Cellulase activity mapping of Trichoderma reesei cultivated in sugar mixtures under fed-batch conditions

Biotechnology for Biofuels

Etienne Jourdier¹  Céline Cohen¹  Laurent Poughon²  Christian Larroche²  Frédéric Monot¹  Fadhel Ben Chaabane¹ 

[1] IFP Energies nouvelles, 1 et 4 avenue de Bois-Préau, 92852 Rueil-Malmaison, France

[2] Clermont Université, Université Blaise Pascal, Labex IMobS3, Institut Pascal, Polytech Clermont-Ferrand, 24 av. des Landais, BP 20206, 63174 Aubière cedex, France

关键词: Bioethanol;    On-site enzyme production;    β-glucosidase;    Xylanase;    Cellulase;    Inducer;    Industrial protocol;    Carbon flux limitation;    Fed-batch cultivation;    Sugar mixture;    Trichoderma reesei;   

Others  :  798030
DOI  :  10.1186/1754-6834-6-79

 received in 2013-03-22, accepted in 2013-05-15,  发布年份 2013

【 摘 要 】

Background

On-site cellulase production using locally available lignocellulosic biomass (LCB) is essential for cost-effective production of 2^nd-generation biofuels. Cellulolytic enzymes (cellulases and hemicellulases) must be produced in fed-batch mode in order to obtain high productivity and yield. To date, the impact of the sugar composition of LCB hydrolysates on cellulolytic enzyme secretion has not been thoroughly investigated in industrial conditions.

Results

The effect of sugar mixtures (glucose, xylose, inducer) on the secretion of cellulolytic enzymes by a glucose-derepressed and cellulase-hyperproducing mutant strain of Trichoderma reesei (strain CL847) was studied using a small-scale protocol representative of the industrial conditions. Since production of cellulolytic enzymes is inducible by either lactose or cellobiose, two parallel mixture designs were performed separately. No significant difference between inducers was observed on cellulase secretion performance, probably because a common induction mechanism occurred under carbon flux limitation. The characteristics of the enzymatic cocktails did not correlate with productivity, but instead were rather dependent on the substrate composition. Increasing xylose content in the feed had the strongest impact. It decreased by 2-fold cellulase, endoglucanase, and cellobiohydrolase activities and by 4-fold β-glucosidase activity. In contrast, xylanase activity was increased 6-fold. Accordingly, simultaneous high β-glucosidase and xylanase activities in the enzymatic cocktails seemed to be incompatible. The variations in enzymatic activity were modelled and validated with four fed-batch cultures performed in bioreactors. The overall enzyme production was maintained at its highest level when substituting up to 75% of the inducer with non-inducing sugars.

Conclusions

The sugar substrate composition strongly influenced the composition of the cellulolytic cocktail secreted by T. reesei in fed-batch mode. Modelling can be used to predict cellulolytic activity based on the sugar composition of the culture-feeding solution, or to fine tune the substrate composition in order to produce a desired enzymatic cocktail.

