RESUMO
MOTIVATION: The cellular system of a living organism is composed of interacting bio-molecules that control cellular processes at multiple levels. Their correspondences are represented by tightly regulated molecular networks. The increase of omics technologies has favored the generation of large-scale disparate data and the consequent demand for simultaneously using molecular and functional interaction networks: gene co-expression, protein-protein interaction (PPI), genetic interaction and metabolic networks. They are rich sources of information at different molecular levels, and their effective integration is essential to understand cell functioning and their building blocks (proteins). Therefore, it is necessary to obtain informative representations of proteins and their proximity, that are not fully captured by features extracted directly from a single informational level. We propose BraneMF, a novel random walk-based matrix factorization method for learning node representation in a multilayer network, with application to omics data integration. RESULTS: We test BraneMF with PPI networks of Saccharomyces cerevisiae, a well-studied yeast model organism. We demonstrate the applicability of the learned features for essential multi-omics inference tasks: clustering, function and PPI prediction. We compare it to the state-of-the-art integration methods for multilayer networks. BraneMF outperforms baseline methods by achieving high prediction scores for a variety of downstream tasks. The robustness of results is assessed by an extensive parameter sensitivity analysis. AVAILABILITY AND IMPLEMENTATION: BraneMF's code is freely available at: https://github.com/Surabhivj/BraneMF, along with datasets, embeddings and result files. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Proteínas , Saccharomyces cerevisiae , Proteínas/metabolismo , Análise por Conglomerados , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: The 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. RESULT: Deletion 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. CONCLUSION: These 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.
Assuntos
Celulase , Trichoderma , Lactose/metabolismo , Deleção de Genes , Celulase/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Trichoderma/genéticaRESUMO
BACKGROUND: Gene expression is regulated at different molecular levels, including chromatin accessibility, transcription, RNA maturation, and transport. These regulatory mechanisms have strong connections with cellular metabolism. In order to study the cellular system and its functioning, omics data at each molecular level can be generated and efficiently integrated. Here, we propose BRANENET, a novel multi-omics integration framework for multilayer heterogeneous networks. BRANENET is an expressive, scalable, and versatile method to learn node embeddings, leveraging random walk information within a matrix factorization framework. Our goal is to efficiently integrate multi-omics data to study different regulatory aspects of multilayered processes that occur in organisms. We evaluate our framework using multi-omics data of Saccharomyces cerevisiae, a well-studied yeast model organism. RESULTS: We test BRANENET on transcriptomics (RNA-seq) and targeted metabolomics (NMR) data for wild-type yeast strain during a heat-shock time course of 0, 20, and 120 min. Our framework learns features for differentially expressed bio-molecules showing heat stress response. We demonstrate the applicability of the learned features for targeted omics inference tasks: transcription factor (TF)-target prediction, integrated omics network (ION) inference, and module identification. The performance of BRANENET is compared to existing network integration methods. Our model outperforms baseline methods by achieving high prediction scores for a variety of downstream tasks.
Assuntos
RNA , Saccharomyces cerevisiae , Cromatina , RNA-Seq , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
DNA methyltransferases are ubiquitous enzymes conserved in bacteria, plants and opisthokonta. These enzymes, which methylate cytosines, are involved in numerous biological processes, notably development. In mammals and higher plants, methylation patterns established and maintained by the cytosine DNA methyltransferases (DMTs) are essential to zygotic development. In fungi, some members of an extensively conserved fungal-specific DNA methyltransferase class are both mediators of the Repeat Induced Point mutation (RIP) genome defense system and key players of sexual reproduction. Yet, no DNA methyltransferase activity of these purified RID (RIP deficient) proteins could be detected in vitro. These observations led us to explore how RID-like DNA methyltransferase encoding genes would play a role during sexual development of fungi showing very little genomic DNA methylation, if any. To do so, we used the model ascomycete fungus Podospora anserina. We identified the PaRid gene, encoding a RID-like DNA methyltransferase and constructed knocked-out ΔPaRid defective mutants. Crosses involving P. anserina ΔPaRid mutants are sterile. Our results show that, although gametes are readily formed and fertilization occurs in a ΔPaRid background, sexual development is blocked just before the individualization of the dikaryotic cells leading to meiocytes. Complementation of ΔPaRid mutants with ectopic alleles of PaRid, including GFP-tagged, point-mutated and chimeric alleles, demonstrated that the catalytic motif of the putative PaRid methyltransferase is essential to ensure proper sexual development and that the expression of PaRid is spatially and temporally restricted. A transcriptomic analysis performed on mutant crosses revealed an overlap of the PaRid-controlled genetic network with the well-known mating-types gene developmental pathway common to an important group of fungi, the Pezizomycotina.
