Familiar Stranger: Ecological Genomics of the Model Saprotroph and Industrial Enzyme Producer Trichoderma reesei Breaks the Stereotypes.

The filamentous fungus Trichoderma reesei (Hypocreales, Ascomycota) has properties of an efficient cell factory for protein production that is exploited by the enzyme industry, particularly with respect to cellulase and hemicellulase formation. Under conditions of industrial fermentations it yields more than 100g secreted protein L(-1). Consequently, T. reesei has been intensively studied in the 20th century. Most of these investigations focused on the biochemical characteristics of its cellulases and hemicellulases, on the improvement of their properties by protein engineering, and on enhanced enzyme production by recombinant strategies. However, as the fungus is rare in nature, its ecology remained unknown. The breakthrough in the understanding of the fundamental biology of T. reesei only happened during 2000s-2010s. In this review, we compile the current knowledge on T. reesei ecology, physiology, and genomics to present a holistic view on the natural behavior of the organism. This is not only critical for science-driven further improvement of the biotechnological applications of this fungus, but also renders T. reesei as an attractive model of filamentous fungi with superior saprotrophic abilities.

Green Mold Diseases of Agaricus and Pleurotus spp. Are Caused by Related but Phylogenetically Different Trichoderma Species.

ABSTRACT Producers of champignon (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus) are facing recent incidents of green mold epidemics in Hungary. We examined 66 Trichoderma strains isolated from Agaricus compost and Pleurotus substrate samples from three Hungarian mushroom producing companies by a polymerase chain reaction-based diagnostic test for T. aggressivum, sequence analysis of the internal transcribed spacer region 1 (ITS1) and ITS2 and (selectively) of the fourth and fifth intron of translation elongation factor 1alpha (tef1alpha), and restriction fragment length polymorphism of mitochondrial DNA. Seven Trichoderma species were identified: T. aggressivum f. europaeum (17 isolates), T. harzianum (three isolates), T. longibrachiatum (four isolates), T. ghanense (one isolate), T. asperellum (four isolates), T. atroviride (nine isolates), and a still undescribed phylogenetic species, Trichoderma sp. DAOM 175924 (28 isolates). T. aggressivum f. europaeum was exclusively derived from A. bisporus compost, whereas Trichoderma sp. DAOM 175924 exclusively occurred in the substrate for Pleurotus cultivation. Sequences of the latter strains were co-specific with those for Trichoderma pathogens of P. ostreatus in Korea. The widespread occurrence of this new species raises questions as to why infections by it have just only recently been observed. Our data document that (i) green mold disease by T. aggressivum f. europaeum has geographically expanded to Central Europe; (ii) the green mold disease of P. ostreatus in Hungary is due to the same Trichoderma species as in Korea and the worldwide distribution of the new species indicates the possibility of spreading epidemics; and (iii) on mushroom farms, the two species are specialized on their different substrates.

Regulation of citrate synthase from the citric acid-accumulating fungus, Aspergillus niger.

Citrate synthase (citrate oxaloacetate-lyase (pro-3S-CH2cOO leads to acetate-CoA), EC 4.1.3.7) was purified 66-fold from cell-free extracts of a citric acid producing strain of Aspergillus niger. The enzyme is labile at low ionic strength, but can effectively be stabilized by K+, oxaloacetate or glycerol. It has a molecular weight of 80 000 and an optimum pH of 8.5. The enzyme is activated by monovalent cations in dilute buffer solutions, and inhibited by Mg2+ independent of the buffer molarity. Kinetic analysis indicated that the reaction proceeds by an ordered sequential mechanism. The Michaelis constants are: 5 microM for oxaloacetic acid at all concentrations of acetyl-CoA; 10 microM for acetyl-CoA at infinite concentrations of oxaloacetate. Coenzyme A is inhibitory, being competitive with acetyl-CoA (Ki = 0.15 mM) and non-competitive with oxaloacetate. Citrate has no effect. Among various metabolites tested, only ATP can inhibit the enzyme. The inhibition is competitive with acetyl-CoA (Ki = 1.0 mM), and non-competitive with oxaloacetate. Mg2+ partially relieves this inhibition. Other adenine nucleotides are also inhibitory, but to a lesser extent. It is proposed that citrate synthase from Aspergillus niger is only weakly regulated, its activity being mainly controlled by oxaloacetate availability.

