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Jens Nielsen - One of the best experts on this subject based on the ideXlab platform.
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lipase production by recombinant strains of aspergillus niger expressing a lipase encoding gene from thermomyces lanuginosus
Applied Microbiology and Biotechnology, 2004Co-Authors: Wai Prathumpai, Simon J Flitter, Mhairi Mcintyre, Jens NielsenAbstract:Two recombinant strains of Aspergillus niger (NW 297-14 and NW297-24) producing a heterologous lipase from Thermomyces lanuginosus were constructed. The heterologous lipase was expressed using the TAKA amylase promoter from Aspergillus oryzae. The production kinetics of the two strains on different carbon sources in batch and carbon-limited chemostat cultivations were evaluated. In batch cultivations, the highest total product yield coefficient (Yxp total), given as the sum of extracellular and intracellular yields, was obtained during growth on glucose for the transformant strain NW297-24 (5.7±0.65 KU/g DW), whereas the highest total product yield coefficient was obtained during growth on maltose for the transformant strain NW297-14 (6.3±0.02 KU/g DW). Both transformants were evaluated in glucose-limited chemostat cultures. Strain NW297-14 was found to be the best producer and was thus employed for further analysis of the influence of carbon source in chemostat cultures. Here, the highest total specific lipase productivity (rp total, the sum of extracellular and intracellular lipase productivity) was found to be 1.60±0.81 KU/g DW/h in maltose-limited Chemostats at a dilution rate of 0.08 h−1, compared with a total specific lipase productivity of 1.10±0.41 KU/g DW/h in glucose-limited Chemostats. At the highest specific productivity obtained in this study, the heterologous enzyme accounted for about 1% of all cellular protein being produced by the cells, which shows that it is possible to obtain high productivities of heterologous fungal enzymes in A. niger. However, SDS-PAGE analysis showed that most of the produced lipase was bound to the cell wall.
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lipase production by recombinant strains of aspergillus niger expressing a lipase encoding gene from thermomyces lanuginosus
Applied Microbiology and Biotechnology, 2004Co-Authors: Wai Prathumpai, Simon J Flitter, Mhairi Mcintyre, Jens NielsenAbstract:Two recombinant strains of Aspergillus niger (NW 297-14 and NW297-24) producing a heterologous lipase from Thermomyces lanuginosus were constructed. The heterologous lipase was expressed using the TAKA amylase promoter from Aspergillus oryzae. The production kinetics of the two strains on different carbon sources in batch and carbon-limited chemostat cultivations were evaluated. In batch cultivations, the highest total product yield coefficient (Y(xp total)), given as the sum of extracellular and intracellular yields, was obtained during growth on glucose for the transformant strain NW297-24 (5.7+/-0.65 KU/g DW), whereas the highest total product yield coefficient was obtained during growth on maltose for the transformant strain NW297-14 (6.3+/-0.02 KU/g DW). Both transformants were evaluated in glucose-limited chemostat cultures. Strain NW297-14 was found to be the best producer and was thus employed for further analysis of the influence of carbon source in chemostat cultures. Here, the highest total specific lipase productivity (r(p total), the sum of extracellular and intracellular lipase productivity) was found to be 1.60+/-0.81 KU/g DW/h in maltose-limited Chemostats at a dilution rate of 0.08 h(-1), compared with a total specific lipase productivity of 1.10+/-0.41 KU/g DW/h in glucose-limited Chemostats. At the highest specific productivity obtained in this study, the heterologous enzyme accounted for about 1% of all cellular protein being produced by the cells, which shows that it is possible to obtain high productivities of heterologous fungal enzymes in A. niger. However, SDS-PAGE analysis showed that most of the produced lipase was bound to the cell wall.
Frederic Mazenc - One of the best experts on this subject based on the ideXlab platform.