Assuntos
Proteínas de Bactérias/fisiologia , Metilases de Modificação do DNA/fisiologia , Redes Reguladoras de Genes/genética , Podospora/fisiologia , Citosina/metabolismo , Metilação de DNA/fisiologia , Epigênese Genética/fisiologia , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Fúngicos Tipo Acasalamento/genética , Genoma BacterianoRESUMO
BACKGROUND: The degradation of cellulose and hemicellulose molecules into simpler sugars such as glucose is part of the second generation biofuel production process. Hydrolysis of lignocellulosic substrates is usually performed by enzymes produced and secreted by the fungus Trichoderma reesei. Studies identifying transcription factors involved in the regulation of cellulase production have been conducted but no overview of the whole regulation network is available. A transcriptomic approach with mixtures of glucose and lactose, used as a substrate for cellulase induction, was used to help us decipher missing parts in the network of T. reesei Rut-C30. RESULTS: Experimental results on the Rut-C30 hyperproducing strain confirmed the impact of sugar mixtures on the enzymatic cocktail composition. The transcriptomic study shows a temporal regulation of the main transcription factors and a lactose concentration impact on the transcriptional profile. A gene regulatory network built using BRANE Cut software reveals three sub-networks related to i) a positive correlation between lactose concentration and cellulase production, ii) a particular dependence of the lactose onto the ß-glucosidase regulation and iii) a negative regulation of the development process and growth. CONCLUSIONS: This work is the first investigating a transcriptomic study regarding the effects of pure and mixed carbon sources in a fed-batch mode. Our study expose a co-orchestration of xyr1, clr2 and ace3 for cellulase and hemicellulase induction and production, a fine regulation of the ß-glucosidase and a decrease of growth in favor of cellulase production. These conclusions provide us with potential targets for further genetic engineering leading to better cellulase-producing strains in industry-like conditions.
Assuntos
Celulase , Trichoderma , Celulase/genética , Redes Reguladoras de Genes , Glucose , Hypocreales , Lactose , Trichoderma/genéticaRESUMO
BACKGROUND: There is a worldwide interest for sustainable and environmentally-friendly ways to produce fuels and chemicals from renewable resources. Among them, the production of acetone, butanol and ethanol (ABE) or Isopropanol, Butanol and Ethanol (IBE) by anaerobic fermentation has already a long industrial history. Isopropanol has recently received a specific interest and the best studied natural isopropanol producer is C. beijerinckii DSM 6423 (NRRL B-593). This strain metabolizes sugars into a mix of IBE with only low concentrations of ethanol produced (< 1 g/L). However, despite its relative ancient discovery, few genomic details have been described for this strain. Research efforts including omics and genetic engineering approaches are therefore needed to enable the use of C. beijerinckii as a microbial cell factory for production of isopropanol. RESULTS: The complete genome sequence and a first transcriptome analysis of C. beijerinckii DSM 6423 are described in this manuscript. The combination of MiSeq and de novo PacBio sequencing revealed a 6.38 Mbp chromosome containing 6254 genomic objects. Three Mobile Genetic Elements (MGE) were also detected: a linear double stranded DNA bacteriophage (Ï6423) and two plasmids (pNF1 and pNF2) highlighting the genomic complexity of this strain. A first RNA-seq transcriptomic study was then performed on 3 independent glucose fermentations. Clustering analysis allowed us to detect some key gene clusters involved in the main life cycle steps (acidogenesis, solvantogenesis and sporulation) and differentially regulated among the fermentation. These putative clusters included some putative metabolic operons comparable to those found in other reference strains such as C. beijerinckii NCIMB 8052 or C. acetobutylicum ATCC 824. Interestingly, only one gene was encoding for an alcohol dehydrogenase converting acetone into isopropanol, suggesting a single genomic event occurred on this strain to produce isopropanol. CONCLUSIONS: We present the full genome sequence of Clostridium beijerinckii DSM 6423, providing a complete genetic background of this strain. This offer a great opportunity for the development of dedicated genetic tools currently lacking for this strain. Moreover, a first RNA-seq analysis allow us to better understand the global metabolism of this natural isopropanol producer, opening the door to future targeted engineering approaches.
