RESUMEN
We have analyzed the role of the three members of the Pex11 protein family in peroxisome formation in the filamentous fungus Penicillium chrysogenum. Two of these, Pex11 and Pex11C, are components of the peroxisomal membrane, while Pex11B is present at the endoplasmic reticulum. We show that Pex11 is a major factor involved in peroxisome proliferation. We also demonstrate that P. chrysogenum cells deleted for known peroxisome fission factors (all Pex11 family proteins and Vps1) still contain peroxisomes. Interestingly, we find that, unlike in mammals, Pex16 is not essential for peroxisome biogenesis in P. chrysogenum, as partially functional peroxisomes are present in a pex16 deletion strain. We also show that Pex16 is not involved in de novo biogenesis of peroxisomes, as peroxisomes were still present in quadruple Δpex11 Δpex11B Δpex11C Δpex16 mutant cells. By contrast, pex3 deletion in P. chrysogenum led to cells devoid of peroxisomes, suggesting that Pex3 may function independently of Pex16. Finally, we demonstrate that the presence of intact peroxisomes is important for the efficiency of ß-lactam antibiotics production by P. chrysogenum. Remarkably, distinct from earlier results with low penicillin producing laboratory strains, upregulation of peroxisome numbers in a high producing P. chrysogenum strain had no significant effect on penicillin production.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Penicillium chrysogenum/ultraestructura , Peroxisomas/fisiología , Proteínas Fúngicas/genética , Técnicas de Inactivación de Genes , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/genética , Penicilina G/metabolismo , Penicillium chrysogenum/genética , Penicillium chrysogenum/metabolismo , Peroxisomas/metabolismo , Peroxisomas/ultraestructura , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Industrial production of semi-synthetic cephalosporins by Penicillium chrysogenum requires supplementation of the growth media with the side-chain precursor adipic acid. In glucose-limited chemostat cultures of P. chrysogenum, up to 88% of the consumed adipic acid was not recovered in cephalosporin-related products, but used as an additional carbon and energy source for growth. This low efficiency of side-chain precursor incorporation provides an economic incentive for studying and engineering the metabolism of adipic acid in P. chrysogenum. Chemostat-based transcriptome analysis in the presence and absence of adipic acid confirmed that adipic acid metabolism in this fungus occurs via ß-oxidation. A set of 52 adipate-responsive genes included six putative genes for acyl-CoA oxidases and dehydrogenases, enzymes responsible for the first step of ß-oxidation. Subcellular localization of the differentially expressed acyl-CoA oxidases and dehydrogenases revealed that the oxidases were exclusively targeted to peroxisomes, while the dehydrogenases were found either in peroxisomes or in mitochondria. Deletion of the genes encoding the peroxisomal acyl-CoA oxidase Pc20g01800 and the mitochondrial acyl-CoA dehydrogenase Pc20g07920 resulted in a 1.6- and 3.7-fold increase in the production of the semi-synthetic cephalosporin intermediate adipoyl-6-APA, respectively. The deletion strains also showed reduced adipate consumption compared to the reference strain, indicating that engineering of the first step of ß-oxidation successfully redirected a larger fraction of adipic acid towards cephalosporin biosynthesis.
Asunto(s)
Cefalosporinas/biosíntesis , Ingeniería Metabólica/métodos , Penicillium chrysogenum/metabolismo , Acil-CoA Deshidrogenasas/genética , Acil-CoA Deshidrogenasas/metabolismo , Acil-CoA Oxidasa/genética , Acil-CoA Oxidasa/metabolismo , Adipatos/metabolismo , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Glucosa/metabolismo , Mitocondrias/enzimología , Mitocondrias/genética , Oxidación-Reducción , Peroxisomas/enzimología , Peroxisomas/genética , TranscriptomaRESUMEN
Hansenula polymorpha is an important yeast in industrial biotechnology. In addition, it is extensively used in fundamental research devoted to unravel the principles of peroxisome biology and nitrate assimilation. Here we present an overview of key components of the genetic toolbox for H. polymorpha. In addition, we present new selection markers that we recently implemented in H. polymorpha. We describe novel strategies for the efficient creation of targeted gene deletions and integrations in H. polymorpha. For this, we generated a yku80 mutant, deficient in non-homologous end joining, resulting in strongly enhanced efficiency of gene targeting relative to the parental strain. Finally, we show the implementation of Gateway technology and a single-step PCR strategy to create deletions in H. polymorpha.
