RESUMO
BACKGROUND: Aspergillus niger is a ubiquitous filamentous fungus widely employed as a cell factory thanks to its abilities to produce a wide range of organic acids and enzymes. Its genome was one of the first Aspergillus genomes to be sequenced in 2007, due to its economic importance and its role as model organism to study fungal fermentation. Nowadays, the genome sequences of more than 20 A. niger strains are available. These, however, do not include the neotype strain CBS 554.65. RESULTS: The genome of CBS 554.65 was sequenced with PacBio. A high-quality nuclear genome sequence consisting of 17 contigs with a N50 value of 4.07 Mbp was obtained. The assembly covered all the 8 centromeric regions of the chromosomes. In addition, a complete circular mitochondrial DNA assembly was obtained. Bioinformatic analyses revealed the presence of a MAT1-2-1 gene in this genome, contrary to the most commonly used A. niger strains, such as ATCC 1015 and CBS 513.88, which contain a MAT1-1-1 gene. A nucleotide alignment showed a different orientation of the MAT1-1 locus of ATCC 1015 compared to the MAT1-2 locus of CBS 554.65, relative to conserved genes flanking the MAT locus. Within 24 newly sequenced isolates of A. niger half of them had a MAT1-1 locus and the other half a MAT1-2 locus. The genomic organization of the MAT1-2 locus in CBS 554.65 is similar to other Aspergillus species. In contrast, the region comprising the MAT1-1 locus is flipped in all sequenced strains of A. niger. CONCLUSIONS: This study, besides providing a high-quality genome sequence of an important A. niger strain, suggests the occurrence of genetic flipping or switching events at the MAT1-1 locus of A. niger. These results provide new insights in the mating system of A. niger and could contribute to the investigation and potential discovery of sexuality in this species long thought to be asexual.
Assuntos
Aspergillus niger , Genes Fúngicos Tipo Acasalamento , Aspergillus niger/genética , Sequência de Bases , Mapeamento Cromossômico , GenômicaRESUMO
A key challenge for bioprocess engineering is the identification of the optimum process conditions for the production of biochemical and biopharmaceutical compounds using prokaryotic as well as eukaryotic cell factories. Shake flasks and bench-scale bioreactor systems are still the golden standard in the early stage of bioprocess development, though they are known to be expensive, time-consuming, and labor-intensive as well as lacking the throughput for efficient production optimizations. To bridge the technological gap between bioprocess optimization and upscaling, we have developed a microfluidic bioreactor array to reduce time and costs, and to increase throughput compared with traditional lab-scale culture strategies. We present a multifunctional microfluidic device containing 12 individual bioreactors (Vt = 15 µl) in a 26 mm × 76 mm area with in-line biosensing of dissolved oxygen and biomass concentration. Following initial device characterization, the bioreactor lab-on-a-chip was used in a proof-of-principle study to identify the most productive cell line for lactic acid production out of two engineered yeast strains, evaluating whether it could reduce the time needed for collecting meaningful data compared with shake flasks cultures. Results of the study showed significant difference in the strains' productivity within 3 hr of operation exhibiting a 4- to 6-fold higher lactic acid production, thus pointing at the potential of microfluidic technology as effective screening tool for fast and parallelizable industrial bioprocess development.
