Biochemical, functional characterization and application of polysaccharide lytic monoxygenases identified in transcriptome and secretoma studies of filamentous fungi cultivated in sugarcane bagasse

• Grant number: 19/22284-7
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Regular Program Grants
• Duration: August 01, 2020 - February 28, 2022
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal Investigator: Fernando Segato
• Grantee: Fernando Segato
• Host Institution: Escola de Engenharia de Lorena (EEL). Universidade de São Paulo (USP). Lorena , SP, Brazil

Abstract

Brazil is the largest producer of sugarcane in the world and generates a large amount of bagasse and crop residues that could be used in the production of second generation ethanol from cellulose, the main source of glucose in this waste. However, like other lignocellulosic substrates, these materials are extremely recalcitrant, which difficult the access to the cellulose in an economically scenario. Several methods have been studied to decrease the recalcitrance of these materials. Pre-treatments with strong acids and bases, high temperatures are efficient, but they require expensive equipments and high operating costs. Cocktails constituted with efficient enzymes for biomass saccharification are able to act under mild conditions, but with insufficient yield. The major commercial cocktails have been composed of three major types of hydrolytic enzymes for the hydrolysis of cellulose, exoglucanases, endoglucanases and ²-glucosidases. However, enzymes capable of degrading cellulose by oxidative mechanism have recently been described and are known as lytic polysaccharide monooxigenases (LPMOs). These enzymes use copper as a cofactor and in a reaction involving molecular oxygen or hydrogen peroxide, and two electrons from a reducing agent can oxidize the C1 and or C4 carbon of the glycosidic bond of several polysaccharides in addition to cellulose, such as chitin, starch and also xyloglucan. Due to its ability to act in the crystalline regions of cellulose these enzymes can promote a synergic effect with hydrolytic enzymes increasing the yield cocktails in addition with the reduction in the dependence of severe and costly pre-treatments. However, recent studies suggest that there are fine adjustments in LPMOs that make them more specific for a kind of substrate. Therefore, enzymatic cocktails should be developed for specific substrates. Transcriptome and secretome studies carried out by our research group with filamentous fungi (Thielavia terrestris, Myceliophthora thermophila, Aspergillus fumigatus var niveus, Malbranchea pulchella, and Laetiporus sulphureus), cultivated in the presence of sugarcane bagasse as carbon source identified genes encoding AA9 LPMOs that have been expressed at higher levels relative to other LPMOs identified in the genome, indicating that they may be more suitable for the degradation of this specific substrate. Due to the fact that no study has been conducted so far with LPMOs identified under these conditions, this project aims to 1) clone the genes coding for these specific LPMOs 2) express heterologously these genes in the filamentous fungus Aspergillus nidulans 3) characterize the produced LPMOs and 4) investigate its synergistic effect on the breakdown of cellulose with other enzymes for the development of a more efficient cocktail in the saccharification process of sugarcane bagasse. (AU)

Scientific publications

(6)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

VELASCO, JOSMAN; PELLEGRINI, VANESSA DE OLIVEIRA ARNOLDI; SEPULCHRO, ANA GABRIELA VEIGA; KADOWAKI, MARCO ANTONIO SEIKI; SANTO, MELISSA CRISTINA ESPIRITO; POLIKARPOV, IGOR; SEGATO, FERNANDO. Comparative analysis of two recombinant LPMOs from Aspergillus fumigatus and their effects on sugarcane bagasse saccharification. Enzyme and Microbial Technology, v. 144, MAR 2021. (19/22284-7, 15/13684-0, 14/06923-6, 14/18714-2, 19/01165-0, 17/50025-0)

BARRIOS-GUTIERREZ, SOLANGE GRACE; VELEZ-MERCADO, MARTHA INES; ORTEGA, JULIA RODRIGUES; LIMA, AWANA DA SILVA; SARAIVA, ANA LUIZA DA ROCHA FORTES; BERTO, GABRIELA LEILA; SEGATO, FERNANDO. Oxidative Machinery of basidiomycetes as potential enhancers in lignocellulosic biorefineries: A lytic polysaccharide monooxygenases approach. Bioresource Technology, v. 386, p. 11-pg., 2023-10-01. (22/00539-6, 22/04227-9, 14/18714-2, 19/22284-7, 22/04713-0, 21/06679-1)

VELASCO, JOSMAN; SEPULCHRO, ANA GABRIELA V.; HIGASI, PAULA M. R.; PELLEGRINI, VANESSA O. A.; CANNELLA, DAVID; DE OLIVEIRA, LEANDRO CRISTANTE; POLIKARPOV, IGOR; SEGATO, FERNANDO. Light Boosts the Activity of Novel LPMO from Aspergillus fumigatus Leading to Oxidative Cleavage of Cellulose and Hemicellulose. ACS SUSTAINABLE CHEMISTRY & ENGINEERING, v. 10, n. 50, p. 16-pg., 2022-12-08. (19/22284-7, 18/22300-0, 19/01165-0, 21/06679-1, 14/18714-2, 15/13684-0)

BERTO, GABRIELA L.; MATTOS, BRUNO D.; VELASCO, JOSMAN; ZHAO, BIN; SEGATO, FERNANDO; ROJAS, ORLANDO J.; ARANTES, VALDEIR. Endoglucanase effects on energy consumption in the mechanical fibrillation of cellulose fibers into nanocelluloses. International Journal of Biological Macromolecules, v. 243, p. 9-pg., 2023-06-13. (15/02862-5, 19/22284-7, 21/07023-2, 21/06679-1)

CAMARGO, SUELEN; MULINARI, EVANDRO J.; DE ALMEIDA, LEONARDO R.; BERNARDES, AMANDA; PRADE, ROLF A.; GARCIA, WANIUS; SEGATO, FERNANDO; MUNIZ, JOAO R. C.. Functional and structural characterization of an alpha-L-arabinofuranosidase from Thermothielavioides terrestris and its exquisite domain-swapped beta-propeller fold crystal packing. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, v. 1868, n. 12, p. 12-pg., 2020-12-01. (17/16291-5, 16/09152-6, 14/18714-2, 14/50897-0, 19/22284-7, 17/50025-0)

QUEIROZ, SARAH S.; OLIVA, BIANCA; SILVA, TATIANE F.; SEGATO, FERNANDO; FELIPE, MARIA G. A.. Integrated bioinformatics, modelling, and gene expression analysis of the putative pentose transporter from Candida tropicalis during xylose fermentation with and without glucose addition. Applied Microbiology and Biotechnology, v. N/A, p. 20-pg., 2022-06-16. (19/22284-7, 19/27092-9, 14/18714-2, 16/22179-0, 19/06663-8)