Lytic Polysaccharide Monooxygenase from Aspergillus fumigatus can Improve Enzymatic Cocktail Activity During Sugarcane Bagasse Hydrolysis

Author(s): Paula Fagundes de Gouvêa, Luis Eduardo Gerolamo, Aline Vianna Bernardi, Lucas Matheus Soares Pereira, Sergio Akira Uyemura, Taisa Magnani Dinamarco*.

Journal Name: Protein & Peptide Letters

Volume 26 , Issue 5 , 2019

Become EABM
Become Reviewer

Graphical Abstract:


Background: Lytic Polysaccharide Monooxygenases (LPMOs) are auxiliary accessory enzymes that act synergistically with cellulases and which are increasingly being used in secondgeneration bioethanol production from biomasses. Several LPMOs have been identified in various filamentous fungi, including Aspergillus fumigatus. However, many LPMOs have not been characterized yet.

Objective: To report the role of uncharacterized A. fumigatus AfAA9_B LPMO.

Methods: qRT-PCR analysis was employed to analyze the LPMO gene expression profile in different carbon sources. The gene encoding an AfAA9_B (Afu4g07850) was cloned into the vector pET- 28a(+), expressed in the E. coli strain RosettaTM (DE3) pLysS, and purified by a Ni2+-nitrilotriacetic (Ni-NTA) agarose resin. To evaluate the specific LPMO activity, the purified protein peroxidase activity was assessed. The auxiliary LPMO activity was investigated by the synergistic activity in Celluclast 1.5L enzymatic cocktail.

Results: LPMO was highly induced in complex biomass like sugarcane bagasse (SEB), Avicel® PH-101, and CM-cellulose. The LPMO gene encoded a protein comprising 250 amino acids, without a CBM domain. After protein purification, the AfAA9_B molecular mass estimated by SDSPAGE was 35 kDa. The purified protein specific peroxidase activity was 8.33 ± 1.9 U g-1. Upon addition to Celluclast 1.5L, Avicel® PH-101 and SEB hydrolysis increased by 18% and 22%, respectively.

Conclusion: A. fumigatus LPMO is a promising candidate to enhance the currently available enzymatic cocktail and can therefore be used in second-generation ethanol production.

Keywords: Lytic polysaccharide monooxygenases, Aspergillus fumigatus, AA9 LPMO, sugarcane bagasse, biomass hydrolysis, bioethanol production.