Assuntos
Reatores Biológicos , Ácido Láctico/metabolismo , Saccharomyces cerevisiae/metabolismo , Desenho de Equipamento , Microbiologia Industrial/instrumentação , Dispositivos Lab-On-A-Chip , Saccharomyces cerevisiae/citologiaRESUMO
Aspergillus niger was engineered using a gene responsible for citric acid transport, which has a significant impact on citric acid secretion when overexpressed. The transport gene was identified by a homology search using an itaconic acid transporter from Ustilago maydis as template. The encoding homologous protein CexA belongs to the major facilitator superfamily subclass DHA1 and members of this family work as drug-H+ antiporter. The disruption of this gene completely abolishes citric acid secretion, which indicates that this protein is the main citric acid transporter in A. niger. In the disruption strain, the metabolism is re-routed mainly to oxalic acid, which is a known by-product during citric acid production. The gene can be heterologously expressed in Saccharomyces cerevisiae, which leads to the secretion of citric acid during the growth on glucose. These results confirm the functionality of CexA as the main transporter for citric acid of A. niger. Overexpression of cexA leads to a significant increase in secreted citric acid. Thereby, striking differences between a strong constitutive expression system using pmbfA as a promoter and an inducible expression system using ptet-on can be observed. The inducible system significantly outcompetes the constitutive expression system yielding up to 109â¯g/L citric acid, which is 5 times higher compared to the parental wild-type strain and 3 times higher compared to the constitutive expression system. These results demonstrate the importance of the cellular transport system for an efficient production of metabolites. By overexpressing a single gene, it is possible to significantly improve the citric acid secretion capability of a moderately producing parental strain.
Assuntos
Aspergillus niger/genética , Aspergillus niger/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ácido Cítrico/metabolismo , Engenharia Metabólica/métodos , Proteínas Associadas a CRISPR , Meios de Cultura , Plasmídeos/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Besides its use for efficient production of recombinant proteins the methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) has been increasingly employed as a platform to produce metabolites of varying origin. We summarize here the impressive methodological developments of the last years to model and analyze the metabolism of P. pastoris, and to engineer its genome and metabolic pathways. Efficient methods to insert, modify or delete genes via homologous recombination and CRISPR/Cas9, supported by modular cloning techniques, have been reported. An outstanding early example of metabolic engineering in P. pastoris was the humanization of protein glycosylation. More recently the cell metabolism was engineered also to enhance the productivity of heterologous proteins. The last few years have seen an increased number of metabolic pathway design and engineering in P. pastoris, mainly towards the production of complex (secondary) metabolites. In this review, we discuss the potential role of P. pastoris as a platform for metabolic engineering, its strengths, and major requirements for future developments of chassis strains based on synthetic biology principles.
Assuntos
Sistemas CRISPR-Cas , Engenharia Metabólica/métodos , Pichia/genética , Pichia/metabolismoRESUMO
In the field of metabolic engineering 13C-based metabolic flux analysis experiments have proven successful in indicating points of action. As every step of this approach is affected by an inherent error, the aim of the present work is the comprehensive evaluation of factors contributing to the uncertainty of nonnaturally distributed C-isotopologue abundances as well as to the absolute flux value calculation. For this purpose, a previously published data set, analyzed in the course of a 13C labeling experiment studying glycolysis and the pentose phosphate pathway in a yeast cell factory, was used. Here, for isotopologue pattern analysis of these highly polar metabolites that occur in multiple isomeric forms, a gas chromatographic separation approach with preceding derivatization was used. This rendered a natural isotope interference correction step essential. Uncertainty estimation of the resulting C-isotopologue distribution was performed according to the EURACHEM guidelines with Monte Carlo simulation. It revealed a significant increase for low-abundance isotopologue fractions after application of the necessary correction step. For absolute flux value estimation, isotopologue fractions of various sugar phosphates, together with the assessed uncertainties, were used in a metabolic model describing the upper part of the central carbon metabolism. The findings pinpointed the influence of small isotopologue fractions as sources of error and highlight the need for improved model curation. Graphical abstract á .
Assuntos
Análise do Fluxo Metabólico/métodos , Pichia/metabolismo , Isótopos de Carbono/análise , Isótopos de Carbono/metabolismo , Simulação por Computador , Engenharia Metabólica , Redes e Vias Metabólicas , Metaboloma , Metabolômica/métodos , Modelos Biológicos , Método de Monte Carlo , Pichia/química , Pichia/citologia , IncertezaRESUMO
Zinc is a crucial mineral for all organisms as it is an essential cofactor for the proper function of a plethora of proteins and depletion of zinc causes oxidative stress. Glutathione is the major redox buffering agent in the cell and therefore important for mitigation of the adverse effects of oxidative stress. In mammalian cells, zinc deficiency is accompanied by a glutathione depletion. In the yeast Saccharomyces cerevisiae, the opposite effect is observed: under low zinc conditions, an elevated glutathione concentration is found. The main regulator to overcome zinc deficiency is Zap1p. However, we show that Zap1p is not involved in this glutathione accumulation phenotype. Furthermore, we found that in glutathione-accumulating strains also the metal ion-binding phytochelatin-2, which is an oligomer of glutathione, is accumulated. This increased phytochelatin concentration correlates with a lower free zinc level in the vacuole. These results suggest that phytochelatin is important for zinc buffering in S. cerevisiae and thus explains how zinc homeostasis is connected with glutathione metabolism.
