Publications

Abstract

RNA–RNA inter- and intramolecular interactions are fundamental for numerous biological processes. While there are reasonable approaches to map RNA secondary structures genome-wide, understanding how different RNAs interact to carry out their regulatory functions requires mapping of intermolecular base pairs. Recently, different strategies to detect RNA–RNA duplexes in living cells, so called direct duplex detection (DDD) methods, have been developed. Common to all is the Psoralen-mediated in vivo RNA crosslinking followed by RNA Proximity Ligation to join the two interacting RNA strands. Sequencing of the RNA via classical RNA-seq and subsequent specialised bioinformatic analyses the result in the prediction of inter- and intramolecular RNA–RNA interactions. Existing approaches adapt standard RNA-seq analysis pipelines, but often neglect inherent features of RNA–RNA interactions that are useful for filtering and statistical assessment. Here we present RNAnue, a general pipeline for the inference of RNA–RNA interactions from DDD experiments that takes into account hybridisation potential and statistical significance to improve prediction accuracy. We applied RNAnue to data from different DDD studies and compared our results to those of the original methods. This showed that RNAnue performs better in terms of quantity and quality of predictions.

Abstract

The function and mode of action of small regulatory RNAs is currently still understudied in archaea. In the halophilic archaeon H. volcanii a plethora of sRNAs have been identified, however, in-depth functional analysis is missing for most of them. We selected a small RNA (s479) from H. volcanii for detailed characterization. The sRNA gene is encoded between a CRISPR RNA locus and the Cas protein gene cluster, the s479 deletion strain is viable and was characterized in detail. Transcriptome studies of wild type Haloferax cells and the deletion mutant revealed up-regulation of six genes in the deletion strain, showing that the sRNA has a clearly defined function. Three of the six up-regulated genes encode potential zinc transporter proteins (ZnuA1, ZnuB1, ZnuC1) suggesting involvement of s479 in regulation of zinc transport. Upregulation of these genes in the deletion strain was confirmed by northern blot and proteome analyses. Furthermore, electrophoretic mobility shift assays demonstrate a direct interaction of s479 with the target znuC1 mRNA. Proteome comparison of wild type and deletion strains further expanded the regulon of s479 deeply rooting this sRNA within the metabolism of H. volcanii especially the regulation of transporter abundance. Interestingly, s479 is not only encoded next to CRISPR-cas genes but the mature s479 contains a crRNA-like 5´ handle and experiments with Cas protein deletion strains indicate maturation by Cas6 and interaction with Cas proteins. Together this might suggest that the CRISPR-Cas system is involved in s479 function.

Abstract

The correct prediction of bacterial sRNA homologs is a prerequisite for many downstream analyses based on comparative genomics, but it is frequently challenging due to the short length and distinct heterogeneity of such homologs. GLASSGo is an efficient tool for the prediction of sRNA homologs from a single input query. To make the algorithm available to a broader community, we offer a Docker container along with a free-access web service. For non-computer scientists, the web service provides a user-friendly interface. However, capabilities were lacking so far for batch processing, version control, and direct interaction with compatible software applications as a workflow management system can provide.Here we present GLASSGo 1.5.2, an updated version that is fully incorporated into the workflow management system Galaxy. The improved version contains a new feature for extracting the upstream regions, allowing the search for conserved promoter elements. Additionally, it supports the use of accession numbers instead of the outdated GI numbers, which widens the applicability of the tool.GLASSGo is available at https://github.com/lotts/GLASSgo/ under the MIT license and is accompanied by instruction and application data. Furthermore, it can be installed into any Galaxy instance using the Galaxy ToolShed.

Abstract

Pseudomonas putida is recognized as a very promising strain for industrial application due to its high redox capacity and frequently observed tolerance towards organic solvents. In this research, we studied the metabolic and transcriptional response of P. putida KT2440 exposed to large-scale heterogeneous mixing conditions in the form of repeated glucose shortage. Cellular responses were mimicked in an experimental setup comprising a stirred tank reactor and a connected plug flow reactor. We deciphered that a stringent response-like transcriptional regulation programme is frequently induced, which seems to be linked to the intracellular pool of 3-hydroxyalkanoates (3-HA) that are known to serve as precursors for polyhydroxyalkanoates (PHA). To be precise, P. putida is endowed with a survival strategy likely to access cellular PHA, amino acids and glycogen in few seconds under glucose starvation to obtain ATP from respiration, thereby replenishing the reduced ATP levels and the adenylate energy charge. Notably, cells only need 0.4\% of glucose uptake to build those 3-HA-based energy buffers. Concomitantly, genes that are related to amino acid catabolism and $\beta$-oxidation are upregulated during the transient absence of glucose. Furthermore, we provide a detailed list of transcriptional short- and long-term responses that increase the cellular maintenance by about 17\% under the industrial-like conditions tested.

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) are essential genetic elements in many archaeal and bacterial genomes, playing a key role in a prokaryote adaptive immune system against invasive foreign elements. In recent years, the CRISPR-Cas system has also been engineered to facilitate target gene editing in eukaryotic genomes. Bioinformatics played an essential role in the detection and analysis of CRISPR systems and here we review the bioinformatics-based efforts that pushed the field of CRISPR-Cas research further. We discuss the bioinformatics tools that have been published over the last few years and, finally, present the most popular tools for the design of CRISPR-Cas9 guides.

Abstract

A successful bioeconomy depends on the manifestation of biorefineries that entirely convert renewable resources to valuable products and energies. Here, the poorly exploited hemicellulose fraction (HF) from beech wood organosolv processing was applied for isobutanol production with Corynebacterium glutamicum. To enable growth of C.glutamicum on HF, we integrated genes required for D-xylose and l-arabinose metabolization into two of 16 systematically identified and novel chromosomal integration loci. Under aerobic conditions, this engineered strain CArXy reached growth rates up to 0.34 +/- 0.02h(-1) on HF. Based on CArXy, we developed the isobutanol producer strain CIsArXy, which additionally (over)expresses genes of the native l-valine biosynthetic and the heterologous Ehrlich pathway. CIsArXy produced 7.2 +/- 0.2mM (0.53 +/- 0.02gL(-1)) isobutanol on HF at a carbon molar yield of 0.31 +/- 0.02 C-mol isobutanol per C-mol substrate (d-xylose + l-arabinose) in an anaerobic zero-growth production process.

Abstract

Bacterial small RNAs (sRNAs) are important post-transcriptional regulators of gene expression. The functional and evolutionary characterization of sRNAs requires the identification of homologs, which is frequently challenging due to their heterogeneity, short length and partly, little sequence conservation. We developed the GLobal Automatic Small RNA Search go (GLASSgo) algorithm to identify sRNA homologs in complex genomic databases starting from a single sequence. GLASSgo combines an iterative BLAST strategy with pairwise identity filtering and a graph-based clustering method that utilizes RNA secondary structure information. We tested the specificity, sensitivity and runtime of GLASSgo, BLAST and the combination RNAlien/cmsearch in a typical use case scenario on 40 bacterial sRNA families. The sensitivity of the tested methods was similar, while the specificity of GLASSgo and RNAlien/cmsearch was significantly higher than that of BLAST. GLASSgo was on average ~87 times faster than RNAlien/cmsearch, and only ~7.5 times slower than BLAST, which shows that GLASSgo optimizes the trade-off between speed and accuracy in the task of finding sRNA homologs. GLASSgo is fully automated, whereas BLAST often recovers only parts of homologs and RNAlien/cmsearch requires extensive additional bioinformatic work to get a comprehensive set of homologs. GLASSgo is available as an easy-to-use web server to find homologous sRNAs in large databases.

Abstract

Industrial bioreactors range from 10.000 to 700.000 L and characteristically show different zones of substrate availabilities, dissolved gas concentrations and pH values reflecting physical, technical and economic constraints of scale-up. Microbial producers are fluctuating inside the bioreactors thereby experiencing frequently changing micro-environmental conditions. The external stimuli induce responses on microbial metabolism and on transcriptional regulation programs. Both may deteriorate the expected microbial production performance in large scale compared to expectations deduced from ideal, well-mixed lab-scale conditions. Accordingly, predictive tools are needed to quantify large-scale impacts considering bioreactor heterogeneities. The review shows that the time is right to combine simulations of microbial kinetics with calculations of large-scale environmental conditions to predict the bioreactor performance. Accordingly, basic experimental procedures and computational tools are presented to derive proper microbial models and hydrodynamic conditions, and to link both for bioreactor modeling. Particular emphasis is laid on the identification of gene regulatory networks as the implementation of such models will surely gain momentum in future studies.

Abstract

Zero-growth processes are a promising strategy for the production of reduced molecules and depict a steady transition from aerobic to anaerobic conditions. To investigate the adaptation of Corynebacterium glutamicum to altering oxygen availabilities, we conceived a triple-phase fermentation process that describes a gradual reduction of dissolved oxygen with a shift from aerobiosis via microaerobiosis to anaerobiosis. The distinct process phases were clearly bordered by the bacteria’s physiologic response such as reduced growth rate, biomass substrate yield and altered yield of fermentation products. During the process, sequential samples were drawn at six points and analyzed via RNA-sequencing, for metabolite concentrations and for enzyme activities. We found transcriptional alterations of almost 50% (1421 genes) of the entire protein coding genes and observed an upregulation of fermentative pathways, a rearrangement of respiration, and mitigation of the basic cellular mechanisms such as transcription, translation and replication as a transient response related to the installed oxygen dependent process phases. To investigate the regulatory regime, 18 transcriptionally altered (putative) transcriptional regulators were deleted, but none of the deletion strains showed noticeable growth kinetics under an oxygen restricted environment. However, the described transcriptional adaptation of C. glutamicum resolved to varying oxygen availabilities provides a useful basis for future process and strain engineering.

Abstract

Cell-free (in vitro) protein synthesis (CFPS) systems provide a versatile tool that can be used to investigate different aspects of the transcription-translation machinery by reducing cells to the basic functions of protein formation. Recent improvements in reaction stability and lysate preparation offer the potential to expand the scope of in vitro biosynthesis from a research tool to a multifunctional and versatile platform for protein production and synthetic biology. To date, even the best-performing CFPS systems are drastically slower than in vivo references. Major limitations are imposed by ribosomal activities that progress in an order of magnitude slower on the mRNA template. Owing to the complex nature of the ribosomal machinery, conventional ``trial and error'' experiments only provide little insight into how the desired performance could be improved. By applying a DNA-sequence-oriented mechanistic model, we analyzed the major differences between cell-free in vitro and in vivo protein synthesis. We successfully identified major limiting elements of in vitro translation, namely the supply of ternary complexes consisting of EFTu and tRNA. Additionally, we showed that diluted in vitro systems suffer from reduced ribosome numbers. On the basis of our model, we propose a new experimental design predicting 90\% increased translation rates, which were well achieved in experiments. Furthermore, we identified a shifting control in the translation rate, which is characterized by availability of the ternary complex under in vitro conditions and the initiation of translation in a living cell. Accordingly, the model can successfully be applied to sensitivity analyses and experimental design.

Abstract

Aerobic production-scale processes are constrained by the technical limitations of maximum oxygen transfer and heat removal. Consequently, microbial activity is often controlled via limited nutrient feeding to maintain it within technical operability. Here, we present an alternative approach based on a newly engineered Escherichia coli strain. This E. coli HGT (high glucose throughput) strain was engineered by modulating the stringent response regulation program and decreasing the activity of pyruvate dehydrogenase. The strain offers about three-fold higher rates of cell-specific glucose uptake under nitrogen-limitation (0.6 g(Glc) gCDW(-1) h(-1)) compared to that of wild type, with a maximum glucose uptake rate of about 1.8 gGlc gCDW(-1) h(-1) already at a 0.3 h(-1) specific growth rate. The surplus of imported glucose is almost completely available via pyruvate and is used to fuel pyruvate and lactate formation. Thus, E. coli HGT represents a novel chassis as a host for pyruvate-derived products.

Abstract

Rapidly changing concentrations of substrates frequently occur during large-scale microbial cultivations. These changing conditions, caused by large mixing times, result in a heterogeneous population distribution. Here, we present a powerful and efficient modeling approach to predict the influence of varying substrate levels on the transcriptional and translational response of the cell. This approach consists of two parts, a single-cell model to describe transcription and translation for an exemplary operon (trp operon) and a second part to characterize cell distribution during the experimental setup. Combination of both models enables prediction of transcriptional patterns for the whole population. In summary, the resulting model is not only able to anticipate the experimentally observed short-term and long-term transcriptional response, it further allows envision of altered protein levels. Our model shows that locally induced stress responses propagate throughout the bioreactor, resulting in temporal, and spatial population heterogeneity. Stress induced transcriptional response leads to a new population steady-state shortly after imposing fluctuating substrate conditions. In contrast, the protein levels take more than 10 h to achieve steady-state conditions.

Abstract

In large-scale production processes, metabolic control is typically achieved by limited supply of essential nutrients such as glucose or ammonia. With increasing bioreactor dimensions, microbial producers such as Escherichia coli are exposed to changing substrate availabilities due to limited mixing. In turn, cells sense and respond to these dynamic conditions leading to frequent activation of their regulatory programmes. Previously, we characterized short- and long-term strategies of cells to adapt to glucose fluctuations. Here, we focused on fluctuating ammonia supply while studying a continuously running two-compartment bioreactor system comprising a stirred tank reactor (STR) and a plug-flow reactor (PFR). The alarmone ppGpp rapidly accumulated in PFR, initiating considerable transcriptional responses after 70s. About 400 genes were repeatedly switched on/off when E.coli returned to the STR. E.coli revealed highly diverging long-term transcriptional responses in ammonia compared to glucose fluctuations. In contrast, the induction of stringent regulation was a common feature of both short-term responses. Cellular ATP demands for coping with fluctuating ammonia supply were found to increase maintenance by 15\%. The identification of genes contributing to the increased ATP demand together with the elucidation of regulatory mechanisms may help to create robust cells and processes for large-scale application.

Abstract

Efficient optimization of microbial processes is a critical issue for achieving a number of sustainable development goals, considering the impact of microbial biotechnology in agrofood, environment, biopharmaceutical and chemical industries. Many of these applications require scale-up after proof of concept. However, the behaviour of microbial systems remains unpredictable (at least partially) when shifting from laboratory-scale to industrial conditions. The need for robust microbial systems is thus highly needed in this context, as well as a better understanding of the interactions between fluid mechanics and cell physiology. For that purpose, a full scale-up/down computational framework is already available. This framework links computational fluid dynamics (CFD), metabolic flux analysis and agent-based modelling (ABM) for a better understanding of the cell lifelines in a heterogeneous environment. Ultimately, this framework can be used for the design of scale-down simulators and/or metabolically engineered cells able to cope with environmental fluctuations typically found in large-scale bioreactors. However, this framework still needs some refinements, such as a better integration of gas-liquid flows in CFD, and taking into account intrinsic biological noise in ABM.

Abstract

The identification of promising metabolic engineering targets is a key issue in metabolic control analysis (MCA). Conventional approaches make intensive use of model-based studies, such as exploiting post-pulse metabolic dynamics after proper perturbation of the microbial system. Here, we present an easy-to-use, purely data-driven approach, defining pool efflux capacities (PEC) for identifying reactions that exert the highest flux control in linear pathways. Comparisons with linlog-based MCA and data-driven substrate elasticities (DDSE) showed that similar key control steps were identified using PEC. Using the example of L-methionine production with recombinant Escherichia coli, PEC consistently and robustly identified main flux controls using perturbation data after a non-labeled C-12-L-serine stimulus. Furthermore, the application of full-labeled C-13-L-serine stimuli yielded additional insights into stimulus propagation to L-methionine. PEC analysis performed on the C-13 data set revealed the same targets as the C-12 data set. Notably, the typical drawback of metabolome analysis, namely, the omnipresent leakage of metabolites, was excluded using the C-13 PEC approach.