【 授权许可】

   
2013 Jourdier et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 2.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Aro N, Pakula TM, Penttila ME: Transcriptional regulation of plant cell wall degradation by filamentous fungi. FEMS Microbiol Rev 2005, 29:719-739.
• [2]Horn S, Vaaje-Kolstad G, Westereng B, Eijsink VG: Novel enzymes for the degradation of cellulose. Biotechnol Biofuels 2012, 5:45.
• [3]Singhania RR, Sukumaran RK, Patel AK, Larroche C, Pandey A: Advancement and comparative profiles in the production technologies using solid-state and submerged fermentation for microbial cellulases. Enzyme Microb Technol 2010, 46:541-549.
• [4]Barta Z, Kovacs K, Reczey K, Zacchi G: Process design and economics of on-site cellulase production on various carbon sources in a softwood-based ethanol plant. Enzyme Research 2010, 2010:734182.
• [5]Kazi FK, Fortman JA, Anex RP, Hsu DD, Aden A, Dutta A, Kothandaraman G: Techno-economic comparison of process technologies for biochemical ethanol production from corn stover. Fuel 2010, 89:S20-S28.
• [6]Klein-Marcuschamer D, Oleskowicz-Popiel P, Simmons BA, Blanch HW: The challenge of enzyme cost in the production of lignocellulosic biofuels. Biotechnol Bioeng 2012, 109:1083-1087.
• [7]Jourdier E, Ben Chaabane F, Poughon L, Larroche C, Monot F: Simple kinetic model of cellulase production by Trichoderma reesei for productivity or yield maximization. Chem Eng Trans 2012, 27:313-318.
• [8]Tolan J, Foody B: Cellulase from submerged fermentation. In Recent Progress in Bioconversion of Lignocellulosics. 65th edition. Edited by Tsao G. Berlin: Springer; 1999:41-67.
• [9]Jourdier E, Poughon L, Larroche C, Monot F, Ben Chaabane F: A new stoichiometric miniaturization strategy for screening of industrial microbial strains: application to cellulase hyper-producing Trichoderma reesei strains. Microb Cell Fact 2012, 11:70.
• [10]Persson I, Tjerneld F, Hahn-Hägerdal B: Fungal cellulolytic enzyme production: a review. Process Biochem 1991, 26:65-74.
• [11]Pourquié J, Warzywoda M, Chevron F, Thery D, Lonchamp D, Vandecasteele JP: Scale up of cellulase production and utilization. In FEMS Symposium n°43: Biochemistry and Genetics of Cellulose Degradation. Edited by Aubert J-P, Beguin P, Millet J. London: Academic Press; 1988:71-86.
• [12]Warzywoda M, Larbre E, Pourquié J: Production and characterization of cellulolytic enzymes from Trichoderma reesei grown on various carbon sources. Bioresour Technol 1992, 39:125-130.
• [13]Philippidis GP: Cellulase production technology. In Enzymatic Conversion of Biomass for Fuels Production. 566th edition. Edited by Himmel M, Baker JO, Overend RP. Washington, DC: American Chemical Society; 1994:188-217.
• [14]Esterbauer H, Steiner W, Labudova I, Hermann A, Hayn M: Production of Trichoderma cellulase in laboratory and pilot scale. Bioresour Technol 1991, 36:51-65.
• [15]Dashtban M, Maki M, Leung KT, Mao C, Qin W: Cellulase activities in biomass conversion: measurement methods and comparison. Crit Rev Biotechnol 2010, 30:302-309.
• [16]Juhasz T, Szengyel Z, Reczey K, Siika-Aho M, Viikari L: Characterization of cellulases and hemicellulases produced by Trichoderma reesei on various carbon sources. Process Biochem 2005, 40:3519-3525.
• [17]Jun H, Kieselbach T, Jonsson L: Enzyme production by filamentous fungi: analysis of the secretome of Trichoderma reesei grown on unconventional carbon source. Microb Cell Fact 2011, 10:68.
• [18]Olsson L, Christensen T, Hansen KP, Palmqvist EA: Influence of the carbon source on production of cellulases, hemicellulases and pectinases by Trichoderma reesei Rut C-30. Enzyme Microb Technol 2003, 33:612-619.
• [19]Sipos B, Benko Z, Dienes D, Reczey K, Viikari L, Siika-Aho M: Characterisation of specific activities and hydrolytic properties of cell-wall-degrading enzymes produced by Trichoderma reesei Rut C30 on different carbon sources. Appl Biochem Biotechnol 2010, 161:347-364.
