In this paper, an approach for the monitoring of biotechnological process kinetics is proposed. T... more In this paper, an approach for the monitoring of biotechnological process kinetics is proposed. The kinetics of each process state variable is presented as a function of two time-varying unknown parameters. For their estimation, a general software sensor is derived with on-line measurements as inputs that are accessible in practice. The stability analysis with a different number of inputs shows that stability can be guaranteed for fourth- and fifth-order software sensors only. As a case study, the monitoring of the kinetics of processes carried out in stirred tank reactors is investigated. A new tuning procedure is derived that results in a choice of only one design parameter. The effectiveness of the proposed procedure is demonstrated with experimental data from Bacillus subtilis fed-batch cultivations.
In bioprocess development, the host and the genetic construct for a new biomanufacturing process ... more In bioprocess development, the host and the genetic construct for a new biomanufacturing process are selected in the early developmental stages. This decision, made at the screening scale with very limited information about the performance in larger reactors, has a major influence on the efficiency of the final process. To overcome this, scale-down approaches during screenings that show the real cell factory performance at industrial-like conditions are essential. We present a fully automated robotic facility with 24 parallel mini-bioreactors that is operated by a model-based adaptive input design framework for the characterization of clone libraries under scale-down conditions. The cultivation operation strategies are computed and continuously refined based on a macro-kinetic growth model that is continuously re-fitted to the available experimental data. The added value of the approach is demonstrated with 24 parallel fed-batch cultivations in a mini-bioreactor system with eight di...
The combination of extreme miniaturization with a high sensitivity and the potential to be integr... more The combination of extreme miniaturization with a high sensitivity and the potential to be integrated in an array form on a chip has made silicon-based photonic microring resonators a very attractive research topic. As biosensors are approaching the nanoscale, analyte mass transfer and bonding kinetics have been ascribed as crucial factors that limit their performance. One solution may be a system that applies dielectrophoretic forces, in addition to microfluidics, to overcome the diffusion limits of conventional biosensors. Dielectrophoresis, which involves the migration of polarized dielectric particles in a non-uniform alternating electric field, has previously been successfully applied to achieve a 1000-fold improved detection efficiency in nanopore sensing and may significantly increase the sensitivity in microring resonator biosensing. In the current work, we designed microring resonators with integrated electrodes next to the sensor surface that may be used to explore the eff...
In conditional microbial screening, a limited number of candidate strains are tested at different... more In conditional microbial screening, a limited number of candidate strains are tested at different conditions searching for the optimal operation strategy in production (e.g., temperature and pH shifts, media composition as well as feeding and induction strategies). To achieve this, cultivation volumes of >10 mL and advanced control schemes are required to allow appropriate sampling and analyses. Operations become even more complex when the analytical methods are integrated into the robot facility. Among other multivariate data analysis methods, principal component analysis (PCA) techniques have especially gained popularity in high throughput screening. However, an important issue specific to high throughput bioprocess development is the lack of so-called golden batches that could be used as a basis for multivariate analysis. In this study, we establish and present a program to monitor dynamic parallel cultivations in a high throughput facility. PCA was used for process monitoring...
Milk kefir is a traditional fermented milk product whose consumption is becoming increasingly pop... more Milk kefir is a traditional fermented milk product whose consumption is becoming increasingly popular. The natural starter for kefir production is kefir grain, which consists of various bacterial and yeast species. At the industrial scale, however, kefir grains are rarely used due to their slow growth, complex application, bad reproducibility and high costs. Instead, mixtures of defined lactic acid bacteria and sometimes yeasts are applied, which alter sensory and functional properties compared to natural grain-based milk kefir. In order to be able to mimic natural starter cultures for authentic kefir production, it is a prerequisite to gain deep knowledge about the nature of kefir grains, its microbial composition, morphologic structure, composition of strains on grains and the impact of environmental parameters on kefir grain characteristics. In addition, it is very important to deeply investigate the numerous multi-dimensional interactions among different species, which play impo...
