人工锌指蛋白介导调控的里氏木霉纤维素酶生产
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摘要
里氏木霉Trichoderma reesei Rut-C30是目前研究最广泛的纤维素酶生产菌,选育高产纤维素酶的里氏木霉菌株有助于提高木质纤维素资源生物炼制的经济性。利用人工锌指蛋白文库转化T.reeseiRut-C30,筛选获得了两株高产纤维素酶的突变株T. reesei M1和M2,与出发菌株比较,突变株M1和M2滤纸酶活分别提高100%和53%,且M1突变株外泌蛋白量提高69%,M2内切纤维素酶活提高64%。实时定量PCR分析结果表明,与对照菌株相比,突变株M1和M2中主要纤维素酶基因转录均上调,但不同酶基因在两株菌中有不同的变化特征。此外,纤维素酶抑制转录因子基因ace1在两株突变株中都转录下调,而纤维素酶正调控转录因子基因xyr1仅在M1突变株中上调。以上结果表明,不同人工锌指蛋白对纤维素酶活性的影响具有多样性。对这些突变体中人工锌指蛋白靶基因进行深入分析,为进一步深入探究里氏木霉纤维素酶合成调控的机理,以及利用代谢工程选育更高效的产酶菌株提供了基础。
Trichoderma reesei Rut-C30 is widely used in industrial cellulase production,and development of cellulase hyper-producer is of great importance for economic lignocellulosic biorefinery.In this study,T.reesei Rut-C30 wasengineered with an artificial zinc finger proteins(AZFPs) library.Two mutants T.reesei M1 and M2 with improved cellulase production were obtained.Compared to the parent strain,the filter paper activity(FPase) of T.reesei M1 and M2 increased 100% and 53%,respectively.In addition,the total amount of extracellular protein from the M1 mutant increased 69%,whereas the endo-β-glucanase(CMCase) activity of the M2 mutant is 64% higher compared to the parental strain.Furthermore,RT-qPCR analysis showed that the major cellulase genes exhibited significantly increased expression in both mutants,but different patterns were observed in the two mutants.On the other hand,the cellulase transcriptional repressor ace1 was down-regulated in both mutants,but the transcription level of the activator xyr1 was only up-regulated in the strain M1.These results demonstrated that different AZFPs exert diverse regulatory mechanisms on cellulase production in T.reesei.Analysis of the target genes of AZFPs from T.reesei M1 and M2 will not only benefit further exploration of the regulatory mechanisms of cellulase biosynthesis in T.reesei,but also enable development of cellulase hyper-producing strains by metabolic engineering.
Trichoderma reesei Rut-C30 is widely used in industrial cellulase production,and development of cellulase hyper-producer is of great importance for economic lignocellulosic biorefinery.In this study,T.reesei Rut-C30 wasengineered with an artificial zinc finger proteins(AZFPs) library.Two mutants T.reesei M1 and M2 with improved cellulase production were obtained.Compared to the parent strain,the filter paper activity(FPase) of T.reesei M1 and M2 increased 100% and 53%,respectively.In addition,the total amount of extracellular protein from the M1 mutant increased 69%,whereas the endo-β-glucanase(CMCase) activity of the M2 mutant is 64% higher compared to the parental strain.Furthermore,RT-qPCR analysis showed that the major cellulase genes exhibited significantly increased expression in both mutants,but different patterns were observed in the two mutants.On the other hand,the cellulase transcriptional repressor ace1 was down-regulated in both mutants,but the transcription level of the activator xyr1 was only up-regulated in the strain M1.These results demonstrated that different AZFPs exert diverse regulatory mechanisms on cellulase production in T.reesei.Analysis of the target genes of AZFPs from T.reesei M1 and M2 will not only benefit further exploration of the regulatory mechanisms of cellulase biosynthesis in T.reesei,but also enable development of cellulase hyper-producing strains by metabolic engineering.
引文
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[8]Cao YL,Zheng FL,Wang L,et al.Rce1,a novel transcriptional repressor,regulates cellulase gene expression by antagonizing the transactivator Xyr1 in Trichoderma reesei.Mol Microbiol,2017,105(1):65-83.