Abstract

Background: A future bioeconomy relies on the efficient use of renewable resources for energy and material product supply. In this context, biorefineries have been developed and play a key role in converting lignocellulosic residues. Although a holistic use of the biomass feed is desired, side streams evoke in current biorefinery approaches. To ensure profitability, efficiency, and sustainability of the overall conversion process, a meaningful valorization of these materials is needed. Here, a so far unexploited side stream derived from fast pyrolysis of wheat straw-pyrolysis water-was used for production of 1,2-propanediol in microbial fermentation with engineered Corynebacterium glutamicum. Results: A protocol for pretreatment of pyrolysis water was established and enabled growth on its major constituents, acetate and acetol, with rates up to 0.36 +/- 0.04 h(-1). To convert acetol to 1,2-propanediol, the plasmid pJULgldA expressing the glycerol dehydrogenase from Escherichia coli was introduced into C. glutamicum. 1,2-propanediol was formed in a growth-coupled biotransformation and production was further increased by construction of C. glutamicum.pqo.aceE.ldhA.mdh pJULgldA. In a two-phase aerobic/microaerobic fed-batch process with pyrolysis water as substrate, this strain produced 18.3 +/- 1.2 mM 1,2-propanediol with a yield of 0.96 +/- 0.05 mol 1,2-propanediol per mol acetol and showed an overall volumetric productivity of 1.4 +/- 0.1 mmol 1,2-propanediol -L-1 h(-1). Conclusions: This study implements microbial fermentation into a biorefinery based on pyrolytic liquefaction of lignocellulosic biomass and accesses a novel value chain by valorizing the side stream pyrolysis water for 1,2-PDO production with engineered C. glutamicum. The established bioprocess operated at maximal product yield and accomplished the so far highest overall volumetric productivity for microbial 1,2-PDO production with an engineered producer strain. Besides, the results highlight the potential of microbial conversion of this biorefinery side stream to other valuable products.

Abstract

Cell-free protein synthesis is a versatile protein production system. Performance of the protein synthesis depends on highly active cytoplasmic extracts. Extracts from E. coli are believed to work best; they are routinely obtained from exponential growing cells, aiming to capture the most active translation system. Here, we report an active cell-free protein synthesis system derived from cells harvested at non-growth, stressed conditions. We found a downshift of ribosomes and proteins. However, a characterization revealed that the stoichiometry of ribosomes and key translation factors was conserved, pointing to a fully intact translation system. This was emphasized by synthesis rates, which were comparable to those of systems obtained from fast-growing cells. Our approach is less laborious than traditional extract preparation methods and multiplies the yield of extract per cultivation. This simplified growth protocol has the potential to attract new entrants to cell-free protein synthesis and to broaden the pool of applications. In this respect, a translation system originating from heat stressed, non-growing E. coli enabled an extension of endogenous transcription units. This was demonstrated by the sigma factor depending activation of parallel transcription. Our cell-free expression platform adds to the existing versatility of cell-free translation systems and presents a tool for cell-free biology.

Abstract

The productivity of industrial fermentation processes is essentially limited by the biomass-specific substrate consumption rate (q(S)) of the applied microbial production system. Since q(S) depends on the growth rate (mu), we highlight the potential of the fastest-growing nonpathogenic bacterium, Vibrio natriegens, as a novel candidate for future biotechnological processes. V. natriegens grows rapidly in BHIN complex medium with a mu of up to 4.43 h(-1) (doubling time of 9.4 min) as well as in minimal medium supplemented with various industrially relevant substrates. Bioreactor cultivations in minimal medium with glucose showed that V. natriegens possesses an exceptionally high q(S) under aerobic (3.90 +/- 0.08 g g(-1) h(-1)) and anaerobic (7.81 +/- 0.71 g g(-1) h(-1)) conditions. Fermentations with resting cells of genetically engineered V. natriegens under anaerobic conditions yielded an overall volumetric productivity of 0.56 +/- 0.10 g alanine liter(-1) min(-1) (i.e., 34 g liter(-1) h(-1)). These inherent properties render V. natriegens a promising new microbial platform for future industrial fermentation processes operating with high productivity. IMPORTANCE Low conversion rates are one major challenge to realizing microbial fermentation processes for the production of commodities operating competitively with existing petrochemical approaches. For this reason, we screened for a novel platform organism possessing characteristics superior to those of traditionally employed microbial systems. We identified the fast-growing V. natriegens, which exhibits a versatile metabolism and shows striking growth and conversion rates, as a solid candidate to reach outstanding productivities. Due to these inherent characteristics, V. natriegens can speed up common laboratory routines, is suitable for already existing production procedures, and forms an excellent foundation for engineering nextgeneration bioprocesses.

Abstract

Transcriptional control under nitrogen and carbon-limitation conditions have been well analyzed for Escherichia colt. However, the transcriptional dynamics that underlie the shift in regulatory programs from nitrogen to carbon limitation is not well studied. In the present study, cells were cultivated at steady state under nitrogen (ammonia)-limited conditions then shifted to carbon (glucose) limitation to monitor changes in transcriptional dynamics. Nitrogen limitation was found to be dominated by sigma 54 (RpoN) and sigma 38 (RpoS), whereas the ``housekeeping'' sigma factor 70 (RpoD) and sigma 38 regulate cellular status under glucose limitation. During the transition, nitrogen-mediated control was rapidly redeemed and mRNAs that encode active uptake systems, such as ptsG and manXYZ, were quickly amplified. Next, genes encoding facilitators such as lamB were overexpressed, followed by high affinity uptake systems such as mglABC and non-specific porins such as ompF. These regulatory programs are complex and require well-equilibrated and superior control. At the metabolome level, 2-oxoglutarate is the likely component that links carbon- and nitrogen-mediated regulation by interacting with major regulatory elements. In the case of dual glucose and ammonia limitation, sigma 24 (RpoE) appears to play a key role in orchestrating these complex regulatory networks.

Abstract

Saccharomyces cerevisiae is often applied in large-scale bioreactors where gradients of dissolved CO2 exist. Under high CO2 pressure, the dissolved gas enters the microbe, causing multifold intracellular responses such as decrease of pH, increase of HCO3- and changes of ion balance. Effects of varying CO2 concentrations are multifold, hard to scale and hardly investigated. Hence, the multi-level response to CO2 shifts was summarized in a predicting ODE model with mass action kinetics, balancing electrochemical charges in steady-state growth conditions. Compared to experimental observations, the simulated dynamics of ion concentrations were found to be consistent. During CO2 shifts, the model predicts the initial depolarization of the membrane potential, the temporal pH drop and the activation of countermeasures such as Pma1-mediated H+ export and Trk1,2-mediated K+ import. In conclusion, extracellular cation concentrations and the cellular pH regulation are critical factors that determine physiology and cellular energy management. Consequently, pressure-induced CO2 gradients cause peaks of ATP demand which may occur in cells circulating in large-scale industrial bioreactors.

Abstract

Ribosomes are a crucial component of the physiological state of a cell. Therefore, we aimed to monitor ribosome dynamics using a fast and easy fluorescence readout. Using fluorescent-labeled ribosomal proteins, the dynamics of ribosomes during batch cultivation and during nutritional shift conditions was investigated. The fluorescence readout was compared to the cellular rRNA content determined by capillary gel electrophoresis with laser-induced fluorescence detection during exponentially accelerating and decelerating growth. We found a linear correlation between the observed fluorescence and the extracted rRNA content throughout cultivation, demonstrating the applicability of this method. Moreover, the results show that ribosome dynamics, as a result of slowing growth, are accompanied by the passive effect of dilution of preexisting ribosomes, de novo ribosome synthesis and ribosome degradation. In light of the challenging task of deciphering ribosome regulatory mechanisms, our approach of using fluorescence to follow ribosome dynamics will allow more comprehensive studies of biological systems.

Abstract

Microbial fermentation of renewable feedstocks is an established technology in industrial biotechnology. Besides strict aerobic or anaerobic modes of operation, novel innovative and industrially applicable fermentation processes were developed connecting the advantages of aerobic and anaerobic conditions in a combined production approach. As a consequence, rapid aerobic biomass formation to high cell densities and subsequent anaerobic high-yield and zero-growth production is realized. Following this strategy, bioprocesses operating with substantial overall yield and productivity can be obtained. Here, we summarize the current knowledge and achievements in such microbial zero-growth production processes and pinpoint to challenges due to the complex adaptation of the cellular metabolism during the cell's passage from aerobiosis to anaerobiosis.

Abstract

Microbial producers such as Escherichia coli are evolutionarily trained to adapt to changing substrate availabilities. Being exposed to large-scale production conditions, their complex, multilayered regulatory programs are frequently activated because they face changing substrate supply due to limited mixing. Here, we show that E. coli can adopt both short- and long-term strategies to withstand these stress conditions. Experiments in which glucose availability was changed over a short time scale were performed in a two-compartment bioreactor system. Quick metabolic responses were observed during the first 30 s of glucose shortage, and after 70 s, fundamental transcriptional programs were initiated. Since cells are fluctuating under simulated large-scale conditions, this scenario represents a continuous on/off switching of about 600 genes. Furthermore, the resulting ATP maintenance demands were increased by about 40-50\%, allowing us to conclude that hyper-producing strains could become ATP-limited under large-scale production conditions. Based on the observed transcriptional patterns, we identified a number of candidate gene deletions that may reduce unwanted ATP losses. In summary, we present a theoretical framework that provides biological targets that could be used to engineer novel E. coli strains such that large-scale performance equals laboratory-scale expectations. (C) 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Abstract

Cell-free protein synthesis, which mimics the biological protein production system, allows rapid expression of proteins without the need to maintain a viable cell. Nevertheless, cell free protein expression relies on active in vivo translation machinery including ribosomes and translation factors. Here, we examined the integrity of the protein synthesis machinery, namely the functionality of ribosomes, during (i) the cell-free extract preparation and (ii) the performance of in vitro protein synthesis by analyzing crucial components involved in translation. Monitoring the 16S rRNA, 23S rRNA, elongation factors and ribosomal protein Si, we show that processing of a cell-free extract results in no substantial alteration of the translation machinery. Moreover, we reveal that the 16S rRNA is specifically cleaved at helix 44 during in vitro translation reactions, resulting in the removal of the anti-Shine-Dalgarno sequence. These defective ribosomes accumulate in the cell-free system. We demonstrate that the specific cleavage of the 16S rRNA is triggered by the decreased concentrations of Mg2+. In addition, we provide evidence that helix 44 of the 30S ribosomal subunit serves as a point-of-entry for ribosome degradation in Escherichia coli. Our results suggest that Mg2+ homeostasis is fundamental to preserving functional ribosomes in cell-free protein synthesis systems, which is of major importance for cell-free protein synthesis at preparative scale, in order to create highly efficient technical in vitro systems.

Abstract

In industrial cell culture engineering, the production process consists of a multiscale seed train from lab scale to large scale. The oxygen demand of the cells has to be satisfied in all scales. Computational fluid dynamics (CFD) simulations can provide a tool to predict the mass transfer between the gas phase and the liquid phase. In this work, CFD was applied using an Euler-Lagrange approach for the prediction of the mass transfer coefficient (kLa) in stirred tank bioreactors for a wide range of operating conditions. The turbulent dissipation was studied for two different scales that show similar flow behavior. Breakup and coalescence of bubbles was not considered. A standard k-epsilon model was used for the simulation of turbulence and the mass transfer was assumed to be isotropic and turbulence driven. A minimalistic model was found, which was able to successfully predict the mass transfer behavior with high accuracy for the lab-scale bioreactor (2.3L) covering a wide range of typical operating conditions. In the given setup, bubbles remained close to the sparger, almost not interfering with the impellers. This supports the assumption of monodisperse bubbles for stirrer speeds between 140 and 260rpm. Simulation results of an 80L stirred tank reactor (STR) revealed the need to integrate physical phenomena like breakup and coalescence and a more sophisticated prediction of the initial bubble size distribution. Two-phase Euler-Lagrange CFD simulations were performed for two differently scaled STRs and the mass transfer coefficient kLa was calculated and compared to experiments in order to evaluate the applied models.

Abstract

Biopharmaceuticals are predominantly produced by Chinese hamster ovary (CHO) cells cultivated in fed-batch mode. Hyperosmotic culture conditions ( 350 mOsmol kg(Sigma 1)) resulting from feeding of nutrients may enhance specific product formation rates (q(p)). As an improved ATP supply was anticipated to enhance q(p) this study focused on the identification of suitable miRNA/mRNA targets to increase ATP levels. Therefor next generation sequencing and a compartment specific metabolomics approach were applied to analyze the response of an antibody (mAB) producing CHO cell line upon osmotic shift (280 430 mOsmol kg(-1)). Hyperosmotic culture conditions caused a approximate to 2.6-fold increase of specific ATP formation rates together with a approximate to 1.7-fold rise in cytosolic and mitochondrial ATP-pools, thus showing increased ATP supply. mRNA expression analysis identified several genes encoding glycosylated proteins with strictly tissue related function. In addition, hyperosmotic culture conditions induced an upregulation of miR-132-3p, miR-132-5p, miR-182, miR-183, miR-194, miR-215-3p, miR-215-5p which have all been related to cell cycle arrest/proliferation in cancer studies. In relation to a previous independent CHO study miR-183 may be the most promising target to enhance q(p) by stable overexpression. Furthermore, deletion of genes with presumably dispensable function in suspension growing CHO cells may enhance mAB formation by increased ATP levels.

Abstract

Market demands for monoclonal antibodies (mAbs) are steadily increasing worldwide. As a result, production processes using Chinese hamster ovary cells (CHO) are in the focus of ongoing intensification studies for maximizing cell-specific and volumetric productivities. This includes the optimization of animal-derived component free (ADCF) cultivation media as part of good cell culture practice. Dipeptides are known to improve CHO culture performance. However, little or even conflicting assumptions exist about their putative import and functionality inside the cells. A set of well-known performance boosters and new dipeptide prospects was evaluated. The present study revealed that dipeptides are indeed imported in the cells, where they are decomposed to the amino acids building blocks. Subsequently, they are metabolized or, unexpectedly, secreted to the medium. Monoclonal antibody production boosting additives like l-alanine-l-glutamine (AQ) or glycyl-l-glutamine (GQ) can be assigned to fast or slow dipeptide uptake, respectively, thus pinpointing to the need to study dipeptide kinetics and to adjust their feeding individually for optimizing mAb production.

Abstract

Cellular response to different types of stress is the hallmark of the cell's strategy for survival. How organisms adjust their cell cycle dynamics to compensate for changes in environmental conditions is an important unanswered question in bacterial physiology. A cell using binary fission for reproduction passes through three stages during its cell cycle: a stage from cell birth to initiation of replication, a DNA replication phase and a period of cell division. We present a detailed analysis of durations of cell cycle phases, investigating their dynamics under environmental stress conditions. Applying continuous steady state cultivations (chemostats), the DNA content of a Pseudomonas putida KT2440 population was quantified with flow cytometry at distinct growth rates. Data-driven modeling revealed that under stress conditions, such as oxygen deprivation, solvent exposure and decreased iron availability, DNA replication was accelerated correlated to the severity of the imposed stress (up to 1.9-fold). Cells maintained constant growth rates by balancing the shortened replication phase with extended cell cycle phases before and after replication. Transcriptome data underpin the transcriptional upregulation of crucial genes of the replication machinery. Hence adaption of DNA replication speed appears to be an important strategy to withstand environmental stress.

Abstract

Bioprocess engineering is a highly interdisciplinary field of study which is strongly benefited by practical courses where students can actively experience the interconnection between biology, engineering, and physical sciences. This work describes a lab course developed for 2nd year undergraduate students of bioprocess engineering and related disciplines, where students are challenged with a real-life bioprocess-engineering application, the production of recombinant protein in a fed-batch process. The lab course was designed to introduce students to the subject of operating and supervising an experiment in a bioreactor, along with the analysis of collected data and a final critical evaluation of the experiment. To provide visual feedback of the experimental outcome, the organism used during class was Escherichia coli which carried a plasmid to recombinantly produce enhanced green fluorescent protein (eGFP) upon induction. This can easily be visualized in both the bioreactor and samples by using ultraviolet light. The lab course is performed with bioreactors of the simplest design, and is therefore highly flexible, robust and easy to reproduce. As part of this work the implementation and framework, the results, the evaluation and assessment of student learning combined with opinion surveys are presented, which provides a basis for instructors intending to implement a similar lab course at their respective institution. (c) 2015 by the International Union of Biochemistry and Molecular Biology, 43(3):189-202, 2015.

