Expression of Saccharomyces cerevisiae RER2 Gene Encoding Cis-Prenyltransferase in Trichoderma atroviride Increases the Activity of Secretory Hydrolases and Enhances Antimicrobial Features.

Some Trichoderma spp. exhibit natural abilities to reduce fungal diseases of plants through their mycoparasitic and antagonistic properties. In this study, we created new Trichoderma atroviride strains with elevated antifungal activity. This effect was achieved by improving the activity of cis-prenyltransferase, the main enzyme in dolichol synthesis, by expressing the RER2 gene from Saccharomyces cerevisiae. Since dolichyl phosphate is the carrier of carbohydrate residues during protein glycosylation, activation of its synthesis enhanced the activities of dolichyl-dependent enzymes, DPM synthase and N-acetylglucosamine transferase, as well as stimulated glycosylation of secretory proteins. Cellulases secreted by the transformants revealed significantly higher levels or activities compared to the control strain. Consequently, the resulting Trichoderma strains were more effective against the plant pathogens Pythium ultimum.

Metabolic Potential, Ecology and Presence of Associated Bacteria Is Reflected in Genomic Diversity of Mucoromycotina.

Mucoromycotina are often considered mainly in pathogenic context but their biology remains understudied. We describe the genomes of six Mucoromycotina fungi representing distant saprotrophic lineages within the subphylum (i.e., Umbelopsidales and Mucorales). We selected two Umbelopsis isolates from soil (i.e., U. isabellina, U. vinacea), two soil-derived Mucor isolates (i.e., M. circinatus, M. plumbeus), and two Mucorales representatives with extended proteolytic activity (i.e., Thamnidium elegans and Mucor saturninus). We complement computational genome annotation with experimental characteristics of their digestive capabilities, cell wall carbohydrate composition, and extensive total lipid profiles. These traits inferred from genome composition, e.g., in terms of identified encoded enzymes, are in accordance with experimental results. Finally, we link the presence of associated bacteria with observed characteristics. Thamnidium elegans genome harbors an additional, complete genome of an associated bacterium classified to Paenibacillus sp. This fungus displays multiple altered traits compared to the remaining isolates, regardless of their evolutionary distance. For instance, it has expanded carbon assimilation capabilities, e.g., efficiently degrades carboxylic acids, and has a higher diacylglycerol:triacylglycerol ratio and skewed phospholipid composition which suggests a more rigid cellular membrane. The bacterium can complement the host enzymatic capabilities, alter the fungal metabolism, cell membrane composition but does not change the composition of the cell wall of the fungus. Comparison of early-diverging Umbelopsidales with evolutionary younger Mucorales points at several subtle differences particularly in their carbon source preferences and encoded carbohydrate repertoire. Nevertheless, all tested Mucoromycotina share features including the ability to produce 18:3 gamma-linoleic acid, use TAG as the storage lipid and have fucose as a cell wall component.

Synthesis of Dolichols in Candida albicans Is Co-Regulated with Elongation of Fatty Acids.

Protein glycosylation requires dolichyl phosphate as a carbohydrate carrier. Dolichols are α-saturated polyprenols, and their saturation in S. cerevisiae is catalyzed by polyprenyl reductase Dfg10 together with some other unknown enzymes. The aim of this study was to identify such enzymes in Candida. The Dfg10 polyprenyl reductase from S. cerevisiae comprises a C-terminal 3-oxo-5-alpha-steroid 4-dehydrogenase domain. Alignment analysis revealed such a domain in two ORFs (orf19.209 and orf19.3293) from C. albicans, which were similar, respectively, to Dfg10 polyprenyl reductase and Tsc13 enoyl-transferase from S. cerevisiae. Deletion of orf19.209 in Candida impaired saturation of polyprenols. The Tsc13 homologue turned out not to be capable of saturating polyprenols, but limiting its expression reduce the cellular level of dolichols and polyprenols. This reduction was not due to a decreased expression of genes encoding cis-prenyltransferases from the dolichol branch but to a lower expression of genes encoding enzymes of the early stages of the mevalonate pathway. Despite the resulting lower consumption of acetyl-CoA, the sole precursor of the mevalonate pathway, it was not redirected towards fatty acid synthesis or elongation. Lowering the expression of TSC13 decreased the expression of the ACC1 gene encoding acetyl-CoA carboxylase, the key regulatory enzyme of fatty acid synthesis and elongation.