Sheldon, R.A. Engineering a more sustainable world through catalysis and green chemistry. J. R. Soc. Interface, 2016, 13, 20160087.
Santos, F.A.; De Queiróz, J.H.; Colodette, J.L.; Fernandes, S.A.; Guimaraes, V.M.; Rezende, S.T. Potencial da palha de cana-de-açúcar para produção de etanol. Quim. Nova, 2012, 35, 1004-1010.
Farzad, S.; Mandegari, M.A.; Guo, M.; Haigh, K.F.; Shah, N.; Görgens, J.F. Multi-product biorefineries from lignocelluloses: A pathway to revitalisation of the sugar industry? Biotechnol. Biofuels, 2017, 10, 87.
Guo, Z.P.; Duquesne, S.; Bozonnet, S.; Nicaud, J.M.; Marty, A.; O’Donohue, M.J. Expressing accessory proteins in cellulolytic Yarrowia lipolytica to improve the conversion yield of recalcitrant cellulose. Biotechnol. Biofuels, 2017, 10, 298.
Glass, N.L.; Schmoll, M.; Cate, J.H.D.; Coradetti, S. Plant cell wall deconstruction by Ascomycete fungi. Annu. Rev. Microbiol., 2013, 67, 477-498.
Wang, Y.; Fan, C.; Hu, H.; Li, Y.; Sun, D.; Wang, Y.; Peng, L. Genetic modification of plant cell walls to enhance biomass yield and biofuel production in bioenergy crops. Biotechnol. Adv., 2016, 34, 997-1017.
Sun, Y.; Cheng, J. Hydrolysis of lignocellulosic materials for ethanol production: A review. Bioresour. Technol., 2002, 83(1), 1-11.
Souza, G.M.; Victoria, R.L.; Joly, C.A.; Verdade, L.M. Bioenergy and Sustainability: Bridging the gaps; SCOPE: Paris, 2015.
Cota, J.; Corrêa, T.L.R.; Damásio, A.R.L.; Diogo, J.A.; Hoffmam, Z.B.; Garcia, W.; Oliveira, L.C.; Prade, R.A.; Squina, F.M. Comparative analysis of three hyperthermophilic GH1 and GH3 family members with industrial potential. N. Biotechnol., 2015, 32, 13-20.
Carvalho, W.; Canilha, L.; Ferraz, A.; Ferreira, M. Uma visão sobre a estrutura, composição e biodegradação da madeira. Quim. Nova, 2009, 32, 2191-2195.
Whitaker, J.R.; Stauffer, C.E. Principles of enzymology for the food sciences, 2nd ed; CRC Press: New York, 1994.
Busk, P.K.; Lange, L. Classification of fungal and bacterial lytic polysaccharide monooxygenases. BMC Genomics, 2015, 16, 368.
Müller, G.; Chylenski, P.; Bissaro, B.; Eijsink, V.G.H.; Horn, S.J. The impact of hydrogen peroxide supply on LPMO activity and overall saccharification efficiency of a commercial cellulase cocktail. Biotechnol. Biofuels, 2018, 11, 209.
Vaaje-kolstad, G. An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides. Science, 2010, 330, 219-222.
Quinlan, R.J.; Sweeney, M.D.; Lo Leggio, L.; Otten, H.; Poulsen, J-C.N.; Johansen, K.S.; Krogh, K.B.R.M.; Jorgensen, C.I.; Tovborg, M.; Anthonsen, A.; Tryfona, T.; Walter, C.P.; Dupree, P.; Xu, F.; Davies, G.J.; Walton, P.H. Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. Proc. Natl. Acad. Sci., 2011, 108, 15079-15084.
Bomble, Y.J.; Lin, C.Y.; Amore, A.; Wei, H.; Holwerda, E.K.; Ciesielski, P.N.; Donohoe, B.S.; Decker, S.R.; Lynd, L.R.; Himmel, M.E. Lignocellulose deconstruction in the biosphere. Curr. Opin. Chem. Biol., 2017, 41, 61-70.
Agger, J.W.; Isaksen, T.; Varnai, A.; Vidal-Melgosa, S.; Willats, W.G.T.; Ludwig, R.; Horn, S.J.; Eijsink, V.G.H.; Westereng, B. Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation. Proc. Natl. Acad. Sci., 2014, 111, 6287-6292.
Berrin, J.G.; Rosso, M.N.; Abou Hachem, M. Fungal secretomics to probe the biological functions of lytic polysaccharide monooxygenases. Carbohydr. Res., 2017, 448, 155-160.
Monclaro, A.V.; Filho, E.X.F. Fungal lytic polysaccharide monooxygenases from family AA9: Recent developments and application in lignocelullose breakdown. Int. J. Biol. Macromol., 2017, 102, 771-778.
Bennati-Granier, C.; Garajova, S.; Champion, C.; Grisel, S.; Haon, M.; Zhou, S.; Fanuel, M.; Ropartz, D.; Rogniaux, H.; Gimbert, I.; Record, E.; Berrin, J.G. Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina. Biotechnol. Biofuels, 2015, 8, 90.