Abstract

Intracellular proteolysis in mammalian cells is a native cellular strategy to recycle proteins and peptides. Whether or not this mechanism may hamper monoclonal antibody (mAb) formation in Chinese hamster ovary (CHO) cells was the key driver for this study. Exponentially growing, anti-interleukin (IL)-8 producing CHO cells were fed with C-13-labeled L-lysine. The fate of the labeling signal was tracked in intracellular peptides, which were the proteolytic fragments of the mAb. Signal analysis was performed in samples after cell disruption, anion exchange SPE and Q-ToF mass detection. Four degradation peptides were found, with HYTQKSLSLSPGK and HYTQKSLSLSPG containing two and one L-lysine unit (K), respectively. Labeling dynamics were used for model-based identification of the degradation rate in four biological replicates. Degradation rates of 22-25 pmol/10(8)cells/h were estimated, representing about 3\% of the net mAb production. Hence, intracellular mAb degradation occurs even under the rather smooth production conditions installed.

Abstract

A variety of approaches has been published to enhance specific productivity (q(p)) of recombinant Chinese hamster ovary (CHO) cells. Changes in culture conditions, e. g. temperature shifts, sodium butyrate treatment and hyperosmolality, were shown to improve q(p). To contribute to a better understanding of the correlation between hyperosmolality and enhanced q(p), we analyzed cellular kinetics and intracellular adenine nucleotide pools during osmotic shift periods. Known phenotypes like increased formation rates for lactate and product (anti-IL-8 antibody; q(lactate), q(p)) as well as increased cell specific uptake rate for glucose (q(glucose)) were foundbesides inhibition of cell growth and G1-arrest occurred during batch cultivations with osmotic shift. The analysis of intracellular AXP pools revealed enlarged ATP amounts for cells as response to hyperosmolality while energy charges remained unchanged. Enhanced ATP-pools coincided with severely increased ATP formation rates (q(ATP)) which outweighed by far the putative requirements attributed to regulatory volume increase. Therefore elevated q(ATP) mirrored an increased cellular demand for energy while experiencing hyperosmotic shift. (c) 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1212-1216, 2015

Abstract

Modeling of metabolic networks as part of systems metabolic engineering requires reliable quantitative experimental data of intracellular concentrations. The hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry (HILIC-ESI-MS/MS) method was used for quantitative profiling of more than 50 hydrophilic key metabolites of cellular metabolism. Without prior derivatization, sugar phosphates, organic acids, nucleotides, and amino acids were measured under alkaline and acidic mobile phase conditions with pre-optimized multiple reaction monitoring (MRM) transitions. Irrespective of the polarity mode of the acquisition method used, alkaline conditions achieved the best quantification limits and linear dynamic ranges. Fully 90\% of the analyzed metabolites presented detection limits better than 0.5 pmol (on column), and 70\% presented 1.5-fold higher signal intensities under alkaline mobile phase conditions. The quality of the method was further demonstrated by absolute quantification of selected metabolites in intracellular extracts of Escherichia coli. In addition, quantification bias caused by matrix effects was investigated by comparison of calibration strategies: standard-based external calibration, isotope dilution, and standard addition with internal standards. Here, we recommend the use of alkaline mobile phase with polymer-based zwitterionic hydrophilic interaction chromatography (ZIC-pHILIC) as the most sensitive scenario for absolute quantification for a broad range of metabolites. (C) 2015 Elsevier Inc. All rights reserved.

Abstract

To smoothen the process of n-butanol formation in Pseudomonas putida KT2440, detailed knowledge of the impact of this organic solvent on cell physiology and regulation is of outmost importance. Here, we conducted a detailed systems biology study to elucidate cellular responses at the metabolic, proteomic, and transcriptional level. Pseudomonas putida KT2440 was cultivated in multiple chemostat fermentations using n-butanol either as sole carbon source or together with glucose. Pseudomonas putida KT2440 revealed maximum growth rates (mu) of 0.3 h(-1) with n-butanol as sole carbon source and of 0.4 h(-1) using equal C-molar amounts of glucose and n-butanol. While C-mole specific substrate consumption and biomass/substrate yields appeared equal at these growth conditions, the cellular physiology was found to be substantially different: adenylate energy charge levels of 0.85 were found when n-butanol served as sole carbon source (similar to glucose as sole carbon source), but were reduced to 0.4 when n-butanol was coconsumed at stable growth conditions. Furthermore, characteristic maintenance parameters changed with increasing n-butanol consumption. C-13 flux analysis revealed that central metabolism was split into a glucose-fueled Entner-Doudoroff/pentose-phosphate pathway and an n-butanol-fueled tricarboxylic acid cycle when both substrates were coconsumed. With the help of transcriptome and proteome analysis, the degradation pathway of n-butanol could be unraveled, thus representing an important basis for rendering P. putida KT2440 from an n-butanol consumer to a producer in future metabolic engineering studies.

Abstract

Background: The implementation of novel platform organisms to be used as microbial cell factories in industrial applications is currently the subject of intense research. Ongoing efforts include the adoption of Pseudomonas putida KT2440 variants with a reduced genome as the functional chassis for biotechnological purposes. In these strains, dispensable functions removed include flagellar motility (1.1\% of the genome) and a number of open reading frames expected to improve genotypic and phenotypic stability of the cells upon deletion (3.2\% of the genome). Results: In this study, two previously constructed multiple-deletion P. putida strains were systematically evaluated as microbial cell factories for heterologous protein production and compared to the parental bacterium (strain KT2440) with regards to several industrially-relevant physiological traits. Energetic parameters were quantified at different controlled growth rates in continuous cultivations and both strains had a higher adenosine triphosphate content, increased adenylate energy charges, and diminished maintenance demands than the wild-type strain. Under all the conditions tested the mutants also grew faster, had enhanced biomass yields and showed higher viability, and displayed increased plasmid stability than the parental strain. In addition to small-scale shaken-flask cultivations, the performance of the genome-streamlined strains was evaluated in larger scale bioreactor batch cultivations taking a step towards industrial growth conditions. When the production of the green fluorescent protein (used as a model heterologous protein) was assessed in these cultures, the mutants reached a recombinant protein yield with respect to biomass up to 40\% higher than that of P. putida KT2440. Conclusions: The two streamlined-genome derivatives of P. putida KT2440 outcompeted the parental strain in every industrially-relevant trait assessed, particularly under the working conditions of a bioreactor. Our results demonstrate that these genome-streamlined bacteria are not only robust microbial cell factories on their own, but also a promising foundation for further biotechnological applications.

Abstract

Mammalian cells show a compartmented metabolism. Getting access to subcellular metabolite pools is of high interest to understand the cells' metabolomic state. Therefore a protocol is developed and applied for monitoring compartment-specific metabolite and nucleotide pool sizes in Chinese hamster ovary (CHO) cells. The approach consists of a subtracting filtering method separating cytosolic components from physically intact mitochondrial compartments. The internal standards glucose-6-phosphate and cis-aconitate were chosen to quantify cytosolic secession and mitochondrial membrane integrity. Extracts of related fractions were studied by liquid chromatography-isotope dilution mass spectrometry for the absolute quantification of a subset of glycolytic and tricarboxylic acid cycle intermediates together with the adenylate nucleotides ATP, ADP and AMP. The application of the protocol revealed highly dynamic changes in the related pool sizes as a function of distinct cultivation periods of IgG1 producing CHO cells. Mitochondrial and cytosolic pool dynamics were in agreement with anticipated metabolite pools of independent studies. The analysis of adenosine phosphate levels unraveled significantly higher ATP levels in the cytosol leading to the hypothesis that mitochondria predominantly serve for fueling ATP into the cytosol where it is tightly controlled at physiological adenylate energy charges about 0.9.

Abstract

What defines central carbon metabolism? The classic textbook scheme of central metabolism includes the Embden-Meyerhof-Parnas (EMP) pathway of glycolysis, the pentose phosphate pathway, and the citric acid cycle. The prevalence of this definition of central metabolism is, however, equivocal without experimental validation. We address this issue using a general experimental approach that combines the monitoring of transcriptional and metabolic flux changes between steady states on alternative carbon sources. This approach is investigated by using the model bacterium Pseudomonas putida with glucose, fructose, and benzoate as carbon sources. The catabolic reactions involved in the initial uptake and metabolism of these substrates are expected to show a correlated change in gene expressions and metabolic fluxes. However, there was no correlation for the reactions linking the 12 biomass precursor molecules, indicating a regulation mechanism other than mRNA synthesis for central metabolism. This result substantiates evidence for a (re) definition of central carbon metabolism including all reactions that are bound to tight regulation and transcriptional invariance. Contrary to expectations, the canonical Entner-Doudoroff and EMP pathways sensu stricto are not a part of central carbon metabolism in P. putida, as they are not regulated differently from the aromatic degradation pathway. The regulatory analyses presented here provide leads on a qualitative basis to address the use of alternative carbon sources by deregulation and overexpression at the transcriptional level, while rate improvements in central carbon metabolism require careful adjustment of metabolite concentrations, as regulation resides to a large extent in posttranslational and/or metabolic regulation.

Abstract

Prokaryotic production systems have been widely used to manufacture recombinant therapeutic proteins. Economically, the prokaryotic production - especially of small therapeutic molecules - is advantageous compared to eukaryotic production strategies. However, due to the potential endotoxin and host cell protein contamination, the requirements for the purification process are disproportionately higher and therefore more expensive and elaborate to circumvent. For this reason, the goal of this work was to develop and establish a rapid, simple, inexpensive and `up-scalable' production and purification process, using the therapeutic relevant protein anti-EGFR scFv hu225 as model molecule. Configuring high cell density cultivation of Escherichia coli - using the rha-BAD expression system as production platform - a specific product concentration up to 20 mg(scFv)/g(CDW) was obtained. By combining freeze-and-thaw, osmotic shock and pH induced host cell protein precipitation, almost 70\% of the product was extracted from the biomass. In a novel approach a mixed mode chromatography was implemented as a capturing and desalting step, which allowed the direct application of further ion exchange chromatography steps for purification up to pharmaceutical grade. Thereby, 50\% of the produced scFv could be purified within 10 h while maintaining the biological activity. (C) 2014 Elsevier B.V. All rights reserved.

Abstract

The exploration of scale-down models to imitate the influence of large scale bioreactor inhomogeneities on cellular metabolism is a topic with increasing relevance. While gradients of substrates, pH, or dissolved oxygen are often investigated, oscillating CO2/HCO3 (-) levels, a typical scenario in large industrial bioreactors, is rarely addressed. Hereby, we investigate the metabolic and transcriptional response in Corynebacterium glutamicum wild type as well as the impact on l-lysine production in a model strain exposed to pCO(2) gradients of (75-315) mbar. A three-compartment cascade bioreactor system was developed and characterized that offers high flexibility for installing gradients and residence times to mimic industrial-relevant conditions and provides the potential of accurate carbon balancing. The phenomenological analysis of cascade fermentations imposed to the pCO(2) gradients at industry-relevant residence times of about 3.6 min did not significantly impair the process performance, with growth and product formation being similar to control conditions. However, transcriptional analysis disclosed up to 66 differentially expressed genes already after 3.6 min under stimulus exposure, with the overall change in gene expression directly correlateable to the pCO(2) gradient intensity and the residence time of the cells.

Abstract

Carbon balancing of microbial fermentations is a valuable tool for the evaluation of the process performance and to identify the presence of undesired by-products. In this study, we demonstrate the relevance of total carbon (TC) analysis for carbon balancing in fermentations with the wild-type of Corynebacterium glutamicum by (i) quantifying significant amounts of dissolved inorganic carbonic species (TIC) in the culture medium and (ii) determining the effective (mass) carbon content of the biomass fraction (M-C,M-X). In principle, TC based carbon balancing yielded at fully matching carbon balances. Thus, the application of our TC approach for the accurate detection of TIC and M-C,M-X increased the total carbon recovery in standard batch fermentations with C. glutamicum on glucose from about 76\% to carbon closures of 94-100\% in contrast to conventional approaches. Besides, the origin of the missing 6\%-gap could be attributed to incomplete quantification of all carbon sources in the liquid phase. To conclude this study, the concept of TC-based balancing was transferred to an L-lysine production process, successfully quantifying relevant system carbon fractions, which resulted in matched carbon recoveries. (C) 2014 Elsevier B.V. All rights reserved.

Abstract

A combined computational fluid dynamics (CFD) and population balance model (PBM) approach has been applied to simulate hydrodynamics and mass transfer in a 0.18 m(3) gas-liquid stirred bioreactor agitated by (1) a Rushton turbine, and (2) a new pitched blade geometry with rotating cartridges. The operating conditions chosen were motivated by typical settings used for culturing mammalian cells. The effects of turbulence, rotating flow, bubbles breakage and coalescence were simulated using the k-epsilon, multiple reference frame (MRF), Sliding mesh (SM) and PBM approaches, respectively. Considering the new pitched blade geometry with rotating aeration microspargers, mass transfer was estimated to be 34 times higher than the conventional Rushton turbine set-up. Notably, the impeller power consumption was modeled to be about 50 \% lower. Independent measurements applying the same operational conditions confirmed this finding. Motivated by these simulated and experimental results, the new aeration and stirring device is qualified as a very promising tool especially useful for cell culture applications which are characterized by the challenging problem of achieving relatively high mass transfer conditions while inserting only low stirrer energy.

Abstract

Population heterogeneity occurring in industrial microbial bioprocesses is regarded as a putative effector causing performance loss in large scale. While the existence of subpopulations is a commonly accepted fact, their appearance and impact on process performance still remains rather unclear. During cell cycling, distinct subpopulations differing in cell division state and DNA content appear which contribute individually to the efficiency of the bioprocess. To identify stressed or impaired subpopulations, we analyzed the interplay of growth rate, cell cycle and phenotypic profile of subpopulations by using flow cytometry and cell sorting in conjunction with mass spectrometry based global proteomics. Adjusting distinct growth rates in chemostats with the model strain Pseudomonas putida KT2440, cells were differentiated by DNA content reflecting different cell cycle stages. The proteome of separated subpopulations at given growth rates was found to be highly similar, while different growth rates caused major changes of the protein inventory with respect to e.g. carbon storage, motility, lipid metabolism and the translational machinery. In conclusion, cells in various cell cycle stages at the same growth rate were found to have similar to identical proteome profiles showing no significant population heterogeneity on the proteome level. In contrast, the growth rate clearly determines the protein composition and therefore the metabolic strategy of the cells.

Abstract

Phosphate starvation is often applied as a tool to limit cell growth in microbial production processes without hampering carbon and/or nitrogen supply alternatively. This contribution focuses on the interplay of process induced phosphate starvation and microbial performance studying an l-tryptophan overproducing Escherichia coli strain as a model for highly ATP demanding processes in comparison with an E. coli wildtype strain. To enable a time-resolved analysis, constant phosphate feeding strategies were applied to elongate the transition from phosphate saturated to phosphate limited cell growth. With increasing phosphate limitation, a reduced cellular efficiency of ATP formation via respiratory chain activity and the ATP synthase complex was found for both strains. Process balancing, transcriptome analysis and flux balance analysis are pointing toward a multi-stage decoupling scenario, which in essence deteriorates the stoichiometric ratio of ATP formation to proton translocation, thereby affecting ATP availability from respiration and carbon usage. Starting off with a potential influence on ATP-synthase efficiency (stage 1), decoupling is further increased by modified respiratory activity (stage 2) and by product overflow (stage 3) finally resulting in a metabolic breakdown entering complete phosphate depletion (stage 4). The decoupling is initiated by phosphate limitation; further effects are mainly mediated on metabolic level through ATP availability and energy charge, additionally affected by ATP demanding product synthesis. (C) 2014 Elsevier B.V. All rights reserved.