Assuntos
2-Propanol/metabolismo , Clostridium beijerinckii/genética , Genoma Bacteriano , Transcriptoma , Reatores Biológicos/microbiologia , Clostridium beijerinckii/metabolismo , Clostridium beijerinckii/fisiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Análise de Sequência de RNA , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismoRESUMO
Following the publication of this article [1], the authors noticed that Figs. 2, 3 and 4 were in the incorrect order and thus had incorrect captions.
RESUMO
The ascomycete Trichoderma reesei is an industrial producer of cellulolytic and hemicellulolytic enzymes, and serves as a prime model for their genetic regulation. Most of its (hemi-)cellulolytic enzymes are obligatorily dependent on the transcriptional activator XYR1. Here, we investigated the nucleo-cytoplasmic shuttling mechanism that transports XYR1 across the nuclear pore complex. We identified 14 karyopherins in T. reesei, of which eight were predicted to be involved in nuclear import, and produced single gene-deletion mutants of all. We found KAP8, an ortholog of Aspergillus nidulansâ KapI, and Saccharomyces cerevisiaeâ Kap121/Pse1, to be essential for nuclear recruitment of GFP-XYR1 and cellulase gene expression. Transformation with the native gene rescued this effect. Transcriptomic analyses of Δkap8 revealed that under cellulase-inducing conditions 42 CAZymes, including all cellulases and hemicellulases known to be under XYR1 control, were significantly down-regulated. Δkap8 strains were capable of forming fertile fruiting bodies but exhibited strongly reduced conidiation both in light and darkness, and showed enhanced sensitivity towards abiotic stress, including high osmotic pressure, low pH and high temperature. Together, these data underscore the significance of nuclear import of XYR1 in cellulase and hemicellulase gene regulation in T. reesei, and identify KAP8 as the major karyopherin required for this process.
Assuntos
Núcleo Celular/metabolismo , Celulase/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Esporos Fúngicos/crescimento & desenvolvimento , Trichoderma/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/enzimologia , Núcleo Celular/genética , Celulase/metabolismo , Proteínas Fúngicas/genética , Transporte Proteico , Reprodução Assexuada , Esporos Fúngicos/enzimologia , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Trichoderma/enzimologia , Trichoderma/genética , Trichoderma/crescimento & desenvolvimento , beta Carioferinas/genéticaRESUMO
BACKGROUND: Inferring gene networks from high-throughput data constitutes an important step in the discovery of relevant regulatory relationships in organism cells. Despite the large number of available Gene Regulatory Network inference methods, the problem remains challenging: the underdetermination in the space of possible solutions requires additional constraints that incorporate a priori information on gene interactions. METHODS: Weighting all possible pairwise gene relationships by a probability of edge presence, we formulate the regulatory network inference as a discrete variational problem on graphs. We enforce biologically plausible coupling between groups and types of genes by minimizing an edge labeling functional coding for a priori structures. The optimization is carried out with Graph cuts, an approach popular in image processing and computer vision. We compare the inferred regulatory networks to results achieved by the mutual-information-based Context Likelihood of Relatedness (CLR) method and by the state-of-the-art GENIE3, winner of the DREAM4 multifactorial challenge. RESULTS: Our BRANE Cut approach infers more accurately the five DREAM4 in silico networks (with improvements from 6% to 11%). On a real Escherichia coli compendium, an improvement of 11.8% compared to CLR and 3% compared to GENIE3 is obtained in terms of Area Under Precision-Recall curve. Up to 48 additional verified interactions are obtained over GENIE3 for a given precision. On this dataset involving 4345 genes, our method achieves a performance similar to that of GENIE3, while being more than seven times faster. The BRANE Cut code is available at: http://www-syscom.univ-mlv.fr/~pirayre/Codes-GRN-BRANE-cut.html. CONCLUSIONS: BRANE Cut is a weighted graph thresholding method. Using biologically sound penalties and data-driven parameters, it improves three state-of-the art GRN inference methods. It is applicable as a generic network inference post-processing, due to its computational efficiency.
