Balaji Balagurunathan - Academia.edu (original) (raw)

Papers by Balaji Balagurunathan

Current Opinion in Biotechnology

Singapore is one of the major centers for oil, gas, and petrochemicals in the world. The centerpi... more Singapore is one of the major centers for oil, gas, and petrochemicals in the world. The centerpiece of Singapore’s petrochemical industry is the Jurong Island complex, which houses many of the world’s leading petroleum and petrochemical companies. Currently, the total refining capacity of Singapore is 1.3 million bpd [1] and the ethylene capacity is 2.1 million tons per year, projected to reach 4 million tons per year by 2014 [2]. The petrochemical industry in Singapore is heavily dependent on fossil fuel-based feedstock, mostly crude oil and natural gas. Use of fossil fuels has negative environmental impact due to the greenhouse gas emission. Another crisis arising from overdependence on fossil fuels is their rapid depletion. Fossil fuels are non-renewable, which means they are finite and might well run out in the near future – a study found that fossil fuels reserve depletion times for oil, coal, and gas are approximately 35, 107, and 37 years, respectively [3]. This rapid deplet...

Computer Aided Chemical Engineering, 2012

Abstract Singapore's petrochemical industry plays an integral part in the nation's econom... more Abstract Singapore's petrochemical industry plays an integral part in the nation's economy. The industry is highly dependent on crude oil as the major feedstock. This could prove increasingly unsustainable with depleting fossil fuels resources and growing environmental concerns over greenhouse gases emissions. Using renewable feedstocks such as biomass is one possible solution. This paper evaluates the potential for bio-based chemicals production in Singapore's petrochemical cluster, focusing on polylactic acid (PLA). Cost and carbon footprint assessment of the PLA supply chain using various bio-renewable feedstocks sourced from the region is presented.

Modeling and analysis of genetic networks have become increasingly important in the investigation... more Modeling and analysis of genetic networks have become increasingly important in the investigation of cellular processes. The genetic networks involved in cellular stress response can have a critical effect on the productivity of recombinant proteins. In this work, it was found that the temperature-inducible expression system for the production of soluble recombinant streptokinase in Escherichia coli resulted in a lower productivity compared to the chemically-induced system. To investigate the effect of the induced cellular response due to temperature up-shift a model-based approach is adopted. The role played by the major molecular chaperone teams DnaK–DnaJ–GrpE and GroEL–GroES on the productivity of recombinant streptokinase was experimentally determined. Based on these investigations, a detailed mechanistic mathematical model was developed for the cellular response during the temperature-induced recombinant streptokinase production. The model simulations were found to have a good ...

We describe here an approach for rapidly producing scar-free and precise gene deletions in S. cer... more We describe here an approach for rapidly producing scar-free and precise gene deletions in S. cerevisiae with high efficiency. Preparation of the disruption gene cassette in this approach was simply performed by overlap extension-PCR of an invert repeat of a partial or complete sequence of the targeted gene with URA3. Integration of the prepared disruption gene cassette to the designated position of a target gene leads to the formation of a mutagenesis cassette within the yeast genome, which consists of a URA3 gene flanked by the targeted gene and its inverted repeat between two short identical direct repeats. The inherent instability of the inverted sequences in close proximity facilitates the self-excision of the entire mutagenesis cassette deposited in the genome and promotes homologous recombination resulting in a seamless deletion via a single transformation. This rapid assembly circumvents the difficulty during preparation of disruption gene cassettes composed of two inverted ...

