Biomedical subjects

Sven Panke

Publications and source records attributed to Sven Panke.

15 recordsLinked to original sources

PubMed2007

Characterization of the AlkS/P(alkB)-expression system as an efficient tool for the production of recombinant proteins in Escherichia coli fed-batch fermentations.

The availability of suitable, well-characterized, and robust expression systems remains an essential requirement for successful metabolic engineering and recombinant protein production. We investigated the suitability of the Pseudomonas putida GPo1-derived AlkS/P(alkB) expression system in strictly aqueous cultures. By applying the apolar inducer dicyclopropylketone (DCPK) to express green fluorescent protein (GFP) from this system in Escherichia coli and analyzing the resulting cultures on single-cell level by flow cytometry, we found that this expression system gives rise to a homogeneous population of cells, even though the overall system is expected to have a positive feed-back element in the expression of the regulatory gene alkS. Overexpressing E. coli's serine hydroxymethyltransferase gene glyA, we showed that the system was already fully turned on at inducer concentrations as low as 0.005% (v/v). This allows efficient mass production of recombinant enzymes even though DCPK concentrations decreased from 0.05% to 0.01% over the course of a fully aerated cultivation in aqueous medium. Therefore, we elaborated the optimum induction procedure for production of the biocatalytically promising serine hydroxymethyltransferase and found volumetric and specific productivity to increase with specific growth rate in glucose-limited fed-batch cultures. Acetate excretion as a result of recombinant protein production could be avoided in an optimized fermentation protocol by switching earlier to a linear feed. This protocol resulted in a production of a final cell dry weight (CDW) concentration of 52 g/L, producing recombinant GlyA with a maximum specific activity of 6.3 U/mg total protein.

Base Sequence↗

PubMed2007

Quantitative analysis of bacterial medium-chain-length poly([R]-3-hydroxyalkanoates) by gas chromatography.

Standard chromatographic methods for the quantification of bacterial poly(3-hydroxyalkanoate) (PHA) proved to be inappropriate for the analysis of medium-chain-length PHA (mcl-PHA). Transesterification catalyzed by protic acids is not quantitative for mcl-PHA under common conditions due to slow reaction kinetics and formation of side-products in case of functionalized side-chains. To circumvent these limitations, an improved method for the quantification of mcl-PHA by GC-FID was developed. Boron trifluoride in methanol was successfully applied to quantitatively methanolyse different mcl-PHA (recovery >94%). This novel method is well-suited for the analysis of purified mcl-PHA as well as for mcl-PHA in biomass.

Alkanes↗

PubMed2006

Systematic assignment of thermodynamic constraints in metabolic network models.

BACKGROUND: The availability of genome sequences for many organisms enabled the reconstruction of several genome-scale metabolic network models. Currently, significant efforts are put into the automated reconstruction of such models. For this, several computational tools have been developed that particularly assist in identifying and compiling the organism-specific lists of metabolic reactions. In contrast, the last step of the model reconstruction process, which is the definition of the thermodynamic constraints in terms of reaction directionalities, still needs to be done manually. No computational method exists that allows for an automated and systematic assignment of reaction directions in genome-scale models. RESULTS: We present an algorithm that - based on thermodynamics, network topology and heuristic rules - automatically assigns reaction directions in metabolic models such that the reaction network is thermodynamically feasible with respect to the production of energy equivalents. It first exploits all available experimentally derived Gibbs energies of formation to identify irreversible reactions. As these thermodynamic data are not available for all metabolites, in a next step, further reaction directions are assigned on the basis of network topology considerations and thermodynamics-based heuristic rules. Briefly, the algorithm identifies reaction subsets from the metabolic network that are able to convert low-energy co-substrates into their high-energy counterparts and thus net produce energy. Our algorithm aims at disabling such thermodynamically infeasible cyclic operation of reaction subnetworks by assigning reaction directions based on a set of thermodynamics-derived heuristic rules. We demonstrate our algorithm on a genome-scale metabolic model of E. coli. The introduced systematic direction assignment yielded 130 irreversible reactions (out of 920 total reactions), which corresponds to about 70% of all irreversible reactions that are required to disable thermodynamically infeasible energy production. CONCLUSION: Although not being fully comprehensive, our algorithm for systematic reaction direction assignment could define a significant number of irreversible reactions automatically with low computational effort. We envision that the presented algorithm is a valuable part of a computational framework that assists the automated reconstruction of genome-scale metabolic models.