Asunto(s)
Biotecnología/métodos , Proteínas Fúngicas/genética , Vectores Genéticos/genética , Pichia/genética , Recombinación Genética , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Expresión Génica , Ingeniería Genética/métodos , Plásmidos/genética , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: In microbial production of non-catabolic products such as antibiotics a loss of production capacity upon long-term cultivation (for example chemostat), a phenomenon called strain degeneration, is often observed. In this study a systems biology approach, monitoring changes from gene to produced flux, was used to study degeneration of penicillin production in a high producing Penicillium chrysogenum strain during prolonged ethanol-limited chemostat cultivations. RESULTS: During these cultivations, the biomass specific penicillin production rate decreased more than 10-fold in less than 22 generations. No evidence was obtained for a decrease of the copy number of the penicillin gene cluster, nor a significant down regulation of the expression of the penicillin biosynthesis genes. However, a strong down regulation of the biosynthesis pathway of cysteine, one of the precursors of penicillin, was observed. Furthermore the protein levels of the penicillin pathway enzymes L-α-(δ-aminoadipyl)-L-α-cystenyl-D-α-valine synthetase (ACVS) and isopenicillin-N synthase (IPNS), decreased significantly. Re-cultivation of fully degenerated cells in unlimited batch culture and subsequent C-limited chemostats did only result in a slight recovery of penicillin production. CONCLUSIONS: Our findings indicate that the observed degeneration is attributed to a significant decrease of the levels of the first two enzymes of the penicillin biosynthesis pathway, ACVS and IPNS. This decrease is not caused by genetic instability of the penicillin amplicon, neither by down regulation of the penicillin biosynthesis pathway. Furthermore no indications were obtained for degradation of these enzymes as a result of autophagy. Possible causes for the decreased enzyme levels could be a decrease of the translation efficiency of ACVS and IPNS during degeneration, or the presence of a culture variant impaired in the biosynthesis of functional proteins of these enzymes, which outcompeted the high producing part of the population.
Asunto(s)
Reactores Biológicos , Microbiología Industrial/métodos , Modelos Biológicos , Penicilinas/biosíntesis , Penicillium chrysogenum/metabolismo , Biología de Sistemas/métodos , Biomasa , Etanol , Dosificación de Gen/genética , Familia de Multigenes/genética , TranscriptomaRESUMEN
In large-scale production reactors the combination of high broth viscosity and large broth volume leads to insufficient liquid-phase mixing, resulting in gradients in, for example, the concentrations of substrate and oxygen. This often leads to differences in productivity of the full-scale process compared with laboratory scale. In this scale-down study of penicillin production, the influence of substrate gradients on process performance and cell physiology was investigated by imposing an intermittent feeding regime on a laboratory-scale culture of a high yielding strain of Penicillium chrysogenum. It was found that penicillin production was reduced by a factor of two in the intermittently fed cultures relative to constant feed cultivations fed with the same amount of glucose per hour, while the biomass yield was the same. Measurement of the levels of the intermediates of the penicillin biosynthesis pathway, along with the enzyme levels, suggested that the reduction of the flux through the penicillin pathway is mainly the result of a lower influx into the pathway, possibly due to inhibitory levels of adenosine monophosphate and pyrophosphate and lower activating levels of adenosine triphosphate during the zero-substrate phase of each cycle of intermittent feeding.
