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. When grown on either poplar or spruce wood blocks, several laccases were detected in the secretome. Two of them (FpLcc1 and FpLcc2) were heterologously produced using Trichoderma reesei QM9414 Δxyr1 as expression host and purified to homogeneity by consecutive steps of hydrophobic interaction, anion exchange and size exclusion chromatography. With the substrates 2,2-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol (2,6-DMP) and guaiacol both laccases showed similar, low pH-optima below 3 for ABTS and 2,6-DMP and at pH 3.5 for guaiacol which is at the acidic end of laccases isolated from white rot fungi. The determined KM values were low while kcat values measured at acidic conditions were comparable to those reported for other laccases from white rot fungi. While both enzymes showed a moderate decrease in activity in the presence of oxalic and citric acid FpLcc2 was activated by acetic acid up to 3.7 times. This activation effect is much more pronounced at pH 5.0 compared to pH 3.0 and could already be observed at a concentration of 1 mM acetic acid.

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. chrysosporium using the well-established methanolytic yeast Pichia pastoris and the filamentous fungus Trichoderma reesei as expression hosts with subsequent purification by anion exchange and hydrophobic interaction chromatography. Both processes were shown to be suitable for the production of the target protein at high levels. GLOX produced in T. reesei carries mainly Man5 glycosylation while the enzyme produced in P. pastoris exhibits the typical high-mannose type N-glycosylation. The enzyme expressed in P. pastoris showed slightly higher specific activities which correlates with the higher copper loading of 65.5 % compared to 51.9 % for the protein from T. reesei. The pH optimum for both recombinant proteins was 6.0, however, GLOX activity was found to be highly affected by different buffer species. Both enzymes showed very similar substrate affinities and turnover numbers with the highest catalytic efficiency observed for methylglyoxal. GLOX from both expression hosts is therefore a suitable enzyme for further mechanistic characterization and application studies.

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. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDHTr) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDHPp). RESULTS: PcCDHTr was recombinantly produced with a yield of 600 U L-1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDHTr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDHTr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDHTr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-D-glucosamine additions at the six potential N-glycosylation sites of PcCDHTr, which indicates the presence of an endo-N-acetyl-ß-D-glucosaminidase in the supernatant. CONCLUSIONS: Heterologous production of PcCDHTr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDHTr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDHTr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells.

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. For the design of these oligonucleotides, the coding region of the gene of interest and the choice of expression vector must be considered. Here, a number of basic techniques for gene analysis and oligonucleotide design based on specific features of expression vectors are discussed, as well as different methods for introduction of the amplified gene 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 ß-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. The evaluation of the LAE1 impact to induce the GHs used soluble and lignocellulose inexpensive carbon sources in a stirred-tank bioreactor. Using sugarcane bagasse with sucrose, the overexpression of lae1 resulted in significantly increment of gh61b (31x), cel7a (25x), bgl1(20x) and xyn3 (20x) genes expression. Reducing sugar released from pretreated sugarcane bagasse, which hydrolyzed by recombinant crude enzyme cocktail, achieved 41% improvement. Therefore, lae1 overexpression effectively is a promising improving GHs target for biomass degradation by T. harzianum.

Deletion of the small GTPase rac1 in Trichoderma reesei provokes hyperbranching and impacts growth and cellulase production.

BACKGROUND: Trichoderma reesei 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. There are several genetic modifications which can cause hyperbranching, for example the deletion of the small Rho GTPase rac. Rac plays a crucial role in actin dynamics and is involved in polarisation of the cell during germination and apical extension of the hyphae. RESULTS: We deleted rac1 in a T. reesei strain with an ectopically overexpressed endoglucanase, CEL12A, under Pcdna1 control. This deletion provoked a hyperbranching phenotype and strong apolar growth during germination and in mature hyphae. The strains displayed dichotomous branching and shorter total mycelium length with a larger hyphal diameter. Δrac1 strains exhibited a decreased radial growth on solid media. Biomass formation in liquid cultures was carbon source dependent; similar to the reference strain during growth on lactose, increased on d-glucose and slightly enhanced on cellulose. While extracellular cellulase activities remained at parental strain levels on d-glucose and cellulose, the specific activity on lactose cultures was increased up to three times at 72 h accompanied by an upregulation of transcription of the main cellulases. Although the morphology of the Δrac1 strains was considerably altered, the viscosity of the culture broth in fed-batch cultivations were not significantly different in comparison to the parental strain. CONCLUSIONS: Deletion of the small Rho GTPase rac1 changes the morphology of the hyphae and provokes hyperbranching without affecting viscosity, independent of the carbon source. In contrast, biomass formation and cellulase production are altered in a carbon source dependent manner in the Δrac1 strains.

