RESUMO
In the present work the impact of large production scale was investigated for Bacillus megaterium expressing green fluorescent protein (GFP). Specifically designed scale-down studies, mimicking the intermittent and continuous nutrient supply of large- and small-scale processes, were carried out for this purpose. The recombinant strain revealed a 40% reduced GFP yield for the large-scale conditions. In line with extended carbon loss via formation of acetate and carbon dioxide, this indicated obvious limitations in the underlying metabolism of B. megaterium under the large-scale conditions. Quantitative analysis of intracellular amino acids via validated fast filtration protocols revealed that their level strongly differed between the two scenarios. During cultivation in large-scale set-up, the availability of most amino acids, serving as key building blocks of the recombinant protein, was substantially reduced. This was most pronounced for tryptophan, aspartate, histidine, glutamine, and lysine. In contrast alanine was increased, probably related to a bottleneck at the level of pyruvate which also triggered acetate overflow metabolism. The pre-cursor quantifications could then be exploited to verify the presumed bottlenecks and improve recombinant protein production under large-scale conditions. Addition of only 5 mM tryptophan, aspartate, histidine, glutamine, and lysine to the feed solution increased the GFP yield by 100%. This rational concept of driving the lab scale productivity of recombinant microorganisms under suboptimal feeding conditions emulating large scale can easily be extended to other processes and production hosts.
Assuntos
Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Biotecnologia/métodos , Meios de Cultura/química , Metabolômica , Acetatos/metabolismo , Aminoácidos/metabolismo , Bacillus megaterium/crescimento & desenvolvimento , Reatores Biológicos/microbiologia , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
We present the development of microbioreactors (MBRs) based on poly(dimethylsiloxane) (PDMS) segmented waveguides (SWG) for local absorbance measurements. Two different MBRs were studied, either using symmetric or asymmetric SWG (being defined as MBR-S and MBR-A, respectively). Their optical and fluidic performances were numerically analyzed, showing robustness from an optical point of view and distinct fluid flow profile. The optical characterization was done in two steps. Initially, the experimental limit of detection (LOD) and the sensitivity were determined for two different analytes (fluorescein and methylorange). With both systems, a similar limit of detection for both analytes was obtained, being in the micromolar level. Their sensitivities were 20.2±0.3 (×10⻳) A.U./µM and 5.5±0.2 (×10⻳) A.U./µM for fluorescein and methylorange, respectively. Once validated its applicability for local absorbance measurements, a continuous cultivation of Saccharomyces cerevisiae was done to test the viability of the proposed systems for photonic MBRs. Concretely, the cell growth was locally monitored inside the MBR during 33 h. Spectral analysis showed that the determination of the culture parameters were wavelength dependant, with a growth rate of 0.39±0.02 h⻹ and a doubling time of 1.65±0.09 h at an optimal wavelength of 469.9±0.3 nm. Besides the easy and monolithic integration of the SWG into poly(dimethylsiloxane) microfluidic systems, the results presented here are very promising for the application in any disposable photonic lab-on-a-chip systems used for online analysis or photonic MBRs.
Assuntos
Reatores Biológicos/microbiologia , Dimetilpolisiloxanos/química , Microfluídica/instrumentação , Óptica e Fotônica/métodos , Leveduras/citologia , Absorção , Desenho de Equipamento , Análise de Falha de Equipamento , Fluoresceína , Dispositivos Lab-On-A-Chip , Microfluídica/métodos , Miniaturização/métodos , Fótons , Saccharomyces cerevisiae , Leveduras/crescimento & desenvolvimentoRESUMO
BACKGROUND: Single cell analysis for bioprocess monitoring is an important tool to gain deeper insights into particular cell behavior and population dynamics of production processes and can be very useful for discrimination of the real bottleneck between product biosynthesis and secretion, respectively. RESULTS: Here different dyes for viability estimation considering membrane potential (DiOC2(3), DiBAC4(3), DiOC6(3)) and cell integrity (DiBAC4(3)/PI, Syto9/PI) were successfully evaluated for Bacillus megaterium cell characterization. It was possible to establish an appropriate assay to measure the production intensities of single cells revealing certain product secretion dynamics. Methods were tested regarding their sensitivity by evaluating fluorescence surface density and fluorescent specific concentration in relation to the electronic cell volume. The assays established were applied at different stages of a bioprocess where the antibody fragment D1.3 scFv production and secretion by B. megaterium was studied. CONCLUSIONS: It was possible to distinguish between live, metabolic active, depolarized, dormant, and dead cells and to discriminate between high and low productive cells. The methods were shown to be suitable tools for process monitoring at single cell level allowing a better process understanding, increasing robustness and forming a firm basis for physiology-based analysis and optimization with the general application for bioprocess development.
