Interactive visualization of clusters in microarray data: an efficient tool for improved metaboli... more Interactive visualization of clusters in microarray data: an efficient tool for improved metabolic analysis of E. coli
For optimization of recombinant fermentations both the type of expression system and the metaboli... more For optimization of recombinant fermentations both the type of expression system and the metabolic capacity of the host cell have to considered. The estimation of the metabolic load on the host cell during expression of recombinant protein is a crucial parameter and gives significant information to approach optimum yield. Metabolic control of microbial cells is based on a hierarchical organisation comprising a multitude of regulatory networks with specific signal molecules linked to specific transduction pathways. Thus the concentration of these specific signal molecules reflects the state of individual regulatory blocks and monitoring of these molecules offers novel tools to optimize control of recombinant ferrnentations. The signal molecule of the stringent control network, guanosinetetraphosphate (ppGpp) was analysed to estimate the metabolic load of different expression systems on the host cell during production of recombinant human superoxidedismutase. The ppGpp concentration was quantitated by ion pair HPLC. Due to the high turnover of ppGpp the development of a special sampling procedure was essential. The formation of ppGpp in relation to various environmental changes and to recombinant protein expression is shown.
A novel approach to bioprocess optimization is to focus on the signal processing capabilities of ... more A novel approach to bioprocess optimization is to focus on the signal processing capabilities of the cell. The analysis of specific signals, such as Guanosinetetraphosphate, enables better understanding of the relations between metabolic load and recombinant protein production, to make use of the cell's synthetic capacity. Due to the fact that analysis of these signal molecules is a difficult task, it would be highly beneficial to model their appearance and concentration. Kohonen's Self Organizing Maps were used for input data selection improvement, furtheron Neural Network models (Radial Basis Function Networks) representing omine data were developed and first results are presented.
The overall strategy for process optimisation of recombinant protein production is aiming at maxi... more The overall strategy for process optimisation of recombinant protein production is aiming at maximal exploitation of the cell factory's potential over a prolonged period of time by means of optimal control of the flux-ratios between biosynthesis of host cell proteins and recombinant proteins. To achieve these goals methods for (1) monitoring of the metabolic load of host cell, (2) methods to monitor and control plasmid copy number and (3) measures for tuning the expression rate to the metabolic load limits of the host cell must be established.
Escherichia coli K-12 and B strains are among the most frequently used bacterial hosts for produc... more Escherichia coli K-12 and B strains are among the most frequently used bacterial hosts for production of recombinant proteins on an industrial scale. To improve existing processes and to accelerate bioprocess development, we performed a detailed host analysis. We investigated the different behaviors of the E. coli production strains BL21, RV308, and HMS174 in response to high-glucose concentrations. Tightly controlled cultivations were conducted under defined environmental conditions for the in-depth analysis of physiological behavior. In addition to acquisition of standard process parameters, we also used DNA microarray analysis and differential gel electrophoresis (Ettan TM DIGE). Batch cultivations showed different yields of the distinct strains for cell dry mass and growth rate, which were highest for BL21. In addition, production of acetate, triggered by excess glucose supply, was much higher for the K-12 strains compared to the B strain. Analysis of transcriptome data showed significant alteration in 347 of 3882 genes common among all three hosts. These differentially expressed genes included, for example, those involved in transport, iron acquisition, and motility. The investigation of proteome patterns additionally revealed a high number of differentially expressed proteins among the investigated hosts. The subsequently selected 38 spots included proteins involved in transport and motility. The results of this comprehensive analysis delivered a full genomic picture of the three investigated strains. Differentially expressed groups for targeted host modification were identified like glucose transport or iron acquisition, enabling potential optimization of strains to improve yield and process quality. Dissimilar growth profiles of the strains confirm different genotypes. Furthermore, distinct transcriptome patterns support differential regulation at the genome level. The identified proteins showed high agreement with the transcriptome data and suggest similar regulation within a host at both levels for the identified groups. Such host attributes need to be considered in future process design and operation.
Recombinant garlic alliin lyase was produced in Escherichia coli, Saccharomyces cerevisiae, and P... more Recombinant garlic alliin lyase was produced in Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. A cDNA clone was obtained from garlic bulbs by PCR and introduced into suitable bacterial and yeast expression vectors. The recombinant alliin lyase forms inclusion bodies in all three host organisms, which are deposited in the cytoplasm. After cell lysis and harvesting by centrifugation, the inclusion bodies were solubilized in Zwittergent 3-14 solution and refolded by stepwise dilution. Specific alliin lyase activity could be recovered by this procedure.