The influence of feedstock characteristics on enzyme production in Trichoderma reesei: 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. Thus, cultivation of Trichoderma reesei, the principal producer of lignocellulolytic enzymes, on the lignocellulosic biomass processed on-site can reduce the cost of enzyme manufacturing. Further, due to a complex gene regulation machinery, the fungus can adjust the gene expression of the lignocellulolytic enzymes towards the characteristics of the feedstock, increasing the hydrolytic efficiency of the produced enzyme cocktail. Despite extensive research over decades, the underlying regulatory mechanisms are not fully elucidated. One aspect that has received relatively little attention in literature is the influence the characteristics of a lignocellulosic substrate, i.e., its chemical and physical composition, has on the produced enzyme mixture. Considering that the fungus is dependent on efficient enzymatic degradation of the lignocellulose for continuous supply of carbon and energy, a relationship between feedstock characteristics and secretome composition can be expected. The aim of this review was to systematically collect, appraise, and aggregate data and integrate results from studies analyzing enzyme production by T. reesei on insoluble cellulosic model substrates and lignocellulosic biomass. The results show that there is a direct effect of the substrate's complexity (rated by structure, composition of the lignin-carbohydrate complex, and recalcitrance in enzymatic saccharification) on enzyme titers and the composition of specific activities in the secretome. It further shows that process-related factors, such as substrate loading and cultivation set-up, are direct targets for increasing enzyme yields. The literature on transcriptome and secretome composition further supports the proposed influence of substrate-related factors on the expression of lignocellulolytic enzymes. This review provides insights into the interrelation between the characteristics of the substrate and the enzyme production by T. reesei, which may help to advance integrated enzyme manufacturing of substrate-specific enzymes cocktails at scale.

Deletion of either the regulatory gene ara1 or metabolic gene xki1 in Trichoderma reesei leads to increased CAZyme gene expression on crude plant biomass.

BACKGROUND: Trichoderma reesei is one of the major producers of enzymes for the conversion of plant biomass to sustainable fuels and chemicals. Crude plant biomass can induce the production of CAZymes in T. reesei, but there is limited understanding of how the transcriptional response to crude plant biomass is regulated. In addition, it is unknown whether induction on untreated recalcitrant crude plant biomass (with a large diversity of inducers) can be sustained for longer. We investigated the transcriptomic response of T. reesei to the two industrial feedstocks, corn stover (CS) and soybean hulls (SBH), over time (4 h, 24 h and 48 h), and its regulatory basis using transcription factor deletion mutants (Δxyr1 and Δara1). We also investigated whether deletion of a xylulokinase gene (Δxki1) from the pentose catabolic pathway that converts potential inducers could lead to increased CAZyme gene expression. RESULTS: By analyzing the transcriptomic responses using clustering as well as differential and cumulative expression of plant biomass degrading CAZymes, we found that corn stover induced a broader range and higher expression of CAZymes in T. reesei, while SBH induced more pectinolytic and mannanolytic transcripts. XYR1 was the major TF regulating CS utilization, likely due to the significant amount of d-xylose in this substrate. In contrast, ARA1 had a stronger effect on SBH utilization, which correlates with a higher abundance of l-arabinose in SBH that activates ARA1. Blocking pentose catabolism by deletion of xki1 led to higher expression of CAZyme encoding genes on both substrates at later time points. Surprisingly, this was also observed for Δara1 at later time points. Many of these genes were XYR1 regulated, suggesting that inducers for this regulator accumulated over time on both substrates. CONCLUSION: Our data demonstrates the complexity of the regulatory system related to plant biomass degradation in T. reesei and the effect the feedstock composition has on this. Furthermore, this dataset provides leads to improve the efficiency of a T. reesei enzyme cocktail, such as by the choice of substrate or by deleting xki1 to obtain higher production of plant biomass degrading CAZymes.

