A plant host, Nicotiana benthamiana, enables the production and study of fungal lignin-degrading enzymes

Khlystov, Nikita A.; Yoshikuni, Yasuo; Deutsch, Samuel; Sattely, Elizabeth S.

doi:10.1038/s42003-021-02464-9

A plant host, Nicotiana benthamiana, enables the production and study of fungal lignin-degrading enzymes

Journal Article · Wed Sep 01 00:00:00 UTC 2021 · Communications Biology

DOI:https://doi.org/10.1038/s42003-021-02464-9· OSTI ID:1818165

; ; Deutsch, Samuel;

Abstract

Lignin has significant potential as an abundant and renewable source for commodity chemicals yet remains vastly underutilized. Efforts towards engineering a biochemical route to the valorization of lignin are currently limited by the lack of a suitable heterologous host for the production of lignin-degrading enzymes. Here, we show that expression of fungal genes in Nicotiana benthamiana enables production of members from seven major classes of enzymes associated with lignin degradation (23 of 35 tested) in soluble form for direct use in lignin activity assays. We combinatorially characterized a subset of these enzymes in the context of model lignin dimer oxidation, revealing that fine-tuned coupling of peroxide-generators to peroxidases results in more extensive C-C bond cleavage compared to direct addition of peroxide. Comparison of peroxidase isoform activity revealed that the extent of C-C bond cleavage depends on peroxidase identity, suggesting that peroxidases are individually specialized in the context of lignin oxidation. We anticipate the use of N. benthamiana as a platform to rapidly produce a diverse array of fungal lignin-degrading enzymes will facilitate a better understanding of their concerted role in nature and unlock their potential for lignin valorization, including within the plant host itself.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE; USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231; SC0014112

OSTI ID:: 1818165

Alternate ID(s):: OSTI ID: 1825264

Journal Information:: Communications Biology, Journal Name: Communications Biology Journal Issue: 1 Vol. 4; ISSN 2399-3642

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United Kingdom

Language:: English

References (52)