Changes in the concentration of fructose 2,6-bisphosphate in Aspergillus niger during stimulation of acidogenesis by elevated sucrose concentration.

The presence of fructose 2,6-bisphosphate (Fru-2,6-P2) and phosphofructokinase 2 (PFK 2) were established in the citric-acid-producing filamentous fungus Aspergillus niger. Fru-2,6-P2 levels were around 3.0 (+/- 0.8) nmol per g dry weight during growth on sucrose, and half of this in mycelia grown on citrate as a carbon source. PFK 2 was detected with a specific activity of 150 mU/mg protein and a Km for fructose 6-phosphate of 40 microM. Induction of citric acid accumulation (acidogenesis) in A. niger by cultivation on high concentrations of sucrose, or replacement on 14% (w/v) sucrose correlated with an increase in the intracellular concentration of Fru-2,6-P2. A similar correlation was obtained when A. niger was cultivated on different carbon sources, which induced different rates of acidogenesis. The increase in Fru-2,6-P2 during transfer to 14% (w/v) sucrose was not correlated with the behaviour of mycelial concentrations of cyclic AMP, a potential regulator of Fru-2,6-P2 formation in other organisms, nor with that of Fru-6-P and ATP, the precursors of its formation. The extracellular addition of cyclic AMP and theophylline, an inhibitor of cellular cyclic AMP breakdown, increased both Fru-2,6-P2 concentration and acidogenesis in mycelia cultivated in 1% (w/v) sucrose medium. It is concluded that Fru-2,6-P2 controls citric acid accumulation by enabling increased rates of glucolysis, a prerequisite to acidogenesis.

Characterization and regulatory properties of a single hexokinase from the citric acid accumulating fungus Aspergillus niger.

A single glucose-phosphorylating enzyme has been detected and purified from the citric acid accumulating fungus Aspergillus niger. The enzyme was formed constitutively, and high activities were formed on glucose and sucrose as carbon sources. Highest activities were formed during growth on high concentrations of glucose or sucrose. The enzyme, purified about 600-fold from cell-free extracts prepared from glucose-grown mycelia, gave a double band in denaturing (SDS)-polyacrylamide gel electrophoresis. Tryptic peptide patterns suggest that the lower molecular weight band was the product of either C- or N-terminal truncation. The specific activity of the enzyme was about 40 and 35 mumol/min and mg protein with glucose and fructose as substrates, respectively. The affinity for glucose was about 10(3)-fold higher than for fructose. The subunit molecular weight was 50,000 and the molecular weight of the native protein was 100,000 by gel permeation chromatography. Of the reaction products ADP, but not glucose 6-phosphate, inhibited hexokinase activity. Citrate inhibited (K1 0.15 mM) non-competitively with respect to both glucose and ATP, which was not due to Mg(2+)-chelation. 2-Deoxyglucose resistant mutant strains of A. niger were isolated which showed decreased growth rate and activity of hexokinase during growth on glucose, while their growth on fructose and hexokinase activities were comparable to the parent strain. They displayed a reduced rate of citric acid accumulation. It is concluded that the synthesis of very high hexokinase activities may counteract citrate inhibition, thereby guaranteeing a high glycolytic flux during citric acid accumulation.

Uptake and export of citric acid by Aspergillus niger is reciprocally regulated by manganese ions.

The uptake as well as the export of citric acid by Aspergillus niger occur by active, deltapH-driven, H(+)-symport dependent systems. They are inhibited by nonmetabolizable tricarboxylic acid analogues and phthalic acid, and by several other mono-, di- and tribasic organic acids. However, citrate export could only be demonstrated in a mycelium cultivated under manganese-deficient growth conditions, whereas the uptake of citrate from the medium was only detectable upon precultivation of A. niger in a medium supplemented with Mn2+ ions. In addition, the uptake of citrate was dependent on the presence of Mn2+ ions in the assay, and inhibited by EDTA. This requirement for Mn2+ could also be partially fulfilled by Mg2+, Fe2+ or Zn2+, whereas Cu2+ ions inhibited citrate transport. The observed divergent effects of manganese ions on citrate uptake and citrate export may be a major reason for the well documented requirement for manganese deficiency of citric acid accumulation.

