27 results on '"Magnus Carlquist"'
Search Results
2. Assessment of the TRX2p-yEGFP Biosensor to Monitor the Redox Response of an Industrial Xylose-Fermenting Saccharomyces cerevisiae Strain during Propagation and Fermentation
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Raquel Perruca Foncillas, Miguel Sanchis Sebastiá, Ola Wallberg, Magnus Carlquist, and Marie F. Gorwa-Grauslund
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Microbiology (medical) ,on-line flow cytometry ,redox imbalance ,TRX2p-GFP ,yeast propagation ,lignocellulosic bioethanol ,wheat-straw hydrolysate ,furfural ,Plant Science ,Ecology, Evolution, Behavior and Systematics - Abstract
The commercial production of bioethanol from lignocellulosic biomass such as wheat straw requires utilizing a microorganism that can withstand all the stressors encountered in the process while fermenting all the sugars in the biomass. Therefore, it is essential to develop tools for monitoring and controlling the cellular fitness during both cell propagation and sugar fermentation to ethanol. In the present study, on-line flow cytometry was adopted to assess the response of the biosensor TRX2p-yEGFP for redox imbalance in an industrial xylose-fermenting strain of Saccharomyces cerevisiae during cell propagation and the following fermentation of wheat-straw hydrolysate. Rapid and transient induction of the sensor was recorded upon exposure to furfural and wheat straw hydrolysate containing up to 3.8 g/L furfural. During the fermentation step, the induction rate of the sensor was also found to correlate to the initial ethanol production rate, highlighting the relevance of redox monitoring and the potential of the presented tool to assess the ethanol production rate in hydrolysates. Three different propagation strategies were also compared, and it was confirmed that pre-exposure to hydrolysate during propagation remains the most efficient method for high ethanol productivity in the following wheat-straw hydrolysate fermentations.
- Published
- 2023
3. Muconic Acid Production Using Engineered Pseudomonas putida KT2440 and a Guaiacol-Rich Fraction Derived from Kraft Lignin
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Henrique Veras, Nádia Skorupa Parachin, Javier Garcia Hidalgo, Marie F. Gorwa-Grauslund, Henrik Almqvist, Magnus Carlquist, Kena Li, and Christian Hulteberg
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Muconic acid ,biology ,Renewable Energy, Sustainability and the Environment ,Depolymerization ,Chemistry ,Bioconversion ,General Chemical Engineering ,Extraction (chemistry) ,General Chemistry ,biology.organism_classification ,complex mixtures ,Pseudomonas putida ,chemistry.chemical_compound ,Environmental Chemistry ,Organic chemistry ,Lignin ,Fermentation ,Guaiacol - Abstract
Industrial lignin such as kraft lignin is an abundant feedstock for renewable chemicals and materials. In this study, a process was developed for depolymerization of kraft lignin followed by an upgrading separation step and further bioconversion of the obtained monoaromatic compounds to muconic acid. First, industrial kraft lignin, Indulin AT, was processed into a guaiacol-rich stream using base-catalyzed depolymerization. This stream was subsequently upgraded using liquid-liquid extraction and evaporation to yield a more concentrated and less inhibitory stream, adapted for bioconversion. Finally, guaiacol was quantitatively converted to muconic acid through bioconversion using an engineered Pseudomonas putida strain containing cytochrome P450 and ferredoxin reductase for guaiacol assimilation and deletion of the native catBC genes for muconic acid production. Isomerization of muconic acid in a fermentation medium depending on pH was also studied. (Less)
- Published
- 2021
4. Engineering Saccharomyces cerevisiae for production of the capsaicinoid nonivamide
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Nina Muratovska, Carl Grey, and Magnus Carlquist
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Ligases ,Fruit ,Coenzyme A ,Bioengineering ,Saccharomyces cerevisiae ,Capsaicin ,Capsicum ,Applied Microbiology and Biotechnology ,Acyltransferases ,Biotechnology - Abstract