Directed evolution of a secretory leader for the improved expression of heterologous proteins and full-length antibodies in Saccharomyces cerevisiae Rakestraw, J. Andy; Sazinsky, Stephen L.; Piatesi, Andrea Biotechnology and Bioengineering, Vol. 103, Issue 6 https://doi.org/10.1002/bit.22338	journal	August 2009
Functional expression of aryl-alcohol oxidase in Saccharomyces cerevisiae and Pichia pastoris by directed evolution Viña-Gonzalez, Javier; Elbl, Katarina; Ponte, Xavier Biotechnology and Bioengineering, Vol. 115, Issue 7 https://doi.org/10.1002/bit.26585	journal	March 2018
Peroxidases in the genus Nicotiana Sheen, S. J. Theoretical and Applied Genetics, Vol. 40, Issue 1 https://doi.org/10.1007/BF00280982	journal	January 1970
Hydrogen-peroxide-producing system ofPleurotus eryngii involving the extracellular enzyme aryl-alcohol oxidase Guillén, F.; Martínez, A. T.; Martínez, M. J. Applied Microbiology and Biotechnology, Vol. 41, Issue 4 https://doi.org/10.1007/BF00939037	journal	June 1994
Fungal aryl-alcohol oxidase: a peroxide-producing flavoenzyme involved in lignin degradation Hernández-Ortega, Aitor; Ferreira, Patricia; Martínez, Angel T. Applied Microbiology and Biotechnology, Vol. 93, Issue 4 https://doi.org/10.1007/s00253-011-3836-8	journal	January 2012
Biotechnological production of vanillin Priefert, H.; Rabenhorst, J.; Steinbüchel, A. Applied Microbiology and Biotechnology, Vol. 56, Issue 3-4 https://doi.org/10.1007/s002530100687	journal	August 2001
Lignin peroxidase of Phanerochaete chrysosporium Tien, Ming; Kirk, T. Kent Methods in Enzymology, Vol. 161, p. 238-249 https://doi.org/10.1016/0076-6879(88)61025-1	book	January 1988
Ligninolysis by a purified lignin peroxidase Hammel, K. E.; Jensen, K. A.; Mozuch, M. D. Journal of Biological Chemistry, Vol. 268, Issue 17 https://doi.org/10.1016/S0021-9258(18)31385-1	journal	June 1993
Manganese(II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium. Kinetic mechanism and role of chelators. Wariishi, H.; Valli, K.; Gold, M. H. Journal of Biological Chemistry, Vol. 267, Issue 33 https://doi.org/10.1016/S0021-9258(18)35893-9	journal	November 1992
Lignin peroxidase compound III. Mechanism of formation and decomposition. Wariishi, H.; Gold, M. H. Journal of Biological Chemistry, Vol. 265, Issue 4 https://doi.org/10.1016/S0021-9258(19)39941-7	journal	February 1990
Crystal structures of Phanerochaete chrysosporium pyranose 2-oxidase suggest that the N-terminus acts as a propeptide that assists in homotetramer assembly Hassan, Noor; Tan, Tien-Chye; Spadiut, Oliver FEBS Open Bio, Vol. 3, Issue 1 https://doi.org/10.1016/j.fob.2013.10.010	journal	January 2013
Role of fungal peroxidases in biological ligninolysis Hammel, K.; Cullen, D. Current Opinion in Plant Biology, Vol. 11, Issue 3 https://doi.org/10.1016/j.pbi.2008.02.003	journal	June 2008
Functional Expression of Horseradish Peroxidase in E. coli by Directed Evolution Lin, Z.; Thorsen, T.; Arnold, F.H. Biotechnology Progress, Vol. 15, Issue 3, p. 467-471 https://doi.org/10.1021/bp990037r	journal	June 1999
The Oxidation of Methoxybenzenes Zweig, Arnold.; Hodgson, William G.; Jura, Walter H. Journal of the American Chemical Society, Vol. 86, Issue 19 https://doi.org/10.1021/ja01073a043	journal	October 1964
Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78 Martinez, Diego; Larrondo, Luis F.; Putnam, Nik Nature Biotechnology, Vol. 22, Issue 6 https://doi.org/10.1038/nbt967	journal	May 2004
Reducing biomass recalcitrance by heterologous expression of a bacterial peroxidase in tobacco (Nicotiana benthamiana) Ligaba-Osena, Ayalew; Hankoua, Bertrand; DiMarco, Kay Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/s41598-017-16909-x	journal	December 2017
Development of novel assays for lignin degradation: comparative analysis of bacterial and fungal lignin degraders Ahmad, Mark; Taylor, Charles R.; Pink, David Molecular BioSystems, Vol. 6, Issue 5 https://doi.org/10.1039/b908966g	journal	January 2010
Lignocellulosic biomass: a sustainable platform for the production of bio-based chemicals and polymers Isikgor, Furkan H.; Becer, C. Remzi Polymer Chemistry, Vol. 6, Issue 25 https://doi.org/10.1039/c5py00263j	journal	January 2015
