Development of tools for quantitative intracellular metabolomics of Aspergillus niger chemostat cultures.
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ABSTRACT:
SUBMITTER: Lameiras F
PROVIDER: S-EPMC4559092 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes, particularly glucoamylase. Although a variety of genetic techniques have been successfully used in wild-type A. niger, the transformation of industrially used strains with few conidia (e.g., A. niger N1) or that are even aconidial (e.g., A. niger O1) remains laborious. Herein, we developed genetic tools, including the protoplast-mediated transformation and Agrobacterium tumefaciens-mediated transformation of the A. niger strains N1 and O1 using green fluorescent protein as a reporter marker. Following the optimization of various factors for protoplast release from mycelium, the protoplast-mediated transformation efficiency reached 89.3% (25/28) for N1 and 82.1% (32/39) for O1. The A. tumefaciens-mediated transformation efficiency was 98.2% (55/56) for N1 and 43.8% (28/64) for O1. We also developed a marker-free CRISPR/Cas9 genome editing system using an AMA1-based plasmid to express the Cas9 protein and sgRNA. Out of 22 transformants, 9 albA deletion mutants were constructed in the A. niger N1 background using the protoplast-mediated transformation method and the marker-free CRISPR/Cas9 system developed here. The genome editing methods improved here will accelerate the elucidation of the mechanism of glucoamylase hyperproduction in these industrial fungi and will contribute to the use of efficient targeted mutation in other industrial strains of A. niger.
| S-EPMC9599018 | biostudies-literature
Project description:The fungus Aspergillus niger forms (sub)millimeter microcolonies within a liquid shaken culture. Here, we show that such microcolonies are heterogeneous with respect to size and gene expression. Microcolonies of strains expressing green fluorescent protein (GFP) from the promoter of the glucoamlyase gene glaA or the ferulic acid esterase gene faeA were sorted on the basis of diameter and fluorescence using the Complex Object Parametric Analyzer and Sorter (COPAS) technology. Statistical analysis revealed that the liquid shaken culture consisted of two populations of microcolonies that differ by 90 μm in diameter. The population of small microcolonies of strains expressing GFP from the glaA or faeA promoter comprised 39% and 25% of the culture, respectively. Two populations of microcolonies could also be distinguished when the expression of GFP in these strains was analyzed. The population expressing a low level of GFP consisted of 68% and 44% of the culture, respectively. We also show that mRNA accumulation is heterogeneous within microcolonies of A. niger. Central and peripheral parts of the mycelium were isolated with laser microdissection and pressure catapulting (LMPC), and RNA from these samples was used for quantitative PCR analysis. This analysis showed that the RNA content per hypha was about 45 times higher at the periphery than in the center of the microcolony. Our data imply that the protein production of A. niger can be improved in industrial fermentations by reducing the heterogeneity within the culture.
| S-EPMC3067247 | biostudies-literature
Project description:1. Quantitative determination of the anionic polymers present in the walls of Bacillus subtilis var. niger organisms undergoing transition, in a chemostat culture, from either Mg(2+)-limitation to PO(4) (3-)-limitation or K(+)-limitation to PO(4) (3-)-limitation showed that teichuronic acid synthesis started immediately the culture became PO(4) (3-)-limited and proceeded at a rate substantially faster than the rate of biomass synthesis. 2. Simultaneously, the cell-wall teichoic acid content diminished at a rate greater than that due to dilution by newly synthesized wall material, and fragments of teichoic acid and mucopeptide accumulated in the culture extracellular fluid. 3. Equally rapid reverse changes occurred when a PO(4) (3-)-limited B. subtilis var. niger culture was returned to being Mg(2+)-limited. 4. It is concluded that in this organism both teichoic acid and teichuronic acid syntheses are expressions of a single genotype, and a mechanism for the control of synthesis of both polymers is suggested. 5. These results are discussed with reference to the constantly changing environmental conditions that obtain in a batch culture and the variation in bacterial cell-wall composition that is reported to occur throughout the growth cycle.