Abstract

Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the L-valine biosynthetic genes ilvBNCE, all strains produced L-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate: quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Delta pqo Delta ppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) L-valine with an overall yield (Y-P/S) of 0.36 mol per mol of glucose and a volumetric productivity (Q(P)) of 13.6 mM per h 1.6 g/(liter x h). Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the L-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303mM(35 g/liter) 2-ketoisovalerate with a Y-P/S of 0.24 mol per mol of glucose and a Q(P) of 6.9 mM per h 0.8 g/(liter x h). The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum L-lysine producers DM1800 and DM1933 improved the production by 100\% and 44\%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

Abstract

A two-phase biotransformation process for selective hydroxylation of n-octane to 1-octanol via Pseudomonas putida KT2440 harboring heterologously expressed P450 monooxygenase from Mycobacterium marinum is presented. Maximum cell-specific conversion rates of 12.7mg(octanol)g(CDW)h(-1) were observed not only in shaking flasks but also in 3.7-L-bioreactor studies. The bioreactor experiments were performed avoiding explosive gas mixtures by lowering volumetric power input, aeration rates and substrate concentrations. Based on a stoichiometric network of P. putida KT2440 topological studies were carried out. As a conclusion, potential limitations of NAD(P)H and/or ATP supply at production conditions can be excluded. Hence, the great potential of the host for further increasing conversion is outlined.

Abstract

AimsThe aim of this study was to engineer Escherichia coli strains that efficiently produce succinate from glycerol under anaerobic conditions after an aerobic growth phase. Methods and ResultsWe constructed E.coli strain ss195 with deletions of pykA and pykF, which resulted in slow growth on glycerol as sole carbon source. This growth defect was overcome by the selection of fast-growing mutants. Whole-genome resequencing of the evolved mutant ss251 identified the mutation A595S in PEP carboxylase (Ppc). Reverse metabolic engineering by introducing the wild-type allele revealed that this mutation is crucial for the described phenotype. Strain ss251 and derivatives thereof produced succinate with high yields above 80\% molmol(-1) from glycerol under nongrowth conditions. ConclusionsThe results show that during the aerobic growth of ss251, the formation of pyruvate proceeds via the proposed POMP pathway, starting with the carboxylation of PEP by Ppc. The resulting oxaloacetate is reduced by malate dehydrogenase (Mdh) to malate, which is then decarboxylated back to pyruvate by a malic enzyme (MaeA or MaeB). Mutation of ppc is crucial for fast growth of pykAF mutants on glycerol. Significance and Impact of StudyAn E.coli mutant that is capable of achieving high yields of succinate (a top valued-added chemical) from glycerol (an abundant carbon source) was constructed. The identified ppc mutation could be applied to other production strains that require strong PEP carboxylation fluxes.

Abstract

We investigated the growth kinetics and transcriptional responses of Corynebacterium glutamicum in environments with low (pCO(2) < 40 mbar) and high (pCO(2) >= 300 mbar) CO2/HCO3 levels compared to standard conditions. When cultivated at high CO2/HCO3--levels, C. glutamicum showed increased (63\%) biomass to substrate yields during the initial growth phase. Other kinetic parameters such as growth rate (mu), specific glucose consumption rate (q(s)), and selected enzymatic activities of anaplerotic reactions, the pentose phosphate pathway and the tricarboxylic acid cycle were similar to standard conditions. However, microarray hybridization disclosed a complex transcriptional response involving 117 differentially expressed genes. Among those, 60 genes were assigned to the complete DtxR/RipA regulon controlling iron homeostasis in C. glutamicum. Impaired growth of a Delta dtxR mutant at high CO2/HCO3- levels validated the relevance of this master regulator to cope with excessive CO2/HCO3- availability. At low CO2/HCO3- levels, C. glutamicum grew in a bi-level manner with three distinct growth phases. Differential analyses revealed approximately doubled activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase accompanied by the formation of L-alanine and L-valine during the lowest mu occurring in mid-phase of the cultivation. DNA microarray analysis revealed more than 100 differentially expressed genes in growth phase II compared to phase I including almost all thiamin pyrophosphate (TPP) biosynthesis genes, which were significantly up regulated. Concluding, we hypothesize that C. glutamicum counteracts the lack of CO2/HCO3- by triggering TPP biosynthesis for increasing the activities of TPP-dependent enzymes involved in CO2 formation. (C) 2013 Elsevier B.V. All rights reserved.

Abstract

Industrial biotechnological production is developing rapidly worldwide. Consequently, more and novel bioprocesses need to perform optimally not only in small lab- but also in large production scales. This article shortly reviews typical impacts found when cells are exposed to micro environmental heterogeneities typically occurring in poorly mixed large scale production reactors. The current state-of-the-art of tool development is presented for analyzing these phenomena. Finally, still open questions are formulated and needs for future research are outlined to further support the expansion of biotech industries by successful research results. (C) 2011 Elsevier B.V. All rights reserved.

Abstract

A novel technically compliant expression system was developed for heterologous protein production in Bacillus subtilis with the aim of increasing product yields at the same time as decreasing production costs. Standard systems involve the positively regulated manP promoter of the mannose operon, which led to relatively high product yields of 5.3\% (5.3 g enhanced green fluorescent protein eGFP per 100 g cell dry weight CDW) but required large quantities of mannose to induce the reactions, thus rendering the system's technical application rather expensive. To improve this situation, mutant B. subtilis strains were used: the Delta manA (mannose metabolism) strain TQ281 and the Delta manP (mannose uptake) strain TQ356. The total amount of inducer could be reduced with TQ281, which, however, displayed sensitivity to mannose. An inducer-independent self-induction system was developed with TQ356 to further improve the cost efficiency and product yield of the system, in which glucose prevents induction by carbon catabolite repression. To create optimal self-induction conditions, a glucose-limited process strategy, namely, a fed-batch process, was utilized as follows. The initiation of self-induction at the beginning of the glucose-restricted transition phase between the batch and fed-batch phase of fermentation and its maintenance throughout the glucose-limiting fed-batch phase led to a nearly 3-fold increase of product yield, to 14.6\%. The novel B. subtilis self-induction system thus makes a considerable contribution to improving product yield and reducing the costs associated with its technical application.

Abstract

In the field of metabolomics, GC-MS has rather established itself as a tool for semi-quantitative strategies like metabolic fingerprinting or metabolic profiling. Absolute quantification of intra- or extracellular metabolites is nowadays mostly accomplished by application of diverse LC-MS techniques. Only few groups have so far adopted GC-MS technology for this exceptionally challenging task. Besides numerous and deeply investigated problems related to sample generation, the pronounced matrix effects in biological samples have led to the almost mandatory application of isotope dilution mass spectrometry (IDMS) for the accurate determination of absolute metabolite concentrations. Nevertheless, access to stable isotope labeled internal standards (ILIS), which are in many cases commercially unavailable, is quite laborious and very expensive. Here we present an improved and simplified gas chromatography-isotope dilution mass spectrometry (GC-IDMS) protocol for the absolute determination of intra- and extracellular metabolite levels. Commercially available C-13-labeled algal cells were used as a convenient source for the preparation of internal standards. Advantages as well as limitations of the described method are discussed. (C) 2011 Elsevier B.V. All rights reserved.

Abstract

Current messenger RNA (mRNA) quantification methods are sophisticated tools for the analysis of gene regulation. However, these methods are not suitable for more complex quantitative approaches such as the mathematical modeling of the in vivo regulation of transcription where dynamic cytosolic mRNA concentrations need to be taken into consideration. In the current study, the ``standard curve method'' for quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR) was extended by including an internal RNA standard. This standard enables transcript losses that occur during the process, as well as variations resulting from nonquantitative processes, to be accounted for. The use of an internal standard yielded transcript concentration estimates that were on average seven times higher than those in cases where an internal standard is omitted. Choosing the cra modulon in Escherichia coli as an example, the method applied shows that the regulation of the Cra protein, as well as the growth rate-dependent regulation, need to be taken into consideration. The new method, which enables the determination of cytosolic mRNA concentrations, allows the quantitative representation of transcriptional dynamics. This is an important aspect of the analysis of the complex interactions of metabolism and regulation and in the application of mathematical modeling for systems biology. (C) 2009 Elsevier Inc. All rights reserved.

Abstract

The field of systems biology is often held back by difficulties in obtaining comprehensive, high-quality, quantitative data sets. In this paper, we undertook an interlaboratory effort to generate such a data set for a very large number of cellular components in the yeast Saccharomyces cerevisiae, a widely used model organism that is also used in the production of fuels, chemicals, food ingredients and pharmaceuticals. With the current focus on biofuels and sustainability, there is much interest in harnessing this species as a general cell factory. In this study, we characterized two yeast strains, under two standard growth conditions. We ensured the high quality of the experimental data by evaluating a wide range of sampling and analytical techniques. Here we show significant differences in the maximum specific growth rate and biomass yield between the two strains. On the basis of the integrated analysis of the high-throughput data, we hypothesize that differences in phenotype are due to differences in protein metabolism.

Abstract

The majority of dynamic gene regulatory network (GRN) models are comprised of only a few genes and do not take multiple transcription regulation into account. The models are conceived in this way in order to minimize the number of kinetic parameters. In this paper, we propose a new approach for predicting kinetic parameters from DNA-binding site sequences by correlating the protein-DNA binding affinities with nucleotide sequence conservation. We present the dynamic modeling of the cra modulon transcription in Escherichia coli during glucose-limited fed-batch cultivation. The concentration of the Cra regulator protein inhibitor, fructose1,6-bis(phosphate), decreases sharply, eventually leading to the repression of transcription. Total RNA concentration data indicate a strong regulation of transcription through the availability of RNA polymerase. A critical assessment of the results of the model simulations supports this finding. This new approach for the prediction of transcription dynamics may improve the metabolic engineering of gene regulation processes. (C) 2009 Published by Elsevier Inc.

Abstract

The enzyme targets for the rational optimization of a Corynebacterium glutamicum strain constructed for valine production are identified by analyzing the control of flux in the valine/leucine pathway. The control analysis is based on measurements of the intracellular metabolite concentrations and on a kinetic model of the reactions in the investigated pathway. Data-driven and model-based methods are used and evaluated against each other. The approach taken gives a quantitative evaluation of the flux control and it is demonstrated how the understanding of flux control is used to reach specific recommendations for strain optimization. The flux control coefficients (FCCs) with respect to the valine excretion rate were calculated, and it was found that the control is distributed mainly between the acetohydroxyacid synthase enzyme (FCC = 0.32), the branched chain amino acid transaminase (FCC = 0.27), and the exporting translocase (FCC = 0.43). The availability of the precursor pyruvate has substantial influence on the valine flux, whereas the cometabolites are less important as demonstrated by the calculation of the respective response coefficients. The model is further used to make in-silico predictions of the change in valine flux following a change in enzyme level. A doubling of the enzyme level of valine translocase will result in. an. increase in valine flux of 31\%. By optimizing the enzyme levels with respect to valine flux it was found that the valine flux can be increased by a factor 2.5 when the optimal enzyme levels are implemented. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 754-762, 2009

Abstract

A time series of whole-genome transcription profiling of Escherichia coli K-12 W3110 was performed during a carbon-limited fed-batch process. The application of a constant feed rate led to the identification of a dynamic sequence of diverse carbon limitation responses ( e. g., the hunger response) and at the same time provided a global view of how cellular and extracellular resources are used: the synthesis of high-affinity transporters guarantees maximal glucose influx, thereby preserving the phosphoenolpyruvate pool, and energy-dependent chemotaxis is reduced in order to provide a more economic ``work mode.'' sigma(S)-mediated stress and starvation responses were both found to be of only minor relevance. Thus, the experimental setup provided access to the hunger response and enabled the differentiation of the hunger response from the general starvation response. Our previous topological model of the global regulation of the E. coli central carbon metabolism through the crp, cra, and relA/spoT modulons is supported by correlating transcript levels and metabolic fluxes and can now be extended. The substrate is extensively oxidized in the tricarboxylic acid (TCA) cycle to enhance energy generation. However, the general rate of oxidative decarboxylation within the pentose phosphate pathway and the TCA cycle is restricted to a minimum. Fine regulation of the carbon flux through these pathways supplies sufficient precursors for biosyntheses. The pools of at least three precursors are probably regulated through activation of the (phosphoenolpyruvate-)glyoxylate shunt. The present work shows that detailed understanding of the genetic regulation of bacterial metabolism provides useful insights for manipulating the carbon flux in technical production processes.

Abstract

The intracellular alarmone guanosine 3 `,5 `-bis(diphosphate) (ppGpp) has been thoroughly investigated over the past 40 years and has become one of the best-known effectors in bacterial physiology. ppGpp is also of great importance for biotechnological applications. Systems biology research, involving experimental and mathematical approaches, has contributed a great deal to uncovering the alarmone's complex regulatory effects. HPLC analysis and UV detection are used to quantify intracellular ppGpp. The samples analyzed also contain other phosphorylated guanine nucleotides and, therefore, are spiked with a standard ppGpp solution. A rapidly growing number of laboratories are turning to synthesizing the nucleotide in vitro involving time-consuming protocols and yielding only low amounts of ppGpp. The current article provides a protocol for the preparation of large quantities of a ribosome extract that contains high ppGpp synthesis activity. The demonstrated upscaling from shaking flask to bioreactor cultivation involves the continuous and refrigerated harvest of the biomass. C-13 NMR analysis enabled the structural characterization of the synthesis product and was complemented by mass spectrometry and methods that are commonly used to identify ppGpp. (C) 2008 Elsevier Inc. All rights reserved.

Abstract

A novel approach to C-13 metabolic flux analysis (MFA) is presented using cytosolic metabolite pool sizes and their C-13 labeling data from an isotopically non-stationary C-13 labeling experiment (INST-CLE). The procedure is demonstrated with an E. coli wild type strain grown at fed batch conditions. The intra cellular labeling dynamics are excited by a sudden step increase of the C-13 portion in the substrate feed. Due to unchanged saturation of the substrate uptake system, the metabolic fluxes remain constant during the following sampling time period of only 16 s, in which 20 samples are taken by an automated rapid sampling device immediately stopping metabolism by methanol quenching. Subsequent cell disruptive sample preparation and LC-MS/MS enabled simultaneous determination of pool sizes and mass isotopomers of intra cellular metabolites requiring detection limits in the nM range. Based on this data the new computational flux analysis tool 13CFLUX/INST is used to determine the intra cellular fluxes based on a complex carbon labeling network model. The measured data is in good agreement with the model predictions, thus proving the applicability of the new isotopically non-stationary C-13 metabolic flux analysis (INST-C-13-MFA) concept. Moreover, it is shown that significant new information with respect to flux identifiability, non-measurable pool sizes, data consistency, or large storage pools can be taken from the novel kind of experimental data. This offers new insight into the biological operation of the metabolic network in vivo. (c) 2006 Elsevier B.V. All rights reserved.

Abstract

Systems biology is attracting significant interest finding applications not only in pharmaceutical development but also for basic studies on microbial systems. The latter often concentrate on the quantitative understanding of global regulation phenomena. So far. these activities are dominated by academic groups basically mirroring the necessity to prepare the sound scientific understanding first, before industrial applications can be derived later. However, this short-term view may not be sufficient because systems biology already offers numerous benefits for industrial applications, provided that special constraints are considered. This contribution indicates some of the constraints worth noticing when industrial systems biology projects are carried out. Consequently, differences in project structure and goals between purely academic and industrial systems biology projects are outlined. (c) 2007 Elsevier B.V All rights reserved.