Systems and Synthetic Biology, Jan 24, 2009

Modeling and analysis of genetic networks have become increasingly important in the investigation... more Modeling and analysis of genetic networks have become increasingly important in the investigation of cellular processes. The genetic networks involved in cellular stress response can have a critical effect on the productivity of recombinant proteins. In this work, it was found that the temperature-inducible expression system for the production of soluble recombinant streptokinase in Escherichia coli resulted in a lower productivity compared to the chemically-induced system. To investigate the effect of the induced cellular response due to temperature up-shift a model-based approach is adopted. The role played by the major molecular chaperone teams DnaK-DnaJ-GrpE and GroEL-GroES on the productivity of recombinant streptokinase was experimentally determined. Based on these investigations, a detailed mechanistic mathematical model was developed for the cellular response during the temperature-induced recombinant streptokinase production. The model simulations were found to have a good qualitative agreement with the experimental results. The mechanistic mathematical model was validated with the experiments conducted on a r 32 mutant strain. Detailed analysis of the parameter sensitivities of the model indicated that the level of free DnaK chaperone in the cell has the major effect on the productivity of recombinant streptokinase during temperature induction. Analysis of the model simulations also shows that down regulation or selective redirection of the heat shock proteins could be a better way of manipulating the cellular stress response than overexpression or deletion. In other words, manipulating the system properties resulting from the interaction of the components is better than manipulating the individual components. Although our results are specific to a recombinant protein (streptokinase) and the expression system (E. coli), we believe that such a systems-biological approach has several advantages over conventional experimental approaches and could be in principle extended to bigger genetic networks as well as other recombinant proteins and expression systems.

Enzyme and microbial technology, 2018

For the sustainable production of acetaldehyde, a key raw-material for a large number of chemical... more For the sustainable production of acetaldehyde, a key raw-material for a large number of chemical products, microbial production is a promising alternative. We have engineered an Escherichia coli strain for acetaldehyde production from glucose by introducing the pyruvate decarboxylase (Pdc) from Zymomonas mobilis and NADH oxidase (Nox) from Lactococcus lactis. Acetaldehyde production was systematically improved by knocking out the competing metabolic pathways. Multiple knockout strains were created and a final acetaldehyde titre of 0.73g/L was achieved using a quadruple knockout strain E. coli MC4100 ΔadhE ΔldhA ΔfrdC ΔackA-pta. In addition to acetaldehyde, about 0.37g/L acetoin was produced by these strains due to the additional carboligase activity exhibited by pyruvate decarboxylase resulting in a total carbon yield of 0.27g/g glucose.

Improvement of biological strains through targeted modification of metabolism is essential for su... more Improvement of biological strains through targeted modification of metabolism is essential for successful development of bioprocesses. The computational complexity of optimization procedures routinely used for identifying genetic targets limits their application to genome-scale metabolic networks. In this study, we combined graph theoretic approaches with mixed-integer liner programming (MILP) to reduce the search space and thus reducing computational time. Specifically, we used cut-sets (minimal set of reactions that cuts metabolic networks) as additional constraints to reduce the search space. The efficacy of proposed approach is illustrated by identifying minimal reaction set for Saccharomyces Cerevisiae.

Applied biochemistry and biotechnology, Jan 15, 2015

The stress response of Escherichia coli to 3-hydroxypropanoic acid (3-HP) was elucidated through ... more The stress response of Escherichia coli to 3-hydroxypropanoic acid (3-HP) was elucidated through global transcriptomic analysis. Around 375 genes showed difference of more than 2-fold in 3-HP-treated samples. Further analysis revealed that the toxicity effect of 3-HP was due to the cation and anion components of this acid and some effects-specific to 3-HP. Genes related to the oxidative stress, DNA protection, and repair were upregulated in treated cells due to the lowered cytoplasmic pH caused by accumulated cations. 3-HP-treated E. coli used the arginine acid tolerance mechanism to increase the cytoplasmic pH. Additionally, the anion effects were manifested as imbalance in the osmotic pressure. Analysis of top ten highly upregulated genes suggests the formation of 3-hydroxypropionaldehyde under 3-HP stress. The transcriptomic analysis shed light on the global genetic reprogramming due to 3-HP stress and suggests strategies for increasing the tolerance of E. coli toward 3-HP.

