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1.
Microb Cell Fact ; 23(1): 177, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38879507

RESUMO

BACKGROUND: Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii. RESULTS: In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system. CONCLUSIONS: In this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.


Assuntos
Reatores Biológicos , Oxigenases de Função Mista , Regiões Promotoras Genéticas , Proteínas Recombinantes , Saccharomycetales , Saccharomycetales/genética , Saccharomycetales/metabolismo , Saccharomycetales/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Metanol/metabolismo
2.
Appl Microbiol Biotechnol ; 108(1): 92, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38204136

RESUMO

Application of filamentous fungi for the production of commercial enzymes such as amylase, cellulase, or xylanase is on the rise due to the increasing demand to degrade several complex carbohydrates as raw material for biotechnological processes. Also, protein production by fungi for food and feed gains importance. In any case, the protein production involves both cellular synthesis and secretion outside of the cell. Unfortunately, the secretion of proteins or enzymes can be hampered due to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) as a result of too high synthesis of enzymes or (heterologous) protein expression. To cope with this ER stress, the cell generates a response known as unfolded protein response (UPR). Even though this mechanism should re-establish the protein homeostasis equivalent to a cell under non-stress conditions, the enzyme expression might still suffer from repression under secretory stress (RESS). Among eukaryotes, Saccharomyces cerevisiae is the only fungus, which is studied quite extensively to unravel the UPR pathway. Several homologs of the proteins involved in this signal transduction cascade are also found in filamentous fungi. Since RESS seems to be absent in S. cerevisiae and was only reported in Trichoderma reesei in the presence of folding and glycosylation inhibitors such as dithiothreitol and tunicamycin, more in-depth study about this mechanism, specifically in filamentous fungi, is the need of the hour. Hence, this review article gives an overview on both, protein secretion and associated stress responses in fungi. KEY POINTS: • Enzymes produced by filamentous fungi are crucial in industrial processes • UPR mechanism is conserved among many fungi, but mediated by different proteins • RESS is not fully understood or studied in industrially relevant filamentous fungi.


Assuntos
Fungos , Saccharomyces cerevisiae , Transporte Proteico , Transporte Biológico , Proteostase
3.
Appl Microbiol Biotechnol ; 107(15): 4745-4758, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37341752

RESUMO

Fungi are widely exploited for large-scale production in the biotechnological industry to produce a diverse range of substances due to their versatility and relative ease of growing on various substrates. The occurrence of a phenomenon-the so-called fungal strain degeneration-leads to the spontaneous loss or decline of production capacity and results in an economic loss on a tremendous scale. Some of the most commonly applied genera of fungi in the biotechnical industry, such as Aspergillus, Trichoderma, and Penicillium, are threatened by this phenomenon. Although fungal degeneration has been known for almost a century, the phenomenon and its underlying mechanisms still need to be understood. The proposed mechanisms causing fungi to degenerate can be of genetic or epigenetic origin. Other factors, such as culture conditions, stress, or aging, were also reported to have an influence. This mini-review addresses the topic of fungal degeneration by describing examples of productivity losses in biotechnical processes using Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, and Penicillium chrysogenum. Further, potential reasons, circumvention, and prevention methods are discussed. This is the first mini-review which provides a comprehensive overview on this phenomenon in biotechnologically used fungi, and it also includes a collection of strategies that can be useful to minimize economic losses which can arise from strain degeneration. KEY POINTS: • Spontaneous loss of productivity is evident in many fungi used in biotechnology. • The properties and mechanisms underlying this phenomenon are very versatile. • Only studying these underlying mechanisms enables the design of a tailored solution.


Assuntos
Aspergillus oryzae , Penicillium chrysogenum , Penicillium , Trichoderma , Aspergillus niger/genética , Penicillium/genética , Penicillium chrysogenum/genética , Fungos/genética , Biotecnologia , Trichoderma/genética
4.
PLoS Comput Biol ; 17(9): e1009372, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34570757

