Dataset Information

Comparative analysis of Chrysoporthe cubensis exoproteomes and their specificity for saccharification of sugarcane bagasse

ABSTRACT: The phytopathogenic fungus Chrysoporthe cubensis is a relevant source of lignocellulolytic enzymes. This work aimed to compare the profile of lignocellulose- degrading proteins secreted by C. cubensis grown under semi-solid state fermentation using wheat bran and sugarcane bagasse. The proteins from the fungus extract grown in wheat bran (WBE) and sugarcane bagasse (SBE) were qualitative and quantitatively analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). Label-free proteomic analysis of WBE and SBE showed that the fungus produced a spectrum of carbohydrate-active enzymes (CAZymes) with exclusive characteristics from each extract. While SBE resulted in an enzymatic profile directed towards the depolymerization of cellulose, the enzymes in WBE were more adaptable to the degradation of biomass rich in hemicellulose and other non-lignocellulosic polymers. Saccharification of alkaline pre-treated sugarcane bagasse with SBE promoted glucose release higher than commercial cocktails (8.11 g L -1 ), while WBE promoted the higher release of xylose (5.71 g L -1 ). Our results allowed an in-depth knowledge of the complex set of enzymes secreted by C. cubensis responsible for its high lignocellulolytic activity and still provided the identification of promising target proteins for biotechnological applications in the context of biorefinery.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Fungi Chrysoporthe Cubensis

SUBMITTER: William Borges

LAB HEAD: William Castro-Borges

PROVIDER: PXD046075 | Pride | 2024-01-26

REPOSITORIES: Pride

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Publications

Comparative analysis of Chrysoporthe cubensis exoproteomes and their specificity for saccharification of sugarcane bagasse.

Tavares Murillo Peterlini MP Morgan Túlio T Gomes Riziane Ferreira RF Mendes Jean Pierre Rocha JPR Castro-Borges William W Maitan-Alfenas Gabriela Piccolo GP Guimarães Valéria Monteze VM

Enzyme and microbial technology 20231122

The phytopathogenic fungus Chrysoporthe cubensis is a relevant source of lignocellulolytic enzymes. This work aimed to compare the profile of lignocellulose-degrading proteins secreted by C. cubensis grown under semi-solid state fermentation using wheat bran (WB) and sugarcane bagasse (SB). The exoproteomes of the fungus grown in wheat bran (WBE) and sugarcane bagasse (SBE) were qualitative and quantitatively analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC- ...[more]

PMID: 38043248

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Project description:Background: Wood-decay basidiomycetes are effective for the degradation of highly lignified and recalcitrant substrates. Brown-rot strain produces carbohydrate-active enzymes involved in the degradation of lignocellulosic materials, along with a non-enzymatic mechanism, via Fenton reaction. Differences in the lignocellulose metabolism occurring even among closely related brown-rots are not completely understood, bringing attention to a multi-omics study of brown-rot L. sulphureus. Results: To evidence the oxidative-hydrolytic mechanism, the Laetiporus sulphureus ATCC 52600 genome was sequenced and the response to lignocellulosic substrates was analyzed by transcriptomics and proteomics. The transcriptomic profile in response to a short cultivation period on in natura sugarcane bagasse revealed 128 out of 12,802 upregulated transcripts. The high upregulated transcripts included a set of redox enzymes along with hemicellulases. The exoproteome in response to extended-time cultivation with Avicel, and steam-exploded sugarcane bagasse, sugarcane straw, and Eucalyptus grandis revealed 121 proteins. Contrasting to the mainly oxidative profile observed in the transcriptome, the secretomes showed a diverse hydrolytic repertoire including constitutive cellulases and hemicellulases, in addition to 19 proteins upregulated relative to glucose. The secretome produced on sugarcane bagasse was evaluated in the saccharification of pretreated sugarcane straw by supplementing a commercial cocktail. Additionally, growth analysis revealed that L. sulphureus ATCC 52600 has higher efficiency to assimilate glucose than other mono and disaccharides. Conclusion: This study shows the singularity of L. sulphureus ATCC 52600 relative to other Polyporales brown-rots, regarding the presence of cellobiohydrolase and peroxidase class II. The multi-omic analysis reinforces the oxidative-hydrolytic metabolism involved in lignocellulose deconstruction, providing insights into the overall mechanisms as well as specific proteins of each step.