Background Capsaicinoids are produced by plants in the Capsicum genus and are the main reason for the pungency of chili pepper fruits. They are strong agonists of TRPV1 (the transient receptor potential cation channel subfamily V member 1) and used as active ingredients in pharmaceuticals for the treatment of pain. The use of bioengineered microorganisms in a fermentation process may be an efficient route for their preparation, as well as for the discovery of (bio-)synthetic capsaicinoids with improved or novel bioactivities. Results Saccharomyces cerevisiae was engineered to over-express a selection of amide-forming N-acyltransferase and CoA-ligase enzyme cascades using a combinatorial gene assembly method, and was screened for nonivamide production from supplemented vanillylamine and nonanoic acid. Data from this work demonstrate that Tyramine N-hydroxycinnamoyl transferase from Capsicum annuum (CaAT) was most efficient for nonivamide formation in yeast, outcompeting the other candidates including AT3 (Pun1) from Capsicum spp. The CoA-ligase partner with highest activity from the ones evaluated here were from Petunia hybrida (PhCL) and Spingomonas sp. Ibu-2 (IpfF). A yeast strain expressing CaAT and IpfF produced 10.6 mg L−1 nonivamide in a controlled bioreactor setup, demonstrating nonivamide biosynthesis by S. cerevisiae for the first time. Conclusions Baker’s yeast was engineered for production of nonivamide as a model capsaicinoid, by expressing N-acyltransferases and CoA-ligases of plant and bacterial origin. The constructed yeast platform holds potential for in vivo biocatalytic formation of capsaicinoids and could be a useful tool for the discovery of novel drugs.
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- 2022
5. Towards engineered yeast as production platform for capsaicinoids
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Nina Muratovska, Paulo Silva, Tatiana Pozdniakova, Humberto Pereira, Carl Grey, Björn Johansson, and Magnus Carlquist
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Fruit ,Humans ,Bioengineering ,Saccharomyces cerevisiae ,Capsaicin ,Capsicum ,Applied Microbiology and Biotechnology ,Biotechnology - Abstract
Capsaicinoids are bioactive alkaloids produced by the chili pepper fruit and are known to be the most potent agonists of the human pain receptor TRPV1 (Transient Receptor Potential Cation Channel Subfamily V Member 1). They are currently produced by extraction from chili pepper fruit or by chemical synthesis. Transfer of the biosynthetic route to a microbial host could enable more efficient capsaicinoid production by fermentation and may also enable the use of synthetic biology to create a diversity of new compounds with potentially improved properties. This review summarises the current state of the art on the biosynthesis of capsaicinoid precursors in baker's yeast, Saccharomyces cerevisiae, and discusses bioengineering strategies for achieving total synthesis from sugar.
- Published
- 2022
6. Flow cytometric analysis reveals culture condition dependent variations in phenotypic heterogeneity of Limosilactobacillus reuteri
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Nikhil Seshagiri Rao, Ludwig Lundberg, Shuai Palmkron, Sebastian Håkansson, Björn Bergenståhl, and Magnus Carlquist
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Limosilactobacillus reuteri ,Bacteriological Techniques ,Multidisciplinary ,Probiotics ,Science ,Temperature ,Hydrogen-Ion Concentration ,Flow Cytometry ,Industrial microbiology ,Bacterial physiology ,Article ,Oxygen ,Applied microbiology ,Freeze Drying ,Phenotype ,Stress, Physiological ,Microscopy, Electron, Scanning ,Humans ,Medicine ,Cell and Molecular Biology ,Food Science - Abstract
Optimisation of cultivation conditions in the industrial production of probiotics is crucial to reach a high-quality product with retained probiotic functionality. Flow cytometry-based descriptors of bacterial morphology may be used as markers to estimate physiological fitness during cultivation, and can be applied for online monitoring to avoid suboptimal growth. In the current study, the effects of temperature, initial pH and oxygen levels on cell growth and cell size distributions of Limosilactobacillus reuteri DSM 17938 were measured using multivariate flow cytometry. A pleomorphic behaviour was evident from the measurements of light scatter and pulse width distributions. A pattern of high growth yielding smaller cells and less heterogeneous populations could be observed. Analysis of pulse width distributions revealed significant morphological heterogeneities within the bacterial cell population under non-optimal growth conditions, and pointed towards low temperature, high initial pH, and high oxygen levels all being triggers for changes in morphology towards cell chain formation. However, cell size did not correlate to survivability after freeze-thaw or freeze-drying stress, indicating that it is not a key determinant for physical stress tolerance. The fact that L. reuteri morphology varies depending on cultivation conditions suggests that it can be used as marker for estimating physiological fitness and responses to its environment.
