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Discovery and Expression of Thermostable LPMOs from Thermophilic Fungi for Producing Efficient Lignocellulolytic Enzyme Cocktails

  • Dhruv Agrawal
  • Neha Basotra
  • Venkatesh Balan
  • Adrian Tsang
  • Bhupinder Singh ChadhaEmail author
Article
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Abstract

In this study, two novel thermostable lytic polysaccharide monooxygenases (LPMOs) were cloned from thermophilic fungus Scytalidium thermophilum (PMO9D_SCYTH) and Malbranchea cinnamomea (PMO9D_MALCI) and expressed in the methylotrophic yeast Pichia pastoris X33. The purified PMO9D_SCYTH was active at 60 °C (t1/2 = 60.58 h, pH 7.0), whereas, PMO9D_MALCI was optimally active at 50 °C (t1/2 = 144 h, pH 7.0). The respective catalytic efficiency (kcat/Km) of PMO9D_SCYTH and PMO9D_MALCI determined against avicel in presence of H2O2 was (6.58 × 10-3 and 1.79 × 10-3 mg-1 ml min-1) and carboxy-methylcellulose (CMC) (1.52 × 10-1 and 2.62 × 10-2 mg-1 ml min-1). The HRMS analysis of products obtained after hydrolysis of avicel and CMC showed the presence of both C1 and C4 oxidized oligosaccharides, in addition to phylogenetic tree constructed with other characterized type 1 and 3 LPMOs demonstrated that both LPMOs belongs to type-3 family of AA9s. The release of sugars during saccharification of acid/alkali pretreated sugarcane bagasse and rice straw was enhanced upon replacing one part of commercial enzyme Cellic CTec2 with these LPMOs.

Keywords

Thermophilic fungi Heterologous expression Lytic polysaccharide monooxygenases (LPMOs) H2O2 Hydrolysis 
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Notes

Acknowledgments

This research was supported by the Department of Biotechnology, India. Projects (BT/PR15271/PBD/26/509/2015) entitled “Bioprospecting for novel lignocellulolytic gylcosyl hydrolases and auxiliary enzymes from diverse thermophilic fungal strains using proteome based approaches” and (BT/PR31115/PBD/26/766/2019) entitled “Novel concepts for developing efficient cellulolytic cocktail for hydrolysis of bio-refinery relevant pre-treated lignocellulosics” are highly acknowledged. Dr. Balan would like to thank the University of Houston for small equipment grant and State of Texas for his startup funds.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no competing interests.

