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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Prochlorococcus is a genus of abundant and ecologically important marine cyanobacteria. Here, we present a comprehensive comparison of the structure and composition of the transcriptomes of two Prochlorococcus strains, which, despite their similarities, have adapted their gene pool to specific environmental constraints. We present genome-wide maps of transcriptional start sites (TSS) for both organisms, which are representatives of the two most diverse clades within the two major ecotypes adapted to high- and low-light conditions, respectively. Our data suggest antisense transcription for three-quarters of all genes, which is substantially more than that observed in other bacteria. We discovered hundreds of TSS within genes, most notably within 16 of the 29 prochlorosin genes, in strain MIT9313. A direct comparison revealed very little conservation in the location of TSS and the nature of non-coding transcripts between both strains. We detected extremely short 5′ untranslated regions with a median length of only 27 and 29 nt for MED4 and MIT9313, respectively, and for 8\% of all protein-coding genes the median distance to the start codon is only 10 nt or even shorter. These findings and the absence of an obvious Shine–Dalgarno motif suggest that leaderless translation and ribosomal protein S1-dependent translation constitute alternative mechanisms for translation initiation in Prochlorococcus. We conclude that genome-wide antisense transcription is a major component of the transcriptional output from these relatively small genomes and that a hitherto unrecognized high degree of complexity and variability of gene expression exists in their transcriptional architecture.

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

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

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

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

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

Abstract

RNA-seq and its variant differential RNA-seq (dRNA-seq) are today routine methods for transcriptome analysis in bacteria. While expression profiling and transcriptional start site prediction are standard tasks today, the problem of identifying transcriptional units in a genome-wide fashion is still not solved for prokaryotic systems. PMID: 24780064

Abstract

RNA molecules, especially non-coding RNAs, play vital roles in the cell and their biological functions are mostly determined by structural properties. Often, these properties are related to dynamic changes in the structure, as in the case of riboswitches, and thus the analysis of RNA folding kinetics is crucial for their study. Exact approaches to kinetic folding are computationally expensive and, thus, limited to short sequences. In a previous study, we introduced a position-specific abstraction based on helices which we termed helix index shapes (hishapes) and a hishape-based algorithm for near-optimal folding pathway computation, called HiPath. The combination of these approaches provides an abstract view of the folding space that offers information about the global features. PMID: 24575751

Abstract

RNA-seq and especially differential RNA-seq-type transcriptomic analyses (dRNA-seq) are powerful analytical tools, as they not only provide insights into gene expression changes but also provide detailed information about all promoters active at a given moment, effectively giving a deep insight into the transcriptional landscape. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a unicellular model cyanobacterium that is widely used in research fields from ecology, photophysiology to systems biology, modelling and biotechnology. Here, we analysed the response of the Synechocystis 6803 primary transcriptome to different, environmentally relevant stimuli. We established genome-wide maps of the transcriptional start sites active under 10 different conditions relevant for photosynthetic growth and identified 4091 transcriptional units, which provide information about operons, 5′ and 3′ untranslated regions (UTRs). Based on a unique expression factor, we describe regulons and relevant promoter sequences at single-nucleotide resolution. Finally, we report several sRNAs with an intriguing expression pattern and therefore likely function, specific for carbon depletion (CsiR1), nitrogen depletion (NsiR4), phosphate depletion (PsiR1), iron stress (IsaR1) or photosynthesis (PsrR1). This dataset is accompanied by comprehensive information providing extensive visualization and data access to allow an easy-to-use approach for the design of experiments, the incorporation into modelling studies of the regulatory system and for comparative analyses.