Vu, V.V.; Beeson, W.T.; Span, E.A.; Farquhar, E.R.; Marletta, M.A. A family of starch-active polysaccharide monooxygenases. Proc. Natl. Acad. Sci., 2014, 111, 13822-13827.
Couturier, M.; Ladevèze, S.; Sulzenbacher, G.; Ciano, L.; Fanuel, M.; Moreau, C.; Villares, A.; Cathala, B.; Chaspoul, F.; Frandsen, K.E.; Labourel, A.; Herpoël-Gimbert, I.; Grisel, S.; Haon, M.; Lenfant, N.; Rogniaux, H.; Ropartz, D.; Davies, G.J.; Rosso, M-N.N.; Walton, P.H.; Henrissat, B.; Berrin, J-G.G. Lytic xylan oxidases from wood-decay fungi unlock biomass degradation. Nat. Chem. Biol., 2018, 14, 306-310.
Cantarel, B.L.; Coutinho, P.M.; Rancurel, C.; Bernard, T.; Lombard, V.; Henrissat, B. The Carbohydrate-Active EnZymes database (CAZy): An expert resource for glycogenomics. Nucleic Acids Res., 2009, 37, D233-D238.
Levasseur, A.; Drula, E.; Lombard, V.; Coutinho, P.M.; Henrissat, B. Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes. Biotechnol. Biofuels, 2013, 6, 41.
Sabbadin, F.; Hemsworth, G.R.; Ciano, L.; Henrissat, B.; Dupree, P.; Tryfona, T.; Marques, R.D.S.S.; Sweeney, S.T.; Besser, K.; Elias, L.; Pesante, G.; Li, Y.; Dowle, A.A.; Bates, R.; Gomez, L.D.; Simister, R.; Davies, G.J.; Walton, P.H.; Bruce, N.C.; Mcqueen-Mason, S.J. An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion. Nat. Commun., 2018, 9, 756.
Cragg, S.M.; Beckham, G.T.; Bruce, N.C.; Bugg, T.D.H.; Distel, D.L.; Dupree, P.; Etxabe, A.G.; Goodell, B.S.; Jellison, J.; McGeehan, J.E.; McQueen-Mason, S.J.; Schnorr, K.; Walton, P.H.; Watts, J.E.M.; Zimmer, M. Lignocellulose degradation mechanisms across the tree of life. Curr. Opin. in Chem. Biol., 2015, 29, 108-119.
Sharma, R.K.; Arora, D.S. Fungal degradation of lignocellulosic residues: An aspect of improved nutritive quality. Crit. Rev. Microbiol., 2015, 41, 52-60.
Borin, G.P.; Sanchez, C.C.; De Souza, A.P.; De Santana, E.S.; De Souza, A.T.; Leme, A.F.P.; Squina, F.M.; Buckeridge, M.; Goldman, G.H.; De Castro Oliveira, J.V. Comparative secretome analysis of Trichoderma reesei and Aspergillus niger during growth on sugarcane biomass. PLoS One, 2015, 10, e0129275.
de Gouvêa, P.F.; Bernardi, A.V.; Gerolamo, L.E.; Santos, E.S.; Riano-Pachon, D.; Uyemura, S.A.; Dinamarco, T.M. Transcriptome and secretome analysis of Aspergillus fumigatus in the presence of sugarcane bagasse. BMC Genomics, 2018, 19, 232.
Lo Leggio, L.; Weihe, C.D.; N., Poulsen J.-C.; Sweeney, M.; Rasmussen, F.; Lin, J.; De Maria, L.; Wogulis, M. Structure of a lytic polysaccharide monooxygenase from Aspergillus fumigatus and an engineered thermostable variant. Carbohydr. Res., 2018, 469, 55-59.
Semighini, C.P.; Marins, M.; Goldman, M.H.S.; Goldman, G.H. Quantitative analysis of the relative transcript levels of ABC transporter Atr genes in Aspergillus nidulans by real-time reverse transcription-PCR assay. Appl. Environ. Microbiol., 2002, 68, 1351-1357.
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, 402-408.
Schmittgen, T.D.; Livak, K.J. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc., 2008, 3, 1101-1108.
Hammond, J.B.; Kruger, N.J. The bradford method for protein quantitation. Methods Mol. Biol., 1988, 3, 25-32.
Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227, 680-685.
Towbin, H.; Staehelin, T.; Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci., 1979, 76, 4350-4354.
Breslmayr, E.; Hanžek, M.; Hanrahan, A.; Leitner, C.; Kittl, R.; Šantek, B.; Oostenbrink, C.; Ludwig, R. A fast and sensitive activity assay for lytic polysaccharide monooxygenase. Biotechnol. Biofuels, 2018, 11, 79.
Miller, G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem., 1959, 31, 426-428.