Assuntos
Bacillus megaterium/citologia , Citometria de Fluxo/métodos , Análise de Célula Única/métodos , Anticorpos de Cadeia Única/metabolismo , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Corantes Fluorescentes/química , Expressão Gênica , Viabilidade Microbiana , Anticorpos de Cadeia Única/genética , Coloração e Rotulagem/métodosRESUMO
Cell damage during sampling and quenching for metabolome analysis have been investigated at whole sample level using an OD-based method and ATP loss investigation, and at single cell level by means of flow cytometry. Escherichia coli was cultivated in shake flasks and sampled into several cold quenching solutions during exponential growth phase varying quenching solution composition and sampling temperature. For single cell analysis, the samples were incubated with selective propidium iodide dye and analysed via flow cytometry to differentiate between intact and damaged cells. It was found that every combination of quenching solution, temperature, or cooling rate tested influenced the E. coli cell membrane integrity indicating rupture which will not only let the dye in, but also intracellular ATP out of the cells, which is not desired in in vivo metabolome analysis.
Assuntos
Escherichia coli/citologia , Escherichia coli/metabolismo , Metaboloma , Viabilidade Microbiana , Trifosfato de Adenosina/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Temperatura Baixa , Escherichia coli/química , Escherichia coli/crescimento & desenvolvimento , Citometria de FluxoRESUMO
Bacillus megaterium was used for production of the lysozyme-specific recombinant scFv D1.3 antibody fragment. Key process parameters like the temperature and the hydromechanical stress play a very important role for significant product formation during process development or scale-up. In this study, the influence of these two variables on growth and recombinant antibody fragment production in a 2-L lab-scale bioreactor system was investigated using a central composite design. Especially a significant influence of the hydromechanical stress on antibody fragment production was detected in batch cultivations. While volumetric power inputs of about 0.5 kW/m(3) (agitation rates around 500 min(-1)) are usually employed in batch cultivations, in this work maximal product concentration was found at a volumetric power input of about 0.06 kW/m(3) (agitation rate around 250 min(-1)) and at a high cultivation temperature of 41 °C. The influence of the two process variables at single-cell level was estimated using flow cytometry too. The characterization was done by estimating the membrane potential giving a hint on bioprocess productivity and secretion capability: the best production was obtained through big cells with low specific membrane potential, which grew at low volumetric power inputs and high cultivation temperatures.
Assuntos
Bacillus megaterium/crescimento & desenvolvimento , Bacillus megaterium/metabolismo , Microbiologia Industrial/métodos , Anticorpos de Cadeia Única/biossíntese , Bacillus megaterium/química , Bacillus megaterium/genética , Fenômenos Biomecânicos , Reatores Biológicos/microbiologia , Meios de Cultura , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Anticorpos de Cadeia Única/genética , TemperaturaRESUMO
The Bacillus megaterium protein production system based on the inducible promoter of the xyl operon (P(xylA)) was systematically optimized. Multiple changes in basic promoter elements, such as the -10 and -35 region and the ribosome-binding site, resulted in an 18-fold increase of protein production compared to the production of the previously established system. The production in shaking-flask culture of green fluorescent protein (Gfp) as a model product led to 82.5 mg per g cell dry weight (g(CDW)) or 124 mg liter(-1). In fed-batch cultivation, the volumetric protein yield was increased 10-fold to 1.25 g liter(-1), corresponding to 36.8 mg protein per g(CDW). Furthermore, novel signal peptides for Sec-dependent protein secretion were predicted in silico using the B. megaterium genome. Subsequently, leader peptides of Vpr, NprM, YngK, YocH, and a computationally designed artificial peptide were analyzed experimentally for their potential to facilitate the secretion of the heterologous model protein Thermobifida fusca hydrolase (Tfh). The best extracellular protein production, 5,000 to 6,200 U liter(-1) (5.3 to 6.6 mg liter(-1)), was observed for strains where the Tfh export was facilitated by a codon-optimized leader peptide of YngK and by the signal peptide of YocH. Further increases in extracellular protein production were achieved when leader peptides were used in combination with the optimized expression system. In this case, the greatest extracellular enzyme amount of 7,200 U liter(-1), 7.7 mg liter(-1), was achieved by YocH leader peptide-mediated protein export. Nevertheless, the observed principal limitations in protein export might be related to components of the Sec-dependent protein transport system.