Assuntos
Glutationa/metabolismo , Homeostase , Fitoquelatinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Zinco/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The mitochondrial carrier protein MttA is involved in the biosynthesis of itaconic acid in Aspergillus terreus. In this paper, the transport specificity of MttA is analyzed making use of different metabolically engineered Aspergillus niger strains. Furthermore, the mitochondrial localization of this protein is confirmed using fluorescence microscopy. It was found that MttA preferentially transports cis-aconitic acid over citric acid and does not transport itaconic acid. The expression of MttA in selected A. niger strains results in secretion of aconitic acid. MttA can be used in further strain engineering strategies to transport cis-aconitic acid to the cytosol to produce itaconic acid or related metabolites. The microbial production of aconitic acid (9g/L) is achieved in strains expressing this transport protein. Thus, metabolic engineering can be used for both the in vivo characterization of transport protein function like MttA and to make use of this protein by creating aconitic acid producing strains.
Assuntos
Ácido Aconítico/metabolismo , Aspergillus , Proteínas Fúngicas , Proteínas de Membrana Transportadoras , Engenharia Metabólica , Proteínas Mitocondriais , Aspergillus/genética , Aspergillus/metabolismo , Transporte Biológico Ativo/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/genética , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genéticaRESUMO
Efficient conversion of hexoses and pentoses into value-added chemicals represents one core step for establishing economically feasible biorefineries from lignocellulosic material. While extensive research efforts have recently provided advances in the overall process performance, the quest for new microbial cell factories and novel enzymes sources is still open. As demonstrated recently the yeast Sugiyamaella lignohabitans (formerly Candida lignohabitans) represents a promising microbial cell factory for the production of organic acids from lignocellulosic hydrolysates. We report here the de novo genome assembly of S. lignohabitans using the Single Molecule Real-Time platform, with gene prediction refined by using RNA-seq. The sequencing revealed a 15.98 Mb genome, subdivided into four chromosomes. By phylogenetic analysis, Blastobotrys (Arxula) adeninivorans and Yarrowia lipolytica were found to be close relatives of S. lignohabitans Differential gene expression was evaluated in typical growth conditions on glucose and xylose and allowed a first insight into the transcriptional response of S. lignohabitans to different carbon sources and different oxygenation conditions. Novel sequences for enzymes and transporters involved in the central carbon metabolism, and therefore of potential biotechnological interest, were identified. These data open the way for a better understanding of the metabolism of S. lignohabitans and provide resources for further metabolic engineering.