Yil102c-A is a Functional Homologue of the DPMII Subunit of Dolichyl Phosphate Mannose Synthase in Saccharomyces cerevisiae.

In a wide range of organisms, dolichyl phosphate mannose (DPM) synthase is a complex of tree proteins Dpm1, Dpm2, and Dpm3. However, in the yeast Saccharomyces cerevisiae, it is believed to be a single Dpm1 protein. The function of Dpm3 is performed in S. cerevisiae by the C-terminal transmembrane domain of the catalytic subunit Dpm1. Until present, the regulatory Dpm2 protein has not been found in S. cerevisiae. In this study, we show that, in fact, the Yil102c-A protein interacts directly with Dpm1 in S. cerevisiae and influences its DPM synthase activity. Deletion of the YIL102c-A gene is lethal, and this phenotype is reversed by the dpm2 gene from Trichoderma reesei. Functional analysis of Yil102c-A revealed that it also interacts with glucosylphosphatidylinositol-N-acetylglucosaminyl transferase (GPI-GnT), similar to DPM2 in human cells. Taken together, these results show that Yil102c-A is a functional homolog of DPMII from T. reesei and DPM2 from humans.

Internalization of the Aspergillus nidulans AstA Transporter into Mitochondria Depends on Growth Conditions, and Affects ATP Levels and Sulfite Oxidase Activity.

The astA gene encoding an alternative sulfate transporter was originally cloned from the genome of the Japanese Aspergillus nidulans isolate as a suppressor of sulfate permease-deficient strains. Expression of the astA gene is under the control of the sulfur metabolite repression system. The encoded protein transports sulfate across the cell membrane. In this study we show that AstA, having orthologs in numerous pathogenic or endophytic fungi, has a second function and, depending on growth conditions, can be translocated into mitochondria. This effect is especially pronounced when an astA-overexpressing strain grows on solid medium at 37 °C. AstA is also recruited to the mitochondria in the presence of mitochondria-affecting compounds such as menadione or antimycin A, which are also detrimental to the growth of the astA-overexpressing strain. Disruption of the Hsp70-Porin1 mitochondrial import system either by methylene blue, an Hsp70 inhibitor, or by deletion of the porin1-encoding gene abolishes AstA translocation into the mitochondria. Furthermore, we observed altered ATP levels and sulfite oxidase activity in the astA-overexpressing strain in a manner dependent on sulfur sources. The presented data indicate that AstA is also involved in the mitochondrial sulfur metabolism in some fungi, and thereby indirectly manages redox potential and energy state.

Identification of bacteria and fungi inhabiting fruiting bodies of Burgundy truffle (Tuber aestivum Vittad.).

Tuber species may be regarded as complex microhabitats hosting diverse microorganisms inside their fruiting bodies. Here, we investigated the structure of microbial communities inhabiting the gleba of wild growing (in stands) T. aestivum, using Illumina sequencing and culture-based methods. The two methods used in combination allowed to extract more information on complex microbiota of Tuber aestivum gleba. Analysis of the V3-V4 region of 16S rDNA identified nine phyla of bacteria present in the gleba of T. aestivum ascomata, mostly Proteobacteria from the family Bradyrhizobiaceae. Our results ideally match the earlier data for other Tuber species where the family Bradyrhizobiaceae was the most represented. The ITS1 region of fungal rDNA represented six alien fungal species belonging to three phyla. To complement the metagenomic analysis, cultivable fungi and bacteria were obtained from the gleba of the same T. aestivum fruiting bodies. The identified fungi mostly belong to the phylum Basidiomycota and same to Ascomycota. Analysis of cultivable bacteria revealed that all the specimens were colonized by different strains of Bacillus. Fungal community inhabiting T. aestivum fruiting bodies was never shown before.

Increased activity of the sterol branch of the mevalonate pathway elevates glycosylation of secretory proteins and improves antifungal properties of Trichoderma atroviride.

