|
|
|
| The Influence of Homologous Overexpression of BglR on β-glucosidase Activities in Myceliophthora thermophila |
| LAI Ya-peng1,2, DENG Ting-ting1,2, LIU Gang1, WANG Juan1 |
1. College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen 518060, China;
2. Shenzhen Key Laboratory of Marine Bioresources and Ecology, Shenzhen 518060, China |
|
|
|
Abstract Objective: The influence of overexpression of β-glucosidase regulator BglR on beta-glucosidase activities in Myceliophthora thermophila ATCC42464 by cloning bglr gene and constructing Mtbglr overexpression vector were focused on. Methods: The SLIC was adopted to construct Mtbglr overexpression vector and the promoter of MtPpdc and terminator of MtTpdc was used for overexpress bglr gene. The gene expression and β-glucosidase activities were observed by protoplast transformation, real-time quantitative PCR and enzymatic determination. Results: The bglr gene was overexpressed in M. thermophila successfully. The result showed that the β-glucosidase activity and secreted protein concentration of transformant strain Mt8 were 1.7 and 1.9 fold higher, respectively, than that of wild type WT. Conclusion: The expression of bglr increased the β-glucosidase activity in M. thermophila under the inducing condition, which laid the theoretical foundation for the regulation of β-glucosidase gene of thermophilic fungi.
|
|
Received: 17 January 2017
Published: 25 July 2017
|
|
|
|
|
[1] 周庆新,戴炳业,陈蕾蕾,等. 瑞氏木霉中β-葡萄糖苷酶基因功能研究进展.中国农业科技导报,2014,16(2):74-78. Zhou Q X,Dai B Y, Chen L L, et al.Progress on functional studies of β-glucosidase genes in Trichoderma reesei. Journal of Agricultural Science and Technology, 2014,16(2):74-78.
[2] Singhania R R, Patel A K, Sukumaran R K, et al. Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol production. Bioresource Technology, 2013, 127: 500-507.
[3] Bhatia Y, Mishra S, Bisaria V S. Microbial β-glucosidases: cloning, properties, and applications. Critical Reviews in Biotechnology, 2002, 22(4): 375-407.
[4] Chandra M, Kalra A, Sangwan N S, et al. Biochemical and proteomic characterization of a novel extracellular β-glucosidase from Trichoderma citrinoviride. Molecular Biotechnology, 2013, 53(3): 289-299.
[5] Handa C L, Couto U R, Vicensoti A H, et al. Optimisation of soy flour fermentation parameters to produce β-glucosidase for bioconversion into aglycones. Food Chemistry, 2014, 152: 56-65.
[6] Abedinifar S, Karimi K, Khanahmadi M, et al. Ethanol production by Mucor indicus and Rhizopus oryzae from rice straw by separate hydrolysis and fermentation. Biomass and Bioenergy, 2009, 33(5): 828-833.
[7] Pal S, Banik S P, Ghorai S, et al. Purification and characterization of a thermostable intra-cellular β-glucosidase with transglycosylation properties from filamentous fungus Termitomyces clypeatus. Bioresource Technology, 2010, 101(7): 2412-2420.
[8] Cantarel B L, Coutinho P M, Rancurel C, et al. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Research, 2009, 37(suppl 1): D233-D238.
[9] Dan S, Marton I, Dekel M, et al. Cloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger β-glucosidase. Journal of Biological Chemistry, 2000, 275(7): 4973-4980.
[10] Liu D, Zhang R, Yang X, et al. Characterization of a thermostable β-glucosidase from Aspergillus fumigatus Z5, and its functional expression in Pichia pastoris X33. Microbial Cell Factories, 2012, 11(25):1-15.
[11] Gao L, Gao F, Zhang D, et al. Purification and characterization of a new β-glucosidase from Penicillium piceum and its application in enzymatic degradation of delignified corn stover. Bioresource Technology, 2013, 147: 658-661.
[12] Chen P, Fu X, Ng T B, et al. Expression of a secretory β-glucosidase from Trichoderma reesei in Pichia pastoris and its characterization. Biotechnology Letters, 2011, 33(12): 2475-2479.
[13] Haki G D, Rakshit S K. Developments in industrially important thermostable enzymes: a review. Bioresource Technology, 2003, 89(1): 17-34.
[14] Morgenstern I, Powlowski J, Ishmael N, et al. A molecular phylogeny of thermophilic fungi. Fungal Biology, 2012, 116(4): 489-502.
[15] Basotra N, Kaur B, Di Falco M, et al. Mycothermus thermophilus (Syn. Scytalidium thermophilum): Repertoire of a diverse array of efficient cellulases and hemicellulases in the secretome revealed. Bioresource Technology, 2016, 222: 413-421.
[16] Xu X, Li J, Shi P, et al. The use of T-DNA insertional mutagenesis to improve cellulase production by the thermophilic fungus Humicola insolens Y1. Scientific Reports, 2016, 6:31108.
[17] Mallek-Fakhfakh H, Belghith H. Physicochemical properties of thermotolerant extracellular β-glucosidase from Talaromyces thermophilus and enzymatic synthesis of cello-oligosaccharides. Carbohydrate Research, 2016, 419: 41-50.
[18] Matsakas L, Antonopoulou I, Christakopoulos P. Evaluation of Myceliopthora thermophila as an enzyme factory for the production of thermophilic cellulolytic enzymes. Bio Resources, 2015, 10(3): 5140-5158.
[19] Berka R M, Grigoriev I V, Otillar R, et al. Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris. Nat Biotechnol, 2011, 29(10):922-927.