Ca2+-calmodulin antagonists interfere with xylanase formation and secretion in Trichoderma reesei.

The addition of Ca2+-antagonizers (La2+), Ca2+-ionophores (A23187) and Ca2+-complexing agents (EGTA) inhibited the formation of xylanase activity in resting mycelia of Trichoderma reesei. The inhibition by the ionophore was reversed by the addition of Ca2+ ions. A similar inhibitory effect was obtained by the addition of the calmodulin inhibitors, trifluoroperazine, chlorpromazine and quinacrine, hence suggesting that the observed effect of Ca2+ on xylanase formation occurred via calmodulin. The inhibition of xylanase formation by trifluoroperazine was accompanied by an inhibition of formation of the xyn2 transcript, and of the hph (hygromycin B-phosphotransferase-encoding) gene when fused downstream of the 5'-regulatory signals of the T. reesei xyn2 gene, indicating that calmodulin is required for xyn2 induction. At trifluoroperazine concentrations, which inhibited extracellular xylanase formation only slightly (about 30%), the cell-free extracts exhibited slightly increased xylanase activities. Subcellular fractionation showed that in these mycelia, the XYN II protein was distributed over a range of light vesicular fractions. This accumulated XYN II protein had the same Mr as the secreted, extracellular enzyme, indicating that it had already passed Golgi-located preprotein processing. Trifluoroperazine also specifically interfered with the endogenous, Ca2+-dependent phosphorylation of a 20-kDa protein, which was predominantly observed in cell-free extracts from mycelia growing on xylan. From these data, we conclude that calmodulin is required for xylanase II formation by T. reesei both at a transcriptional level as well as at a post-Golgi step of the secretory pathway. We also suggest that at least one of these two steps may be mediated via Ca2+-calmodulin-dependent phosphorylation.

A re-appraisal of multiplicity of endoglucanase I from Trichoderma reesei using monoclonal antibodies and plasma desorption mass spectrometry.

An endo beta-1,4-glucanase (EC 3.2.1.4, 1.4-(1,3;1,4)-beta-D-glucan 4 glucanhydrolase) was purified to apparent homogeneity from culture filtrates of Trichoderma reesei QM 9414. Identity of the protein with endoglucanase I (EG I) was examined by subjecting CNBr fragments of the protein to analysis by plasma desorption mass spectrometry. Seven non-glycosylated fragments, mapped on the eg1 gene sequence, could be identified, hence proving at least 39.4% identity of the amino acid sequence. No sign for microheterogeneity was observed. Purified EG I was used to prepare monoclonal antibodies. 17 stable clones were obtained, of which one--Mab EG 3--was used to analyze several commercial T. reesei cellulase preparations as well as culture filtrates from T. pseudokoningii and T. longibrachiatum for the presence of EG I. Most of them contained immunoreactive material migrating as a prominent 50-55 kDa band on SDS-PAGE, resembling EG I, but in some instances additional lower molecular weight bands were also observed. Cultivation of T. reesei at low pH led to an increase of these lower molecular weight bands. EG I was rather stable against proteolysis by papain in vitro, but after prolonged treatment, immunopositive products of 50 and 45 kDa were produced at the expense of the 55 kDa band. Our monoclonal antibodies failed to react with a low-molecular-weight endoglucanase, which was previously shown to be detectable with polyclonal antiserum against EG I. However, all monoclonals reacted with a 118 kDa protein which is most probably a dimer of EG I. These results are discussed with respect to the occurrence of multiple forms of EG I in T. reesei cellulase preparations.

Monoclonal antibodies against different domains of cellobiohydrolase I and II from Trichoderma reesei.

