Lukas Hartl

The Hypocrea jecorina (Trichoderma reesei) hypercellulolytic mutant RUT C30 lacks a 85 kb (29 gene-encoding) region of the wild-type genome

BackgroundThe 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-processing β-glucosidase II encoding gene are the only known genetic differences in strain RUT C30.ResultsIn the present paper we show that H. jecorina RUT C30 lacks an 85 kb genomic fragment, and consequently misses additional 29 genes comprising transcription factors, enzymes of the primary metabolism and transport proteins. This loss is already present in the ancestor of RUT C30 – NG 14 – and seems to have occurred in a palindromic AT-rich repeat (PATRR) typically inducing chromosomal translocations, and is not linked to the cre1 locus. The mutation of the cre1 locus has specifically occurred in RUT C30. Some of the genes that are lacking in RUT C30 could be correlated with pronounced alterations in its phenotype, such as poor growth on α-linked oligo- and polyglucosides (loss of maltose permease), or disturbance of osmotic homeostasis.ConclusionOur data place a general caveat on the use of H. jecorina RUT C30 for further basic research.

The CRE1 carbon catabolite repressor of the fungus Trichoderma reesei: A master regulator of carbon assimilation

BackgroundThe identification and characterization of the transcriptional regulatory networks governing the physiology and adaptation of microbial cells is a key step in understanding their behaviour. One such wide-domain regulatory circuit, essential to all cells, is carbon catabolite repression (CCR): it allows the cell to prefer some carbon sources, whose assimilation is of high nutritional value, over less profitable ones. In lower multicellular fungi, the C2H2 zinc finger CreA/CRE1 protein has been shown to act as the transcriptional repressor in this process. However, the complete list of its gene targets is not known.ResultsHere, we deciphered the CRE1 regulatory range in the model cellulose and hemicellulose-degrading fungus Trichoderma reesei (anamorph of Hypocrea jecorina) by profiling transcription in a wild-type and a delta-cre1 mutant strain on glucose at constant growth rates known to repress and de-repress CCR-affected genes. Analysis of genome-wide microarrays reveals 2.8% of transcripts whose expression was regulated in at least one of the four experimental conditions: 47.3% of which were repressed by CRE1, whereas 29.0% were actually induced by CRE1, and 17.2% only affected by the growth rate but CRE1 independent. Among CRE1 repressed transcripts, genes encoding unknown proteins and transport proteins were overrepresented. In addition, we found CRE1-repression of nitrogenous substances uptake, components of chromatin remodeling and the transcriptional mediator complex, as well as developmental processes.ConclusionsOur study provides the first global insight into the molecular physiological response of a multicellular fungus to carbon catabolite regulation and identifies several not yet known targets in a growth-controlled environment.

The putative protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei

Trichoderma reesei is an industrial producer of enzymes that degrade lignocellulosic polysaccharides to soluble monomers, which can be fermented to biofuels. Here we show that the expression of genes for lignocellulose degradation are controlled by the orthologous T. reesei protein methyltransferase LAE1. In a lae1 deletion mutant we observed a complete loss of expression of all seven cellulases, auxiliary factors for cellulose degradation, β‐glucosidases and xylanases were no longer expressed. Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Lae1‐modulated cellulase gene expression was dependent on the function of the general cellulase regulator XYR1, but also xyr1 expression was LAE1‐dependent. LAE1 was also essential for conidiation of T. reesei. Chromatin immunoprecipitation followed by high‐throughput sequencing (‘ChIP‐seq’) showed that lae1 expression was not obviously correlated with H3K4 di‐ or trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZyme coding regions, suggesting that LAE1 does not affect CAZyme gene expression by directly modulating H3K4 or H3K9 methylation. Our data demonstrate that the putative protein methyltransferase LAE1 is essential for cellulase gene expression in T. reesei through mechanisms that remain to be identified.

Gene targeting in a nonhomologous end joining deficient Hypocrea jecorina

The industrially applied ascomycete Hypocrea jecorina (synonym: Trichoderma reesei) exhibits a low rate of exogenous DNA integration by homologous recombination (HR). This hinders the high-throughput generation of strains by gene replacement and is therefore impeding systematic functional gene analyses towards, e.g. strain improvement for protein or enzyme production. To increase the rate of HR events during fungal transformation we identified and deleted the orthologue of the human KU70 in H. jecorina, which is required for the nonhomologous end joining (NHEJ) pathway and responsible for ectopic DNA integration. The effect of the absence of the H. jecorina tku70 on gene targeting was tested by deletion of two so far uncharacterized genes encoding a short chain dehydrogenase and a fungal specific transcription factor. Efficiency of gene targeting for both genes was >95% in a Deltatku70 strain when 1kb homologous flanking regions were used in the deletion construct. This is a significant increase in targeting efficiency compared to the parental - non-tku70 deleted - strain TU-6 where a gene knock-out frequency of only 5-10% was observed. Together with the recently annotated genomic sequence of H. jecorina, this system provides a useful tool for a genome-wide functional gene analysis on a high-throughput scale to improve the biotechnological potential of this fungus.