[20] Visser H, Joosten V, Punt P J, et al. RESEARCH: Development of a mature fungal technology and production platform for industrial enzymes based on a Myceliophthora thermophila isolate, previously known as Chrysosporium lucknowense C1. Industrial Biotechnology, 2011, 7(3): 214-223.
[21] Karnaouri A, Topakas E, Paschos T, et al. Cloning, expression and characterization of an ethanol tolerant GH3β-glucosidase from Myceliophthora thermophila. Peer J, 2013, 1: e46.
[22] Zhao J, Guo C, Tian C, et al. Heterologous expression and characterization of a GH3β-glucosidase from thermophilic fungi Myceliophthora thermophila in Pichia pastoris. Applied Biochemistry and Biotechnology, 2015, 177(2): 511-527.
[23] Murray P, Aro N, Collins C, et al. Expression in Trichoderma reesei and characterisation of a thermostable family 3β-glucosidase from the moderately thermophilic fungus Talaromyces emersonii. Protein Expression and Purification, 2004, 38(2): 248-257.
[24] Hong J, Tamaki H, Kumagai H. Cloning and functional expression of thermostable β-glucosidase gene from Thermoascus aurantiacus. Applied Microbiology and Biotechnology, 2007, 73(6): 1331-1339.
[25] Yang X, Ma R, Shi P, et al. Molecular characterization of a highly-active thermophilic β-glucosidase from Neosartorya fischeri P1 and its application in the hydrolysis of soybean isoflavone glycosides. PLoS One, 2014, 9(9): e106785.
[26] Guo Y, Yan Q, Yang Y, et al. Expression and characterization of a novel β-glucosidase, with transglycosylation and exo-β-1, 3-glucanase activities, from Rhizomucor miehei. Food Chemistry, 2015, 175: 431-438.
[27] Xia W, Xu X, Qian L, et al. Engineering a highly active thermophilic β-glucosidase to enhance its pH stability and saccharification performance. Biotechnology for Biofuels, 2016, 9(1): 147.
[28] Nitta M, Furukawa T, Shida Y, et al. A new Zn (Ⅱ) 2 Cys 6-type transcription factor BglR r-egulates β-glucosidase expression in Trichoderma reesei. Fungal Genetics and Biology, 2012, 49(5): 388-397.
[29] Tani S, Kawaguchi T, Kobayashi T. Complex regulation of hydrolytic enzyme genes for cellulosic biomass degradation in filamentous fungi. Applied Microbiology and Biotechnology, 2014, 98(11): 4829-4837.
[30] Wang J, Wu Y, Gong Y, et al. Enhancing xylanase production in the thermophilic fungus Myceliophthora thermophila by homologous overexpression of Mtxyr1. Journal of Industrial Microbiology & Biotechnology, 2015, 42(9): 1233-1241.
[31] Li M Z, Elledge S J. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nature Methods, 2007, 4(3): 251-256.
[32] Jeong J Y, Yim H S, Ryu J Y, et al. One-step sequence-and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies. Applied and Environmental Microbiology, 2012, 78(15): 5440-5443.
[33] Penttilä M, Nevalainen H, Rättö M, et al. A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene, 1987, 61(2): 155-164.
[34] Yang F, Gong Y, Liu G, et al. Enhancing cellulase production in thermophilic fungus Myceliophthora thermophila ATCC42464 by RNA interference of cre1 gene expression. Journal of Microbiology and Biotechnology, 2015, 25(7): 1101-1107.
[35] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2- ΔΔCT method. Methods, 2001, 25(4): 402-408.
[36] 王宝林,韩燕峰,王玉荣,等.一株嗜热毁丝霉菌株产纤维素酶条件优化. 酿酒科技,2012,(10):27-31. Wang B L, Han Y F, Wang Y R,et al.Optimization of cellulase-producing conditions of A Thermophilic myceliophthora sp. H127-1 strain. Liquor-Making Science & Tcchnology, 2012,(10):27-31.
[37] Eveleigh D E, Mandels M, Andreotti R, et al. Measurement of saccharifying cellulase. Biotechnology for Biofuels, 2009, 2(1): 21.
[38] Mansour A A, Da Costa A, Arnaud T, et al. Review of lignocellulolytic enzyme activity analyses and scale-down to microplate-based assays. Talanta, 2016, 150: 629-637.
[39] Aro N, Saloheimo A, Ilmén M, et al. 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.
[40] Zeilinger S, Ebner A, Marosits T, et al. The Hypocrea jecorina HAP 2/3/5 protein complex binds to the inverted CCAAT-box (ATTGG) within the cbh2 (cellobiohydrolase Ⅱ-gene) activating element. Molecular Genetics and Genomics, 2001, 266(1): 56-63.
[41] Stricker A R, Grosstessner-Hain K, Würleitner E, et al. Xyr1(xylanase regulator 1) regulates both the hydrolytic enzyme system and D-xylose metabolism in Hypocrea jecorina. Eukaryotic Cell, 2006, 5(12): 2128-2137.
[42] Aro N, Ilmén M, Saloheimo A, et al. ACEI of Trichoderma reesei is a repressor of cellulase and xylanase expression. Applied and Environmental Microbiology, 2003, 69(1): 56-65.
[43] Portnoy T, Margeot A, Linke R, et al. The CRE1 carbon catabolite repressor of the fungus Trichoderma reesei: a master regulator of carbon assimilation. BMC Genomics, 2011, 12(1): 269.
[44] Wang S, Liu G, Yu J, et al. RNA interference with carbon catabolite repression in Trichoderma koningii for enhancing cellulase production. Enzyme and Microbial Technology, 2013, 53(2): 104-109. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