We describe a prokaryotic expression system using the autoproteolytic function of N pro from clas... more We describe a prokaryotic expression system using the autoproteolytic function of N pro from classical swine fever virus. Proteins or peptides expressed as N pro fusions are deposited as inclusion bodies. On in vitro refolding by switching from chaotropic to kosmotropic conditions, the fusion partner is released from the C-terminal end of the autoprotease by self-cleavage, leaving the target protein with an authentic N terminus. A tailor-made N pro mutant called EDDIE, with increased in vitro and decreased in vivo cleavage rates, has enabled us to express proinsulin, domain-D of staphylococcal protein A, hepcidin, interferon-a1, keratin-associated protein 10-4, green fluorescent protein, inhibitorial peptide of senescence-evasion-factor, monocyte chemoattractant protein-1 and toxic gyrase inhibitor, among others. This N pro expression system can be used as a generic tool for the high-level production of recombinant toxic peptides and proteins (up to 12 g/l) in Escherichia coli without the need for chemical or enzymatic removal of the fusion tag.
The accomplishment of the quantification of the recombinant protein content of whole bacterial ce... more The accomplishment of the quantification of the recombinant protein content of whole bacterial cells by FT-IR spectroscopy by application of chemometrics is shown. Recombinant Escherichia coli cells expressing an inclusion body forming fusion protein were dried on a 96-well silicon plate for the analysis in a high-throughput FT-IR spectrometer. Acquired spectra of additionally conventionally quantified samples were used to establish a multivariate calibration. The obtained method was tested by predicting inclusion body contents of samples not used for the multivariate model. Results from FT-IR spectra coincided well with the data of universalized electrophoresis analysis. Hence FT-IR spectroscopy could prove as a fast and simple alternative to conventional quantification methods.
The main goal of this work was to develop a strategy that enables tuning of recombinant gene expr... more The main goal of this work was to develop a strategy that enables tuning of recombinant gene expression relative to the metabolic capacity of the host cell synthesis machinery. In the past, strong expression systems have been developed in order to maximize recombinant gene expression. However, these systems exert an extremely high metabolic burden onto the host cell, which may even lead to cell death. Hence, the period of recombinant gene expression is significantly reduced, and therefore, maximal yield cannot be attained. To extend the production phase and to achieve optimal yields, adjustment of recombinant gene expression by modulation of the transcription rate is required. To control transcription, we designed a feed regime, which continuously supplies limiting amounts of inducer in a constant ratio to biomass. For the accurate determination of appropriate amounts of inducer, a time shifted exponential substrate and inducer feed strategy has been developed. The potential of this metabolic and engineering integrated approach was proven in fed-batch cultivation experiments using E. coli HMS174(DE3)(pET11ahSOD) as model system. Furthermore, our strategy enables the use of lactose as inducer, since its consumption can be compensated by appropriate feed profiles. The attained results fully comply with all requirements of industrial large scale cultivation and improve the applicability of strong expression systems.
Background: Interpretation of comprehensive DNA microarray data sets is a challenging task for bi... more Background: Interpretation of comprehensive DNA microarray data sets is a challenging task for biologists and process engineers where scientific assistance of statistics and bioinformatics is essential. Interdisciplinary cooperation and concerted development of software-tools for simplified and accelerated data analysis and interpretation is the key to overcome the bottleneck in dataanalysis workflows. This approach is exemplified by gcExplorer an interactive visualization toolbox based on cluster analysis. Clustering is an important tool in gene expression data analysis to find groups of co-expressed genes which can finally suggest functional pathways and interactions between genes. The visualization of gene clusters gives practitioners an understanding of the cluster structure of their data and makes it easier to interpret the cluster results. Results: In this study the interactive visualization toolbox gcExplorer is applied to the interpretation of E. coli microarray data. The data sets derive from two fedbatch experiments conducted in order to investigate the impact of different induction strategies on the host metabolism and product yield. The software enables direct graphical comparison of these two experiments. The identification of potentially interesting gene candidates or functional groups is substantially accelerated and eased. Conclusion: It was shown that gcExplorer is a very helpful tool to gain a general overview of microarray experiments. Interesting gene expression patterns can easily be found, compared among different experiments and combined with information about gene function from publicly available databases.