Assuntos
Algoritmos , Escherichia coli/genética , Redes Reguladoras de Genes , Área Sob a Curva , Simulação por Computador , Bases de Dados Genéticas , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Trichoderma reesei is the main industrial source of cellulases and hemicellulases required for the hydrolysis of biomass to simple sugars, which can then be used in the production of biofuels and biorefineries. The highly productive strains in use today were generated by classical mutagenesis. As byproducts of this procedure, mutants were generated that turned out to be unable to produce cellulases. In order to identify the mutations responsible for this inability, we sequenced the genome of one of these strains, QM9136, and compared it to that of its progenitor T. reesei QM6a. RESULTS: In QM9136, we detected a surprisingly low number of mutagenic events in the promoter and coding regions of genes, i.e. only eight indels and six single nucleotide variants. One of these indels led to a frame-shift in the Zn2Cys6 transcription factor XYR1, the general regulator of cellulase and xylanase expression, and resulted in its C-terminal truncation by 140 amino acids. Retransformation of strain QM9136 with the wild-type xyr1 allele fully recovered the ability to produce cellulases, and is thus the reason for the cellulase-negative phenotype. Introduction of an engineered xyr1 allele containing the truncating point mutation into the moderate producer T. reesei QM9414 rendered this strain also cellulase-negative. The correspondingly truncated XYR1 protein was still able to enter the nucleus, but failed to be expressed over the basal constitutive level. CONCLUSION: The missing 140 C-terminal amino acids of XYR1 are therefore responsible for its previously observed auto-regulation which is essential for cellulases to be expressed. Our data present a working example of the use of genome sequencing leading to a functional explanation of the QM9136 cellulase-negative phenotype.
Assuntos
Celulase/genética , Proteínas Fúngicas/genética , Genoma Fúngico , Fatores de Transcrição/genética , Trichoderma/enzimologia , Trichoderma/genética , Alelos , Núcleo Celular/metabolismo , Celulase/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Fenótipo , Polimorfismo de Nucleotídeo Único , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Análise de Sequência de DNA , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Trichoderma reesei, the main filamentous fungus used for industrial cellulase production, was long considered to be asexual. The recent discovery of the mating type locus in the natural isolate QM6a and the possibility to cross this sterile female strain with a fertile natural female strain opened up a new avenue for strain optimization. We crossed the hyperproducer RutC30 with a compatible female ascospore-derived isolate of the wild-type strain CBS999.97 and analyzed about 300 offspring. A continuous distribution of secreted protein levels was observed in the progeny, confirming the involvement of several mutated loci in the hyperproductive phenotype. A bias toward MAT1-2 strains was identified for higher producers, but not directly linked to the Mating-type locus itself. Transgressive phenotypes were observed in terms of both productivity and secretome quality, with offspring that outperform their parents for three enzymatic activities. Genomic sequences of the 10 best producers highlighted the genetic diversity generated and the involvement of parental alleles in hyperproduction and fertility. IMPORTANCE: The filamentous fungus Trichoderma reesei produces cellulolytic enzymes that are essential for the hydrolysis of lignocellulosic biomass into monomerics sugars. The filamentous fungus T. reesei produces cellulolytic enzymes that are essential for the hydrolysis of lignocellulosic biomass into monomerics sugars, which can in turn be fermented to produce second-generation biofuels and bioproducts. Production performance improvement, which is essential to reduce production cost, relies on classical mutagenesis and genetic engineering techniques. Although sexual reproduction is a powerful tool for improving domesticated species, it is often difficult to apply to industrial fungi since most of them are considered asexual. In this study, we demonstrated that outbreeding is an efficient strategy to optimize T. reesei. Crossing between a natural isolate and a mutagenized strain generated a biodiverse progeny with some offspring displaying transgressive phenotype for cellulase activities.