Assuntos
Bacillus megaterium/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Expressão Gênica , Bacillus megaterium/genética , Proteínas de Bactérias/genética , Reatores Biológicos , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Óperon , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcrição GênicaRESUMO
In this study a proteomic approach was used to investigate the steady-state response of Escherichia coli to temperature up-shifts in a cascade of two continuously operated bioreactors. The first reactor served as cell source with optimal settings for microbial growth, while in the second chemostat the cells were exposed to elevated temperatures. By using this reactor configuration, which has not been reported to be used for the study of bacterial stress responses so far, it is possible to study temperature stress under well-defined, steady-state conditions. Specifically the effect on the cellular adaption to temperature stress using two-dimensional gel electrophoresis was examined and compared at the cultivation temperatures of 37 degrees C and 47.5 degrees C. As expected, the steady-state study with the double bioreactor configuration delivered a different protein spectrum compared to that obtained with standard batch experiments in shaking flasks and bioreactors. Setting a high cut-out spot-to-spot size ratio of 5, proteins involved in defence against oxygen stress, functional cell envelope proteins, chaperones and proteins involved in protein biosynthesis, the energy metabolism and the amino acid biosynthesis were found to be differently expressed at high cultivation temperatures. The results demonstrate the complexity of the stress response in a steady-state culture not reported elsewhere to date.
RESUMO
A number of rapid sampling devices for metabolic engineering applications have been developed over the last years with the purpose of the estimation of in vivo metabolic concentrations and dynamics. This review outlines the designs and characteristics as well as the developments and changes in diverse approaches over the years. Primary performance parameters for these constructions are sampling time and rate and, for an accurate representation of the in vivo condition in cells, the reproducibility of results and easy handling throughout the sampling operation.
Assuntos
Técnicas de Cultura/instrumentação , Microbiologia Industrial/instrumentação , Reatores Biológicos/microbiologia , Técnicas de Cultura/métodos , Desenho de Equipamento , Microbiologia Industrial/métodos , Fatores de TempoRESUMO
The recombinant Bacillus megaterium strain WH323 was employed for the inducible production and secretion of recombinant Thermobifida fusca hydrolase (TFH). Continuous cultivations were carried out in a chemostat using either glucose or pyruvate as sole carbon source. A remarkable increase of produced TFH was detected for the pyruvate-dependent cultivation compared to glucose-dependent growth. Estimation of intracellular carbon fluxes through the central metabolism for both growth conditions using (13)C-labelled substrates revealed noticeable changes of the fluxes through the tricarboxylic acid cycle, the pentose phosphate pathway and around the pyruvate node when protein production was induced. With pyruvate as sole carbon source the observed alterations of the fluxes yielded an increased production of ATP and NADPH both required for the anabolism. Additionally, the analysis of the corresponding secretome revealed significantly reduced amounts of extracellular proteases in the medium compared to glucose-grown cultivations. Thus, pyruvate-dependent chemostat cultivation was identified as a favourable condition for production and secretion of recombinant TFH using B. megaterium as production host.
Assuntos
Actinomycetales/enzimologia , Bacillus megaterium/enzimologia , Hidrolases/metabolismo , Ácido Pirúvico/metabolismo , Proteínas Recombinantes/metabolismo , Bacillus megaterium/genética , Reatores Biológicos , Ciclo do Ácido Cítrico/fisiologia , Metabolismo Energético , Hidrolases/biossíntese , Hidrolases/genética , Via de Pentose Fosfato/fisiologia , Proteínas Recombinantes/biossínteseRESUMO
Aspergillus niger AB1.13 cultures with glucoamylase production (with D-glucose as substrate) and without glucoamylase production (with D-xylose as substrate) were characterized by metabolic flux analysis. Two comprehensive metabolic models for d-glucose- as well as for D-xylose-consumption were used to quantify and compare the metabolic fluxes through the central pathways of carbon metabolism at different pH-values. The models consist of the most relevant metabolic pathways for A. niger including glycolysis, pentose-phosphate pathway, citrate cycle, energy metabolism and anaplerotic reactions comprising two intracellular compartments, the cytoplasm and mitochondrion. When D-xylose was used as the sole carbon source, the relative flux of the substrate through the oxidative pentose-phosphate pathway (PPP) via G6P-dehydrogenase was unaffected by the pH-value of the culture medium. About 30% of D-xylose consumed was routed through the oxidative PPP. In contrast, the flux of D-glucose (i.e., under glucoamylase-producing conditions) through the oxidative PPP was remarkably higher and, in addition was significantly affected by the pH-value of the culture medium (40% at pH 5.5, 56% at pH 3.7, respectively). Summarizing, the flux through the PPP under glucoamylase producing conditions was 30-90% higher than for non-producing conditions.