Summary Biotechnology, with the main applications in food and nutrition, dates back to the early ... more Summary Biotechnology, with the main applications in food and nutrition, dates back to the early times of mankind. In the recent decades the progress in natural sciences, mathematics and computer science has led to a new branch termed molecular biotechnology, which finally developed as an autonomous scientific discipline. The field of biotechnology, in the past generally empirically driven, now largely benefits from molecular biotechnology by improved systems, knowledge and understanding. Thereby, compliance with the recently published initiatives of the regulatory authorities to accelerate the approval process for the manufacturing of biopharmaceuticals can be gained.
ABSTRACT The stimulating effect of some heavy metals, when added to cheese whey, on growth of the... more ABSTRACT The stimulating effect of some heavy metals, when added to cheese whey, on growth of the yeast Candida intermedia was investigated. With copper, manganese, zinc, iron, and molybdemum addition to whey, biomass productivity was increased enormously. The process was implemented by an Austrian dairy. In a 14-m 3 jet fermentor 30,000 liters of whey were processed daily. The mash leaving the fermentor was concentrated to 12% yeast dry matter by a vacuum evaporator, and after supplementation with other nutrients was fed directly to pigs. Based on Euro- pean prices, the yeast product was about 20% cheaper than soy meal. Because the process is simple and can be run under nonaseptic conditions, it can be integrated fully into an existing dairy without additional operating per- sonnel.
A new strategy for controlling recombinant gene expression improves efficiency, maximizes host ve... more A new strategy for controlling recombinant gene expression improves efficiency, maximizes host vector exploitation, reduces costs, improves product consistency, and accelerates product development. Continuous feeds of limited amounts of inducer proportional to biomass growth grant optimal control over the ratio between gene expression and host cell metabolism, providing stable, prolonged recombinant protein production.
The model expression vector pET30arhSOD was used to trigger metabolic stress upon induction of re... more The model expression vector pET30arhSOD was used to trigger metabolic stress upon induction of recombinant protein production. With the 2-plasmid-system a significant increase of fluorescence of the induced fermentation in comparison with the non induced control fermentation was found. The concept of fusing stress relevant promoters with GFP for monitoring of the overburden of the cell was proven. To cope with the weak fluorescence signal of the genome integrated monitoring host amplification of the GFP signal is planned.
Interactive visualization of clusters in microarray data: an efficient tool for improved metaboli... more Interactive visualization of clusters in microarray data: an efficient tool for improved metabolic analysis of E. coli
For optimization of recombinant fermentations both the type of expression system and the metaboli... more For optimization of recombinant fermentations both the type of expression system and the metabolic capacity of the host cell have to considered. The estimation of the metabolic load on the host cell during expression of recombinant protein is a crucial parameter and gives significant information to approach optimum yield. Metabolic control of microbial cells is based on a hierarchical organisation comprising a multitude of regulatory networks with specific signal molecules linked to specific transduction pathways. Thus the concentration of these specific signal molecules reflects the state of individual regulatory blocks and monitoring of these molecules offers novel tools to optimize control of recombinant ferrnentations. The signal molecule of the stringent control network, guanosinetetraphosphate (ppGpp) was analysed to estimate the metabolic load of different expression systems on the host cell during production of recombinant human superoxidedismutase. The ppGpp concentration was quantitated by ion pair HPLC. Due to the high turnover of ppGpp the development of a special sampling procedure was essential. The formation of ppGpp in relation to various environmental changes and to recombinant protein expression is shown.
A novel approach to bioprocess optimization is to focus on the signal processing capabilities of ... more A novel approach to bioprocess optimization is to focus on the signal processing capabilities of the cell. The analysis of specific signals, such as Guanosinetetraphosphate, enables better understanding of the relations between metabolic load and recombinant protein production, to make use of the cell's synthetic capacity. Due to the fact that analysis of these signal molecules is a difficult task, it would be highly beneficial to model their appearance and concentration. Kohonen's Self Organizing Maps were used for input data selection improvement, furtheron Neural Network models (Radial Basis Function Networks) representing omine data were developed and first results are presented.
The overall strategy for process optimisation of recombinant protein production is aiming at maxi... more The overall strategy for process optimisation of recombinant protein production is aiming at maximal exploitation of the cell factory's potential over a prolonged period of time by means of optimal control of the flux-ratios between biosynthesis of host cell proteins and recombinant proteins. To achieve these goals methods for (1) monitoring of the metabolic load of host cell, (2) methods to monitor and control plasmid copy number and (3) measures for tuning the expression rate to the metabolic load limits of the host cell must be established.