Bioprocess and biosystems engineering, Jan 28, 2016

Integrated approaches using in silico model-based design and advanced genetic tools have enabled ... more Integrated approaches using in silico model-based design and advanced genetic tools have enabled efficient production of fuels, chemicals and functional ingredients using microbial cell factories. In this study, using a recently developed genome-scale metabolic model for Escherichia coli iJO1366, a mutant strain has been designed in silico for the anaerobic growth-coupled production of a simple polyol, glycerol. Computational complexity was significantly reduced by systematically reducing the target reactions used for knockout simulations. One promising penta knockout E. coli mutant (E. coli ΔadhE ΔldhA ΔfrdC ΔtpiA ΔmgsA) was selected from simulation study and was constructed experimentally by sequentially deleting five genes. The penta mutant E. coli bearing the Saccharomyces cerevisiae glycerol production pathway was able to grow anaerobically and produce glycerol as the major metabolite with up to 90% of theoretical yield along with stoichiometric quantities of acetate and format...

nt.ntnu.no, 2005

Recombinant protein over-expression in Escherichia coli often elicits a stress response that lead... more Recombinant protein over-expression in Escherichia coli often elicits a stress response that leads to the up-regulation of stress genes and their products. The localization of the recombinant protein within the cell plays a crucial role in the response of the cell to stress. Studies on the mechanisms employed by Escherichia coli to sense and respond to stress have led to a greater understanding of protein processing within the cell. Separate cellular stress response systems serve each compartment in Escherichia coli, the cytoplasm and the envelope (which includes the inner membrane, periplasm and the outer membrane). The cytoplasmic heat shock response is controlled by the regulated proteolysis and chaperone-mediated inactivation of alternative sigma factor Sigma32 [1-3]. Escherichia coli senses and responds to extracytoplasmic stress via at least two overlapping, but distinct, transduction pathways, the Cpx twocomponent system and the heat shock sigmaE pathway. The Cpx pathway is induced by elevated pH, altered inner membrane composition and overproduction of envelope proteins or pili subunits. The sigmaE envelope stress response is induced by heat shock, ethanol shock or by perturbations to outer membrane protein folding .

Applied biochemistry …, 2013

An extremely simple and effective colony PCR procedure is established for both gram-negative and ... more An extremely simple and effective colony PCR procedure is established for both gram-negative and gram-positive bacteria, yeasts, and microalgae. Among the four lysis buffers examined, Y-PER is observed to be more effective than Tris/EDTA, 0.2 % SDS, and 10 mM EDTA in the extraction of PCR-quality genomic DNA from those microorganisms. Vortexing or pipetting agitation of the cells in Y-PER for 5-10 s was sufficient to release genomic DNA for all the test bacteria and yeasts, and most microalgae. Additional incubation at 98 °C for 5 min for further cell disruption was essential only for Chlorella vulgaris due to its notoriously rigid cell wall.

Systems and Synthetic Biology, 2008

Modeling and analysis of genetic networks have become increasingly important in the investigation... more Modeling and analysis of genetic networks have become increasingly important in the investigation of cellular processes. The genetic networks involved in cellular stress response can have a critical effect on the productivity of recombinant proteins. In this work, it was found that the temperature-inducible expression system for the production of soluble recombinant streptokinase in Escherichia coli resulted in a lower productivity compared to the chemically-induced system. To investigate the effect of the induced cellular response due to temperature up-shift a model-based approach is adopted. The role played by the major molecular chaperone teams DnaK–DnaJ–GrpE and GroEL–GroES on the productivity of recombinant streptokinase was experimentally determined. Based on these investigations, a detailed mechanistic mathematical model was developed for the cellular response during the temperature-induced recombinant streptokinase production. The model simulations were found to have a good qualitative agreement with the experimental results. The mechanistic mathematical model was validated with the experiments conducted on a σ32 mutant strain. Detailed analysis of the parameter sensitivities of the model indicated that the level of free DnaK chaperone in the cell has the major effect on the productivity of recombinant streptokinase during temperature induction. Analysis of the model simulations also shows that down regulation or selective redirection of the heat shock proteins could be a better way of manipulating the cellular stress response than overexpression or deletion. In other words, manipulating the system properties resulting from the interaction of the components is better than manipulating the individual components. Although our results are specific to a recombinant protein (streptokinase) and the expression system (E. coli), we believe that such a systems-biological approach has several advantages over conventional experimental approaches and could be in principle extended to bigger genetic networks as well as other recombinant proteins and expression systems.