The Promoter Toolbox for Recombinant Gene Expression in Trichoderma reesei.

The ascomycete Trichoderma reesei 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 T. reesei to facilitate genetic engineering and improve the production of heterologous proteins grew. An important instrument to modify the expression of key genes are promoters to initiate and control their transcription. To date, the most commonly used promoter for T. reesei is the strong inducible promoter of the main cellobiohydrolase cel7a. Beside this one, there is a number of alternative inducible promoters derived from other cellulase- and xylanase encoding genes and a few constitutive promoters. With the advances in genomics and transcriptomics the identification of new constitutive and tunable promoters with different expression strength was simplified. In this review, we will discuss new developments in the field of promoters and compare their advantages and disadvantages. Synthetic expression systems constitute a new option to control gene expression and build up complex gene circuits. Therefore, we will address common structural features of promoters and describe options for promoter engineering and synthetic design of promoters. The availability of well-characterized gene expression control tools is essential for the analysis of gene function, detection of bottlenecks in gene networks and yield increase for biotechnology applications.

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. reesei ortholog of ARA1 from Pyricularia oryzae that regulates l-arabinose releasing and catabolic genes was deleted and characterized by growth profiling and transcriptomics along with a xyr1 mutant and xyr1/ara1 double mutant. Our results show that in addition to the l-arabinose-related role, T. reesei ARA1 is essential for expression of d-galactose releasing and catabolic genes, while XYR1 is not involved in this process.

Genome sequencing and transcriptome analysis of Trichoderma reesei QM9978 strain reveals a distal chromosome translocation to be responsible for loss of vib1 expression and loss of cellulase induction.

BACKGROUND: The hydrolysis of biomass to simple sugars used for the production of biofuels in biorefineries requires the action of cellulolytic enzyme mixtures. During the last 50 years, the ascomycete Trichoderma reesei, the main source of industrial cellulase and hemicellulase cocktails, has been subjected to several rounds of classical mutagenesis with the aim to obtain higher production levels. During these random genetic events, strains unable to produce cellulases were generated. Here, whole genome sequencing and transcriptomic analyses of the cellulase-negative strain QM9978 were used for the identification of mutations underlying this cellulase-negative phenotype. RESULTS: Sequence comparison of the cellulase-negative strain QM9978 to the reference strain QM6a identified a total of 43 mutations, of which 33 were located either close to or in coding regions. From those, we identified 23 single-nucleotide variants, nine InDels, and one translocation. The translocation occurred between chromosomes V and VII, is located upstream of the putative transcription factor vib1, and abolishes its expression in QM9978 as detected during the transcriptomic analyses. Ectopic expression of vib1 under the control of its native promoter as well as overexpression of vib1 under the control of a strong constitutive promoter restored cellulase expression in QM9978, thus confirming that the translocation event is the reason for the cellulase-negative phenotype. Gene deletion of vib1 in the moderate producer strain QM9414 and in the high producer strain Rut-C30 reduced cellulase expression in both cases. Overexpression of vib1 in QM9414 and Rut-C30 had no effect on cellulase production, most likely because vib1 is already expressed at an optimal level under normal conditions. CONCLUSION: We were able to establish a link between a chromosomal translocation in QM9978 and the cellulase-negative phenotype of the strain. We identified the transcription factor vib1 as a key regulator of cellulases in T. reesei whose expression is absent in QM9978. We propose that in T. reesei, as in Neurospora crassa, vib1 is involved in cellulase induction, although the exact mechanism remains to be elucidated. The data presented here show an example of a combined genome sequencing and transcriptomic approach to explain a specific trait, in this case the QM9978 cellulase-negative phenotype, and how it helps to better understand the mechanisms during cellulase gene regulation. When focusing on mutations on the single base-pair level, changes on the chromosome level can be easily overlooked and through this work we provide an example that stresses the importance of the big picture of the genomic landscape during analysis of sequencing data.