Escherichia coli K-12 and B strains are among the most frequently used bacterial hosts for produc... more Escherichia coli K-12 and B strains are among the most frequently used bacterial hosts for production of recombinant proteins on an industrial scale. To improve existing processes and to accelerate bioprocess development, we performed a detailed host analysis. We investigated the different behaviors of the E. coli production strains BL21, RV308, and HMS174 in response to high-glucose concentrations. Tightly controlled cultivations were conducted under defined environmental conditions for the in-depth analysis of physiological behavior. In addition to acquisition of standard process parameters, we also used DNA microarray analysis and differential gel electrophoresis (Ettan TM DIGE). Batch cultivations showed different yields of the distinct strains for cell dry mass and growth rate, which were highest for BL21. In addition, production of acetate, triggered by excess glucose supply, was much higher for the K-12 strains compared to the B strain. Analysis of transcriptome data showed significant alteration in 347 of 3882 genes common among all three hosts. These differentially expressed genes included, for example, those involved in transport, iron acquisition, and motility. The investigation of proteome patterns additionally revealed a high number of differentially expressed proteins among the investigated hosts. The subsequently selected 38 spots included proteins involved in transport and motility. The results of this comprehensive analysis delivered a full genomic picture of the three investigated strains. Differentially expressed groups for targeted host modification were identified like glucose transport or iron acquisition, enabling potential optimization of strains to improve yield and process quality. Dissimilar growth profiles of the strains confirm different genotypes. Furthermore, distinct transcriptome patterns support differential regulation at the genome level. The identified proteins showed high agreement with the transcriptome data and suggest similar regulation within a host at both levels for the identified groups. Such host attributes need to be considered in future process design and operation.
Recombinant garlic alliin lyase was produced in Escherichia coli, Saccharomyces cerevisiae, and P... more Recombinant garlic alliin lyase was produced in Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. A cDNA clone was obtained from garlic bulbs by PCR and introduced into suitable bacterial and yeast expression vectors. The recombinant alliin lyase forms inclusion bodies in all three host organisms, which are deposited in the cytoplasm. After cell lysis and harvesting by centrifugation, the inclusion bodies were solubilized in Zwittergent 3-14 solution and refolded by stepwise dilution. Specific alliin lyase activity could be recovered by this procedure.
We describe a prokaryotic expression system using the autoproteolytic function of N pro from clas... more We describe a prokaryotic expression system using the autoproteolytic function of N pro from classical swine fever virus. Proteins or peptides expressed as N pro fusions are deposited as inclusion bodies. On in vitro refolding by switching from chaotropic to kosmotropic conditions, the fusion partner is released from the C-terminal end of the autoprotease by self-cleavage, leaving the target protein with an authentic N terminus. A tailor-made N pro mutant called EDDIE, with increased in vitro and decreased in vivo cleavage rates, has enabled us to express proinsulin, domain-D of staphylococcal protein A, hepcidin, interferon-a1, keratin-associated protein 10-4, green fluorescent protein, inhibitorial peptide of senescence-evasion-factor, monocyte chemoattractant protein-1 and toxic gyrase inhibitor, among others. This N pro expression system can be used as a generic tool for the high-level production of recombinant toxic peptides and proteins (up to 12 g/l) in Escherichia coli without the need for chemical or enzymatic removal of the fusion tag.
The accomplishment of the quantification of the recombinant protein content of whole bacterial ce... more The accomplishment of the quantification of the recombinant protein content of whole bacterial cells by FT-IR spectroscopy by application of chemometrics is shown. Recombinant Escherichia coli cells expressing an inclusion body forming fusion protein were dried on a 96-well silicon plate for the analysis in a high-throughput FT-IR spectrometer. Acquired spectra of additionally conventionally quantified samples were used to establish a multivariate calibration. The obtained method was tested by predicting inclusion body contents of samples not used for the multivariate model. Results from FT-IR spectra coincided well with the data of universalized electrophoresis analysis. Hence FT-IR spectroscopy could prove as a fast and simple alternative to conventional quantification methods.
