多靶向沉默里氏木霉碳代谢阻遏物对纤维素酶活性和表达的调控研究
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摘要
【目的】构建多靶向siRNA表达载体对里氏木霉碳阻遏抑制因子CRE1、CRE2、CRE3和CRE4进行同时多靶向siRNA干扰,以研究其对里氏木霉纤维素酶基因表达的调控作用。【方法】根据此前研究筛选出沉默cre1、cre2、cre3和cre4基因的4个最佳siRNA序列,设计并构建了A多靶向表达载体,另根据cre1、cre2、cre3和cre4基因中所含有的5个共有序列设计并构建了B多靶向表达载体,将两者转化至里氏木霉QM9414。经筛选后分别在48 h和120 h对各转化子进行纤维素酶酶活力测试(CMC活力测试和滤纸酶酶活力测试)及利用qPCR检测相关基因的表达。【结果】通过RT-qPCR测定结果表明,两种表达载体均可同时抑制里氏木霉的分解代谢物阻遏基因cre1、cre2、cre3和cre4的表达,纤维素酶活力比出发菌株明显升高,多靶向抑制菌株的CMC酶活和滤纸酶活比出发菌株平均提高了1.95倍和2.66倍。纤维素酶基因cbh1和egl1的表达水平比出发菌株也有明显提升,平均提高了3.83倍和3.95倍。纤维素酶相关基因xyr1的表达水平与出发菌株相比也明显上升,平均提高了2.78倍。【结论】多靶向沉默里氏木霉的碳代谢阻遏蛋白有利于解除葡萄糖效应,提高非还原糖的利用,从而提高纤维素酶的产量,使纤维素酶的表达得到更大的提升,为里氏木霉表达纤维素酶在分解代谢物阻遏基因调控方面提供了实验依据和新的技术思路。
[Objective] In order to study carbon metabolic repressors on cellulase activities and expression regulation in Trichoderma reesei, we constructed multi-targeting siRNA expression vectors to perform silencing carbon metabolic repressor cre1, cre2, cre3 and cre4 by simultaneous producing multi-targeting siRNAs. [Methods] According to previous studies and screening, the four best siRNAs targeting the cre1, cre2, cre3 and cre4 were selected and they were constructed a multi-targeting siRNA expression vector A. Additionally, according to 5 overlap common sequence in the cre1, cre2, cre3 and cre4, we designed and constructed the vector B. The two vectors were transformed the protoplasm of Trichoderma reesei QM9414 and selected on the hygromycin selection medium. Cellulase activities(CMC activity and filter paper activity) of transformants were detected and related gene expressions were also detected by RT-qPCR after incubation for 48 and 120 hours respectively. [Results] RT-qPCR results showed that the cre1, cre2, cre3 and cre4 expression levels in Trichoderma reesei were simultaneously silenced, and the cellulase activities were much higher than that of the staring strain. The CMC activity and filter paper activity of the multi-targeted inhibitor strains were 1.95-fold and 2.66-fold higher than those of the original strains. The cellulase-related genes expression levels were also increased significantly. The expression levels of cbh1, egl1 and xyr1 were 3.83-fold, 3.95-fold and 2.78-fold higher than those of the original strain. [Conclusion] Our results indicated that simultaneous silencing multi-targets of the carbon metabolic repressors in Trichoderma reesei is beneficial to release the glucose repressor effects and increase the expression and production of cellulase. These results provide evidence and techniques for study of regulation of carbon catabolite repressor genes on cellulase expression in Trichoderma reesei.
[Objective] In order to study carbon metabolic repressors on cellulase activities and expression regulation in Trichoderma reesei, we constructed multi-targeting siRNA expression vectors to perform silencing carbon metabolic repressor cre1, cre2, cre3 and cre4 by simultaneous producing multi-targeting siRNAs. [Methods] According to previous studies and screening, the four best siRNAs targeting the cre1, cre2, cre3 and cre4 were selected and they were constructed a multi-targeting siRNA expression vector A. Additionally, according to 5 overlap common sequence in the cre1, cre2, cre3 and cre4, we designed and constructed the vector B. The two vectors were transformed the protoplasm of Trichoderma reesei QM9414 and selected on the hygromycin selection medium. Cellulase activities(CMC activity and filter paper activity) of transformants were detected and related gene expressions were also detected by RT-qPCR after incubation for 48 and 120 hours respectively. [Results] RT-qPCR results showed that the cre1, cre2, cre3 and cre4 expression levels in Trichoderma reesei were simultaneously silenced, and the cellulase activities were much higher than that of the staring strain. The CMC activity and filter paper activity of the multi-targeted inhibitor strains were 1.95-fold and 2.66-fold higher than those of the original strains. The cellulase-related genes expression levels were also increased significantly. The expression levels of cbh1, egl1 and xyr1 were 3.83-fold, 3.95-fold and 2.78-fold higher than those of the original strain. [Conclusion] Our results indicated that simultaneous silencing multi-targets of the carbon metabolic repressors in Trichoderma reesei is beneficial to release the glucose repressor effects and increase the expression and production of cellulase. These results provide evidence and techniques for study of regulation of carbon catabolite repressor genes on cellulase expression in Trichoderma reesei.