Monoclonal antibodies have been produced against two functionally different domains present in two cellobiohydrolases from Trichoderma reesei (CBH I and CBH II). Four groups of antibodies were obtained, which specifically recognized (Western blotting, ELISA) (a) the core protein within CBH I, (b) the core protein within CBH II, (c) the BA region of CBH I, and (d) the ABB' region of CBH II. No cross-reactivities within these four groups were observed. The antibodies reacted also specifically with proteins of similar size to CBH I and CBH II (SDS-PAGE) from other Trichoderma strains (Western blotting), whereas no reaction was observed with cellulases from other fungal sources. Analysis of culture filtrates of T. reesei QM 9414 harvested at various times of growth on cellulose under buffered conditions (pH 5-6) indicated the presence of only single bands of CBH I and CBH II, even after prolonged cultivation (160 h). Cultivation on cellulose in unbuffered media, however, showed the appearance (Western blotting) of additional lower molecular weight proteins, which reacted with the monoclonal antibodies directed against the cores of CBH I and II, but not with those recognizing the respective BA and ABB' regions. The appearance of these lower molecular weight bands was most pronounced in unbuffered media, supplemented with a 3-fold (w/w) amount of organic nitrogen (peptone). Analysis of some commercial cellulase preparations from T. harzianum revealed the same pattern of lower molecular weight proteins, in contrast to samples from other fungal cellulases. Those samples or preparations, showing a multiple pattern of CBH I and CBH II, exhibited higher activities of an acid proteinase. These results imply that the use of unbuffered, high nitrogen-supplemented culture conditions for production of cellulases may lead to considerable proteolytic modification of the secreted cellobiohydrolases.

Sequence of the pyr4 gene encoding orotidine-5'-phosphate decarboxylase from the biocontrol fungus Trichoderma harzianum.

The pyr4 gene, encoding orotidine-5'-phosphate decarboxylase (OMP decarboxylase) from the biocontrol fungus Trichoderma harzianum, has been isolated by hybridization, using a polymerase chain reaction (PCR)-derived fragment as a probe. The PCR primers corresponded to conserved regions of OMP decarboxylase-encoding genes from other filamentous fungi. A 2490-bp genomic fragment, which complemented a pyr4-auxotrophic T. reesei mutant, was sequenced. The gene showed high homology to pyr4 genes from other pyrenomycetes.

A protein kinase-encoding gene, pkt1, from Trichoderma reesei, homologous to the yeast YPK1 and YPK2 (YKR2) genes.

A gene (pkt1) was isolated from the filamentous fungus Trichoderma reesei, which exhibits high homology with the yeast YPK1 and YKR2 (YPK2) genes. It contains a 2123-bp ORF that is interrupted by two introns, and it encodes a 662-amino-acid protein with a calculated M(r) of 72,820. During active growth, pkt1 is expressed as two mRNAs of 3.1 and 2.8 kb which differ in the 3' untranslated region due to the use of two different polyadenylation sites.

Characterization of the pyruvate kinase-encoding gene (pki1) of Trichoderma reesei.

The pyruvate kinase-encoding gene (pki1) from Trichoderma reesei was isolated by hybridization to the corresponding Aspergillus nidulans pkiA gene. The 1614-bp nucleotide (nt) sequence of the cloned gene codes for a 538-amino-acid protein. The coding sequence contains a single intron of 246 nt at a position identical to that of intron E in the A. nidulans gene. The PKI protein shows extensive homology to the PKIs of A. nidulans and A. niger (67%) and Saccharomyces cerevisiae (59%). The 5' non-coding sequence contains a number of motifs typical for yeast glycolytic genes, but so far only rarely found in filamentous fungi.

Amino acid sequence similarities between low molecular weight endo-1,4-beta-xylanases and family H cellulases revealed by clustering analysis.

The amino acid sequences of seventeen family G xylanases and the two known family H cellulases have been compared by hydrophobic cluster analysis. A weak but significant similarity was demonstrated between these two families suggesting that these enzymes share the same molecular mechanism and catalytic residues and that they have related 3D folds. The major differences were found in the N-terminal regions.