Journal of Biotechnology, 2008

Computer Aided …, 2009

Improvement of biological strains through targeted modification of metabolism is essential for su... more Improvement of biological strains through targeted modification of metabolism is essential for successful development of bioprocesses. The computational complexity of optimization procedures routinely used for identifying genetic targets limits their application to genome-scale metabolic networks. In this study, we combined graph theoretic approaches with mixed-integer liner programming (MILP) to reduce the search space and thus reducing computational time. Specifically, we used cut-sets (minimal set of reactions that cuts metabolic networks) as additional constraints to reduce the search space. The efficacy of proposed approach is illustrated by identifying minimal reaction set for Saccharomyces Cerevisiae.

Systems and synthetic biology, 2008

Modeling and analysis of genetic networks have become increasingly important in the investigation... more Modeling and analysis of genetic networks have become increasingly important in the investigation of cellular processes. The genetic networks involved in cellular stress response can have a critical effect on the productivity of recombinant proteins. In this work, it was found that the temperature-inducible expression system for the production of soluble recombinant streptokinase in Escherichia coli resulted in a lower productivity compared to the chemically-induced system. To investigate the effect of the induced cellular response due to temperature up-shift a model-based approach is adopted. The role played by the major molecular chaperone teams DnaK–DnaJ–GrpE and GroEL–GroES on the productivity of recombinant streptokinase was experimentally determined. Based on these investigations, a detailed mechanistic mathematical model was developed for the cellular response during the temperature-induced recombinant streptokinase production. The model simulations were found to have a good qualitative agreement with the experimental results. The mechanistic mathematical model was validated with the experiments conducted on a σ32 mutant strain. Detailed analysis of the parameter sensitivities of the model indicated that the level of free DnaK chaperone in the cell has the major effect on the productivity of recombinant streptokinase during temperature induction. Analysis of the model simulations also shows that down regulation or selective redirection of the heat shock proteins could be a better way of manipulating the cellular stress response than overexpression or deletion. In other words, manipulating the system properties resulting from the interaction of the components is better than manipulating the individual components. Although our results are specific to a recombinant protein (streptokinase) and the expression system (E. coli), we believe that such a systems-biological approach has several advantages over conventional experimental approaches and could be in principle extended to bigger genetic networks as well as other recombinant proteins and expression systems.Electronic supplementary materialThe online version of this article (doi:10.1007/s11693-009-9021-z) contains supplementary material, which is available to authorized users.

Current Opinion in Biotechnology

Singapore is one of the major centers for oil, gas, and petrochemicals in the world. The centerpi... more Singapore is one of the major centers for oil, gas, and petrochemicals in the world. The centerpiece of Singapore’s petrochemical industry is the Jurong Island complex, which houses many of the world’s leading petroleum and petrochemical companies. Currently, the total refining capacity of Singapore is 1.3 million bpd [1] and the ethylene capacity is 2.1 million tons per year, projected to reach 4 million tons per year by 2014 [2]. The petrochemical industry in Singapore is heavily dependent on fossil fuel-based feedstock, mostly crude oil and natural gas. Use of fossil fuels has negative environmental impact due to the greenhouse gas emission. Another crisis arising from overdependence on fossil fuels is their rapid depletion. Fossil fuels are non-renewable, which means they are finite and might well run out in the near future – a study found that fossil fuels reserve depletion times for oil, coal, and gas are approximately 35, 107, and 37 years, respectively [3]. This rapid deplet...

Computer Aided Chemical Engineering, 2012

Abstract Singapore's petrochemical industry plays an integral part in the nation's econom... more Abstract Singapore's petrochemical industry plays an integral part in the nation's economy. The industry is highly dependent on crude oil as the major feedstock. This could prove increasingly unsustainable with depleting fossil fuels resources and growing environmental concerns over greenhouse gases emissions. Using renewable feedstocks such as biomass is one possible solution. This paper evaluates the potential for bio-based chemicals production in Singapore's petrochemical cluster, focusing on polylactic acid (PLA). Cost and carbon footprint assessment of the PLA supply chain using various bio-renewable feedstocks sourced from the region is presented.

