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The N-acetylglucosamine catabolic gene cluster in Trichoderma reesei is controlled by the Ndt80-like transcription factor RON1.

ABSTRACT: Chitin is an important structural constituent of fungal cell walls composed of N-acetylglucosamine (GlcNAc) monosaccharides, but catabolism of GlcNAc has not been studied in filamentous fungi so far. In the yeast Candida albicans, the genes encoding the three enzymes responsible for stepwise conversion of GlcNAc to fructose-6-phosphate are clustered. In this work, we analysed GlcNAc catabolism in ascomycete filamentous fungi and found that the respective genes are also clustered in these fungi. In contrast to C. albicans, the cluster often contains a gene for an Ndt80-like transcription factor, which we named RON1 (regulator of N-acetylglucosamine catabolism 1). Further, a gene for a glycoside hydrolase 3 protein related to bacterial N-acetylglucosaminidases can be found in the GlcNAc gene cluster in filamentous fungi. Functional analysis in Trichoderma reesei showed that the transcription factor RON1 is a key activator of the GlcNAc gene cluster and essential for GlcNAc catabolism. Furthermore, we present an evolutionary analysis of Ndt80-like proteins in Ascomycota. All GlcNAc cluster genes, as well as the GlcNAc transporter gene ngt1, and an additional transcriptional regulator gene, csp2, encoding the homolog of Neurospora crassa?CSP2/GRHL, were functionally characterised by gene expression analysis and phenotypic characterisation of knockout strains in T.?reesei.

SUBMITTER: Kappel L

PROVIDER: S-EPMC4950302 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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The N-acetylglucosamine catabolic gene cluster in Trichoderma reesei is controlled by the Ndt80-like transcription factor RON1.

Kappel Lisa L Gaderer Romana R Flipphi Michel M Seidl-Seiboth Verena V

Molecular microbiology 20151119 4

Chitin is an important structural constituent of fungal cell walls composed of N-acetylglucosamine (GlcNAc) monosaccharides, but catabolism of GlcNAc has not been studied in filamentous fungi so far. In the yeast Candida albicans, the genes encoding the three enzymes responsible for stepwise conversion of GlcNAc to fructose-6-phosphate are clustered. In this work, we analysed GlcNAc catabolism in ascomycete filamentous fungi and found that the respective genes are also clustered in these fungi. ...[more]

PMID: 26481444

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Project description:Sorbicillinoids are a diverse group of yellow secondary metabolites that are produced by a range of not closely related ascomycetes, including Penicillium chrysogenum, Acremonium chrysogenum, and Trichoderma reesei. They share a similarity to the name-giving compound sorbicillin, a hexaketide. Previously, a conserved gene cluster containing two polyketide synthases has been identified as the source of sorbicillin, and a model for the biosynthesis of sorbicillin in P. chrysogenum has been proposed. In this study, we deleted the major genes of interest of the cluster in T. reesei, namely sor1, sor3, and sor4. Sor1 is the homolog of P. chrysogenum SorA, which is the first polyketide synthase of the proposed biosynthesis pathway. Sor3 is a flavin adenine dinucleotide (FAD)-dependent monooxygenase, and its homolog in P. chrysogenum, SorC, was shown to oxidize sorbicillin and 2',3'-dihydrosorbicillin to sorbicillinol and 2',3'-dihydrosorbicillinol, respectively, in vitro. Sor4 is an FAD/flavin mononucleotide-containing dehydrogenase with an unknown function. We measured the amounts of synthesized sorbicillinoids throughout growth and could verify the roles of Sor1 and Sor3 in vivo in T. reesei. In the absence of Sor4, two compounds annotated to dihydrosorbicillinol accumulate in the supernatant and only small amounts of sorbicillinol are synthesized. Therefore, we suggest extending the current biosynthesis model about Sor4 reducing 2',3'-dihydrosorbicillin and 2',3'-dihydrosorbicillinol to sorbicillinol and sorbicillinol, respectively. Sorbicillinol turned out to be the main chemical building block for most sorbicillinoids, including oxosorbicillinol, bisorbicillinol, and bisvertinolon. Further, we detected the sorbicillinol-dependent synthesis of 5-hydroxyvertinolide at early time points, which contradicts previous models for biosynthesis of 5-hydroxyvertinolide. Finally, we investigated whether sorbicillinoids from T. reesei have a growth limiting effect on the fungus itself or on plant pathogenic fungi or on pathogenic bacteria.

Project description:BackgroundThe fungus Trichoderma reesei is one of the most used industrial cellulase producers due to its high capacity of protein secretion. Strains of T. reesei with enhanced protein secretion capacity, such as Rut-C30, have been obtained after several rounds of random mutagenesis. The strain was shown to possess an expanded endoplasmic reticulum, but the genetic factors responsible for this phenotype remain still unidentified. Recently, three new transcription factors were described in Neurospora crassa which were demonstrated to be involved in protein secretion. One of them, RES2, was involved in upregulation of secretion-related genes. The aim of our present study was therefore to analyze the role of RES2, on protein secretion in the T. reesei Rut-C30 strain.ResultDeletion of the res2 gene in Rut-C30 resulted in slightly slower growth on all substrates tested, and lower germination rate as well as lower protein secretion compared to the parental strain Rut-C30. Transcriptomic analysis of the Rut-C30 and the Δres2 mutant strain in secretion stress conditions showed remarkably few differences : 971 genes were differentially expressed (DE) in both strains while 192 genes out of 1163 (~ 16.5%) were DE in Rut-C30 only and 693 out of 1664 genes (~ 41.6%) displayed differential expression solely in Δres2. Notably, induction of protein secretion by cultivating on lactose and addition of secretion stress inducer DTT induced many genes of the secretion pathway similarly in both strains. Among the differentially expressed genes, those coding for amino acid biosynthesis genes, transporters and genes involved in lipid metabolism were found to be enriched specifically in the Δres2 strain upon exposure to lactose or DTT. Besides, redox homeostasis and DNA repair genes were specifically upregulated in the Δres2 strain, indicating an altered stress response.ConclusionThese results indicate that in the T. reesei Rut-C30 strain, RES2 does not act as a master regulator of the secretion pathway, but it contributes to a higher protein secretion by adjusting the expression of genes involved in different steps of protein synthesis and the secretion pathway.

Dataset Information

The N-acetylglucosamine catabolic gene cluster in Trichoderma reesei is controlled by the Ndt80-like transcription factor RON1.

Publications

The N-acetylglucosamine catabolic gene cluster in Trichoderma reesei is controlled by the Ndt80-like transcription factor RON1.

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