RESUMO

Secondary metabolites (SMs) are a vast group of compounds with different structures and properties that have been utilized as drugs, food additives, dyes, and as monomers for novel plastics. In many cases, the biosynthesis of SMs is catalysed by enzymes whose corresponding genes are co-localized in the genome in biosynthetic gene clusters (BGCs). Notably, BGCs may contain so-called gap genes, that are not involved in the biosynthesis of the SM. Current genome mining tools can identify BGCs, but they have problems with distinguishing essential genes from gap genes. This can and must be done by expensive, laborious, and time-consuming comparative genomic approaches or transcriptome analyses. In this study, we developed a method that allows semi-automated identification of essential genes in a BGC based on co-evolution analysis. To this end, the protein sequences of a BGC are blasted against a suitable proteome database. For each protein, a phylogenetic tree is created. The trees are compared by treeKO to detect co-evolution. The results of this comparison are visualized in different output formats, which are compared visually. Our results suggest that co-evolution is commonly occurring within BGCs, albeit not all, and that especially those genes that encode for enzymes of the biosynthetic pathway are co-evolutionary linked and can be identified with FunOrder. In light of the growing number of genomic data available, this will contribute to the studies of BGCs in native hosts and facilitate heterologous expression in other organisms with the aim of the discovery of novel SMs.


Assuntos
Vias Biossintéticas/genética , Evolução Molecular , Genes Essenciais , Família Multigênica , Software , Aspergillus/genética , Aspergillus/metabolismo , Biologia Computacional , Bases de Dados de Proteínas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/genética , Fungos/metabolismo , Genes Sintéticos , Genoma Fúngico , Genômica , Lovastatina/biossíntese , Lovastatina/genética , Filogenia , Proteoma/genética
5.
Appl Microbiol Biotechnol ; 105(10): 4017-4031, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33950280

RESUMO

The pentose phosphate pathway (PPP) is one of the most targeted pathways in metabolic engineering. This pathway is the primary source of NADPH, and it contributes in fungi to the production of many compounds of interest such as polyols, biofuels, carotenoids, or antibiotics. However, the regulatory mechanisms of the PPP are still not fully known. This review provides an insight into the current comprehension of the PPP in fungi and the limitations of this current understanding. It highlights how this knowledge contributes to targeted engineering of the PPP and thus to better performance of industrially used fungal strains. KEY POINTS: • Type of carbon and nitrogen source as well as oxidative stress influence the PPP. • A complex network of transcription factors regulates the PPP. • Improved understanding of the PPP will allow to increase yields of bioprocesses.


Assuntos
Fungos , Via de Pentose Fosfato , Biocombustíveis , Fungos/genética , Fungos/metabolismo , Engenharia Metabólica , NADP/metabolismo
6.
BMC Genomics ; 21(1): 258, 2020 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-32216757

RESUMO

BACKGROUND: Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a highly diverse group of secondary metabolites (SM) of bacterial and fungal origin. While RiPPs have been intensively studied in bacteria, little is known about fungal RiPPs. In Fungi only six classes of RiPPs are described. Current strategies for genome mining are based on these six known classes. However, the genes involved in the biosynthesis of theses RiPPs are normally organized in biosynthetic gene clusters (BGC) in fungi. RESULTS: Here we describe a comprehensive strategy to mine fungal genomes for RiPPs by combining and adapting existing tools (e.g. antiSMASH and RiPPMiner) followed by extensive manual curation based on conserved domain identification, (comparative) phylogenetic analysis, and RNASeq data. Deploying this strategy, we could successfully rediscover already known fungal RiPPs. Further, we analysed four fungal genomes from the Trichoderma genus. We were able to find novel potential RiPP BGCs in Trichoderma using our unconventional mining approach. CONCLUSION: We demonstrate that the unusual mining approach using tools developed for bacteria can be used in fungi, when carefully curated. Our study is the first report of the potential of Trichoderma to produce RiPPs, the detected clusters encode novel uncharacterized RiPPs. The method described in our study will lead to further mining efforts in all subdivisions of the fungal kingdom.


Assuntos
Biologia Computacional/métodos , Mineração de Dados/métodos , Proteínas Fúngicas/genética , Trichoderma/genética , Curadoria de Dados , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Filogenia , Metabolismo Secundário , Análise de Sequência de RNA , Sequenciamento Completo do Genoma
7.
Anal Chem ; 92(24): 15719-15725, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33259186

RESUMO

Determination of the intracellular location of proteins is one of the fundamental tasks of microbiology. Conventionally, label-based microscopy and super-resolution techniques are employed. In this work, we demonstrate a new technique that can determine intracellular protein distribution at nanometer spatial resolution. This method combines nanoscale spatial resolution chemical imaging using the photothermal-induced resonance (PTIR) technique with multivariate modeling to reveal the intracellular distribution of cell components. Here, we demonstrate its viability by imaging the distribution of major cellulases and xylanases in Trichoderma reesei using the colocation of a fluorescent label (enhanced yellow fluorescence protein, EYFP) with the target enzymes to calibrate the chemometric model. The obtained partial least squares model successfully shows the distribution of these proteins inside the cell and opens the door for further studies on protein secretion mechanisms using PTIR.