Asunto(s)
Glucosa/metabolismo , Microbiología Industrial , Penicilinas/biosíntesis , Penicillium chrysogenum/metabolismo , Ciclo del Carbono , Coenzima A Ligasas/metabolismo , Redes y Vías Metabólicas , Oxidorreductasas/metabolismo , Penicillium chrysogenum/química , Péptido Sintasas/metabolismoRESUMEN
We have investigated the significance of autophagy in the production of the ß-lactam antibiotic penicillin (PEN) by the filamentous fungus Penicillium chrysogenum. In this fungus PEN production is compartmentalized in the cytosol and in peroxisomes. We demonstrate that under PEN-producing conditions significant amounts of cytosolic and peroxisomal proteins are degraded via autophagy. Morphological analysis, based on electron and fluorescence microscopy, revealed that this phenomenon might contribute to progressive deterioration of late subapical cells. We show that deletion of the P. chrysogenum ortholog of Saccharomyces cerevisiae serine-threonine kinase atg1 results in impairment of autophagy. In P. chrysogenum atg1 cells, a distinct delay in cell degeneration is observed relative to wild-type cells. This phenomenon is associated with an increase in the enzyme levels of the PEN biosynthetic pathway and enhanced production levels of this antibacterial compound.
Asunto(s)
Autofagia , Penicilinas/biosíntesis , Penicillium chrysogenum/fisiología , Proteínas Serina-Treonina Quinasas/genética , Autofagia/genética , Citosol/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Espectroscopía de Resonancia Magnética , Microscopía Electrónica , Microscopía Fluorescente , Penicilinas/metabolismo , Penicillium chrysogenum/enzimología , Penicillium chrysogenum/genética , Penicillium chrysogenum/metabolismo , Peroxisomas/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Eliminación de SecuenciaRESUMEN
Autophagy (macroautophagy) is a bulk degradative pathway by which cytoplasmic components are delivered to the vacuole for recycling. This process is conserved from yeast to human, where it is implicated in cancer and neurodegenerative diseases. During the last decade, many ATG genes involved in autophagy have been identified, initially in Saccharomyces cerevisiae. This review summarizes the knowledge on the molecular mechanisms of autophagy using yeast as model system. Although many of the core components involved in autophagy are conserved from yeast to human, there are, nevertheless, significant differences between these organisms, for example, during autophagy initiation. Autophagy also plays an essential role in filamentous fungi especially during differentiation. Remarkably, in these species autophagy may reflect features of both yeast and mammals. This is exemplified by the finding that filamentous fungi lack the S. cerevisiae clade-specific Atg31 protein, but contain Atg101, which is absent in this clade. A reappraisal of genome data further suggests that, similar to yeast and mammals, filamentous fungi probably also contain two distinct phosphatidylinositol 3-kinase complexes. This review also summarizes the state of knowledge on the role of autophagy in filamentous fungi during differentiation, such as pathogenic development, programmed cell death during heteroincompatibility, and spore formation.
Asunto(s)
Autofagia , Hongos/fisiología , Secuencia de Aminoácidos , Animales , Hongos/crecimiento & desarrollo , Interacciones Huésped-Patógeno , Humanos , Datos de Secuencia Molecular , Micosis/metabolismo , Micosis/microbiología , Oryza/microbiología , Fosfatidilinositol 3-Quinasas/metabolismo , Enfermedades de las Plantas/microbiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de SecuenciaRESUMEN
Pex11 is a key player in peroxisome proliferation, but the molecular mechanisms of its function are still unknown. Here, we show that Pex11 contains a conserved sequence at the N-terminus that can adopt the structure of an amphipathic helix. Using Penicillium chrysogenum Pex11, we show that this amphipathic helix, termed Pex11-Amph, associates with liposomes in vitro. This interaction is especially evident when negatively charged liposomes are used with a phospholipid content resembling that of peroxisomal membranes. Binding of Pex11-Amph to negatively charged membrane vesicles resulted in strong tubulation. This tubulation of vesicles was also observed when the entire soluble N-terminal domain of Pex11 was used. Using mutant peptides, we demonstrate that maintaining the amphipathic properties of Pex11-Amph in conjunction with retaining its α-helical structure are crucial for its function. We show that the membrane remodelling capacity of the amphipathic helix in Pex11 is conserved from yeast to man. Finally, we demonstrate that mutations abolishing the membrane remodelling activity of the Pex11-Amph domain also hamper the function of full-length Pex11 in peroxisome fission in vivo.