Assuntos
Aspergillus niger/enzimologia , Glucana 1,4-alfa-Glucosidase/metabolismo , Glucose/metabolismo , Modelos Químicos , Xilose/metabolismo , Modelos Biológicos , Via de Pentose Fosfato/fisiologia , Especificidade por SubstratoRESUMO
This work describes the adsorption-desorption behavior of a histidine-tagged single-chain Fragment variable antibody (D1.3 scFv) on a commercial immobilized metal ion affinity chromatography (IMAC) column. A clarified cell culture supernatant originating from Bacillus megaterium was characterized using single column experiments in a pH-gradient elution mode. The cell culture supernatant containing the antibody fragment D1.3 scFv could be treated in the chromatographic separation process as a pseudo-binary mixture. Adsorption equilibrium constants of the antibody fragment fraction (ABF) and the non-specifically retained protein impurity fraction (IMP) were determined experimentally at constant pH by reinjecting pulses of pooled fractions collected in preliminary batch gradient elution runs. Based on the estimated adsorption equilibrium constants a possible multicolumn open-loop three-zone two-step pH-gradient simulated moving bed (SMB) process is suggested and designed, which possesses the potential to isolate continuously the antibody fragment fraction (ABF) containing the single-chain antibody fragment D1.3 scFv.
Assuntos
Cromatografia de Afinidade/métodos , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/isolamento & purificação , Bacillus megaterium/química , Bacillus megaterium/metabolismo , Simulação por Computador , Eletroforese em Gel de Poliacrilamida , Histidina/química , Concentração de Íons de Hidrogênio , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismoRESUMO
This perspective article is based on an expert panel review on microbioreactor applications in biochemical and biomedical engineering that was organized by the M³C (measurement, monitoring, modelling and control) Working Group of the European Section of Biochemical Engineering Science (ESBES) in the European Federation of Biotechnology (EFB). The aim of the panel was to provide an updated view on the present status of the subject and to identify critical needs and issues for furthering the successful development of microbioreactor monitoring and control. This will benefit future bioprocess development and in vitro toxicity testing. The article concludes with a set of recommendations for extended use and further development of microbioreactors.
Assuntos
Reatores Biológicos , Biotecnologia , Animais , Bioengenharia , Pesquisa Biomédica , Ensaios de Triagem em Larga Escala , Humanos , Técnicas Analíticas MicrofluídicasRESUMO
Bacillus megaterium was used as an alternative high potential microbial production system for the production of antibody fragment D1.3 scFv. The aim of the study was to follow a holistic optimization approach from medium screening in small scale microtiter platforms, gaining deeper process understanding in the bioreactor scale and implementing advanced process strategies at larger scales (5-100 L). Screening and optimization procedures were supported by statistical design of experiments and a genetic algorithm approach. The process control relied on a soft-sensor for biomass estimation to establish a µ-oscillating time-dependent fed-batch strategy. Several cycles of growth phases and production phases, equal to starving phases, were performed in one production. Flow cytometry was used to monitor and characterize the dynamics of secretion and cell viability. Besides the biosynthesis of the product, secretion was optimized by an appropriate medium design considering different carbon sources, metal ions, (NH(4))(2)SO(4), and inductor concentrations. For bioprocess design, an adapted oscillating fed-batch strategy was conceived and successfully implemented at an industrially relevant scale of 100 L. In comparison to common methods for controlling fed-batch profiles, the developed process delivered increased overall productivities. Thereby measured process parameters such as growth stagnation or productivity fluctuations were directly linked to single cell or population behavior leading to a more detailed process understanding. Above all, the importance of single cell analysis as key scale-free tool to characterize and optimize recombinant protein production is highlighted, since this can be applied to all development stages independently of the cultivation platform.