引文
[1]Mach RL,Strauss J,Zeilinger S,Schindler M,Kubicek CP.Carbon catabolite repression of xylanase I(xyn1)gene expression in Trichoderma reesei.Molecular Microbiology,1996,21(6):1273-1281.
[2]Joutsjoki VV,Kuittinen M,Torkkeli TK,Suominen PL.Secretion of the Hormoconis resinae glucoamylase p enzyme from Trichoderma reesei directed by the natural and the cbh1 gene secretion signal.FEMS Microbiology Letters,1993,112(3):281-286.
[3]Zhang GT,Hartl L,Schuster A,Polak S,Schmoll M,Wang TH,Seidl V,Seiboth B.Gene targeting in a nonhomologous end joining deficient Hypocrea jecorina.Journal of Biotechnology,2009,139(2):146-151.
[4]Gao YY,Zhong LY,Dong GY,She WY,Zhou JJ,Liu SY,Tian SL.Trichoderma reseei constitutive type of expression vector siRNA interfering cre1 gene on cellulases expression regulation.Journal of Microbiology,2018,38(1):12-19.(in Chinese)高云雨,钟路遥,董冠园,佘伟怡,周娇娇,刘思远,田生礼.里氏木霉组成型表达sirna干扰cre1基因对纤维素酶表达的调控作用.微生物学杂志,2018,38(1):12-19.
[5]Foreman PK,Brown D,Dankmeyer L,Dean R,Diener S,Dunn-Coleman NS,Goedegebuur F,Houfek TD,England GJ,Kelley AS,Meerman HJ,Mitchell T,Mitchinson C,Olivares HA,Teunissen PJM,Yao J,Ward M.Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei.Journal of Biological Chemistry,2003,278(34):31988-31997.
[6]Kumar R,Singh S,Singh OV.Bioconversion of lignocellulosic biomass:Biochemical and molecular perspectives.Journal of Industrial Microbiology&Biotechnology,2008,35(5):377-391.
[7]Boase NA,Kelly JM.A role for cre D,a carbon catabolite repression gene from Aspergillus nidulans,in ubiquitination.Molecular Microbiology,2004,53(3):929-940.
[8]Kubicek CP,Mikus M,Schuster A,Schmoll M,Seiboth B.Metabolic engineering strategies for the improvement of cellulase production by hypocrea jecorina.Biotechnology for Biofuels,2009,2:19.
[9]Lockington RA,Kelly JM.The WD40-repeat protein creCinteracts with and stabilizes the deubiquitinating enzyme creB in vivo in Aspergillus nidulans.Molecular Microbiology,2002,43(5):1173-1182.
[10]Denton JA,Kelly JM.Disruption of trichoderma reesei cre2,encoding an ubiquitin C-terminal hydrolase,results in increased cellulase activity.BMC Biotechnology,2011,11:103.
[11]Saloheimo A,Aro N,Ilmén M,Penttil?M.Isolation of the ace1 gene encoding a cys2-his2 transcription factor involved in regulation of activity of the cellulase promoter cbh1 of Trichoderma reesei.Journal of Biological Chemistry,2000,275(8):5817-5825.
[12]Aro N,Ilmén M,Saloheimo A,Penttil?M.Acei of Trichoderma reesei is a repressor of cellulase and xylanase expression.Applied and Environmental Microbiology,2003,69(1):56-65.
[13]Qin LN,Cai FR,Dong XR,Huang ZB,Tao Y,Huang JZ,Dong ZY.Improved production of heterologous lipase in Trichoderma reesei by RNAi mediated gene silencing of an endogenic highly expressed gene.Bioresource Technology,2012,109:116-122.
[14]He RL,Guo W,Wang LX,Zhang DY.Construction of an efficient rnai system in the cellulolytic fungus Trichoderma reesei.Journal of Microbiological Methods,2015,108:70-73.