Modeling and analysis of genetic networks have become increasingly important in the investigation... more Modeling and analysis of genetic networks have become increasingly important in the investigation of cellular processes. The genetic networks involved in cellular stress response can have a critical effect on the productivity of recombinant proteins. In this work, it was found that the temperature-inducible expression system for the production of soluble recombinant streptokinase in Escherichia coli resulted in a lower productivity compared to the chemically-induced system. To investigate the effect of the induced cellular response due to temperature up-shift a model-based approach is adopted. The role played by the major molecular chaperone teams DnaK–DnaJ–GrpE and GroEL–GroES on the productivity of recombinant streptokinase was experimentally determined. Based on these investigations, a detailed mechanistic mathematical model was developed for the cellular response during the temperature-induced recombinant streptokinase production. The model simulations were found to have a good ...

We describe here an approach for rapidly producing scar-free and precise gene deletions in S. cer... more We describe here an approach for rapidly producing scar-free and precise gene deletions in S. cerevisiae with high efficiency. Preparation of the disruption gene cassette in this approach was simply performed by overlap extension-PCR of an invert repeat of a partial or complete sequence of the targeted gene with URA3. Integration of the prepared disruption gene cassette to the designated position of a target gene leads to the formation of a mutagenesis cassette within the yeast genome, which consists of a URA3 gene flanked by the targeted gene and its inverted repeat between two short identical direct repeats. The inherent instability of the inverted sequences in close proximity facilitates the self-excision of the entire mutagenesis cassette deposited in the genome and promotes homologous recombination resulting in a seamless deletion via a single transformation. This rapid assembly circumvents the difficulty during preparation of disruption gene cassettes composed of two inverted ...

Systems and Synthetic Biology, Jan 24, 2009

Modeling and analysis of genetic networks have become increasingly important in the investigation... more Modeling and analysis of genetic networks have become increasingly important in the investigation of cellular processes. The genetic networks involved in cellular stress response can have a critical effect on the productivity of recombinant proteins. In this work, it was found that the temperature-inducible expression system for the production of soluble recombinant streptokinase in Escherichia coli resulted in a lower productivity compared to the chemically-induced system. To investigate the effect of the induced cellular response due to temperature up-shift a model-based approach is adopted. The role played by the major molecular chaperone teams DnaK-DnaJ-GrpE and GroEL-GroES on the productivity of recombinant streptokinase was experimentally determined. Based on these investigations, a detailed mechanistic mathematical model was developed for the cellular response during the temperature-induced recombinant streptokinase production. The model simulations were found to have a good qualitative agreement with the experimental results. The mechanistic mathematical model was validated with the experiments conducted on a r 32 mutant strain. Detailed analysis of the parameter sensitivities of the model indicated that the level of free DnaK chaperone in the cell has the major effect on the productivity of recombinant streptokinase during temperature induction. Analysis of the model simulations also shows that down regulation or selective redirection of the heat shock proteins could be a better way of manipulating the cellular stress response than overexpression or deletion. In other words, manipulating the system properties resulting from the interaction of the components is better than manipulating the individual components. Although our results are specific to a recombinant protein (streptokinase) and the expression system (E. coli), we believe that such a systems-biological approach has several advantages over conventional experimental approaches and could be in principle extended to bigger genetic networks as well as other recombinant proteins and expression systems.

Enzyme and microbial technology, 2018

For the sustainable production of acetaldehyde, a key raw-material for a large number of chemical... more For the sustainable production of acetaldehyde, a key raw-material for a large number of chemical products, microbial production is a promising alternative. We have engineered an Escherichia coli strain for acetaldehyde production from glucose by introducing the pyruvate decarboxylase (Pdc) from Zymomonas mobilis and NADH oxidase (Nox) from Lactococcus lactis. Acetaldehyde production was systematically improved by knocking out the competing metabolic pathways. Multiple knockout strains were created and a final acetaldehyde titre of 0.73g/L was achieved using a quadruple knockout strain E. coli MC4100 ΔadhE ΔldhA ΔfrdC ΔackA-pta. In addition to acetaldehyde, about 0.37g/L acetoin was produced by these strains due to the additional carboligase activity exhibited by pyruvate decarboxylase resulting in a total carbon yield of 0.27g/g glucose.