The main goal of this work was to develop a strategy that enables tuning of recombinant gene expr... more The main goal of this work was to develop a strategy that enables tuning of recombinant gene expression relative to the metabolic capacity of the host cell synthesis machinery. In the past, strong expression systems have been developed in order to maximize recombinant gene expression. However, these systems exert an extremely high metabolic burden onto the host cell, which may even lead to cell death. Hence, the period of recombinant gene expression is significantly reduced, and therefore, maximal yield cannot be attained. To extend the production phase and to achieve optimal yields, adjustment of recombinant gene expression by modulation of the transcription rate is required. To control transcription, we designed a feed regime, which continuously supplies limiting amounts of inducer in a constant ratio to biomass. For the accurate determination of appropriate amounts of inducer, a time shifted exponential substrate and inducer feed strategy has been developed. The potential of this metabolic and engineering integrated approach was proven in fed-batch cultivation experiments using E. coli HMS174(DE3)(pET11ahSOD) as model system. Furthermore, our strategy enables the use of lactose as inducer, since its consumption can be compensated by appropriate feed profiles. The attained results fully comply with all requirements of industrial large scale cultivation and improve the applicability of strong expression systems.
Background: Interpretation of comprehensive DNA microarray data sets is a challenging task for bi... more Background: Interpretation of comprehensive DNA microarray data sets is a challenging task for biologists and process engineers where scientific assistance of statistics and bioinformatics is essential. Interdisciplinary cooperation and concerted development of software-tools for simplified and accelerated data analysis and interpretation is the key to overcome the bottleneck in dataanalysis workflows. This approach is exemplified by gcExplorer an interactive visualization toolbox based on cluster analysis. Clustering is an important tool in gene expression data analysis to find groups of co-expressed genes which can finally suggest functional pathways and interactions between genes. The visualization of gene clusters gives practitioners an understanding of the cluster structure of their data and makes it easier to interpret the cluster results. Results: In this study the interactive visualization toolbox gcExplorer is applied to the interpretation of E. coli microarray data. The data sets derive from two fedbatch experiments conducted in order to investigate the impact of different induction strategies on the host metabolism and product yield. The software enables direct graphical comparison of these two experiments. The identification of potentially interesting gene candidates or functional groups is substantially accelerated and eased. Conclusion: It was shown that gcExplorer is a very helpful tool to gain a general overview of microarray experiments. Interesting gene expression patterns can easily be found, compared among different experiments and combined with information about gene function from publicly available databases.
Summary Biotechnology, with the main applications in food and nutrition, dates back to the early ... more Summary Biotechnology, with the main applications in food and nutrition, dates back to the early times of mankind. In the recent decades the progress in natural sciences, mathematics and computer science has led to a new branch termed molecular biotechnology, which finally developed as an autonomous scientific discipline. The field of biotechnology, in the past generally empirically driven, now largely benefits from molecular biotechnology by improved systems, knowledge and understanding. Thereby, compliance with the recently published initiatives of the regulatory authorities to accelerate the approval process for the manufacturing of biopharmaceuticals can be gained.
ABSTRACT The stimulating effect of some heavy metals, when added to cheese whey, on growth of the... more ABSTRACT The stimulating effect of some heavy metals, when added to cheese whey, on growth of the yeast Candida intermedia was investigated. With copper, manganese, zinc, iron, and molybdemum addition to whey, biomass productivity was increased enormously. The process was implemented by an Austrian dairy. In a 14-m 3 jet fermentor 30,000 liters of whey were processed daily. The mash leaving the fermentor was concentrated to 12% yeast dry matter by a vacuum evaporator, and after supplementation with other nutrients was fed directly to pigs. Based on Euro- pean prices, the yeast product was about 20% cheaper than soy meal. Because the process is simple and can be run under nonaseptic conditions, it can be integrated fully into an existing dairy without additional operating per- sonnel.
A new strategy for controlling recombinant gene expression improves efficiency, maximizes host ve... more A new strategy for controlling recombinant gene expression improves efficiency, maximizes host vector exploitation, reduces costs, improves product consistency, and accelerates product development. Continuous feeds of limited amounts of inducer proportional to biomass growth grant optimal control over the ratio between gene expression and host cell metabolism, providing stable, prolonged recombinant protein production.
The model expression vector pET30arhSOD was used to trigger metabolic stress upon induction of re... more The model expression vector pET30arhSOD was used to trigger metabolic stress upon induction of recombinant protein production. With the 2-plasmid-system a significant increase of fluorescence of the induced fermentation in comparison with the non induced control fermentation was found. The concept of fusing stress relevant promoters with GFP for monitoring of the overburden of the cell was proven. To cope with the weak fluorescence signal of the genome integrated monitoring host amplification of the GFP signal is planned.
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Papers by Karl Bayer