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Stability and Stabilization for Models of Chemostats with Multiple Limiting Substrates, in "Journal of Biological Dynamics
2020Co-Authors: Frederic Mazenc, Michael MalisoffAbstract:Abstract We study chemostat models in which multiple species compete for two or more limiting nutrients. First we consider the case where the nutrient flow and species removal rates and input nutrient concentrations are all given positive constants. In that case, we use Brouwer degree theory to give conditions guaranteeing that the models admit globally asymptotically stable componentwise positive equilibrium points, from all componentwise positive initial states. Then we use the results to develop stabilization theory for a class of controlled Chemostats with two or more limiting nutrients. For cases where the dilution rate and input nutrient concentrations can be selected as controls, we prove that many different componentwise positive equilibria can be made globally asymptotically stable. This extends the existing control results for Chemostats with one limiting nutrient. We demonstrate our methods in simulations
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stability analysis of mathematical model of competition in a chain of Chemostats in series with delay
Applied Mathematical Modelling, 2019Co-Authors: Frederic Mazenc, Silviuiulian Niculescu, Gonzalo RobledoAbstract:Abstract We study a nonlinear system of differential delay equations describing a model of a chain of two Chemostats, where one contains two microbial species in competition for a single limiting nutrient and receives an external input of the less advantaged competitor, which is cultivated in an external chemostat. We obtain sufficient conditions ensuring coexistence of all the species in competition which consist in upper delay bounds.
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Stability and stabilization for models of Chemostats with multiple limiting substrates
Journal of Biological Dynamics, 2012Co-Authors: Frederic Mazenc, Michael MalisoffAbstract:We study chemostat models in which multiple species compete for two or more limiting nutrients. First, we consider the case where the nutrient flow and species removal rates and input nutrient concentrations are all given as positive constants. In that case, we use Brouwer degree theory to give conditions guaranteeing that the models admit globally asymptotically stable componentwise positive equilibrium points, from all componentwise positive initial states. Then we use the results to develop stabilization theory for a class of controlled Chemostats with two or more limiting nutrients. For cases where the dilution rate and input nutrient concentrations can be selected as controls, we prove that many different componentwise positive equilibria can be made globally asymptotically stable. This extends the existing control results for Chemostats with one limiting nutrient. We demonstrate our methods in simulations.
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On stability and stabilization for Chemostats with many limiting nutrients
2011 50th IEEE Conference on Decision and Control and European Control Conference, 2011Co-Authors: Frederic Mazenc, Michael MalisoffAbstract:We study models of Chemostats where the species compete for multiple limiting nutrients. We first consider cases where the input nutrient concentrations, nutrient flow, and species removal rates are all given positive constants. For such cases, we use Brouwer degree theory to find conditions guaranteeing that the models admit globally asymptotically stable componentwise positive equilibria, starting from all componentwise positive initial states. Then we use our results to prove stabilization results for controlled Chemostats with two or more limiting nutrients. When the dilution rate and input nutrient concentrations can be taken as controls, we show that many possible componentwise positive equilibrium points can be rendered globally asymptotically stable. This extends existing control results for Chemostats with one limiting nutrient. We illustrate our methods in simulations.
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stabilization of a chemostat model with haldane growth functions and a delay in the measurements
Automatica, 2010Co-Authors: Frederic Mazenc, Michael MalisoffAbstract:The stabilization of equilibria in Chemostats with measurement delays is a complex and challenging problem, and is of significant ongoing interest in bioengineering and population dynamics. In this paper, we solve an output feedback stabilization problem for chemostat models having two species, one limiting substrate, and either Haldane or Monod growth functions. Our feedback stabilizers depend on a given linear combination of the species concentrations, which are both measured with a constant time delay. The values of the delays are unknown. Instead, one only knows an upper bound on the delays, and we allow the upper bound to be arbitrarily large. The stabilizing feedback depends on the known upper bound for the delays as well. Our work is based on a Lyapunov-Krasovskii argument.
Wai Prathumpai - One of the best experts on this subject based on the ideXlab platform.
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lipase production by recombinant strains of aspergillus niger expressing a lipase encoding gene from thermomyces lanuginosus
Applied Microbiology and Biotechnology, 2004Co-Authors: Wai Prathumpai, Simon J Flitter, Mhairi Mcintyre, Jens NielsenAbstract:Two recombinant strains of Aspergillus niger (NW 297-14 and NW297-24) producing a heterologous lipase from Thermomyces lanuginosus were constructed. The heterologous lipase was expressed using the TAKA amylase promoter from Aspergillus oryzae. The production kinetics of the two strains on different carbon sources in batch and carbon-limited chemostat cultivations were evaluated. In batch cultivations, the highest total product yield coefficient (Yxp total), given as the sum of extracellular and intracellular yields, was obtained during growth on glucose for the transformant strain NW297-24 (5.7±0.65 KU/g DW), whereas the highest total product yield coefficient was obtained during growth on maltose for the transformant strain NW297-14 (6.3±0.02 KU/g DW). Both transformants were evaluated in glucose-limited chemostat cultures. Strain NW297-14 was found to be the best producer and was thus employed for further analysis of the influence of carbon source in chemostat cultures. Here, the highest total specific lipase productivity (rp total, the sum of extracellular and intracellular lipase productivity) was found to be 1.60±0.81 KU/g DW/h in maltose-limited Chemostats at a dilution rate of 0.08 h−1, compared with a total specific lipase productivity of 1.10±0.41 KU/g DW/h in glucose-limited Chemostats. At the highest specific productivity obtained in this study, the heterologous enzyme accounted for about 1% of all cellular protein being produced by the cells, which shows that it is possible to obtain high productivities of heterologous fungal enzymes in A. niger. However, SDS-PAGE analysis showed that most of the produced lipase was bound to the cell wall.