Fungal chitinases: diversity, mechanistic properties and biotechnological potential

Chitin derivatives, chitosan and substituted chito-oligosaccharides have a wide spectrum of applications ranging from medicine to cosmetics and dietary supplements. With advancing knowledge about the substrate-binding properties of chitinases, enzyme-based production of these biotechnologically relevant sugars from biological resources is becoming increasingly interesting. Fungi have high numbers of glycoside hydrolase family 18 chitinases with different substrate-binding site architectures. As presented in this review, the large diversity of fungal chitinases is an interesting starting point for protein engineering. In this review, recent data about the architecture of the substrate-binding clefts of fungal chitinases, in connection with their hydrolytic and transglycolytic abilities, and the development of chitinase inhibitors are summarized. Furthermore, the biological functions of chitinases, chitin and chitosan utilization by fungi, and the effects of these aspects on biotechnological applications, including protein overexpression and autolysis during industrial processes, are discussed in this review.

The galactokinase of Hypocrea jecorina is essential for cellulase induction by lactose but dispensable for growth on d-galactose.

Lactose is the only soluble carbon source which can be used economically for the production of cellulases or heterologous proteins under cellulase expression signals by Hypocrea jecorina (=Trichoderma reesei). Towards an understanding of lactose metabolism and its role in cellulase formation, we have cloned and characterized the gal1 (galactokinase) gene of H. jecorina, which catalyses the first step in d‐galactose catabolism. It exhibits a calculated Mr of 57 kDa, and shows moderate identity (about 40%) to its putative homologues of Saccharomyces cerevisiae and Kluyveromyces lactis. Gal1 is a member of the GHMP family, shows conservation of a Gly/Ser rich region involved in ATP binding and of amino acids (Arg 51, Glu 57, Asp 60, Asp 214, Tyr 270) responsible for galactose binding. A single transcript was formed constitutively during the rapid growth phase on all carbon sources investigated and accumulated to about twice this level during growth on d‐galactose, l‐arabinose and their corresponding polyols. Deletion of gal1 reduces growth on d‐galactose but does only slightly affect growth on lactose. This is the result of the operation of a second pathway for d‐galactose catabolism, which involves galactitol as an intermediate, and whose transient concentration is strongly enhanced in the delta‐gal1 strain. In this pathway, galactitol is catabolised by the lad1‐encoded l‐arabinitol‐4‐dehydrogenase, because a gal1/lad1 double delta‐mutant failed to grow on d‐galactose. In the delta‐gal1 strain, induction of the Leloir pathway gene gal7 (encoding galactose‐1‐phosphate uridylyltransferase) by d‐galactose, but not by l‐arabinose, is impaired. Induction of cellulase gene expression by lactose is also impaired in a gal1 deleted strain, whereas their induction by sophorose (the putative cellulose‐derived inducer) was shown to be normal, thus demonstrating that galactokinase is a key enzyme for cellulase induction during growth on lactose, and that induction by lactose and sophorose involves different mechanisms.

The d‐xylose reductase of Hypocrea jecorina is the major aldose reductase in pentose and d‐galactose catabolism and necessary for β‐galactosidase and cellulase induction by lactose

The Hypocrea jecorinad‐xylose reductase encoding gene xyl1 shows low basal transcript levels, and is induced by d‐xylose, l‐arabinose and l‐arabinitol and, to a lesser extent, by lactose, d‐galactose, galactitol and xylitol. The recombinantly expressed XYL1 catalyzes the NADPH‐dependent reduction of the pentoses d‐xylose and l‐arabinose and the hexose d‐galactose. Deletion of xyl1 slightly reduces growth on all carbon sources, but a significant decrease is found on d‐xylose, l‐arabinose and d‐galactose. Similar to pentose degradation, XYL1 reduces d‐galactose to galactitol in a recently identified second d‐galactose pathway. Strains impaired in both d‐galactose pathways are almost unable to grow on d‐galactose. Δxyl1 strains show reduced growth on lactose and are impaired in β‐galactosidase expression and induction of the major cellobiohydrolase gene cbh1. A strain deleted in the cellulase regulator XYR1 is even more severely impaired in growth and β‐galactosidase expression on lactose, and does not produce any cbh1 transcript at all. In this strain, only a low basal level of xyl1 transcription is found on lactose. Galactitol, but not d‐galactose is able to induce xyl1 transcription in a XYR1‐independent manner. Our results show that the role of the H. jecorina XYL1 is not restricted to d‐xylose catabolism and demonstrates its importance for induction of cellulases and β‐galactosidases.

