Effect of different carbon sources on the production of succinic acid using metabolically engineered Escherichia coli

Succinic acid (SA) is an important platform molecule in the synthesis of a number of commodity and specialty chemicals. In the present work, dual-phase batch fermentations with the E. coli strain AFP184 were performed using a medium suited for large-scale industrial production of SA. The ability of the strain to ferment different sugars was investigated. The sugars studied were sucrose, glucose, fructose, xylose, and equal mixtures of glucose and fructose and glucose and xylose at a total initial sugar concentration of 100 g L-1. AFP184 was able to utilize all sugars and sugar combinations except sucrose for biomass generation and succinate production. For sucrose as a substrate no succinic acid was produced and none of the sucrose was metabolized. The succinic acid yield from glucose (0.83 g succinic acid per gram glucose consumed anaerobically) was higher than the yield from fructose (0.66 g g-1). When using xylose as a carbon source, a yield of 0.50 g g-1 was obtained. In the mixed-sugar fermentations no catabolite repression was detected. Mixtures of glucose and xylose resulted in higher yields (0.60 g g-1) than use of xylose alone. Fermenting glucose mixed with fructose gave a lower yield (0.58 g g-1) than fructose used as the sole carbon source. The reason is an increased pyruvate production. The pyruvate concentration decreased later in the fermentation. Final succinic acid concentrations were in the range of 25-40 g L-1. Acetic and pyruvic acid were the only other products detected and accumulated to concentrations of 2.7-6.7 and 0-2.7 g L-1. Production of succinic acid decreased when organic acid concentrations reached approximately 30 g L-1. This study demonstrates that E. coli strain AFP184 is able to produce succinic acid in a low cost medium from a variety of sugars with only small amounts of byproducts formed.     

Development of both colorimetric and fluorescence heparinase activity assays using fondaparinux as substrate

Because tumors and other diseases are characterized by increased heparanase levels, human heparanase is a promising drug target and diagnostic marker. Therefore, methods are needed to determine heparanase activity and to examine potential inhibitors. Because of substrate comparability, we used the bacterial enzyme heparinase II (heparinase) for the assay development. Usually the substrate of heparanase assays is heparan sulfate, which has several disadvantages. Because of that, we used fondaparinux, which is being cleaved by both heparanase and heparinase. Two concepts to detect its degradation were examined: measurement of anti-factor Xa activity of fondaparinux and its direct quantification with the fluorescent sensor polymer-H. Using fondaparinux as substrate, the anti-factor Xa assay was shsown to be appropriate to determine heparinase activity. The detection with polymer-H was easier and even faster to perform. Linearity was given with fondaparinux as well as heparan sulfate, and heparin as substrates, but fondaparinux turned out to be most suitable. By modifications (incubation time, fondaparinux concentration, and polymer-H concentration), the limit of quantification and the linear range can be adapted to the respective requirements. In conclusion, a simple, accurate, and robust heparinase assay was developed. It is suitable for heparinase quality control and testing heparinase inhibitors and could be adapted to heparanase.     

Affinity pulldown of γ‐secretase and associated proteins from human and rat brain

γ‐Secretase is a transmembrane protease complex responsible for the processing of a multitude of type 1 transmembrane proteins, including amyloid precursor protein (APP) and Notch. A functional complex is dependent on the assembly of four proteins: presenilin (PS), nicastrin, Aph‐1 and Pen‐2. Little is known about how the substrates are selected by γ‐secretase, but it has been suggested that γ‐secretase associated proteins (GSAPs) could be of importance. For instance, it was recently reported from studies in cell lines that TMP21, a transmembrane protein involved in trafficking, binds to γ‐secretase and regulates the processing of APP‐derived substrates without affecting Notch cleavage. Here, we present an efficient and selective method for purification and analysis of γ‐secretase and GSAPs. Microsomal membranes were prepared from rat or human brain and incubated with a γ‐secretase inhibitor coupled to biotin via a long linker and a S‐S bridge. After pulldown using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin. The tryptic peptides were subjected to LC‐MS/MS analysis, and proteins were identified by sequence data from MS/MS spectra. All of the known γ‐secretase components were identified. Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to γ‐secretase in rat brain. We suggest that the present method can be used for further studies on the composition of the γ‐secretase complex.     

Genetic engineering of the major timothy grass pollen allergen, Phl p 6, to reduce allergenic activity and preserve immunogenicity

On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1-33, 1-57, and 31-110 of the major timothy grass pollen allergen Phl p 6 aa 1-110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical alpha-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the alpha helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31-110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31-110 inhibited patients' IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.     