Supplementary material

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References

  1. 1.
    Aguilar, D. L., Rodríguez-Jasso, R. M., Zanuso, E., Lara-Flores, A. A., Aguilar, C. N., Sanchez, A., & Ruiz, H. A. (2018). Operational strategies for enzymatic hydrolysis in a biorefinery. In S. Kumar & R. K. Sani (Eds.), Biorefining of Biomass to Biofuels (pp. 223–248). Cham: Springer.  https://doi.org/10.1007/978-3-319-67678-4_10.CrossRefGoogle Scholar
  2. 2.
    Merino, S. T., & Cherry, J. (2007). Progress and challenges in enzyme development for biomass utilization. In L. Olsson (Ed.), Biofuels (pp. 95–120). Berlin, Heidelberg: Springer.  https://doi.org/10.1007/10_2007_066.CrossRefGoogle Scholar
  3. 3.
    Fang, X., Shen, Y., Zhao, J., Bao, X., & Qu, Y. (2010). Status and prospect of lignocellulosic bioethanol production in China. Bioresour Technol, 101(13), 4814–4819.  https://doi.org/10.1016/j.biortech.2009.11.050.CrossRefPubMedGoogle Scholar
  4. 4.
    Fujii, T., Fang, X., Inoue, H., Murakami, K., & Sawayama, S. (2009). Enzymatic hydrolyzing performance of Acremonium cellulolyticus and Trichoderma reesei against three lignocellulosic materials. Biotechnol Biofuels, 2(1), 24.  https://doi.org/10.1186/1754-6834-2-24.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    McClendon, S. D., Batth, T., Petzold, C. J., Adams, P. D., Simmons, B. A., & Singer, S. W. (2012). Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions. Biotechnol Biofuels, 5(1), 54.  https://doi.org/10.1186/1754-6834-5-54.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Visser, H., Joosten, V., Punt, P. J., Gusakov, A. V., Olson, P. T., Joosten, R., Bartels, J., Visser, J., Sinitsyn, A. P., Emalfarb, M. A., & Verdoes, J. C. (2011). Development of a mature fungal technology and production platform for industrial enzymes based on a Myceliophthora thermophila isolate, previously known as Chrysosporium lucknowense C1. Ind Biotechnol, 7, 214–223.  https://doi.org/10.1089/ind.2011.7.214.CrossRefGoogle Scholar
  7. 7.
    Balan, V. (2014). Current challenges in commercially producing biofuels from lignocellulosic biomass. ISRN Biotechnol, 2014, 31.  https://doi.org/10.1155/2014/463074.CrossRefGoogle Scholar
  8. 8.
    Chundawat, S. P., Uppugundla, N., Gao, D., Curran, P. G., Balan, V., & Dale, B. E. (2017). Shotgun approach to increasing enzymatic saccharification yields of ammonia fiber expansion pretreated cellulosic biomass. Front Energy Res, 7, 9.  https://doi.org/10.3389/fenrg.2017.00009.CrossRefGoogle Scholar
  9. 9.
    Vaaje-Kolstad, G., Westereng, B., Horn, S. J., Liu, Z., Zhai, H., Sørlie, M., & Eijsink, V. G. (2010). An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides. Science., 330(6001), 219–222.  https://doi.org/10.1126/science.1192231.CrossRefPubMedGoogle Scholar
  10. 10.
    Beckham, G. T., Matthews, J. F., Peters, B., Bomble, Y. J., Himmel, M. E., & Crowley, M. F. (2011). Molecular-level origins of biomass recalcitrance: decrystallization free energies for four common cellulose polymorphs. J Phys Chem B, 115(14), 4118–4127.  https://doi.org/10.1021/jp1106394.CrossRefPubMedGoogle Scholar
  11. 11.
    Horn, S. J., Vaaje-Kolstad, G., Westereng, B., & Eijsink, V. (2012). Novel enzymes for the degradation of cellulose. Biotechnol Biofuels, 5(1), 45.  https://doi.org/10.1186/1754-6834-5-45.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Gao, D., Uppugundla, N., Chundawat, S. P., Yu, X., Hermanson, S., Gowda, K., Brumm, P., Mead, D., Balan, V., & Dale, B. E. (2011). Hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides. Biotechnol Biofuels, 4, 5.  https://doi.org/10.1186/1754-6834-4-5.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Forsberg, Z., Sørlie, M., Petrović, D., Courtade, G., Aachmann, F. L., Vaaje-Kolstad, G., Bissaro, B., Røhr, Å. K., & Eijsink, V. G. (2019). Polysaccharide degradation by lytic polysaccharide monooxygenases. Curr Opin Struct Biol, 59, 54–64.  https://doi.org/10.1016/j.sbi.2019.02.015.CrossRefPubMedGoogle Scholar
