Comparative characterization of nine novel GH51, GH54 and GH62 α-l-arabinofuranosidases from Penicillium subrubescens

α-l-Arabinofuranosidases (ABFs) are important enzymes in plant biomass degradation with a wide range of applications. The ascomycete fungus Penicillium subrubescens has more α-l-arabinofuranosidase-encoding genes in its genome compared to other Penicillia. We characterized nine ABFs from glycoside hydrolase (GH) families GH51, GH54 and GH62 from this fungus and demonstrated that they have highly diverse specificity and activity levels, indicating that the expansion was accompanied by diversification of the enzymes. Comparison of the substrate preference of the enzymes to the expression of the corresponding genes when the fungus was grown on either of two plant biomass substrates did not show a clear correlation, suggesting a more complex regulatory system governing l-arabinose release from plant biomass by P. subrubescens

The presence of trace components significantly broadens the molecular response of Aspergillus niger to guar gum

Guar gum consists mainly of galactomannan and constitutes the endosperm of guar seeds that acts as a reserve polysaccharide for germination. Due to its molecular structure and physical properties, this biopolymer has been considered as one of the most important and widely used gums in industry. However, for many of these applications this (hemi-) cellulosic structure needs to be modified or (partially) depolymerized in order to customize and improve its physicochemical properties. In this study, transcriptome, exoproteome and enzyme activity analyses were employed to decipher the complete enzymatic arsenal for guar gum depolymerization by Aspergillus niger. This multi-omic analysis revealed a set of 46 genes encoding carbohydrate-active enzymes (CAZymes) responding to the presence of guar gum, including CAZymes not only with preferred activity towards galactomannan, but also towards (arabino-) xylan, cellulose, starch and pectin, likely due to trace components in guar gum. This demonstrates that the purity of substrates has a strong effect on the resulting enzyme mixture produced by A. niger and probably by other fungi as well, which has significant implications for the commercial production of fungal enzyme cocktails.Peer reviewe

Recombinant production and characterization of six novel GH27 and GH36 α-galactosidases from Penicillium subrubescens and their synergism with a commercial mannanase during the hydrolysis of lignocellulosic biomass

α-Galactosidases are important industrial enzymes for hemicellulosic biomass degradation or modification. In this study, six novel extracellular α-galactosidases from Penicillium subrubescens were produced in Pichia pastoris and characterized. All α-galactosidases exhibited high affinity to pNPαGal, and only AglE was not active towards galacto-oligomers. Especially AglB and AglD released high amounts of galactose from guar gum, carob galactomannan and locust bean, but combining α-galactosidases with an endomannanase dramatically improved galactose release. Structural comparisons to other α-galactosidases and homology modelling showed high sequence similarities, albeit significant differences in mechanisms of productive binding, including discrimination between various galactosides. To our knowledge, this is the first study of such an extensive repertoire of extracellular fungal α-galactosidases, to demonstrate their potential for degradation of galactomannan-rich biomass. These findings contribute to understanding the differences within glycoside hydrolase families, to facilitate the development of new strategies to generate tailor-made enzymes for new industrial bioprocesses

Comparative characterization of nine novel GH51, GH54 and GH62 α-l-arabinofuranosidases from Penicillium subrubescens

α-l-Arabinofuranosidases (ABFs) are important enzymes in plant biomass degradation with a wide range of applications. The ascomycete fungus Penicillium subrubescens has more α-l-arabinofuranosidase-encoding genes in its genome compared to other Penicillia. We characterized nine ABFs from glycoside hydrolase (GH) families GH51, GH54 and GH62 from this fungus and demonstrated that they have highly diverse specificity and activity levels, indicating that the expansion was accompanied by diversification of the enzymes. Comparison of the substrate preference of the enzymes to the expression of the corresponding genes when the fungus was grown on either of two plant biomass substrates did not show a clear correlation, suggesting a more complex regulatory system governing l-arabinose release from plant biomass by P. subrubescens