Asunto(s)
Proteínas Fúngicas/metabolismo , Membranas Intracelulares/metabolismo , Liposomas/metabolismo , Proteínas de la Membrana/metabolismo , Penicillium chrysogenum/metabolismo , Peroxisomas/metabolismo , Secuencia de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Membranas Intracelulares/química , Liposomas/química , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Penicillium chrysogenum/química , Penicillium chrysogenum/genética , Peroxisomas/química , Fosfolípidos/química , Fosfolípidos/metabolismo , Estructura Secundaria de Proteína , Alineación de SecuenciaRESUMEN
By genome analysis, we previously identified Pex14/17p as a putative novel peroxin of Penicillium chrysogenum. Here, we show that Pex14/17p is a component of the peroxisomal membrane that is essential for efficient peroxisomal targeting signal 1 and peroxisomal targeting signal 2 matrix protein import, implying that the protein is indeed a genuine peroxin. Additionally, a PEX14/17 deletion strain is affected in conidiospore formation. Pex14/17p has properties of both Pex14p and Pex17p, in that the N-terminus of this protein is similar to the highly conserved Pex5p-binding region present in the N-termini of Pex14p proteins, whereas its C-terminus shows weak similarity to yeast Pex17p proteins. We have identified a novel motif in both Pex17p and Pex14/17p that is absent in Pex14p. We show that an N-terminally truncated, but not a C-terminally truncated, Pex14/17p is able to complement both the matrix protein import and sporulation defects of a Delta pex14/17 strain, implying that it is the Pex17p-related portion of the protein that is crucial for its function as a peroxin. Possibly, this compensates for the fact that P. chrysogenum lacks an authenthic Pex17p. We also show that, in P. chrysogenum, Pex14/17p plays a role in making the penicillin biosynthesis process more efficient.
Asunto(s)
Penicilinas/biosíntesis , Penicillium chrysogenum/química , Peroxisomas/química , Proteínas Fúngicas , Membranas Intracelulares/química , Proteínas de la Membrana , Proteínas de Transporte de Membrana , Peroxisomas/ultraestructuraRESUMEN
In the fungus Penicillium chrysogenum, penicillin (PEN) production is compartmentalized in the cytosol and in peroxisomes. Here we show that intact peroxisomes that contain the two final enzymes of PEN biosynthesis, acyl coenzyme A (CoA):6-amino penicillanic acid acyltransferase (AT) as well as the side-chain precursor activation enzyme phenylacetyl CoA ligase (PCL), are crucial for efficient PEN synthesis. Moreover, increasing PEN titers are associated with increasing peroxisome numbers. However, not all conditions that result in enhanced peroxisome numbers simultaneously stimulate PEN production. We find that conditions that lead to peroxisome proliferation but simultaneously interfere with the normal physiology of the cell may be detrimental to antibiotic production. We furthermore show that peroxisomes develop in germinating conidiospores from reticule-like structures. During subsequent hyphal growth, peroxisome proliferation occurs at the tip of the growing hyphae, after which the organelles are distributed over newly formed subapical cells. We observed that the organelle proliferation machinery requires the dynamin-like protein Dnm1.
Asunto(s)
Penicilinas/biosíntesis , Penicillium chrysogenum/metabolismo , Peroxisomas/metabolismo , Aciltransferasas/metabolismo , Coenzima A Ligasas/metabolismo , Dinamina I/metabolismo , Proteínas Fúngicas/metabolismo , Hifa/enzimología , Hifa/crecimiento & desarrollo , Hifa/ultraestructura , Proteínas de Unión a las Penicilinas/metabolismo , Penicillium chrysogenum/enzimología , Peroxisomas/enzimología , Esporas Fúngicas/enzimología , Esporas Fúngicas/ultraestructuraRESUMEN
Cells need a constant supply of precursors to enable the production of macromolecules to sustain growth and survival. Unlike metazoans, unicellular eukaryotes depend exclusively on the extracellular medium for this supply. When environmental nutrients become depleted, existing cytoplasmic components will be catabolized by (macro)autophagy in order to re-use building blocks and to support ATP production. In many cases, autophagy takes care of cellular housekeeping to sustain cellular viability. Autophagy encompasses a multitude of related and often highly specific processes that are implicated in both biogenetic and catabolic processes. Recent data indicate that in some unicellular eukaryotes that undergo profound differentiation during their life cycle (e.g. kinetoplastid parasites and amoebes), autophagy is essential for the developmental change that allows the cell to adapt to a new host or form spores. This review summarizes the knowledge on the molecular mechanisms of autophagy as well as the cytoplasm-to-vacuole-targeting pathway, pexophagy, mitophagy, ER-phagy, ribophagy and piecemeal microautophagy of the nucleus, all highly selective forms of autophagy that have first been uncovered in yeast species. Additionally, a detailed analysis will be presented on the state of knowledge on autophagy in non-yeast unicellular eukaryotes with emphasis on the role of this process in differentiation.