The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and efficient ... more The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and efficient alternative to chemical synthesis routes. In this study, dihalogenated nucleoside analogues were produced by thermostable nucleoside phosphorylases in transglycosylation reactions using uridine or thymidine as sugar donors. Prior to the enzymatic process, ideal maximum product yields were calculated after the determination of equilibrium constants through monitoring the equilibrium conversion in analytical-scale reactions. Equilibrium constants for dihalogenated nucleosides were comparable to known purine nucleosides, ranging between 0.071 and 0.081. To achieve 90% product yield in the enzymatic process, an approximately five-fold excess of sugar donor was needed. Nucleoside analogues were purified by semi-preparative HPLC, and yields of purified product were approximately 50% for all target compounds. To evaluate the impact of halogen atoms in positions 2 and 6 on the antiproliferative ...
Microbial cells in industrial large-scale bioreactors are exposed to fluctuating conditions, e.g.... more Microbial cells in industrial large-scale bioreactors are exposed to fluctuating conditions, e.g., nutrient concentration, dissolved oxygen, temperature, and pH. These inhomogeneities can influence the cell physiology and metabolism, e.g., decelerate cell growth and product formation. Microfluidic systems offer new opportunities to study such effects in great detail by examining responses to varying environmental conditions at single-cell level. However, the possibility to reproduce large-scale bioreactor conditions in microscale cultivation systems has not yet been systematically investigated. Hence, we apply computational fluid dynamics (CFD) simulations to analyze and compare three commonly used microfluidic single-cell trapping and cultivation devices that are based on (i) mother machines (MM), (ii) monolayer growth chambers (MGC), and (iii) negative dielectrophoresis (nDEP). Several representative time-variant nutrient concentration profiles are applied at the chip entry. Respo...
Background: The circular economy closes loops in industrial manufacturing processes and minimizes... more Background: The circular economy closes loops in industrial manufacturing processes and minimizes waste. A bio-based economy aims to replace fossil-based resources and processes by sustainable alternatives which exploits renewable biomass for the generation of products used in our daily live. A current trend in fungal biotechnologythe production of fungal-based biomaterials-will contribute to both.
Streptomyces clavuligerus is a gram-positive filamentous bacterium notable for producing clavulan... more Streptomyces clavuligerus is a gram-positive filamentous bacterium notable for producing clavulanic acid (CA), an inhibitor of β-lactamase enzymes, which confers resistance to bacteria against several antibiotics. Here we present a comparative analysis of the morphological and metabolic response of S. clavuligerus linked to the CA production under low and high shear stress conditions in a 2D rocking-motion single-use bioreactor (CELL-tainer ®) and stirred tank bioreactor (STR), respectively. The CELL-tainer® guarantees high turbulence and enhanced volumetric mass transfer at low shear stress, which (in contrast to bubble columns) allows the investigation of the impact of shear stress without oxygen limitation. The results indicate that high shear forces do not compromise the viability of S. clavuligerus cells; even higher specific growth rate, biomass, and specific CA production rate were observed in the STR. Under low shear forces in the CELL-tainer® the mycelial diameter increased...
Polyhydroxyalkanoates (PHAs) are biodegradable plastic-like materials with versatile properties. ... more Polyhydroxyalkanoates (PHAs) are biodegradable plastic-like materials with versatile properties. Plant oils are excellent carbon sources for a cost-effective PHA production, due to their high carbon content, large availability, and comparatively low prices. Additionally, efficient process development and control is required for competitive PHA production, which can be facilitated by on-line or in-line monitoring devices. To this end, we have evaluated photon density wave (PDW) spectroscopy as a new process analytical technology for Ralstonia eutropha (Cupriavidus necator) H16 plant oil cultivations producing polyhydroxybutyrate (PHB) as an intracellular polymer. PDW spectroscopy was used for in-line recording of the reduced scattering coefficient µs’ and the absorption coefficient µa at 638 nm. A correlation of µs’ with the cell dry weight (CDW) and µa with the residual cell dry weight (RCDW) was observed during growth, PHB accumulation, and PHB degradation phases in batch and pulse...