Algorithms↗

PubMed2006

The efficiency of recombinant Escherichia coli as biocatalyst for stereospecific epoxidation.

Styrene is efficiently converted into (S)-styrene oxide by growing Escherichia coli expressing the styrene monooxygenase genes styAB of Pseudomonas sp. strain VLB120 in an organic/aqueous emulsion. Now, we investigated factors influencing the epoxidation activity of recombinant E. coli with the aim to improve the process in terms of product concentration and volumetric productivity. The catalytic activity of recombinant E. coli was not stable and decreased with reaction time. Kinetic analyses and the independence of the whole-cell activity on substrate and biocatalyst concentrations indicated that the maximal specific biocatalyst activity was not exploited under process conditions and that substrate mass transfer and enzyme inhibition did not limit bioconversion performance. Elevated styrene oxide concentrations, however, were shown to promote acetic acid formation, membrane permeabilization, and cell lysis, and to reduce growth rate and colony-forming activity. During biotransformations, when cell viability was additionally reduced by styAB overexpression, such effects coincided with decreasing specific epoxidation rates and metabolic activity. This clearly indicated that biocatalyst performance was reduced as a result of product toxicity. The results point to a product toxicity-induced biological energy shortage reducing the biocatalyst activity under process conditions. By reducing exposure time of the biocatalyst to the product and increasing biocatalyst concentrations, volumetric productivities were increased up to 1,800 micromol/min/liter aqueous phase (with an average of 8.4 g/L(aq) x h). This represents the highest productivity reported for oxygenase-based whole-cell biocatalysis involving toxic products.

Biotransformation↗

PubMed2006

Synthetic biology--putting engineering into biology.

Synthetic biology is interpreted as the engineering-driven building of increasingly complex biological entities for novel applications. Encouraged by progress in the design of artificial gene networks, de novo DNA synthesis and protein engineering, we review the case for this emerging discipline. Key aspects of an engineering approach are purpose-orientation, deep insight into the underlying scientific principles, a hierarchy of abstraction including suitable interfaces between and within the levels of the hierarchy, standardization and the separation of design and fabrication. Synthetic biology investigates possibilities to implement these requirements into the process of engineering biological systems. This is illustrated on the DNA level by the implementation of engineering-inspired artificial operations such as toggle switching, oscillating or production of spatial patterns. On the protein level, the functionally self-contained domain structure of a number of proteins suggests possibilities for essentially Lego-like recombination which can be exploited for reprogramming DNA binding domain specificities or signaling pathways. Alternatively, computational design emerges to rationally reprogram enzyme function. Finally, the increasing facility of de novo DNA synthesis-synthetic biology's system fabrication process-supplies the possibility to implement novel designs for ever more complex systems. Some of these elements have merged to realize the first tangible synthetic biology applications in the area of manufacturing of pharmaceutical compounds.

Animals↗

PubMed2006

Putative regulatory sites unraveled by network-embedded thermodynamic analysis of metabolome data.

As one of the most recent members of the omics family, large-scale quantitative metabolomics data are currently complementing our systems biology data pool and offer the chance to integrate the metabolite level into the functional analysis of cellular networks. Network-embedded thermodynamic analysis (NET analysis) is presented as a framework for mechanistic and model-based analysis of these data. By coupling the data to an operating metabolic network via the second law of thermodynamics and the metabolites' Gibbs energies of formation, NET analysis allows inferring functional principles from quantitative metabolite data; for example it identifies reactions that are subject to active allosteric or genetic regulation as exemplified with quantitative metabolite data from Escherichia coli and Saccharomyces cerevisiae. Moreover, the optimization framework of NET analysis was demonstrated to be a valuable tool to systematically investigate data sets for consistency, for the extension of sub-omic metabolome data sets and for resolving intracompartmental concentrations from cell-averaged metabolome data. Without requiring any kind of kinetic modeling, NET analysis represents a perfectly scalable and unbiased approach to uncover insights from quantitative metabolome data.