  14. 14.
    Kim, S., Ståhlberg, J., Sandgren, M., Paton, R. S., & Beckham, G. T. (2014). Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl, oxygen-rebound mechanism. Proc Natl Acad Sci U S A, 111(1), 149–154.  https://doi.org/10.1073/pnas.1316609111.CrossRefPubMedGoogle Scholar
  15. 15.
    Bissaro, B., Røhr, Å. K., Müller, G., Chylenski, P., Skaugen, M., Horn, S. J., Vaaje-kolstad, G., & Eijsink, V. G. H. (2017). Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2. Nat Chem Biol, 13, 1123–1128.  https://doi.org/10.1038/nchembio.2470.CrossRefPubMedGoogle Scholar
  16. 16.
    Frommhagen, M., Koetsier, M. J., Westphal, A. H., Visser, J., Hinz, S. W., Vincken, J. P., Van Berkel, W. J., Kabel, M. A., & Gruppen, H. (2016). Lytic polysaccharide monooxygenases from Myceliophthora thermophila C1 differ in substrate preference and reducing agent specificity. Biotechnol Biofuels, 9(1), 186.  https://doi.org/10.1186/s13068-016-0594-y.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Quinlan, R. J., Sweeney, M. D., Leggio, L. L., Otten, H., Poulsen, J. C. N., Johansen, K. S., Krogh, K. B., Jørgensen, C. I., Tovborg, M., Anthonsen, A., & Tryfona, T. (2011). Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. Proc Natl Acad Sci U S A, 108(37), 15079–15084.  https://doi.org/10.1073/pnas.1105776108.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Kracher, D., Andlar, M., Furtmüller, P. G., & Ludwig, R. (2018). Active-site copper reduction promotes substrate binding of fungal lytic polysaccharide monooxygenase and reduces stability. J Biol Chem, 293(5), 1676–1687.  https://doi.org/10.1074/jbc.RA117.000109.CrossRefPubMedGoogle Scholar
  19. 19.
    Phillips, C. M., Beeson IV, W. T., Cate, J. H., & Marletta, M. A. (2011). Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa. ACS Chem Biol, 6, 1399–1406.  https://doi.org/10.1021/cb200351y.CrossRefPubMedGoogle Scholar
  20. 20.
    Beeson, W. T., Phillips, C. M., Cate, J. H., & Marletta, M. A. (2011). Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases. J Am Chem Soc, 134, 890–892.  https://doi.org/10.1021/ja210657t.CrossRefPubMedGoogle Scholar
  21. 21.
    Mahajan, C., Chadha, B. S., Nain, L., & Kaur, A. (2014). Evaluation of glycosyl hydrolases from thermophilic fungi for their potential in bioconversion of alkali and biologically treated Parthenium hysterophorus weed and rice straw into ethanol. Bioresour Technol, 163, 300–307.  https://doi.org/10.1016/j.biortech.2014.04.057.CrossRefPubMedGoogle Scholar
  22. 22.
    Basotra, N., Dhiman, S. S., Agrawal, D., Sani, R. K., Tsang, A., & Chadha, B. S. (2019). Characterization of a novel lytic polysaccharide monooxygenase from Malbranchea cinnamomea exhibiting dual catalytic behavior. Carbohydr Res, 478, 46–53.  https://doi.org/10.1016/j.carres.2019.04.006.CrossRefPubMedGoogle Scholar
  23. 23.
    Rai, R., Kaur, B., Singh, S., Di Falco, M., Tsang, A., & Chadha, B. S. (2016). Evaluation of secretome of highly efficient lignocellulolytic Penicillium sp. Dal 5 isolated from rhizosphere of conifers. Bioresour Technol, 216, 958–967.  https://doi.org/10.1016/j.biortech.2016.06.040.CrossRefPubMedGoogle Scholar
  24. 24.