Abstract

With the increasing use of metabolomics as a means to study a large number of different biological research questions, there is a need for a minimal set of reporting standards that allow the scientific community to evaluate, understand, repeat, compare and re-investigate metabolomics studies. Here we propose, a first draft of minimal requirements to effectively describe the biological context of metabolomics studies that involve microbial or in vitro biological subjects. This recommendation has been produced by the microbiology and in vitro biology working subgroup of the Metabolomics Standards Initiative in collaboration with the yeast systems biology network as part of a wider standardization initiative led by the Metabolomics Society. Microbial and in vitro biology metabolomics is defined by this sub-working group as studies with any cell or organism that require a defined external medium to facilitate growth and propagation. Both a minimal set and a best practice set of reporting standards for metabolomics experiments have been defined. The minimal set of reporting standards for microbial or in vitro biology metabolomics experiments includes those factors that are specific for metabolomics experiments and that critically determine the outcome of the experiments. The best practice set of reporting standards contains both the factors that are specific for metabolomics experiments and general aspects that critically determine the outcome of any microbial or in vitro biological experiment.

Abstract

A highly selective and sensitive method for identification and quantification of intracellular metabolites involved in central carbon metabolism (including glycolysis, pentose phosphate pathway and tricarboxylic acid cycle) by means of liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) was developed. The volatile ion pair modifier tributylammonium acetate (TBAA) was employed in the mobile phase for simultaneously separation of 29 negatively charged compounds including sugar phosphates, nucleotides, and carboxylic acids on a common C 18 reversed-phase column. Method validation results displayed that limits of detection (LODs) calculated according to DIN (German Institute for Standardization) 32645 are mostly below 60 nM, only with the exception of pyruvate and malate. The calibration curves showed excellent linearity mainly over three orders of magnitude with correlation coefficients R-2 > 0.9982. This LC-MS/MS method was successfully applied to determine these metabolites in cell extracts of Escherichia coli. Most of the intracellular metabolites were found within the detection range and the relative standard deviations of the measurements were smaller than 5.65\% (n = 5) for a cell extract sample. (c) 2007 Elsevier B.V. All rights reserved.

Abstract

One fundamental shortcoming of biotechnological processes operating under carbon-limiting conditions is the high-energy demand (maintenance) of the cells. Although the function of the central carbon metabolism in supplying precursors and energy for biosynthesis has been thoroughly characterized, its regulation and dynamic behaviour during carbon-limited growth has not yet been revealed. The current work demonstrates a time series of metabolic flux distributions during fed-batch cultivation of Escherichia coli K-12 W3110 applying a constant feed rate. The fluxes in glycolysis, pentose phosphate pathway and biosynthesis fell significantly, whereas TCA cycle fluxes remained constant. The flux redistribution resulted in an enhanced energy generation in the TCA cycle and consequently, in a 20\% lower biomass yield. The intracellular alarmones ppGpp and cAMP accumulated in large quantities after the onset of nutrient limitation, subsequently declining to basal levels. The network topology of the regulation of the central metabolic pathways was identified so that the observed metabolic and regulatory behaviour can be described. This provides novel aspects of global regulation of the metabolism by the cra, crp and relA/spoT modulons. The work constitutes an important step towards dynamic mathematical modelling of regulation and metabolism, which is needed for the rational optimization of biotechnological processes. (c) 2007 Elsevier B.V. All rights reserved.

Abstract

Two new concepts, ``Limitation Potential'' and ``Constraint Limitation Sensitivity'' are introduced that use definitions derived from metabolic flux analysis (MFA) and metabolic network analysis (MNA). They are applied to interpret a measured flux distribution in the context of all possible flux distributions and thus combine MFA with MNA. The proposed measures are used to quantify and compare the influence of intracellular fluxes on the production yield. The methods are purely based on the stoichiometry of the network and constraints that are given from irreversible fluxes. In contrast to metabolic control analysis (MCA), within this approach no information about the kinetic mechanisms are needed. A limitation potential (LP) is defined as the reduction, of the reachable (theoretical) maximum by a measured flux. Measured fluxes that strongly narrow the reachable maximum are assumed to be limiting as the network has no ability to counterbalance the restriction due to the observed flux. In a second step, the sensitivity of the reduced maximum is regarded. This measure provides information about the necessitated changes to reach higher yields. The methods are applied to interpret the capabilities of a network based on measured fluxes for a L-phenylalanine producer. The strain was examined by a series of experiments and three flux maps of the production phase are analyzed. It can be shown that the reachable, yield is drastically reduced by the measured efflux into the TCA cycle, while the oxidative pentose-phosphate pathway only plays a secondary role on the reachable maximum. (c) 2006 Wiley Periodicals, Inc.

Abstract

The intracellular concentrations of the valine and leucine pathway intermediates in a Corynebacterium glutamicum strain were measured during a transient state. The data were obtained by performing a glucose stimulus-response experiment with the use of a rapid sampling device and advanced mass spectrometry. The glucose stimulus resulted in a 3-fold increase in the intracellular pyruvate concentration within less than a second, demonstrating the very fast interactions in metabolic networks. The samples were taken at subsecond intervals for a time period of 25 s. The time courses of the metabolite concentrations formed the experimental basis of a mathematical model simulating the fluxes and concentrations in the valine/leucine pathway. The implementation of a model selection criterion based on the second law of thermodynamics is demonstrated to be essential for the identification of realistic and unique models. Large differences between the enzyme properties determined in vitro and those determined in vivo by the model were observed with the in vivo maximal rates being almost an order of magnitude larger than the in vitro maximal rates. The transamination of ketoisovalerate (KIV) to valine is carried out mainly by the transaminase B enzyme, with the transaminase C enzyme playing a minor role. The availability of the cofactors NADP and NADPH only has modest influence on the flux through the valine pathway, while the influence of NAD and NADH on the flux through the leucine pathway is negligible.

Abstract

The application of functionalised magnetic adsorbent particles in combination with magnetic separation techniques has received considerable attention in recent years. The magnetically responsive nature of such adsorbent particles permits their selective manipulation and separation in the presence of other suspended solids. Thus, it becomes possible to magnetically separate selected target species directly out of crude biological process liquors (e.g. fermentation broths, cell disruptates, plasma, milk, whey and plant extracts) simply by binding them on magnetic adsorbents before application of a magnetic field. By using magnetic separation in this way, the several stages of sample pretreatment (especially centrifugation, filtration and membrane separation) that are normally necessary to condition an extract before its application on packed bed chromatography columns, may be eliminated. Magnetic separations are fast, gentle, scaleable, easily automated, can achieve separations that would be impossible or impractical to achieve by other techniques, and have demonstrated credibility in a wide range of disciplines, including minerals processing, wastewater treatment, molecular biology, cell sorting and clinical diagnostics. However, despite the highly attractive qualities of magnetic methods on a process scale, with the exception of wastewater treatment, few attempts to scale up magnetic operations in biotechnology have been reported thus far. The purpose of this review is to summarise the current state of development of protein separation using magnetic adsorbent particles and identify the obstacles that must be overcome if protein purification with magnetic adsorbent particles is to find its way into industrial practice.

Abstract

Motivation: Supporting the evolutionary modeling process of dynamic biochemical networks based on sampled in vivo data requires more than just simulation. In the course of the modeling process, the modeler is typically concerned not only with a single model but also with sequences, alternatives and structural variants of models. Powerful automatic methods are then required to assist the modeler in the organization and the evaluation of alternative models. Moreover, the structure and peculiarities of the data require dedicated tool support. Summary: To support all stages of an evolutionary modeling process, a new general formalism for the combinatorial specification of large model families is introduced. It allows for automatic navigation in the space of models and excludes biologically meaningless models on the basis of elementary flux mode analysis. An incremental usage of the measured data is supported by using splined data instead of state variables. With MMT2, a versatile tool has been developed as a computational engine intended to be built into a tool chain. Using automatic code generation, automatic differentiation for sensitivity analysis and grid computing technology, a high performance computing environment is achieved. MMT2 supplies XML model specification and several software interfaces. The performance of MMT2 is illustrated by several examples from ongoing research projects.

Abstract

So far it is mainly transcriptome and proteome analysis that has been applied to elucidate the correlation between genotype and phenotype although thorough metabolome studies can provide substantial information on the control of the metabolism at the biochemical level. Stimulus-response experiments, i.e. the investigation of metabolism dynamics after a glucose pulse (pulse experiment), can be used to study the in vivo enzyme kinetics offering insight into underlying reaction mechanisms. Usually, this requires rapid cell quenching combined with cell inactivation to `freeze' the microbial metabolism response at a definite time-lag after pulse stimulation. To access the `frozen' metabolic reply, adequate analytical methods are needed to measure intracellular metabolite concentrations in the cell extract. As shown in the introductory review part, stimulus-response experiments were usually applied to study central metabolism dynamics in wildtype strains. Our own results, presented in the second part of the contribution, indicate that stimulus-response experiments should also be applied to analyse pathway dynamics in anabolic routes. Using the example of the aromatic amino acid pathway, an LC-MS/MS technique is presented that allows the quantification of intracellular pools of central metabolism as well as of the aromatic amino acid pathway. Based on the analytical approach metabolic profiling is performed to monitor the metabolism dynamics after a glucose pulse experiment allowing the conclusion that pulse stimulation is transmitted to the anabolic pathway of interest.

Abstract

A model discriminating experimental design approach for fed-batch processes has been developed and applied to the fermentative production Of L-valine by a genetically modified Corynebacterium glutamicum strain possessing multiple auxotrophies as an example. Being faced with the typical situation of uncertain model information based on preliminary experiments, model discriminating design was successfully applied to improve discrimination between five competing models. Within the same modeling and experimental design framework, also the planning of an optimized production process with respect to the total volumetric productivity is shown. Simulation results were experimentally affirmed, yielding an increased total volumetric productivity of 6.2 mM L-valine per hour. However, also so far unknown metabolic mechanisms were observed in the optimized process, underlining the importance of process optimization during modeling to avoid problems of extreme extrapolation of model predictions during the final process optimization. (C) 2005 Wiley Periodicals, Inc.

Abstract

A dynamic model of prokaryotic gene expression is developed that makes considerable use of gene sequence information. The main contribution arises from the fact that the combined gene expression model allows us to access the impact of altering a nucleotide sequence on the dynamics of gene expression rates mechanistically. The high level of detail of the mathematical model is considered as an important step towards bringing together the tremendous amount of biological in-depth knowledge that has been accumulated at the molecular level, using a systems level analysis (in the sense of a bottom-up, inductive approach). This enables to the model to provide highly detailed insights into the various steps of the protein expression process and it allows us to access possible targets for model-based design. Taken as a whole, the mathematical gene expression model presented in this study provides a comprehensive framework for a thorough analysis of sequence-related effects on the stages of mRNA synthesis, mRNA degradation and ribosomal translation, as well as their nonlinear interconnectedness. Therefore, it may be useful in the rational design of recombinant bacterial protein synthesis systems, the modulation of enzyme activities in pathway design, in vitro protein biosynthesis, and RNA-based vaccination.

Abstract

Using a concerted approach of biochemical standard preparation, analytical access via LC-MS/MS, glucose pulse, metabolic profiling, and statistical data analysis, the metabolism dynamics in the aromatic amino acid pathway has been stimulated, monitored, and analyzed in different tyrosine-auxotrophic L-phenylalanine-producing Escherichia coli strains. During the observation window from -4 s (before) up to 27 s after the glucose pulse, the dynamics of the first five enzymatic reactions in the aromatic amino acid pathway was observed by measuring intracellular concentrations of 3-deoxy-D-arabino-heptulosonate 7-phosphate DAH(P), 3-dehydroquinate (3-DHQ), 3-dehydroshikimate (3-DHS), shikimate 3-phosphate (S3P), and shikimate (SHI), together with the pathway precursors phosphoenolpyruvate (PEP) and P5P, the lumped pentose phosphate pool as an alternative to the nondetectable erythrose 4-phosphate (E4P). Provided that a sufficient fortification of the carbon flux into the pathway of interest is ensured, respective metabolism dynamics can be observed. On the basis of the intracellular pool measurements, the standardized pool velocities were calculated, and a simple, data-driven criterion-called ``pool efflux capacity'' (PEC)-is derived. Despite its simplifying system description, the criterion managed to identify the well-known AroB limitation in the E. coli strain A (genotype Delta(pheA tyrA aroF)/pJF119EH aroF(fbr) pheA(fbr) amp) and it also succeeded to identify AroL and AroA (in strain B, genotype Delta(pheA tyrA aroF)/pJF119EH aroF(fbr) pheA(fbr) aroB amp) as promising metabolic engineering targets to alleviate respective flux control in subsequent L-Phe producing strains. Furthermore, using of a simple correlation analysis, the reconstruction of the metabolite sequence of the observed pathway was enabled. The results underline the necessity to extend the focus of glucose pulse experiments by studying not only the central metabolism but also anabolic pathways.

Abstract

Using our recently developed sensor reactor approach, lysine-producing, nongrowing Corynebacterium glutamicum MH20-22B cells were subjected to serial (13)C-labeling experiments for flux analysis during the leucine-limited fed-batch production phase in a 300-L bioreactor. Based on two-dimensional (2D) nuclear magnetic resonance (NMR) measurements of (13)C-labeling patterns of cytoplasmic free metabolites, metabolic flux distributions in the central metabolism were successfully determined. Focusing on the highly concentrated metabolite L-glutamate, the working hypothesis was validated that the equilibration of labeling patterns in intracellular pools was much faster (up to 9.45 min) than the labeling period (3 h) used in the experiments. Analysis of anaplerotic reactions revealed that highly selective lysine production was accompanied by a significant reduction of decarboxylating reactions from 10 mol\% to only 2 mol\%, whereas PEP/pyruvate-carboxylating fluxes remained constant at about 40 mol\% of consumed glucose. These results support the conclusion that an optimized C. glutamicum L-lysine producer should possess increased PEP carboxylase and/or pyruvate carboxylase activity combined with downregulated, decarboxylating fluxes consuming oxaloacetate/malate. The findings also illustrate the usefulness of the sensor reactor approach in the study of industrial fermentations. (C) 2004 Wiley Periodicals, Inc.

Abstract

Using the pyruvate production strain Escherichia coli YYC202 IdhA::Kan different process alternatives are studied with the aim of preventing potential product inhibition by appropriate product separation. This strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. Continuous experiments with cell retention, repetitive fed-batch, and an in situ product recovery (ISPR) process with fully integrated electrodialysis were tested. Although the continuous approach achieved a high volumetric productivity (Q(P)) of 110 g L(-1) d(-1), this approach was not pursued because of long-term production strain instabilities. The highest pyruvate/glucose molar yield of up to 1.78 mol mol(-1) together with high Q(P) 145 g L(-1) d(-1) and high pyruvate titers was achieved by the repetitive fed-batch approach. To separate pyruvate from fermentation broth a fully integrated continuous process was developed. In this process electrodialysis was used as a separation unit. Under optimum conditions a (calculated) final pyruvate titer of >900 mmol L(-1) (79 g L(-1)) was achieved. (C) 2004 Wiley Periodicals, Inc.

Abstract

The contribution intends to elucidate chances and pitfalls of in situ product removal (ISPR) approaches by focusing on microbial processes for the production of low molecular products. With the aid of illustrative examples of industry-like and industrial processes, common characteristics are outlined which are hold to be important for successful commercialization. Finally, the attempt of a future prognosis for ISPR processes is given.

Abstract

A novel in situ product recovery (ISPR) approach for the (fully) integrated separation of L-phenylalanine (L-phe) from a 20 l fed-batch process with the recombinant L-tyrosine auxotrophic strain E. coli F-4/pF81 is presented. The strain was rationally constructed for the production of the aromatic amino acid. Glucose and tyrosine control is used. A reactive extraction system consisting of kerosene, the cation-selective carrier D(2)EHPA and sulphuric acid, all circulating in liquid-liquid centrifuges, is applied for the on-line L-phe separation from cell- and protein-free permeate. Permeate is drained off from the bioreactor bypass. Using the novel ISPR approach, a significantly extended product formation period at 0.25 mmol/(g*h) together with a reduced by-product formation and a 28\% relative glucose/L-phe yield increase is observed. Thus, the ISPR approach is superior to the reference non-ISPR process and even offers extraction rates approximately three times higher than the published membrane-based process.