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

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

RNA has many pivotal functions especially in the regulation of gene expression by ncRNAs. Identification of their structure is an important requirement for understanding their function. Structure prediction alone is often insufficient for this task, due to algorithmic problems, parameter inaccuracies, and biological peculiarities. Among the latter, there are base modifications, cotranscriptional folding leading to folding traps, and conformational switching as in the case of riboswitches. All these require more in-depth analysis of the folding space. The major drawback, which all methods have to cope with, is the exponential growth of the folding space. Therefore, methods are often limited in the sequence length they can analyze, or they make use of heuristics, sampling, or abstraction. Our approach adopts the abstraction strategy and remedies some problems of existing methods. We introduce a position-specific abstraction based on helices that we term helix index shapes, or hishapes for short. Utilizing a dynamic programming framework, we have implemented this abstraction in the program RNAHeliCes. Furthermore, we developed two hishape-based methods, one for energy barrier estimation, called HiPath, and one for abstract structure comparison, termed HiTed. We demonstrate the superior performance of HiPath compared to other existing methods and the competitive accuracy of HiTed. RNAHeliCes, together with HiPath and HiTed, are available for download at http://www.cyanolab.de/software/RNAHeliCes.htm.

Abstract

Regulatory small RNAs (sRNAs) have crucial roles in the adaptive responses of bacteria to changes in the environment. Thus far, potential regulatory RNAs have been studied mainly in marine picocyanobacteria in genetically intractable Prochlorococcus, rendering their molecular analysis difficult. Synechococcus sp. WH7803 is a model cyanobacterium, representative of the picocyanobacteria from the mesotrophic areas of the ocean. Similar to the closely related Prochlorococcus it possesses a relatively streamlined genome and a small number of genes, but is genetically tractable. Here, a comparative genome analysis was performed for this and four additional marine Synechococcus to identify the suite of possible sRNAs and other RNA elements. Based on the prediction and on complementary microarray profiling, we have identified several known as well as 32 novel sRNAs. Some sRNAs overlap adjacent coding regions, for instance for the central photosynthetic gene psbA. Several of these novel sRNAs responded specifically to environmentally relevant stress conditions. Among them are six sRNAs changing their accumulation level under cold stress, six responding to high light and two to iron limitation. Target predictions suggested genes encoding components of the light-harvesting apparatus as targets of sRNAs originating from genomic islands and that one of the iron-regulated sRNAs might be a functional homolog of RyhB. These data suggest that marine Synechococcus mount adaptive responses to these different stresses involving regulatory sRNAs.

Abstract

Synechocystis sp. PCC 6803 is a widely used model cyanobacterium for studying photosynthesis, phototaxis, the production of biofuels and many other aspects. Here we present a re-sequencing study of the genome and seven plasmids of one of the most widely used Synechocystis sp. PCC 6803 substrains, the glucose tolerant and motile Moscow or ‘PCC-M’ strain, revealing considerable evidence for recent microevolution. Seven single nucleotide polymorphisms (SNPs) specifically shared between ‘PCC-M’ and the ‘PCC-N and PCC-P’ substrains indicate that ‘PCC-M’ belongs to the ‘PCC’ group of motile strains. The identified indels and SNPs in ‘PCC-M’ are likely to affect glucose tolerance, motility, phage resistance, certain stress responses as well as functions in the primary metabolism, potentially relevant for the synthesis of alkanes. Three SNPs in intergenic regions could affect the promoter activities of two protein-coding genes and one cis-antisense RNA. Two deletions in ‘PCC-M’ affect parts of clustered regularly interspaced short palindrome repeats-associated spacer-repeat regions on plasmid pSYSA, in one case by an unusual recombination between spacer sequences.

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

There has been an increasing interest in cyanobacteria because these photosynthetic organisms convert solar energy into biomass and because of their potential for the production of biofuels. However, the exploitation of cyanobacteria for bioengineering requires knowledge of their transcriptional organization. Using differential RNA sequencing, we have established a genome-wide map of 3,527 transcriptional start sites (TSS) of the model organism Synechocystis sp. PCC6803. One-third of all TSS were located upstream of an annotated gene; another third were on the reverse complementary strand of 866 genes, suggesting massive antisense transcription. Orphan TSS located in intergenic regions led us to predict 314 noncoding RNAs (ncRNAs). Complementary microarray-based RNA profiling verified a high number of noncoding transcripts and identified strong ncRNA regulations. Thus, ∼64\% of all TSS give rise to antisense or ncRNAs in a genome that is to 87\% protein coding. Our data enhance the information on promoters by a factor of 40, suggest the existence of additional small peptide-encoding mRNAs, and provide corrected 5′ annotations for many genes of this cyanobacterium. The global TSS map will facilitate the use of Synechocystis sp. PCC6803 as a model organism for further research on photosynthesis and energy research.