Cre1, the carbon catabolite repressor protein from Trichoderma reesei.

In order to investigate the mechanism of carbon catabolite repression in the industrially important fungus Trichoderma reesei, degenerated PCR-primers were designed to amplify a 0.7-bp fragment of the cre1 gene, which was used to clone the entire gene. It encodes a 402-amino acid protein with a calculated M(r) of 43.6 kDa. Its aa-sequence shows 55.6% and 54.7% overall similarity to the corresponding genes of Aspergillus nidulans and A. niger, respectively. Similarity was restricted to the aa-region containing the C2H2 zinc finger and several aa-regions rich in proline and basic amino acids, which may be involved in the interaction with other proteins. Another aa-region rich in the SPXX-motif that has been considered analogous to a region of yeast RGR1p, was instead identified as a domain occurring in several eucaryotic transcription factors. The presence of the cre1 translation product was demonstrated with polyclonal antibodies against Cre1, which identified a protein of 43 (+/- 2) kDa in cell-free extracts from T. reesei. A Cre1 protein fragment from the two zinc fingers to the region similar to the aa-sequence of eucaryotic transcription factors, was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. EMSA and in vitro footprinting revealed binding of the fusion protein to the sequence 5'-GCGGAG-3', which matches well with the A. nidulans consensus sequence for CreA binding (5'-SYGGRG-3'). Cell-free extracts of T. reesei formed different complexes with DNA-fragments carrying this binding sites, and the presence of Cre1 and additional proteins in these complexes was demonstrated. We conclude that T. reesei Cre1 is the functional homologue of Aspergillus CreA and that it binds to its target sequence probably as a protein complex.

The role of the citric acid cycle in fungal organic acid fermentations.

Filamentous fungi are well known for their potential to accumulate organic acids in the medium when supplied with large amounts of sugar. Commercial applications of this are the production of citric and itaconic acids. The present review attempts to present the current state of knowledge on the biochemical basis of organic acid accumulation by filamentous fungi (citric, itaconic, fumaric and oxalic acids), particularly with respect to the role of citric acid cycle reactions. The explanations offered are based on recent advances in understanding the compartmentation of the fungal cell, and regulation of some key enzymes. The general conclusion is that fungi accumulate organic acids by mechanisms which avoid the channeling of substrates into the citric acid cycle under conditions of strongly active glycolysis.

Regulation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N biosynthesis in Penicillium chrysogenum by the alpha-aminoadipate pool size.

The effect of changes in the intracellular concentration of alpha-aminoadipate on the formation of alpha-aminoadipyl-cysteinyl-valine (ACV) and isopenicillin N (IPN)--two intermediates of penicillin biosynthesis--by strains of Penicillium chrysogenum has been investigated by measuring the incorporation of radioactivity from (6-14C)-alpha-aminoadipate into cellular 14C-ACV and 14C-IPN. No ACV or IPN were found in any strain during cultivation on glucose, but were clearly detected in all three strains during growth on lactose, displaying increased formation in strains exhibiting increased penicillin productivity and increased intracellular alpha-aminoadipate pools. ACV and IPN formation was affected by subjected P. chrysogenum mycelia to either general amino acid control (by addition of amitrol) or by exogenous addition of 5 mM L-lysine. In all cases, the changes observed paralleled the changes in the intracellular alpha-aminoadipate pool. These results are consistent with the alpha-aminoadipate pool limiting the biosynthesis of ACV and IPN and hence penicillin biosynthesis in the present strains of P. chrysogenum.

Randomly amplified polymorphic DNA fingerprinting identifies subgroups of Trichoderma viride and other Trichoderma sp. capable of chestnut blight biocontrol.

Eleven strains of Trichoderma viride, 2 strains of the putative teleomorph Hypocrea rufa and 9 of several other Trichoderma sp. were characterized by random polymorphic DNA amplification (RAPD) fingerprinting and screened for their ability to antagonize growth of European strains of the chestnut blight causing fungus Cryphonectria parasitica, using a dual-culture assay. The best strains were found in the species T. harzianum, T. parceramosum, a distinguishable subgroup of T. viride and a not named Trichoderma sp. The successful application of these strains against chestnut blight in vivo is demonstrated.