• [20]Xiong H, Turunen O, Pastinen O, Leisola M, von Weymarn N: Improved xylanase production by Trichoderma reesei grown on L-arabinose and lactose or D-glucose mixtures. Appl Microbiol Biotechnol 2004, 64:353-358.
• [21]Xiong HR, von Weymarn N, Turunen O, Leisola M, Pastinen O: Xylanase production by Trichoderma reesei Rut C-30 grown on L-arabinose-rich plant hydrolysates. Bioresour Technol 2005, 96:753-759.
• [22]Ballerini D, Desmarquest JP, Pourquié J, Nativel F, Rebeller M: Ethanol production from Lignocellulosics: large scale experimentation and economics. Bioresour Technol 1994, 50:17-23.
• [23]Ike M, Park JY, Tabuse M, Tokuyasu K: Controlled preparation of cellulases with xylanolytic enzymes from Trichoderma reesei (Hypocrea jecorina) by continuous-feed cultivation using soluble sugars. Biosci Biotechnol Biochem 2013, 77:161-166.
• [24]Durand H, Clanet M, Tiraby G: Genetic improvement of Trichoderma reesei for large scale cellulase production. Enzyme Microb Technol 1988, 10:341-346.
• [25]Ghose TK: Measurement of cellulase activities. Pure Appl Chem 1987, 59:257-268.
• [26]Ghose TK, Bisaria VS: Measurement of hemicellulase activities - Part 1: Xylanases. Pure Appl Chem 1987, 59:1739-1752.
• [27]Seiboth B, Herold S, Kubicek CP: Metabolic engineering of inducer formation for cellulase and hemicellulase gene expression in Trichoderma reesei. In Reprogramming Microbial Metabolic Pathways. 64th edition. Edited by Wang X, Chen J, Quinn P. Netherlands: Springer; 2012:367-390.
• [28]Mandels M, Reese ET: Induction of cellulase in fungi by cellobiose. J Bacteriol 1960, 79:816-826.
• [29]Fritscher C, Messner R, Kubicek CP: Cellobiose metabolism and cellobiohydrolase I biosynthesis by Trichoderma reesei. Exp Mycol 1990, 14:405-415.
• [30]Kubicek CP, Messner R, Gruber F, Mandels M, Kubicek-Pranz EM: Triggering of cellulase biosynthesis by cellulose in Trichoderma reesei. Involvement of a constitutive, sophorose-inducible, glucose-inhibited beta-diglucoside permease. J Biol Chem 1993, 268:19364-19368.
• [31]Zhou Q, Xu J, Kou Y, Lv X, Zhang X, Zhao G, Zhang W, Chen G, Liu W: Differential involvement of beta-Glucosidases from Hypocrea jecorina in rapid induction of cellulase genes by cellulose and cellobiose. Eukaryot Cell 2012, 11:1371-1381.
• [32]Morikawa Y, Ohashi T, Mantani O, Okada H: Cellulase induction by lactose in Trichoderma reesei PC-3-7. Appl Microbiol Biotechnol 1995, 44:106-111.
• [33]Ilmen M, Saloheimo A, Onnela ML, Penttila ME: Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei. Appl Environ Microbiol 1997, 63:1298-1306.
• [34]Pakula TM, Salonen K, Uusitalo J, Penttila ME: The effect of specific growth rate on protein synthesis and secretion in the filamentous fungus Trichoderma reesei. Microbiology 2005, 151:135-143.
• [35]Bura R, Chandra R, Saddler J: Influence of xylan on the enzymatic hydrolysis of steam-pretreated corn stover and hybrid poplar. Biotechnol Prog 2009, 25:315-322.
• [36]Hu J, Arantes V, Saddler J: The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes such as xylanase: is it an additive or synergistic effect? Biotechnol Biofuels 2011, 4:36.
• [37]Zhang J, Tang M, Viikari L: Xylans inhibit enzymatic hydrolysis of lignocellulosic materials by cellulases. Bioresour Technol 2012, 121:8-12.
• [38]Benko Z, Siika-Aho M, Viikari L, Reczey K: Evaluation of the role of xyloglucanase in the enzymatic hydrolysis of lignocellulosic substrates. Enzyme Microb Technol 2008, 43:109-114.
• [39]Kumar R, Wyman CE: Effects of cellulase and xylanase enzymes on the deconstruction of solids from pretreatment of poplar by leading technologies. Biotechnol Prog 2009, 25:302-314.
• [40]Shi J, Ebrik MA, Yang B, Garlock RJ, Balan V, Dale BE, Ramesh Pallapolu V, Lee YY, Kim Y, Mosier NS, Ladisch MR, Holtzapple MT, Falls M, Sierra-Ramirez R, Donohoe BS, Vinzant TB, Elander RT, Hames B, Thomas S, Warner RE, Wyman CE: Application of cellulase and hemicellulase to pure xylan, pure cellulose, and switchgrass solids from leading pretreatments. Bioresour Technol 2011, 102:11080-11088.