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- 2021
7. Flow Cytometric Analysis Reveals Culture-condition Dependent Variations in Phenotypic Heterogeneity of Limosilactobacillus Reuteri
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Ludwig Lundberg, Sebastian Håkansson, Nikhil Seshagiri Rao, Shuai Palmkron, Björn Bergenståhl, and Magnus Carlquist
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Text mining ,Flow (mathematics) ,business.industry ,Genetic heterogeneity ,Computational biology ,Biology ,business ,Condition dependent - Abstract
Optimisation of cultivation conditions in the industrial production of probiotics is crucial to reach a high-quality product with retained probiotic functionality. Flow cytometry-based descriptors of bacterial morphology may be used as markers to estimate physiological fitness during cultivation, and can be applied for online monitoring to avoid suboptimal growth. In the current study, the effects of temperature, pH and oxygen levels on cell growth and cell size distributions of Limosilactobacillus reuteri DSM 17938 were measured using multivariate flow cytometry. A pleomorphic behaviour was evident from the measurement of light scatter and pulse width distributions. A pattern of high growth yielding smaller cells and less heterogeneous populations could be observed. Analysis of pulse width distributions revealed significant morphological heterogeneities within the bacterial cell population under non-optimal growth conditions, and pointed towards low temperature, high pH, and high oxygen levels all being triggers for changes in morphology towards cell chain formation. However, cell size did not correlate to survivability after freeze-thaw or freeze-drying stress, indicating that it is not a key determinant for physical stress tolerance. The fact that L. reuteri morphology varies depending on cultivation conditions suggests that it can be used as marker for estimating physiological fitness and responses to its environment.
- Published
- 2021
8. Assessment of fluorescent protein candidates for multi-color flow cytometry analysis of Saccharomyces cerevisiae
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Raquel Perruca-Foncillas, Johan Davidsson, Magnus Carlquist, and Marie F. Gorwa-Grauslund
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Applied Microbiology and Biotechnology ,Biotechnology - Published
- 2022
9. Boronic Acid Modified Polymer Nanoparticles for Enhanced Bacterial Deactivation
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Lei Ye, Weifeng Liu, Magnus Carlquist, and Haiyue Gong
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inorganic chemicals ,Staphylococcus aureus ,Polymers ,Nanoparticle ,010402 general chemistry ,01 natural sciences ,Biochemistry ,chemistry.chemical_compound ,Antimicrobial polymer ,medicine ,Escherichia coli ,Molecular Biology ,chemistry.chemical_classification ,Microbial Viability ,biology ,010405 organic chemistry ,Chloramphenicol ,Organic Chemistry ,Polymer ,biology.organism_classification ,Combinatorial chemistry ,Boronic Acids ,0104 chemical sciences ,Anti-Bacterial Agents ,chemistry ,Drug delivery ,Molecular Medicine ,Nanoparticles ,Molecular imprinting ,Boronic acid ,Bacteria ,medicine.drug - Abstract
A new method has been developed to enhance the antibacterial efficiency of traditional antibiotics. Chloramphenicol-imprinted polymer particles were decorated with boronic acid to improve their binding to both Gram-negative and -positive bacteria. The polymer particles have a high antibiotic loading and provide a slow release of the antibiotic payload to deactivate the target bacteria. The boronic acid modified polymer particles not only contribute to enhanced antibacterial efficiency, but also have the potential to act as scavengers to remove unused antibiotic from the environment.