[15]Li JX.RNA interference on cellulase expression in Trichoderma reesei catabolite repressor cre2.Master Dissertation of Shenzhen University,2014.(in Chinese)李洁璇.RNA干扰里氏木霉分解代谢阻遏物cre2对纤维素酶表达的研究.深圳大学硕士学位论文,2014.
[16]Liang XY.RNA interference with the catabolic repressor cre3 of Trichoderma reesei and its effect on cellulase expression.Master Dissertation of Shenzhen University,2015.(in Chinese)梁秀怡.RNA干扰里氏木霉分解代谢阻遏物cre3及对纤维素酶表达影响的研究.深圳大学硕士学位论文,2015.
[17]Liu WL.Cre4 gene regulation of cellulase expression in Trichoderma reesei.Master Dissertation of Shenzhen University,2016.(in Chinese)刘雯莉.Cre4基因在里氏木霉中对纤维素酶表达的调控研究.深圳大学硕士学位论文,2016.
[18]Cong W,Cui SQ,Chen JL,Zuo XP,Lu YG,Yan WY,Zheng ZX.Construction of a multiple targeting RNAi plasmid that inhibits target gene expression and FMDV replication in BHK-21 cells and suckling mice.Veterinary Research Communications,2010,34(4):335-346.
[19]Penttil?M,Nevalainen H,R?tt?M,Salminen E,Knowles J.A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei.Gene,1987,61(2):155-164.
[20]Ghose TK,Bisaria VS.Measurement of hemicellulase activities:Part I xylanases.Pure and Applied Chemistry,1987,59(12):1739-1751.
[21]Aro N,Saloheimo A,IlmeéM,Penttil?M.Aceii,a novel transcriptional activator involved in regulation of cellulase and xylanase genes of Trichoderma reesei.Journal of Biological Chemistry,2001,276(26):24309-24314.
[22]Stricker AR,Mach RL,de Graaff LH.Regulation of transcription of cellulases-and hemicellulases-encoding genes in Aspergillus niger and Hypocrea jecorina(Trichoderma reesei).Applied Microbiology and Biotechnology,2008,78(2):211-220.
[23]Silva-Rocha R,Castro LDS,Antoniêto ACC,Guazzaroni ME,Persinoti GF,Silva RN.Deciphering the cis-regulatory elements for xyr1 and cre1 regulators in Trichoderma reesei.PLoS ONE,2014,9(6):e99366.
[24]Lin CT,Chen YC,Jinn TR,Wu CC,Hong YM,Wu WH.Role of the camp-dependent carbon catabolite repression in capsular polysaccharide biosynthesis in Klebsiella pneumoniae.PLoS ONE,2013,8(2):e54430.
[25]Borirak O,Bekker M,Hellingwerf KJ.Molecular physiology of the dynamic regulation of carbon catabolite repression in Escherichia coli.Microbiology,2014,160(6):1214-1223.
[2]Joutsjoki VV,Kuittinen M,Torkkeli TK,Suominen PL.Secretion of the Hormoconis resinae glucoamylase p enzyme from Trichoderma reesei directed by the natural and the cbh1 gene secretion signal.FEMS Microbiology Letters,1993,112(3):281-286.
[3]Zhang GT,Hartl L,Schuster A,Polak S,Schmoll M,Wang TH,Seidl V,Seiboth B.Gene targeting in a nonhomologous end joining deficient Hypocrea jecorina.Journal of Biotechnology,2009,139(2):146-151.
[4]Gao YY,Zhong LY,Dong GY,She WY,Zhou JJ,Liu SY,Tian SL.Trichoderma reseei constitutive type of expression vector siRNA interfering cre1 gene on cellulases expression regulation.Journal of Microbiology,2018,38(1):12-19.(in Chinese)高云雨,钟路遥,董冠园,佘伟怡,周娇娇,刘思远,田生礼.里氏木霉组成型表达sirna干扰cre1基因对纤维素酶表达的调控作用.微生物学杂志,2018,38(1):12-19.
[5]Foreman PK,Brown D,Dankmeyer L,Dean R,Diener S,Dunn-Coleman NS,Goedegebuur F,Houfek TD,England GJ,Kelley AS,Meerman HJ,Mitchell T,Mitchinson C,Olivares HA,Teunissen PJM,Yao J,Ward M.Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei.Journal of Biological Chemistry,2003,278(34):31988-31997.
[6]Kumar R,Singh S,Singh OV.Bioconversion of lignocellulosic biomass:Biochemical and molecular perspectives.Journal of Industrial Microbiology&Biotechnology,2008,35(5):377-391.
[7]Boase NA,Kelly JM.A role for cre D,a carbon catabolite repression gene from Aspergillus nidulans,in ubiquitination.Molecular Microbiology,2004,53(3):929-940.