Some Trichoderma spp. have an ability to inhibit proliferation of fungal plant pathogens in the soil. Numerous compounds with a proven antifungal activity are synthesized via the terpene pathway. Here, we stimulated the activity of the mevalonate pathway in T. atroviride P1 by expressing the Saccharomyces cerevisiae ERG20 gene coding for farnesyl pyrophosphate (FPP) synthase, a key enzyme of this pathway. ERG20-expressing Trichoderma strains showed higher activities of FPP synthase and squalene synthase, the principal recipient of FPP in the mevalonate pathway. We also observed activation of dolichyl phosphate mannose (DPM) synthase, an enzyme in protein glycosylation, and significantly increased O- and N-glycosylation of secreted proteins. The hyper-glycosylation of secretory hydrolases could explain their increased activity observed in the ERG20 transformants. Analysis of the antifungal properties of the new strains revealed that the hydrolases secreted by the transformants inhibited growth of a plant pathogen, Pythium ultimum more efficiently compared to the control strain. Consequently, the biocontrol activity of the transgenic strains, determined as their ability to protect bean seeds and seedlings against harmful action of P. ultimum, was also improved substantially.

Inhibition of Dephosphorylation of Dolichyl Diphosphate Alters the Synthesis of Dolichol and Hinders Protein N-Glycosylation and Morphological Transitions in Candida albicans.

The essential role of dolichyl phosphate (DolP) as a carbohydrate carrier during protein N-glycosylation is well established. The cellular pool of DolP is derived from de novo synthesis in the dolichol branch of the mevalonate pathway and from recycling of DolPP after each cycle of N-glycosylation, when the oligosaccharide is transferred from the lipid carrier to the protein and DolPP is released and then dephosphorylated. In Saccharomyces cerevisiae, the dephosphorylation of DolPP is known to be catalyzed by the Cwh8p protein. To establish the role of the Cwh8p orthologue in another distantly related yeast species, Candida albicans, we studied its mutant devoid of the CaCWH8 gene. A double Cacwh8∆/Cacwh8∆ strain was constructed by the URA-blaster method. As in S. cerevisiae, the mutant was impaired in DolPP recycling. This defect, however, was accompanied by an elevation of cis-prenyltransferase activity and higher de novo production of dolichols. Despite these compensatory changes, protein glycosylation, cell wall integrity, filamentous growth, and biofilm formation were impaired in the mutant. These results suggest that the defects are not due to the lack of DolP for the protein N-glycosylation but rather that the activity of oligosacharyltransferase could be inhibited by the excess DolPP accumulating in the mutant.

Poly-Saturated Dolichols from Filamentous Fungi Modulate Activity of Dolichol-Dependent Glycosyltransferase and Physical Properties of Membranes.

 Mono-saturated polyprenols (dolichols) have been found in almost all Eukaryotic cells, however, dolichols containing additional saturated bonds at the ω-end, have been identified in A. fumigatus and A. niger. Here we confirm using an LC-ESI-QTOF-MS analysis, that poly-saturated dolichols are abundant in other filamentous fungi, Trichoderma reesei, A. nidulans and Neurospora crassa, while the yeast Saccharomyces cerevisiae only contains the typical mono-saturated dolichols. We also show, using differential scanning calorimetry (DSC) and fluorescence anisotropy of 1,6-diphenyl-l,3,5-hexatriene (DPH) that the structure of dolichols modulates the properties of membranes and affects the functioning of dolichyl diphosphate mannose synthase (DPMS). The activity of this enzyme from T. reesei and S. cerevisiae was strongly affected by the structure of dolichols. Additionally, the structure of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) model membranes was more strongly disturbed by the poly-saturated dolichols from Trichoderma than by the mono-saturated dolichols from yeast. By comparing the lipidome of filamentous fungi with that from S. cerevisiae, we revealed significant differences in the PC/PE ratio and fatty acids composition. Filamentous fungi differ from S. cerevisiae in the lipid composition of their membranes and the structure of dolichols. The structure of dolichols profoundly affects the functioning of dolichol-dependent enzyme, DPMS.