- Published
- 2019
10. Assessing the effect of d-xylose on the sugar signaling pathways of Saccharomyces cerevisiae in strains engineered for xylose transport and assimilation
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Lisa Wasserstrom, Magnus Carlquist, Daniel P. Brink, Celina Borgström, Karen Ofuji Osiro, and Marie F. Gorwa-Grauslund
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0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Genotype ,Population ,Saccharomyces cerevisiae ,Biosensing Techniques ,Xylose ,Applied Microbiology and Biotechnology ,Microbiology ,Metabolic engineering ,03 medical and health sciences ,chemistry.chemical_compound ,Oxidoreductase ,education ,Sugar ,chemistry.chemical_classification ,education.field_of_study ,biology ,Biological Transport ,Assimilation (biology) ,General Medicine ,biology.organism_classification ,Glucose ,030104 developmental biology ,Metabolic Engineering ,Biochemistry ,chemistry ,Galactose ,Mutation ,Sugars ,Plasmids ,Signal Transduction - Abstract
One of the challenges of establishing an industrially competitive process to ferment lignocellulose to value-added products using Saccharomyces cerevisiae is to get efficient mixed sugar fermentations. Despite successful metabolic engineering strategies, the xylose assimilation rates of recombinant S. cerevisiae remain significantly lower than for the preferred carbon source, glucose. Previously, we established a panel of in vivo biosensor strains (TMB371X) where different promoters (HXT1/2/4p; SUC2p, CAT8p; TPS1p/2p, TEF4p) from the main sugar signaling pathways were coupled with the yEGFP3 gene, and observed that wild-type S. cerevisiae cannot sense extracellular xylose. Here, we expand upon these strains by adding a mutated galactose transporter (GAL2-N376F) with improved xylose affinity (TMB372X), and both the transporter and an oxidoreductase xylose pathway (TMB375X). On xylose, the TMB372X strains displayed population heterogeneities, which disappeared when carbon starvation was relieved by the addition of the xylose assimilation pathway (TMB375X). Furthermore, the signal in the TMB375X strains on high xylose (50 g/L) was very similar to the signal recorded on low glucose (≤5 g/L). This suggests that intracellular xylose triggers a similar signal to carbon limitation in cells that are actively metabolizing xylose, in turn causing the low assimilation rates.
- Published
- 2018
11. Increased lignocellulosic inhibitor tolerance of Saccharomyces cerevisiae cell populations in early stationary phase
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Jenny Schelin, Marie F. Gorwa-Grauslund, Venkatachalam Narayanan, Magnus Carlquist, and Ed W. J. van Niel
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0301 basic medicine ,lcsh:Biotechnology ,Intracellular pH ,030106 microbiology ,Saccharomyces cerevisiae ,Cell ,Population ,Stress tolerance ,Management, Monitoring, Policy and Law ,Acetic acid ,Applied Microbiology and Biotechnology ,lcsh:Fuel ,Flow cytometry ,03 medical and health sciences ,lcsh:TP315-360 ,lcsh:TP248.13-248.65 ,medicine ,Carbon starvation ,Viability assay ,education ,education.field_of_study ,medicine.diagnostic_test ,biology ,Renewable Energy, Sustainability and the Environment ,biology.organism_classification ,Furfural ,Yeast ,General Energy ,medicine.anatomical_structure ,Biochemistry ,Vanillin ,Fermentation ,Reactive oxygen species ,Biotechnology - Abstract
Background Production of second-generation bioethanol and other bulk chemicals by yeast fermentation requires cells that tolerate inhibitory lignocellulosic compounds at low pH. Saccharomyces cerevisiae displays high plasticity with regard to inhibitor tolerance, and adaptation of cell populations to process conditions is essential for reaching efficient and robust fermentations. Results In this study, we assessed responses of isogenic yeast cell populations in different physiological states to combinations of acetic acid, vanillin and furfural at low pH. We found that cells in early stationary phase (ESP) exhibited significantly increased tolerance compared to cells in logarithmic phase, and had a similar ability to initiate growth in the presence of inhibitors as pre-adapted cells. The ESP cultures consisted of subpopulations with different buoyant cell densities which were isolated with flotation and analysed separately. These so-called quiescent (Q) and non-quiescent (NQ) cells were found to possess similar abilities to initiate growth in the presence of lignocellulosic inhibitors at pH 3.7, and had similar viabilities under static conditions. Therefore, differentiation into Q-cells was not the cause for increased tolerance of ESP cultures. Flow cytometry analysis of cell viability, intracellular pH and reactive oxygen species levels revealed that tolerant cell populations had a characteristic response upon inhibitor perturbations. Growth in the presence of a