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

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 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

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

Retrograde signaling is a pathway of communication from mitochondria and plastids to the nucleus in the context of cell differentiation, development and stress response. In Chlamydomonas reinhardtii, the tetrapyrroles Mg-protoporphyrin IX (MgProto) and heme are only synthesized within the chloroplast, and they have been implicated in the retrograde control of nuclear gene expression in this unicellular green alga. Feeding the two tetrapyrroles to Chlamydomonas cultures was previously shown to transiently induce five nuclear genes, three of which encode the heat shock proteins HSP70A, B, and E. In contrast, controversial results exist on the possible role of MgProto in the repression of genes for light harvesting proteins in higher plants, raising the question of how important this mode of regulation is. Here we used genome-wide transcriptional profiling to measure the global impact of these tetrapyrroles on gene regulation and the scope of the response. We identified almost 1000 genes whose expression level changed transiently but significantly. Among them were only a few genes for photosynthetic proteins but several encoding enzymes of the TCA cycle, heme-binding proteins, stress-response proteins, as well as proteins involved in protein folding and degradation. More than \textgreater50\% of the latter class of genes were also regulated by heat shock. The observed drastic fold changes at RNA level did not correlate with similar changes of protein concentrations under the tested experimental conditions. Phylogenetic profiling revealed that genes of putative endosymbiontic origin are not overrepresented among the responding genes. This and the transient nature of changes in gene expression suggest a signaling role of both tetrapyrroles as secondary messengers for adaptive responses affecting the entire cell and not only organellar proteins.

Abstract

The ability of some RNA molecules to switch between different metastable conformations plays an important role in cellular processes. In order to identify such molecules and to predict their conformational changes one has to investigate the refolding pathways. As a qualitative measure of these transitions, the barrier height marks the energy peak along such refolding paths. We introduce a meta-heuristic to estimate such barriers, which is an NP-complete problem. To guide an arbitrary path heuristic, the method uses RNA shape representative structures as intermediate checkpoints for detours. This enables a broad but estimationcient search for refolding pathways. The resulting Shape Triples meta-heuristic enables a close to optimal estimation of the barrier height that outperforms the precision of the employed path heuristic.

Abstract

In response to nitrogen deficiency, some cyanobacteria develop heterocysts, a terminally differentiated cell type, specialized for the fixation of atmospheric nitrogen. In Nostocales, this differentiation process is controlled by two major regulators, NtcA and HetR, but additional unknown factors are likely to be involved as well. In the context of a genome-wide search for potential non-coding RNAs, we identified an array of 12 tandem repeats that is transcribed in large amounts when cells enter conditions that trigger cell differentiation and switch to nitrogen fixation. The main accumulating transcript, which we suggest designating nitrogen stress-induced RNA 1 (NsiR1), has properties similar to regulatory non-coding RNAs. In Anabaena sp. PCC 7120, it is about 60 nt in length, has a very distinct predicted secondary structure, and is expressed very early and transiently after nitrogen step-down. Moreover, its expression requires HetR and NtcA and is restricted to cells that are differentiating into heterocysts, clearly placing NsiR1 within the regulon that controls the switch to nitrogen fixation and heterocyst formation. The genomic arrangement of NsiR1, located upstream of hetF, a gene whose product is involved in heterocyst formation, is conserved in all five Nostocales whose genomes are completely sequenced. Additionally, we detected NsiR1 expression in 19 different heterocyst-forming cyanobacteria. Our data suggest that every repeat is a complete transcriptional unit furnished with a cell-type-specific promoter and a Rho-independent terminator, which gives rise to a very high NsiR1 transcript level. NsiR1 is the first known bacterial non-coding RNA that is specifically upregulated in response to nitrogen step-down.