Recombinant production and characterization of six novel GH27 and GH36 α-galactosidases from Penicillium subrubescens and their synergism with a commercial mannanase during the hydrolysis of lignocellulosic biomass

α-Galactosidases are important industrial enzymes for hemicellulosic biomass degradation or modification. In this study, six novel extracellular α-galactosidases from Penicillium subrubescens were produced in Pichia pastoris and characterized. All α-galactosidases exhibited high affinity to pNPαGal, and only AglE was not active towards galacto-oligomers. Especially AglB and AglD released high amounts of galactose from guar gum, carob galactomannan and locust bean, but combining α-galactosidases with an endomannanase dramatically improved galactose release. Structural comparisons to other α-galactosidases and homology modelling showed high sequence similarities, albeit significant differences in mechanisms of productive binding, including discrimination between various galactosides. To our knowledge, this is the first study of such an extensive repertoire of extracellular fungal α-galactosidases, to demonstrate their potential for degradation of galactomannan-rich biomass. These findings contribute to understanding the differences within glycoside hydrolase families, to facilitate the development of new strategies to generate tailor-made enzymes for new industrial bioprocesses

The presence of trace components significantly broadens the molecular response of Aspergillus niger to guar gum

Guar gum consists mainly of galactomannan and constitutes the endosperm of guar seeds that acts as a reserve polysaccharide for germination. Due to its molecular structure and physical properties, this biopolymer has been considered as one of the most important and widely used gums in industry. However, for many of these applications this (hemi-)cellulosic structure needs to be modified or (partially) depolymerized in order to customize and improve its physicochemical properties. In this study, transcriptome, exoproteome and enzyme activity analyses were employed to decipher the complete enzymatic arsenal for guar gum depolymerization by Aspergillus niger. This multi-omic analysis revealed a set of 46 genes encoding carbohydrate-active enzymes (CAZymes) responding to the presence of guar gum, including CAZymes not only with preferred activity towards galactomannan, but also towards (arabino-)xylan, cellulose, starch and pectin, likely due to trace components in guar gum. This demonstrates that the purity of substrates has a strong effect on the resulting enzyme mixture produced by A. niger and probably by other fungi as well, which has significant implications for the commercial production of fungal enzyme cocktails

Myceliophthora thermophila Xyr1 is predominantly involved in xylan degradation and xylose catabolism

Background: Myceliophthora thermophila is a thermophilic ascomycete fungus that is used as a producer of enzyme cocktails used in plant biomass saccharification. Further development of this species as an industrial enzyme factory requires a detailed understanding of its regulatory systems driving the production of plant biomass-degrading enzymes. In this study, we analyzed the function of MtXlr1, an ortholog of the (hemi-)cellulolytic regulator XlnR first identified in another industrially relevant fungus, Aspergillus niger. Results: The Mtxlr1 gene was deleted and the resulting strain was compared to the wild type using growth profiling and transcriptomics. The deletion strain was unable to grow on xylan and d-xylose, but showed only a small growth reduction on l-arabinose, and grew similar to the wild type on Avicel and cellulose. These results were supported by the transcriptome analyses which revealed reduction of genes encoding xylan-degrading enzymes, enzymes of the pentose catabolic pathway and putative pentose transporters. In contrast, no or minimal effects were observed for the expression of cellulolytic genes. Conclusions: Myceliophthora thermophila MtXlr1 controls the expression of xylanolytic genes and genes involved in pentose transport and catabolism, but has no significant effects on the production of cellulases. It therefore resembles more the role of its ortholog in Neurospora crassa, rather than the broader role described for this regulator in A. niger and Trichoderma reesei. By revealing the range of genes controlled by MtXlr1, our results provide the basic knowledge for targeted strain improvement by overproducing or constitutively activating this regulator, to further improve the biotechnological value of M. thermophila