Lysine biosynthesis in Penicillium chrysogenum is regulated by feedback inhibition of alpha-aminoadipate reductase.

A partially purified preparation of alpha-aminoadipate reductase (EC 1.2.1.31) from Penicillium chrysogenum is competitively inhibited by lysine (Ki of 0.26 mM). Exogenous addition of 10 mM L-lysine to resting mycelia of P. chrysogenum increased the intracellular lysine pool concentration 2-fold, but decreased the incorporation of (6-14C)-alpha-aminoadipate into protein-bound lysine to a fifth. The distribution of radioactivity in the pathway metabolites alpha-aminoadipate, saccharopine and lysine was consistent with the assumption of a lysine sensitive enzyme step in vivo between alpha-aminoadipate and saccharopine. Hence lysine inhibition of alpha-aminoadipate reductase may be of physiologic importance.

Trehalose-6-phosphate synthase A affects citrate accumulation by Aspergillus niger under conditions of high glycolytic flux.

Accumulation of citric acid by Aspergillus niger depends on a high flux through glycolysis. We have investigated the possibility of control of this flux by trehalose 6-phosphate, an inhibitor of hexokinase of Saccharomyces cerevisiae and other eukaryotes (Blasquez et al., FEBS Lett. (1993) 329, 51-54). Hexokinase of A. niger was shown in vitro to be only weakly inhibited by trehalose 6-phosphate (KI 1.5-2 mM). To investigate the in vivo relevance of this inhibition, we used isogenic strains of A. niger, carrying either a disruption or an amplification of the trehalose-6-phosphate synthase A (T6PSA)-encoding gene (ggsA) and exhibiting corresponding differences in T6PSA activity. These strains produced citric acid at comparable rates and with similar yields on 1 or 2.5% (w/v) sucrose. At 5-14% (w/v) sucrose, the ggsA disrupted strain initiated citric acid accumulation earlier, whereas the multicopy strain showed the reverse effect. When sucrose was replaced by lactose, which enabled only low rates of catabolism irrespective of its concentration (1-8%), no differences in the initiation or rate of citric acid accumulation were observed between the three strains. The possible mechanisms by which ggsA controls glycolytic flux in A. niger in the presence of high sugar concentrations are discussed.

Modulation of mannosylphosphodolichol synthase and dolichol kinase activity in Trichoderma, related to protein secretion.

It has been postulated that exoprotein secretion in Trichoderma is related to their O-glycosylation. In the present paper the involvement of phosphodolichol in this process is described and the key role of mannosylphosphodolichol (MPD) synthase in protein O-mannosylation is discussed. The effect of water soluble phospholipid precursors such as choline and Tween 80, known also to increase secretion of cellulases when added to the medium, on MPD-synthase activity is presented. This effect is positive in the Trichoderma reesei QM 9414 (a low producing strain) but has no influence on the enzyme activity from the RUT C-30 strain selected to overproduce secretion of exoproteins and known to contain an increased cellular amount of endoplasmic reticulum. The positive effect of addition of choline and Tween to the medium on the level of dolichol kinase activity is also demonstrated. The influence of cultivation temperature on the activity of the various enzymes involved in dolichol-dependent protein glycosylation i.e. MPD-synthase, dolichyl kinase and MPD/Protein mannosyl transferase was tested. For all enzymes cultivation at 35 degrees C led to the elevated activity, which was most striking for dolichol kinase, whereas for MPD-synthase and MPD/Protein mannosyl transferase the difference was only apparent in the assay when endogenous phosphodolichol was used as a substrate. Furthermore, lipid extract from the membranes cultivated at elevated temperature, when added to the enzyme obtained from Trichoderma grown at 25 degrees C, enhanced the dolichol kinase activity measured in the absence of exogenous dolichol.(ABSTRACT TRUNCATED AT 250 WORDS)