    Carninci, P., Nishiyama, Y., Westover, A., Itoh, M., Nagaoka, S., Sasaki, N., Okazaki, Y., Muramatsu, M., & Hayashizaki, Y. (1998). Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA. Proc Natl Acad Sci U S A, 95(2), 520–524.  https://doi.org/10.1073/pnas.95.2.520.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Henke, W., Herdel, K., Jung, K., Schnorr, D., & Loening, S. A. (1997). Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res, 25, 3957–3958.  https://doi.org/10.1093/nar/25.19.3957.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Cregg, J. M., Tolstorukov, I., Kusari, A., Sunga, J., Madden, K., & Chappell, T. (2009). In R. R. Burgess & M. P. Deutscher (Eds.), Methods in enzymology (pp. 169–189). Cambridge: Elsevier, Academic Press.  https://doi.org/10.1016/S0076-6879(09)63013-5.CrossRefGoogle Scholar
  27. 27.
    Saloheimo, M., Nakari-SetäLä, T., Tenkanen, M., & Penttilä, M. (1997). cDNA cloning of a Trichoderma reesei cellulase and demonstration of endoglucanase activity by expression in yeast. Eur J Biochem, 249(2), 584–591.  https://doi.org/10.1111/j.1432-1033.1997.00584.x.CrossRefPubMedGoogle Scholar
  28. 28.
    Karlsson, J., Saloheimo, M., Siika-aho, M., Tenkanen, M., Penttilä, M., & Tjerneld, F. (2001). Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei. Eur J Biochem, 268(24), 6498–6507.  https://doi.org/10.1046/j.0014-2956.2001.02605.x.CrossRefPubMedGoogle Scholar
  29. 29.
    Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem, 31, 426–428.  https://doi.org/10.1021/ac60147a030.CrossRefGoogle Scholar
  30. 30.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J Biol Chem, 193, 265–275 http://www.jbc.org/content/193/1/265.citation.PubMedGoogle Scholar
  31. 31.
    Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature., 227, 680 https://www.nature.com/articles/227680a0.CrossRefGoogle Scholar
  32. 32.
    Zhang, R., Liu, Y., Zhang, Y., Feng, D., Hou, S., Guo, W., Niu, K., Jiang, Y., Han, L., Sindhu, L., & Fang, X. (2019). Identification of a thermostable fungal lytic polysaccharide monooxygenase and evaluation of its effect on lignocellulosic degradation. Appl Microbial Biot, 1-12(14), 5739–5750.  https://doi.org/10.1007/s00253-019-09928-3.CrossRefGoogle Scholar
  33. 33.
    Bey, M., Zhou, S., Poidevin, L., Henrissat, B., Coutinho, P. M., Berrin, J. G., & Sigoillot, J. C. (2013). Cello-oligosaccharide oxidation reveals differences between two lytic polysaccharide monooxygenases (family GH61) from Podospora anserine. Appl Environ Microbiol, 79, 488–496.  https://doi.org/10.1128/AEM.02942-12.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Kuusk, S., Bissaro, B., Kuusk, P., Forsberg, Z., Eijsink, V. G., Sørlie, M., & Väljamäe, P. (2018). Kinetics of H2O2-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenases. J Biol Chem, 293(2), 523–531.  https://doi.org/10.1074/jbc.M117.817593.CrossRefPubMedGoogle Scholar
  35. 35.
    Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol, 35(6), 1547–1549.  https://doi.org/10.1093/molbev/msy096.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Kittl, R., Kracher, D., Burgstaller, D., Haltrich, D., & Ludwig, R. (2012). Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay. Biotechnol Biofuels, 5(1), 79.  https://doi.org/10.1186/1754-6834-5-79.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Breslmayr, E., Hanžek, M., Hanrahan, A., Leitner, C., Kittl, R., Šantek, B., Oostenbrink, C., & Ludwig, R. (2018). A fast and sensitive activity assay for lytic polysaccharide monooxygenase. Biotechnol Biofuels, 11(1), 79–13.  https://doi.org/10.1186/s13068-018-1063-6.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Langston, J. A., Shaghasi, T., Abbate, E., Xu, F., Vlasenko, E., & Sweeney, M. D. (2011). Oxidoreductive cellulose depolymerization by the enzymes cellobiose dehydrogenase and glycoside hydrolase 61. Appl Environ Microbiol, 77(19), 7007–7015.  https://doi.org/10.1128/AEM.05815-11.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Sharma, M., Chadha, B. S., & Saini, H. S. (2010). Purification and characterization of two thermostable xylanases from Malbranchea flava active under alkaline conditions. Bioresour Technol, 101(22), 8834–8842.  https://doi.org/10.1016/j.biortech.2010.06.071.CrossRefPubMedGoogle Scholar