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

Regulatory RNA plays a pivotal role in the regulation of bacterial gene expression. Here, five small RNAs were studied in Sinorhizobium meliloti - SmrC15, SmrC16, Sra33, 6S and the signal recognition particle (SRP) RNA, which are conserved among at least seven different Rhizobium and Sinorhizobium species. The amount of SmrC16 decreased in stationary phase, while the other RNAs were up-regulated. The smallest changes, maximally 2-fold, were observed for 6S RNA. In the distantly related Bradyrhizobium japonicum, the amount of 6S RNA was not increased in stationary phase, suggesting some functional divergence in the roles of this molecule in Rhizobiales in comparison to Escherichia coli. Different decay rates were observed for SmrC15 and SmrC16 of S. meliloti upon rifampicin treatment, revealing posttranscriptional regulation during growth. The use of a Deltahfq mutant showed that Hfq protects full-length SmrC16 from degradation and stabilises its specific degradation products.

Abstract

BACKGROUND:In bacteria, non-coding RNAs (ncRNA) are crucial regulators of gene expression, controlling various stress responses, virulence, and motility. Previous work revealed a relatively high number of ncRNAs in some marine cyanobacteria. However, for efficient genetic and biochemical analysis it would be desirable to identify a set of ncRNA candidate genes in model cyanobacteria that are easy to manipulate and for which extended mutant, transcriptomic and proteomic data sets are available.RESULTS:Here we have used comparative genome analysis for the biocomputational prediction of ncRNA genes and other sequence/structure-conserved elements in intergenic regions of the three unicellular model cyanobacteria Synechocystis PCC6803, Synechococcus elongatus PCC6301 and Thermosynechococcus elongatus BP1 plus the toxic Microcystis aeruginosa NIES843. The unfiltered numbers of predicted elements in these strains is 383, 168, 168, and 809, respectively, combined into 443 sequence clusters, whereas the numbers of individual elements with high support are 94, 56, 64, and 406, respectively. Removing also transposon-associated repeats, finally 78, 53, 42 and 168 sequences, respectively, are left belonging to 109 different clusters in the data set. Experimental analysis of selected ncRNA candidates in Synechocystis PCC6803 validated new ncRNAs originating from the fabF-hoxH and apcC-prmA intergenic spacers and three highly expressed ncRNAs belonging to the Yfr2 family of ncRNAs. Yfr2a promoter-luxAB fusions confirmed a very strong activity of this promoter and indicated a stimulation of expression if the cultures were exposed to elevated light intensities.CONCLUSION:Comparison to entries in Rfam and experimental testing of selected ncRNA candidates in Synechocystis PCC6803 indicate a high reliability of the current prediction, despite some contamination by the high number of repetitive sequences in some of these species. In particular, we identified in the four species altogether 8 new ncRNA homologs belonging to the Yfr2 family of ncRNAs. Modelling of RNA secondary structures indicated two conserved single-stranded sequence motifs that might be involved in RNA-protein interactions or in the recognition of target RNAs. Since our analysis has been restricted to find ncRNA candidates with a reasonable high degree of conservation among these four cyanobacteria, there might be many more, requiring direct experimental approaches for their identification.

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 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

Prochlorococcus is the most abundant phototroph in the vast, nutrient-poor areas of the ocean. It plays an important role in the ocean carbon cycle, and is a key component of the base of the food web. All cells share a core set of about 1,200 genes, augmented with a variable number of “flexible” genes. Many of the latter are located in genomic islands—hypervariable regions of the genome that encode functions important in differentiating the niches of “ecotypes.” Of major interest is how cells with such a small genome regulate cellular processes, as they lack many of the regulatory proteins commonly found in bacteria. We show here that contrary to the regulatory proteins, ncRNAs are present at levels typical of bacteria, revealing that they might have a disproportional regulatory role in Prochlorococcus—likely an adaptation to the extremely low-nutrient conditions of the open oceans, combined with the constraints of a small genome. Some of the ncRNAs were differentially expressed under stress conditions, and a high number of them were found to be associated with genomic islands, suggesting functional links between these RNAs and the response of Prochlorococcus to particular environmental challenges.