Ligninase of Phanerochaete chrysosporium. Mechanism of its degradation of the non-phenolic arylglycerol β-aryl ether substructure of lignin Kirk, T. K.; Tien, M.; Kersten, P. J. Biochemical Journal, Vol. 236, Issue 1 https://doi.org/10.1042/bj2360279	journal	May 1986
Comparison of lignin peroxidase, horseradish peroxidase and laccase in the oxidation of methoxybenzenes Kersten, P. J.; Kalyanaraman, B.; Hammel, K. E. Biochemical Journal, Vol. 268, Issue 2 https://doi.org/10.1042/bj2680475	journal	June 1990
Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases Michener, J. K.; Nielsen, J.; Smolke, C. D. Proceedings of the National Academy of Sciences, Vol. 109, Issue 47 https://doi.org/10.1073/pnas.1212287109	journal	November 2012
Extensive sampling of basidiomycete genomes demonstrates inadequacy of the white-rot/brown-rot paradigm for wood decay fungi Riley, Robert; Salamov, Asaf A.; Brown, Daren W. Proceedings of the National Academy of Sciences, Vol. 111, Issue 27 https://doi.org/10.1073/pnas.1400592111	journal	June 2014
Evolutionary convergence in lignin-degrading enzymes Ayuso-Fernández, Iván; Ruiz-Dueñas, Francisco J.; Martínez, Angel T. Proceedings of the National Academy of Sciences, Vol. 115, Issue 25 https://doi.org/10.1073/pnas.1802555115	journal	June 2018
Cellulosome assembly revealed by the crystal structure of the cohesin-dockerin complex Carvalho, A. L.; Dias, F. M. V.; Prates, J. A. M. Proceedings of the National Academy of Sciences, Vol. 100, Issue 24 https://doi.org/10.1073/pnas.1936124100	journal	November 2003
Glyoxal oxidase of Phanerochaete chrysosporium: its characterization and activation by lignin peroxidase. Kersten, P. J. Proceedings of the National Academy of Sciences, Vol. 87, Issue 8 https://doi.org/10.1073/pnas.87.8.2936	journal	April 1990
Oxidation of a Tetrameric Nonphenolic Lignin Model Compound by Lignin Peroxidase Mester, Tünde; Ambert-Balay, Katia; Ciofi-Baffoni, Simone Journal of Biological Chemistry, Vol. 276, Issue 25 https://doi.org/10.1074/jbc.M010739200	journal	April 2001
Lignin-degrading Peroxidases from Genome of Selective Ligninolytic Fungus Ceriporiopsis subvermispora Fernández-Fueyo, Elena; Ruiz-Dueñas, Francisco J.; Miki, Yuta Journal of Biological Chemistry, Vol. 287, Issue 20 https://doi.org/10.1074/jbc.M112.356378	journal	March 2012
Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A TRANSIENT-STATE KINETICS, DIRECTED MUTAGENESIS, EPR, AND NMR STUDY Sáez-Jiménez, Verónica; Baratto, Maria Camilla; Pogni, Rebecca Journal of Biological Chemistry, Vol. 290, Issue 38 https://doi.org/10.1074/jbc.M115.665919	journal	August 2015
Progress and obstacles in the production and application of recombinant lignin-degrading peroxidases Lambertz, Camilla; Ece, Selin; Fischer, Rainer Bioengineered, Vol. 7, Issue 3 https://doi.org/10.1080/21655979.2016.1191705	journal	April 2016
Lignin degradation: microorganisms, enzymes involved, genomes analysis and evolution Janusz, Grzegorz; Pawlik, Anna; Sulej, Justyna FEMS Microbiology Reviews, Vol. 41, Issue 6 https://doi.org/10.1093/femsre/fux049	journal	October 2017
Multiple Molecular Forms of Peroxidases and Esterases Among Nicotiana Species and Amphiploids Smith, H. H.; Hamill, D. E.; Weaver, E. A. Journal of Heredity, Vol. 61, Issue 5 https://doi.org/10.1093/oxfordjournals.jhered.a108085	journal	September 1970
Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris Morawski, Birgit; Lin, Zhanglin; Cirino, Pat Protein Engineering, Design and Selection, Vol. 13, Issue 5 https://doi.org/10.1093/protein/13.5.377	journal	May 2000
Nicotiana benthamiana : Its History and Future as a Model for Plant–Pathogen Interactions Goodin, Michael M.; Zaitlin, David; Naidu, Rayapati A. Molecular Plant-Microbe Interactions, Vol. 21, Issue 8 https://doi.org/10.1094/MPMI-21-8-1015	journal	August 2008
Purification and Catalytic Properties of Two Manganese Peroxidase Isoenzymes from Pleurotus eryngii Martinez, Maria J.; Ruiz-Duenas, Francisco J.; Guillen, Francisco European Journal of Biochemistry, Vol. 237, Issue 2 https://doi.org/10.1111/j.1432-1033.1996.0424k.x	journal	April 1996
pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants Sainsbury, Frank; Thuenemann, Eva C.; Lomonossoff, George P. Plant Biotechnology Journal, Vol. 7, Issue 7 https://doi.org/10.1111/j.1467-7652.2009.00434.x	journal	September 2009
