Functional expression and characterization of two laccases from the brown rot Fomitopsis pinicola.

Laccase is predominantly found in lignin degrading filamentous white rot fungi, where it is involved in the oxidative degradation of this recalcitrant heteropolymer. In brown rot fungi it is much less prevalent: laccases from only a few brown rots have been detected and only two have been characterized. This study tries to understand the role of this ligninolytic enzyme in brown rots by investigating the catalytic properties of laccases secreted by Fomitopsis pinicola FP58527 SS1.

Comparative characterization of glyoxal oxidase from Phanerochaete chrysosporium expressed at high levels in Pichia pastoris and Trichoderma reesei.

In the secretome of Phanerochaete chrysosporium, a white-rot fungus serving as a model organism to elucidate lignocellulose deconstruction, the copper containing metalloprotein glyoxal oxidase (GLOX) is potentially involved in the crucial production of hydrogen peroxide to fuel and initiate oxidative biomass degradation by lignin-degrading peroxidases. Its ability to oxidize a variety of aldehydes and α-hydroxy carbonyls with the concomitant reduction of dioxygen to hydrogen peroxide has attracted attention for its application as green biocatalyst in different industrial fields. Here we report and compare two efficient processes for the heterologous production of GLOX from P.

Heterologous expression of Phanerochaete chrysosporium cellobiose dehydrogenase in Trichoderma reesei.

Background: Cellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme.

In Silico Gene Analysis and Oligonucleotide Design for the Construction of Expression Vectors.

Recombinant expression of a gene of interest is a convenient method to study the function of the encoded protein. To introduce a gene into an expression vector, it is necessary to analyze the structure of the gene and determine some of its basic features including the exact start and end of the coding region or its exon/intron boundaries. Based on this gene analysis, oligonucleotides are then designed for amplification of the coding region by PCR and insertion into the expression vector.

The impact of putative methyltransferase overexpression on the Trichoderma harzianum cellulolytic system for biomass conversion.

Trichoderma harzianum has attracting attention for its potential alternative use in biofuel production, due to a recognized competence for high diversity glycoside hydrolases (GH) enzyme complex, including higher β-glucosidases and auxiliary proteins, using low-cost carbon sources. This strain constitutively overexpressed the global regulator putative methyltransferase - LAE1, in order to improve the GHs production. The recombinant strain achieved 79-fold increase in lae1 expression and high GHs productivity.

Deletion of the small GTPase in provokes hyperbranching and impacts growth and cellulase production.

Background: is widely known for its enormous protein secretion capacity and as an industrially relevant producer of cellulases and hemicellulases. Over the last decades, rational strain engineering was applied to further enhance homologous and heterologous enzyme yields. The introduction of hyperbranching is believed to increase protein secretion, since most exocytosis is located at the hyphal apical tip.

The influence of feedstock characteristics on enzyme production in : a review on productivity, gene regulation and secretion profiles.

Biorefineries, designed for the production of lignocellulose-based chemicals and fuels, are receiving increasing attention from the public, governments, and industries. A major obstacle for biorefineries to advance to commercial scale is the high cost of the enzymes required to derive the fermentable sugars from the feedstock used. As summarized in this review, techno-economic studies suggest co-localization and integration of enzyme manufacturing with the cellulosic biorefinery as the most promising alternative to alleviate this problem.

The Promoter Toolbox for Recombinant Gene Expression in .

The ascomycete is one of the main fungal producers of cellulases and xylanases based on its high production capacity. Its enzymes are applied in food, feed, and textile industry or in lignocellulose hydrolysis in biofuel and biorefinery industry. Over the last years, the demand to expand the molecular toolbox for to facilitate genetic engineering and improve the production of heterologous proteins grew.

ARA1 regulates not only l-arabinose but also d-galactose catabolism in Trichoderma reesei.

Trichoderma reesei is used to produce saccharifying enzyme cocktails for biofuels. There is limited understanding of the transcription factors (TFs) that regulate genes involved in release and catabolism of l-arabinose and d-galactose, as the main TF XYR1 is only partially involved. Here, the T.

N-acetylglucosamine, the building block of chitin, inhibits growth of Neurospora crassa.

N-acetylglucosamine (GlcNAc) is the monomer of the polysaccharide chitin, an essential structural component of the fungal cell wall and the arthropod exoskeleton. We recently showed that the genes encoding the enzymes for GlcNAc catabolism are clustered in several ascomycetes. In the present study we tested these fungi for growth on GlcNAc and chitin.

Regulators of plant biomass degradation in ascomycetous fungi.

