabolites analysis in fermentation broth showed that the recombinant strain produced more protective compounds, such as glycerol, trehalose and succinic acid, than the control strain, which could be the reason for enhancing strain tolerance to these inhibitors from pretreatment process of lignocellulose. The results indicated that overexpression of LCB4 gene could significantly improve ethanol fermentation in S. cerevisiae S288 C under acetic acid, furfural and vanillin stresses.
By-products released from pretreatment process of lignocellulose seriously hinder the development of cellulosic fuel ethanol. Therefore, the great way to increase the efficiency of cellulosic ethanol production is improvement of Saccharomyces cerevisiae tolerance to these inhibitors. In this work, the effects of LCB4 gene overexpression on cell growth and ethanol fermentation in S. cerevisiae S288 C under acetic acid, furfural and vanillin stresses were studied. Compared to the control strain S288 C-HO, the recombinant strain S288 C-LCB4 grew better on YPD solid medium containing 10 g/L acetic acid, 1.5 g/L furfural and 1 g/L vanillin. Ethanol yields of recombinant strain S288 C-LCB4 were 0.85 g/(L·h), 0.76 g/(L·h) and 1.12 g/(L·h) when 10 g/L acetic acid, 3 g/L furfural and 2 g/L vanillin were supplemented into the fermentation medium respectively, which increased by 34.9%, 85.4% and 330.8% than the control strain S288 C-HO. Meanwhile, ethanol fermentation time was reduced by 30 h and 44 h under furfural and vanillin stresses respectively. Further metabolites analysis in fermentation broth showed that the recombinant strain produced more protective compounds, such as glycerol, trehalose and succinic acid, than the control strain, which could be the reason for enhancing strain tolerance to these inhibitors from pretreatment process of lignocellulose. The results indicated that overexpression of LCB4 gene could significantly improve ethanol fermentation in S. cerevisiae S288 C under acetic acid, furfural and vanillin stresses.
引文
[1]Zhao XQ,Zi LH,Bai FW,et al.Bioethanol from lignocellulosic biomass.Adv Biochem Eng Biotechnol,2012,128(1):25-51.
[2]Larsson S,Palmqvist E,Hahn-H?gerdal B,et al.The generation of fermentation inhibitors during dilute acid hydrolysis of softwood.Enzyme Microb Technol,1999,24(3/4):151-159.
[3]J?nsson LJ,Alriksson B,Nilvebrant NO.Bioconversion of lignocellulose:inhibitors and detoxification.Biotechnol Biofuels,2013,6:16.
[4]Almeida JR,Modig T,Petersson A,et al.Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae.J Chem Technol Biotechnol,2007,82(4):340-349.
[5]Parawira W,Tekere M.Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production:review.Crit Rev Biotechnol,2011,31(1):20-31.
[6]Do?an A,Demirci S,Aytekin A?,et al.Improvements of tolerance to stress conditions by genetic engineering in Saccharomyces cerevisiae during ethanol production.Appl Biochem Biotechnol,2014,174(1):28-42.
[7]Kim D,Hahn JS.Roles of the Yap1 Transcription factor and antioxidants in Saccharomyces cerevisiae’s tolerance to furfural and 5-hydroxymethylfurfural,which function as thiol-reactive electrophiles generating oxidative stress.Appl Environ Microbiol,2013,79(16):5069-5077.
[8]Liu ZL,Ma MG,Song MZ.Evolutionarily engineered ethanologenic yeast detoxifies lignocellulosic biomass conversion inhibitors by reprogrammed pathways.Mol Genet Genom,2009,282(3):233-244.
[9]Nagiec MM,Skrzypek M,Nagiec EE,et al.The LCB4(YOR171c)and LCB5(YLR260w)genes of Saccharomyces encode sphingoid long chain base kinases.J Biol Chem,1998,273(31):19437-19442.
[10]Hait NC,Fujita K,Lester RL,et al.Lcb4p sphingoid base kinase localizes to the Golgi and late endosomes.FEBS Lett,2002,532(1/2):97-102.
[11]Funato K,Lombardi R,Vallée B,et al.Lcb4p is a key regulator of ceramide synthesis from exogenous long chain sphingoid base in Saccharomyces cerevisiae.J Biol Chem,2003,278(9):7325-7334.
[12]Rivera J,Proia RL,Olivera A.The alliance of sphingosine-1-phosphate and its receptors in immunity.Nat Rev Immunol,2008,8(10):753-763.
[13]Olivera A,Kohama T,Edsall T,et al.Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival.J Cell Biol,1999,147(3):545-558.
[14]Pyne S,Pyne NJ.Sphingosine-1-phosphate signaling in mammalian cells.Biochem J,2000,349(2):385-402.
[15]Jenkins GM,Hannun YA.Role for de Novo sphingoid base biosynthesis in the heat-induced transient cell cycle arrest of Saccharomyces cerevisiae.J Biol Chem,2001,276(11):8574-8581.
