Konstantin Schneider

Metabolic flux analysis in eukaryotes.

Metabolic flux analysis (MFA) represents a powerful tool for systems biology research on eukaryotic cells. This review describes recent advances, the challenges as well as applications of metabolic flux analysis comprising fungi, mammalian cells and plants. While MFA is widely established and applied in microorganisms, it remains still a challenge to adapt these methods to eukaryotic cell systems having a higher complexity particularly concerning compartmentation or media composition. In fungi MFA was used in the past few years to analyze a variety of conditions and factors and their effects on cellular metabolism. In mammalian cells MFA was applied mainly in cell culture technology and in medical and toxicological research. (13)C metabolic studies on native whole plants are additionally challenging by the fact that CO(2) is usually the only carbon source.

Optical device for parallel online measurement of dissolved oxygen and pH in shake flask cultures

We describe a new device with parallel optical measurement of dissolved oxygen (DO) and pH in up to nine shake flasks applicable in any conventional shaking incubator. Measurement ranges are 0–500% of air saturation for oxygen and 5.5–8.5 for pH. It was used to characterize growth profiles of different l-lysine producing strains of Corynebacterium glutamicum, of Saccharomyces cerevisiae and of Escherichia coli. Cultures in unbaffled flasks were highly reproducible. Oxygen limitation was indicated online which is particularly important when cultivating fast growing cells as E. coli. C. glutamicum strains showed distinct characteristic patterns of DO and pH indicating biological events. During the cultivation of S. cerevisiae on glucose, fructose and galactose, oxygen uptake rate was determined using the predetermined value of kLa. pH measurement was used to determine the minimum buffer requirement for a culture of C. glutamicum.

Novel peptidoglycan-based diagnostic devices for detection of wound infection

Detection of wound infection is based on evaluation of the well-known signs of inflammation like rubor (redness), calor (heat), tumor (swelling), and dolor (pain) by medical doctors and/or time-consuming procedures requiring special machinery. There is currently no rapid diagnostic device available for the indication of wound infection, which would especially be helpful in home care of chronic ulcer patients. In this study, a new concept for a fast diagnostic tool for wound infection based on lysozyme and elastase triggered release of dye from a peptidoglycan matrix was investigated. The matrix consisted of alginate/agarose and peptidoglycan covalently labeled with Remazol brilliant blue. Lysozyme activity in postoperative wounds and decubitus wound fluids was significantly elevated upon infection (4830 ± 1848 U mL(-1)) compared to noninfected wounds (376 ± 240 U mL(-1)). Consequently, incubation of 8% (w/v) labeled agarose/peptidoglycan blend layers with infected wound fluid samples for 2 h at 37 °C resulted in a 4-fold higher amount of dye released than measured for noninfected wounds. For alginate/peptidoglycan beads, a 7-fold higher amount of dye was released in case of infected wound fluid samples compared to noninfected ones. Apart from lysozyme, proteases [i.e., gelatinase matrix metalloproteinase MMP-2 and MMP-9 and elastase] were detected in wound fluids (e.g., using Western blotting). When dosed in ratios typical for wounds, a slight synergistic effect was measured for peptidoglycan hydrolysis (i.e., dye release) between lysozyme and these proteases. Incubation of a double-layer system consisting of stained and nonstained peptidoglycan with infected wound fluids resulted in a color change from yellow to blue, thus allowing simple visual detection of wound infection.

Sensor materials for the detection of human neutrophil elastase and cathepsin G activity in wound fluid

Abstract:  Human neutrophil elastase (HNE) and cathepsin G (CatG) are involved in the pathogenesis of a number of inflammatory disorders. These serine proteinases are released by neutrophils and monocytes in case of infection. Wound infection is a severe complication regarding wound healing causing diagnostic and therapeutic problems. In this study we have shown the potential of HNE and CatG to be used as markers for early detection of infection. Significant differences in HNE and CatG levels in infected and non‐infected wound fluids were observed. Peptide substrates for these two enzymes were successfully immobilised on different surfaces, including collagen, modified collagen, polyamide polyesters and silica gel. HNE and CatG activities were monitored directly in wound fluid via hydrolysis of the chromogenic substrates. Infected wound fluids led to significant higher substrate hydrolysis compared with non‐infected ones. These different approaches could be used for the development of devices which are able to detect elevated enzyme activities before manifestation of infection directly on bandages. This would allow a timely intervention by medical doctors thus preventing severe infections.