Ruminococcin-A (RumA) is a peptide antibiotic with post-translational modifications including thi... more Ruminococcin-A (RumA) is a peptide antibiotic with post-translational modifications including thioether cross-links formed from non-canonical amino acids, called lanthionines, synthesized by a dedicated lanthionine-generating enzyme RumM. RumA is naturally produced by Ruminococcus gnavus, which is part of the normal bacterial flora in the human gut. High activity of RumA against pathogenic Clostridia has been reported, thus allowing potential exploitation of RumA for clinical applications. However, purifying RumA from R. gnavus is challenging due to low production yields (<1 µg L −1 ) and difficulties to cultivate the obligately anaerobic organism. We recently reported the reconstruction of the RumA biosynthesis machinery in Escherichia coli where the fully modified and active peptide was expressed as a fusion protein together with GFP. In the current study we developed a scale-up strategy for the biotechnologically relevant heterologous production of RumA, aimed at overproducing the peptide under conditions comparable with those in industrial production settings. To this end, glucose-limited fed-batch cultivation was used. Firstly, parallel cultivations were performed in 24microwell plates using the enzyme-based automated glucose-delivery cultivation system EnPresso R B to determine optimal conditions for IPTG induction. We combined the bioprocess development with ESI-MS and tandem ESI-MS to monitor modification of the precursor peptide (preRumA) during bioreactor cultivation. Dehydration of threonine and serine residues in the core peptide, catalyzed by RumM, occurs within 1 h after IPTG induction while formation of thioether cross-bridges occur around 2.5 h after induction. Our data also supplies important information on modification kinetics especially with respect to the fluctuations observed in the various dehydrated precursor peptide versions or intermediates produced at different time points during bioreactor cultivation. Overall, protein yields obtained from the bioreactor cultivations were >120 mg L −1 for the chimeric construct and >150 mg L −1 for RumM. The correlation observed between microscale and lab-scale bioreactor cultivations suggests that the process is robust and realistically applicable to industrial-scale conditions.
Pyrimidine-nucleoside phosphorylases (Py-NPases) have a significant potential to contribute to th... more Pyrimidine-nucleoside phosphorylases (Py-NPases) have a significant potential to contribute to the economic and ecological production of modified nucleosides. These can be produced via pentose-1-phosphates, an interesting but mostly labile and expensive precursor. Thus far, no dynamic model exists for the production process of pentose-1-phosphates, which involves the equilibrium state of the Py-NPase catalyzed reversible reaction. Previously developed enzymological models are based on the understanding of the structural principles of the enzyme and focus on the description of initial rates only. The model generation is further complicated, as Py-NPases accept two substrates which they convert to two products. To create a well-balanced model from accurate experimental data, we utilized an improved high-throughput spectroscopic assay to monitor reactions over the whole time course until equilibrium was reached. We examined the conversion of deoxythymidine and phosphate to deoxyribose-...
Depending on the environmental conditions, cells adapt their metabolism and specific growth rate.... more Depending on the environmental conditions, cells adapt their metabolism and specific growth rate. Rearrangements occur on many different levels such as macromolecular composition, gene and protein expression, morphology and metabolic flux patterns. As the interplay of these processes also determines the output of a recombinant protein producing system, having control over specific growth rate of the culture is advantageous. Continuous culture methods were developed to grow cells in a constant environment and have been used for decades to study basic microbial physiology in a controlled and reproducible manner. Our review summarizes the uses of continuous cultures in cell physiology studies and process development, with a focus on recombinant protein-producing microorganisms.
Ruminococcin A (RumA) is a lanthipeptide with high activity against pathogenic clostridia and is ... more Ruminococcin A (RumA) is a lanthipeptide with high activity against pathogenic clostridia and is naturally produced by the strict anaerobic bacterium E1, isolated from human intestine. Cultivating E1 is challenging, limiting high-quality production, further biotechnological development and therapeutic exploitation of RumA. To supply an alternative production system, the gene encoding RumA-modifying enzyme (RumM) and the gene encoding the unmodified precursor peptide (preRumA) were amplified from the chromosome of E1 and coexpressed in . Our results show that the ruminococcin-A lanthionine synthetase RumM catalyzed dehydration of threonine and serine residues and subsequently installed thioether bridges into the core structure of a mutant version of preRumA (preRumA). These modifications were achieved when the peptide was expressed as a fusion protein together with green fluorescence protein (GFP), demonstrating that a larger attachment to the N-terminus of the leader peptide does no...