Role of the bga1-Encoded Extracellular β-Galactosidase of Hypocrea jecorina in Cellulase Induction by Lactose

ABSTRACT Lactose is the only soluble and economically feasible carbon source for the production of cellulases or heterologous proteins regulated by cellulase expression signals by Hypocrea jecorina (Trichoderma reesei). We investigated the role of the major β-galactosidase of H. jecorina in lactose metabolism and cellulase induction. A genomic copy of the bga1 gene was cloned, and this copy encodes a 1,023-amino-acid protein with a 20-amino-acid signal sequence. This protein has a molecular mass of 109.3 kDa, belongs to glycosyl hydrolase family 35, and is the major extracellular β-galactosidase during growth on lactose. Its transcript was abundant during growth on l-arabinose and l-arabinitol but was much less common when the organism was grown on lactose, d-galactose, galactitol, d-xylose, and xylitol. Δbga1 strains grow more slowly and accumulate less biomass on lactose, but the cellobiohydrolase I and II gene expression and the final cellulase yields were comparable to those of the parental strain. Overexpression of bga1 under the control of the pyruvate kinase promoter reduced the lag phase, increased growth on lactose, and limited transcription of cellobiohydrolases. We detected an additional extracellular β-galactosidase activity that was not encoded by bga1 but no intracellular β-galactosidase activity. In conclusion, cellulase production on lactose occurs when β-galactosidase activity levels are low but decreases as the β-galactosidase activities increase. The data indicate that bga1-encoded β-galactosidase activity is a critical factor for cellulase production on lactose.

d-Xylose Metabolism in Hypocrea jecorina: Loss of the Xylitol Dehydrogenase Step Can Be Partially Compensated for by lad1-Encoded l-Arabinitol-4-Dehydrogenase

ABSTRACT With the goal of the genetic characterization of the d-xylose pathway in Hypocrea jecorina (anamorph: Trichoderma reesei), we cloned the xdh1 gene, encoding NAD-xylitol dehydrogenase, which catalyzes the second step of fungal d-xylose catabolism. This gene encodes a 363-amino-acid protein which has a mass of 38 kDa, belongs to the zinc-containing alcohol dehydrogenase family, exhibits high sequence identity to the published sequences of xylitol dehydrogenases from yeast origins, but contains a second, additional binding site for Zn2+. The enzyme catalyzed the NAD-dependent oxidation of xylitol and d-sorbitol and the NADH-dependent reduction of d-xylulose and d-fructose. No activity was observed with NADP, l-arabinose, or l-arabinitol. A single 1.4-kb transcript was formed during growth on xylan, d-xylose, l-arabinose, l-arabinitol and, at a lower abundance, xylitol, d-galactose, galactitol, and lactose but not on d-glucose and glycerol. xdh1 deletion mutants exhibited 50% reduced growth rates on d-xylose, whereas growth rates on xylitol remained unaltered. These mutants contained 30% of the xylitol dehydrogenase activity of the parent strain, indicating the presence of a second xylitol dehydrogenase. This activity was shown to be due to lad1-encoded l-arabinitol-4-dehydrogenase, because H. jecorina xdh1 lad1 double-deletion strains failed to grow on d-xylose or xylitol. In contrast, lad1 deletion strains of H. jecorina grew normally on these carbon sources. These results show that H. jecorina contains a single xylitol dehydrogenase which is encoded by xdh1 and is involved in the metabolism of d-xylose and that lad1-encoded l-arabinitol-4-dehydrogenase can compensate for it partially in mutants with a loss of xdh1 function.

Induction of the gal pathway and cellulase genes involves no transcriptional inducer function of the galactokinase in Hypocrea jecorina.

The Saccharomyces cerevisiae galactokinase ScGal1, a key enzyme for d-galactose metabolism, catalyzes the conversion of d-galactose to d-galactose 1-phosphate, whereas its catalytically inactive paralogue, ScGal3, activates the transcription of the GAL pathway genes. In Kluyveromyces lactis the transcriptional inducer function and the galactokinase activity are encoded by a single bifunctional KlGal1. Here, we investigated the cellular function of the single galactokinase GAL1 in the multicellular ascomycete Hypocrea jecorina (= Trichoderma reesei) in the induction of the gal genes and of the galactokinase-dependent induction of the cellulase genes by lactose (1,4-O-β-d-galactopyranosyl-d-glucose). A comparison of the transcriptional response of a strain deleted in the gal1 gene (no putative transcriptional inducer and no galactokinase activity), a strain expressing a catalytically inactive GAL1 version (no galactokinase activity but a putative inducer function), and a strain expressing the Escherichia coli galK (no putative transcriptional inducer but galactokinase activity) showed that, in contrast to the two yeasts, both the GAL1 protein and the galactokinase activity are fully dispensable for induction of the Leloir pathway gene gal7 by d-galactose and that only the galactokinase activity is required for cellulase induction by lactose. The data document a fundamental difference in the mechanisms by which yeasts and multicellular fungi respond to the presence of d-galactose, showing that the Gal1/Gal3-Gal4-Gal80-dependent regulatory circuit does not operate in multicellular fungi.