Polymerisation of lignins by ligninases from Phanerochaete chrysosporium Haemmerli, Stephan D.; Leisola, Matti S. A.; Fiechter, Armin FEMS Microbiology Letters, Vol. 35, Issue 1 https://doi.org/10.1111/j.1574-6968.1986.tb01494.x	journal	June 1986
Degradation of lignin β-aryl ether units in Arabidopsis thaliana expressing LigD , LigF and LigG from Sphingomonas paucimobilis SYK-6 Mnich, Ewelina; Vanholme, Ruben; Oyarce, Paula Plant Biotechnology Journal, Vol. 15, Issue 5 https://doi.org/10.1111/pbi.12655	journal	November 2016
HEx: A heterologous expression platform for the discovery of fungal natural products Harvey, Colin J. B.; Tang, Mancheng; Schlecht, Ulrich Science Advances, Vol. 4, Issue 4 https://doi.org/10.1126/sciadv.aar5459	journal	April 2018
The Paleozoic Origin of Enzymatic Lignin Decomposition Reconstructed from 31 Fungal Genomes Floudas, Dimitrios; Binder, Manfred; Riley, Robert Science, Vol. 336, Issue 6089 https://doi.org/10.1126/science.1221748	journal	June 2012
Lignin Valorization: Improving Lignin Processing in the Biorefinery Ragauskas, A. J.; Beckham, G. T.; Biddy, M. J. Science, Vol. 344, Issue 6185, p. 1246843-1246843 https://doi.org/10.1126/science.1246843	journal	May 2014
Lignin-Degrading Enzyme from the Hymenomycete Phanerochaete chrysosporium Burds Tien, M.; Kirk, T. K. Science, Vol. 221, Issue 4611 https://doi.org/10.1126/science.221.4611.661	journal	August 1983
Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone Lau, Warren; Sattely, Elizabeth S. Science, Vol. 349, Issue 6253 https://doi.org/10.1126/science.aac7202	journal	September 2015
Moderate Expression of SEC16 Increases Protein Secretion by Saccharomyces cerevisiae Bao, Jichen; Huang, Mingtao; Petranovic, Dina Applied and Environmental Microbiology, Vol. 83, Issue 14 https://doi.org/10.1128/AEM.03400-16	journal	July 2017
Studies on the Production of Fungal Peroxidases in Aspergillus niger Conesa, Ana; van den Hondel, Cees A. M. J. J.; Punt, Peter J. Applied and Environmental Microbiology, Vol. 66, Issue 7 https://doi.org/10.1128/AEM.66.7.3016-3023.2000	journal	July 2000
Rapid Degradation of Isolated Lignins by Phanerochaete chrysosporium Ulmer, Duane C.; Leisola, Matti S. A.; Schmidt, Brigitte H. Applied and Environmental Microbiology, Vol. 45, Issue 6 https://doi.org/10.1128/aem.45.6.1795-1801.1983	journal	June 1983
Pyranose Oxidase, a Major Source of H ₂ O ₂ during Wood Degradation by Phanerochaete chrysosporium, Trametes versicolor, and Oudemansiella mucida Daniel, Geoffrey; Volc, Jindrich; Kubatova, Elena Applied and Environmental Microbiology, Vol. 60, Issue 7 https://doi.org/10.1128/aem.60.7.2524-2532.1994	journal	July 1994
Lignin Peroxidase Isozymes from Phanerochaete chrysosporium Can Be Enzymatically Dephosphorylated Rothschild, N.; Hadar, Y.; Dosoretz, C. G. Applied and Environmental Microbiology, Vol. 63, Issue 3 https://doi.org/10.1128/aem.63.3.857-861.1997	journal	March 1997
Ligninolytic peroxidase genes in the oyster mushroom genome: heterologous expression, molecular structure, catalytic and stability properties, and lignin-degrading ability Fernández-Fueyo, Elena; Ruiz-Dueñas, Francisco J.; Martínez, María Biotechnology for Biofuels, Vol. 7, Issue 1 https://doi.org/10.1186/1754-6834-7-2	journal	January 2014
New methods and reagents in organic synthesis. 46. Trimethylsilyldiazomethane: A convenient reagent for the O-methylation of phenols and enols. Aoyama, Toyohiko; Terasawa, Satomi; Sudo, Kimio Chemical and Pharmaceutical Bulletin, Vol. 32, Issue 9 https://doi.org/10.1248/cpb.32.3759	journal	January 1984
Pyridine Hemochromagen Assay for Determining the Concentration of Heme in Purified Protein Solutions Barr, Ian; Guo, Feng BIO-PROTOCOL, Vol. 5, Issue 18 https://doi.org/10.21769/bioprotoc.1594	journal	January 2015
Lignin Engineering in Forest Trees Chanoca, Alexandra; de Vries, Lisanne; Boerjan, Wout Frontiers in Plant Science, Vol. 10 https://doi.org/10.3389/fpls.2019.00912	journal	July 2019
The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example O'Leary, Brendan M.; Rico, Arantza; McCraw, Sarah Journal of Visualized Experiments, Issue 94 https://doi.org/10.3791/52113	journal	January 2014

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