Filamentous fungi including Aspergillus niger are cell factories for the production of organic ac... more Filamentous fungi including Aspergillus niger are cell factories for the production of organic acids, proteins and bioactive compounds. Traditionally, stirred-tank reactors (STRs) are used to cultivate them under highly reproducible conditions ensuring optimum oxygen uptake and high growth rates. However, agitation via mechanical stirring causes high shear forces, thus affecting fungal physiology and macromorphologies. Two-dimensional rocking-motion wave-mixed bioreactor cultivations could offer a viable alternative to fungal cultivations in STRs, as comparable gas mass transfer is generally achievable while deploying lower friction and shear forces. The aim of this study was thus to investigate for the first time the consequences of wave-mixed cultivations on the growth, macromorphology and product formation of A. niger. We investigated the impact of hydrodynamic conditions on A. niger cultivated at a 5 L scale in a disposable two-dimensional rocking motion bioreactor (CELL-tainer)...
Nucleoside phosphorylases catalyze the reversible phosphorolysis of pyrimidine and purine nucleos... more Nucleoside phosphorylases catalyze the reversible phosphorolysis of pyrimidine and purine nucleosides in the presence of phosphate. They are relevant to the appropriate function of the immune system in mammals and interesting drug targets for cancer treatment. Next to their role as drug targets nucleoside phosphorylases are used as catalysts in the synthesis of nucleosides and their analogs that are widely applied as pharmaceuticals. Based on their substrates nucleoside phosphorylases are classified as pyrimidine and purine nucleoside phosphorylases. This article describes the substrate spectra of nucleoside phosphorylases and structural properties that influence their activity. Substrate ranges are summarized and relations between members of pyrimidine or purine nucleoside phosphorylases are elucidated. Nucleoside phosphorylases accept a broad spectrum of substrates: they accept both base and sugar modified nucleosides. The most widely studied nucleoside phosphorylases are those of...
In this paper, an approach for the monitoring of biotechnological process kinetics is proposed. T... more In this paper, an approach for the monitoring of biotechnological process kinetics is proposed. The kinetics of each process state variable is presented as a function of two time-varying unknown parameters. For their estimation, a general software sensor is derived with on-line measurements as inputs that are accessible in practice. The stability analysis with a different number of inputs shows that stability can be guaranteed for fourth- and fifth-order software sensors only. As a case study, the monitoring of the kinetics of processes carried out in stirred tank reactors is investigated. A new tuning procedure is derived that results in a choice of only one design parameter. The effectiveness of the proposed procedure is demonstrated with experimental data from Bacillus subtilis fed-batch cultivations.
In bioprocess development, the host and the genetic construct for a new biomanufacturing process ... more In bioprocess development, the host and the genetic construct for a new biomanufacturing process are selected in the early developmental stages. This decision, made at the screening scale with very limited information about the performance in larger reactors, has a major influence on the efficiency of the final process. To overcome this, scale-down approaches during screenings that show the real cell factory performance at industrial-like conditions are essential. We present a fully automated robotic facility with 24 parallel mini-bioreactors that is operated by a model-based adaptive input design framework for the characterization of clone libraries under scale-down conditions. The cultivation operation strategies are computed and continuously refined based on a macro-kinetic growth model that is continuously re-fitted to the available experimental data. The added value of the approach is demonstrated with 24 parallel fed-batch cultivations in a mini-bioreactor system with eight di...
The combination of extreme miniaturization with a high sensitivity and the potential to be integr... more The combination of extreme miniaturization with a high sensitivity and the potential to be integrated in an array form on a chip has made silicon-based photonic microring resonators a very attractive research topic. As biosensors are approaching the nanoscale, analyte mass transfer and bonding kinetics have been ascribed as crucial factors that limit their performance. One solution may be a system that applies dielectrophoretic forces, in addition to microfluidics, to overcome the diffusion limits of conventional biosensors. Dielectrophoresis, which involves the migration of polarized dielectric particles in a non-uniform alternating electric field, has previously been successfully applied to achieve a 1000-fold improved detection efficiency in nanopore sensing and may significantly increase the sensitivity in microring resonator biosensing. In the current work, we designed microring resonators with integrated electrodes next to the sensor surface that may be used to explore the eff...