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lipase production by recombinant strains of aspergillus niger expressing a lipase encoding gene from thermomyces lanuginosus
Applied Microbiology and Biotechnology, 2004Co-Authors: Wai Prathumpai, Simon J Flitter, Mhairi Mcintyre, Jens NielsenAbstract:Two recombinant strains of Aspergillus niger (NW 297-14 and NW297-24) producing a heterologous lipase from Thermomyces lanuginosus were constructed. The heterologous lipase was expressed using the TAKA amylase promoter from Aspergillus oryzae. The production kinetics of the two strains on different carbon sources in batch and carbon-limited chemostat cultivations were evaluated. In batch cultivations, the highest total product yield coefficient (Y(xp total)), given as the sum of extracellular and intracellular yields, was obtained during growth on glucose for the transformant strain NW297-24 (5.7+/-0.65 KU/g DW), whereas the highest total product yield coefficient was obtained during growth on maltose for the transformant strain NW297-14 (6.3+/-0.02 KU/g DW). Both transformants were evaluated in glucose-limited chemostat cultures. Strain NW297-14 was found to be the best producer and was thus employed for further analysis of the influence of carbon source in chemostat cultures. Here, the highest total specific lipase productivity (r(p total), the sum of extracellular and intracellular lipase productivity) was found to be 1.60+/-0.81 KU/g DW/h in maltose-limited Chemostats at a dilution rate of 0.08 h(-1), compared with a total specific lipase productivity of 1.10+/-0.41 KU/g DW/h in glucose-limited Chemostats. At the highest specific productivity obtained in this study, the heterologous enzyme accounted for about 1% of all cellular protein being produced by the cells, which shows that it is possible to obtain high productivities of heterologous fungal enzymes in A. niger. However, SDS-PAGE analysis showed that most of the produced lipase was bound to the cell wall.
Michael Malisoff - One of the best experts on this subject based on the ideXlab platform.
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Stability and Stabilization for Models of Chemostats with Multiple Limiting Substrates, in "Journal of Biological Dynamics
2020Co-Authors: Frederic Mazenc, Michael MalisoffAbstract:Abstract We study chemostat models in which multiple species compete for two or more limiting nutrients. First we consider the case where the nutrient flow and species removal rates and input nutrient concentrations are all given positive constants. In that case, we use Brouwer degree theory to give conditions guaranteeing that the models admit globally asymptotically stable componentwise positive equilibrium points, from all componentwise positive initial states. Then we use the results to develop stabilization theory for a class of controlled Chemostats with two or more limiting nutrients. For cases where the dilution rate and input nutrient concentrations can be selected as controls, we prove that many different componentwise positive equilibria can be made globally asymptotically stable. This extends the existing control results for Chemostats with one limiting nutrient. We demonstrate our methods in simulations
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Stability and stabilization for models of Chemostats with multiple limiting substrates
Journal of Biological Dynamics, 2012Co-Authors: Frederic Mazenc, Michael MalisoffAbstract:We study chemostat models in which multiple species compete for two or more limiting nutrients. First, we consider the case where the nutrient flow and species removal rates and input nutrient concentrations are all given as positive constants. In that case, we use Brouwer degree theory to give conditions guaranteeing that the models admit globally asymptotically stable componentwise positive equilibrium points, from all componentwise positive initial states. Then we use the results to develop stabilization theory for a class of controlled Chemostats with two or more limiting nutrients. For cases where the dilution rate and input nutrient concentrations can be selected as controls, we prove that many different componentwise positive equilibria can be made globally asymptotically stable. This extends the existing control results for Chemostats with one limiting nutrient. We demonstrate our methods in simulations.