Assuntos
Celulases/análise , Endo-1,4-beta-Xilanases/análise , Hypocreales/enzimologia , Celulases/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Microscopia de Força Atômica , Tamanho da Partícula , Espectrofotometria Infravermelho , Propriedades de Superfície
8.
RNA Biol ; 17(1): 47-61, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31517564

RESUMO

Long non-coding RNAs (lncRNAs) are crucial factors acting on regulatory processes in eukaryotes. Recently, for the first time in a filamentous fungus, the lncRNA HAX1 was characterized in the ascomycete Trichoderma reesei. In industry, this fungus is widely applied for the high-yield production of cellulases. The lncRNA HAX1 was reported to influence the expression of cellulase-encoding genes; interestingly, this effect is dependent on the presence of its most abundant length. Clearly, HAX1 acts in association with a set of well-described transcription factors to regulate gene expression. In this study, we attempted to elucidate the regulatory strategy of HAX1 and its interactions with the major transcriptional activator Xylanase regulator 1 (Xyr1). We demonstrated that HAX1 interferes with the negative feedback regulatory loop of Xyr1 in a sophisticated manner and thus ultimately has a positive effect on gene expression.


Assuntos
Fungos/genética , Regulação Fúngica da Expressão Gênica , RNA Longo não Codificante/genética , Transativadores/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Modelos Biológicos , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas
9.
Appl Microbiol Biotechnol ; 104(5): 1977-1991, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31965222

RESUMO

As photosynthetic microbes, cyanobacteria are attractive hosts for the production of high-value molecules from CO2 and light. Strategies for genetic engineering and tightly controlled gene expression are essential for the biotechnological application of these organisms. Numerous heterologous or native promoter systems were used for constitutive and inducible expression, yet many of them suffer either from leakiness or from a low expression output. Anyway, in recent years, existing systems have been improved and new promoters have been discovered or engineered for cyanobacteria. Moreover, alternative tools and strategies for expression control such as riboswitches, riboregulators or genetic circuits have been developed. In this mini-review, we provide a broad overview on the different tools and approaches for the regulation of gene expression in cyanobacteria and explain their advantages and disadvantages.


Assuntos
Cianobactérias/genética , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes/genética , Cianobactérias/metabolismo , Expressão Gênica , Engenharia Genética , Regiões Promotoras Genéticas , RNA Interferente Pequeno , Riboswitch , Biologia Sintética
10.
Proc Natl Acad Sci U S A ; 114(4): E560-E569, 2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-28074041

RESUMO

Fungi can produce a wide range of chemical compounds via secondary metabolism. These compounds are of major interest because of their (potential) application in medicine and biotechnology and as a potential source for new therapeutic agents and drug leads. However, under laboratory conditions, most secondary metabolism genes remain silent. This circumstance is an obstacle for the production of known metabolites and the discovery of new secondary metabolites. In this study, we describe the dual role of the transcription factor Xylanase promoter binding protein 1 (Xpp1) in the regulation of both primary and secondary metabolism of Trichoderma reesei Xpp1 was previously described as a repressor of xylanases. Here, we provide data from an RNA-sequencing analysis suggesting that Xpp1 is an activator of primary metabolism. This finding is supported by our results from a Biolog assay determining the carbon source assimilation behavior of an xpp1 deletion strain. Furthermore, the role of Xpp1 as a repressor of secondary metabolism is shown by gene expression analyses of polyketide synthases and the determination of the secondary metabolites of xpp1 deletion and overexpression strains using an untargeted metabolomics approach. The deletion of Xpp1 resulted in the enhanced secretion of secondary metabolites in terms of diversity and quantity. Homologs of Xpp1 are found among a broad range of fungi, including the biocontrol agent Trichoderma atroviride, the plant pathogens Fusarium graminearum and Colletotrichum graminicola, the model organism Neurospora crassa, the human pathogen Sporothrix schenckii, and the ergot fungus Claviceps purpurea.