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[11]Lee JY,Sung BH,Yu BJ,et al.Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli.Nucleic Acids Res,2008,36(16):e102.
[12]Zhang F,Bai FW,Zhao XQ.Enhanced cellulase production from Trichoderma reesei Rut-C30 by engineering with an artificial zinc finger protein library.Biotech J,2016,11(10):1282-1290.
[13]Wang MY,Zhao QS,Yang JH,et al.Amitogen-activated protein kinase Tmk3 participates in high osmolarity resistance,cell wall integrity maintenance and cellulase production regulation in Trichoderma reesei.PLoS ONE,2013,8(8):e72189.
[14]Zhou GQ,LüJ,Li ZH,et al.Enhanced cellulase production of Penicillium decumbens by knocking out CreB encoding a deubiquitination enzyme.Chin JBiotech,2012,28(8):959-972(in Chinese).周广麒,吕晶,李忠海,等.斜卧青霉去泛素化蛋白酶CREB的缺失提高纤维素酶的生产.生物工程学报,2012,28(8):959-972.
[15]Liu YG,Chen YL.High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences.BioTechniques,2007,43(5):649-656.
[16]Rebar EJ,Pabo CO.Zinc finger phage:affinity selection of fingers with new DNA-binding specificities.Science,1994,263(5147):671-673.
[17]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method.Methods,25(4):402-408.
[18]Su XY,Chu X,Dong ZY.Identification of elevated transcripts in a Trichoderma reesei strain expressing a chimeric transcription activator using suppression subtractive hybridization.World J Microb Biot,2009,25(6):1075-1084.
[19]Nitta M,Furukawa T,Shida Y et al.A new Zn(II)2Cys6-type transcription factor BglR regulatesβ-glucosidase expression in Trichoderma reesei.Fungal Genet Biol,2012,49(5):388-397.
[20]Jiang Y,Su N,Fang X.Genetic modification and application of lignocellulose degrading fungus.Acta Microbiol Sin,2017,57(8):1235-1248(in Chinese).蒋艺,苏宁,方诩.木质纤维素降解酶生产菌株的遗传改造及应用.微生物学报,2017,57(8):1235-1248.
[21]Chen L,Zou G,Wang JZ,et al.Characterization of the Ca2+-responsive signaling pathway in regulating the expression and secretion of cellulases in Trichoderma reesei Rut-C30.Mol Microbiol,2016,100(3),560-575.
[22]Zhang F,Zhao XQ,Bai FW.Improvement of cellulase production in Trichoderma reesei Rut-C30by overexpression of a novel regulatory gene Trvib-1.Bioresour Technol,2018,247:676-683.
[23]Li JX,Wang J,Wang SW,et al.Achieving efficient protein expression in Trichoderma reesei by using strong constitutive promoters.Microb Cell Fact,2012,11:84.
[24]Gao LW,Li ZH,Xia CQ,et al.Combining manipulation of transcription factors and overexpression of the target genes to enhance lignocellulolytic enzyme production in Penicillium oxalicum.Biotechnol Biofuels,2017,10:100.
[2]Taha M,Foda M,Shahsavari E,et al.Commercial feasibility of lignocellulose biodegradation:possibilities and challenges.Curr Opin Biotechnol,2016,38:190-197.
[3]Peterson R,Nevalainen H.Trichoderma reesei RUT-C30-thirty years of strain improvement.Microbiology,2012,158(1):58-68.
[4]Zhang F,Bai FW,Zhao XQ.Induction and regulation of cellulase expression in filamentous fungi:a review.Chin J Biotech,2016,32(11):1481-1495(in Chinese).张飞,白凤武,赵心清.丝状真菌纤维素酶合成诱导及转录调控.生物工程学报,2016,32(11):1481-1495.
[5]Druzhinina IS,Kubicek CP.Genetic engineering of Trichoderma reesei cellulases and their production.Microb Biotechnol,2017,10(6):1485-1499.
[6]Gao J,Qian YC,Wang YF,Qu,et al.Production of the versatile cellulase for cellulose bioconversion and cellulase inducer synthesis by genetic improvement of Trichoderma reesei.Biotechnol Biofuels,2017,10:272.