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

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

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

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

Different forms of the 6S non-coding RNA (ncRNA) exist in enterobacteria and in B. subtilis but there is only limited information about this RNA from other groups of bacteria. Prochlorococcus is an oceanic, ecologically important, cyanobacterium. It possesses the most streamlined genome within the cyanobacterial phylum, lacking many regulatory proteins and mechanisms well-known from other bacteria. Here we show the accumulation of two distinct types of 6S RNA in Prochlorococcus MED4. One of these RNAs is transcribed from a specific promoter located 23 nucleotides downstream the terminal codon of the purK gene, whereas the longer transcript is produced by processing from a purK-6S RNA precursor. The expression of both 6S transcripts is under diel control, reaching maxima during the day and minima coinciding with the S- and G2-like phases which are typical for synchronized cultures of this prokaryote. Based on data from four closely related Prochlorococcus strains and 11 environmental sequences from the Sargasso Sea, a previously unknown structural element is predicted within the 6S RNA 5' domain by comparative computational analysis. The divergent expression in synchronized cultures and unusual structural domains that were detected based on metagenomic data sets indicate that 6S RNA is an extremely important global regulator in these marine cyanobacteria.

Abstract

BACKGROUND:MicroRNAs (miRNAs) are regulatory RNA molecules that are specified by their mode of action, the structure of primary transcripts, and their typical size of 20-24 nucleotides. Frequently, not only single miRNAs but whole families of closely related miRNAs have been found in animals and plants. Some families are widely conserved among different plant taxa. Hence, it is evident that these conserved miRNAs are of ancient origin and indicate essential functions that have been preserved over long evolutionary time scales. In contrast, other miRNAs seem to be species-specific and consequently must possess very distinct functions. Thus, the analysis of an early-branching species provides a window into the early evolution of fundamental regulatory processes in plants.RESULTS:Based on a combined experimental-computational approach, we report on the identification of 48 novel miRNAs and their putative targets in the moss Physcomitrella patens. From these, 18 miRNAs and two targets were verified in independent experiments. As a result of our study, the number of known miRNAs in Physcomitrella has been raised to 78. Functional assignments to mRNAs targeted by these miRNAs revealed a bias towards genes that are involved in regulation, cell wall biosynthesis and defense. Eight miRNAs were detected with different expression in protonema and gametophore tissue. The miRNAs 1-50 and 2-51 are located on a shared precursor that are separated by only one nucleotide and become processed in a tissue-specific way.CONCLUSION:Our data provide evidence for a surprisingly diverse and complex miRNA population in Physcomitrella. Thus, the number and function of miRNAs must have significantly expanded during the evolution of early land plants. As we have described here within, the coupled maturation of two miRNAs from a shared precursor has not been previously identified in plants.

Abstract

BACKGROUND:Non-coding RNAs (ncRNA) are regulators of gene expression in all domains of life. They control growth and differentiation, virulence, motility and various stress responses. The identification of ncRNAs can be a tedious process due to the heterogeneous nature of this molecule class and the missing sequence similarity of orthologs, even among closely related species. The small ncRNA Yfr1 has previously been found in the Prochlorococcus/Synechococcus group of marine cyanobacteria.RESULTS:Here we show that screening available genome sequences based on an RNA motif and followed by experimental analysis works successfully in detecting this RNA in all lineages of cyanobacteria. Yfr1 is an abundant ncRNA between 54 and 69 nt in size that is ubiquitous for cyanobacteria except for two low light-adapted strains of Prochlorococcus, MIT 9211 and SS120, in which it must have been lost secondarily. Yfr1 consists of two predicted stem-loop elements separated by an unpaired sequence of 16-20 nucleotides containing the ultraconserved undecanucleotide 5'-ACUCCUCACAC-3'.CONCLUSION:Starting with an ncRNA previously found in a narrow group of cyanobacteria only, we show here the highly specific and sensitive identification of its homologs within all lineages of cyanobacteria, whereas it was not detected within the genome sequences of E. coli and of 7 other eubacteria belonging to the alpha-proteobacteria, chlorobiaceae and spirochaete. The integration of RNA motif prediction into computational pipelines for the detection of ncRNAs in bacteria appears as a promising step to improve the quality of such predictions.