The role of Alg13 N-acetylglucosaminyl transferase in the expression of pathogenic features of Candida albicans.

BACKGROUND: The pathogenic potential of Candida albicans depends on adhesion to the host cells mediated by highly glycosylated adhesins, hyphae formation and growth of biofilm. These factors require effective N-glycosylation of proteins. Here, we present consequences of up- and down-regulation of the newly identified ALG13 gene encoding N-acetylglucosaminyl transferase, a potential member of the Alg7p/Alg13p/Alg14p complex catalyzing the first two initial reactions in the N-glycosylation process. METHODS: We constructed C. albicans strain alg13∆::hisG/TRp-ALG13 with one allele of ALG13 disrupted and the other under the control of a regulatable promoter, TRp. Gene expression and enzyme activity were measured using RT-qPCR and radioactive substrate. Cell wall composition was estimated by HPLC DIONEX. Protein glycosylation status was analyzed by electrophoresis of HexNAcase, a model N-glycosylated protein in C. albicans. RESULTS: Both decreased and elevated expression of ALG13 changed expression of all members of the complex and resulted in a decreased activity of Alg7p and Alg13p and under-glycosylation of HexNAcase. The alg13 strain was also defective in hyphae formation and growth of biofilm. These defects could result from altered expression of genes encoding adhesins and from changes in the carbohydrate content of the cell wall of the mutant. GENERAL SIGNIFICANCE: This work confirms the important role of protein N-glycosylation in the pathogenic potential of C. albicans.

Diversity of Cell Wall Related Proteins in Human Pathogenic Fungi.

The cell wall is one of the major keys to fungal identity. Fungi use their cell wall to sense the environment, and localize nutrients and competing microorganism. Pathogenic species additionally modify their cell walls to hide from a host's immune system. With the growing number of fungal infections and alarming shortage of available drugs, we are in need of new approaches to fight pathogens. The cell wall seems to be a natural target, since animal host cells are devoid of it. The current knowledge about fungal cell wall components is often limited, and there is huge diversity both in structure and composition between species. In order to compare the distribution of diverse proteins involved in cell wall biosynthesis and maintenance, we performed sequence homology searches against 24 fungal proteomes from distinct taxonomic groups, all reported as human pathogens. This approach led to identification of 4014 cell wall proteins (CWPs), and enabled us to speculate about cell wall composition in recently sequenced pathogenic fungi with limited experimental information. We found large expansions of several CWP families, in particular taxa, and a number of new CWPs possibly involved in evading host immune recognition. Here, we present a comprehensive evolutionary history of fungal CWP families in the context of the fungal tree of life.

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species.

The genus Trichoderma contains fungi with high relevance for humans, with applications in enzyme production for plant cell wall degradation and use in biocontrol. Here, we provide a broad, comprehensive overview of the genomic content of these species for "hot topic" research aspects, including CAZymes, transport, transcription factors, and development, along with a detailed analysis and annotation of less-studied topics, such as signal transduction, genome integrity, chromatin, photobiology, or lipid, sulfur, and nitrogen metabolism in T. reesei, T. atroviride, and T. virens, and we open up new perspectives to those topics discussed previously. In total, we covered more than 2,000 of the predicted 9,000 to 11,000 genes of each Trichoderma species discussed, which is >20% of the respective gene content. Additionally, we considered available transcriptome data for the annotated genes. Highlights of our analyses include overall carbohydrate cleavage preferences due to the different genomic contents and regulation of the respective genes. We found light regulation of many sulfur metabolic genes. Additionally, a new Golgi 1,2-mannosidase likely involved in N-linked glycosylation was detected, as were indications for the ability of Trichoderma spp. to generate hybrid galactose-containing N-linked glycans. The genomic inventory of effector proteins revealed numerous compounds unique to Trichoderma, and these warrant further investigation. We found interesting expansions in the Trichoderma genus in several signaling pathways, such as G-protein-coupled receptors, RAS GTPases, and casein kinases. A particularly interesting feature absolutely unique to T. atroviride is the duplication of the alternative sulfur amino acid synthesis pathway.

Fusarium sambucinum astA gene expressed during potato infection is a functional orthologue of Aspergillus nidulans astA.