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On stability and stabilization for Chemostats with many limiting nutrients
2011 50th IEEE Conference on Decision and Control and European Control Conference, 2011Co-Authors: Frederic Mazenc, Michael MalisoffAbstract:We study models of Chemostats where the species compete for multiple limiting nutrients. We first consider cases where the input nutrient concentrations, nutrient flow, and species removal rates are all given positive constants. For such cases, we use Brouwer degree theory to find conditions guaranteeing that the models admit globally asymptotically stable componentwise positive equilibria, starting from all componentwise positive initial states. Then we use our results to prove stabilization results for controlled Chemostats with two or more limiting nutrients. When the dilution rate and input nutrient concentrations can be taken as controls, we show that many possible componentwise positive equilibrium points can be rendered globally asymptotically stable. This extends existing control results for Chemostats with one limiting nutrient. We illustrate our methods in simulations.
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stabilization of a chemostat model with haldane growth functions and a delay in the measurements
Automatica, 2010Co-Authors: Frederic Mazenc, Michael MalisoffAbstract:The stabilization of equilibria in Chemostats with measurement delays is a complex and challenging problem, and is of significant ongoing interest in bioengineering and population dynamics. In this paper, we solve an output feedback stabilization problem for chemostat models having two species, one limiting substrate, and either Haldane or Monod growth functions. Our feedback stabilizers depend on a given linear combination of the species concentrations, which are both measured with a constant time delay. The values of the delays are unknown. Instead, one only knows an upper bound on the delays, and we allow the upper bound to be arbitrarily large. The stabilizing feedback depends on the known upper bound for the delays as well. Our work is based on a Lyapunov-Krasovskii argument.
Wenying Shou - One of the best experts on this subject based on the ideXlab platform.
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Intracellular lysine content and lysine release rate of A−L+ vary with environment.
2019Co-Authors: Samuel F M Hart, Robin Green, Li Xie, Jose Mario Bello Pineda, Babak Momeni, Wenying ShouAbstract:(A) Intracellular lysine content increases upon hypoxanthine limitation. A−L+ cells grown to exponential phase in SD plus excess hypoxanthine were washed and diluted into SD at time zero. Cells were either starved further (“starve batch,” dashed lines) or inoculated into hypoxanthine-limited Chemostats after 24 h of prestarvation (“8-h chemo.,” solid lines). At various times, cells were harvested, and intracellular lysine was extracted (Methods, “Extraction of intracellular metabolites”) and measured via yield bioassay (Methods, “Bioassays”). Intracellular lysine content increased by 6-fold during the 24-h prestarvation, even though the average cell size increased by only about 20% (S17 Fig). Intracellular lysine content continued to increase, reaching a higher level in 8-h Chemostats than in starvation. Different colors represent different experiments. Data can be found in S6 Data. (B) Lysine release rate varies with the environment. Lysine release rates were quantified for cells at different growth rates (doubling times marked above) and during starvation (e.g., S15 Fig). The time window for phenotype measurement is similar to that for CoSMO growth rate measurement to ensure similar evolutionary effects (S9 Fig). Mean release rates and their 2 SEMs were plotted. Lysine release rate in an exponential batch culture in excess hypoxanthine was below the level of detection (red dotted line = 0.003 fmole/cell/h; Methods, “Measuring the upper bound of release rate in excess metabolites”). The green dotted regression line in B was used in analytical calculation (Eq 5; Methods, “Calculating steady-state community growth rate”), while the black and the green dotted regression lines in B were used in spatial simulations. Lysine release rates were summarized in S8 Table and plotted in greater detail in S20 Fig. chemo., chemostat; CoSMO, Cooperation that is Synthetic and Mutually Obligatory; Lys, lysine; SD, Synthetic Dextrose minimal medium; SEM, standard error of the mean.
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Uncovering and resolving challenges of quantitative modeling in a simplified community of interacting cells.