Assuntos
Bacillus megaterium/imunologia , Reatores Biológicos , Engenharia Celular/métodos , Fragmentos de Imunoglobulinas/biossíntese , Microbiologia Industrial/métodos , Sulfato de Amônio , Bacillus megaterium/genética , Carbono , Meios de Cultura , Citometria de Fluxo , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/isolamento & purificação , Projetos Piloto , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Over the last two decades the Gram-positive bacterium Bacillus megaterium was systematically developed to a useful alternative protein production host. Multiple vector systems for high yield intra- and extracellular protein production were constructed. Strong inducible promoters were combined with DNA sequences for optimised ribosome binding sites, various leader peptides for protein export and N- as well as C-terminal affinity tags for affinity chromatographic purification of the desired protein. High cell density cultivation and recombinant protein production were successfully tested. For further system biology based control and optimisation of the production process the genomes of two B. megaterium strains were completely elucidated, DNA arrays designed, proteome, fluxome and metabolome analyses performed and all data integrated using the bioinformatics platform MEGABAC. Now, solid theoretical and experimental bases for primary modeling attempts of the production process are available.
Assuntos
Bacillus megaterium/fisiologia , Proteínas de Bactérias/fisiologia , Melhoramento Genético/métodos , Modelos Genéticos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Biologia de Sistemas/métodos , Simulação por ComputadorRESUMO
Polyelectrolyte multilayers (PEMs) based on the combinations poly(diallyldimethylammonium chloride)∕poly(acrylic acid) (PDADMAC∕PAA) and poly(allylamine hydrochloride)∕PAA (PAH∕PAA) were adsorbed on poly(dimethylsiloxane) (PDMS) and tested for nonspecific surface attachment of hydrophobic yeast cells using a parallel plate flow chamber. A custom-made graft copolymer containing poly(ethylene glycol) (PEG) side chains (PAA-g-PEG) was additionally adsorbed on the PEMs as a terminal layer. A suitable PEM modification effectively decreased the adhesion strength of Saccharomyces cerevisiae DSM 2155 to the channel walls. However, a further decrease in initial cell attachment and adhesion strength was observed after adsorption of PAA-g-PEG copolymer onto PEMs from aqueous solution. The results demonstrate that a facile layer-by-layer surface functionalization from aqueous solutions can be successfully applied to reduce cell adhesion strength of S. cerevisiae by at least two orders of magnitude compared to bare PDMS. Therefore, this method is potentially suitable to promote planktonic growth inside capped PDMS-based microfluidic devices if the PEM deposition is completed by a dynamic flow-through process.
RESUMO
A diffusion-based microreactor system operated with a reaction volume of 8 µL is presented and characterized to intensify the process understanding in microscale cultivations. Its potential as screening tool for biological processes is evaluated. The advantage of the designed microbioreactor is the use for the continuous cultivation mode by integrating online measurement technique for dissolved oxygen (DO) and optical density (OD). A further advantage is the broaden application for biological systems. The bioreactor geometry was chosen to achieve homogeneous flow during continuous process operation. The device consisted of a microstructured top layer made of poly(dimethylsiloxane) (PDMS), which was designed and fabricated using UV-depth and soft lithography assembled with a glass bottom. CFD simulation data used for geometry design were verified via microparticle-image-velocimetry (µPIV). In the used microreactor geometry no concentration gradients occurred along the entire reaction volume because of rapid diffusive mixing, the homogeneous medium flow inside the growth chamber of the microreactor could be realized. Undesirable bubble formation before and during operation was reduced by using degassed medium as well as moistened and moderate incident air flow above the gas permeable PDMS membrane. Because of this a passive oxygen supply of the culture medium in the device is ensured by diffusion through the PDMS membrane. The oxygen supply itself was monitored online via integrated DO sensors based on a fluorescent dye complex. An adequate overall volumetric oxygen transfer coefficient K(L)a as well as mechanical stability of the device were accomplished for a membrane thickness of 300 µm. Experimental investigations considering measurements of OD (online) and several metabolite concentrations (offline) in a modified Verduyn medium. The used model organism Saccharomyces cerevisiae DSM 2155 tended to strong reactor wall growth resembling a biofilm.