Kim, I.J.; Seo, N.; An, H.J.; Kim, J.H.; Harris, P.V.; Kim, K.H. Type-dependent action modes of TtAA9E and TaAA9A acting on cellulose and differently pretreated lignocellulosic substrates. Biotechnol. Biofuels, 2017, 10, 46.
Frommhagen, M.; Sforza, S.; Westphal, A.H.; Visser, J.; Hinz, S.W.A.; Koetsier, M.J.; Van Berkel, W.J.H.; Gruppen, H.; Kabel, M.A. Discovery of the combined oxidative cleavage of plant xylan and cellulose by a new fungal polysaccharide monooxygenase. Biotechnol. Biofuels, 2015, 8, 101.
Hemsworth, G.R.; Johnston, E.M.; Davies, G.J.; Walton, P.H. Lytic polysaccharide monooxygenases in biomass conversion. Trends Biotechnol., 2015, 33, 747-761.
Rodrigues, K.B.; Macêdo, J.K.A.; Teixeira, T.; Barros, J.S.; Araújo, A.C.B.; Santos, F.P.; Quirino, B.F.; Brasil, B.S.A.F.; Salum, T.F.C.; Abdelnur, P.V.; Fávaro, L.C.L. Recombinant expression of Thermobifida fusca E7 LPMO in Pichia pastoris and Escherichia coli and their functional characterization. Carbohydr. Res., 2017, 448, 175-181.
Zhang, H.; Zhao, Y.; Cao, H.; Mou, G.; Yin, H. Expression and characterization of a lytic polysaccharide monooxygenase from Bacillus thuringiensis. Int. J. Biol. Macromol., 2015, 79, 72-75.
Liu, B.; Olson, Å.; Wu, M.; Broberg, A.; Sandgren, M. Biochemical studies of two lytic polysaccharide monooxygenasesfrom the white-rot fungus Heterobasidion irregulare and their roles in lignocellulose degradation. PLoS One, 2017, 12, e0189479.
Chylenski, P.; Petrović, D.M.; Müller, G.; Dahlström, M.; Bengtsson, O.; Lersch, M.; Siika-Aho, M.; Horn, S.J.; Eijsink, V.G.H. Enzymatic degradation of sulfite-pulped softwoods and the role of LPMOs. Biotechnol. Biofuels, 2017, 10, 177.
Nekiunaite, L.; Arntzen, M.; Svensson, B.; Vaaje-Kolstad, G.; Hachem, M.A. Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by Aspergillus nidulans grown on different starches. Biotechnol. Biofuels, 2016, 19, 187.
Miao, Y.; Li, J.; Xiao, Z.; Shen, Q.; Zhang, R. Characterization and identification of the xylanolytic enzymes from Aspergillus fumigatus Z5. BMC Microbiol., 2015, 15, 126.
Emy, G.; Midorikawa, O.; Correa, C.L.; Noronha, E.F.; Ximenes, E.; Filho, F.; Togawa, R.C.; Mota, M.; Silva-junior, O.B.; Grynberg, P.; Neil, R.; Miller, G.; Silva, R. Analysis of the transcriptome in Aspergillus tamarii during enzymatic degradation of sugarcane bagasse. Front. Bioeng. Biotechnol., 2018, 6, 123.
Hüttner, S.; Nguyen, T.T.; Granchi, Z.; Chin-A-Woeng, T.; Ahrén, D.; Larsbrink, J.; Thanh, V.N.; Olsson, L. Combined genome and transcriptome sequencing to investigate the plant cell wall degrading enzyme system in the thermophilic fungus Malbranchea cinnamomea. Biotechnol. Biofuels, 2017, 10, 265.
Petrovi, D.M.; Bissaro, B.; Chylenski, P.; Skaugen, M.; Sørlie, M.; Jensen, M.S.; Aachmann, F.L.; Courtade, G.; Várnai, A.; Eijsink, V.G.H. Methylation of the N-terminal histidine protects a lytic polysaccharide monooxygenase from auto-oxidative inactivation. Protein Sci., 2018, 27, 16360-1650.
Kojima, Y.; Várnai, A.; Ishida, T.; Sunagawa, N.; Petrovic, D.M.; Igarashi, K.; Jellison, J.; Goodell, B.; Alfredsen, G.; Westereng, B.; Eijsink, V.G.H.; Yoshida, M. A lytic polysaccharide monooxygenase with broad xyloglucan specificity from the brown-rot fungus Gloeophyllum trabeum and its action on cellulose-xyloglucan complexes. Appl. Environ. Microbiol., 2016, 82, 6557-6572.
Miao, Y.; Liu, D.; Li, G.; Li, P.; Xu, Y.; Shen, Q.; Zhang, R. Genome-wide transcriptomic analysis of a superior biomass-degrading strain of A. fumigatus revealed active lignocellulose-degrading genes. BMC Genomics, 2015, 16, 459.
Yang, B.; Wyman, C.E. Effect of xylan and lignin removal by batch and flowthrough pretreatment on the enzymatic digestibility of corn stover cellulose. Biotechnol. Bioeng., 2004, 86, 88-95.