Assuntos
Proteínas Fúngicas/metabolismo , Metabolismo Secundário , Fatores de Transcrição/metabolismo , Trichoderma/metabolismo , Proteínas Fúngicas/genética , Análise de Sequência de RNA , Fatores de Transcrição/genética , Trichoderma/genética
11.
Appl Environ Microbiol ; 84(1)2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-29079620

RESUMO

Trichoderma reesei can produce up to 100 g/liter of extracellular proteins. The major and industrially relevant products are cellobiohydrolase I (CBHI) and the hemicellulase XYNI. The genes encoding both enzymes are transcriptionally activated by the regulatory protein Xyr1. The first 850 nucleotides of the cbh1 promoter contain 14 Xyr1-binding sites (XBS), and 8 XBS are present in the xyn1 promoter. Some of these XBS are arranged in tandem and others as inverted repeats. One such cis element, an inverted repeat, plays a crucial role in the inducibility of the xyn1 promoter. We investigated the impact of the properties of such cis elements by shuffling them by insertion, exchange, deletion, and rearrangement of cis elements in both the cbh1 and xyn1 promoter. A promoter-reporter assay using the Aspergillus nigergoxA gene allowed us to measure changes in the promoter strength and inducibility. Most strikingly, we found that an inverted repeat of XBS causes an important increase in cbh1 promoter strength and allows induction by xylan or wheat straw. Furthermore, evidence is provided that the distances of cis elements to the transcription start site have important influence on promoter activity. Our results suggest that the arrangement and distances of cis elements have large impacts on the strength of the cbh1 promoter, whereas the sheer number of XBS has only secondary importance. Ultimately, the biotechnologically important cbh1 promoter can be improved by cis element rearrangement.IMPORTANCE In the present study, we demonstrate that the arrangement of cis elements has a major impact on promoter strength and inducibility. We discovered an influence on promoter activity by the distances of cis elements to the transcription start site. Furthermore, we found that the configuration of cis elements has a greater effect on promoter strength than does the sheer number of transactivator binding sites present in the promoter. Altogether, the arrangement of cis elements is an important factor that should not be overlooked when enhancement of gene expression is desired.


Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Trichoderma/genética , Sítios de Ligação , Celulose 1,4-beta-Celobiosidase/genética , Celulose 1,4-beta-Celobiosidase/metabolismo , Proteínas Fúngicas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Trichoderma/enzimologia
12.
Appl Microbiol Biotechnol ; 102(17): 7319-7331, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29974182

RESUMO

Long noncoding RNAs (lncRNAs) are crucial players in epigenetic regulation. They were initially discovered in human, yet they emerged as common factors involved in a number of central cellular processes in several eukaryotes. For example, in the past decade, research on lncRNAs in yeast has steadily increased. Several examples of lncRNAs were described in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Also, screenings for lncRNAs in ascomycetes were performed and, just recently, the first full characterization of a lncRNA was performed in the filamentous fungus Trichoderma reesei. In this review, we provide a broad overview about currently known fugal lncRNAs. We make an attempt to categorize them according to their functional context, regulatory strategies or special properties. Moreover, the potential of lncRNAs as a biotechnological tool is discussed.


Assuntos
RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Saccharomyces cerevisiae , Schizosaccharomyces , Trichoderma , Epigênese Genética/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Trichoderma/genética , Trichoderma/metabolismo
13.
J Basic Microbiol ; 58(2): 144-153, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29193198

RESUMO

Humicola grisea var. thermoidea (Hgvt) is a thermophilic ascomycete that produces lignocellulolytic enzymes and it is proposed for the conversion of agricultural residues into useful byproducts. Drugs that inhibit the DNA methyltransferases (DNMTs) activity are employed in epigenetic studies but nothing is known about a possible effect on the production of fungal enzymes. We evaluated the effect of 5-aza-2'-deoxycytidine (5-Aza; a chemical inhibitor of DNMTs activity) on the secreted enzyme activity and on the transcription of cellulase and xylanase genes from Hgvt grown in agricultural residues and in glucose. Upon cultivation on wheat bran (WB), the drug provoked an increase in the xylanase activity at 96 h. When Hgvt was grown in glucose (GLU), a repressor of Hgvt glycosyl hydrolase genes, 5-Aza led to increased transcript accumulation for the cellobiohydrolases and for the xyn2 xylanase genes. In WB, 5-Aza enhanced the expression of the transcription factor CreA gene. Growth on WB or GLU, in presence of 5-Aza, led to a significant increase in transcripts of the pH-response regulator PacC gene. To our knowledge, this is the first report on the effect of a DNMT inhibitor in the production of fungal plant cell wall degradation enzymes.