Abstract

BACKGROUND:Soon after the first algorithms for RNA folding became available, it was recognised that the prediction of only one energetically optimal structure is insufficient to achieve reliable results. An in-depth analysis of the folding space as a whole appeared necessary to deduce the structural properties of a given RNA molecule reliably. Folding space analysis comprises various methods such as suboptimal folding, computation of base pair probabilities, sampling procedures and abstract shape analysis. Common to many approaches is the idea of partitioning the folding space into classes of structures, for which certain properties can be derived.RESULTS:In this paper we extend the approach of abstract shape analysis. We show how to compute the accumulated probabilities of all structures that share the same shape. While this implies a complete (non-heuristic) analysis of the folding space, the computational effort depends only on the size of the shape space, which is much smaller. This approach has been integrated into the tool RNAshapes, and we apply it to various RNAs.CONCLUSION:Analyses of conformational switches show the existence of two shapes with probabilities approximately 23 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaadaWcaaqaaiabikdaYaqaaiabiodaZaaaaaa@2EA2@ vs. 13 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaadaWcaaqaaiabigdaXaqaaiabiodaZaaaaaa@2EA0@, whereas the analysis of a microRNA precursor reveals one shape with a probability near to 1.0. Furthermore, it is shown that a shape can outperform an energetically more favourable one by achieving a higher probability. From these results, and the fact that we use a complete and exact analysis of the folding space, we conclude that this approach opens up new and promising routes for investigating and understanding RNA secondary structure.

Abstract

Computational analysis of RNA secondary structure is a classical field of biosequence analysis, which has recently gained momentum due to the manyfold regulatory functions of RNA that have become apparent. We present five recent computational approaches that address the problems of synoptic folding space analysis, pseudoknot prediction, structure alignment, comparative structure prediction, and miRNA target prediction. All these programs are in current use and are available via the Bielefeld Bioinformatics Server at http://bibiserv.techfak.uni-bielefeld.de.

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

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

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

Micro-RNAs (miRNAs) are one class of small non-coding RNAs that have important regulatory roles in higher plants. Much less is known about their prevalence and function in lower land plants. Previously we cloned 100 non-structural small RNAs from the moss Physcomitrella patens but could annotate only 11 as miRNAs. To identify additional moss miRNAs among cloned small RNAs we have analyzed their genomic sequences for a characteristic miRNA precursor-like structure. This analysis revealed 19 new moss miRNAs that are predicted to be encoded by 22 putative foldbacks. Northern blot analysis confirmed the expression of 14 new miRNA representatives. Half of these were gametophore specific, the rest were detected at low levels in the protonema. We predicted 12 genes as targets of nine new miRNAs. Three of these show homology to transcription factors and the others appear to play roles in diverse physiological processes including light and cytokine signaling, which have not to date been shown to be regulated by a miRNA in flowering plants. Four target genes, which show homology to ATN1-like protein kinase, NAC transcription factors and a cytokinin receptor, have been validated by miRNA-mediated mRNA cleavage. In addition, our analysis revealed that seven small RNAs represent miRNA* and three represent intermediates of pre-miRNA processing, providing evidence for specific DICER-like cleavage steps during miRNA biogenesis in moss. Our findings suggest that miRNAs are common in mosses and set the stage for the elucidation of their varied biological functions.

Abstract

Summary: We introduce RNAshapes, a new software package that integrates three RNA analysis tools based on the abstract shapes approach: the analysis of shape representatives, the calculation of shape probabilities and the consensus shapes approach. This new package is completely reimplemented in C and outruns the original implementations significantly in runtime and memory requirements. Additionally, we added a number of useful features like suboptimal folding with correct dangling energies, structure graph output, shape matching and a sliding window approach. Availability: RNAshapes is freely available at http://bibiserv.techfak.uni-bielefeld.de/rnashapes/ as C source code, and as compiled binaries for the most common computer architectures. For Microsoft Windows, we also offer a graphical user interface with convenient access to the complete functionality of the package. Contact: psteffen@techfak.uni-bielefeld.de