| S-EPMC1187486 | biostudies-other
Project description:BackgroundSaccharomyces cerevisiae is the most relevant yeast species conducting the alcoholic fermentation that takes place during winemaking. Although the physiology of this model organism has been extensively studied, systematic quantitative physiology studies of this yeast under winemaking conditions are still scarce, thus limiting the understanding of fermentative metabolism of wine yeast strains and the systematic description, modelling and prediction of fermentation processes. In this study, we implemented and validated the use of chemostat cultures as a tool to simulate different stages of a standard wine fermentation, thereby allowing to implement metabolic flux analyses describing the sequence of metabolic states of S. cerevisae along the wine fermentation.ResultsChemostat cultures mimicking the different stages of standard wine fermentations of S. cerevisiae EC1118 were performed using a synthetic must and strict anaerobic conditions. The simulated stages corresponded to the onset of the exponential growth phase, late exponential growth phase and cells just entering stationary phase, at dilution rates of 0.27, 0.04, 0.007 h-1, respectively. Notably, measured substrate uptake and product formation rates at each steady state condition were generally within the range of corresponding conversion rates estimated during the different batch fermentation stages.Moreover, chemostat data were further used for metabolic flux analysis, where biomass composition data for each condition was considered in the stoichiometric model. Metabolic flux distributions were coherent with previous analyses based on batch cultivations data and the pseudo-steady state assumption.ConclusionsSteady state conditions obtained in chemostat cultures reflect the environmental conditions and physiological states of S. cerevisiae corresponding to the different growth stages of a typical batch wine fermentation, thereby showing the potential of this experimental approach to systematically study the effect of environmental relevant factors such as temperature, sugar concentration, C/N ratio or (micro) oxygenation on the fermentative metabolism of wine yeast strains.
| S-EPMC4070652 | biostudies-literature
Project description:The industrially important fungus Aspergillus niger feeds naturally on decomposing plant material, for which it is equipped with a range of enzyme systems. A significant proportion of plant material are lipids that might be available either as for energy storage or as membrane building blocks. With 63 potential lipase-encoding genes in its genome, A. niger has the tools to degrade these extracellular lipids. In contrast to polysaccharide-degrading enzyme networks not much is known about the signalling and regulatory processes that control lipase expression and activity in fungi both under laboratory and natural occurring conditions. A pulse of 1 mM of various oils was applied to four bioreactor-grown A. niger cultures to examine (i) whether A. niger responds at the level of gene transcription, (ii) at what time point this effect is detected most accurately, and (iii) whether differences between the response towards oils are observed. The triglyceride olive oil induces genes encoding peroxins and enzymes of fatty acid metabolism. A complex oil mixture extracted from wheat gluten, which is enriched for digalactosyl-diglycerides, induces genes encoding peroxins as well as enzymes of fatty acid metabolism, but with different expression profile when compared to olive oil. Pure digalactosyldiglyceride, a proxy for plant membrane lipids, does not trigger a transcriptional response. Keywords: time course; induction experiment
2009-09-01 | GSE14285 | GEO
Project description:A novel subfamily of putative intracellular invertase enzymes (glycoside hydrolase family 32) has previously been identified in fungal genomes. Here, we report phylogenetic, molecular, and biochemical characteristics of SucB, one of two novel intracellular invertases identified in Aspergillus niger. The sucB gene was expressed in Escherichia coli and an invertase-negative strain of Saccharomyces cerevisiae. Enzyme purified from E. coli lysate displayed a molecular mass of 75 kDa, judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Its optimum pH and temperature for sucrose hydrolysis were determined to be 5.0 and 37 to 40 degrees C, respectively. In addition to sucrose, the enzyme hydrolyzed 1-kestose, nystose, and raffinose but not inulin and levan. SucB produced 1-kestose and nystose from sucrose and 1-kestose, respectively. With nystose as a substrate, products up to a degree of polymerization of 4 were observed. SucB displayed typical Michaelis-Menten kinetics with substrate inhibition on sucrose (apparent K(m), K(i), and V(max) of 2.0 +/- 0.2 mM, 268.1 +/- 18.1 mM, and 6.6 +/- 0.2 mumol min(-1) mg(-1) of protein [total activity], respectively). At sucrose concentrations up to 400 mM, transfructosylation (FTF) activity contributed approximately 20 to 30% to total activity. At higher sucrose concentrations, FTF activity increased to up to 50% of total activity. Disruption of sucB in A. niger resulted in an earlier onset of sporulation on solid medium containing various carbon sources, whereas no alteration of growth in liquid culture medium was observed. SucB thus does not play an essential role in inulin or sucrose catabolism in A. niger but may be needed for the intracellular conversion of sucrose to fructose, glucose, and small oligosaccharides.