Public Library of Science (PLoS), 2019Co-Authors: Samuel F M Hart, Robin Green, Li Xie, Jose Mario Bello Pineda, Babak Momeni, Wenying ShouAbstract:Quantitative modeling is useful for predicting behaviors of a system and for rationally constructing or modifying the system. The predictive power of a model relies on accurate quantification of model parameters. Here, we illustrate challenges in parameter quantification and offer means to overcome these challenges, using a case example in which we quantitatively predict the growth rate of a cooperative community. Specifically, the community consists of two Saccharomyces cerevisiae strains, each engineered to release a metabolite required and consumed by its partner. The initial model, employing parameters measured in batch monocultures with zero or excess metabolite, failed to quantitatively predict experimental results. To resolve the model-experiment discrepancy, we chemically identified the correct exchanged metabolites, but this did not improve model performance. We then remeasured strain phenotypes in Chemostats mimicking the metabolite-limited community environments, while mitigating or incorporating effects of rapid evolution. Almost all phenotypes we measured, including death rate, metabolite release rate, and the amount of metabolite consumed per cell birth, varied significantly with the metabolite environment. Once we used parameters measured in a range of community-like chemostat environments, prediction quantitatively agreed with experimental results. In summary, using a simplified community, we uncovered and devised means to resolve modeling challenges that are likely general to living systems
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Metabolite consumption is sensitive to the environment.
2019Co-Authors: Samuel F M Hart, Robin Green, Li Xie, Jose Mario Bello Pineda, Babak Momeni, Wenying ShouAbstract:(A) Consumption in excess lysine. L−A+ population density and lysine concentration remaining in the medium were measured over time (Methods, “Measuring consumption in batch cultures”). Consumption per birth was calculated from the slope of the lavender line. (B) Consumption in cultures grown to saturation. L−A+ cells were inoculated into SD supplemented with various concentrations of lysine. After cultures had reached saturation, total cell densities were measured by flow cytometry. Lysine consumed per birth was quantified from 1/(slope of the orange line). This value was used in Models i and ii. (C) Consumption per birth in different environments. Lysine consumption was measured in lysine-limited Chemostats at various doubling times (Methods, “Quantifying phenotypes in Chemostats,” and Eq 12), and data were jittered slightly along the x axis to facilitate visualization. For chemostat measurements, error bars represent 2 standard deviations caused by fluctuations in steady-state population density. For exponential and saturation consumption, error bars mark 2 SEMs for slope estimation. The black dashed line marks the average lysine consumption per L−A+ birth in Chemostats (Table 1; S5 Table), which we used in Model iii. All plotted data can be found in S4 Data. Lys, lysine; SD, Synthetic Dextrose minimal medium; SEM, standard error of the mean.
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developing a low cost milliliter scale chemostat array for precise control of cellular growth
Quantitative biology (Beijing China), 2018Co-Authors: David Skelding, Samuel F M Hart, Thejas Vidyasagar, Alexander E Pozhitkov, Wenying ShouAbstract:Background Multiplexed milliliter-scale Chemostats are useful for measuring cell physiology under various degrees of nutrient limitation and for carrying out evolution experiments. In each chemostat, fresh medium containing a growth rate-limiting metabolite is pumped into the culturing chamber at a constant rate, while culture effluent exits at an equal rate. Although such devices have been developed by various labs, key parameters — the accuracy, precision, and operational range of flow rate — are not explicitly characterized.
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developing a low cost milliliter scale chemostat array for precise control of cellular growth
bioRxiv, 2017Co-Authors: David Skelding, Samuel F M Hart, Thejas Vidyasagar, Alexander E Pozhitkov, Wenying ShouAbstract:Multiplexed milliliter-scale Chemostats are useful for measuring cell physiology under various degrees of nutrient limitation and for experimental evolution. In each chemostat, fresh medium containing a growth rate-limiting metabolite is pumped into the culturing chamber at a constant rate, while culture effluent exits at an equal rate. Although such devices have been developed by various labs, key parameters - the accuracy and precision of flow rate and the operational range - are not explicitly characterized. Here we report the development of multiplexed milliliter-scale Chemostats where flow rates for eight chambers can be independently controlled to vary within a wide range, corresponding to population doubling times of 3~ 13 hours. Importantly, flow rates are precise and accurate without the use of expensive feedback systems. Among the eight chambers, the maximal coefficient of variation in flow rate is less than 3%, and average flow rates are only slightly below targets, i.e., 3-6% for 13-hour and 0.6-1.0% for 3-hour doubling times. This deficit is largely due to evaporation and should be correctable. We experimentally demonstrate that our device allows accurate and precise quantification of population phenotypes.