BackgroundRenewable lignocellulosic biomass is an advantageous resource for the production of second generation biofuels and other biorefinery products. In Middle Europe, wheat straw is one of the most abundant low-cost sources of lignocellulosic biomass. For its efficient use, an efficient mix of cellulases and hemicellulases is required. In this paper, we investigated how cellulase production by T. reesei on wheat straw compares to that on lactose, the only soluble and also cheap inducing carbon source for enzyme production.ResultsWe have examined and compared the transcriptome of T. reesei growing on wheat straw and lactose as carbon sources under otherwise similar conditions. Gene expression on wheat straw exceeded that on lactose, and 1619 genes were found to be only induced on wheat straw but not on lactose. They comprised 30% of the CAZome, but were also enriched in genes associated with phospholipid metabolism, DNA synthesis and repair, iron homeostatis and autophagy. Two thirds of the CAZome was expressed both on wheat straw as well as on lactose, but 60% of it at least >2-fold higher on the former. Major wheat straw specific genes comprised xylanases, chitinases and mannosidases. Interestingly, the latter two CAZyme families were significantly higher expressed in a strain in which xyr1 encoding the major regulator of cellulase and hemicellulase biosynthesis is non-functional.ConclusionsOur data reveal several major differences in the transcriptome between wheat straw and lactose which may be related to the higher enzyme formation on the former and their further investigation could lead to the development of methods for increasing enzyme production on lactose.

Background: The hypercellulolytic mutant Hypocrea jecorina (anamorph Trichoderma reesei) RUT C30 is the H. jecorina strain most frequently used for cellulase fermentations and has also often been employed for basic research on cellulase regulation. This strain has been reported to contain a truncated carbon catabolite repressor gene cre1 and is consequently carbon catabolite derepressed. To date this and an additional frame-shift mutation in the glycoprotein-processingglucosidase II encoding gene are the only known genetic differences in strain RUT C30.

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BackgroundHydrophobins are proteins containing eight conserved cysteine residues that occur uniquely in mycelial fungi. Their main function is to confer hydrophobicity to fungal surfaces in contact with air or during attachment of hyphae to hydrophobic surfaces of hosts, symbiotic partners or themselves resulting in morphogenetic signals. Based on their hydropathy patterns and solubility characteristics, hydrophobins are divided into two classes (I and II), the latter being found only in ascomycetes.ResultsWe have investigated the mechanisms driving the evolution of the class II hydrophobins in nine species of the mycoparasitic ascomycetous genus Trichoderma/Hypocrea, using three draft sequenced genomes (H. jecorina = T. reesei, H. atroviridis = T. atroviride; H. virens = T. virens) an additional 14,000 ESTs from six other Trichoderma spp. (T. asperellum, H. lixii = T. harzianum, T. aggressivum var. europeae, T. longibrachiatum, T. cf. viride). The former three contained six, ten and nine members, respectively. Ten is the highest number found in any ascomycete so far. All the hydrophobins we examined had the conserved four beta-strands/one helix structure, which is stabilized by four disulfide bonds. In addition, a small number of these hydrophobins (HFBs)contained an extended N-terminus rich in either proline and aspartate, or glycine-asparagine. Phylogenetic analysis reveals a mosaic of terminal clades containing duplicated genes and shows only three reasonably supported clades. Calculation of the ratio of differences in synonymous vs. non-synonymous nucleotide substitutions provides evidence for strong purifying selection (KS/Ka >> 1). A genome database search for class II HFBs from other ascomycetes retrieved a much smaller number of hydrophobins (2–4) from each species, and most were from Sordariomycetes. A combined phylogeny of these sequences with those of Trichoderma showed that the Trichoderma HFBs mostly formed their own clades, whereas those of other Sordariomycetes occurred in shared clades.ConclusionOur study shows that the genus Trichoderma/Hypocrea has a proliferated arsenal of class II hydrophobins which arose by birth-and-death evolution followed by purifying selection.