RESUMO
An F(1)F(O) ATP synthase in the inner mitochondrial membrane catalyzes the late steps of ATP production via the process of oxidative phosphorylation. A small protein subunit (subunit c or ATP9) of this enzyme shows a substantial genetic diversity, and its gene can be found in both the mitochondrion and/or nucleus. In a representative set of 26 species of fungi for which the genomes have been entirely sequenced, we found five Atp9 gene repartitions. The phylogenetic distribution of nuclear and mitochondrial Atp9 genes suggests that their evolution has included two independent transfers to the nucleus followed by several independent episodes of the loss of the mitochondrial and/or nuclear gene. Interestingly, we found that in Podospora anserina, subunit c is exclusively produced from two nuclear genes (PaAtp9-5 and PaAtp9-7), which display different expression profiles through the life cycle of the fungus. The PaAtp9-5 gene is specifically and strongly expressed in germinating ascospores, whereas PaAtp9-7 is mostly transcribed during sexual reproduction. Consistent with these observations, deletion of PaAtp9-5 is lethal, whereas PaAtp9-7 deletion strongly impairs ascospore production. The P. anserina PaAtp9-5 and PaAtp9-7 genes are therefore nonredundant. By swapping the 5' and 3' flanking regions between genes we demonstrated, however, that the PaAtp9 coding sequences are functionally interchangeable. These findings show that after transfer to the nucleus, the subunit c gene in Podospora became a key target for the modulation of cellular energy metabolism according to the requirements of the life cycle.
Assuntos
Proteínas Fúngicas/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , ATPases Mitocondriais Próton-Translocadoras/genética , Podospora/genética , Sequência de Bases , Núcleo Celular , Proteínas Fúngicas/metabolismo , Deleção de Genes , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Dados de Sequência Molecular , Micélio/genética , Micélio/crescimento & desenvolvimento , Fenótipo , Filogenia , Podospora/enzimologia , Podospora/crescimento & desenvolvimento , Subunidades Proteicas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
Transcription pattern during mycelium growth of Podospora anserina was assayed by microarray analysis in wild type and in mutants affected in the MAP kinase genes PaMpk1 and PaMpk2 and in the NADPH oxidase gene PaNox1. 15% of the genes have their expression modified by a factor two or more as growth proceeds in wild type. The genes whose expression is modified during growth in P. anserina are either not conserved or differently regulated in Neurospora crassa and Aspergillus niger, two fungi for which transcriptome data during growth are available. The P. anserina mutants display a similar alteration of their transcriptome profile, with nearly 1000 genes affected similarly in the three mutants, accounting for their similar growth phenotypes. Yet, each mutant has its specific set of modified transcripts, in line with particular phenotypes exhibited by each mutant. Again, there is limited conservation during evolution of the genes regulated at the transcription level by MAP kinases, as indicated by the comparison the P. anserina data, with those of Aspergillus fumigatus and N. crassa, two fungi for which gene expression data are available for mutants of the MAPK pathways. Among the genes regulated in wild type and affected in the mutants, those involved in carbohydrate and secondary metabolisms appear prominent. The vast majority of the genes differentially expressed are of unknown function. Availability of their transcription profile at various stages of development should help to decipher their role in fungal physiology and development.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/genética , Podospora/genética , Transdução de Sinais , Transcriptoma , Evolução Biológica , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Anotação de Sequência Molecular , Podospora/metabolismo , Ativação TranscricionalRESUMO
NADPH oxidases are enzymes that produce reactive oxygen species. Studies in mammals, plants and fungi have shown that they play important roles in differentiation, defence, host/pathogen interaction and mutualistic symbiosis. In this paper, we have identified a Podospora anserina mutant strain impaired for processes controlled by PaNox1 and PaNox2, the two Nox isoforms characterized in this model ascomycete. We show that the gene mutated is PaNoxR, the homologue of the gene encoding the regulatory subunit p67(phox), conserved in mammals and fungi, and that PaNoxR regulates both PaNox1 and PaNox2. Genome sequence analysis of P. anserina reveals that this fungus posses a third Nox isoform, PaNox3, related to human Nox5/Duox and plant Rboh. We have generated a knock-out mutant of PaNox3 and report that PaNox3 plays a minor role in P. anserina, if any. We show that PaNox1 and PaNox2 play antagonist roles in cellulose degradation. Finally, we report for the first time that a saprobic fungus, P. anserina, develops special cell structures dedicated to breach and to exploit a solid cellulosic substrate, cellophane. Importantly, as for similar structures present in some plant pathogens, their proper differentiation requires PaNox1, PaNox2, PaNoxR and the tetraspanin PaPls1.