combination of inhibitors at low pH correlated with pre-cultures having a high frequency of cells with low pHi and low ROS levels. Furthermore, only a subpopulation of ESP cultures was able to tolerate lignocellulosic inhibitors at low pH, while pre-adapted cell populations displayed an almost uniform high tolerance to the adverse condition. This was in stark contrast to cell populations growing exponentially in non-inhibitory medium that were uniformly sensitive to the inhibitors at low pH. Conclusions ESP cultures of S. cerevisiae were found to have high tolerance to lignocellulosic inhibitors at low pH, and were able to initiate growth to the same degree as cells that were pre-adapted to inhibitors at a slightly acidic pH. Carbon starvation may thus be a potential strategy to prepare cell populations for adjacent stressful environments which may be beneficial from a process perspective for fermentation of non-detoxified lignocellulosic substrates at low pH. Furthermore, flow cytometry analysis of pHi and ROS level distributions in ESP cultures revealed responses that were characteristic for populations with high tolerance to lignocellulosic inhibitors. Measurement of population distribution responses as described herein may be applied to predict the outcome of environmental perturbations and thus can function as feedback for process control of yeast fitness during lignocellulosic fermentation.
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- 2017
12. Increased lignocellulosic inhibitor tolerance of
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Venkatachalam, Narayanan, Jenny, Schelin, Marie, Gorwa-Grauslund, Ed Wj, van Niel, and Magnus, Carlquist
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Research ,Vanillin ,Stress tolerance ,Carbon starvation ,Quiescence ,Acetic acid ,Reactive oxygen species ,Population heterogeneity ,Furfural ,Intracellular pH - Abstract
Background Production of second-generation bioethanol and other bulk chemicals by yeast fermentation requires cells that tolerate inhibitory lignocellulosic compounds at low pH. Saccharomyces cerevisiae displays high plasticity with regard to inhibitor tolerance, and adaptation of cell populations to process conditions is essential for reaching efficient and robust fermentations. Results In this study, we assessed responses of isogenic yeast cell populations in different physiological states to combinations of acetic acid, vanillin and furfural at low pH. We found that cells in early stationary phase (ESP) exhibited significantly increased tolerance compared to cells in logarithmic phase, and had a similar ability to initiate growth in the presence of inhibitors as pre-adapted cells. The ESP cultures consisted of subpopulations with different buoyant cell densities which were isolated with flotation and analysed separately. These so-called quiescent (Q) and non-quiescent (NQ) cells were found to possess similar abilities to initiate growth in the presence of lignocellulosic inhibitors at pH 3.7, and had similar viabilities under static conditions. Therefore, differentiation into Q-cells was not the cause for increased tolerance of ESP cultures. Flow cytometry analysis of cell viability, intracellular pH and reactive oxygen species levels revealed that tolerant cell populations had a characteristic response upon inhibitor perturbations. Growth in the presence of a combination of inhibitors at low pH correlated with pre-cultures having a high frequency of cells with low pHi and low ROS levels. Furthermore, only a subpopulation of ESP cultures was able to tolerate lignocellulosic inhibitors at low pH, while pre-adapted cell populations displayed an almost uniform high tolerance to the adverse condition. This was in stark contrast to cell populations growing exponentially in non-inhibitory medium that were uniformly sensitive to the inhibitors at low pH. Conclusions ESP cultures of S. cerevisiae were found to have high tolerance to lignocellulosic inhibitors at low pH, and were able to initiate growth to the same degree as cells that were pre-adapted to inhibitors at a slightly acidic pH. Carbon starvation may thus be a potential strategy to prepare cell populations for adjacent stressful environments which may be beneficial from a process perspective for fermentation of non-detoxified lignocellulosic substrates at low pH. Furthermore, flow cytometry analysis of pHi and ROS level distributions in ESP cultures revealed responses that were characteristic for populations with high tolerance to lignocellulosic inhibitors. Measurement of population distribution responses as described herein may be applied to predict the outcome of environmental perturbations and thus can function as feedback for process control of yeast fitness during lignocellulosic fermentation. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0794-0) contains supplementary material, which is available to authorized users.