Project description:The thermophilic fungus Malbranchea cinnamomea belongs to the order of Onygenales and is a promising source of thermostable, industrially relevant biocatalysts, as it can grow at temperatures of more than 50°C and is able to utilise many different types of plant biomass. Enzymes from M. cinnamomea that have been characterised so far include an α-amylase, an α-glucosidase, xylanases and an alkaline β-1,3-1,4-glucanase (lichenase), all of which have been reported to have temperature optima between 50°C and 80°C. With this study, we complement the knowledge of the enzymatic repertoire of M. cinnamomea with a transcriptomic analysis of strain FCH 10.5 to provide a more comprehensive view of its lignocellulolytic enzyme system. Genes differentially expressed during growth on two different polymeric substrates, beechwood xylan and wheat bran, point to differences in the fungal response to the deconstruction of a hardwood hemicellulose (beechwood xylan) and a cereal hemicellulose (wheat bran). The data presented here will form the basis for a systematic exploration of the full potential of this fungus as a source of thermostable enzymes. We sequenced the genome of M. cinnamomea FCH 10.5, which was isolated from the compost of a waste treatment plant in Hanoi, Vietnam (PMC5604768, https://www.ncbi.nlm.nih.gov/nuccore/FQSS02000000). For RNAseq, the fungus was grown on three different carbon sources (glucose, wheat bran, beechwood xylan) at 50°C. Mycelium was harvested after 4h and 48h and RNA was extracted. For RNAseq analysis, the RNA of 4h and 48h samples was mixed 1:1, to get information about both early- and late-response genes during growth on the different carbon sources. Two independent duplicate experiments were done for each substrate. Total RNA was extracted using TRIzol (Invitrogen) and chloroform, and further purified with the RNeasy Plant RNA Kit with on-column DNAse digestion (QIAGEN). The quality of the purified RNA was verified by agarose gel electrophoresis, Nanodrop (Thermo Scientific) and Qubit (Life Technologies). The NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) was used to process the samples according to the manufacturer’s instructions. Briefly, mRNA was isolated from total RNA using oligo-dT magnetic beads and used to synthesise cDNA. The cDNA was ligated with sequencing adapters and PCR amplified. The quality and yield after sample preparation were determined with the Fragment Analyzer (Advanced Analytical). The size of the resulting products was consistent with the expected size distribution (a broad peak between 300-500 bp). Standard Illumina primers for Illumina cBot and HiSeq 2500, and the HiSeq control software HCS v2.2.58 were used according to manufacturer’s protocols for clustering and DNA sequencing with a concentration of 16.0 pM. The Illumina data analysis pipelines RTA v1.18.64 and Bcl2fastq v2.17 were used for image analysis, base calling, and quality check. Sequencing was performed on an Illumina HiSeq 2500 sequencer. The assembled genome from the DNA sequencing was used as a reference to map the reads using the packages Tophat (v2.0.14. Linux_x86_64) and Bowtie (v2-2.1.0) with a default mismatch rate of 2%. The frequency with which a read was mapped on a transcript was determined based on the mapped locations from the alignment. To normalise for transcript length, fpkm (fragments per kilobase of transcript per million mapped reads) were calculated. For differential expression analysis, the read counts were loaded into the DESeq package v 1.10.1. Genes were considered differentially expressed if they showed a log2 fold change ≥ 1 and the adjusted p-value was < 0.05.

Dataset Information

Comparative analysis of Chrysoporthe cubensis exoproteomes and their specificity for saccharification of sugarcane bagasse

Publications

Comparative analysis of Chrysoporthe cubensis exoproteomes and their specificity for saccharification of sugarcane bagasse.

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