[16]Ferguson-Yankey SR,Skrzypek MS,Lester RL,et al.Mutant analysis reveals complex regulation of sphingolipid long chain base phosphates and long chain bases during heat stress in yeast.Yeast,2002,19(7):573-586.
[17]Fang Q,Zhang MM,Chen HQ,et al.Improvement of acetic acid tolerance of Saccharomyces cerevisiae by overexpressing glutaredoxin encoding gene GRX5.CIESC J,2015,66(4):1434-1439(in Chinese).方青,张明明,陈洪奇,等.过表达谷氧还蛋白基因GRX5提高酿酒酵母乙酸耐性.化工学报,2015,66(4):1434-1439.
[18]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.Appl Energy,2012,100:33-40.
[19]Voth WP,Richards JD,Shaw JM,et al.Yeast vectors for integration at the HO locus.Nucleic Acids Res,2001,29(12):E59.
[20]Wei XW,Ma C,Xiong L,et al.Effect of vacuolar proteinase B on high temperature ethanol fermentation of Saccharomyces cerevisiae.Microbiol China,2015,42(10):1841-1846(in Chinese).魏小文,马翠,熊亮,等.液泡蛋白酶B对酿酒酵母高温乙醇发酵效率的影响.微生物学通报,2015,42(10):1841-1846.
[21]Fang Q.Effect of overexpression of key enzyme genes on stress tolerance of Saccharomyces cerevisiae[D].Dalian:Dalian University of Technology,2016(in Chinese).方青.过表达关键酶基因对酿酒酵母胁迫耐性的影响[D].大连:大连理工大学,2016.
[22]Teste MA,Duquenne M,Fran?ois JM,et al.Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae.BMC Mol Biol,2009,10:99.
[23]Geng P,Zhang L,Shi GY.Omics analysis of acetic acid tolerance in Saccharomyces cerevisiae.World JMicrobiol Biotechnol,2017,33(5):94.
[24]Mira NP,Palma M,Guerreiro JF,et al.Genome-wide identification of Saccharomyces cerevisiae genes required for tolerance to acetic acid.Microb Cell Factor,2010,9:79.
[25]Wang X,Li BZ,Ding MZ,et al.Metabolomic analysis reveals key metabolites related to the rapid adaptation of Saccharomyce cerevisiae to multiple inhibitors of furfural,acetic acid,and phenol.OMICS,2013,17(3):150-159.
[26]Wiemken A.Trehalose in yeast,stress protectant rather than reserve carbohydrate.Antonie van Leeuwenhoek,1990,58(3):209-217.
[27]Yoshiyama Y,Tanaka K,Yoshiyama K,et al.Trehalose accumulation enhances tolerance of Saccharomyces cerevisiae to acetic acid.J Biosci Bioeng,2015,119(2):172-175.
[28]Cao J,Barbosa JM,Singh NK,et al.GABA shunt mediates thermotolerance in Saccharomyces cerevisiae,by reducing reactive oxygen production.Yeast,2013,30(4):129-144.
[29]Voit EO.Biochemical and genomic regulation of the trehalose cycle in yeast:review of observations and canonical model analysis.J Theoret Biol,2003,223(1):55-78.
[30]Wang XN,Liang ZZ,Jin H,et al.Identification and functional evaluation of the reductases and dehydrogenases from Saccharomyces cerevisiae involved in vanillin resistance.BMC Biotechnol,2016,16:31.
[2]Larsson S,Palmqvist E,Hahn-H?gerdal B,et al.The generation of fermentation inhibitors during dilute acid hydrolysis of softwood.Enzyme Microb Technol,1999,24(3/4):151-159.
[3]J?nsson LJ,Alriksson B,Nilvebrant NO.Bioconversion of lignocellulose:inhibitors and detoxification.Biotechnol Biofuels,2013,6:16.
[4]Almeida JR,Modig T,Petersson A,et al.Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae.J Chem Technol Biotechnol,2007,82(4):340-349.
[5]Parawira W,Tekere M.Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production:review.Crit Rev Biotechnol,2011,31(1):20-31.
[6]Do?an A,Demirci S,Aytekin A?,et al.Improvements of tolerance to stress conditions by genetic engineering in Saccharomyces cerevisiae during ethanol production.Appl Biochem Biotechnol,2014,174(1):28-42.
[7]Kim D,Hahn JS.Roles of the Yap1 Transcription factor and antioxidants in Saccharomyces cerevisiae’s tolerance to furfural and 5-hydroxymethylfurfural,which function as thiol-reactive electrophiles generating oxidative stress.Appl Environ Microbiol,2013,79(16):5069-5077.
[8]Liu ZL,Ma MG,Song MZ.Evolutionarily engineered ethanologenic yeast detoxifies lignocellulosic biomass conversion inhibitors by reprogrammed pathways.Mol Genet Genom,2009,282(3):233-244.