Assuntos
Azacitidina/análogos & derivados , Repressão Catabólica/efeitos dos fármacos , Celulase/biossíntese , Inibidores Enzimáticos/metabolismo , Enzimas/metabolismo , Sordariales/efeitos dos fármacos , Xilosidases/biossíntese , Azacitidina/metabolismo , Decitabina , Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Sordariales/crescimento & desenvolvimento , Triticum/metabolismo , Triticum/microbiologia
14.
Appl Environ Microbiol ; 82(20): 6247-6257, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27520818

RESUMO

The industrially used ascomycete Trichoderma reesei secretes a typical yellow pigment during cultivation, while other Trichoderma species do not. A comparative genomic analysis suggested that a putative secondary metabolism cluster, containing two polyketide-synthase encoding genes, is responsible for the yellow pigment synthesis. This cluster is conserved in a set of rather distantly related fungi, including Acremonium chrysogenum and Penicillium chrysogenum In an attempt to silence the cluster in T. reesei, two genes of the cluster encoding transcription factors were individually deleted. For a complete genetic proof-of-function, the genes were reinserted into the genomes of the respective deletion strains. The deletion of the first transcription factor (termed yellow pigment regulator 1 [Ypr1]) resulted in the full abolishment of the yellow pigment formation and the expression of most genes of this cluster. A comparative high-pressure liquid chromatography (HPLC) analysis of supernatants of the ypr1 deletion and its parent strain suggested the presence of several yellow compounds in T. reesei that are all derived from the same cluster. A subsequent gas chromatography/mass spectrometry analysis strongly indicated the presence of sorbicillin in the major HPLC peak. The presence of the second transcription factor, termed yellow pigment regulator 2 (Ypr2), reduces the yellow pigment formation and the expression of most cluster genes, including the gene encoding the activator Ypr1. IMPORTANCE: Trichoderma reesei is used for industry-scale production of carbohydrate-active enzymes. During growth, it secretes a typical yellow pigment. This is not favorable for industrial enzyme production because it makes the downstream process more complicated and thus increases operating costs. In this study, we demonstrate which regulators influence the synthesis of the yellow pigment. Based on these data, we also provide indication as to which genes are under the control of these regulators and are finally responsible for the biosynthesis of the yellow pigment. These genes are organized in a cluster that is also found in other industrially relevant fungi, such as the two antibiotic producers Penicillium chrysogenum and Acremonium chrysogenum The targeted manipulation of a secondary metabolism cluster is an important option for any biotechnologically applied microorganism.


Assuntos
Proteínas Fúngicas/metabolismo , Pigmentos Biológicos/biossíntese , Fatores de Transcrição/metabolismo , Trichoderma/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Família Multigênica , Fatores de Transcrição/genética , Trichoderma/genética
16.
Curr Genomics ; 17(2): 145-52, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27226770

RESUMO

The ascomycete Trichoderma reesei is used for the production of plant cell wall-degrading enzymes in industrial scale. The interplay of the transactivator Xyr1 and the repressor Cre1 mainly regulates the expression of these enzymes. During induc-ing conditions, such as in the presence of sophorose, the transcription of the two major cellulase-encoding genes, cbh1 and cbh2, is activated as well as the expression of xyr1. In the presence of D-glucose carbon catabolite repression mediated by Cre1 takes place and the expression of Xyr1 and the plant cell wall-degrading enzymes is down-regulated. In this study we compare the chromatin status of xyr1, cbh1, and cbh2 promoters in the wild-type strain and the Cre1-deficient strain Rut-C30. Chromatin rearrangement occurs in the xyr1 promoter during induction on sophorose. Chromatin opening and protein-DNA interactions in the xyr1 promoter were detected especially in a region located 0.9 kb upstream the translation start co-don, which bears several putative Cre1-binding sites and a CCAAT-box. Moreover, the xyr1 promoter is overall more acces-sible in a cre1-truncated background, no matter which carbon source is present. This makes the xyr1 regulatory sequence a good target for promoter engineering aiming at the enhancement of cellulase production.

17.
Nucleic Acids Res ; 42(1): e1, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24097437

RESUMO

Knowing which regions of a gene are targeted by transcription factors during induction or repression is essential for understanding the mechanisms responsible for regulation. Therefore, we re-designed the traditional in vivo footprinting method to obtain a highly sensitive technique, which allows identification of the cis elements involved in condition-dependent gene regulation. Data obtained through DMS methylation, HCl DNA cleavage and optimized ligation-mediated PCR using fluorescent labelling followed by capillary gel electrophoresis are analysed by ivFAST. In this work we have developed this command line-based program, which is designed to ensure automated and fast data processing and visualization. The new method facilitates a quantitative, high-throughput approach because it enables the comparison of any number of in vivo footprinting results from different conditions (e.g. inducing, repressing, de-repressing) to one another by employing an internal standard. For validation of the method the well-studied upstream regulatory region of the Trichoderma reesei xyn1 (endoxylanase 1) gene was used. Applying the new method we could identify the motives involved in condition-dependent regulation of the cbh2 (cellobiohydrolase 2) and xyn2 (endoxylanase 2) genes.