Assuntos
Perfilação da Expressão Gênica , Genoma Fúngico , Redes e Vias Metabólicas/genética , Pentoses/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Cromossomos Fúngicos , Glucose/metabolismo , Filogenia , Saccharomycetales/classificação , Saccharomycetales/crescimento & desenvolvimento , Homologia de Sequência , Xilose/metabolismoRESUMO
Production of heterologous proteins in Pichia pastoris (syn. Komagataella sp.) has been shown to exert a metabolic burden on the host metabolism. This burden is associated with metabolite drain, which redirects nucleotides and amino acids from primary metabolism. On the other hand, recombinant protein production affects energy and redox homeostasis of the host cell. In a previous study, we have demonstrated that overexpression of single genes of the oxidative pentose phosphate pathway (PPP) had a positive influence on recombinant production of cytosolic human superoxide dismutase (hSOD). In this study, different combinations of these genes belonging to the oxidative PPP were generated and analyzed. Thereby, a 3.8-fold increase of hSOD production was detected when glucose-6-phosphate dehydrogenase (ZWF1) and 6-gluconolactonase (SOL3) were simultaneously overexpressed, while the combinations of other genes from PPP had no positive effect on protein production. By measuring isotopologue patterns of (13)C-labelled metabolites, we could detect an upshift in the flux ratio of PPP to glycolysis upon ZWF1 and SOL3 co-overexpression, as well as increased levels of 6-phosphogluconate. The substantial improvement of hSOD production by ZWF1 and SOL3 co-overexpression appeared to be connected to an increase in PPP flux. In conclusion, we show that overexpression of SOL3 together with ZWF1 enhanced both the PPP flux ratio and hSOD accumulation, providing evidence that in P. pastoris Sol3 limits the flux through PPP and recombinant protein production.
Assuntos
Expressão Gênica , Via de Pentose Fosfato , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Superóxido Dismutase/biossíntese , Glucose/metabolismo , Humanos , Pichia/genética , Proteínas Recombinantes/genética , Superóxido Dismutase/genéticaRESUMO
The objective of this study is to analyze the accuracy of computed tomography in detecting malignant thyroid cartilage invasion. In a retrospective chart review, 120 patients with carcinoma of the larynx and hypopharynx underwent computed tomography before total laryngectomy. These data were compared with the histological specimens. Multidetector computed tomography (MDCT) scan had a positive predictive value (PPV) of 76 % and a negative predictive value (NPV) of 69 %. The specificity of MDCT was 89 % and sensitivity was 46 %. Comparison between radiologic suspected cartilage invasion and histologic results showed a significant correlation (p < 0.02). We found no significant impact of cartilage invasion concerning survival rates (5-year overall survival p = 0.683; 5-year disease-free survival p = 0.711). Preoperative CT scan is an important instrument in detecting neoplastic cartilage invasion.
Assuntos
Carcinoma de Células Escamosas/cirurgia , Neoplasias Hipofaríngeas/cirurgia , Neoplasias Laríngeas/cirurgia , Laringectomia , Tomografia Computadorizada Multidetectores , Cartilagem Tireóidea/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/patologia , Feminino , Seguimentos , Humanos , Neoplasias Hipofaríngeas/diagnóstico por imagem , Neoplasias Hipofaríngeas/patologia , Neoplasias Laríngeas/diagnóstico por imagem , Neoplasias Laríngeas/patologia , Laringectomia/métodos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Cuidados Pré-Operatórios , Estudos Retrospectivos , Sensibilidade e Especificidade , Cartilagem Tireóidea/diagnóstico por imagemRESUMO
BACKGROUND: Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia pastoris (syn. Komagataella sp.) is frequently used for the production of heterologous proteins and also serves as a model organism for organelle research. Our current knowledge of methylotrophic lifestyle mainly derives from sophisticated biochemical studies which identified many key methanol utilization enzymes such as alcohol oxidase and dihydroxyacetone synthase and their localization to the peroxisomes. C1 assimilation is supposed to involve the pentose phosphate pathway, but details of these reactions are not known to date. RESULTS: In this work we analyzed the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 reactions of P. pastoris comparing growth on glucose and on a methanol/glycerol mixed medium, respectively. Contrary to previous assumptions, we found that the entire methanol assimilation pathway is localized to peroxisomes rather than employing part of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration. For this purpose, P. pastoris (and presumably also other methylotrophic yeasts) have evolved a duplicated methanol inducible enzyme set targeted to peroxisomes. This compartmentalized cyclic C1 assimilation process termed xylose-monophosphate cycle resembles the principle of the Calvin cycle and uses sedoheptulose-1,7-bisphosphate as intermediate. The strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase leads to high demand of their cofactors riboflavin, thiamine, nicotinamide, and heme, respectively, which is reflected in strong up-regulation of the respective synthesis pathways on methanol. Methanol-grown cells have a higher protein but lower free amino acid content, which can be attributed to the high drain towards methanol metabolic enzymes and their cofactors. In context with up-regulation of many amino acid biosynthesis genes or proteins, this visualizes an increased flux towards amino acid and protein synthesis which is reflected also in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation. CONCLUSIONS: Taken together, our work illustrates how concerted interpretation of multiple levels of systems biology data can contribute to elucidation of yet unknown cellular pathways and revolutionize our understanding of cellular biology.
Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Glicerol/metabolismo , Metanol/metabolismo , Pichia/genética , Proteínas Fúngicas/metabolismo , Pichia/metabolismoRESUMO
For the first time an analytical work flow based on accurate mass gas chromatography-quadrupole time-of-flight mass spectrometry (GC-QTOFMS) with chemical ionization for analysis providing a comprehensive picture of (13)C distribution along the primary metabolism is elaborated. The method provides a powerful new toolbox for (13)C-based metabolic flux analysis, which is an emerging strategy in metabolic engineering. In this field, stable isotope tracer experiments based on, for example, (13)C are central for providing characteristic patterns of labeled metabolites, which in turn give insights into the regulation of metabolic pathway kinetics. The new method enables the analysis of isotopologue fractions of 42 free intracellular metabolites within biotechnological samples, while tandem mass isotopomer information is also accessible for a large number of analytes. Hence, the method outperforms previous approaches in terms of metabolite coverage, while also providing rich isotopomer information for a significant number of key metabolites. Moreover, the established work flow includes novel evaluation routines correcting for isotope interference of naturally distributed elements, which is crucial following derivatization of metabolites. Method validation in terms of trueness, precision, and limits of detection was performed, showing excellent analytical figures of merit with an overall maximum bias of 5.8%, very high precision for isotopologue and tandem mass isotopomer fractions representing >10% of total abundance, and absolute limits of detection in the femtomole range. The suitability of the developed method is demonstrated on a flux experiment of Pichia pastoris employing two different tracers, i.e., 1,6(13)C2-glucose and uniformly labeled (13)C-glucose.
Assuntos
Isótopos de Carbono/análise , Isótopos de Carbono/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Análise do Fluxo Metabólico , Isótopos de Carbono/química , Humanos , Pichia/genética , Pichia/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Fatores de TempoRESUMO
Metabolomics can be defined as the quantitative assessment of a large number of metabolites of a biological system. A prerequisite for the accurate determination of intracellular metabolite concentrations is a reliable and reproducible sample preparation method, which needs to be optimized for each organism individually. Here, we compare the performance of rapid filtration and centrifugation after quenching of Pichia pastoris cells in cold methanol. During incubation in the quenching solution, metabolites are lost from the cells with a half-life of 70-180 min. Metabolites with lower molecular weights showed lower half-lifes compared to metabolites with higher molecular weight. Rapid filtration within 2 min after quenching leads to only minor losses below 2%, and is thus the preferred method for cell separation.
Assuntos
Filtração/métodos , Metaboloma , Metabolômica/métodos , Técnicas Microbiológicas/métodos , Pichia/química , Centrifugação/métodos , Congelamento , Fatores de TempoRESUMO
Promoters are indispensable elements of a standardized parts collection for synthetic biology. Regulated promoters of a wide variety of well-defined induction ratios and expression strengths are highly interesting for many applications. Exemplarily, we discuss the application of published genome scale transcriptomics data for the primary selection of methanol inducible promoters of the yeast Pichia pastoris (Komagataella sp.). Such a promoter collection can serve as an excellent toolbox for cell and metabolic engineering, and for gene expression to produce heterologous proteins.
