Metabolic flux analysis using stoichiometric models for Aspergillus niger: comparison under glucoamylase-producing and non-producing conditions.

Aspergillus niger AB1.13 cultures with glucoamylase production (with D-glucose as substrate) and without glucoamylase production (with D-xylose as substrate) were characterized by metabolic flux analysis. Two comprehensive metabolic models for d-glucose- as well as for D-xylose-consumption were used to quantify and compare the metabolic fluxes through the central pathways of carbon metabolism at different pH-values. The models consist of the most relevant metabolic pathways for A. niger including glycolysis, pentose-phosphate pathway, citrate cycle, energy metabolism and anaplerotic reactions comprising two intracellular compartments, the cytoplasm and mitochondrion. When D-xylose was used as the sole carbon source, the relative flux of the substrate through the oxidative pentose-phosphate pathway (PPP) via G6P-dehydrogenase was unaffected by the pH-value of the culture medium. About 30% of D-xylose consumed was routed through the oxidative PPP. In contrast, the flux of D-glucose (i.e., under glucoamylase-producing conditions) through the oxidative PPP was remarkably higher and, in addition was significantly affected by the pH-value of the culture medium (40% at pH 5.5, 56% at pH 3.7, respectively). Summarizing, the flux through the PPP under glucoamylase producing conditions was 30-90% higher than for non-producing conditions.

JCat: a novel tool to adapt codon usage of a target gene to its potential expression host.

A novel method for the adaptation of target gene codon usage to most sequenced prokaryotes and selected eukaryotic gene expression hosts was developed to improve heterologous protein production. In contrast to existing tools, JCat (Java Codon Adaptation Tool) does not require the manual definition of highly expressed genes and is, therefore, a very rapid and easy method. Further options of JCat for codon adaptation include the avoidance of unwanted cleavage sites for restriction enzymes and Rho-independent transcription terminators. The output of JCat is both graphically and as Codon Adaptation Index (CAI) values given for the pasted sequence and the newly adapted sequence. Additionally, a list of genes in FASTA-format can be uploaded to calculate CAI values. In one example, all genes of the genome of Caenorhabditis elegans were adapted to Escherichia coli codon usage and further optimized to avoid commonly used restriction sites. In a second example, the Pseudomonas aeruginosa exbD gene codon usage was adapted to E.coli codon usage with parallel avoidance of the same restriction sites. For both, the degree of introduced changes was documented and evaluated. JCat is integrated into the PRODORIC database that hosts all required information on the various organisms to fulfill the requested calculations. JCat is freely accessible at http://www.prodoric.de/JCat.

Influence of growth conditions on biofilm development and mass transfer at the bulk/biofilm interface.

In a long-term study on heterotrophic biofilms in tube reactors, this investigation focused on mass transfer at the bulk/biofilm interface, biofilm density and substrate conversion rates. Several biofilms were cultivated under different substrate and hydrodynamic conditions. Oxygen concentration profiles were measured with microelectrodes in the biofilm and in the boundary layer directly in the biofilm tube reactors. The thickness of the concentration boundary layer was found to depend on the surface structure of the biofilm. The hydrodynamic conditions and the substrate load during the growth phase of the biofilm in biofilm systems are two key parameters that influence the biofilm growth, particularly the structure, density and thickness. The measured substrate conversion rates, biofilm densities and the boundary layer thickness were used to formulate an equation for the mass transfer in biofilm tube reactors.

Characterization and control of fungal morphology for improved production performance in biotechnology.

Filamentous fungi have been widely applied in industrial biotechnology for many decades. In submerged culture processes, they typically exhibit a complex morphological life cycle that is related to production performance--a link that is of high interest for process optimization. The fungal forms can vary from dense spherical pellets to viscous mycelia. The resulting morphology has been shown to be influenced strongly by process parameters, including power input through stirring and aeration, mass transfer characteristics, pH value, osmolality and the presence of solid micro-particles. The surface properties of fungal spores and hyphae also play a role. Due to their high industrial relevance, the past years have seen a substantial development of tools and techniques to characterize the growth of fungi and obtain quantitative estimates on their morphological properties. Based on the novel insights available from such studies, more recent studies have been aimed at the precise control of morphology, i.e., morphology engineering, to produce superior bio-processes with filamentous fungi.