Assuntos
Pegada de DNA/métodos , Elementos Reguladores de Transcrição , Celulose 1,4-beta-Celobiosidase/genética , Clivagem do DNA , Metilação de DNA , Endo-1,4-beta-Xilanases/genética , Reação em Cadeia da Polimerase , Software , Trichoderma/genética
18.
BMC Genomics ; 16: 588, 2015 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-26248555

RESUMO

BACKGROUND: Trichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging. RESULTS: Comparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1. CONCLUSIONS: The regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status.


Assuntos
Celulase/genética , Montagem e Desmontagem da Cromatina/genética , Cromatina/genética , Trichoderma/genética , Celulases/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Glucose/genética , Regiões Promotoras Genéticas/genética , Transativadores/genética , Transcrição Gênica/genética , Transcriptoma/genética
19.
Appl Environ Microbiol ; 81(18): 6314-23, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26150462

RESUMO

The state-of-the-art procedure for gene insertions into Trichoderma reesei is a cotransformation of two plasmids, one bearing the gene of interest and the other a marker gene. This procedure yields up to 80% transformation efficiency, but both the number of integrated copies and the loci of insertion are unpredictable. This can lead to tremendous pleiotropic effects. This study describes the development of a novel transformation system for site-directed gene insertion based on auxotrophic markers. For this purpose, we tested the applicability of the genes asl1 (encoding an enzyme of the l-arginine biosynthesis pathway), the hah1 (encoding an enzyme of the l-lysine biosynthesis pathway), and the pyr4 (encoding an enzyme of the uridine biosynthesis pathway). The developed transformation system yields strains with an additional gene at a defined locus that are prototrophic and ostensibly isogenic compared to their parental strain. A positive transformation rate of 100% was achieved due to the developed split-marker system. Additionally, a double-auxotrophic strain that allows multiple genomic manipulations was constructed, which facilitates metabolic engineering purposes in T. reesei. By employing goxA of Aspergillus niger as a reporter system, the influence on the expression of an inserted gene caused by the orientation of the insertion and the transformation strategy used could be demonstrated. Both are important aspects to be considered during strain engineering.


Assuntos
Genoma Fúngico , Mutagênese Insercional/métodos , Transformação Genética , Trichoderma/genética , Arginina/biossíntese , Aspergillus niger/genética , Southern Blotting , Genes Fúngicos , Genes Reporter , Engenharia Genética/métodos , Lisina/biossíntese , Plasmídeos , Trichoderma/metabolismo , Trichoderma/ultraestrutura , Uridina/biossíntese
20.
Fungal Genet Biol ; 72: 73-81, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25064064

RESUMO

The transcriptional activator XlnR (Xlr1/Xyr1) is a major regulator in fungal xylan and cellulose degradation as well as in the utilization of d-xylose via the pentose catabolic pathway. XlnR homologs are commonly found in filamentous ascomycetes and often assumed to have the same function in different fungi. However, a comparison of the saprobe Aspergillus niger and the plant pathogen Magnaporthe oryzae showed different phenotypes for deletion strains of XlnR. In this study wild type and xlnR/xlr1/xyr1 mutants of five fungi were compared: Fusarium graminearum, M. oryzae, Trichoderma reesei, A. niger and Aspergillus nidulans. Growth profiling on relevant substrates and a detailed analysis of the secretome as well as extracellular enzyme activities demonstrated a common role of this regulator in activating genes encoding the main xylanolytic enzymes. However, large differences were found in the set of genes that is controlled by XlnR in the different species, resulting in the production of different extracellular enzyme spectra by these fungi. This comparison emphasizes the functional diversity of a fine-tuned (hemi-)cellulolytic regulatory system in filamentous fungi, which might be related to the adaptation of fungi to their specific biotopes. Data are available via ProteomeXchange with identifier PXD001190.


Assuntos
Fungos/crescimento & desenvolvimento , Fungos/genética , Transativadores/genética , Transativadores/metabolismo , Meios de Cultura/química , Proteínas Fúngicas/metabolismo , Fungos/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Proteoma/análise , Regulon , Xilanos/metabolismo
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