Abstract

With the aid of the recently developed Sensor reactor system, a series of three subsequent C-13 labeling experiments was performed mirroring the L-phenylalanine (L-Phe) production phase of a recombinant E. coli strain that was cultivated under industry-like conditions in a 300 L bioreactor. On the basis of the data from NMR labeling analysis, three subsequent flux patterns were successfully derived monitoring the L-Phe formation (luring an observation window from 14 to 23.3 h process time. Linear programming was performed to identify optimal flux patterns for L-Phe formation. Additionally, flux sensitivity analysis was used to identify the most promising metabolic engineering target. As a result, high rates of phosphoenolpyruvate (PEP) to pyruvate (PYR) conversion were identified as the most important reason for deterioration of the L-Phe/glucose yield from 20 to finally 11 mol \%. Considering the characteristics of the enzyme kinetics involved, the working hypothesis was formulated that phosphoenolpyruvate synthase activity was increasingly hampered by rising oxaloacetate and 2-oxoglutarate concentrations, while at the same time pyruvate kinase activity arose due to activation by fructose 1,6-diphosphate. Hence, pps overexpression should be performed to optimize the existing production strain.

Abstract

Two different model reduction strategies are studied in order to test their applicability to reduce complex metabolism models. Using a model of one pre-identified model set describing complex metabolic dynamics after glucose pulse stimulation, a model reduction method based on the parameter tuning importance is compared with a pea based approach. Up to 49 of 122 parameters are rejected without significant changes of the simulated trajectories and of the flux distribution. Applying the reduction procedure to 12 other dynamic models reveals a general model structure inconsistency within the description of the pentose phosphate pathway. That points out the need of additional experiments to reproduce metabolite courses especially of this metabolic pathway. Thus the sensitivity based model reduction procedure is qualified as a promising tool for the model structure check and can be very useful for the entire model validation process which also includes the critical analysis of the data sets underlying the models. (C) 2004 Elsevier Ltd. All rights reserved.

Abstract

A continuous-flow technique has been developed to analyse the deltaD and delta(13)C values for CH4 from gas samples, in a single run. This is achieved by splitting the sample gas stream and directing the streams simultaneously through a CuNiPt combustion reactor and an alumina pyrolysis reactor. The CO2 from CH4 combustion is trapped in a liquid nitrogen trap while the H-2 exiting the pyrolysis reactor is directed to the mass spectrometer for deltaD(CH4) determination. The CO2 is then sublimed and directed to the mass spectrometer for delta(13)C(CH4) determination. Sample runs take approximately 10 minutes. This technique gives accurate delta(13)C(CH4) results to within +/-0.3-0.5parts per thousand and deltaD(CH4) results to within +/-2-5parts per thousand. Injection volumes between 0.5 and 2.5 muL of CH4, equivalent to between 20 and 100 nmol CH4, are required for accurate delta(13)C and deltaD analyses, respectively, using sample injection into a split flow with a split ratio of 10. This method provides rapid, accurate and reproducible results on multiple sample runs and is, therefore, an ideal method for analysing natural gas samples from a variety of sources. Copyright (C) 2003 John Wiley Sons, Ltd.

Abstract

Arthrobacter crystallopoietes produces a D-specific hydantoinase and D-N-carbamoylase useful for the industrial production of enantiomerically pure D-amino acids. The D-hydantoinase was purified by conventional chromatographic methods. The enzyme was digested by trypsine and some of the oligopeptides sequenced. The amino acid sequence information was used to isolate a DNA fragment of the corresponding gene. By several steps of inverse PCR a region of 6011 bp was obtained from the A. crystallopoietes genome surrounding the hydantoinase gene fragment. DNA sequence analysis revealed the presence of a D-hydantoinase, the D-N-carbamoylase and a putative L-N-carbamoylase on the 6011 bp fragment. In addition, two incomplete open reading frames (ORFs) showed similarity to LacI type transcriptional regulators and transmembrane proteins responsible for the uptake of nucleosides and allantoin, respectively. The D-hydantoinase gene and the D-N-carbamoylase gene were expressed in Escherichia coli and the enzyme activity was determined. From the putative L-N-carbamoylase gene, expressed in the same way in E. coli, only insoluble protein was obtained and no carbamoylase activity was detected.

Abstract

The contribution intends to elucidate chances and pitfalls of in situ product removal (ISPR) approaches by focusing on microbial processes for the production of low molecular products. With the aid of illustrative examples of industry-like and industrial processes, common characteristics are outlined which are hold to be important for successful commercialization. Finally, the attempt of a future prognosis for ISPR processes is given.

Abstract

Based on experimental data from fermentation runs, as well as from L-phenylalanine (L-Phe) separation studies, a simple model is presented that describes the total ISPR approach for on-line L-Phe separation. While fermentation process modeling via a macrokinetic model revealed an L-Phe inhibition constant of 20 +/- 1.35 g/L using recombinant E. coli cells, the reactive-extraction process modeling identified the L-Phe cation diffusion in the aqueous donor film and the transport of the lowly soluble carrier/ L-Phe complex in the aqueous acceptor film as the most dominant transfer steps. The corresponding mass transfer coefficients were estimated as k(PheD) = 128 x 10(-7) cm/s (extraction) and k(CPheA) = 178 x 10(-5) cm/s (back-extraction). Simulation studies were performed for the total ISPR approach, which gave hints for strategies of further process optimization.

Abstract

A family of 10 competing, unstructured models has been developed to model cell growth, substrate consumption, and product formation of the pyruvate producing strain Escherichia coli YYC202 ldhA::Kan strain used in fed-batch processes. The strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate (using glucose as the carbon source) resulting in an acetate auxotrophy during growth in glucose minimal medium. Parameter estimation was carried out using data from fed-batch fermentation performed at constant glucose feed rates of q(VG)=10 mL h(-1). Acetate was fed according to the previously developed feeding strategy. While the model identification was realized by least-square fit, the model discrimination was based on the model selection criterion (MSC). The validation of model parameters was performed applying data from two different fed-batch experiments with glucose feed rate q(VG)=20 and 30 mL h(-1), respectively. Consequently, the most suitable model was identified that reflected the pyruvate and biomass curves adequately by considering a pyruvate inhibited growth (Jerusalimsky approach) and pyruvate inhibited product formation (described by modified Luedeking-Piret/Levenspiel term).

Abstract

A novel Sensor Reactor technology is presented which permits (13)C labeling experiments for metabolic flux analysis during large-scale, semi-industrial, (fed-) batch fermentation processes deriving a series of flux maps that document fermentation courses in detail. The small-scale Sensor Reactor can be inoculated within 1.50-1.20 s via a special inoculation unit with an inoculation volume accuracy of 1.025 +/- 0.021 L. The large-scale production reactor (here: 300 L) and the Sensor Reactor were run in parallel master/ slave modes to control the current pH, temperature, pressure and dissolved oxygen values as changing set points for the Sensor Reactor. Using an automated pulsing technology, glucose pulses of 5 g/L could be realized within 0.51 s. The similarity of fermentations in the Sensor Reactor with the production process was demonstrated by studying L-lysine production with C. glutamicum during multiple, `simulated' labeling experiments each lasting 2.5 h. `Real' labeling experiments are presented in Part II. (C) 2003 Elsevier Science (USA). All rights reserved.

Abstract

Cyclohexadiene-trans-5,6-diols such as (SS)-2,3-dihydroxy-2,3-dihydrobenzoic acid (2,3-trans-CHD) have been shown to be of importance as chiral starting materials for the syntheses of bioactive substances, especially for the syntheses of carbasugars. By using methods of metabolic-pathway engineering, the Escherichia coli genes entB and entC, which encode isochorismatase and isochorismate synthase, were cloned and over-expressed in E. coli strains with a deficiency of entA, which encodes 2,3-dihydroxybenzoate synthase. A 30-fold increase in the corresponding EntB/EmC enzyme activities affects the accumulation of 2,3-trans-CHD in the cultivation medium. Although the strains did not contain deletions in chorismate-utilising pathways towards aromatic amino acids, neither chorismate nor any other metabolic intermediates were found as by-products. Fermentation of these strains in a 30 L pH-controlled stirred tank reactor showed that 2,3-trans-CHD could be obtained in concentrations of up to 4.6 g L-1. This demonstrates that post-chorismate metabolites are accessible on a preparative scale by using techniques of metabolic-pathway engineering. Isolation and separation from fermentation salts could be performed economically in one step through anion-exchange chromatography or, alternatively, by reactive extraction. Starting from 2,3-trans-CHD as an example, we established short syntheses towards new carbasugar derivatives.

Abstract

Corynebacterium glutamicum is intensively used for the industrial large-scale (fed-) batch production of amino acids, especially glutamate and lysine. However, metabolic flux analyses based on C-13-labeling experiments of this organism have hitherto been restricted to small-scale batch conditions and carbon-limited chemostat cultures, and are therefore of questionable relevance for industrial fermentations. To lever flux analysis to the industrial level, a novel Sensor Reactor approach was developed (E1 Massaoudi et al., Metab. Eng., submitted), in which a 300-L production reactor and a 1-L Sensor Reactor are run in parallel master/slave modus, thus enabling C-13-based metabolic flux analysis to generate a series of flux maps that document large-scale fermentation courses in detail. We describe the successful combination of this technology with nuclear magnetic resonance (NMR) analysis, metabolite balancing methods and a mathematical description of C-13-isotope labelings resulting in a powerful tool for quantitative pathway analysis during a batch fermentation. As a first application, C-13-based metabolic flux analysis was performed on exponentially growing, lysine-producing C. glutamicum MH20-22B during three phases of a pilot-scale batch fermentation. By studying the growth, (co-) substrate consumption and (by-) product formation, the similarity of the fermentations in production and Sensor Reactor was verified. Applying a generally applicable mathematical model, which included metabolite and carbon labeling balances for the analysis of proteinogenic amino acid C-13-isotopomer labeling data, the in vivo metabolic flux distribution was investigated during subsequent phases of exponential growth. It was shown for the first time that the in vivo reverse C-4-decarboxylation flux at the anaplerotic node in C. glutamicum significantly decreased (70\%) in parallel with threefold increased lysine formation during the investigated subsequent phases of exponential growth. (C) 2003 Elsevier Inc. All rights reserved.

Abstract

Fed-batch fermentation was applied to the production of pyruvate by using a recombinant Escherichia coli YYC202 strain. This strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. By controlling acetate and glucose feed rate, a series of lab-scale fed-batch experiments were performed at pH 7 and 37 degrees C. CO2 production rate (CTR) was used for on-line regulation of the acetate feed rate. The correlation between CTR and acetate consumption rate (ACR) was determined experimentally. At optimal process conditions a final pyruvate concentration higher than 62 g/L, a space-time yield of up to 42 g/L/d and pyruvate/glucose molar yield of 1.11 mol/mol were achieved. Experimental evidence was gathered that pyruvate export is active.

Abstract

The atrazine-specific single-chain variable antibody fragments (scFv) K411B was produced by expression in either the cytoplasm or the periplasm of Escherichia coli BL21 (DE3). For periplasmic production, the pelB leader was N-terminally fused to scFv, whereas the unfused variant resulted in cytoplasmic expression. The extent of protein accumulation differed significantly. Expression of scFv with leader was 2.3 times higher than that of the protein without leader. This was further investigated by generating the respective translation profiles using coupled in vitro transcription/translation assays, the results of which were in agreement. This comparative approach was also applied to functionality: Periplasmic expression and in vitro expression resulted in only 10\% correctly folded scFv, indicating that the oxidizing environment of the periplasm did not increase proper folding. Thus, the data obtained in vitro confirmed the findings observed in vivo and suggested that the discrepancy in expression levels was due to different translation efficiencies. However, the in vivo production of scFv with enhanced green fluorescent protein (EGFP) fused C-terminally (scFv-EGFP) was only successful in the cytoplasm, although in vitro the expression with and without the leader rendered the same production profile as for scFv. This indicated that neither the translation efficiency nor the solubility but other factors impeded periplasmic expression of the fusion protein.

Abstract

A fully integrated process for the microbial production and recovery of the aromatic amino acid L-phenylalanine is presented. Using a recombinant L-tyrosine (L-Tyr) auxotrophic Escherichia coli production strain, a fed-batch fermentation process was developed in a 20-1-scale bioreactor. Concentrations of glucose and L-Tyr were closed-loop-controlled in a fed-batch process. After achieving final L-phenylalanine (L-Phe) titres >30 g/l the process strategy was scaled up to 300-1 pilot scale. In technical scale fermentation L-phenylalanine was continuously recovered via a fully integrated reactive extraction system achieving a maximum extraction rate of 110 g/h (final purity >99\%). It was thus possible to increase L-Phe/glucose selectivity from 15 mol\% without to 20.3 mol\% with integrated product separation.

Abstract

L-Hydantoinase from Arthrobacter aurescens (L-Hyd) is a member of the dihydropyrimidinases which in turn belong to the cyclic amidases. Dihydropyrimidinases catalyze the reversible hydrolytic ring opening of dihydropyrimidines as the second step in the catabolism of pyrimidines. In biotechnology, their hydroloytic activity on five-membered cyclic diamides (hydantoins) is used in the enantio-specific production of amino acids from racemic hydantoins. L-Hyd differs from most of the other dihydropyrimidinases by an L-enantio specificity and by lacking activity on possible natural substrates such as dihydropyrimidines. In this paper, we describe the three-dimensional structure of L-Hyd which Was solved by molecular replacement using a homology model and subsequently refined to 2.6 Angstrom resolution. Each subunit of the tetrameric L-Hyd consists of an elliptically distorted (alpha/beta)(s)-barrel domain, which hosts the active site, and a beta-sheet domain. In the active site, a binuclear zinc center activates a Water molecule for nucleophilic attack on the substrates' amide bond. L-Hyd shows a strong homology both in fold and in metal coordination in the active site to another dihydropyrimidinase from Thermus sp. (D-hydantoinase) and to a slightly lesser degree to ureases, dihydroorotase and phosphotriesterase. Using the homology to ureases, a model for the transition state was modeled in the active site of L-Hyd and D-hydantoinase. This model could provide an explanation for the different substrate and enantio selectivities of both dihydropyrimidinases.

Abstract

Pilot-scale reactive-extraction technology for fully integrated L-phenylalanine (L-Phe) separation in Escherichia coli fed-batch fermentations was investigated in order to prevent an inhibition of microbial L-Phe production by-product accumulation. An optimal reactive-extraction system, consisting of an organic kerosene phase with the cation-selective carrier DEHPA (di-2-ethylhexyl phosphonic acid) and an aqueous stripping phase including sulphuric acid, was found particularly efficient. Using this system with two membrane contactors, mass-transfer coefficients of up to 288x10(-7) cm s(-1) for the aqueous/organic and 77x10(-7) cm s(-1) for the organic/stripping phase were derived from experimental data using a simple modelling approach. Concentration factors higher than 4 were achieved in the stripping phase as compared to the aqueous donor phase. Reactive extraction enabled a 98\% cation portion of L-Phe in the stripping phase, leading to final product purity higher than 99\% after L-Phe precipitation. A doubling of L-Phe/glucose yield was observed when kerosene/DEHPA was added to the fermentation solution in the bioreactor to experimentally simulate a fully integrated L-Phe separation process.