[7]Liu Z,Zhang F,Zhao XQ,et al.Effects of zinc-finger proteins and artificial zinc-finger proteins on microbial metabolisms-a review.Chin J Biotech,2014,30(3):331-340(in Chinese).刘卓,张飞,赵心清,等.锌指蛋白及人工锌指蛋白对微生物代谢影响的研究进展.生物工程学报,2014,30(3):331-340.
[8]Cao YL,Zheng FL,Wang L,et al.Rce1,a novel transcriptional repressor,regulates cellulase gene expression by antagonizing the transactivator Xyr1 in Trichoderma reesei.Mol Microbiol,2017,105(1):65-83.
[9]Park KS,Lee DK,Lee H,et al.Phenotypic alteration of eukaryotic cells using randomized libraries of artificial transcription factors.Nat Biotechnol,2003,21(10):1208-1214.
[10]Ma C,Wei XW,Sun CH,et al.Improvement of acetic acid tolerance of Saccharomyces cerevisiae using a zinc-finger-based artificial transcription factor and identification of novel genes involved in acetic acid tolerance.Appl Microbiol Biotechnol,2015,99(5):2441-2449.
[11]Lee JY,Sung BH,Yu BJ,et al.Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli.Nucleic Acids Res,2008,36(16):e102.
[12]Zhang F,Bai FW,Zhao XQ.Enhanced cellulase production from Trichoderma reesei Rut-C30 by engineering with an artificial zinc finger protein library.Biotech J,2016,11(10):1282-1290.
[13]Wang MY,Zhao QS,Yang JH,et al.Amitogen-activated protein kinase Tmk3 participates in high osmolarity resistance,cell wall integrity maintenance and cellulase production regulation in Trichoderma reesei.PLoS ONE,2013,8(8):e72189.
[14]Zhou GQ,LüJ,Li ZH,et al.Enhanced cellulase production of Penicillium decumbens by knocking out CreB encoding a deubiquitination enzyme.Chin JBiotech,2012,28(8):959-972(in Chinese).周广麒,吕晶,李忠海,等.斜卧青霉去泛素化蛋白酶CREB的缺失提高纤维素酶的生产.生物工程学报,2012,28(8):959-972.
[15]Liu YG,Chen YL.High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences.BioTechniques,2007,43(5):649-656.
[16]Rebar EJ,Pabo CO.Zinc finger phage:affinity selection of fingers with new DNA-binding specificities.Science,1994,263(5147):671-673.
[17]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method.Methods,25(4):402-408.
[18]Su XY,Chu X,Dong ZY.Identification of elevated transcripts in a Trichoderma reesei strain expressing a chimeric transcription activator using suppression subtractive hybridization.World J Microb Biot,2009,25(6):1075-1084.
[19]Nitta M,Furukawa T,Shida Y et al.A new Zn(II)2Cys6-type transcription factor BglR regulatesβ-glucosidase expression in Trichoderma reesei.Fungal Genet Biol,2012,49(5):388-397.
[20]Jiang Y,Su N,Fang X.Genetic modification and application of lignocellulose degrading fungus.Acta Microbiol Sin,2017,57(8):1235-1248(in Chinese).蒋艺,苏宁,方诩.木质纤维素降解酶生产菌株的遗传改造及应用.微生物学报,2017,57(8):1235-1248.
[21]Chen L,Zou G,Wang JZ,et al.Characterization of the Ca2+-responsive signaling pathway in regulating the expression and secretion of cellulases in Trichoderma reesei Rut-C30.Mol Microbiol,2016,100(3),560-575.
[22]Zhang F,Zhao XQ,Bai FW.Improvement of cellulase production in Trichoderma reesei Rut-C30by overexpression of a novel regulatory gene Trvib-1.Bioresour Technol,2018,247:676-683.
[23]Li JX,Wang J,Wang SW,et al.Achieving efficient protein expression in Trichoderma reesei by using strong constitutive promoters.Microb Cell Fact,2012,11:84.
[24]Gao LW,Li ZH,Xia CQ,et al.Combining manipulation of transcription factors and overexpression of the target genes to enhance lignocellulolytic enzyme production in Penicillium oxalicum.Biotechnol Biofuels,2017,10:100.
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