Production of epoxide hydrolases in batch fermentations of Botryosphaeria rhodina.

The filamentous fungus Botryosphaeria rhodina (ATCC 9055) was investigated related to its ability for epoxide hydrolase (EH) production. Epoxide hydrolase activity is located at two different sites of the cells. The larger part is present in the cytosol (70%), while the smaller part is associated to membranes (30%). In media optimization experiments, an activity of 3.5 U/gDW for aromatic epoxide hydrolysis of para-nitro-styrene oxide (pNSO) could be obtained. Activity increased by 30% when pNSO was added to the culture during exponential growth. An increase of enzyme activity up to 6 U/gDW was achieved during batch-fermentations in a bioreactor with 2.7 l working volume. Evaluation of fermentations with 30 l working volume revealed a relation of oxygen uptake rate to EH expression. Oxygen limitation resulted in a decreased EH activity. Parameter estimation by the linearization method of Hanes yielded Km values of 2.54 and 1.00 mM for the substrates S-pNSO and R-pNSO, respectively. vmax was 3.4 times higher when using R-pNSO. A protein purification strategy leading to a 47-fold increase in specific activity (940 U/mgProtein) was developed as a first step to investigate molecular and structural characteristics of the EH.

Structural characterization of the exopolysaccharide PS-EDIV from Sphingomonas pituitosa strain DSM 13101.

Members of the bacterial genus Sphingomonas are known to produce highly viscous polysaccharides in solution. The exopolysaccharide PS-EDIV was produced by Sphingomonas pituitosa strain DSM 13101, purified using centrifugation, and precipitation and its structure was elucidated by 1D and 2D NMR techniques and chemical microderivatization combined with various mass spectrometric techniques. The following repeating unit of the polysaccharide could be identified: [Formula: see text]. In addition, the polysaccharide also contains acetyl and glyceryl groups whose exact positions were not determined. PS-EDIV is similar in structure to a known exopolysaccharide but differs in being the first bacterial polysaccharide in which two different glucuronic acids are combined. It caused a high viscosity of the culture broth after cultivation for 48 h, although a gelation was not observed.

Structure and shear strength of microbial biofilms as determined with confocal laser scanning microscopy and fluid dynamic gauging using a novel rotating disc biofilm reactor.

The cohesive strength of microbial biofilms cultivated on a rotating disc has been measured using fluid dynamic gauging (FDG). The thickness of heterotrophic mixed culture biofilms was found to depend on substrate concentration and shear force at the biofilm surface during the cultivation. For high substrate concentrations and low shear forces the biofilm thickness increased to several 100 microm within 7 days. Low substrate concentration and higher shear forces yielded thin biofilms of about 100 microm thickness. Independent from cultivation conditions and thickness of the biofilms their cohesive strength ranged between 6.0 and 7.7 N m(-2). The ratio between cohesive strength measured with FDG and shear forces applied during biofilm cultivation have ranged from 200 to 1,100. Higher concentrations of iron in the cultivation media has a positive effect on the stability of the biofilms cultivated. By using the CLSM technique a stable base biofilm with a high amount of stained EPS glycoconjugates could be visualized after gauging. The thickness of the base biofilm was about 100 microm for all biofilms cultivated and was not removable under the applied shear conditions used during FDG.

Agitation effects on submerged growth and product formation of Aspergillus niger.

Product formation of mycelial organisms, like Aspergillus niger, is intimately connected with their morphology. Pellet morphology is often requested for product formation. Therefore, it is important to reveal the influence of the hydrodynamic conditions on the morphological development. In the present study, pellet morphology and glucoamylase formation were studied under different agitation intensities of A. niger AB1.13. For pellet formation inside the bioreactor, without the use of precultures, it is necessary to work at low energy dissipation rates. Biomass growth and glucoamylase activity were correlated with energy dissipation. Furthermore, product yield was analysed in dependence of pellet size and concentration. The present work shows that simple equations based on Monod-kinetics can describe growth and product formation, in general, also in mycelian organisms. All measured morphological data, like pellet concentration, as well as glucoamylase formation, strongly depend on the hydrodynamic conditions.