Abstract

Glucose pulse experiments were performed to elucidate their effects on the carbon flux into the aromatic amino acid pathway in different Escherichia coli strains. Using a 3-deoXy-D-arabino-heptulosonate 7-phosphate (DAHP, aroB(-))-producing strain, a fed-batch fermentation strategy specialized for glucose pulse experiments was developed and further applied for 3-dehydroshikimate (DHS, aroE(-))- and shikimate 3-phosphate (S3P, aroA(-))-producing E. coli strains. The strains overexpress a feedback-resistant DAHP synthase and additional enzymes to prevent rate-limiting steps in the aromatic amino acid pathway. Changes of carbon flux into the aromatic amino acid pathway were determined via extracellular metabolite accumulations using (1)H NMR and HPLC measurements. As an important result, a close relationship between pulse intensity and aromatic metabolite formation rates was identified. The more downstream an aromatic pathway intermediate was located, the stronger the glucose pulse intensity had to be in order to detect significant changes in product formation. However, with the experimental conditions chosen, changes after pulse were detected even for shikimate 3-phosphate, the most downstream accumulating metabolite of this experimental series. Hence glucose pulse experiments are assumed to be a promising tool even for the analysis of final pathway products such as, for example, L-phenylalanine.

Abstract

A novel fed-batch approach for the production of L-phenylalanine (L-Phe) with recombinant E. coli is presented concerning the on-line control of the key fermentation parameters glucose and tyrosine. Two different production strains possessing either the tyrosine feedback resistant aroF(fbr) (encoding tyrosine feedback resistant DAHP-synthase (3-desoxy-D-arabinoheptusonate-7-phosphate)) or the wild-type aroF(wt) were used as model systems to elucidate the necessity of finding an individual process optimum for each genotype. With the aid of tyrosine control, wild-type aroF(wt) could be used for L-Phe production achieving higher final L-Phe titers (34 g/L) than the aroF(fbr) strain (28 g/L) and providing higher DAHP-synthase activities. With on-line glucose control, an optimum glucose concentration of 5 g/L could be identified that allowed a sufficient carbon supply for L-Phe production while at the same time an overflow metabolism leading to acetate by-product formation was avoided. The process approach is suitable for other production strains not only in lab-scale but also in pilot-scale bioreactors. (C) 2002 Wiley Periodicals, Inc.

Abstract

A cascade of hydantoinase, N-carbamoylase and hydantoinracemase can be used for the production of natural and unnatural chiral D- and L-amino acids from chemically synthesized hydantoin derivatives. Potentially, 100\% conversion and 100\% optically pure amino acids can be obtained at the same time if racemic substrates are used. Recent research activities concentrate on newly isolated or improved enzymes and include directed evolution techniques, structure elucidation, studies of fusion proteins and the use of specially designed whole cell biocatalysts.

Abstract

A high-cell-density fed-batch fermentation for the production of heterologous proteins in Escherichia coli was developed using the positively regulated Escherichia coli rhaBAD promoter. The expression system was improved by reducing of the amount of expensive L-rhamnose necessary for induction of the rhamnose promoter and by increasing the vector stability. Consumption of the inducer L-rhamnose was inhibited by inactivation of L-rhamnulose kinase encoding gene rhaB of Escherichia coli W3110, responsible for the first irreversible step in rhamnose catabolism. Plasmid instability caused by multimerization of the expression vector in the recombination-proficient W3110 was prevented by insertion of the multimer resolution site cer from the ColE1 plasmid into the vector. Fermentation experiments with the optimized system resulted in the production of 100 g L-1 cell dry weight and 3.8 g L-1 of recombinant L-N-carbamoylase, an enzyme, which is needed for the production of enantiomeric pure amino acids in a two-step reaction from hydantoins, (C) 2001 John Wiley & Sons, Inc.

Abstract

Continuous fermentation was applied to the production of recombinant human chymotrypsinogen B (hCTRB) by the methylotrophic yeast Pichia pastoris as a tool for the kinetic analysis of growth and product formation. Using methanol as the sole source of carbon, energy, and induction, cell growth could be described by a non-competitive M ON approach. The maximum growth rate p a,, was determined to be 0.084 h(-1) and the K(M)-value for methanol to 0.22 g L(-1), respectively. With respect to product formation a similar model was established exhibiting a methanol concentration of 0.13 g L(-1) as the K(M)-value and a maximum biomass-specific product-formation rate Of pi(max) = 0.23 mg g(-1) h(-1). The production of hCTRB was strictly growth-coupled. The data provided covers the range of methanol concentrations between 0 and 4 g L-1. Substrate concentrations exceeding this upper value led to a complete collapse of product formation. This change in phenotype turned out to be irreversible indicating a genetic instability of transformed Pichia pastoris caused by excess methanol.

Abstract

The quantitative comprehension of microbial metabolic networks is a prerequisite for an efficient rational strain improvement (''metabolic engineering''). It is therefore necessary to accurately determine the concentration of a large number of reactants (i.e., metabolites, nucleotides, cofactors) in order to understand ``in vivo'' reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates and nucleotides in Escherichia coli K12 using a perchloric acid extraction and an LC-ESI-MS method was achieved. Intracellular metabolites (e.g., glucose 6-phosphate, fructose 1,6-bisphosphate, 6-phospho gluconate, acetyl-CoA, adenine nucleotides) were quantified under defined (glucose-limited steady-state) growth conditions. The method was verified by comparing the intracellular metabolite concentrations measured via LC-ESI-MS with enzymatic determinations. It is thus possible to identify and quantify more than 15 intracellular metabolites in parallel with a minimal amount of sample volume. (C) 2001 Academic Press.

Abstract

Based on experimental results of eight fed-batch fermentations using a recombinant L-phenylalanine-producing Escherichia coli strain, the applicability of principal-component analysis (PCA) for fermentation analysis was studied. Three principal components were identified, representing approximately 90\% of total variance. Among them, concentrations of tyrosine and acetate were identified as key fermentation parameters. Their significance was also confirmed when measurement errors were taken into consideration by Monte-Carlo estimations. The error estimation approach was also used to investigate PCA suitability for the time-specific analysis of different fermentation phases. Relatively large principal-component score errors were calculated that limit the applicability of PCA for detailed fermentation course analysis.

Abstract

This study provides a mathematical model of T7 RNA polymerase (T7 RNAP) kinetics under in vitro conditions targeted at application of this model to simulation of dynamic transcription performance. A functional dependence of transcript synthesis rate is derived based on: (a) essential reactant concentrations, including T7 RNAP and its promoter, substrate nucleotides, and the inhibitory byproduct inorganic pyrophosphate; (b) a distinction among vector characteristics such as recognition sequences regulating transcription initiation and termination, respectively; and (c) specific properties of the nucleotide sequence including both transcript length and nucleotide composition. Inactivation kinetics showed a half-life of T7 RNAP activity of 50 min under the conditions applied in vitro using the isolated enzyme. Model parameters and their precision are estimated using dynamic simulation and nonlinear regression analysis. The particular novelty of this model is its capability to incorporate linear genomic sequence information for simulation of nonlinear in vitro transcription kinetics. (C) 2001 John Wiley & Sons, Inc.

Abstract

The regioselective oxidation of terfenadine with the fungi cunninghamella blakesleeana was studied as a biochemical alternative for the chemical synthesis of the antihistaminic drug fexofenadine. It was demonstrated that C. blakesleeana oxidises the tert-butyl group of terfenadine to the corresponding alcohol 1-4-(1,1-dimethyl-2-hydroxyethyl)phenyl-4-4-(hydroxydiphenylmethy l)-1-piperidinyl1-butanol. A continuous process for regioselective oxidation of terfenadine was developed. Terfenadine was supplied micro-crystalline due to the low solubility in water. Optimum reaction conditions with respect to medium composition, temperature, pH, pO(2), co-substrate and feeding rates were found by means of reaction engineering studies. A cross-flow microfiltration unit was operated in a by-pass of a lab-scale stirred tank reactor for retention of the biocatalysts and the micro-crystalline substrate. The alcohol was continuously removed with the filtrate to minimise product inhibition. Continuous biotransformation of micro-crystalline terfenadine with C. blakesleeana in the membrane reactor system with a dilution rate of 33 h at co-substrate concentrations of about 1 up to 3 g/l glycerol in the reactor resulted in a space-time yield of 145 mg of alcohol/l/day and an alcohol yield of 71\%. The produced alcohol was easily isolated from the filtrate by adsorption on XAD-4 resin followed by eluation with methanol (concentration factor 7). (C) 2000 Elsevier Science B.V. All rights reserved.

Abstract

Cell-free extracts (lysates) from Escherichia coli were used for protein synthesis in vitro. Essential steps of the lysate preparation were modified and analyzed with respect to their impact on in vitro protein synthesis capacity, using the green fluorescent protein (GFP) as a target protein. Variably manufactured lysates of low, medium and higher protein synthesis activity, were examined by high resolution two-dimensional get electrophoresis to determine whether the modifications result in substantial alterations in protein composition of the final lysate. The total number of proteins calculated from the get maps did not vary for lysates with different activity and thus cannot serve as an evaluation parameter. Ribosomal proteins RP-S1, RP-L9, and RP-L10 were found in stoichiometric amounts for each of these lysates and in equal concentrations in comparison among the different lysates. Conversely, depending on the activity profiles, up to 7 different isoforms of the elongation factor EF-Ts were detected in the gel maps.

Abstract

Hydantoinases are valuable enzymes for the production of optically pure D- and L-amino acids. They catalyse the reversible hydrolytic ring cleavage of hydantoin or 5'-monosubstituted hydantoins and are therefore classified in the EC nomenclature as cyclic amidases (EC 3.5.2.). In the EC nomenclature, four different hydantoin-cleaving enzymes are described: dihydropyrimidinase (3,5.2.2), allantoinase (EC 3.5.2.5), carboxymethylhydantoinase (EC 3.5.2.4), and N-methylhydantoinase (EC 3.5.2.14). Beside these, other hydantoinases with known metabolic functions, such as imidase and carboxyethylhydantoinase and enzymes with unknown metabolic function, are described in the literature and have not yet been classified. An important question is whether the distinct hydantoinases, which are frequently classified as L-, D-, and non-selective hydantoinases depending on their substrate specificity and stereoselectivity, are related to each other. In order to investigate the evolutionary relationship, amino acid sequence data can be used for a phylogenetic analysis. Although most of these enzymes only share limited sequence homology (identity <15\%) and therefore are only distantly related, it can be shown (i) that most of them are members of a broad set of amidases with similarities to ureases and build a protein superfamily, whereas ATP-dependent hydantoinases are not related, (ii) that the urease-related amidases have evolved divergently from a common ancestor and (iii) that they share a metal-binding motif consisting of conserved histidine residues. The difference in enantioselectivity used for the classification of hydantoinases on the basis of their biotechnological value does not reflect their evolutionary relationship, which is to a more diverse group of enzymes than was assumed earlier. This protein superfamily probably has its origin in the prebiotic conditions of the primitive earth.

Abstract

A D-specific hydantoinase has been purified to homogeneity from Arthrobacter crystallopoietes DSM 20117 with a yield of 5\% related to the crude extract. The active enzyme is a tetramer of 257 kDa consisting of four identical subunits, each with a molecular mass of 60 kDa. Incubation of the enzyme with the metal-chelating agent EDTA had no inhibitory effect, while 8-hydroxyquinoline-5-sulfonic acid resulted in a complete and irreversible inactivation. The purified enzyme contains zinc as cofactor, which could be detected by subjection to direct analysis using inductive/coupled plasma-atomic emission spectrometry. The hydantoinase has a wide substrate specificity for the D-selective cleavage of 5-monosubstituted hydantoin derivatives with aliphatic and aromatic side chains. The V-max-value for phenylhydantoin is 217 U/mg, the K-m-value is 8 mM. Dihydrouracil was found to be a natural substrate (V-max = 35 U/mg). The N-terminal amino acid sequence of the enzyme shows distinct homologies to other metal-dependent cyclic amidases involved in the nucleotide metabolism especially to dihydropyrimidinases as well as to ureases, L- and unselective hydantoinases. Due to these findings, this enzyme has to be considered as a possible link in the evolution to related L-selective and unselective hydantoinases from the genus of Arthrobacter. (C) 1999 Elsevier Science B.V. All rights reserved.

Abstract

An automated sampling device coupled to a stirred tank reactor was developed for monitoring intracellular metabolite dynamics. Sample flasks fixed in transport magazines were moved by a step engine in a way that each sample flask was filled within 220 ms, resulting in a sampling rate of 4.5 s(-1). Rapid inactivation of the metabolism was achieved by spraying the samples into 60\% methanol at -50 degrees C. After centrifugation of the quenched cells at -20 degrees C the metabolites were extracted with perchloric acid and analyzed biochemically or with HPLC. The automated sampling device was applied for investigation of the intracellular metabolite dynamics of glycolysis in Escherichia coli after rapid glucose addition to a glucose-limited steady-state culture. For the first time oscillations of intracellular metabolite concentrations like glucose-6-phosphate, phosphoenolpyruvate, glyceraldehyde 3-phosphate, dihydroxyacetonphosphate, 3-phosphoglycerate, and pyruvate were quantified on a subseconds to seconds scale in E. coli. As an example, the kinetics of the decomposition of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetonphosphate were investigated by use of a well-known mechanistic kinetic model and the measured in vivo metabolite dynamics. (C) 1999 Academic Press.

Abstract

A cell-free extract from Escherichia coli, generated through a routine procedure according to Chen and Zubay (Methods Enzymol. 1983, 101, 674-690), was used for an in vitro protein synthesis. High-resolution two-dimensional gel electrophoresis (2-DE) was exploited to investigate the protein composition of the cell-extract and its dynamic development during a 24 h-period of cell-free protein synthesis performed in a membrane reactor device. Green fluorescent protein (GFP) was chosen as a target protein to be produced in a cell-free reactor because of its functional activity, which can easily be monitored by measurement of fluorescence, and because of its high sensitivity. GFP synthesis was observed by a standard fluorescence assay and was correlated to a quantitative assessment of the silver-stained GFP spot appearing on 2-DE gel maps. A constant protein synthesis rate was obtained for at least 8 h of process operation. While declining continuously, protein synthesis stopped entirely after 24 h. Both, the total protein content and total number of detectable spots were found to decrease over the reaction time, due to proteolytic digestion and protein precipitation. Certain proteins taking part in the translation process, such as the elongation factors (EF-Tu, EF-Ts) and the ribosomal protein RP-L9, were identified by Edman N-terminal sequencing and have thus been considered for reaction evaluation. The dynamics obtained during the entire process suggest that these translational factors were likewise affected by proteolytic decay.

Abstract

Metal dependency of the hydantoin amidohydrolase (hydantoinase) from Arthrobacter aurescens DSM 3745 has been analyzed based on kinetic studies of metal/chelator-caused enzyme inactivation, denaturation and reactivation, accompanied by the identification of specific metal binding ligands. The enzyme can be inactivated by metal chelating agents and-apart from the loss of its activity-completely dissociates into its subunits. Enzyme activity can be restored from recollected monomers by the addition of cobalt, manganese or zinc-ions, whereas nickel and magnesia remain ineffective. Subjection of the hydantoinase to metal analysis reveals a content of 10 mol zinc per mol enzyme. Zinc plays an essential role not only for the catalytic activity but also for the stabilization of the active quarternary structure of the hydantoinase. Histidine-specific chemical modification of the enzyme causes a complete loss of the catalytic activity and reveals histidine residues as putative zinc binding ligands. Both, the metal/chelator-caused enzyme inactivation as well as the metal-caused enzyme reactivation, can be reduced in the presence of the substrate. Therefore, it is very likely that at least one metal-ion acts specifically near or at the active site of the enzyme. (C) 1998 Elsevier Science B.V. All rights reserved.