Computational Biology↗

PubMed2006

Integrated operation of continuous chromatography and biotransformations for the generic high yield production of fine chemicals.

The rapid progress in biocatalysis in the identification and development of enzymes over the last decade has enormously enlarged the chemical reaction space that can be addressed not only in research applications, but also on industrial scale. This enables us to consider even those groups of reactions that are very promising from a synthetic point of view, but suffer from drawbacks on process level, such as an unfavourable position of the reaction equilibrium. Prominent examples stem from the aldolase-catalyzed enantioselective carbon-carbon bond forming reactions, reactions catalyzed by isomerising enzymes, and reactions that are kinetically controlled. On the other hand, continuous chromatography concepts such as the simulating moving bed technology have matured and are increasingly realized on industrial scale for the efficient separation of difficult compound mixtures - including enantiomers - with unprecedented efficiency. We propose that coupling of enzyme reactor and continuous chromatography is a very suitable and potentially generic process concept to address the thermodynamic limitations of a host of promising biotransformations. This way, it should be possible to establish novel in situ product recovery processes of unprecedented efficiency and selectivity that represent a feasible way to recruit novel biocatalysts to the industrial portfolio.

Biotechnology↗

PubMed2006

Suitability of teicoplanin-aglycone bonded stationary phase for simulated moving bed enantioseparation of racemic amino acids employing composition-constrained eluents.

The suitability of a teicoplanin-aglycone based chiral stationary phase for the simulated moving bed (SMB) enantioseparation of amino acids under enzyme-compatible conditions was shown following a procedure that is based solely on model-based simulations and HPLC experiments. A set of eight amino acids could be separated employing aqueous solvent containing only 10% (v/v) methanol, five of them with baseline resolution. The impact of type and concentration of organic modifier and pH modifier and pH on the separation characteristics of racemic methionine was investigated. Invariant elution profiles of repetitive adsorption/desorption of large amounts of methionine representing SMB-like conditions suggest stable adsorption behavior. Competitive loading capacity (20 mg of methionine per g of chiral stationary phase (CSP)) and SMB productivity (1 g of D-methionine per g of CSP per day) were predicted. The applied transport-dispersive model based on a competitive Bi-Langmuir isotherm was validated and its parameter estimated by model-based experimental analysis.

Adsorption↗

PubMed2005

In silico genome-scale reconstruction and validation of the Staphylococcus aureus metabolic network.

A genome-scale metabolic model of the Gram-positive, facultative anaerobic opportunistic pathogen Staphylococcus aureus N315 was constructed based on current genomic data, literature, and physiological information. The model comprises 774 metabolic processes representing approximately 23% of all protein-coding regions. The model was extensively validated against experimental observations and it correctly predicted main physiological properties of the wild-type strain, such as aerobic and anaerobic respiration and fermentation. Due to the frequent involvement of S. aureus in hospital-acquired bacterial infections combined with its increasing antibiotic resistance, we also investigated the clinically relevant phenotype of small colony variants and found that the model predictions agreed with recent findings of proteome analyses. This indicates that the model is useful in assisting future experiments to elucidate the interrelationship of bacterial metabolism and resistance. To help directing future studies for novel chemotherapeutic targets, we conducted a large-scale in silico gene deletion study that identified 158 essential intracellular reactions. A more detailed analysis showed that the biosynthesis of glycans and lipids is rather rigid with respect to circumventing gene deletions, which should make these areas particularly interesting for antibiotic development. The combination of this stoichiometric model with transcriptomic and proteomic data should allow a new quality in the analysis of clinically relevant organisms and a more rationalized system-level search for novel drug targets.

Computational Biology↗

PubMed2005

Advances in biocatalytic synthesis of pharmaceutical intermediates.

Biocatalytic synthesis of enantiomerically pure compounds for pharmaceutical intermediates is gaining momentum. This is the result of advances in genomics, screening and evolution technologies leading to the increased availability of new and robust biocatalysts suited for industrial-scale application, and is stimulated by an increased demand for catalysts that are able to address the increased complexity of active pharmaceutical ingredients. The vast majority of biotransformation reactions for the manufacturing of optically active pharmaceutical intermediates are still based on enantioselective ketone reductions and enantiospecific hydrolyses. This review aims to point at alternative reaction types and integrated multi-enzymatic steps that are emerging in large-scale applications.