Abstract

The analysis of RNA secondary structure has become more and more important throughout the last decades after it was recognised that RNA does not only serve as a passive messenger (mRNA), but also as a functional compound of the cell. Furthermore, it was elucidated that mainly the structure rather than the sequence determines the function of such non-protein-coding RNA. This means that two RNA molecules which have low sequence similarity but high structure similarity are likely to have a similar function. The prediction of RNA secondary structure is based on parameters that have been measured in vitro. This results in rather static parameters, that do not incorporate the dynamic change of environment occurring in living organisms. Nevertheless, the use of these parameters, that are summarised in the energy model, gave valuable results, especially for short sequences. Several refinements throughout the years improved the predictions, but still the calculated optimal structure is not guaranteed to correspond to the native one. In this case, and due to the fact that the native structure is feasible under the energy model, it is common practice to additionally calculate suboptimal structures and incorporate these in the study. The set of all suboptimal structures is referred to as the structure space, which actually holds the information needed to answer questions such as: Is the optimal structure also the native one? Are there more than one structure an RNA molecule can adopt? How well-defined is the optimal structure? Major problems in the analysis of the structure space are its size and its shape. The number of suboptimal structures is exponential in the sequence length, which means that for sequences of moderate length the size quickly exceeds several billion. Besides the size, the appearance of the structure space complicates its study. The structure space can be imagined as a rough landscape with valleys, holding local optimal structures, separated by mountains and saddles. This landscape is not smooth but cliffy and complex, which prevents the development of a practical and still intuitive visualisation. In general, the intention of structure space analysis is not its visualisation, but its complexity also hampers approaches to derive specific features hidden in the structure space. Despite these problems, several tools exist that analyse the complete structure space or at least a part of it to answer the aforementioned questions. Among these are MFOLD which produces a subset of all possible structures according to a threshold of structural similarity, SFOLD which samples the structures in a probabilistic fashion and provides a method to identify alternating structures, RNAsubopt to produce all suboptimal structures within a given energy threshold, barriers to identify valleys, mountains and saddles of the structure landscape, and others. My contribution to this area of research is twofold: First, I present paRNAss (prediction of alternating RNA secondary structures) which focuses on the detection of conformational switches and analyses the structure space based on pairwise comparisons. paRNAss has been available since 1997 and I could improve its predictive power as well as its speed which made possible a systematic evaluation. During this evaluation it turned out that paRNAss can even be used to identify more than two competing structures and hence get a deeper insight into the structure space. The second tool I introduce is RNAshapes which facilitates different kinds of analyses. The algorithm makes use of abstract representations of the secondary structure to compute only those that are morphologically dissimilar, i.e. are composed of different structural elements. Structures being morphologically similar are pooled in a class of structures and each class is represented by its best member. The list of these representatives gives a general overview of what is there in the structure space. In addition to this, I introduce an algorithm to compute probabilities of the aforementioned classes of structures. This gives hints to properties such as alternating secondary structures (two classes with similar probabilities) and structural well-definedness (one class with very high probability).

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

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

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

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

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 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

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

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

The function of a non-protein-coding RNA is often determined by its structure. Since experimental determination of RNA structure is time-consuming and expensive, its computational prediction is of great interest, and efficient solutions based on thermodynamic parameters are known. Frequently, however, the predicted minimum free energy structures are not the native ones, leading to the necessity of generating suboptimal solutions. While this can be accomplished by a number of programs, the user is often confronted with large outputs of similar structures, although he or she is interested in structures with more fundamental differences, or, in other words, with different abstract shapes. Here, we formalize the concept of abstract shapes and introduce their efficient computation. Each shape of an RNA molecule comprises a class of similar structures and has a representative structure of minimal free energy within the class. Shape analysis is implemented in the program RNAshapes. We applied RNAshapes to the prediction of optimal and suboptimal abstract shapes of several RNAs. For a given energy range, the number of shapes is considerably smaller than the number of structures, and in all cases, the native structures were among the top shape representatives. This demonstrates that the researcher can quickly focus on the structures of interest, without processing up to thousands of near-optimal solutions. We complement this study with a large-scale analysis of the growth behaviour of structure and shape spaces. RNAshapes is available for download and as an online version on the Bielefeld Bioinformatics Server.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 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

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

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 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

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

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

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. 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

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.