In conditional microbial screening, a limited number of candidate strains are tested at different... more In conditional microbial screening, a limited number of candidate strains are tested at different conditions searching for the optimal operation strategy in production (e.g., temperature and pH shifts, media composition as well as feeding and induction strategies). To achieve this, cultivation volumes of >10 mL and advanced control schemes are required to allow appropriate sampling and analyses. Operations become even more complex when the analytical methods are integrated into the robot facility. Among other multivariate data analysis methods, principal component analysis (PCA) techniques have especially gained popularity in high throughput screening. However, an important issue specific to high throughput bioprocess development is the lack of so-called golden batches that could be used as a basis for multivariate analysis. In this study, we establish and present a program to monitor dynamic parallel cultivations in a high throughput facility. PCA was used for process monitoring...
Milk kefir is a traditional fermented milk product whose consumption is becoming increasingly pop... more Milk kefir is a traditional fermented milk product whose consumption is becoming increasingly popular. The natural starter for kefir production is kefir grain, which consists of various bacterial and yeast species. At the industrial scale, however, kefir grains are rarely used due to their slow growth, complex application, bad reproducibility and high costs. Instead, mixtures of defined lactic acid bacteria and sometimes yeasts are applied, which alter sensory and functional properties compared to natural grain-based milk kefir. In order to be able to mimic natural starter cultures for authentic kefir production, it is a prerequisite to gain deep knowledge about the nature of kefir grains, its microbial composition, morphologic structure, composition of strains on grains and the impact of environmental parameters on kefir grain characteristics. In addition, it is very important to deeply investigate the numerous multi-dimensional interactions among different species, which play impo...
The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and efficient ... more The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and efficient alternative to chemical synthesis routes. In this study, dihalogenated nucleoside analogues were produced by thermostable nucleoside phosphorylases in transglycosylation reactions using uridine or thymidine as sugar donors. Prior to the enzymatic process, ideal maximum product yields were calculated after the determination of equilibrium constants through monitoring the equilibrium conversion in analytical-scale reactions. Equilibrium constants for dihalogenated nucleosides were comparable to known purine nucleosides, ranging between 0.071 and 0.081. To achieve 90% product yield in the enzymatic process, an approximately five-fold excess of sugar donor was needed. Nucleoside analogues were purified by semi-preparative HPLC, and yields of purified product were approximately 50% for all target compounds. To evaluate the impact of halogen atoms in positions 2 and 6 on the antiproliferative ...
Microbial cells in industrial large-scale bioreactors are exposed to fluctuating conditions, e.g.... more Microbial cells in industrial large-scale bioreactors are exposed to fluctuating conditions, e.g., nutrient concentration, dissolved oxygen, temperature, and pH. These inhomogeneities can influence the cell physiology and metabolism, e.g., decelerate cell growth and product formation. Microfluidic systems offer new opportunities to study such effects in great detail by examining responses to varying environmental conditions at single-cell level. However, the possibility to reproduce large-scale bioreactor conditions in microscale cultivation systems has not yet been systematically investigated. Hence, we apply computational fluid dynamics (CFD) simulations to analyze and compare three commonly used microfluidic single-cell trapping and cultivation devices that are based on (i) mother machines (MM), (ii) monolayer growth chambers (MGC), and (iii) negative dielectrophoresis (nDEP). Several representative time-variant nutrient concentration profiles are applied at the chip entry. Respo...
Background: The circular economy closes loops in industrial manufacturing processes and minimizes... more Background: The circular economy closes loops in industrial manufacturing processes and minimizes waste. A bio-based economy aims to replace fossil-based resources and processes by sustainable alternatives which exploits renewable biomass for the generation of products used in our daily live. A current trend in fungal biotechnologythe production of fungal-based biomaterials-will contribute to both.
