Modeling isotopomer distributions in biochemical networks using isotopomer mapping matrices.

Within the last decades NMR spectroscopy has undergone tremendous development and has become a powerful analytical tool for the investigation of intracellular flux distributions in biochemical networks using (13)C-labeled substrates. Not only are the experiments much easier to conduct than experiments employing radioactive tracer elements, but NMR spectroscopy also provides additional information on the labeling pattern of the metabolites. Whereas the maximum amount of information obtainable with (14)C-labeled substrates is the fractional enrichment in the individual carbon atom positions, NMR spectroscopy can also provide information on the degree of labeling at neighboring carbon atom positions by analyzing multiplet patterns in NMR spectra or using 2-dimensional NMR spectra.

Mycotoxin production and postharvest storage rot of ginger (Zingiber officinale) by Penicillium brevicompactum.

Twenty naturally infected ginger (Zingiber officinale) rhizomes displaying visible mold growth were examined to identify the fungi and to evaluate the presence of fungal secondary metabolites. Penicillium brevicompactum was the predominant species isolated from 85% of the samples. Mycophenolic acid was identified from corresponding tissue extracts.

Manipulation of malic enzyme in Saccharomyces cerevisiae for increasing NADPH production capacity aerobically in different cellular compartments.

The yeast Saccharomyces cerevisiae is an attractive cell factory, but in many cases there are constraints related with balancing the formation and consumption of redox cofactors. In this work, we studied the effect of having an additional source of NADPH in the cell. In order to do this, two strains were engineered by overexpression of malic enzyme.

Fractionation of whey proteins with high-capacity superparamagnetic ion-exchangers.

In this study we describe the design, preparation and testing of superparamagnetic anion-exchangers, and their use together with cation-exchangers in the fractionation of bovine whey proteins as a model study for high-gradient magnetic fishing. Adsorbents prepared by attachment of trimethyl amine to particles activated in sequential reactions with allyl bromide and N-bromosuccinimide yielded a maximum bovine serum albumin binding capacity of 156 mg g(-1) combined with a dissociation constant of 0.60 microM, whereas ion-exchangers created by linking polyethylene imine through superficial aldehydes bound up to 337 mg g(-1) with a dissociation constant of 0.

Diversity in metabolite production by Fusarium langsethiae, Fusarium poae, and Fusarium sporotrichioides.

The production of mycotoxins and other metabolites by 109 strains of Fusarium langsethiae, Fusarium poae, Fusarium sporotrichioides, and F. kyushuense was investigated independently in four laboratories by liquid or gas chromatography analyses of cultural extracts with UV diode array, electron capture, or mass spectrometric detection systems. From the compiled results, it was found that F.

Superparamagnetic adsorbents for high-gradient magnetic fishing of lectins out of legume extracts.

This work presents the development, testing, and application in high-gradient magnetic fishing of superparamagnetic supports for adsorption of lectins. Various approaches were examined to produce affinity, mixed mode, and hydrophobic charge induction type adsorbents. In clean monocomponent systems affinity supports created by direct attachment of glucose or maltose to amine-terminated iron oxide particles could bind concanavalin A at levels of up to approximately 280 mg g(-1) support with high affinity ( approximately 1 microM dissociation constants).

Use of genome-scale microbial models for metabolic engineering.

Metabolic engineering serves as an integrated approach to design new cell factories by providing rational design procedures and valuable mathematical and experimental tools. Mathematical models have an important role for phenotypic analysis, but can also be used for the design of optimal metabolic network structures. The major challenge for metabolic engineering in the post-genomic era is to broaden its design methodologies to incorporate genome-scale biological data.

Identification of the Entner-Doudoroff pathway in an antibiotic-producing actinomycete species.

The metabolic network of the central carbon metabolism represents the backbone of cellular metabolism and provides the precursors and cofactors required for synthesis of secondary metabolites. It is therefore pivotal to map the operating metabolic network in the central carbon metabolism in order to design metabolic engineering strategies towards construction of more efficient producers of specific metabolites. In this context, methods that allow rapid and reliable mapping of the central carbon metabolism are valuable.

Growth and enzyme production by three Penicillium species on monosaccharides.

The growth and preference for utilisation of various sugar by the Penicillium species Penicillium pinophilum IBT 4186, Penicillium persicinum IBT 13226 and Penicillium brasilianum IBT 20888 was studied in batch cultivations using various monosaccharides as carbon source, either alone or in mixtures. P. pinophilum IBT 4186 and P.

Screening genus Penicillium for producers of cellulolytic and xylanolytic enzymes.

For enzymatic hydrolysis of lignocellulosic material, cellulolytic enzymes from Trichoderma reesei are most commenly used, but, there is a need for more efficient enzyme cocktails. In this study, the production of cellulolytic and xylanolytic enzymes was investigated in 12 filamentous fungi from genus Penicillium and compared with that of T. reesei.

Genome-wide transcriptional response of a Saccharomyces cerevisiae strain with an altered redox metabolism.

The genome-wide transcriptional response of a Saccharomyces cerevisiae strain deleted in GDH1 that encodes a NADP(+)-dependent glutamate dehydrogenase was compared to a wild-type strain under anaerobic steady-state conditions. The GDH1-deleted strain has a significantly reduced NADPH requirement, and therefore, an altered redox metabolism. Identification of genes with significantly changed expression using a t-test and a Bonferroni correction yielded only 16 transcripts when accepting two false-positives, and 7 of these were Open Reading Frames (ORFs) with unknown function.

Protein kinase A is involved in the control of morphology and branching during aerobic growth of Mucor circinelloides.

