Bernd Nörtemann

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 .

Influence of physiological conditions on EDTA degradation

Aerobic biodegradation of the chelating agent EDTA by a mixed bacterial culture was investigated. Bacterial growth and degradation of the substrate required the presence of sufficient metal ions in the culture fluid. Uncomplexed EDTA interacted negatively with the cell walls of the bacteria and completely inhibited bacterial growth, whereas Mg(II)/Ca(II)-EDTA was degraded up to an initial concentration of 4.7 g/l. Therefore, concentrations of metal ions must be stoichiometric to that of EDTA or higher. Specific degradation rates ranged between 120 mg EDTA g−1 (cell dry weight) h−1 and 285 mg EDTA g−1 h−1. In contrast, complexes with high thermodynamic stability constants such as Fe(III)-EDTA remained as inert compounds in the solution. Specific growth rates of the mixed culture were found to vary between 0.03 h−1 and 0.07 h−1, which could be explained by population dynamics within the synergistic mixed community. Growth was significantly accelerated by the addition of vitamins.

Metabolism of EDTA and its metal chelates by whole cells and cell-free extracts of strain BNC1

Abstract The influence of metal ions on the metabolism of ethylenediaminetetraacetate (EDTA) by whole cells and cell-free extracts of strain BNC1 was investigated. Metal-EDTA chelates with thermodynamic stability constants below 1012 were readily mineralized by whole cells with maximum specific turnover rates of 15 (MnEDTA) to 20 (Ca-, Mg-, and BaEDTA) μmol g protein−1 min−1. With the exception of ZnEDTA, chelates with stability constants greater than 1012 were not oxidized at a significant rate. However, it was shown for Fe(III)EDTA that even strong complexes can be degraded after pretreatment by addition of calcium and magnesium salts in the pH range 9–11. The range of EDTA chelates converted by cell-free extracts of strain BNC1 did not depend on their thermodynamic stabilities. The EDTA chelates of Ba2+, Co2+, Mg2+, Mn2+, and Zn2+ were oxidized whereas Ca-, Cd-, Cu-, Fe-, Pb-, and SnEDTA were not. The first catabolic enzyme appears to be an EDTA monooxygenase since it requires O2, NADH, and FMN for its activity and yields glyoxylate and ethylenediaminetriacetate as products. The latter is further degraded via N,N′-ethylenediaminediacetate. The maximum specific turnover rate with MgEDTA, the favoured EDTA species, was 50–130 μmol g protein−1 min−1, and the Km value was 120 μmol/l (Ks for whole cells = 8 μmol/l). Whole cells as well as cell-free extracts of strain BNC1 also converted several structural analogues of EDTA.

Morphology of Filamentous Fungi: Linking Cellular Biology to Process Engineering Using Aspergillus niger

In various biotechnological processes, filamentous fungi, e.g. Aspergillus niger, are widely applied for the production of high value-added products due to their secretion efficiency. There is, however, a tangled relationship between the morphology of these microorganisms, the transport phenomena and the related productivity. The morphological characteristics vary between freely dispersed mycelia and distinct pellets of aggregated biomass. Hence, advantages and disadvantages for mycel or pellet cultivation have to be balanced out carefully. Due to this inadequate understanding of morphogenesis of filamentous microorganisms, fungal morphology, along with reproducibility of inocula of the same quality, is often a bottleneck of productivity in industrial production. To obtain an optimisation of the production process it is of great importance to gain a better understanding of the molecular and cell biology of these microorganisms as well as the approaches in biochemical engineering and particle technique, in particular to characterise the interactions between the growth conditions, cell morphology, spore-hyphae-interactions and product formation. Advances in particle and image analysis techniques as well as micromechanical devices and their applications to fungal cultivations have made available quantitative morphological data on filamentous cells. This chapter provides the ambitious aspects of this line of action, focussing on the control and characterisation of the morphology, the transport gradients and the approaches to understand the metabolism of filamentous fungi. Based on these data, bottlenecks in the morphogenesis of A. niger within the complex production pathways from gene to product should be identified and this may improve the production yield.

BIOLOGICAL DEGRADATION OF EDTA: REACTION KINETICS AND TECHNICAL APPROACH

The microbial mineralization of EDTA in waste water by a mixed culture was studied with suspended and immobilized cells. Efficient degradation of EDTA could be achieved, though the chelator is stated not to be biodegradable. A complete set of kinetic parameters was determined that enables the modelling of EDTA degradation and, related to this, bacterial growth, ammonium release, maintenance requirement as well as oxygen uptake. In order to obtain important technical scale-up parameters, the microorganisms were immobilized on different carrier particles and employed in continuously operated three-phase airlift-loop reactors. The reactors could be operated at a dilution rate up to D=1·2 h−1 (D≪μmax) that, at an EDTA concentration of 450 mg dm−3, led to EDTA degradation rates up to 12·8 kg m−3 day−1. The extent of EDTA deg-radation remained constant at 95–99% with increasing values of D. Achieved kinetic parameters of the biofilm systems were compared with those which were obtained from experiments with suspended cells.

METABOLIC FLUX ANALYSIS APPLICATIONS TO ASPERGILLUS NIGER AB1.13 CULTIVATIONS

Abstract A stoichiometric model was developed for Aspergillus niger AB1.13. Metabolic flux analysis (MFA) revealed that the pH of the medium does not affect the flux distribution of the central carbon metabolism. However, one exception could be observed regarding the oxaloacetate hydrolase ( OAAH ) reaction. During D-glucose and D-xylose feeding, a 6-fold and 2-fold increase in flux distribution was observed with increasing pH (ΔpH 2.5), respectively. Differences in flux with D-glucose and D-xylose as substrate were reflected in a higher demand of NADPH during D-xylose consumption. Additionally, a comparison between metabolic models revealed that the ribulose-5-phosphate epimerase ( RPE ) might not be expressed during D-xylose consumption.