Bacteria↗

PubMed2004

Trends and innovations in industrial biocatalysis for the production of fine chemicals.

Biocatalysis has become an established technology for the industrial manufacture of fine chemicals. In recent years, a multitude of chemical companies have embraced biocatalysis for the manufacture of desired stereoisomers, and new or improved methods for the synthesis of enantiomerically pure alpha- and beta-amino acids, amines, amides, peptides, nitriles, alcohols, organic acids and epoxides have emerged. Furthermore, the selectivity and mild operational conditions of biocatalysts are increasingly applied in industry to modify complex target molecules. These recent innovations in the manufacture of industrial fine chemicals using biocatalysis are discussed from an industrial perspective.

Alcohols↗

PubMed2002

Pilot-scale production of (S)-styrene oxide from styrene by recombinant Escherichia coli synthesizing styrene monooxygenase.

Recombinant Escherichia coli JM101(pSPZ10) cells produce the styrene monooxygenase of Pseudomonas sp. strain VLB120, which catalyzes the oxidation of styrene to (S)-styrene oxide at an enantiomeric excess larger than 99%. This biocatalyst was used to produce 388 g of styrene oxide in a two-liquid phase 30-L fed-batch bioconversion. The average overall volumetric activity was 170 U per liter over a period of more than 10 h, equivalent to mass transfer rates of 10.2 mmoles per liter per hour at a phase ratio of 0.5. At this transfer rate, the biotransformation system appeared to be substrate mass-transfer limited. The reactor had an estimated power input in the order of 5 W. L(-1), which is close to values typically obtained with commercially operating units. The product could be easily purified by fractional distillation to a purity in excess of 97%. The process illustrates the feasibility of recombinant whole cell biotransformations in two-liquid phase systems with toxic substrates and products.

Cell Line↗

PubMed2002

Enzyme technology and bioprocess engineering.

The impact of directed evolution and site-specific mutagenesis on the industrial utility of enzymatic catalysis through the modification of enzyme structure and function is clearly an important area of research in bioprocess engineering. High-throughput screening for novel or improved enzyme activities, both by more efficiently exploring nature's diversity and by creating new diversity in the test tube, allows new bioprocesses to be developed. Similarly, innovations in enzyme technology that address novel ways to apply enzymes in bioprocesses also have an impact on bioprocess engineering. Several recent developments have been made in this latter aspect of bioprocess engineering.

Biotechnology↗

PubMed2002

The production of fine chemicals by biotransformations.

Today, biocatalysis is a standard technology for the production of chemicals. An analysis of 134 industrial biotransformations reveals that hydrolases (44%) and redox biocatalysts (30%) are the most prominent categories. Most products are chiral (89%) and are used as fine chemicals. In the chemical industry, successful product developments involve on average a yield of 78%, a volumetric productivity of 15.5 g/(L.h) and a final product concentration of 108 g/L. By contrast, the pharmaceutical industry focuses on time-to-market. The implications of this for future research and development on biocatalysis are discussed.

Biotechnology↗

PubMed2001

Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes.

The Pseudomonas putida GPo1 (commonly known as Pseudomonas oleovorans GPo1) alkBFGHJKL and alkST gene clusters, which encode proteins involved in the conversion of n-alkanes to fatty acids, are located end to end on the OCT plasmid, separated by 9.7 kb of DNA. This DNA segment encodes, amongst others, a methyl-accepting transducer protein (AlkN) that may be involved in chemotaxis to alkanes. In P. putida P1, the alkBFGHJKL and alkST gene clusters are flanked by almost identical copies of the insertion sequence ISPpu4, constituting a class 1 transposon. Other insertion sequences flank and interrupt the alk genes in both strains. Apart from the coding regions of the GPo1 and P1 alk genes (80-92% sequence identity), only the alkB and alkS promoter regions are conserved. Competition experiments suggest that highly conserved inverted repeats in the alkB and alkS promoter regions bind ALKS:

Alkanes↗