Assuntos
Metanol/metabolismo , Pichia/metabolismo , Biologia Sintética/métodos , Regulação Fúngica da Expressão Gênica , Engenharia Metabólica , Pichia/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
The production of recombinant proteins is frequently enhanced at the levels of transcription, codon usage, protein folding and secretion. Overproduction of heterologous proteins, however, also directly affects the primary metabolism of the producing cells. By incorporation of the production of a heterologous protein into a genome scale metabolic model of the yeast Pichia pastoris, the effects of overproduction were simulated and gene targets for deletion or overexpression for enhanced productivity were predicted. Overexpression targets were localized in the pentose phosphate pathway and the TCA cycle, while knockout targets were found in several branch points of glycolysis. Five out of 9 tested targets led to an enhanced production of cytosolic human superoxide dismutase (hSOD). Expression of bacterial ß-glucuronidase could be enhanced as well by most of the same genetic modifications. Beneficial mutations were mainly related to reduction of the NADP/H pool and the deletion of fermentative pathways. Overexpression of the hSOD gene itself had a strong impact on intracellular fluxes, most of which changed in the same direction as predicted by the model. In vivo fluxes changed in the same direction as predicted to improve hSOD production. Genome scale metabolic modeling is shown to predict overexpression and deletion mutants which enhance recombinant protein production with high accuracy.
Assuntos
Engenharia Metabólica , Metaboloma/genética , Modelos Biológicos , Pichia , Ciclo do Ácido Cítrico/genética , Expressão Gênica , Glicólise/genética , Humanos , NAD/genética , NAD/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
Non-homologous end joining (NHEJ) and homology-directed repair (HDR) are two mechanisms in filamentous fungi to repair DNA damages. NHEJ is the dominant response pathway to rapidly join DNA double-strand breaks, but often leads to insertions or deletions. On the other hand, HDR is more precise and utilizes a homologous DNA template to restore the damaged sequence. Both types are exploited in genetic engineering approaches ranging from knock-out mutations to precise sequence modifications.In this study, we evaluated the efficiency of an HDR based gene integration system designed for the pyrG locus of Aspergillus niger. While gene integration was achieved at a rate of 91.4%, we also discovered a mixed-type repair (MTR) mechanism with simultaneous repair of a Cas9-mediated double-strand break by both NHEJ and HDR. In 20.3% of the analyzed transformants the donor DNA was integrated by NHEJ at the 3' end and by HDR at the 5' end of the double-strand break. Furthermore, sequencing of the locus revealed different DNA repair mechanisms at the site of the NHEJ event.Together, the results support the applicability of the genome integration system and a novel DNA repair type with implication on the diversity of genetic modifications in filamentous fungi.
RESUMO
The ability to acquire three-dimensional (3D) information of cellular structures without the need for fluorescent tags or staining makes holotomographic imaging a powerful tool in cellular biology. It provides valuable insights by measuring the refractive index (RI), an optical parameter describing the phase delay of light that passes through the living cell. Here, we demonstrate holotomographic imaging on industrial relevant ascomycete fungi and study their development and morphogenesis. This includes conidial germination, subcellular dynamics, and cytoplasmic flow during hyphal growth in Aspergillus niger. In addition, growth and budding of Aureobasidium pullulans cells are captured using holotomographic microscopy. Coupled to fluorescence imaging, lipid droplets, vacuoles, the mitochondrial network, and nuclei are targeted and analyzed in the 3D RI reconstructed images. While lipid droplets and vacuoles can be assigned to a specific RI pattern, mitochondria and nuclei were not pronounced. We show, that the lower sensitivity of RI measurements derives from the fungal cell wall that acts as an additional barrier for the illumination light of the microscope. After cell wall digest of hyphae and protoplast formation of A. niger expressing GFP-tagged histone H2A, location of nuclei could be determined by non-invasive RI measurements. Furthermore, we used coupled fluorescence microscopy to observe migration of nuclei in unperturbed hyphal segments and duplication during growth on a single-cell level. Detailed micromorphological studies in Saccharomyces cerevisiae and Trichoderma reesei are challenging due to cell size restrictions. Overall, holotomography opens up new avenues for exploring dynamic cellular processes in real time and enables the visualization of fungi from a new perspective.