[9]Nagiec MM,Skrzypek M,Nagiec EE,et al.The LCB4(YOR171c)and LCB5(YLR260w)genes of Saccharomyces encode sphingoid long chain base kinases.J Biol Chem,1998,273(31):19437-19442.
[10]Hait NC,Fujita K,Lester RL,et al.Lcb4p sphingoid base kinase localizes to the Golgi and late endosomes.FEBS Lett,2002,532(1/2):97-102.
[11]Funato K,Lombardi R,Vallée B,et al.Lcb4p is a key regulator of ceramide synthesis from exogenous long chain sphingoid base in Saccharomyces cerevisiae.J Biol Chem,2003,278(9):7325-7334.
[12]Rivera J,Proia RL,Olivera A.The alliance of sphingosine-1-phosphate and its receptors in immunity.Nat Rev Immunol,2008,8(10):753-763.
[13]Olivera A,Kohama T,Edsall T,et al.Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival.J Cell Biol,1999,147(3):545-558.
[14]Pyne S,Pyne NJ.Sphingosine-1-phosphate signaling in mammalian cells.Biochem J,2000,349(2):385-402.
[15]Jenkins GM,Hannun YA.Role for de Novo sphingoid base biosynthesis in the heat-induced transient cell cycle arrest of Saccharomyces cerevisiae.J Biol Chem,2001,276(11):8574-8581.
[16]Ferguson-Yankey SR,Skrzypek MS,Lester RL,et al.Mutant analysis reveals complex regulation of sphingolipid long chain base phosphates and long chain bases during heat stress in yeast.Yeast,2002,19(7):573-586.
[17]Fang Q,Zhang MM,Chen HQ,et al.Improvement of acetic acid tolerance of Saccharomyces cerevisiae by overexpressing glutaredoxin encoding gene GRX5.CIESC J,2015,66(4):1434-1439(in Chinese).方青,张明明,陈洪奇,等.过表达谷氧还蛋白基因GRX5提高酿酒酵母乙酸耐性.化工学报,2015,66(4):1434-1439.
[18]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.Appl Energy,2012,100:33-40.
[19]Voth WP,Richards JD,Shaw JM,et al.Yeast vectors for integration at the HO locus.Nucleic Acids Res,2001,29(12):E59.
[20]Wei XW,Ma C,Xiong L,et al.Effect of vacuolar proteinase B on high temperature ethanol fermentation of Saccharomyces cerevisiae.Microbiol China,2015,42(10):1841-1846(in Chinese).魏小文,马翠,熊亮,等.液泡蛋白酶B对酿酒酵母高温乙醇发酵效率的影响.微生物学通报,2015,42(10):1841-1846.
[21]Fang Q.Effect of overexpression of key enzyme genes on stress tolerance of Saccharomyces cerevisiae[D].Dalian:Dalian University of Technology,2016(in Chinese).方青.过表达关键酶基因对酿酒酵母胁迫耐性的影响[D].大连:大连理工大学,2016.
[22]Teste MA,Duquenne M,Fran?ois JM,et al.Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae.BMC Mol Biol,2009,10:99.
[23]Geng P,Zhang L,Shi GY.Omics analysis of acetic acid tolerance in Saccharomyces cerevisiae.World JMicrobiol Biotechnol,2017,33(5):94.
[24]Mira NP,Palma M,Guerreiro JF,et al.Genome-wide identification of Saccharomyces cerevisiae genes required for tolerance to acetic acid.Microb Cell Factor,2010,9:79.
[25]Wang X,Li BZ,Ding MZ,et al.Metabolomic analysis reveals key metabolites related to the rapid adaptation of Saccharomyce cerevisiae to multiple inhibitors of furfural,acetic acid,and phenol.OMICS,2013,17(3):150-159.
[26]Wiemken A.Trehalose in yeast,stress protectant rather than reserve carbohydrate.Antonie van Leeuwenhoek,1990,58(3):209-217.
[27]Yoshiyama Y,Tanaka K,Yoshiyama K,et al.Trehalose accumulation enhances tolerance of Saccharomyces cerevisiae to acetic acid.J Biosci Bioeng,2015,119(2):172-175.
[28]Cao J,Barbosa JM,Singh NK,et al.GABA shunt mediates thermotolerance in Saccharomyces cerevisiae,by reducing reactive oxygen production.Yeast,2013,30(4):129-144.
[29]Voit EO.Biochemical and genomic regulation of the trehalose cycle in yeast:review of observations and canonical model analysis.J Theoret Biol,2003,223(1):55-78.
[30]Wang XN,Liang ZZ,Jin H,et al.Identification and functional evaluation of the reductases and dehydrogenases from Saccharomyces cerevisiae involved in vanillin resistance.BMC Biotechnol,2016,16:31.