alpha-Amylase production in high cell density submerged cultivation of Aspergillus oryzae and A. nidulans.

The effect of biomass concentration on the formation of Aspergillus oryzae alpha-amylase during submerged cultivation with A. oryzae and recombinant A. nidulans strains has been investigated.

Dynamic responses of Pseudomonas fluorescens DF57 to nitrogen or carbon source addition.

Cultures of Pseudomonas fluorescens DF57 were grown on different carbon and nitrogen sources. Glucose, succinate and acetate were used as carbon source and pulsed to an aerobic steady-state cultivation of P. fluorescens DF57 at D = 0.

Quantitative analysis of Penicillium chrysogenum Wis54-1255 transformants overexpressing the penicillin biosynthetic genes.

The low penicillin-producing, single gene copy strain Wis54-1255 was used to study the effect of overexpressing the penicillin biosynthetic genes in Penicillium chrysogenum. Transformants of Wis54-1255 were obtained with the amdS expression-cassette using the four combinations: pcbAB, pcbC, pcbC-penDE, and pcbAB-pcbC-penDE of the three penicillin biosynthetic genes. Transformants showing an increased penicillin production were investigated during steady-state continuous cultivations with glucose as the growth-limiting substrate.

Network identification and flux quantification in the central metabolism of Saccharomyces cerevisiae under different conditions of glucose repression.

The network structure and the metabolic fluxes in central carbon metabolism were characterized in aerobically grown cells of Saccharomyces cerevisiae. The cells were grown under both high and low glucose concentrations, i.e.

Increasing galactose consumption by Saccharomyces cerevisiae through metabolic engineering of the GAL gene regulatory network.

Increasing the flux through central carbon metabolism is difficult because of rigidity in regulatory structures, at both the genetic and the enzymatic levels. Here we describe metabolic engineering of a regulatory network to obtain a balanced increase in the activity of all the enzymes in the pathway, and ultimately, increasing metabolic flux through the pathway of interest. By manipulating the GAL gene regulatory network of Saccharomyces cerevisiae, which is a tightly regulated system, we produced prototroph mutant strains, which increased the flux through the galactose utilization pathway by eliminating three known negative regulators of the GAL system: Gal6, Gal80, and Mig1.

Simultaneous overexpression of enzymes of the lower part of glycolysis can enhance the fermentative capacity of Saccharomyces cerevisiae.

Recombinant S. cerevisiae strains, with elevated levels of the enzymes of lower glycolysis (glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase, phosphoglycerate kinase, enolase, pyruvate kinase, pyruvate decarboxylase and alcohol dehydrogenase) were physiologically characterized. During growth on glucose the enzyme levels in the recombinant strains (YHM4 and YHM7) were 1.

Antisense silencing of the creA gene in Aspergillus nidulans.

Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in Aspergillus nidulans resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect was approximately one-half of that achieved in a null creA mutant. Unlike results for that mutant, however, growth parameters and colony morphology in the antisense transformants were not affected.

Metabolic characterization of high- and low-yielding strains of Penicillium chrysogenum.

A recently developed method for analyzing metabolic networks using 13C-labels was employed for investigating the metabolism of a high- and a low-yielding strain of Penicillium chrysogenum. Under penicillin-producing conditions, the flux through the pentose phosphate (PP) pathway in the high- and the low-yielding strains was estimated to 70 and 66, respectively. When the high-yielding strain was cultivated in a medium without the penicillin side chain precursor, phenoxyacetic acid, the PP pathway flux was estimated as 71.

Synthesis and posttranslational regulation of pyruvate formate-lyase in Lactococcus lactis.

The enzyme pyruvate formate-lyase (PFL) from Lactococcus lactis was produced in Escherichia coli and purified to obtain anti-PFL antibodies that were shown to be specific for L. lactis PFL. It was demonstrated that activated L.

Isotopomer analysis using GC-MS.

Knowledge of the complete isotopomer distribution represents the ultimate amount of information on the labeling pattern of a metabolite. One technique for measuring the isotopomer distributions is the analysis of the multiplet intensities arising from the 13C-13C couplings in NMR spectroscopy. While this technique has proven to be very valuable in the elucidation of labeling patterns of C2 and C3 units of various amino acids, fragments larger than C3 are very difficult to measure.

Construction and characterization of an oxalic acid nonproducing strain of Aspergillus niger.

Aspergillus niger produces oxalic acid as a by-product which causes problems with downstream processing of industrial enzymes. To overcome this problem the oah gene encoding oxaloacetate hydrolase (EC 3.7.

Quantification of intracellular metabolic fluxes from fractional enrichment and 13C-13C coupling constraints on the isotopomer distribution in labeled biomass components.

A method for the quantification of intracellular metabolic flux distributions from steady-state mass balance constraints and from the constraints posed by the measured 13C labeling state of biomass components is presented. Two-dimensional NMR spectroscopy is used to analyze the labeling state of cell protein hydrolysate and cell wall components. No separation of the biomass hydrolysate is required to measure the degree of 13C-13C coupling and the fractional 13C enrichment in various carbon atom positions.

The role of metabolic engineering in the improvement of Saccharomyces cerevisiae: utilization of industrial media.