Improvement of biological strains through targeted modification of metabolism is essential for su... more Improvement of biological strains through targeted modification of metabolism is essential for successful development of bioprocesses. The computational complexity of optimization procedures routinely used for identifying genetic targets limits their application to genome-scale metabolic networks. In this study, we combined graph theoretic approaches with mixed-integer liner programming (MILP) to reduce the search space and thus reducing computational time. Specifically, we used cut-sets (minimal set of reactions that cuts metabolic networks) as additional constraints to reduce the search space. The efficacy of proposed approach is illustrated by identifying minimal reaction set for Saccharomyces Cerevisiae.

Applied biochemistry and biotechnology, Jan 15, 2015

The stress response of Escherichia coli to 3-hydroxypropanoic acid (3-HP) was elucidated through ... more The stress response of Escherichia coli to 3-hydroxypropanoic acid (3-HP) was elucidated through global transcriptomic analysis. Around 375 genes showed difference of more than 2-fold in 3-HP-treated samples. Further analysis revealed that the toxicity effect of 3-HP was due to the cation and anion components of this acid and some effects-specific to 3-HP. Genes related to the oxidative stress, DNA protection, and repair were upregulated in treated cells due to the lowered cytoplasmic pH caused by accumulated cations. 3-HP-treated E. coli used the arginine acid tolerance mechanism to increase the cytoplasmic pH. Additionally, the anion effects were manifested as imbalance in the osmotic pressure. Analysis of top ten highly upregulated genes suggests the formation of 3-hydroxypropionaldehyde under 3-HP stress. The transcriptomic analysis shed light on the global genetic reprogramming due to 3-HP stress and suggests strategies for increasing the tolerance of E. coli toward 3-HP.

Bioprocess and biosystems engineering, Jan 28, 2016

Integrated approaches using in silico model-based design and advanced genetic tools have enabled ... more Integrated approaches using in silico model-based design and advanced genetic tools have enabled efficient production of fuels, chemicals and functional ingredients using microbial cell factories. In this study, using a recently developed genome-scale metabolic model for Escherichia coli iJO1366, a mutant strain has been designed in silico for the anaerobic growth-coupled production of a simple polyol, glycerol. Computational complexity was significantly reduced by systematically reducing the target reactions used for knockout simulations. One promising penta knockout E. coli mutant (E. coli ΔadhE ΔldhA ΔfrdC ΔtpiA ΔmgsA) was selected from simulation study and was constructed experimentally by sequentially deleting five genes. The penta mutant E. coli bearing the Saccharomyces cerevisiae glycerol production pathway was able to grow anaerobically and produce glycerol as the major metabolite with up to 90% of theoretical yield along with stoichiometric quantities of acetate and format...

nt.ntnu.no, 2005

Recombinant protein over-expression in Escherichia coli often elicits a stress response that lead... more Recombinant protein over-expression in Escherichia coli often elicits a stress response that leads to the up-regulation of stress genes and their products. The localization of the recombinant protein within the cell plays a crucial role in the response of the cell to stress. Studies on the mechanisms employed by Escherichia coli to sense and respond to stress have led to a greater understanding of protein processing within the cell. Separate cellular stress response systems serve each compartment in Escherichia coli, the cytoplasm and the envelope (which includes the inner membrane, periplasm and the outer membrane). The cytoplasmic heat shock response is controlled by the regulated proteolysis and chaperone-mediated inactivation of alternative sigma factor Sigma32 [1-3]. Escherichia coli senses and responds to extracytoplasmic stress via at least two overlapping, but distinct, transduction pathways, the Cpx twocomponent system and the heat shock sigmaE pathway. The Cpx pathway is induced by elevated pH, altered inner membrane composition and overproduction of envelope proteins or pili subunits. The sigmaE envelope stress response is induced by heat shock, ethanol shock or by perturbations to outer membrane protein folding .

Applied biochemistry …, 2013

An extremely simple and effective colony PCR procedure is established for both gram-negative and ... more An extremely simple and effective colony PCR procedure is established for both gram-negative and gram-positive bacteria, yeasts, and microalgae. Among the four lysis buffers examined, Y-PER is observed to be more effective than Tris/EDTA, 0.2 % SDS, and 10 mM EDTA in the extraction of PCR-quality genomic DNA from those microorganisms. Vortexing or pipetting agitation of the cells in Y-PER for 5-10 s was sufficient to release genomic DNA for all the test bacteria and yeasts, and most microalgae. Additional incubation at 98 °C for 5 min for further cell disruption was essential only for Chlorella vulgaris due to its notoriously rigid cell wall.