Fungi play a major role in the global carbon cycle because of their ability to utilize plant biomass (polysaccharides, proteins, and lignin) as carbon source. Due to the complexity and heterogenic composition of plant biomass, fungi need to produce a broad range of degrading enzymes, matching the composition of (part of) the prevalent substrate. This process is dependent on a network of regulators that not only control the extracellular enzymes that degrade the biomass, but also the metabolic pathways needed to metabolize the resulting monomers.

Molecular and catalytic properties of fungal extracellular cellobiose dehydrogenase produced in prokaryotic and eukaryotic expression systems.

Background: Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by lignocellulolytic fungi. cdh gene expression is high in cellulose containing media, but relatively low CDH concentrations are found in the supernatant of fungal cultures due to strong binding to cellulose. Therefore, heterologous expression of CDH in Pichia pastoris was employed in the last 15 years, but the obtained enzymes were over glycosylated and had a reduced specific activity.

Trichoderma reesei xylanase 5 is defective in the reference strain QM6a but functional alleles are present in other wild-type strains.

Trichoderma reesei is a paradigm for the regulation and industrial production of plant cell wall-degrading enzymes. Among these, five xylanases, including the glycoside hydrolase (GH) family 11 XYN1 and XYN2, the GH10 XYN3, and the GH30 XYN4 and XYN6, were described. By genome mining and transcriptome analysis, a further putative xylanase, encoded by xyn5, was identified.

Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus.

Background: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus.

The relation between xyr1 overexpression in Trichoderma harzianum and sugarcane bagasse saccharification performance.

This work investigates the influence of the positive regulator XYR1 of Trichoderma harzianum on the production of cellulolytic enzymes, using sugarcane bagasse as carbon source. Constitutive expression of xyr1 was achieved under the control of the strong Trichoderma reesei pki1 promoter. Five clones with xyr1 overexpression achieved higher xyr1 expression and greater enzymatic productivity when cultivated under submerged fermentation, hence validating the genetic construction for T.

Functional characterization of the native swollenin from Trichoderma reesei: study of its possible role as C1 factor of enzymatic lignocellulose conversion.

Background: Through binding to cellulose, expansin-like proteins are thought to loosen the structural order of crystalline surface material, thus making it more accessible for degradation by hydrolytic enzymes. Swollenin SWO1 is the major expansin-like protein from the fungus Trichoderma reesei. Here, we have performed a detailed characterization of a recombinant native form of SWO1 with respect to its possible auxiliary role in the enzymatic saccharification of lignocellulosic substrates.

Cellulases and beyond: the first 70 years of the enzyme producer Trichoderma reesei.

More than 70 years ago, the filamentous ascomycete Trichoderma reesei was isolated on the Solomon Islands due to its ability to degrade and thrive on cellulose containing fabrics. This trait that relies on its secreted cellulases is nowadays exploited by several industries. Most prominently in biorefineries which use T.

Restoration of female fertility in Trichoderma reesei QM6a provides the basis for inbreeding in this industrial cellulase producing fungus.

Background: Filamentous fungi are frequently used as production platforms in industrial biotechnology. Most of the strains involved were known as reproducing exclusively asexually thereby preventing the application of conventional strain breeding techniques. In the last decade, evidence was obtained that a number of these imperfect fungi possess a sexual life cycle, too.

L-Methionine repressible promoters for tuneable gene expression in Trichoderma reesei.

Background: Trichoderma reesei is the main producer of lignocellulolytic enzymes that are required for plant biomass hydrolysis in the biorefinery industry. Although the molecular toolbox for T. reesei is already well developed, repressible promoters for strain engineering and functional genomics studies are still lacking.

The ß-importin KAP8 (Pse1/Kap121) is required for nuclear import of the cellulase transcriptional regulator XYR1, asexual sporulation and stress resistance in Trichoderma reesei.

The ascomycete Trichoderma reesei is an industrial producer of cellulolytic and hemicellulolytic enzymes, and serves as a prime model for their genetic regulation. Most of its (hemi-)cellulolytic enzymes are obligatorily dependent on the transcriptional activator XYR1. Here, we investigated the nucleo-cytoplasmic shuttling mechanism that transports XYR1 across the nuclear pore complex.

Kinetic transcriptome analysis reveals an essentially intact induction system in a cellulase hyper-producer Trichoderma reesei strain.

Background: The filamentous fungus Trichoderma reesei is the main industrial cellulolytic enzyme producer. Several strains have been developed in the past using random mutagenesis, and despite impressive performance enhancements, the pressure for low-cost cellulases has stimulated continuous research in the field. In this context, comparative study of the lower and higher producer strains obtained through random mutagenesis using systems biology tools (genome and transcriptome sequencing) can shed light on the mechanisms of cellulase production and help identify genes linked to performance.