Lactose (1,4-O-b-D-galactopyranosyl-D-glucose) is a soluble and economic carbon source for the industrial production of cellulases or recombinant proteins by Hypocrea jecorina (anamorph Trichoderma reesei). The mechanism by which lactose induces cellulase formation is not understood. Recent data showed that the galactokinase step is essential for cellulase induction by lactose, but growth on D-galactose alone does not induce cellulases. Consequently, the hypothesis was tested that D-galactose may be an inducer only at a low growth rate, which is typically observed when growing on lactose. Carbon-limited chemostat cultivations of H. jecorina were therefore performed at different dilution rates with D-galactose, lactose, galactitol and D-glucose. Cellulase gene expression was monitored by using a strain carrying a fusion between the cbh2 (encoding cellobiohydrolase 2, Cel6A) promoter region and the Aspergillus niger glucose oxidase gene and by identification of the two major cellobiohydrolases Cel7A and Cel6A. The results show that D-galactose indeed induces cbh2 gene transcription and leads to Cel7A and Cel6A accumulation at a low (D=0?015 h "1 ) but not at higher dilution rates. At the same dilution rate, growth on D-glucose did not lead to cbh2 promoter activation or Cel6A formation but a basal level, lower than that observed on D-galactose, was detected for the carbon-catabolite-derepressible Cel7A. Lactose induced significantly higher cellulase levels at 0?015 h "1 than D-galactose and induced cellulases even at growth rates up to 0?042 h "1. Results of chemostats with an equimolar mixture of D-galactose and D-glucose essentially mimicked the behaviour on D-galactose alone, whereas an equimolar mixture of D-galactose and galactitol, the first intermediate of a recently described second pathway of D-galactose catabolism, led to cellulase induction at D=0?030 h "1 . It is concluded that D-galactose indeed induces cellulases at low growth rate and that the operation of the alternative pathway further increases this induction. However, under those conditions lactose is still a superior inducer for which the mechanism remains to be clarified.

The enzyme b-galactosidase (EC 3.2.1.23) catalyses the hydrolysis of terminal nonreducing b-d-galactose residues in b-d-galactosides as, for example, lactose (1,4-O-b-d-galactopyranosyl-d-glucose) and structurally related compounds. It is found in plants and animals, as well as in a wide variety of microorganisms including yeasts, fungi, bacteria and Archaea. According to the Carbohydrate Active Enzymes database (http://www.cazy.org/)[1], b-galactosidases are members of four different glycoside hydrolase (GH) families (GH1, GH2, GH35 and GH42), indicating their structural diversity. In biotechnology, they are mainly applied for the hydrolysis of lactose to d-glucose and d-galactose in various products of the dairy industry [2,3]. This results in improved quality of the end product (softer texture of ice cream, faster ripening of cheese, etc.) and also lessens the problem of lactose intolerance, which is prevalent in more than half of the world's population [4]. In addition, b-galactosidases catalyse transgalactosylation reactions of various b-d-galactosides including lactose. Recently, these galacto-oligosaccharides have attracted considerable interest because of a proposed beneficial effects on human health [5,6].The filamentous fungus Hypocrea jecorina (anamorph: Trichoderma reesei) is a potent producer of The extracellular bga1-encoded b-galactosidase of Hypocrea jecorina (Trichoderma reesei) was overexpressed under the pyruvat kinase (pki1) promoter region and purified to apparent homogeneity. The monomeric enzyme is a glycoprotein with a molecular mass of 118.8 ± 0.5 kDa (MALDI-MS) and an isoelectric point of 6.6. Bga1 is active with several disaccharides, e.g. lactose, lactulose and galactobiose, as well as with aryland alkyl-b-d-galactosides. Based on the catalytic efficiencies, lactitol and lactobionic acid are the poorest substrates and o-nitrophenyl-b-d-galactoside and lactulose are the best. The pH optimum for the hydrolysis of galactosides is 5.0, and the optimum temperature was found to be 60°C. Bga1 is also capable of releasing d-galactose from b-galactans and is thus actually a galacto-b-d-galactanase. b-Galactosidase is inhibited by its reaction product d-galactose and the enzyme also shows a significant transferase activity which results in the formation of galacto-oligosaccharides.Abbreviations

BackgroundTrichoderma 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. One such promoter that is widely employed for yeasts is that of the l-methionine repressible MET3 gene, encoding ATP sulphurylase.ResultsWe show that the MET3 system can only be applied for T. reesei when the cellulase inducing carbon source lactose is used but not when wheat straw, a relevant lignocellulosic substrate for enzyme production, is employed. We therefore performed a transcriptomic screen for genes that are l-methionine repressible in a wheat straw culture. This analysis retrieved 50 differentially regulated genes of which 33 were downregulated. Among these, genes encoding transport proteins as well as iron containing DszA like monooxygenases and TauD like dioxygenases were strongly overrepresented. We show that the promoter region of one of these dioxygenases can be used for the strongly repressible expression of the Aspergillus niger sucA encoded extracellular invertase in T. reesei wheat straw cultures. This system is also portable to other carbon sources including d-glucose and glycerol as demonstrated by the repressible expression of the Escherichia coli lacZ encoded ß-galactosidase in T. reesei.ConclusionWe describe a novel, versatile set of promoters for T. reesei that can be used to drive recombinant gene expression in wheat straw cultures at different expression strengths and in an l-methionine repressible manner. The dioxygenase promoter that we studied in detail is furthermore compatible with different carbon sources and therefore applicable for manipulating protein production as well as functional genomics with T. reesei.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0308-3) contains supplementary material, which is available to authorized users.

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.