Sulfate assimilation plays a vital role in prototrophic organisms. Orthologues of the alternative sulfate transporter (AstA) gene from Aspergillus nidulans were identified in the fungal plant pathogens Fusarium sambucinum and Fusarium graminearum. By physiological and biochemical analyses, the AstA orthologues were determined to be able to uptake sulfate from the environment. Similarly to astA in A. nidulans, the FsastA gene was found to be regulated by sulfur metabolite repression (SMR) in a sulfur-dependent manner. In contrast, the FgastA transcript was undetectable, however, when the FgastA gene was expressed heterologously in A. nidulans, the translated FgAstA protein acted as a sulfate transporter. Interestingly, F. sambucinum astA expression was remarkably augmented in infected potato tubers, despite the presence abundant sulfate and was found not to be correlated with plant resistance.

Overexpression of erg20 gene encoding farnesyl pyrophosphate synthase has contrasting effects on activity of enzymes of the dolichyl and sterol branches of mevalonate pathway in Trichoderma reesei.

The mevalonate pathway is the most diverse metabolic route resulting in the biosynthesis of at least 30,000 isoprenoid compounds, many of which, such as sterols or dolichols, are indispensable for living cells. In the filamentous fungus Trichoderma of major biotechnological interest isoprenoid metabolites are also involved in the biocontrol processes giving the mevalonate pathway an additional significance. On the other hand, little is known about genes coding for enzymes of the mevalonate pathway in Trichoderma. Here, we present cloning and functional analysis of the erg20 gene from Trichoderma reesei coding for farnesyl pyrophosphate (FPP) synthase (EC 2.5.1.10), an enzyme located at the branching point of the mevalonate pathway. Expression of the gene in a thermosensitive erg20-2 mutant of Saccharomyces cerevisiae impaired in the FPP synthase activity suppressed the thermosensitive phenotype. The same gene overexpressed in T. reesei significantly enhanced the FPP synthase activity and also stimulated the activity of cis-prenyltransferase, an enzyme of the dolichyl branch of the mevalonate pathway. Unexpectedly, the activity of squalene synthase from the other, sterol branch, was significantly decreased without, however, affecting ergosterol level.

Elevated activity of dolichyl phosphate mannose synthase enhances biocontrol abilities of Trichoderma atroviride.

Antagonism of Trichoderma spp. against phytopathogenic fungi is widely exploited for biocontrol of plant diseases. A crucial role in the biocontrol mechanism is attributed to cell-wall-degrading enzymes secreted by Trichoderma spp. Therefore, more efficient production and secretion of the enzymes should elevate the biocontrol abilities of Trichoderma spp. Because the majority of secretory hydrolases are glycoproteins, it has been postulated that the posttranslational modification of these proteins could constitute a bottleneck in their production and secretion. Our previous study showed that improvement of O-glycosylation elevated protein secretion by Trichoderma reesei. In this study, we enhanced the biocontrol abilities of T. atroviride P1 against plant pathogens by overexpressing the Saccharomyces cerevisiae DPM1 gene coding for dolichyl phosphate mannose (DPM) synthase, a key enzyme in the O-glycosylation pathway. The transformants we obtained showed doubled DPM synthase activity and, at the same time, significantly elevated cellulolytic activity. They also revealed an improved antifungal activity against the plant pathogen Pythium ultimum.

Cloning and functional analysis of the dpm2 and dpm3 genes from Trichoderma reesei expressed in a Saccharomyces cerevisiae dpm1Δ mutant strain.

In Trichoderma reesei, dolichyl phosphate mannose (dpm) synthase, a key enzyme in the O-glycosylation process, requires three proteins for full activity. In this study, the dpm2 and dpm3 genes coding for the DPMII and DPMIII subunits of T. reesei DPM synthase were cloned and functionally analyzed after expression in the Saccharomyces cerevisiae dpm1Δ [genotype (BY4743; his3Δ1; /leu2Δ0; lys2Δ0; /ura3Δ0; YPR183w::kanMX4] mutant. It was found that apart from the catalytic subunit DPMI, the DPMIII subunit is also essential to form an active DPM synthase in yeast. Additional expression of the DPMII protein, considered to be a regulatory subunit of DPM synthase, decreased the enzymatic activity. We also characterized S. cerevisiae strains expressing the dpm1, 2, 3 or dpm1, 3 genes and analyzed the consequences of dpm expression on protein O-glycosylation in vivo and on the cell wall composition.

