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

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 whiterot 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 R CTec2. However, the white-rot fungi secretomes were less efficient than Cellic R CTec2 during hydrolysis of more recalcitrant substrates, such as acid or alkaline sulfite-pretreated sugarcane bagasse, likely because Cellic R CTec2 contains an excess of CBHs compared with the white-rot fungi secretomes. General comparison of the white-rot fungi secretomes

Systems biology-guided understanding of white-rot fungi for biotechnological applications: A review

“…(2020a) revealed that the enzyme pools released for the degradation of cellulose, hemicellulose, and pectin were conserved in three different Pycnoporus spp. growing on cellulose, wheat straw, and aspen, whereas Machado et al. (2020) showed that the specific production of orthologous enzymes of cellobiohydrolases Cel7C and Cel7D in P. chrysosporium and Trametes versicolor was conserved in cellulose-containing cultivations.…”

“…In this review, we therefore conducted an additional analysis to identify trends of secreted enzymes across different types of substrates and WRF. We collected 92 secretomic datasets from 31 publications and spanning 19 WRF species, all of which have published genomes ( Couturier et al., 2015 ; del Cerro et al., 2021 ; Fernández-Fueyo et al., 2012 ; Fernández-Fueyo et al., 2016 ; Floudas et al., 2012 ; Hori et al., 2014a ; Hori et al., 2014b ; Iwata et al., 2021 ; Kuuskeri et al., 2016 ; Levasseur et al., 2014 ; Liu et al., 2020 ; Machado et al., 2020 ; Mahajan and Master, 2010 ; Manavalan et al., 2011 ; Miyauchi et al., 2020a ; Miyauchi et al., 2017 ; Miyauchi et al., 2018 ; Moody et al., 2018 ; Peña et al., 2021 ; Presley et al., 2018 ; Ravalason et al., 2008 ; Rytioja et al., 2017 ; Salvachúa et al., 2016 ; Salvachúa et al., 2013a ; Vanden Wymelenberg et al., 2009 ; Vanden Wymelenberg et al., 2010 ; Vanden Wymelenberg et al., 2011 ; Xiao et al., 2019 ; Xie et al., 2021 ; Yoav et al., 2018 ; Zhu et al., 2016 ). Metadata for each dataset can be found in Table S3 , and detailed analytical pipelines can be found in Transparent Methods Supplemental File .…”

“…Another challenge is the lack of consensus in analyzing proteomics data. Some publications utilize relative abundance of total peptide count in each sample for protein quantitation ( Kuuskeri et al., 2016 ; Machado et al., 2020 ; Rytioja et al., 2017 ; Yoav et al., 2018 ). Some others use peptide spectral count as a proxy for relative quantitation ( Fernández-Fueyo et al., 2012 , 2016 ; Peña et al., 2021 ; Presley et al., 2018 ).…”

Produção integrada de monossacarídeos e lignosulfonatos a partir de bagaço de cana-de-açúcar

“…Os pré-tratamentos biológicos utilizam fungos de degradação branca, como Phanerochaete chrysosporium, Lentinula edodes, Pleurotus ostreatus e Ceriporiopsis subvermispora que apresentam a capacidade de degradar lignina seletivamente e preservar parte dos polissacarídeos presentes na biomassa. Apesar de pouco intensivos em termos do emprego de reagentes químicos, estes pré-tratamentos são menos utilizados, pois possuem como desvantagens a necessidade do emprego de processos de biodegradação de lignocelulósicos em grande escala mantidos por longos períodos de pré-tratamento (SALVACHÚA et al, 2011;DONG et al, 2013;MACHADO;FERRAZ, 2017).…”

“…A hidrólise enzimática foi realizada inicialmente em pequena escala em diferentes consistências de sólidos: 2% e 5% (m/v). Nestes ensaios foram utilizados, respectivamente, 20 mg e 50 mg de bagaço de cana pré-tratado (base seca) moído a 20 mesh e suspensos em um volume final de reação de 1,0 mL de solução tampão acetato de sódio 50 mM, pH 4,8 e 0,01% de azida sódica, contendo o preparado comercial de celulases Cellic Ctec2 (131 ± 4 FPU/mL) (Cellic CTec2 -SAE0020, SIGMA) numa carga de 10 FPU/g de sólidos (10,5 mg de proteína/g de material pré-tratado) (MACHADO et al, 2020). As condições experimentais pré-estabelecidas foram agitação de 120 rpm a 45 °C por 72 h.…”