Production of l-lysine on different silage juices using genetically engineered Corynebacterium glutamicum

Corynebacterium glutamicum, the best established industrial producer organism for lysine was genetically modified to allow the production of lysine on grass and corn silages. The resulting strain C. glutamicum lysC(fbr)dld(Psod)pyc(Psod)malE(Psod)fbp(Psod)gapX(Psod) was based on earlier work (Neuner and Heinzle, 2011). That mutant carries a point mutation in the aspartokinase (lysC) regulatory subunit gene as well as overexpression of D-lactate dehydrogenase (dld), pyruvate carboxylase (pyc) and malic enzyme (malE) using the strong Psod promoter. Here, we additionally overexpressed fructose 1,6-bisphosphatase (fbp) and glyceraldehyde 3-phosphate dehydrogenase (gapX) using the same promoter. The resulting strain grew readily on grass and corn silages with a specific growth rate of 0.35 h⁻¹ and lysine carbon yields of approximately 90 C-mmol (C-mol)⁻¹. Lysine yields were hardly affected by oxygen limitation whereas linear growth was observed under oxygen limiting conditions. Overall, this strain seems very robust with respect to the composition of silage utilizing all quantified low molecular weight substrates, e.g. lactate, glucose, fructose, maltose, quinate, fumarate, glutamate, leucine, isoleucine and alanine.

A system of miniaturized stirred bioreactors for parallel continuous cultivation of yeast with online measurement of dissolved oxygen and off‐gas

Chemostat cultivation is a powerful tool for physiological studies of microorganisms. We report the construction and application of a set of eight parallel small‐scale bioreactors with a working volume of 10 mL for continuous cultivation. Hungate tubes were used as culture vessels connected to multichannel‐peristaltic pumps for feeding fresh media and removal of culture broth and off‐gas. Water saturated air is sucked into the bioreactors by applying negative pressure, and small stirrer bars inside the culture vessels allow sufficient mixing and oxygen transfer. Optical sensors are used for non‐invasive online measurement of dissolved oxygen, which proved to be a powerful indicator of the physiological state of the cultures, particularly of steady‐state conditions. Analysis of culture exhaust‐gas by means of mass spectrometry enables balancing of carbon. The capacity of the developed small‐scale bioreactor system was validated using the fission yeast Schizosaccharomyces pombe, focusing on the metabolic shift from respiratory to respiro‐fermentative metabolism, as well as studies on consumption of different substrates such as glucose, fructose, and gluconate. In all cases, an almost completely closed carbon balance was obtained proving the reliability of the experimental setup. Biotechnol. Bioeng. 2013; 110: 535–542.

Analysis of myeloperoxidase activity in wound fluids as a marker of infection

Background Neutrophilic polymorphonuclear leukocytes play a crucial role in the host defence against bacterial and fungal infections. They participate in the inflammatory response through the liberation of peptides and enzymes like myeloperoxidase (MPO). Therefore, MPO has a potential as a marker enzyme for the diagnosis of wound infection. Methods Substrate specificities and reaction pathways of MPO were investigated for new MPO substrates: crystal violet, leuco crystal violet, fast blue RR (4-benzoylamino-2,5-dimethoxybenzenediazonium chloride hemi(zinc chloride) salt) and various systematically substituted model substrates based on 2,7-dihydroxy-1-(4-hydroxyphenylazo)naphtalene-3,6-disulphonic acid. In addition, fast blue RR was covalently bound to siloxanes allowing immobilization of the substrate, while cellobiosedehydrogenase was integrated for generation of hydrogen peroxide required by MPO. Results Elevated concentrations of MPO were found in infected wounds compared with non-infected wounds (92.2 ± 45.0 versus 1.9 ± 1.8 U/mL). Various soluble and immobilized substrates were oxidized by MPO in wound samples and the influence of substrate structure and reaction pathways were elucidated for selected compounds. Conclusions Incubation of different MPO substrates with infected wound fluid samples resulted in a clear colour change in the case of elevated MPO concentrations, thus allowing early diagnosis of wound infection.