Metabolic engineering has become a very important approach to strain improvement in parallel with classical strain development. Although Saccharomyces cerevisiae has been domesticated for ethanol and bread production, there are still some fundamental problems associated with its industrial use. The industrially used carbon sources often consist of a sugar mixture, and due to glucose repression these sugars are utilized sequentially, resulting in prolonged production time.

Metabolic network analysis of Penicillium chrysogenum using (13)C-labeled glucose.

Using (13)C-labeled glucose fed to a penicillin-overproducing strain of Penicillium chrysogenum, the intracellular fluxes were quantified, and the presence of two new pathways, not previously described in this organism, is suggested. Thus, glycine was synthesized not only by serine hydroxymethyltransferase, but also by threonine aldolase. The formation of cytosolic acetyl-CoA was found to be synthesized both via the citrate lyase-catalyzed reaction and by degradation of the penicillin side-chain precursor, phenoxyacetic acid.

Cloning and characterization of oah, the gene encoding oxaloacetate hydrolase in Aspergillus niger.

The enzyme oxaloacetate hydrolase (EC 3.7.1.

The influence of nitrogen sources on the alpha-amylase productivity of Aspergillus oryzae in continuous cultures.

The influence of the nitrogen source on the alpha-amylase productivity of Aspergillus oryzae was quantified in continuous cultivations. Both inorganic and complex nitrogen sources were investigated and glucose was used as the carbon and energy sources. For production of alpha-amylase, nitrate was shown to be inferior to ammonia as a nitrogen source.

Physiological studies in aerobic batch cultivations of Saccharomyces cerevisiae strains harboring the MEL1 gene.

Physiological studies of Saccharomyces cerevisiae strains harboring the MEL1 gene were carried out in aerobic batch cultivations on glucose-galactose mixtures and on the disaccharide melibiose, which is hydrolyzed by the enzyme melibiase (Mel1, EC 3.2.1.

Metabolic engineering of Saccharomyces cerevisiae.

Comprehensive knowledge regarding Saccharomyces cerevisiae has accumulated over time, and today S. cerevisiae serves as a widley used biotechnological production organism as well as a eukaryotic model system. The high transformation efficiency, in addition to the availability of the complete yeast genome sequence, has facilitated genetic manipulation of this microorganism, and new approaches are constantly being taken to metabolicially engineer this organism in order to suit specific needs.

Biosynthetic studies of the glycopeptide teicoplanin by (1)H and (13)C NMR.

The biosynthesis of the glycopeptide antibiotic teicoplanin was studied by growing a teicoplanin producing strain of Actinoplanes teichomyceticus (ATCC 31121) on glucose containing either 34.0% [1-(13)C]glucose or 9.7% [U-(13)C]glucose.

Role of substrate concentration in mitosis and hyphal extension of Aspergillus.

The filamentous fungi Aspergillus oryzae and A. niger grow by apical extension of multinucleate hyphae that are subdivided into compartments by cross-walls called septa. Submerged cultivation, image analysis, and fluorescence microscopy were used to study the role of the carbon source on mitosis and hyphal extension in these fungi.

Genetically structured modeling of protein production in filamentous fungi.

A general framework for a genetically structured model is presented. The framework allows description of the interactions in a system of regulatory and structural genes. The model assumes equilibrium kinetics for the binding of regulatory proteins to the promoter regions of the genes and includes the possible activation of proteins following their synthesis.

Quantitative analysis of metabolic fluxes in Escherichia coli, using two-dimensional NMR spectroscopy and complete isotopomer models.

Complete isotopomer models that simulate distribution of label in 13C tracer experiments are applied to the quantification of metabolic fluxes in the primary carbon metabolism of E. coli under aerobic and anaerobic conditions. The concept of isotopomer mapping matrices (IMMs) is used to simplify the formulation of isotopomer mass balances by expressing all isotopomer mass balances of a metabolite pool in a single matrix equation.

Kinetics of alpha-amylase secretion in Aspergillus oryzae.

Pulse and pulse-chase experiments have been performed to study L-[(35)S] methionine incorporation and protein secretion kinetics in Aspergillus oryzae. Pulse experiments confirmed the mechanism of methionine uptake reported previously for Penicillium chrysogenum (Benko et al., 1967).

Structural and transcriptional analysis of the pyrABCN, pyrD and pyrF genes in Aspergillus nidulans and the evolutionary origin of fungal dihydroorotases.

The six biochemical steps of the de novo pyrimidine biosynthesis pathway are conserved in all known organisms. However, in animals and fungi, unlike prokaryotes, at least the first two activities are grouped on a multifunctional enzyme. Here, we report cloning, mapping and transcriptional characterization of some pyrimidine biosynthesis genes in the filamentous fungus Aspergillus nidulans.

Study of the glucoamylase promoter in Aspergillus niger using green fluorescent protein.

An Aspergillus niger strain expressing a red-shifted green fluorescent protein (GFP) in the cytoplasm under the control of the glucoamylase promoter (PgIaA) was characterized with respect to its physiology and morphology. Although xylose acted as a repressor carbon source during batch cultivations, PgIaA-driven GFP expression by the glucoamylase promoter could be demonstrated in xylose-limited continuous cultures. In these cultivations, the xylose concentration was therefore too low to cause repression.