[8]Kubicek CP,Mikus M,Schuster A,Schmoll M,Seiboth B.Metabolic engineering strategies for the improvement of cellulase production by hypocrea jecorina.Biotechnology for Biofuels,2009,2:19.
[9]Lockington RA,Kelly JM.The WD40-repeat protein creCinteracts with and stabilizes the deubiquitinating enzyme creB in vivo in Aspergillus nidulans.Molecular Microbiology,2002,43(5):1173-1182.
[10]Denton JA,Kelly JM.Disruption of trichoderma reesei cre2,encoding an ubiquitin C-terminal hydrolase,results in increased cellulase activity.BMC Biotechnology,2011,11:103.
[11]Saloheimo A,Aro N,Ilmén M,Penttil?M.Isolation of the ace1 gene encoding a cys2-his2 transcription factor involved in regulation of activity of the cellulase promoter cbh1 of Trichoderma reesei.Journal of Biological Chemistry,2000,275(8):5817-5825.
[12]Aro N,Ilmén M,Saloheimo A,Penttil?M.Acei of Trichoderma reesei is a repressor of cellulase and xylanase expression.Applied and Environmental Microbiology,2003,69(1):56-65.
[13]Qin LN,Cai FR,Dong XR,Huang ZB,Tao Y,Huang JZ,Dong ZY.Improved production of heterologous lipase in Trichoderma reesei by RNAi mediated gene silencing of an endogenic highly expressed gene.Bioresource Technology,2012,109:116-122.
[14]He RL,Guo W,Wang LX,Zhang DY.Construction of an efficient rnai system in the cellulolytic fungus Trichoderma reesei.Journal of Microbiological Methods,2015,108:70-73.
[15]Li JX.RNA interference on cellulase expression in Trichoderma reesei catabolite repressor cre2.Master Dissertation of Shenzhen University,2014.(in Chinese)李洁璇.RNA干扰里氏木霉分解代谢阻遏物cre2对纤维素酶表达的研究.深圳大学硕士学位论文,2014.
[16]Liang XY.RNA interference with the catabolic repressor cre3 of Trichoderma reesei and its effect on cellulase expression.Master Dissertation of Shenzhen University,2015.(in Chinese)梁秀怡.RNA干扰里氏木霉分解代谢阻遏物cre3及对纤维素酶表达影响的研究.深圳大学硕士学位论文,2015.
[17]Liu WL.Cre4 gene regulation of cellulase expression in Trichoderma reesei.Master Dissertation of Shenzhen University,2016.(in Chinese)刘雯莉.Cre4基因在里氏木霉中对纤维素酶表达的调控研究.深圳大学硕士学位论文,2016.
[18]Cong W,Cui SQ,Chen JL,Zuo XP,Lu YG,Yan WY,Zheng ZX.Construction of a multiple targeting RNAi plasmid that inhibits target gene expression and FMDV replication in BHK-21 cells and suckling mice.Veterinary Research Communications,2010,34(4):335-346.
[19]Penttil?M,Nevalainen H,R?tt?M,Salminen E,Knowles J.A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei.Gene,1987,61(2):155-164.
[20]Ghose TK,Bisaria VS.Measurement of hemicellulase activities:Part I xylanases.Pure and Applied Chemistry,1987,59(12):1739-1751.
[21]Aro N,Saloheimo A,IlmeéM,Penttil?M.Aceii,a novel transcriptional activator involved in regulation of cellulase and xylanase genes of Trichoderma reesei.Journal of Biological Chemistry,2001,276(26):24309-24314.
[22]Stricker AR,Mach RL,de Graaff LH.Regulation of transcription of cellulases-and hemicellulases-encoding genes in Aspergillus niger and Hypocrea jecorina(Trichoderma reesei).Applied Microbiology and Biotechnology,2008,78(2):211-220.
[23]Silva-Rocha R,Castro LDS,Antoniêto ACC,Guazzaroni ME,Persinoti GF,Silva RN.Deciphering the cis-regulatory elements for xyr1 and cre1 regulators in Trichoderma reesei.PLoS ONE,2014,9(6):e99366.
[24]Lin CT,Chen YC,Jinn TR,Wu CC,Hong YM,Wu WH.Role of the camp-dependent carbon catabolite repression in capsular polysaccharide biosynthesis in Klebsiella pneumoniae.PLoS ONE,2013,8(2):e54430.
[25]Borirak O,Bekker M,Hellingwerf KJ.Molecular physiology of the dynamic regulation of carbon catabolite repression in Escherichia coli.Microbiology,2014,160(6):1214-1223.