Assuntos
Celofane/metabolismo , Proteínas Fúngicas/metabolismo , NADPH Oxidases/metabolismo , Podospora/genética , Biodegradação Ambiental , Proteínas Fúngicas/genética , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , NADPH Oxidases/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Podospora/enzimologia , Podospora/crescimento & desenvolvimento , RNA Fúngico/genéticaRESUMO
[This corrects the article DOI: 10.1186/s13068-017-0837-6.].
RESUMO
BACKGROUND: The presence of low complexity and repeated regions in genomes often results in difficulties to assemble sequencing data into full chromosomes. However, the availability of full genome scaffolds is essential to several investigations, regarding for instance the evolution of entire clades, the analysis of chromosome rearrangements, and is pivotal to sexual crossing studies. In non-conventional but industrially relevant model organisms, such as the ascomycete Trichoderma reesei, a complete genome assembly is seldom available. RESULTS: The chromosome scaffolds of T. reesei QM6a and Rut-C30 strains have been generated using a contact genomic/proximity ligation genomic approach. The original reference assembly, encompassing dozens of scaffolds, was reorganized into two sets of seven chromosomes. Chromosomal contact data also allowed to characterize 10-40 kb, gene-free, AT-rich (76%) regions corresponding to the T. reesei centromeres. Large chromosomal rearrangements (LCR) in Rut-C30 were then characterized, in agreement with former studies, and the position of LCR breakpoints used to assess the likely chromosome structure of other T. reesei strains [QM9414, CBS999.97 (1-1, re), and QM9978]. In agreement with published results, we predict that the numerous chromosome rearrangements found in highly mutated industrial strains may limit the efficiency of sexual reproduction for their improvement. CONCLUSIONS: The GRAAL program allowed us to generate the karyotype of the Rut-C30 strain, and from there to predict chromosome structure for most T. reesei strains for which sequence is available. This method that exploits proximity ligation sequencing approach is a fast, cheap, and straightforward way to characterize both chromosome structure and centromere sequences and is likely to represent a popular convenient alternative to expensive and work-intensive resequencing projects.
RESUMO
BACKGROUND: The hydrolysis of biomass to simple sugars used for the production of biofuels in biorefineries requires the action of cellulolytic enzyme mixtures. During the last 50 years, the ascomycete Trichoderma reesei, the main source of industrial cellulase and hemicellulase cocktails, has been subjected to several rounds of classical mutagenesis with the aim to obtain higher production levels. During these random genetic events, strains unable to produce cellulases were generated. Here, whole genome sequencing and transcriptomic analyses of the cellulase-negative strain QM9978 were used for the identification of mutations underlying this cellulase-negative phenotype. RESULTS: Sequence comparison of the cellulase-negative strain QM9978 to the reference strain QM6a identified a total of 43 mutations, of which 33 were located either close to or in coding regions. From those, we identified 23 single-nucleotide variants, nine InDels, and one translocation. The translocation occurred between chromosomes V and VII, is located upstream of the putative transcription factor vib1, and abolishes its expression in QM9978 as detected during the transcriptomic analyses. Ectopic expression of vib1 under the control of its native promoter as well as overexpression of vib1 under the control of a strong constitutive promoter restored cellulase expression in QM9978, thus confirming that the translocation event is the reason for the cellulase-negative phenotype. Gene deletion of vib1 in the moderate producer strain QM9414 and in the high producer strain Rut-C30 reduced cellulase expression in both cases. Overexpression of vib1 in QM9414 and Rut-C30 had no effect on cellulase production, most likely because vib1 is already expressed at an optimal level under normal conditions. CONCLUSION: We were able to establish a link between a chromosomal translocation in QM9978 and the cellulase-negative phenotype of the strain. We identified the transcription factor vib1 as a key regulator of cellulases in T. reesei whose expression is absent in QM9978. We propose that in T. reesei, as in Neurospora crassa, vib1 is involved in cellulase induction, although the exact mechanism remains to be elucidated. The data presented here show an example of a combined genome sequencing and transcriptomic approach to explain a specific trait, in this case the QM9978 cellulase-negative phenotype, and how it helps to better understand the mechanisms during cellulase gene regulation. When focusing on mutations on the single base-pair level, changes on the chromosome level can be easily overlooked and through this work we provide an example that stresses the importance of the big picture of the genomic landscape during analysis of sequencing data.