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. All fungi, containing the GlcNAc gene cluster, could grow on GlcNAc with the exception of four independent Neurospora crassa wild-type isolates, which were however able to grow on chitin. GlcNAc even inhibited their growth in the presence of other carbon sources. Genes involved in GlcNAc catabolism were strongly upregulated in the presence of GlcNAc, but during growth on chitin their expression was not increased. Deletion of hxk-3 (encoding the first catabolic enzyme, GlcNAc-hexokinase) and ngt-1 (encoding the GlcNAc transporter) improved growth of N. crassa on GlcNAc in the presence of glycerol. A crucial step in GlcNAc catabolism is enzymatic conversion from glucosamine-6-phosphate to fructose-6-phosphate, catalyzed by the glucosamine-6-phosphate deaminase, DAM-1. To assess, if DAM-1 is compromised in N. crassa, the orthologue from Trichoderma reesei, Trdam1, was expressed in N. crassa. Trdam1 expression partially alleviated the negative effects of GlcNAc in the presence of a second carbon source, but did not fully restore growth on GlcNAc. Our results indicate that the GlcNAc-catabolism pathway is bypassed during growth of N. crassa on chitin by use of an alternative pathway, emphasizing the different strategies that have evolved in the fungal kingdom for chitin utilization.

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. This review will summarize the current knowledge on regulation of plant biomass utilization in fungi and compare the differences between fungal species, focusing in particular on the presence or absence of the regulators involved in this process.

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. RESULTS: We compare the well-established CDH expression host P. pastoris with the less frequently used hosts Escherichia coli, Aspergillus niger, and Trichoderma reesei. The study evaluates the produced quantity and protein homogeneity of Corynascus thermophilus CDH in the culture supernatants, the purification, and finally compares the enzymes in regard to cofactor loading, glycosylation, catalytic constants and thermostability. CONCLUSIONS: Whereas E. coli could only express the catalytic dehydrogenase domain of CDH, all eukaryotic hosts could express full length CDH including the cytochrome domain. The CDH produced by T. reesei was most similar to the CDH originally isolated from the fungus C. thermophilus in regard to glycosylation, cofactor loading and catalytic constants. Under the tested experimental conditions the fungal expression hosts produce CDH of superior quality and uniformity compared to P. pastoris.

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. Analysis of xyn5 from the genome-sequenced reference strain T. reesei QM6a shows that it encodes a non-functional, truncated form of XYN5. However, non-truncated orthologues are present in other genome sequenced Trichoderma spp., and sequencing of xyn5 in other T. reesei wild-type isolates shows that they harbor a putative functional xyn5 allele. In silico analysis and 3D modeling revealed that the encoded XYN5 has significant structural similarities to xylanases of the GH11 family, including a GH-typical substrate binding groove and a carboxylate pair in the active site. The xyn5 of wild-type strain TUCIM1282 was recombinantly expressed in a T. reesei strain with a (hemi)cellulase-free background and the corresponding protein purified to apparent homogeneity. The pH and temperature optima and the kinetic parameters of the purified XYN5 were pH 4, 50 °C, and V max = 2646 nkat/mg with a K m of 9.68 mg/ml. This functional xyn5 allele was used to replace the mutated version which led to an overall increase of the xylanolytic activity. These findings are of particular importance as GH11 xylanases are of high biotechnological relevance, and T. reesei is one of the main industrial producers of such lignocellulose-degrading enzymes.

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. harzianum. Clone 5 presented a relative expression of xyr1 26-fold higher than the parent strain and exhibited 66, 37, and 36% higher values for filter paper activity, xylanase activity, and ß-glucosidase activity, respectively, during cultivation in a stirred-tank bioreactor. The overexpression of xyr1 in T. harzianum resulted in an enzymatic complex with significantly improved performance in sugarcane bagasse saccharification, with an enhancement of 25% in the first 24h. Our results also show that constitutive overexpression of xyr1 leads to the induction of several important players in biomass degradation at early (24h) and also late (48h) timepoints of inoculation. However, we also observed that the carbon catabolite repressor CRE1 was upregulated in xyr1 overexpression mutants. These findings demonstrate the feasibility of improving cellulase production by modifying regulator expression and suggest an attractive approach for increasing total cellulase productivity in T. harzianum.