Streptomyces clavuligerus is a gram-positive filamentous bacterium notable for producing clavulan... more Streptomyces clavuligerus is a gram-positive filamentous bacterium notable for producing clavulanic acid (CA), an inhibitor of β-lactamase enzymes, which confers resistance to bacteria against several antibiotics. Here we present a comparative analysis of the morphological and metabolic response of S. clavuligerus linked to the CA production under low and high shear stress conditions in a 2D rocking-motion single-use bioreactor (CELL-tainer ®) and stirred tank bioreactor (STR), respectively. The CELL-tainer® guarantees high turbulence and enhanced volumetric mass transfer at low shear stress, which (in contrast to bubble columns) allows the investigation of the impact of shear stress without oxygen limitation. The results indicate that high shear forces do not compromise the viability of S. clavuligerus cells; even higher specific growth rate, biomass, and specific CA production rate were observed in the STR. Under low shear forces in the CELL-tainer® the mycelial diameter increased...
Polyhydroxyalkanoates (PHAs) are biodegradable plastic-like materials with versatile properties. ... more Polyhydroxyalkanoates (PHAs) are biodegradable plastic-like materials with versatile properties. Plant oils are excellent carbon sources for a cost-effective PHA production, due to their high carbon content, large availability, and comparatively low prices. Additionally, efficient process development and control is required for competitive PHA production, which can be facilitated by on-line or in-line monitoring devices. To this end, we have evaluated photon density wave (PDW) spectroscopy as a new process analytical technology for Ralstonia eutropha (Cupriavidus necator) H16 plant oil cultivations producing polyhydroxybutyrate (PHB) as an intracellular polymer. PDW spectroscopy was used for in-line recording of the reduced scattering coefficient µs’ and the absorption coefficient µa at 638 nm. A correlation of µs’ with the cell dry weight (CDW) and µa with the residual cell dry weight (RCDW) was observed during growth, PHB accumulation, and PHB degradation phases in batch and pulse...
Ruminococcin-A (RumA) is a peptide antibiotic with post-translational modifications including thi... more Ruminococcin-A (RumA) is a peptide antibiotic with post-translational modifications including thioether cross-links formed from non-canonical amino acids, called lanthionines, synthesized by a dedicated lanthionine-generating enzyme RumM. RumA is naturally produced by Ruminococcus gnavus, which is part of the normal bacterial flora in the human gut. High activity of RumA against pathogenic Clostridia has been reported, thus allowing potential exploitation of RumA for clinical applications. However, purifying RumA from R. gnavus is challenging due to low production yields (<1 µg L −1 ) and difficulties to cultivate the obligately anaerobic organism. We recently reported the reconstruction of the RumA biosynthesis machinery in Escherichia coli where the fully modified and active peptide was expressed as a fusion protein together with GFP. In the current study we developed a scale-up strategy for the biotechnologically relevant heterologous production of RumA, aimed at overproducing the peptide under conditions comparable with those in industrial production settings. To this end, glucose-limited fed-batch cultivation was used. Firstly, parallel cultivations were performed in 24microwell plates using the enzyme-based automated glucose-delivery cultivation system EnPresso R B to determine optimal conditions for IPTG induction. We combined the bioprocess development with ESI-MS and tandem ESI-MS to monitor modification of the precursor peptide (preRumA) during bioreactor cultivation. Dehydration of threonine and serine residues in the core peptide, catalyzed by RumM, occurs within 1 h after IPTG induction while formation of thioether cross-bridges occur around 2.5 h after induction. Our data also supplies important information on modification kinetics especially with respect to the fluctuations observed in the various dehydrated precursor peptide versions or intermediates produced at different time points during bioreactor cultivation. Overall, protein yields obtained from the bioreactor cultivations were >120 mg L −1 for the chimeric construct and >150 mg L −1 for RumM. The correlation observed between microscale and lab-scale bioreactor cultivations suggests that the process is robust and realistically applicable to industrial-scale conditions.