Asunto(s)
Autofagia/fisiología , Eucariontes/citología , Eucariontes/fisiología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Dictyostelium/citología , Dictyostelium/genética , Dictyostelium/crecimiento & desarrollo , Dictyostelium/fisiología , Retículo Endoplásmico/metabolismo , Entamoeba/citología , Entamoeba/genética , Entamoeba/crecimiento & desarrollo , Entamoeba/fisiología , Eucariontes/genética , Eucariontes/crecimiento & desarrollo , Leishmania/citología , Leishmania/genética , Leishmania/crecimiento & desarrollo , Leishmania/fisiología , Modelos Biológicos , Peroxisomas/metabolismo , Fagosomas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Trypanosoma/citología , Trypanosoma/genética , Trypanosoma/crecimiento & desarrollo , Trypanosoma/fisiología , Vacuolas/metabolismoRESUMEN
In budding yeast Saccharomyces cerevisiae, the peroxisomal protein Inp2 is required for inheritance of peroxisomes to the bud, by connecting the organelles to the motor protein Myo2 and the actin cytoskeleton. Recent data suggested that the function of Inp2 may not be conserved in other yeast species. Using in silico analyses we have identified a weakly conserved Inp2-related protein in 18 species of budding yeast and analyzed the role of the identified protein in the methylotrophic yeast Hansenula polymorpha in peroxisome inheritance. Our data show that H. polymorpha Inp2 locates to peroxisomes, interacts with Myo2, and is essential for peroxisome inheritance.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/fisiología , Peroxisomas/metabolismo , Pichia/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Actinas/metabolismo , Citoesqueleto/metabolismo , Proteínas Fúngicas/genética , Pichia/genética , Pichia/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Activation of the cephalosporin side-chain precursor to the corresponding CoA-thioester is an essential step for its incorporation into the beta-lactam backbone. To identify an acyl-CoA ligase involved in activation of adipate, we searched in the genome database of Penicillium chrysogenum for putative structural genes encoding acyl-CoA ligases. Chemostat-based transcriptome analysis was used to identify the one presenting the highest expression level when cells were grown in the presence of adipate. Deletion of the gene renamed aclA, led to a 32% decreased specific rate of adipate consumption and a threefold reduction of adipoyl-6-aminopenicillanic acid levels, but did not affect penicillin V production. After overexpression in Escherichia coli, the purified protein was shown to have a broad substrate range including adipate. Finally, protein-fusion with cyan-fluorescent protein showed co-localization with microbody-borne acyl-transferase. Identification and functional characterization of aclA may aid in developing future metabolic engineering strategies for improving the production of different cephalosporins.
