Special Issue: “Peter Biely, A Pioneering Researcher in the Enzymology of Plant Biomass Degradation”

As it has been outlined on the website of the Special Issue entitled “Peter Biely, a pioneering researcher in the enzymology of plant biomass degradation” in the journal Molecules (section Macromolecular Chemistry, ISSN 1420-3049), plant biomass is a key renewable resource [...]

Novel fungal metal-dependent GH54 α-L-arabinofuranosidase: expanded substrate specificity and potential use for plant biomass degradation

Trichoderma genus fungi present great potential for the production of carbohydrate-active enzymes (CAZYmes), including glycoside hydrolase (GH) family members. From a renewability perspective, CAZYmes can be biotechnologically exploited to convert plant biomass into free sugars for the production of advanced biofuels and other high-value chemicals. GH54 is an attractive enzyme family for biotechnological applications because many GH54 enzymes are bifunctional. Thus, GH54 enzymes are interesting targets in the search for new enzymes for use in industrial processes such as plant biomass conversion. Herein, a novel metal-dependent GH54 arabinofuranosidase (ThABF) from the cellulolytic fungus Trichoderma harzianum was identified and biochemically characterized. Initial in silico searches were performed to identify the GH54 sequence. Next, the gene was cloned and heterologously overexpressed in Escherichia coli. The recombinant protein was purified, and the enzyme’s biochemical and biophysical properties were assessed. The GH54 members show wide functional diversity and specifically remove plant cell decorations including arabinose and galactose, in the presence of a metallic cofactor. Plant cell wall decoration have a major impact on lignocellulosic substrate conversion into high-value chemicals. These results expand the known functional diversity within the GH54 family, showing the potential of a novel arabinofuranosidase for plant biomass degradation.

“Integrative genomic analysis of the bioprospection of regulators and accessory enzymes associated with cellulose degradation in a filamentous fungus (Trichoderma harzianum)”

Background Unveiling fungal genome structure and function reveals the potential biotechnological use of fungi. Trichoderma harzianum is a powerful CAZyme-producing fungus. We studied the genomic regions in T. harzianum IOC3844 containing CAZyme genes, transcription factors and transporters. Results We used bioinformatics tools to mine the T. harzianum genome for potential genomics, transcriptomics, and exoproteomics data and coexpression networks. The DNA was sequenced by PacBio SMRT technology for multiomics data analysis and integration. In total, 1676 genes were annotated in the genomic regions analyzed; 222 were identified as CAZymes in T. harzianum IOC3844. When comparing transcriptome data under cellulose or glucose conditions, 114 genes were differentially expressed in cellulose, with 51 being CAZymes. CLR2, a transcription factor physically and phylogenetically conserved in Trichoderma spp., was differentially expressed under cellulose conditions. The genes induced/repressed under cellulose conditions included those important for plant biomass degradation, including CIP2 of the CE15 family and a copper-dependent LPMO of the AA9 family. Conclusions Our results provide new insights into the relationship between genomic organization and hydrolytic enzyme expression and regulation in T. harzianum IOC3844. Our results can improve plant biomass degradation, which is fundamental for developing more efficient strains and/or enzymatic cocktails to produce hydrolytic enzymes.

The synergistic actions of hydrolytic genes reveal the mechanism of Trichoderma harzianum for cellulose degradation

Bioprospecting genes and proteins related to plant biomass degradation is an attractive approach for the identification of target genes for biotechnological purposes, especially genes with potential applications in the biorefinery industry that can enhance second-generation ethanol production technology. Trichoderma harzianum is a potential candidate for cellulolytic enzyme prospection and production. Herein, the enzymatic activities, transcriptome, exoproteome, and coexpression networks of the T. harzianum strain CBMAI-0179 were examined under biomass degradation conditions. We used RNA-Seq to identify differentially expressed genes (DEGs) and carbohydrate-active enzyme (CAZyme) genes related to plant biomass degradation and compared them with the genes of strains from congeneric species (T. harzianum IOC-3844 and T. atroviride CBMAI-0020). T. harzianum CBMAI-0179 harbors strain- and treatment-specific CAZyme genes and transcription factors. We detected important proteins related to biomass degradation, including β-glucosidases, endoglucanases, cellobiohydrolases, lytic polysaccharide monooxygenases, endo-1,4-β-xylanases and β-mannanases, in the exoproteome under cellulose growth conditions. Coexpression networks were constructed to explore the relationships among the genes and corresponding secreted proteins that act synergistically for cellulose degradation. An enriched cluster with degradative enzymes was described, and the subnetwork of CAZymes revealed strong correlations among the secreted proteins (AA9, GH6, GH10, GH11 and CBM1) and differentially expressed CAZyme genes (GH45, GH7, AA7 and GH1). Our results provide valuable information for future studies on the genetic regulation of plant cell wall-degrading enzymes. This knowledge can be exploited for the improvement of enzymatic reactions in biomass degradation for bioethanol production.Key pointsDifferent biotechnological approaches were used to understand the mechanism of cellulose degradation of Trichoderma spp.T. harzianum CBMAI-0179 is a potential candidate for the production of cellulolytic enzymes.Coexpression networks revealed genes and proteins acting synergistically for cellulose hydrolysis.

Transcriptome analysis of Aspergillus niger xlnR and xkiA mutants grown on corn Stover and soybean hulls reveals a highly complex regulatory network

Background Enzymatic plant biomass degradation by fungi is a highly complex process and one of the leading challenges in developing a biobased economy. Some industrial fungi (e.g. Aspergillus niger) have a long history of use with respect to plant biomass degradation and for that reason have become ‘model’ species for this topic. A. niger is a major industrial enzyme producer that has a broad ability to degrade plant based polysaccharides. A. niger wild-type, the (hemi-)cellulolytic regulator (xlnR) and xylulokinase (xkiA1) mutant strains were grown on a monocot (corn stover, CS) and dicot (soybean hulls, SBH) substrate. The xkiA1 mutant is unable to utilize the pentoses D-xylose and L-arabinose and the polysaccharide xylan, and was previously shown to accumulate inducers for the (hemi-)cellulolytic transcriptional activator XlnR and the arabinanolytic transcriptional activator AraR in the presence of pentoses, resulting in overexpression of their target genes. The xlnR mutant has reduced growth on xylan and down-regulation of its target genes. The mutants therefore have a similar phenotype on xylan, but an opposite transcriptional effect. D-xylose and L-arabinose are the most abundant monosaccharides after D-glucose in nearly all plant-derived biomass materials. In this study we evaluated the effect of the xlnR and xkiA1 mutation during growth on two pentose-rich substrates by transcriptome analysis. Results Particular attention was given to CAZymes, metabolic pathways and transcription factors related to the plant biomass degradation. Genes coding for the main enzymes involved in plant biomass degradation were down-regulated at the beginning of the growth on CS and SBH. However, at a later time point, significant differences were found in the expression profiles of both mutants on CS compared to SBH. Conclusion This study demonstrates the high complexity of the plant biomass degradation process by fungi, by showing that mutant strains with fairly straightforward phenotypes on pure mono- and polysaccharides, have much less clear-cut phenotypes and transcriptomes on crude plant biomass.