过表达MRP8提高酿酒酵母乙酸耐性及乙醇发酵效率
|
摘要
乙酸是木质纤维素水解液中含量较多的抑制物,因此提高酿酒酵母菌株对乙酸的耐受性有助于提高纤维素乙醇生产效率。本文中,笔者利用基于CRISPR/Cas9系统的基因组编辑技术过表达了酿酒酵母(Saccharomyces cerevisiae)S288c线粒体核糖体蛋白编码基因MRP8,并比较了过表达MRP8的菌株与对照菌株的生长和发酵特性。平板耐性检测发现,MRP8过表达明显提高了菌株的乙酸胁迫耐受性;乙醇发酵结果表明,在4.8 g/L乙酸胁迫条件下,过表达菌株MRP8-3在51 h消耗全部的葡萄糖,发酵时间缩短了25 h,显著优于相同时间的对照菌株。本研究结果为构建高效纤维素乙醇发酵的酿酒酵母菌株提供了新思路。
Acetic acid is one of the major inhibitors derived from the hydrolysis of lignocellulosic biomass,and enhancing the tolerance of Saccharomyces cerevisiae to acetic acid is of great importance for improving the lignocellulosic ethanol production. Here,we overexpressed mitochondrial ribosomal protein encoding gene MRP8 via CRISPR/Cas9-based genome editing in S. cerevisiae S288c,and compared the growth and fermentation performance between the mutants with MRP8 and the wild type.The tolerance to acetic acid was increased by overexpression MRP8 on agar plates. Also,mutant MRP8-3 consumed all glucose within 51 h in the presence of 4. 8 g/L acetic acid,and the fermentation time was shortened by 25 h,indicating the mutant was superior to the wild type.Our findings provide a novel strategy for constructing robust yeast strains for efficient lignocellulosic ethanol fermentation.
Acetic acid is one of the major inhibitors derived from the hydrolysis of lignocellulosic biomass,and enhancing the tolerance of Saccharomyces cerevisiae to acetic acid is of great importance for improving the lignocellulosic ethanol production. Here,we overexpressed mitochondrial ribosomal protein encoding gene MRP8 via CRISPR/Cas9-based genome editing in S. cerevisiae S288c,and compared the growth and fermentation performance between the mutants with MRP8 and the wild type.The tolerance to acetic acid was increased by overexpression MRP8 on agar plates. Also,mutant MRP8-3 consumed all glucose within 51 h in the presence of 4. 8 g/L acetic acid,and the fermentation time was shortened by 25 h,indicating the mutant was superior to the wild type.Our findings provide a novel strategy for constructing robust yeast strains for efficient lignocellulosic ethanol fermentation.
引文
[1]ZHAO X,XIONG L,ZHANG M,et al.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.
[2]WEI N,QUARTERMAN J,KIM S R,et al.Enhanced biofuel production through coupled acetic acid and xylose consumption by engineered yeast[J].Nat Commun,2013,4:2580.
[3]赵心清,张明明,徐桂红,等.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.
[4]GRAVES T,NARENDRANATH N V,DAWSON K,et al.Effect of p H and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash[J].J Ind Microbiol Biotechnol,2006,33(6):469-474.
[5]APEL A R,D'ESPAUX L,WEHRS M,et al.A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae[J].Nucleic Acids Res,2017,45(1):496-508.
[6]谢泽雄,李炳志,沈玥,等.酵母基因组工程[J].中国科学:生命科学,2015,45(10):985-992.
[7]ZHANG M M,ZHAO X Q,CHENG C,et al.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.
[8]ZHANG M,ZHANG K,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].Bioresour Technol,2017,doi:10.1016/j.biortech.2017.05.191.
[9]张明明,万青青,张克俞,等.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.
[10]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[J].化工学报,2015,66(4):1434-1439.
[11]ABRAHAM P R,MULDER A,VAN'T RIET J,et al.Molecular cloning and physical analysis of an 8.2 kb segment of chromosome XI of Saccharomyces cerevisiae reveals five tightly linked genes[J].Yeast,1992,8(3):227-238.
[12]BOORSMA A,DE NOBEL H,TER RIET B,et al.Characterization of the transcriptional response to cell wall stress in Saccharomyces cerevisiae[J].Yeast,2004,21(5):413-427.
[13]ZHANG G C,KONG I I,KIM H,et al.Construction of a quadruple auxotrophic mutant of an industrial polyploid Saccharomyces cerevisiae strain by using RNA-guided Cas9nuclease[J].Appl Environ Microbiol,2014,80(24):7694-7701.
[14]GIETZ R D,SCHIESTL R H.High-efficiency yeast transformation using the Li Ac/SS carrier DNA/PEG method[J].Nat Protoc,2007,2(1):31-34.
[15]TESTE M A,DUQUENNE M,FRANCOIS J M,et al.Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae[J].BMC Mol Biol,2009,10:99.
[16]FLAGFELDT D,SIEWERS V,HUANG L,et al.Characterization of chromosomal integration sites for heterologous gene expression in Sacchromyces cerevisiae[J].Yeast,2009,26:545-551.