The cAMP signal transduction pathway controls many processes in fungi. The Mucor circinelloides pkaR and pkaC genes, encoding the regulatory (PKAR) and catalytic (PKAC) subunit of the cAMP-dependent protein kinase A (PKA), have been cloned recently. Expression analysis during the dimorphic shift and colony morphology suggested a role for PKAR in the control of morphology and branching.

Pyruvate decarboxylases from the petite-negative yeast Saccharomyces kluyveri.

Saccharomyces kluyveri is a petite-negative yeast, which is less prone to form ethanol under aerobic conditions than is S. cerevisiae. The first reaction on the route from pyruvate to ethanol is catalysed by pyruvate decarboxylase, and the differences observed between S.

Characterization of the Mucor circinelloides life cycle by on-line image analysis.

Aims: The life cycle of the dimorphic fungus Mucor circinelloides was studied in a temperature-controlled flow-through cell, which constitutes an ideal tool when following the development of individual cells, with a view to understanding the growth and differentiation processes occurring in and between the different morphological forms of the organism.

Methods And Results: Mycelial growth and the transformation of hyphae into chains of arthrospores were characterized by image analysis techniques and described quantitatively. The influence of the nature (glucose and xylose) and concentration of the carbon source on specific growth rate and hyphal growth unit length were studied.

Reconstruction of the central carbon metabolism of Aspergillus niger.

The topology of central carbon metabolism of Aspergillus niger was identified and the metabolic network reconstructed, by integrating genomic, biochemical and physiological information available for this microorganism and other related fungi. The reconstructed network may serve as a valuable database for annotation of genes identified in future genome sequencing projects on aspergilli. Based on the metabolic reconstruction, a stoichiometric model was set up that includes 284 metabolites and 335 reactions, of which 268 represent biochemical conversions and 67 represent transport processes between the different intracellular compartments and between the cell and the extracellular medium.

Aerobic physiology of redox-engineered Saccharomyces cerevisiae strains modified in the ammonium assimilation for increased NADPH availability.

Recombinant strains altered in the ammonium assimilation pathways were constructed with the purpose of increasing NADPH availability. The NADPH-dependent glutamate dehydrogenase encoded by GDH1, which accounts for a major fraction of the NADPH consumption during growth on ammonium, was deleted, and alternative pathways for ammonium assimilation were overexpressed: GDH2 (NADH-consuming) or GLN1 and GLT1 (the GS-GOGAT system). The flux through the pentose phosphate pathway during aerobic growth on glucose decreased to about half that of the reference strain Saccharomyces cerevisiae CEN.

Large-scale evaluation of in silico gene deletions in Saccharomyces cerevisiae.

A large-scale in silico evaluation of gene deletions in Saccharomyces cerevisiae was conducted using a genome-scale reconstructed metabolic model. The effect of 599 single gene deletions on cell viability was simulated in silico and compared to published experimental results. In 526 cases (87.

Solid-supported enzymatic synthesis of pectic oligogalacturonides and their analysis by MALDI-TOF mass spectrometry.

Solid-phase biosynthetic reactions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF), was used to gain insight into the biosynthesis of pectin oligomers. Sepharose supports bearing long pectic oligogalacturonides (OGAs) anchored through a disulfide-containing cleavable linker, were prepared. The OGAs (degrees of polymerization of 13 and 14) were efficiently immobilized through the reducing end via formation of an oxime linkage.

Branching is coordinated with mitosis in growing hyphae of Aspergillus nidulans.

Filamentous fungi like Aspergillus nidulans can effectively colonize their surroundings by the formation of new branches along the existing hyphae. While growth conditions, chemical perturbations, and mutations affecting branch formation have received great attention during the last decades, the mechanisms that regulates branching is still poorly understood. In this study, a possible relation between cell cycle progression and branching was studied by testing the effect of a nuclei distribution mutation, cell cycle inhibitors, and conditional cell cycle mutations in combination with tip-growth inhibitors and varying substrate concentrations on branch initiation.

Metabolic engineering of ammonium assimilation in xylose-fermenting Saccharomyces cerevisiae improves ethanol production.

Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase, and by overexpressing either GDH2, which encodes an NADH-dependent glutamate dehydrogenase, or GLT1 and GLN1, which encode the GS-GOGAT complex.

Metabolic control analysis of xylose catabolism in Aspergillus.

A kinetic model for xylose catabolism in Aspergillus is proposed. From a thermodynamic analysis it was found that the intermediate xylitol will accumulate during xylose catabolism. Use of the kinetic model allowed metabolic control analysis (MCA) of the xylose catabolic pathway to be carried out, and flux control was shown to be dependent on the metabolite levels.

Aerobic and anaerobic ethanol production by Mucor circinelloides during submerged growth.

The dimorphic organism Mucor circinelloides is currently being investigated as a potential host for heterologous protein production. The production of ethanol on pentose and hexose sugars was studied in submerged batch cultivations to further the general knowledge of Mucor physiology, with a view to the minimisation or elimination of the by-product ethanol for future process design. Large amounts of ethanol were produced during aerobic growth on glucose under non-oxygen limiting conditions, which is indicative of M.

Identification of in vivo enzyme activities in the cometabolism of glucose and acetate by Saccharomyces cerevisiae by using 13C-labeled substrates.

A detailed characterization of the central metabolic network of Saccharomyces cerevisiae CEN.PK 113-7D was carried out during cometabolism of different mixtures of glucose and acetate, using aerobic C-limited chemostats in which one of these two substrates was labeled with (13)C. To confirm the role of malic enzyme, an isogenic strain with the corresponding gene deleted was grown under the same conditions.