| S-EPMC1865650 | biostudies-literature
Project description:This work presents an exploration of submerged differentiation of the ubiquitous saprophyte and industrially important fungus, Aspergillus niger, in response to a limited availability of a sole carbon and energy source, maltose. In aspergilli and other mold fungi, asexual reproduction through formation of elaborate conidiogenic structures normally requires an aerial interface. This requirement is bypassed in submerged culture in response to severe nutrient limitation. Continuous cultures with cell retention (retentostat cultures) were applied to generate a fundamental physiological state, where the specific growth rate approaches zero, as the density of the cell population adapts to the supply of the limiting energy source. Temporal differentiation of mycelium structure and commitment to asexual reproduction were major phenomena, apparent on biochemical, morphological, physiological, and transcriptomic level. The severe substrate limitation had a rapid negative impact on cytoplasmic processes, and promoted endo- and exogenous nutrient mobilization, and hyphal compartmentalisation. The first conidiogenic structures appeared after one day with little additional differentiation until Day 4 to 6, where a transition to full commitment to reproductive growth took place. Submerged conidiation in A. niger involved transcriptional regulation of homologs of the regulatory pathway, centered around the Bristle gene (brlA), and structural genes previously described in other aspergilli. Comparison of transcriptomes, revealed a number of co-regulated gene clusters, which appear to encode secondary metabolite biosynthetic potential. We discuss the concept of maintenance energy in the context of differentiation, a possible physiological trigger for sporulation and the special physiological adaptations of the starved mycelium. We also present a simple and efficient method for in situ retention of filamentous organisms.
2010-07-07 | GSE21752 | GEO
Project description:Dry-cured meat products are worldwide food with high-salt content, and filamentous fungi are beneficial to the maturation process. However, some salt-tolerant strains of Aspergillus and Penicillium produce ochratoxin A (OTA) on these products and thus threaten food safety. In our study, proteomic analysis was performed to reveal the mechanism of adaptability to high-salt environment by Aspergillus ochraceus. Twenty g/L and 70 g/L NaCl substrates were used to provide medium- and high-NaCl content environments, respectively. The NaCl addition could induce fungal growth, but only 20 g/L NaCl addition could induce spore production while 70 g/L repressed it. Proteomics analysis identified 2646 proteins in A. ochraceus fc-1, of which 237 and 251 were differentially expressed with 20 g/L and 70 g/L NaCl addition, respectively. Potential factors affecting fungal growth and development were identified by GO and KEGG analyses of biological process, cellular component, and molecular function terms. The results revealed that ergosterol synthesis pathway was significantly upregulated with 20 g/L and 70 g/L NaCl addition. However, fungal growth and development including OTA production were complex processes associated with many factors including nutrient uptake, cell membrane integrity, cell cycle, energy metabolism, intracellular redox homeostasis, protein synthesis and processing, autophagy, and secondary metabolism. Reactive oxygen species may be an important window to understand the mechanism that medium-salt content was conducive to intracellular signal transduction while high-salt content caused oxidative stress. The findings would help to improve the processes and storage conditions of dry-cured meat products.
| S-EPMC7828334 | biostudies-literature
Project description:Recent studies indicate an important role of bacteriophages for successful fecal microbiota transplantation (FMT). However, wider clinical applications of FMT are hampered by to donor variability and concerns of infection risks by bacteria and human viruses. To overcome these challenges, mouse cecal and human fecal material were propagated in a chemostat fermentation setup supporting multiplication of bacteria, and phages, while propagation of eukaryotic viruses will be prevented in the absence of eukaryotic host cells. The results showed decrease of the median relative abundance of viral contigs of classified eukaryotic viruses below 0.01%. The corresponding virome profiles showed dilution rate dependency, a reproducibility between biological replicates, and maintained high diversity regarding both the human and mouse inocula. This proof-of-concept cultivation approach may constitute the first step of developing novel therapeutic tools with high reproducibility and with low risk of infection from the donor material to target gut-related diseases.
| S-EPMC11298661 | biostudies-literature
Project description:Accurate, reliable and reproducible measurement of intracellular metabolite levels has become important for metabolic studies of microbial cell factories. A first critical step for metabolomic studies is the establishment of an adequate quenching and washing protocol, which ensures effective arrest of all metabolic activity and removal of extracellular metabolites, without causing leakage of metabolites from the cells. Five different procedures based on cold methanol quenching and cell separation by filtration were tested for metabolomics of Pichia pastoris regarding methanol content and temperature of the quenching solution as key parameters. Quantitative evaluation of these protocols was carried out through mass balance analysis, based on metabolite measurements in all sample fractions, those are whole broth, quenched and washed cells, culture filtrate and quenching and washing solution. Finally, the optimal method was used to study the time profiles of free amino acid and central carbon metabolism intermediates in glucose-limited chemostat cultures. Acceptable recoveries (>90%) were obtained for all quenching procedures tested. However, quenching at -27°C in 60% v/v methanol performed slightly better in terms of leakage minimization. We could demonstrate that five residence times under glucose limitation are enough to reach stable intracellular metabolite pools. Moreover, when comparing P. pastoris and S. cerevisiae metabolomes, under the same cultivation conditions, similar metabolite fingerprints were found in both yeasts, except for the lower glycolysis, where the levels of these metabolites in P. pastoris suggested an enzymatic capacity limitation in that part of the metabolism. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0308-1) contains supplementary material, which is available to authorized users.
| S-EPMC3291848 | biostudies-literature