• [41]Qing Q, Wyman CE: Hydrolysis of different chain length xylooliogmers by cellulase and hemicellulase. Bioresour Technol 2011, 102:1359-1366.
• [42]Qing Q, Yang B, Wyman CE: Xylooligomers are strong inhibitors of cellulose hydrolysis by enzymes. Bioresour Technol 2010, 101:9624-9630.
• [43]Qing Q, Wyman C: Supplementation with xylanase and beta-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover. Biotechnol Biofuels 2011, 4:18.
• [44]Penttilä PA, Várnai A, Pere J, Tammelin T, Salmén L, Siika-aho M, Viikari L, Serimaa R: Xylan as limiting factor in enzymatic hydrolysis of nanocellulose. Bioresour Technol 2013, 129:135-141.
• [45]Várnai A, Huikko L, Pere J, Siika-aho M, Viikari L: Synergistic action of xylanase and mannanase improves the total hydrolysis of softwood. Bioresour Technol 2011, 102:9096-9104.
• [46]Gao D, Uppugundla N, Chundawat S, Yu X, Hermanson S, Gowda K, Brumm P, Mead D, Balan V, Dale B: Hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides. Biotechnol Biofuels 2011, 4:5.
• [47]Billard H, Faraj A, Lopes Ferreira N, Menir S, Heiss-Blanquet S: Optimization of a synthetic mixture composed of major Trichoderma reesei enzymes for the hydrolysis of steam-exploded wheat straw. Biotechnol Biofuels 2012, 5:9.
• [48]Banerjee G, Car S, Scott-Craig JS, Borrusch MS, Walton JD: Rapid optimization of enzyme mixtures for deconstruction of diverse pretreatment/biomass feedstock combinations. Biotechnol Biofuels 2010, 3:22.
• [49]Singhania RR, Patel AK, Sukumaran RK, Larroche C, Pandey A: Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol production. Bioresour Technol 2013, 127:500-507.
• [50]Herpoel-Gimbert I, Margeot A, Dolla A, Jan G, Molle D, Lignon S, Mathis H, Sigoillot JC, Monot F, Asther M: Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains. Biotechnol Biofuels 2008, 1:18.
• [51]Rahman Z, Shida Y, Furukawa T, Suzuki Y, Okada H, Ogasawara W, Morikawa Y: Application of Trichoderma reesei cellulase and xylanase promoters through homologous recombination for enhanced production of extracellular beta-glucosidase i. Biosci Biotechnol Biochem 2009, 73:1083-1089.
• [52]Zhang J, Zhong Y, Zhao X, Wang T: Development of the cellulolytic fungus Trichoderma reesei strain with enhanced beta-glucosidase and filter paper activity using strong artifical cellobiohydrolase 1 promoter. Bioresour Technol 2010, 101:9815-9818.
• [53]Wang B, Xia L: High efficient expression of cellobiase gene from Aspergillus niger in the cells of Trichoderma reesei. Bioresour Technol 2011, 102:4568-4572.
• [54]Nakazawa H, Kawai T, Ida N, Shida Y, Kobayashi Y, Okada H, Tani S, Sumitani JI, Kawaguchi T, Morikawa Y, Ogasawara W: Construction of a recombinant Trichoderma reesei strain expressing Aspergillus aculeatus beta-glucosidase 1 for efficient biomass conversion. Biotechnol Bioeng 2012, 109:92-99.
• [55]Ma L, Zhang J, Zou G, Wang C, Zhou Z: Improvement of cellulase activity in Trichoderma reesei by heterologous expression of a beta-glucosidase gene from Penicillium decumbens. Enzyme Microb Technol 2011, 49:366-371.
• [56]Dashtban M, Qin W: Overexpression of an exotic thermotolerant beta-glucosidase in trichoderma reesei and its significant increase in cellulolytic activity and saccharification of barley straw. Microb Cell Fact 2012, 11:63.
• [57]Ayrinhac C, Margeot A, Ferreira NL, Ben Chaabane F, Monot F, Ravot G, Sonet JM, Fourage L: Improved saccharification of wheat straw for biofuel production using an Engineered Secretome of Trichoderma reesei. Org Process Res Dev 2011, 15:275-278.
• [58]Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 1951, 193:265-275.
• [59]Miller GL: Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959, 31:426-428.
• [60]Xiao Z, Storms R, Tsang A: Microplate-based filter paper assay to measure total cellulase activity. Biotechnol Bioeng 2004, 88:832-837.