Overcoming the metabolic burden of protein secretion in Schizosaccharomyces pombe – A quantitative approach using 13C-based metabolic flux analysis

Protein secretion in yeast is generally associated with a burden to cellular metabolism. To investigate this metabolic burden in Schizosaccharomyces pombe, we constructed a set of strains secreting the model protein maltase in different amounts. We quantified the influence of protein secretion on the metabolism applying (13)C-based metabolic flux analysis in chemostat cultures. Analysis of the macromolecular biomass composition revealed an increase in cellular lipid content at elevated levels of protein secretion and we observed altered metabolic fluxes in the pentose phosphate pathway, the TCA cycle, and around the pyruvate node including mitochondrial NADPH supply. Supplementing acetate to glucose or glycerol minimal media was found to improve protein secretion, accompanied by an increased cellular lipid content and carbon flux through the TCA cycle as well as increased mitochondrial NADPH production. Thus, systematic metabolic analyses can assist in identifying factors limiting protein secretion and in deriving strategies to overcome these limitations.

Novel protease-based diagnostic devices for detection of wound infection

A gelatinase‐based device for fast detection of wound infection was developed. Collective gelatinolytic activity in infected wounds was 23 times higher (p ≤ 0.001) than in noninfected wounds and blisters according to the clinical and microbiological description of the wounds. Enzyme activities of critical wounds showed 12‐fold elevated enzyme activities compared with noninfected wounds and blisters. Upon incubation of gelatin‐based devices with infected wound fluids, an incubation time of 30 minutes led to a clearly visible dye release. A 32‐fold color increase was measured after 60 minutes. Both matrix metalloproteinases and elastases contributed to collective gelatinolytic enzyme activity as shown by zymography and inhibition experiments. The metalloproteinase inhibitor 1,10‐phenanthroline (targeting matrix metalloproteinases) and the serine protease inhibitor phenylmethlysulfonyl fluoride (targeting human neutrophil elastase) inhibited gelatinolytic activity in infected wound fluid samples by 11–37% and 60–95%, respectively. Staphylococcus aureus and Pseudomonas aeruginosa, both known for gelatinase production, were isolated in infected wound samples.

Bioresponsive systems based on polygalacturonate containing hydrogels

Polysaccharide acid (PSA) based devices (consisting of alginic acid and polygalacturonic acid) were investigated for the detection of contaminating microorganisms. PSA-CaCl(2) hydrogel systems were compared to systems involving covalent cross-linking of PSA with glycidylmethacrylate (PSA-GMA) which was confirmed with Fourier Transformed Infrared (FTIR) analysis. Incubation of PSA-CaCl(2) and PSA-GMA beads loaded with Alizarin as a model ingredient with trigger enzymes (polygalacturonases or pectate lyases) or bacteria lead to a smoothening of the surface and exposure of Alizarin according to Environmental Scanning Electron Microscopy (ESEM) analysis. Enzyme triggered release of Alizarin was demonstrated for a commercial enzyme preparation from Aspergillus niger and with purified polygalacturonase and pectate lyase from S. rolfsii and B. pumilus, respectively. In contrast to the PSA-CaCl(2) beads, cross-linking (PSA-GMA beads) restricted the release of Alizarin in absence of enzymes. There was a linear relation between release of Alizarin (5-348 μM) and enzyme activity in a range of 0-300 U ml(-1) dosed. In addition to enzymes, both PSA-CaCl(2) and PSA-GMA beads were incubated with Bacillus subtilis and Yersinia entercolitica as model contaminating microorganism. After 72 h, a release between 10 μM and 57 μM Alizarin was detected. For protection of the hydrogels, an enzymatically modified PET membrane was covalently attached onto the surface. This lead to a slower release and improve long term storage stability based on less than 1% release of dye after 21 days. Additionally, this allowed simple detection by visual inspection of the device due to a colour change of the white membrane to orange upon enzyme triggered release of the dye.