Abstract

A hydantoinase from Arthrobacter aurescens DSM 3745 has been purified to homogeneity with a yield of 77\% using a three-step purification procedure. The active enzyme is a tetramer consisting of four identical subunits, each with a molecular mass of 49670 Da as determined by mass spectrometry. The N-terminal amino acid sequence of the enzyme indicates sequence identities to cyclic amidases involved in the nucleotide metabolism as the D-hydantoinase from Agrobacterium radiobacter (53\%), the D-selective dihydropyrimidinase from Bacillus stearothermophilus (38\%), the allantoinase from Rana catesbeiana (26\%), as well as to the catalytic subunit of the urease from Heliobacter pylori (50\%). However, all studies based on substrate-dependent growth, induction and catalytic behavior documented the novelty of the bacterial hydantoinase and that its physiological role is not related to any of these enzymes or known metabolic pathways. Its substrate specificity differs from hydantoinases listed in Enzyme Nomenclature and is rather more predominant for the cleavage of aryl-than for alkyl-hydantoin, derivatives. It is shown that the stereoselectivity of this enzyme depends on the substrate used for bioconversion: although it is strictly L-selective for the cleavage of D,L-5-indolylmethylhydantoin, it appears to be D-selective for the hydrolysis of D,L-methylthioethylhydantoin. Due to these findings we conclude that this novel bacterial hydantoinase should be classified as a new member of the EC-group 3.5.2 of cyclic amidases. (C) 1998 Elsevier Science B.V. All rights reserved.

Abstract

The hydantoin amidohydrolase (hydantoinase) from Arthrobacter aurescens DSM 3745 was purified to homogeneity and subjected to metal analysis under atomic absorption spectrometry (AAS) and inductive coupled plasma-atomic emission spectrometry (ICP-AES). Three independent preparations of homogeneous enzyme indicated that 1 mol of the active enzyme contains 10 mol zinc ions. This corresponds to 2.5 mol zinc per mol subunit, since the hydantoinase consists of four identical subunits. Only trace amounts of manganese, magnesia, nickel and cobalt were detected. Other metals were either absent or existed below detection levels. (C) 1998 Elsevier Science B.V. All rights reserved.

Abstract

The complete amino acid sequence of the hydantoinase from Arthrobacter aurescens DSM 3745 has been derived by automated Edman degradation. This is the fi rst ever reported amino acid sequence of a non-ATP-dependent hydantoinase, which hydrolyzes 5'-monosubstituted hydantoin derivatives L-selectively, A homology search performed in protein and nucleic acid databases retrieved only distantly related proteins. All of these are members of the recently described protein superfamily of amidohydrolases related to ureases (Holm and Sander, Proteins 28: 72-82, 1997). Phylogenetic analysis revealed that the novel hydantoinase forms a new branch separate from other hydantoin cleaving enzymes like dihydropyrimidinases (EC 3.5.2.2) and allantoinases (EC 3,5,2.5), Our results suggests that the enzymes of this protein superfamily have evolved from a common ancestor and therefore are the product of divergent evolution, We show further that the enclosed gene families developed very early in evolution, probably prior to the formation of the three domains, Archaea, Eukarya and Bacteria, Hydantoinases related to ATP-depdent N-methylhydantoinases (EC 3.5.2.14) or 5-oxoprolinases (EC 3.5.2.9) do not belong to this superfamily.

Abstract

A new and highly sensitive method was developed for the identification of hydantoinases on acrylamide gels. For this purpose, cell-lysates from different natural isolates are subjected on PAGE under non-denaturating conditions. The respective localisation of the hydantoinase is obtained by in situ product precipitation during the reverse enzyme reaction: in contrast to the used substrate (N-carbamoyltryptophan), the product (indolylmethylhydantoin) is barely soluble and gives a dense precipitation dot caused by crystallisation of the product inside of the polyacrylamide gel at the position corresponding to the location of the enzyme. This method can also be used for the subsequent differentiation between L- and D-selective hydantoinases, since L- or D-carbamoyltryptophan is used as substrate.

Abstract

An experimental design method for the identification of macrokinetic models was developed applying an extended D-optimal design criterion. The D-optimal design criterion was modified to consider variable measurement variances as well as multivariate macrokinetic models. The macrokinetics of formate dehydrogenase (FDH) production with Candida boidinii were thus identified within 10 steady state experiments in a labscale continuous stirred tank reactor (10 model parameters). Closed loop control (nutristat) was applied to set-up the operating states suggested by this experimental design method. After each set of steady state experiments the quality of macrokinetic parameters was characterized statistically. For model discrimination a parameter discrimination algorithm based on entropy formulations was adapted. Again a multivariate criterion considering variable measurement variances was developed. This discrimination algorithm was applied to discriminate the macrokinetic model of FDH production with Candida boidinii out of 10 different macrokinetic approaches. An unequivocal discrimination result could be obtained calculating model specific probabilities. These were compared with commonly used sum of squares values. (C) 1997 John Wiley & Sons, Inc.

Abstract

L-Hydantoinase from Arthrobacter aurescens DSM 3745 has been purified to homogeneity and crystallized from polyethylene glycol solutions in a form suitable for X-ray diffraction analysis. The crystals have been grown by the sitting-drop variant of the vapour-diffusion method. X-ray diffraction studies show that the crystals belong to the monoclinic space group P2(1) with a = 111.2, b = 74.4, c = 146.5 Angstrom and beta = 106.7 degrees. Its asymmetric unit contains four monomers related by 222 symmetry. The crystals diffract to at least 2.6 Angstrom.

Abstract

We show that a simple cell-free translation system from Escherichia coli, programmed with phage MS2 RNA, is able to infect F+ E. coli cells, The plaques appearing on the E. coli host strain are morphologically indistinguishable from those derived from normal phage MS2 infection, This effect is strictly translation-dependent, since an incomplete translation system or the system inhibited by antibiotics leads to no infection, The cell-free based infection is maximal under conditions favouring the highest synthesis of maturation protein (one of the four phage-encoded proteins), The infection is abolished when RNase A or trypsin treatment is included before addition of cells, Similarly, due to RNA and maturation protein degradation, the continued incubation of the translation mixture under protein synthesis conditions significantly decreases infectivity, These findings suggest the formation of `minimal infectious units', simple complexes of MS2 RNA and maturation protein, Here we describe the first example of bacteriophage infectious unit formation directly performed in a cell-free translation system, A possible application of this phenomenon might be the construction of newly designed RNA vector delivery systems and, moreover, could be an approach for molecular evolution studies.

Abstract

Molecular imprinting was applied to the synthesis of methacrylic acid-ethylene glycol dimethacrylate copolymers specific for the herbicide atrazine. In toluene, these imprints bound atrazine with dissociation constant (K-D) values as low as 10(-6) M, and could be used for the development of a ligand binding assay for the detection of atrazine. The selectivity profile of the imprints for triazines structurally related to atrazine was comparable to those of antibodies. The substituent of carbon C1 is the most important feature determining the level of cross-reactivity, and the binding affinity decreased in the order Cl > OCH3 > OH > SCH3 (atrazine, the imprint species, has C1 at this position). No cross-reaction of structurally unrelated herbicides and other compounds was detected. Rebinding experiments in phosphate buffer containing 0.15\% Tween 20 gave similar results. The polymers could be packed into HPLC columns and used for the separation of triazine derivatives. The potential uses of imprinted polymers for the detection of pollutants in environmental analysis and as sorbents for the specific removal of toxic compounds in waste water treatment are discussed.

Abstract

Polyclonal antibodies were produced against the highly purified L-5-aryl-alkyl-hydantoinase and hydantoin-racemase of Arthrobacter aurescens DSM 3747. Serological interaction of the hydantoinase antibody with its corresponding antigenic fraction let to a remarkable improvement of the stability of the hydantoinase. The immobilization of the catalytic active enzyme-antibody complex onto a Sepharose 4B-support underlined this antibody-mediated stabilization effect, while control experiments with preimmunsera or the hydantoin-racemase antibodies gave no or even a negative effect.

Abstract

Polyclonal antibodies were produced against the highly purified enzymes L-hydantoinase, hydantoin-racemase and L-N-carbamoylamino acid amidohydrolase of Arthrobacter aurescens DSM 3747. Using these antibodies as screening tools, a colony transfer procedure allowed the rapid detection of highly active strains. Furthermore, common Western-blot analysis enabled the direct estimation of internal enzyme concentrations, protein turnover, efficiency of different inducers and a molecular comparison of enzymes derived from different sources. Using this method it is demonstrated that of several tested microorganisms, two Arthrobacter strains showed improved production abilities.

Abstract

Polyclonal antibodies were produced against the highly purified enzymes L-hydantoinase, hydantoin-racemase and L-N-carbamoylamino acid amidohydrolase of Arthrobacter aurescens DSM 3747. In order to exploit these antibodies for basic research (molecular biology) or bioengineering (process development), the serological properties had to be characterized. Both, the hydantoinase- and carbamoylase-antibodies were observed to be monofunctional, whereas the hydantoin-racemase-antibody was found to be additionally specific against the L-hydantoinase. Monospecificity was realized after affinity chromatography. Investigations on serological crossreactions with several linear- and cyclic amidases (e.g. hydantoinases) as well as hydantoin-racemases are demonstrated in this paper.

Abstract

Cancer is a group of diseases that involves abnormal cell growth, resulting from genetic perturbations in signaling mechanisms. High resolution RNAseq and microarray assays enable the evaluation of the transcriptional activity of high number of signaling molecules. Furthermore, many signaling pathways are described in publically available databases. Today’s challenge lies in the connection of signaling pathways and signaling data to produce predictive models which have the power to validate and identify targets in disease treatment. Curating networks manually can be exhaustive handiwork. We designed an ensemble approach of gene set enrichment on seven pathway databases. It generates a basic gene set mapping of the complex input data on comprehensive pathways. Using two publically available protein-protein interaction databases, the novel algorithm automatically reconstructs a comprehensive biological system representation from these mappings. The reconstruction was based on a newly shortest path algorithm. Using a microarray data set from hepatocellular cancer cells as input, a network with well-known cancer signaling mechanisms was derived. Furthermore, nodes accounting for hormone signaling were found as being modified in liver cancer that can be used as future research targets. Two recent publically available networks were adequately inferred when testing the method to reconstruct manually curated signaling networks. Finally, our method shows that integration of raw data and publically available knowledge expeditiously generates convenient and analyzable network views.

Abstract

De-novo assembly of a metagenomic dataset obtained from a decaying cyanobacterial Trichodesmium bloom from the New Caledonian lagoon resulted in a complete giant phage genome of 257,908bp, obtained independently with multiple assembly tools. Noteworthy, gammaproteobacteria were an abundant fraction in the sequenced samples. Mapping of the raw reads with 99% accuracy to the giant phage genome resulted in an average coverage of 262X. The closest sequenced relatives, albeit still distant, are the Pseudomonas phages PaBG from Lake Baikal and Lu11 isolated from a soil sample from the Philippines. The phage reported here might belong to the same family within the Myoviridae as PaBG and Lu11 and would thus be its first marine member, indicating a more widespread occurrence of this group. We named this phage NCTB (New Caledonia Trichodesmium Bloom) after its origin.

Abstract

Successful scale-up of bioprocesses requires that laboratory-scale performance is equally achieved during large-scale production to meet economic constraints. In industry, heuristic approaches are often applied, making use of physical scale-up criteria that do not consider cellular needs or properties. As a consequence, large-scale productivities, conversion yields, or product purities are often deteriorated, which may prevent economic success. The occurrence of population heterogeneity in large-scale production may be the reason for underperformance. In this study, an in silico method to predict the formation of population heterogeneity by combining computational fluid dynamics (CFD) with a cell cycle model of Pseudomonas putida KT2440 was developed. The glucose gradient and flow field of a 54,000 L stirred tank reactor were generated with the Euler approach, and bacterial movement was simulated as Lagrange particles. The latter were statistically evaluated using a cell cycle model. Accordingly, 72% of all cells were found to switch between standard and multifork replication, and 10% were likely to undergo massive, transcriptional adaptations to respond to extracellular starving conditions. At the same time, 56% of all cells replicated very fast, with µ ≥ 0.3 h−1 performing multifork replication. The population showed very strong heterogeneity, as indicated by the observation that 52.9% showed higher than average adenosine triphosphate (ATP) maintenance demands (12.2%, up to 1.5 fold). These results underline the potential of CFD linked to structured cell cycle models for predicting large-scale heterogeneity in silico and ab initio.

Abstract

For eukaryotes there seems to be no doubt that differences on the trancriptomic level substantially contribute to the process of species diversification, whereas for bacteria this is thought to be less important. Recent years saw a significant increase in full transcriptome studies for bacteria, which provided deep insight into the architecture of bacterial transcriptomes. Most notably, it became evident that, in contrast to previous scientific consensus, bacterial transcriptomes are quite complex. There exist a large number of cis-antisense RNAs, non-coding RNAs, overlapping transcripts and RNA elements that regulate transcription, such as riboswitches. Furthermore, processing and degradation of RNA has gained interest, because it has a significant impact on the composition of the transcriptome. In this review, we summarize recent findings and put them into a broader context with respect to the complexity of bacterial transcriptomes and its putative biological meanings.

Abstract

Motivation: We developed VisualGraphX, a web-based, interactive visualization tool for large-scale graphs. Current graph visualization tools that follow the rich-internet paradigm lack an interactive and scalable visualization of graph-based data. VisualGraphX aims to provide a universal graph visualization tool that empowers the users to efficiently explore the data for themselves at a large scale. It is available as a visualization plugin for the Galaxy platform, such that VisualGraphX can be integrated into custom analysis pipelines. Availability and Implementation: VisualGraphX has been released as a visualization plugin for the Galaxy platform under AFL 3.0 and is available with instructions and application data at http://gitlab.com/comptrans/VisualGraphX/. Contact: bjoern.voss@ibvt.uni-stuttgart.de

Abstract

The visualization of massive datasets, such as those resulting from comparative metatranscriptome analyses or the analysis of microbial population structures using ribosomal RNA sequences, is a challenging task. We developed a new method called CoVennTree (Comparative weighted Venn Tree) that simultaneously compares up to three multifarious datasets by aggregating and propagating information from the bottom to the top level and produces a graphical output in Cytoscape. With the introduction of weighted Venn structures, the contents and relationships of various datasets can be correlated and simultaneously aggregated without losing information. We demonstrate the suitability of this approach using a dataset of 16S rDNA sequences obtained from microbial populations at three different depths of the Gulf of Aqaba in the Red Sea. CoVennTree has been integrated into the Galaxy ToolShed and can be directly downloaded and integrated into the user instance.

Abstract

Synechocystis sp. PCC 6803 is the most popular cyanobacterial model for prokaryotic photosynthesis and for metabolic engineering to produce biofuels. Genomic and transcriptomic comparisons between closely related bacteria are powerful approaches to infer insights into their metabolic potentials and regulatory networks. To enable a comparative approach, we generated the draft genome sequence of Synechocystis sp. PCC 6714, a closely related strain of 6803 (16S rDNA identity 99.4\%) that also is amenable to genetic manipulation. Both strains share 2838 protein-coding genes, leaving 845 unique genes in Synechocystis sp. PCC 6803 and 895 genes in Synechocystis sp. PCC 6714. The genetic differences include a prophage in the genome of strain 6714, a different composition of the pool of transposable elements, and a ∼40 kb genomic island encoding several glycosyltransferases and transport proteins. We verified several physiological differences that were predicted on the basis of the respective genome sequence. Strain 6714 exhibited a lower tolerance to Zn2+ ions, associated with the lack of a corresponding export system and a lowered potential of salt acclimation due to the absence of a transport system for the re-uptake of the compatible solute glucosylglycerol. These new data will support the detailed comparative analyses of this important cyanobacterial group than has been possible thus far. Genome information for Synechocystis sp. PCC 6714 has been deposited in Genbank (accession no AMZV01000000).

Abstract

Synechocystis sp. strain PCC 6714 is a unicellular cyanobacterium closely related to the popular model organism Synechocystis sp. strain PCC 6803. A combination of PacBio SMRT and Illumina GAIIx data results in a highly accurate finished genome sequence that provides a reliable resource for further comparative analyses.

Abstract

Marine diatoms constitute a major component of eukaryotic phytoplankton and stand at the crossroads of several evolutionary lineages. These microalgae possess peculiar genomic features and novel combinations of genes acquired from bacterial, animal and plant ancestors. Furthermore, they display both DNA methylation and gene silencing activities. Yet, the biogenesis and regulatory function of small RNAs (sRNAs) remain ill defined in diatoms. PMID: 25142710