The Secretome of Phanerochaete chrysosporium and Trametes versicolor Grown in Microcrystalline Cellulose and Use of the Enzymes for Hydrolysis of Lignocellulosic Materials.

Angela S Machado,Fernando Segato,André Ferraz,Fernanda Valadares,Tatiane F Silva,Adriane M F Milagres

doi:10.3389/fbioe.2020.00826

Angela S Machado, Fernando Segato + Show 4 more

Open Access

https://doi.org/10.3389/fbioe.2020.00826

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Abstract

The ability of white-rot fungi to degrade polysaccharides in lignified plant cell walls makes them a suitable reservoir for CAZyme prospects. However, to date, CAZymes from these species are barely studied, which limits their use in the set of choices for biomass conversion in modern biorefineries. The current work joined secretome studies of two representative white-rot fungi, Phanerochaete chrysosporium and Trametes versicolor, with expression analysis of cellobiohydrolase (CBH) genes, and use of the secretomes to evaluate enzymatic conversion of simple and complex sugarcane-derived substrates. Avicel was used to induce secretion of high levels of CBHs in the extracellular medium. A total of 56 and 58 proteins were identified in cultures of P. chrysosporium and T. versicolor, respectively, with 78–86% of these proteins corresponding to plant cell wall degrading enzymes (cellulolytic, hemicellulolytic, pectinolytic, esterase, and auxiliary activity). CBHI predominated among the plant cell wall degrading enzymes, corresponding to 47 and 34% of the detected proteins in P. chrysosporium and T. versicolor, respectively, which confirms that Avicel is an efficient CBH inducer in white-rot fungi. The induction by Avicel of genes encoding CBHs (cel) was supported by high expression levels of cel7D and cel7C in P. chrysosporium and T. versicolor, respectively. Both white-rot fungi secretomes enabled hydrolysis experiments at 10 FPU/g substrate, despite the varied proportions of CBHs and other enzymes present in each case. When low recalcitrance sugarcane pith was used as a substrate, P. chrysosporium and T. versicolor secretomes performed similarly to Cellic® CTec2. However, the white-rot fungi secretomes were less efficient than Cellic® CTec2 during hydrolysis of more recalcitrant substrates, such as acid or alkaline sulfite-pretreated sugarcane bagasse, likely because Cellic® CTec2 contains an excess of CBHs compared with the white-rot fungi secretomes. General comparison of the white-rot fungi secretomes highlighted T. versicolor enzymes for providing high glucan conversions, even at lower proportion of CBHs, probably because the other enzymes present in this secretome and CBHs lacking carbohydrate-binding modules compensate for problems associated with unproductive binding to lignin.

Highlights

Hydrolytic enzymes are major constituents of commercial enzymatic cocktails used in plant biomass conversion of pretreated lignocellulosic materials (Payne et al, 2015; Adsul et al, 2020)
Secretomes produced by P. chrysosporium and T. versicolor grown in microcrystalline cellulose (Avicel) were characterized and used for plant biomass saccharification
The high CBH titers detected in Cellic R Ctec2 (Table 1) seem essential for high glucan conversion to glucose, since the reference cocktail performed significantly better than the P. chrysosporium and T. versicolor secretomes (Figure 5)

Summary

Introduction

Hydrolytic enzymes are major constituents of commercial enzymatic cocktails used in plant biomass conversion of pretreated lignocellulosic materials (Payne et al, 2015; Adsul et al, 2020). Most of these enzymatic cocktails are developed for polysaccharide conversion to monosaccharides and contain diverse cellulolytic and hemicellulolytic enzymes, including glucoside hydrolases (GHs) and auxiliary activity enzymes, such as lytic polysaccharide monooxygenases (LPMOs), that cause oxidative cleavage of polysaccharides (Payne et al, 2015; Adsul et al, 2020) Both enzyme groups can contain carbohydrate-binding modules (CBMs) associated with the catalytic domain that enable efficient adsorption of the enzymes on insoluble polysaccharides, improving the enzymatic catalysis efficiency (Payne et al, 2015). Residual lignin contained in pretreated materials can cause unproductive binding of GHs and LPMOs, especially because CBMs adsorb on lignin surfaces (Rahikainen et al, 2013; Siqueira et al, 2017; Santos et al, 2019)

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