[17]MEDINA V G,ALMERING M J,VAN MARIS A J,et al.Elimination of glycerol production in anaerobic cultures of a Saccharomyces cerevisiae strain engineered to use acetic acid as an electron acceptor[J].Appl Environ Microbiol,2010,76(1):190-195.
[18]MURAKAMI C,DELANEY J R,CHOU,A et al.p H neutralization protects against reduction in replicative lifespan following chronological aging in yeast[J].Cell Cycle,2012,11(16):3087-3096.
[19]WAN C,ZHAO X,ZHANG M,et al.Impact of zinc sulfate addition on dynamic metabolic profiling of Saccharomyces cerevisiae subjected to long term acetic acid stress treatment and identification of key metabolites involved in antioxidant effect of zinc[J].Metallomics,2015,7(2):322-332.
[20]SCHMITT A P,MCENTEE K.Msn2p,a zinc finger DNA-binding protein,is the transcriptional activator of the multistress response in Saccharomyces cerevisiae[J].Proc Natl Acad Sci USA,1996,93(12):5777-5782.
[21]HU Z,KILLION P J,IYER V R.Genetic reconstruction of a functional transcriptional regulatory network[J].Nat Genet,2007,39(5):683-687.
[22]VENTERS B J,WACHI S,MAVRICH T N,et al.A comprehensive genomic binding map of gene and chromatin regulatory proteins in Saccharomyces[J].Mol Cell,2011,41(4):480-492.
[2]WEI N,QUARTERMAN J,KIM S R,et al.Enhanced biofuel production through coupled acetic acid and xylose consumption by engineered yeast[J].Nat Commun,2013,4:2580.
[3]赵心清,张明明,徐桂红,等.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.
[4]GRAVES T,NARENDRANATH N V,DAWSON K,et al.Effect of p H and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash[J].J Ind Microbiol Biotechnol,2006,33(6):469-474.
[5]APEL A R,D'ESPAUX L,WEHRS M,et al.A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae[J].Nucleic Acids Res,2017,45(1):496-508.
[6]谢泽雄,李炳志,沈玥,等.酵母基因组工程[J].中国科学:生命科学,2015,45(10):985-992.
[7]ZHANG M M,ZHAO X Q,CHENG C,et al.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.
[8]ZHANG M,ZHANG K,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].Bioresour Technol,2017,doi:10.1016/j.biortech.2017.05.191.
[9]张明明,万青青,张克俞,等.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.
[10]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[J].化工学报,2015,66(4):1434-1439.
[11]ABRAHAM P R,MULDER A,VAN'T RIET J,et al.Molecular cloning and physical analysis of an 8.2 kb segment of chromosome XI of Saccharomyces cerevisiae reveals five tightly linked genes[J].Yeast,1992,8(3):227-238.
[12]BOORSMA A,DE NOBEL H,TER RIET B,et al.Characterization of the transcriptional response to cell wall stress in Saccharomyces cerevisiae[J].Yeast,2004,21(5):413-427.
[13]ZHANG G C,KONG I I,KIM H,et al.Construction of a quadruple auxotrophic mutant of an industrial polyploid Saccharomyces cerevisiae strain by using RNA-guided Cas9nuclease[J].Appl Environ Microbiol,2014,80(24):7694-7701.
[14]GIETZ R D,SCHIESTL R H.High-efficiency yeast transformation using the Li Ac/SS carrier DNA/PEG method[J].Nat Protoc,2007,2(1):31-34.
[15]TESTE M A,DUQUENNE M,FRANCOIS J M,et al.Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae[J].BMC Mol Biol,2009,10:99.
[16]FLAGFELDT D,SIEWERS V,HUANG L,et al.Characterization of chromosomal integration sites for heterologous gene expression in Sacchromyces cerevisiae[J].Yeast,2009,26:545-551.
[17]MEDINA V G,ALMERING M J,VAN MARIS A J,et al.Elimination of glycerol production in anaerobic cultures of a Saccharomyces cerevisiae strain engineered to use acetic acid as an electron acceptor[J].Appl Environ Microbiol,2010,76(1):190-195.
[18]MURAKAMI C,DELANEY J R,CHOU,A et al.p H neutralization protects against reduction in replicative lifespan following chronological aging in yeast[J].Cell Cycle,2012,11(16):3087-3096.
[19]WAN C,ZHAO X,ZHANG M,et al.Impact of zinc sulfate addition on dynamic metabolic profiling of Saccharomyces cerevisiae subjected to long term acetic acid stress treatment and identification of key metabolites involved in antioxidant effect of zinc[J].Metallomics,2015,7(2):322-332.
[20]SCHMITT A P,MCENTEE K.Msn2p,a zinc finger DNA-binding protein,is the transcriptional activator of the multistress response in Saccharomyces cerevisiae[J].Proc Natl Acad Sci USA,1996,93(12):5777-5782.
[21]HU Z,KILLION P J,IYER V R.Genetic reconstruction of a functional transcriptional regulatory network[J].Nat Genet,2007,39(5):683-687.
[22]VENTERS B J,WACHI S,MAVRICH T N,et al.A comprehensive genomic binding map of gene and chromatin regulatory proteins in Saccharomyces[J].Mol Cell,2011,41(4):480-492.