Wong, D.D.W.S.; Chan, V.J.; McCormack, A.A.; Batt, S.B. A novel xyloglucan-specific endo-β-1,4-glucanase: Biochemical properties and inhibition studies. Appl. Microbiol. Biotechnol., 2010, 86, 1463-1471.
Adav, S.S.; Ravindran, A.; Sze, S.K. Quantitative proteomic study of Aspergillus fumigatus secretome revealed deamidation of secretory enzymes. J. Proteomics, 2015, 119, 154-168.
Frommhagen, M.; Koetsier, M.J.; Westphal, A.H.; Visser, J.; Hinz, S.W.A.; Vincken, J.P.; Van Berkel, W.J.H.; Kabel, M.A.; Gruppen, H. Lytic polysaccharide monooxygenases from Myceliophthora thermophila C1 differ in substrate preference and reducing agent specificity. Biotechnol. Biofuels, 2016, 9, 186.
Jagadeeswaran, G.; Gainey, L.; Prade, R.; Mort, A.J. A family of AA9 lytic polysaccharide monooxygenases in Aspergillus nidulans is differentially regulated by multiple substrates and at least one is active on cellulose and xyloglucan. Appl. Microbiol. Biotechnol., 2016, 100, 4535-4547.
Crouch, L.I.; Labourel, A.; Walton, P.H.; Davies, G.J.; Gilbert, H.J. The contribution of non-catalytic carbohydrate binding modules to the activity of lytic polysaccharide monooxygenases. J. Biol. Chem., 2016, 291, 7439-7449.
Harris, P.V.; Welner, D.; McFarland, K.C.; Re, E.; Navarro Poulsen, J.C.; Brown, K.; Salbo, R.; Ding, H.; Vlasenko, E.; Merino, S.; Xu, F.; Cherry, J.; Larsen, S.; Lo Leggio, L. Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: Structure and function of a large, enigmatic family. Biochemistry, 2010, 49, 3305-3316.
Jagadeeswaran, G.; Gainey, L.; Mort, A.J. An AA9-LPMO containing a CBM1 domain in Aspergillus nidulans is active on cellulose and cleaves cello-oligosaccharides. AMB Express, 2018, 8, 171.
Vuong, T.V.; Liu, B.; Sandgren, M.; Master, E.R. Microplate-based detection of lytic polysaccharide monooxygenase activity by fluorescence-labeling of insoluble oxidized products. Biomacromolecules, 2017, 18, 610-616.
Chabbert, B.; Habrant, A.; Herbaut, M.; Foulon, L.; Aguié-Béghin, V.; Garajova, S.; Grisel, S.; Bennati-Granier, C.; Gimbert-Herpoël, I.; Jamme, F.; Réfrégiers, M.; Sandt, C.; Berrin, J.G.; Paës, G. Action of lytic polysaccharide monooxygenase on plant tissue is governed by cellular type. Sci. Rep., 2017, 7, 17792.
Pierce, B.C.; Agger, J.W.; Zhang, Z.; Wichmann, J.; Meyer, A.S. A comparative study on the activity of fungal lytic polysaccharide monooxygenases for the depolymerization of cellulose in soybean spent flakes. Carbohydr. Res., 2017, 449, 85-94.
Jung, S.; Song, Y.; Kim, H.M.; Bae, H.J. Enhanced lignocellulosic biomass hydrolysis by oxidative Lytic Polysaccharide Monooxygenases (LPMOs) GH61 from Gloeophyllum trabeum. Enz. Mic. Technol., 2015, 77, 38-45.
Kim, I.J.; Nam, K.H.; Yun, E.J.; Kim, S.; Youn, H.J.; Lee, H.J.; Choi, I.G.; Kim, K.H. Optimization of synergism of a recombinant auxiliary activity 9 from Chaetomium globosum with cellulase in cellulose hydrolysis. Appl. Microbiol. Biotechnol., 2015, 99, 8537-8547.
Bernardi, A.V.; Gouvêa, P.F. De; Gerolamo, L.E.; Yonamine, D.K.; Lourdes, L.De; Balico, D.L.; Uyemura, S.A. Functional characterization of GH7 endo-1,4-β-glucanase from Aspergillus fumigatus and its potential industrial application. Protein Expr. Purif., 2018, 150, 1-11.
Karnaouri, A.; Muraleedharan, M.N.; Dimarogona, M.; Topakas, E.; Rova, U.; Sandgren, M.; Christakopoulos, P. Recombinant expression of thermostable processive MtEG5 endoglucanase and its synergism with MtLPMO from Myceliophthora thermophila during the hydrolysis of lignocellulosic substrates. Biotechnol. Biofuels, 2017, 10, 126.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2019
Page: [377 - 385]
Pages: 9
DOI: 10.2174/0929866526666190228163629
Price: $65

Article Metrics

PDF: 26