- Published
- 2017
13. Cell mass and cell cycle dynamics of an asynchronous budding yeast population: Experimental observations, flow cytometry data analysis, and multi-scale modeling
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Abhishek Dutta, Anna-Lena Heins, Luisa Lundin, Magnus Carlquist, Ingmar Nopens, Søren J. Sørensen, Anker Degn Jensen, Krist V. Gernaey, Rita Lencastre Fernandes, and Anna Eliasson Lantz
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education.field_of_study ,biology ,medicine.diagnostic_test ,Ecology ,Cell Cycle ,Population ,Dynamics (mechanics) ,Saccharomyces cerevisiae ,Experimental data ,Bioengineering ,Models, Theoretical ,Cell cycle ,Flow Cytometry ,biology.organism_classification ,Applied Microbiology and Biotechnology ,Multiscale modeling ,Flow cytometry ,Asynchronous communication ,medicine ,education ,Biological system ,Cell Size ,Biotechnology - Abstract
Despite traditionally regarded as identical, cells in a microbial cultivation present a distribution of phenotypic traits, forming a heterogeneous cell population. Moreover, the degree of heterogeneity is notably enhanced by changes in micro-environmental conditions. A major development in experimental single-cell studies has taken place in the last decades. It has however not been fully accompanied by similar contributions within data analysis and mathematical modeling. Indeed, literature reporting, for example, quantitative analyses of experimental single-cell observations and validation of model predictions for cell property distributions against experimental data is scarce. This study focuses on the experimental and mathematical description of the dynamics of cell size and cell cycle position distributions, of a population of Saccharomyces cerevisiae, in response to the substrate consumption observed during batch cultivation. The good agreement between the proposed multi-scale model (a population balance model [PBM] coupled to an unstructured model) and experimental data (both the overall physiology and cell size and cell cycle distributions) indicates that a mechanistic model is a suitable tool for describing the microbial population dynamics in a bioreactor. This study therefore contributes towards the understanding of the development of heterogeneous populations during microbial cultivations. More generally, it consists of a step towards a paradigm change in the study and description of cell cultivations, where average cell behaviors observed experimentally now are interpreted as a potential joint result of various co-existing single-cell behaviors, rather than a unique response common to all cells in the cultivation.
- Published
- 2012
14. Increased availability of NADH in metabolically engineered baker's yeast improves transaminase-oxidoreductase coupled asymmetric whole-cell bioconversion
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Jan Knudsen, Cecilia Hägglöf, Nora Weber, and Magnus Carlquist
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0106 biological sciences ,0301 basic medicine ,Saccharomyces cerevisiae ,Bioengineering ,Glycerolphosphate Dehydrogenase ,01 natural sciences ,Applied Microbiology and Biotechnology ,Cofactor ,Kinetic resolution ,Metabolic engineering ,03 medical and health sciences ,Chiral alcohols ,Oxidoreductase ,010608 biotechnology ,Phenethylamines ,(S)-1-phenylethanol ,Chiral amines ,(R)-1-phenylethylamine ,Benzyl Alcohols ,Transaminases ,Alcohol dehydrogenase ,chemistry.chemical_classification ,Co-factor regeneration ,Whole-cell biocatalysis ,biology ,Research ,Alcohol Dehydrogenase ,Acetophenones ,Stereoisomerism ,biology.organism_classification ,NAD ,Yeast ,030104 developmental biology ,Glycerol-3-phosphate dehydrogenase ,Glucose ,chemistry ,Biochemistry ,Metabolic Engineering ,Benzaldehydes ,biology.protein ,Biocatalysis ,Metabolome ,Oxidoreductases ,Biotechnology - Abstract