Asunto(s)
Adipatos/metabolismo , Cefalosporinas/biosíntesis , Coenzima A Ligasas/metabolismo , Proteínas Fúngicas/metabolismo , Penicillium chrysogenum/enzimología , Coenzima A Ligasas/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Penicillium chrysogenum/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
This study aimed at developing an alternative host for the production of penicillin (PEN). As yet, the industrial production of this beta-lactam antibiotic is confined to the filamentous fungus Penicillium chrysogenum. As such, the yeast Hansenula polymorpha, a recognized producer of pharmaceuticals, represents an attractive alternative. Introduction of the P. chrysogenum gene encoding the non-ribosomal peptide synthetase (NRPS) delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) in H. polymorpha, resulted in the production of active ACVS enzyme, when co-expressed with the Bacillus subtilis sfp gene encoding a phosphopantetheinyl transferase that activated ACVS. This represents the first example of the functional expression of a non-ribosomal peptide synthetase in yeast. Co-expression with the P. chrysogenum genes encoding the cytosolic enzyme isopenicillin N synthase as well as the two peroxisomal enzymes isopenicillin N acyl transferase (IAT) and phenylacetyl CoA ligase (PCL) resulted in production of biologically active PEN, which was efficiently secreted. The amount of secreted PEN was similar to that produced by the original P. chrysogenum NRRL1951 strain (approx. 1 mg/L). PEN production was decreased over two-fold in a yeast strain lacking peroxisomes, indicating that the peroxisomal localization of IAT and PCL is important for efficient PEN production. The breakthroughs of this work enable exploration of new yeast-based cell factories for the production of (novel) beta-lactam antibiotics as well as other natural and semi-synthetic peptides (e.g. immunosuppressive and cytostatic agents), whose production involves NRPS's.
Asunto(s)
Ingeniería Genética , Penicilinas/metabolismo , Pichia/genética , Pichia/metabolismo , Eliminación de Gen , Genes Fúngicos , Penicillium chrysogenum/enzimología , Péptido Sintasas/biosíntesis , Peroxisomas/metabolismo , Pichia/citología , Pichia/ultraestructura , Transporte de Proteínas , Fracciones Subcelulares/ultraestructura , beta-Lactamas/metabolismoRESUMEN
Cellular proteins and organelles such as peroxisomes are under continuous quality control. Upon synthesis in the cytosol, peroxisomal proteins are kept in an import-competent state by chaperones or specific proteins with an analogous function to prevent degradation by the ubiquitin-proteasome system. During protein translocation into the organelle, the peroxisomal targeting signal receptors (Pex5, Pex20) are also continuously undergoing quality control to enable efficient functioning of the translocon (RADAR pathway). Even upon maturation of peroxisomes, matrix enzymes and peroxisomal membranes remain subjected to quality control. As a result of their oxidative metabolism, peroxisomes are producers of reactive oxygen species (ROS), which may damage proteins and lipids. To counteract ROS-induced damage, yeast peroxisomes contain two important antioxidant enzymes: catalase and an organelle-specific peroxiredoxin. Additionally, a Lon-type protease has recently been identified in the peroxisomal matrix, which is capable of degrading nonfunctional proteins. Finally, cellular housekeeping processes keep track of the functioning of peroxisomes so that dysfunctional organelles can be quickly removed via selective autophagy (pexophagy). This review provides an overview of the major processes involved in quality control of yeast peroxisomes.
Asunto(s)
Proteínas Fúngicas/metabolismo , Orgánulos/fisiología , Levaduras/fisiología , ADN de Hongos/química , ADN de Hongos/genética , Proteínas Fúngicas/genética , Modelos Biológicos , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
In the filamentous fungus Penicillium chrysogenum, microbodies are essential for penicillin biosynthesis. To better understand the role of these organelles in antibiotics production, we determined the matrix enzyme contents of P. chrysogenum microbodies. Using a novel in silico approach, we first obtained a catalogue of 200 P. chrysogenum proteins with putative microbody targeting signals (PTSs). This included two orthologs of proteins involved in cephalosporin biosynthesis, which we demonstrate to be bona fide microbody matrix constituents. Subsequently, we performed a proteomics based inventory of P. chrysogenum microbody matrix proteins using nano-LC-MS/MS analysis. We identified 89 microbody proteins, 79 with a PTS, including the two known microbody-borne penicillin biosynthesis enzymes, isopenicillin N:acyl CoA acyltransferase and phenylacetyl-CoA ligase. Comparative analysis revealed that 69 out of 79 PTS proteins identified experimentally were in the reference list. A prominent microbody protein was identified as a novel fumarate reductase-cytochrome b5 fusion protein, which contains an internal PTS2 between the two functional domains. We show that this protein indeed localizes to P. chrysogenum microbodies.