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. RESULTS: The swo1 gene was overexpressed in T. reesei QM9414 Δxyr1 mutant, featuring downregulated cellulase production, and the protein was purified from culture supernatant. SWO1 was N-glycosylated and its circular dichroism spectrum suggested a folded protein. Adsorption isotherms (25 °C, pH 5.0, 1.0 mg substrate/mL) revealed SWO1 to be 120- and 20-fold more specific for binding to birchwood xylan and kraft lignin, respectively, than for binding to Avicel PH-101. The SWO1 binding capacity on lignin (25 µmol/g) exceeded 12-fold that on Avicel PH-101 (2.1 µmol/g). On xylan, not only the binding capacity (22 µmol/g) but also the affinity of SWO1 (K d = 0.08 µM) was enhanced compared to Avicel PH-101 (K d = 0.89 µM). SWO1 caused rapid release of a tiny amount of reducing sugars (<1 % of total) from different substrates (Avicel PH-101, nanocrystalline cellulose, steam-pretreated wheat straw, barley ß-glucan, cellotetraose) but did not promote continued saccharification. Atomic force microscopy revealed that amorphous cellulose films were not affected by SWO1. Also with AFM, binding of SWO1 to cellulose nanocrystallites was demonstrated at the single-molecule level, but adsorption did not affect this cellulose. SWO1 exhibited no synergy with T. reesei cellulases in the hydrolysis of the different celluloses. However, SWO1 boosted slightly (1.5-fold) the reducing sugar release from a native grass substrate. CONCLUSIONS: SWO1 is a strongly glycosylated protein, which has implications for producing it in heterologous hosts. Although SWO1 binds to crystalline cellulose, its adsorption to xylan is much stronger. SWO1 is not an auxiliary factor of the enzymatic degradation of a variety of cellulosic substrates. Effect of SWO1 on sugar release from intact plant cell walls might be exploitable with certain (e.g., mildly pretreated) lignocellulosic feedstocks.

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. reesei enzymes to saccharify lignocellulose from renewable plant biomass in order to produce biobased fuels and chemicals. In this review we summarize important milestones of the development of T. reesei as the leading production host for biorefinery enzymes, and discuss emerging trends in strain engineering. Trichoderma reesei has very recently also been proposed as a consolidated bioprocessing organism capable of direct conversion of biopolymeric substrates to desired products. We therefore cover this topic by reviewing novel approaches in metabolic engineering of T. reesei.

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. Trichoderma reesei, an industrial producer of enzymes for food, feed and biorefinery purposes, is heterothallic and takes a special position among industrially utilized species as all industrial strains are derived from the single MAT1-2 isolate QM6a. Consequently, strain improvement by crossing is not feasible within this strain line as this necessitates a MAT1-1 mating partner. Simply switching the mating type in one of the mating partners to MAT1-1, however, is not sufficient to produce a genotype capable of sexual reproduction with QM6a MAT1-2. RESULTS: We have used a systems biology approach to identify genes restoring sexual reproduction in the QM6a strain line. To this end, T. reesei QM6a was crossed with the MAT1-1 wild-type strain CBS999.97. The descendants were backcrossed 8-times in two lineages with QM6a to obtain mating competent MAT1-1 strains with a minimal set of CBS999.97 specific genes. Comparative genome analysis identified a total of 73 genes of which two-encoding an unknown C2H2/ankyrin protein and a homolog of the WD-protein HAM5-were identified to be essential for fruiting body formation. The introduction of a functional ham5 allele in a mating type switched T. reesei QM6a allowed sexual crossing with the parental strain QM6a. CONCLUSION: The finding that Trichoderma reesei is generally capable of undergoing sexual reproduction even under laboratory conditions raised hope for the applicability of classical breeding techniques with this fungus as known for plants and certain yeasts. The discovery that the wild-type isolate QM6a was female sterile, however, precluded any progress along that line. With the discovery of the genetic cause of female sterility and the creation of an engineered fertile strain we now provide the basis to establish sexual crossing in this fungus and herald a new era of strain improvement in T. reesei.