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BACKGROUND: The filamentous fungus Trichoderma reesei is the main industrial cellulolytic enzyme producer. Several strains have been developed in the past using random mutagenesis, and despite impressive performance enhancements, the pressure for low-cost cellulases has stimulated continuous research in the field. In this context, comparative study of the lower and higher producer strains obtained through random mutagenesis using systems biology tools (genome and transcriptome sequencing) can shed light on the mechanisms of cellulase production and help identify genes linked to performance. Previously, our group published comparative genome sequencing of the lower and higher producer strains NG 14 and RUT C30. In this follow-up work, we examine how these mutations affect phenotype as regards the transcriptome and cultivation behaviour. RESULTS: We performed kinetic transcriptome analysis of the NG 14 and RUT C30 strains of early enzyme production induced by lactose using bioreactor cultivations close to an industrial cultivation regime. RUT C30 exhibited both earlier onset of protein production (3 h) and higher steady-state productivity. A rather small number of genes compared to previous studies were regulated (568), most of them being specific to the NG 14 strain (319). Clustering analysis highlighted similar behaviour for some functional categories and allowed us to distinguish between induction-related genes and productivity-related genes. Cross-comparison of our transcriptome data with previously identified mutations revealed that most genes from our dataset have not been mutated. Interestingly, the few mutated genes belong to the same clusters, suggesting that these clusters contain genes playing a role in strain performance. CONCLUSIONS: This is the first kinetic analysis of a transcriptomic study carried out under conditions approaching industrial ones with two related strains of T. reesei showing distinctive cultivation behaviour. Our study sheds some light on some of the events occurring in these strains following induction by lactose. The fact that few regulated genes have been affected by mutagenesis suggests that the induction mechanism is essentially intact compared to that for the wild-type isolate QM6a and might be engineered for further improvement of T. reesei. Genes from two specific clusters might be potential targets for such genetic engineering.
RESUMO
Pseudo-homothallism is a reproductive strategy elected by some fungi producing heterokaryotic sexual spores containing genetically different but sexually compatible nuclei. This lifestyle appears as a compromise between true homothallism (self-fertility with predominant inbreeding) and complete heterothallism (with exclusive outcrossing). However, pseudohomothallic species face the problem of maintaining heterokaryotic mycelia to fully benefit from this lifestyle, as homokaryons are self-sterile. Here, we report on the structure of chromosome 1 in mat+ and mat- isolates of strain S of the pseudohomothallic fungus Podospora anserina. Chromosome 1 contains either one of the mat+ and mat- mating types of P. anserina, which is mostly found in nature as a mat+/mat- heterokaryotic mycelium harboring sexually compatible nuclei. We identified a "mat" region â¼0.8 Mb long, devoid of meiotic recombination and containing the mating-type idiomorphs, which is a candidate to be involved in the maintenance of the heterokaryotic state, since the S mat+ and S mat- strains have different physiology that may enable hybrid-vigor-like phenomena in the heterokaryons. The mat region contains 229 coding sequences. A total of 687 polymorphisms were detected between the S mat+ and S mat- chromosomes. Importantly, the mat region is colinear between both chromosomes, which calls for an original mechanism of recombination inhibition. Microarray analyses revealed that 10% of the P. anserina genes have different transcriptional profiles in S mat+ and S mat-, in line with their different phenotypes. Finally, we show that the heterokaryotic state is faithfully maintained during mycelium growth of P. anserina, yet mat+/mat+ and mat-/mat- heterokaryons are as stable as mat+/mat- ones, evidencing a maintenance of heterokaryosis that does not rely on fitness-enhancing complementation between the S mat+ and S mat- strains.