The ultrastructure of chemolithoautotrophic Gallionella ferruginea and Thiobacillus ferrooxidans as revealed by chemical fixation and freeze-etching

By using sodium thioglycolate to dissolve the high amount of excreted stalk material in axenic cultures of the chemolithoautotrophic iron bacterium Gallionella ferruginea, the ultrastructure of Gallionella cells from pure cell suspensions could be studied without any loss of viability or disturbance by dense ferric stalk fibers, and compared with Thiobacillus ferrooxidans, also grown chemolithoautotrophically with ferrous iron as energy source. Both organisms were chemically fixed or freeze-etched. Particular structural differences between these iron-bacteria could be ascertained. G. ferruginea possesses intracytoplasmic membranes and soluble d-ribulose-1,5-bisphosphate-carboxylase, whereas T. ferrooxidans contains carboxysomes but no intracytoplasmic membranes; Gallionella forms poly--hydroxybutyrate and glycogen as storage material; T. ferrooxidans produces only glycogen. Both organisms also differ from each other with respect to the freeze fracture behaviour of the cell envelope layers. Whereas the cells of T. ferrooxidans exhibit a characteristic double cleavage, exposing the plasmic fracture face and exoplasmic fracture face of the outer membrane and cytoplasmic membrane, the exceptionally thin multilayered cell envelope of G. ferruginea revealed a particularly intimate association between the layers, resulting in a visualisation of the supramolecular organisation of only the inner fracture face of the cytoplasmic membrane. The results are discussed predominantly in relation to the extremely distinct environments of both organisms.     

Determination of glucose metabolites in stored erythrocytes and in erythrocytes from patients with thalassemia by analytical isotachophoresis

Glycolysis is for some cells, such as erythrocytes, neutrophil granulocytes and many cancer cells, the only or most important source of energy (ATP) production. Based on previous studies we developed an isotachophoretic (ITP) method which allows, in principle, the simultaneous determination of all metabolites of glycolysis. Since glucose metabolites are small anions, mobility of some of them may overlap in isotachophoresis and, therefore, partial mixed zones are generated. By variation of the leading/terminating system, however, it is possible to separate the compounds of interest. In this communication, we describe a method for analysis of glucose metabolites in erythrocytes from healthy donors during storage in blood bags, and from patients with thalassemia, with special respect to intracellular 2,3 bisphosphoglycerate, lactate and ATP/ADP. The well known characteristic changes of glycolysis in erythrocytes during blood storage and in erythrocytes from thalassemia patients, which are often analysed by separate enzymatic assays, could be confirmed with this isotachophoretic procedure. The method is currently adapted for analysis of glycolysis in neutrophil granulocytes and cancer cells which requires some modifications of sample preparation and performance of the isotachophoretic analysis.     

Inherent high correlation of individual motility enhances population dispersal in a heterotrophic, planktonic protist

Quantitative linkages between individual organism movements and the resulting population distributions are fundamental to understanding a wide range of ecological processes, including rates of reproduction, consumption, and mortality, as well as the spread of diseases and invasions. Typically, quantitative data are collected on either movement behaviors or population distributions, rarely both. This study combines empirical observations and model simulations to gain a mechanistic understanding and predictive ability of the linkages between both individual movement behaviors and population distributions of a single-celled planktonic herbivore. In the laboratory, microscopic 3D movements and macroscopic population distributions were simultaneously quantified in a 1L tank, using automated video- and image-analysis routines. The vertical velocity component of cell movements was extracted from the empirical data and used to motivate a series of correlated random walk models that predicted population distributions. Validation of the model predictions with empirical data was essential to distinguish amongst a number of theoretically plausible model formulations. All model predictions captured the essence of the population redistribution (mean upward drift) but only models assuming long correlation times (minute), captured the variance in population distribution. Models assuming correlation times of 8 minutes predicted the least deviation from the empirical observations. Autocorrelation analysis of the empirical data failed to identify a de-correlation time in the up to 30-second-long swimming trajectories. These minute-scale estimates are considerably greater than previous estimates of second-scale correlation times. Considerable cell-to-cell variation and behavioral heterogeneity were critical to these results. Strongly correlated random walkers were predicted to have significantly greater dispersal distances and more rapid encounters with remote targets (e.g. resource patches, predators) than weakly correlated random walkers. The tendency to disperse rapidly in the absence of aggregative stimuli has important ramifications for the ecology and biogeography of planktonic organisms that perform this kind of random walk.