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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 type:Program for Research on Bioenergy (BIOEN) - Regular Program Grants
Duration: August 01, 2020 - July 31, 2022
Field of knowledge:Biological Sciences - Biochemistry - Enzymology
Principal Investigator:Fernando Segato
Grantee:Fernando Segato
Home 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)