Pyrimidine-nucleoside phosphorylases (Py-NPases) have a significant potential to contribute to th... more Pyrimidine-nucleoside phosphorylases (Py-NPases) have a significant potential to contribute to the economic and ecological production of modified nucleosides. These can be produced via pentose-1-phosphates, an interesting but mostly labile and expensive precursor. Thus far, no dynamic model exists for the production process of pentose-1-phosphates, which involves the equilibrium state of the Py-NPase catalyzed reversible reaction. Previously developed enzymological models are based on the understanding of the structural principles of the enzyme and focus on the description of initial rates only. The model generation is further complicated, as Py-NPases accept two substrates which they convert to two products. To create a well-balanced model from accurate experimental data, we utilized an improved high-throughput spectroscopic assay to monitor reactions over the whole time course until equilibrium was reached. We examined the conversion of deoxythymidine and phosphate to deoxyribose-...
Depending on the environmental conditions, cells adapt their metabolism and specific growth rate.... more Depending on the environmental conditions, cells adapt their metabolism and specific growth rate. Rearrangements occur on many different levels such as macromolecular composition, gene and protein expression, morphology and metabolic flux patterns. As the interplay of these processes also determines the output of a recombinant protein producing system, having control over specific growth rate of the culture is advantageous. Continuous culture methods were developed to grow cells in a constant environment and have been used for decades to study basic microbial physiology in a controlled and reproducible manner. Our review summarizes the uses of continuous cultures in cell physiology studies and process development, with a focus on recombinant protein-producing microorganisms.
Ruminococcin A (RumA) is a lanthipeptide with high activity against pathogenic clostridia and is ... more Ruminococcin A (RumA) is a lanthipeptide with high activity against pathogenic clostridia and is naturally produced by the strict anaerobic bacterium E1, isolated from human intestine. Cultivating E1 is challenging, limiting high-quality production, further biotechnological development and therapeutic exploitation of RumA. To supply an alternative production system, the gene encoding RumA-modifying enzyme (RumM) and the gene encoding the unmodified precursor peptide (preRumA) were amplified from the chromosome of E1 and coexpressed in . Our results show that the ruminococcin-A lanthionine synthetase RumM catalyzed dehydration of threonine and serine residues and subsequently installed thioether bridges into the core structure of a mutant version of preRumA (preRumA). These modifications were achieved when the peptide was expressed as a fusion protein together with green fluorescence protein (GFP), demonstrating that a larger attachment to the N-terminus of the leader peptide does no...
Filamentous fungi including Aspergillus niger are cell factories for the production of organic ac... more Filamentous fungi including Aspergillus niger are cell factories for the production of organic acids, proteins and bioactive compounds. Traditionally, stirred-tank reactors (STRs) are used to cultivate them under highly reproducible conditions ensuring optimum oxygen uptake and high growth rates. However, agitation via mechanical stirring causes high shear forces, thus affecting fungal physiology and macromorphologies. Two-dimensional rocking-motion wave-mixed bioreactor cultivations could offer a viable alternative to fungal cultivations in STRs, as comparable gas mass transfer is generally achievable while deploying lower friction and shear forces. The aim of this study was thus to investigate for the first time the consequences of wave-mixed cultivations on the growth, macromorphology and product formation of A. niger. We investigated the impact of hydrodynamic conditions on A. niger cultivated at a 5 L scale in a disposable two-dimensional rocking motion bioreactor (CELL-tainer)...
Nucleoside phosphorylases catalyze the reversible phosphorolysis of pyrimidine and purine nucleos... more Nucleoside phosphorylases catalyze the reversible phosphorolysis of pyrimidine and purine nucleosides in the presence of phosphate. They are relevant to the appropriate function of the immune system in mammals and interesting drug targets for cancer treatment. Next to their role as drug targets nucleoside phosphorylases are used as catalysts in the synthesis of nucleosides and their analogs that are widely applied as pharmaceuticals. Based on their substrates nucleoside phosphorylases are classified as pyrimidine and purine nucleoside phosphorylases. This article describes the substrate spectra of nucleoside phosphorylases and structural properties that influence their activity. Substrate ranges are summarized and relations between members of pyrimidine or purine nucleoside phosphorylases are elucidated. Nucleoside phosphorylases accept a broad spectrum of substrates: they accept both base and sugar modified nucleosides. The most widely studied nucleoside phosphorylases are those of...
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Papers by Peter Neubauer