Assuntos
Reatores Biológicos/microbiologia , Microfluídica/métodos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Dimetilpolisiloxanos/química , Interações Hidrofóbicas e Hidrofílicas , Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: The identification of genetic target genes is a key step for rational engineering of production strains towards bio-based chemicals, fuels or therapeutics. This is often a difficult task, because superior production performance typically requires a combination of multiple targets, whereby the complex metabolic networks complicate straightforward identification. Recent attempts towards target prediction mainly focus on the prediction of gene deletion targets and therefore can cover only a part of genetic modifications proven valuable in metabolic engineering. Efficient in silico methods for simultaneous genome-scale identification of targets to be amplified or deleted are still lacking. RESULTS: Here we propose the identification of targets via flux correlation to a chosen objective flux as approach towards improved biotechnological production strains with optimally designed fluxes. The approach, we name Flux Design, computes elementary modes and, by search through the modes, identifies targets to be amplified (positive correlation) or down-regulated (negative correlation). Supported by statistical evaluation, a target potential is attributed to the identified reactions in a quantitative manner. Based on systems-wide models of the industrial microorganisms Corynebacterium glutamicum and Aspergillus niger, up to more than 20,000 modes were obtained for each case, differing strongly in production performance and intracellular fluxes. For lysine production in C. glutamicum the identified targets nicely matched with reported successful metabolic engineering strategies. In addition, simulations revealed insights, e.g. into the flexibility of energy metabolism. For enzyme production in A.niger flux correlation analysis suggested a number of targets, including non-obvious ones. Hereby, the relevance of most targets depended on the metabolic state of the cell and also on the carbon source. CONCLUSIONS: Objective flux correlation analysis provided a detailed insight into the metabolic networks of industrially relevant prokaryotic and eukaryotic microorganisms. It was shown that capacity, pathway usage, and relevant genetic targets for optimal production partly depend on the network structure and the metabolic state of the cell which should be considered in future metabolic engineering strategies. The presented strategy can be generally used to identify priority sorted amplification and deletion targets for metabolic engineering purposes under various conditions and thus displays a useful strategy to be incorporated into efficient strain and bioprocess optimization.
Assuntos
Aspergillus niger/genética , Aspergillus niger/metabolismo , Biotecnologia/métodos , Biologia Computacional/métodos , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Aspergillus niger/citologia , Aspergillus niger/enzimologia , Biocatálise , Corynebacterium glutamicum/citologia , Epóxido Hidrolases/metabolismo , Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Deleção de Genes , Glucana 1,4-alfa-Glucosidase/metabolismo , Glucose/metabolismo , Cinética , Lisina/biossíntese , Lisina/metabolismo , Redes e Vias Metabólicas , Técnicas de Amplificação de Ácido Nucleico , Oligossacarídeos/biossíntese , Amido/metabolismo , Estereoisomerismo , Biologia de Sistemas/métodosRESUMO
An incubator with up to 16 parallel bubble columns was equipped with artificial light sources assuring a light supply with a homogenous light spectrum directly above the bioreactors. Cylindrical light reflecting tubes were positioned around every single bubble column to avoid light scattering effects and to redirect the light from the top onto the cylindrical outer glass surface of each bubble column. The light reflecting tubes were equipped with light intensity filters to control the total light intensity for every single photo-bioreactor. Parallel cultivations of the unicellular obligate phototrophic cyanobacterium, Synechococcus PCC7942, were studied under different constant light intensities ranging from 20 to 102 microE m(-2)s(-1) at a constant humidified air flow rate supplemented with CO(2).
Assuntos
Reatores Biológicos , Synechococcus/crescimento & desenvolvimentoRESUMO
A priori information or valuable qualitative knowledge can be incorporated explicitly to describe enzyme kinetics making use of fuzzy-logic models. Although restricted to linear relationships, it is shown that fuzzy-logic augmented models are not only able to capture non-linear features of enzyme kinetics but also allow the proper mathematical treatment of metabolic control analysis. The explicit incorporation of valuable qualitative knowledge is crucial, particularly when handling data estimated from in vivo kinetics studies, since this experimental information is scarce and usually contains measurement errors. Therefore, data-driven techniques, such as the one presented in this work, form a serious alternative to established kinetics approaches.
Assuntos
Simulação por Computador , Lógica Fuzzy , Metabolismo , Modelos Estatísticos , Animais , Enzimas/metabolismo , Humanos , Modelos BiológicosRESUMO
The quantitative estimation of intracellular metabolite concentrations (metabolic profiling) is a prerequisite for a better understanding of biological processes and thus inevitable for the rational improvement of microbial production strains and process design. Since pool sizes of substrates regulate flux through different enzymes, the accurate determination of intracellular metabolite concentrations is necessary to understand in vivo reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates in Escherichia coli K12 was achieved by using a novel in situ rapid sampling and quenching procedure. A new extraction procedure using buffered hot water was established. By use of simultaneous multi-substrate feeding with various ratios of glucose, fructose and acetate during continuous cultivations several metabolic states were induced. Metabolic flux analysis and the newly developed metabolic profiling procedure were used to determine in vivo enzyme kinetics as exemplified for fructose 1,6-bisphosphate aldolase and citrate synthase.