RESUMO
Methanol is a toxic alcohol contained in alcoholic beverages as a natural byproduct of fermentation or added intentionally to counterfeits to increase profit. To ensure consumer safety, many countries and the EU have established strict legislation limits for methanol content. Methanol concentration is mostly detected by laboratory instrumentation since mobile devices for routine on-site testing of beverages in distilleries, at border stations or even at home are not available. Here, we validated a handheld methanol detector for beverage analysis in an ISO 5725 interlaboratory trial: a total of 119 measurements were performed by 17 independent participants (distilleries, universities, authorities, and competence centers) from six countries on samples with relevant methanol concentrations (0.1, 1.5 vol%). The detector was based on a microporous separation filter and a nanostructured gas sensor allowing on-site measurement of methanol down to 0.01 vol% (in the liquid) within only 2 min by laymen. The detector showed excellent repeatability (<5.4%), reproducibility (<9.5%) and small bias (<0.012 vol%). Additional measurements on various methanol-spiked alcoholic beverages (whisky, rum, gin, vodka, tequila, port, sherry, liqueur) indicated that the detector is not interfered by environmental temperature and spirit composition, featuring excellent linearity (R2 > 0.99) down to methanol concentrations of 0.01 vol%. This device has been recently commercialized (Alivion Spark M-20) with comparable accuracy to the gold-standard gas chromatography and can be readily applied for final product inspection, intake control of raw materials or to identify toxic counterfeit products.
Assuntos
Bebidas Alcoólicas , Metanol , Metanol/análise , Bebidas Alcoólicas/análise , Reprodutibilidade dos Testes , Análise de Alimentos/instrumentação , Análise de Alimentos/métodos , Laboratórios/normasRESUMO
Itaconic acid is an unsaturated dicarboxylic acid which has a high potential as a biochemical building block. It can be microbially produced from some Aspergillus species, such as Aspergillus itaconicus and Aspergillus terreus. However, the achieved titers are significantly lower as compared to the citric acid production by A. niger. Heterologous expression of cis-aconitate decarboxylase in A. niger leads to the accumulation of small amounts of itaconic acid. Additional expression of aconitase, the second enzyme metabolically linking citric acid and itaconic acid improves productivity. However, proper organelle targeting of the enzymes appears to be an important point to consider. Here we compare the mitochondrial expression with the cytosolic expression of cis-aconitate decarboxylase or aconitase in A. niger. Heterologous expression of both enzymes in the mitochondria doubles the productivity compared to strains which express the enzymes in the cytosol. It is essential to target enzymes to the correct compartment in order to establish a proper flux through a compartmentalized pathway.
Assuntos
Aspergillus niger/metabolismo , Succinatos/metabolismo , Aconitato Hidratase/biossíntese , Aconitato Hidratase/genética , Aspergillus niger/genética , Carboxiliases/biossíntese , Carboxiliases/genética , Ácido Cítrico/metabolismo , Citosol/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Engenharia Metabólica/métodos , Mitocôndrias/enzimologia , Mitocôndrias/genéticaRESUMO
Genetic tools for the fine-tuning of gene expression levels are a prerequisite for rational strain optimization through metabolic engineering. While Aspergillus niger is an industrially important fungus, widely used for production of organic acids and heterologous proteins, the available genetic tool box for this organism is still rather limited. Here, we characterize six novel constitutive promoters of A. niger providing different expression levels. The selection of the promoters was based on published transcription data of A. niger. The promoter strength was determined with the ß-glucuronidase (gusA) reporter gene of Escherichia coli. The six promoters covered a GUS activity range of two to three orders of magnitude depending on the strain background. In order to demonstrate the power of the newly characterized promoters for metabolic engineering, they were used for heterologous expression of the cis-aconitate decarboxylase (cad1) gene of Aspergillus terreus, allowing the production of the building block chemical itaconic acid with A. niger. The CAD activity, dependent on the choice of promoter, showed a positive correlation with the specific productivity of itaconic acid. Product titers from the detection limit to up to 570 mg/L proved that the set of constitutive promoters is a powerful tool for the fine-tuning of metabolic pathways for the improvement of industrial production processes.