Integration of additional copies of Trichoderma reesei gene encoding protein O-mannosyltransferase I results in a decrease of the enzyme activity and alteration of cell wall composition.

In fungi, transfer of the first mannosyl residue to proteins during their O-glycosylation is catalyzed by protein O-mannosyltransferases. Integration of additional copies of the pmt1 gene into Trichoderma reesei genome unexpectedly resulted in the silencing of pmt1 expression. Strains carrying the additional copies of pmt1 gene exhibited lower total activity of protein O-mannosyltransferases, lower O- and N-glycosylation of secreted proteins and showed defects in their cell wall composition. Moreover, the strains grew slowly on solid medium and were hypersensitive to an antifungal reagent, Calcofluor white. These results indicate that protein O-mannosyltransferases are required for proper cell wall formation, and their decreased activity influences not only O- but also N-glycosylation.

Influence of sorbitol on protein production and glycosylation and cell wall formation in Trichoderma reesei.

Sorbitol is often used at 1 mol/liter as an osmotic stabilizer for cultivation of fungi with a fragile cell wall phenotype. On the other hand, at this concentration sorbitol causes an osmotic stress in fungal cells resulting in intensive production of intracellular glycerol. The highly increased consumption of glucose for glycerol synthesis may lead to changes in processes requiring carbohydrate residues. This study provides new information on the consequences of osmotic stress to the cell wall composition, protein production and glycosylation, and cell morphology of Trichoderma reesei. We observed that high osmolarity conditions enhanced biomass production and strongly limited synthesis of cell wall glucans and chitin. Moreover, in these conditions the amount of secreted protein decreased nearly ten-fold and expression of cbh1 and cbh2 genes coding for cellobiohydrolase I and cellobiohydrolase II, the main secretory proteins in T. reesei, was inhibited resulting in a lack of the proteins in the cell and cultivation medium. The activity of DPM synthase, enzyme engaged in both N- and O-glycosylation pathways, was reduced two-fold, suggesting an overall inhibition of protein glycosylation. However, the two modes of glycosylation were affected divergently: O-glycosylation of secreted proteins decreased in the early stages of growth while N-glycosylation significantly increased in the stationary phase.

Alterations in protein secretion caused by metabolic engineering of glycosylation pathways in fungi.

Due to its natural properties, Trichoderma reesei is commonly used in industry-scale production of secretory proteins. Since almost all secreted proteins are O-glycosylated, modulation of the activity of enzymes of the O-glycosylation pathway are likely to affect protein production and secretion or change the glycosylation pattern of the secreted proteins, altering their stability and biological activity. Understanding how the activation of different components of the O-glycosylation pathway influences the glycosylation pattern of proteins and their production and secretion could help in elucidating the mechanism of the regulation of these processes and should facilitate creation of engineered microorganisms producing high amounts of useful proteins. In this review we focus on data concerning Trichoderma, but also present some background information allowing comparison with other fungal species.

Disruption of Trichoderma reesei gene encoding protein O-mannosyltransferase I results in a decrease of the enzyme activity and alteration of cell wall composition.

In fungi transfer of the first mannosyl residue to proteins during their O-glycosylation is catalyzed by protein O-mannosyltransferases encoded by pmt genes. Disruption of the pmt1 gene in Trichoderma caused a significant decrease in the total activity of protein O-mannosyltransferases. Moreover, disruption of the pmt1 gene also led to osmotic sensitivity of the strain, indicating an essential role of the PMTI protein activity for cell wall synthesis. At the same time, the strain was defective in septa formation, producing only half the number of septa per unit length of hypha compared with the wild type. Disruption of the pmt1 gene decreased protein secretion but had no effect on glycosylation of secreted proteins, which suggests that PMTI protein O-mannosyltranferase does not take part in glycosylation of these proteins.