Systems and Synthetic Biology, 2008

Modeling and analysis of genetic networks have become increasingly important in the investigation... more Modeling and analysis of genetic networks have become increasingly important in the investigation of cellular processes. The genetic networks involved in cellular stress response can have a critical effect on the productivity of recombinant proteins. In this work, it was found that the temperature-inducible expression system for the production of soluble recombinant streptokinase in Escherichia coli resulted in a lower productivity compared to the chemically-induced system. To investigate the effect of the induced cellular response due to temperature up-shift a model-based approach is adopted. The role played by the major molecular chaperone teams DnaK–DnaJ–GrpE and GroEL–GroES on the productivity of recombinant streptokinase was experimentally determined. Based on these investigations, a detailed mechanistic mathematical model was developed for the cellular response during the temperature-induced recombinant streptokinase production. The model simulations were found to have a good qualitative agreement with the experimental results. The mechanistic mathematical model was validated with the experiments conducted on a σ32 mutant strain. Detailed analysis of the parameter sensitivities of the model indicated that the level of free DnaK chaperone in the cell has the major effect on the productivity of recombinant streptokinase during temperature induction. Analysis of the model simulations also shows that down regulation or selective redirection of the heat shock proteins could be a better way of manipulating the cellular stress response than overexpression or deletion. In other words, manipulating the system properties resulting from the interaction of the components is better than manipulating the individual components. Although our results are specific to a recombinant protein (streptokinase) and the expression system (E. coli), we believe that such a systems-biological approach has several advantages over conventional experimental approaches and could be in principle extended to bigger genetic networks as well as other recombinant proteins and expression systems.

Journal of Biotechnology, 2008

Computer Aided …, 2009

Improvement of biological strains through targeted modification of metabolism is essential for su... more Improvement of biological strains through targeted modification of metabolism is essential for successful development of bioprocesses. The computational complexity of optimization procedures routinely used for identifying genetic targets limits their application to genome-scale metabolic networks. In this study, we combined graph theoretic approaches with mixed-integer liner programming (MILP) to reduce the search space and thus reducing computational time. Specifically, we used cut-sets (minimal set of reactions that cuts metabolic networks) as additional constraints to reduce the search space. The efficacy of proposed approach is illustrated by identifying minimal reaction set for Saccharomyces Cerevisiae.

Systems and synthetic biology, 2008

Modeling and analysis of genetic networks have become increasingly important in the investigation... more Modeling and analysis of genetic networks have become increasingly important in the investigation of cellular processes. The genetic networks involved in cellular stress response can have a critical effect on the productivity of recombinant proteins. In this work, it was found that the temperature-inducible expression system for the production of soluble recombinant streptokinase in Escherichia coli resulted in a lower productivity compared to the chemically-induced system. To investigate the effect of the induced cellular response due to temperature up-shift a model-based approach is adopted. The role played by the major molecular chaperone teams DnaK–DnaJ–GrpE and GroEL–GroES on the productivity of recombinant streptokinase was experimentally determined. Based on these investigations, a detailed mechanistic mathematical model was developed for the cellular response during the temperature-induced recombinant streptokinase production. The model simulations were found to have a good qualitative agreement with the experimental results. The mechanistic mathematical model was validated with the experiments conducted on a σ32 mutant strain. Detailed analysis of the parameter sensitivities of the model indicated that the level of free DnaK chaperone in the cell has the major effect on the productivity of recombinant streptokinase during temperature induction. Analysis of the model simulations also shows that down regulation or selective redirection of the heat shock proteins could be a better way of manipulating the cellular stress response than overexpression or deletion. In other words, manipulating the system properties resulting from the interaction of the components is better than manipulating the individual components. Although our results are specific to a recombinant protein (streptokinase) and the expression system (E. coli), we believe that such a systems-biological approach has several advantages over conventional experimental approaches and could be in principle extended to bigger genetic networks as well as other recombinant proteins and expression systems.Electronic supplementary materialThe online version of this article (doi:10.1007/s11693-009-9021-z) contains supplementary material, which is available to authorized users.