  40. 40.
    Aachmann, F. L., Sørlie, M., Skjåk-Bræk, G., Eijsink, V. G., & Vaaje-Kolstad, G. (2012). NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions. Proc Natl Acad Sci U S A, 109(46), 18779–18784.  https://doi.org/10.1073/pnas.1208822109.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Semenova, M. V., Gusakov, A. V., Volkov, P. V., Matys, V. Y., Nemashkalov, V. A., Telitsin, V. D., Rozhkova, A. M., & Sinitsyn, A. P. (2019). Enhancement of the enzymatic cellulose saccharification by Penicillium verruculosum multienzyme cocktails containing homologously overexpressed lytic polysaccharide monooxygenases. Mol Biol Rep, 15(2), 1–8.  https://doi.org/10.1007/s11033-019-04693-y.CrossRefGoogle Scholar
  42. 42.
    Scheller, H. V., & Ulvskov, P. (2010). Hemicelluloses. Annu Rev Plant Biol, 4, 61.  https://doi.org/10.1146/annurev-arplant-042809-112315.CrossRefGoogle Scholar
  43. 43.
    Agger, J. W., Isaksen, T., Várnai, A., Vidal-Melgosa, S., Willats, W. G., Ludwig, R., Horn, S. J., Eijsink, V. G., & Westereng, B. (2014). Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation. Proc Natl Acad Sci U S A, 111(17), 6287–6292.  https://doi.org/10.1073/pnas.1323629111.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Bennati-Granier, C., Garajova, S., Champion, C., Grisel, S., Haon, M., Zhou, S., Fanuel, M., Ropartz, D., Rogniaux, H., Gimbert, I., & Record, E. (2015). Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserine. Biotechnol Biofuels, 8, 90.  https://doi.org/10.1186/s13068-015-0274-3.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Morgenstern, I., Powlowski, J., Ishmael, N., Darmond, C., Marqueteau, S., Moisan, M. C., Quenneville, G., & Tsang, A. (2012). A molecular phylogeny of thermophilic fungi. Fungal Biol, 116, 489–502.  https://doi.org/10.1016/j.funbio.2012.01.010.CrossRefPubMedGoogle Scholar
  46. 46.
    Sharma, M., Chadha, B. S., Kaur, M., Ghatora, S. K., & Saini, H. S. (2008). Molecular characterization of multiple xylanase producing thermophilic/thermotolerant fungi isolated from composting materials. Lett Appl Microbiol, 46(5), 526–535.  https://doi.org/10.1111/j.1472-765X.2008.02357.x.CrossRefPubMedGoogle Scholar
  47. 47.
    Müller, G., Várnai, A., Johansen, K. S., Eijsink, V. G., & Horn, S. J. (2015). Harnessing the potential of LPMO-containing cellulase cocktails poses new demands on processing conditions. Biotechnol Biofuels, 8, 187.  https://doi.org/10.1186/s13068-015-0376-y.CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Jagadeeswaran, G., Gainey, L., Prade, R., & Mort, A. J. (2016). 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, 100(10), 4535–4547.  https://doi.org/10.1007/s00253-016-7505-9.CrossRefPubMedGoogle Scholar
  49. 49.
    Jung, S., Song, Y., Kim, H. M., & Bae, H. J. (2015). Enhanced lignocellulosic biomass hydrolysis by oxidative lytic polysaccharide monooxygenases (LPMOs) GH61 from Gloeophyllum trabeum. Enzym Microb Technol, 77, 38–45.  https://doi.org/10.1016/j.enzmictec.2015.05.006.CrossRefGoogle Scholar

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Authors and Affiliations

  1. 1.Department of MicrobiologyGuru Nanak Dev UniversityAmritsarIndia
  2. 2.Department Engineering Technology, Biotechnology Program, College of TechnologyUniversity of HoustonHoustonUSA
  3. 3.Center for Structural and Functional GenomicsConcordia UniversityQuebecCanada

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