Asunto(s)
Microcuerpos/metabolismo , Penicilinas/biosíntesis , Penicillium chrysogenum/genética , Penicillium chrysogenum/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN/genética , ADN de Hongos/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Microcuerpos/ultraestructura , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Penicillium chrysogenum/ultraestructura , Plásmidos/genética , Señales de Clasificación de Proteína/genética , Proteoma , Proteómica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masas en TándemRESUMEN
The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.
Asunto(s)
Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Genoma Fúngico , Genómica , Aspergillus nidulans/fisiologíaRESUMEN
Fungal microbodies (peroxisomes) are inducible organelles that proliferate in response to nutritional cues. Proteins involved in peroxisome biogenesis/proliferation are designated peroxins and are encoded by PEX genes. An autophagy-related process, termed pexophagy, is responsible for the selective removal of peroxisomes from the cell. Several genes involved in pexophagy are also required for autophagy and are collectively known as ATG genes. We have re-analysed the Aspergillus nidulans genome for the presence of PEX and ATG genes and have identified a number of previously missed genes. Also, we manually determined the correct intron positions in each identified gene. The data show that in A. nidulans and related fungi the basic set of genes involved in peroxisome biogenesis or degradation are conserved. However, both processes have features that more closely resemble organelle formation/degradation in mammals rather than yeast. Thus, filamentous fungi like A. nidulans are ideal model systems for peroxisome homeostasis in man.
Asunto(s)
Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Microcuerpos/metabolismo , Secuencia Conservada , IntronesRESUMEN
Industrial penicillin production with the filamentous fungus Penicillium chrysogenum is based on an unprecedented effort in microbial strain improvement. To gain more insight into penicillin synthesis, we sequenced the 32.19 Mb genome of P. chrysogenum Wisconsin54-1255 and identified numerous genes responsible for key steps in penicillin production. DNA microarrays were used to compare the transcriptomes of the sequenced strain and a penicillinG high-producing strain, grown in the presence and absence of the side-chain precursor phenylacetic acid. Transcription of genes involved in biosynthesis of valine, cysteine and alpha-aminoadipic acid-precursors for penicillin biosynthesis-as well as of genes encoding microbody proteins, was increased in the high-producing strain. Some gene products were shown to be directly controlling beta-lactam output. Many key cellular transport processes involving penicillins and intermediates remain to be characterized at the molecular level. Genes predicted to encode transporters were strongly overrepresented among the genes transcriptionally upregulated under conditions that stimulate penicillinG production, illustrating potential for future genomics-driven metabolic engineering.
Asunto(s)
Mapeo Cromosómico/métodos , Proteínas Fúngicas/genética , Genoma Fúngico/genética , Penicilina G/metabolismo , Penicillium chrysogenum/genética , Factores de Transcripción/genética , Secuencia de Bases , Datos de Secuencia Molecular , Análisis de Secuencia de ADN/métodosRESUMEN
We show that Mdv1 and Caf4, two components of the mitochondrial fission machinery in Saccharomyces cerevisiae, also function in peroxisome proliferation. Deletion of MDV1, CAF4 or both, however, had only a minor effect on peroxisome numbers at peroxisome-inducing growth conditions, most likely related to the fact that Vps1--and not Dnm1--is the key player in peroxisome fission in this organism. In contrast, in Hansenula polymorpha, which has only a Dnm1-dependent peroxisome fission machinery, deletion of MDV1 led to a drastic reduction of peroxisome numbers. This phenotype was accompanied by a strong defect in mitochondrial fission. The MDV1 paralog CAF4 is absent in H. polymorpha. In wild-type H. polymorpha, cells Dnm1-mCherry and green fluorescent protein (GFP)-Mdv1 colocalize in spots that associate with both peroxisomes and mitochondria. Furthermore, Fis1 is essential to recruit Mdv1 to the peroxisomal and mitochondrial membrane. However, formation of GFP-Mdv1 spots--and related to this normal organelle fission--is strictly dependent on the presence of Dnm1. In dnm1 cells, GFP-Mdv1 is dispersed over the surface of peroxisomes and mitochondria. Also, in H. polymorpha mdv1 or fis1 cells, the number of Dnm1-GFP spots is strongly reduced. These spots still associate to organelles but are functionally inactive.