Background Saccharomyces cerevisiae can be engineered to perform a multitude of different chemical reactions that are not programmed in its original genetic code. It has a large potential to function as whole-cell biocatalyst for one-pot multistep synthesis of various organic molecules, and it may thus serve as a powerful alternative or complement to traditional organic synthetic routes for new chemical entities (NCEs). However, although the selectivity in many cases is high, the catalytic activity is often low which results in low space-time-yields. In the case for NADH-dependent heterologous reductive reactions, a possible constraint is the availability of cytosolic NADH, which may be limited due to competition with native oxidative enzymes that act to maintain redox homeostasis. In this study, the effect of increasing the availability of cytosolic NADH on the catalytic activity of engineered yeast for transamination-reduction coupled asymmetric one-pot conversion was investigated. Results A series of active whole-cell biocatalysts were constructed by over-expressing the (S)-selective ω-transaminase (VAMT) from Capsicum chinense together with the NADH-dependent (S)-selective alcohol dehydrogenase (SADH) originating from Rhodococcus erythropolis in strains with or without deletion of glycerol-3-phosphate dehydrogenases 1 and 2 (GPD1 and GPD2). The yeast strains were evaluated as catalysts for simultaneous: (a) kinetic resolution of the racemic mixture to (R)-1-phenylethylamine, and (b) reduction of the produced acetophenone to (S)-1-phenylethanol. For the gpd1Δgpd2Δ strain, cell metabolism was effectively used for the supply of both amine acceptors and the co-factor pyridoxal-5′-phosphate (PLP) for the ω-transaminase, as well as for regenerating NADH for the reduction. In contrast, there was nearly no formation of (S)-1-phenylethanol when using the control strain with intact GPDs and over-expressing the VAMT-SADH coupling. It was found that a gpd1Δgpd2Δ strain over-expressing SADH had a 3-fold higher reduction rate and a 3-fold lower glucose requirement than the strain with intact GPDs over-expressing SADH. Conclusions Overall the results demonstrate that the deletion of the GPD1 and GPD2 genes significantly increases activity of the whole-cell biocatalyst, and at the same time reduces the co-substrate demand in a process configuration where only yeast and sugar is added to drive the reactions, i.e. without addition of external co-factors or prosthetic groups. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0430-x) contains supplementary material, which is available to authorized users.
- Published
- 2015
15. Kinetic resolution of racemic 5,6-epoxy-bicyclo[2.2.1]heptane-2-one using genetically engineered Saccharomyces cerevisiae
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Torbjörn Frejd, Cecilia Olsson, Basti Bergdahl, Marie-Francoise Gorwa-Grauslund, Ed W. J. van Niel, and Magnus Carlquist
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Glucose-6-phosphate isomerase ,biology ,Bicyclic molecule ,Chemistry ,Stereochemistry ,Process Chemistry and Technology ,Saccharomyces cerevisiae ,Substrate (chemistry) ,Bioengineering ,Dehydrogenase ,biology.organism_classification ,Biochemistry ,Catalysis ,Kinetic resolution ,Yield (chemistry) ,Racemic mixture - Abstract
(+)-5,6-Epoxy-bicyclo[2.2.1]heptane-2-one, (+)-1, and endo-(−)-5,6-epoxy-bicyclo[2.2.1]heptane-2-ol, endo-(−)-2, were obtained by kinetic resolution of rac-1 by asymmetric bioreduction catalyzed by whole cells of a genetically engineered Saccharomyces cerevisiae yeast strain. The strain, TMB4100, had 1% phosphoglucose isomerase (PGI) activity and overexpressed a specific short-chain dehydrogenase, encoded by the gene YMR226c. The whole cell biocatalystwas demonstrated to be significantly inactivated within 24 h, thus restricting the reaction to lowconcentration. Despite this, the resolution method could be used to produce optically pure (+)-1 and endo-(−)-2 from the racemic mixture at 5 g/L substrate. At optimal conditions, 1 g of rac-1 was kinetically resolved to give (+)-1 in 95% ee and 28% yield and endo-(−)-2 in 74% ee, 80% de and 45% yield. (Less)
- Published
- 2009
16. Flavonoids as inhibitors of human carbonyl reductase 1
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Torbjörn Frejd, Magnus Carlquist, and Marie-Francoise Gorwa-Grauslund
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Models, Molecular ,Carbonyl Reductase ,CBR1 ,Stereochemistry ,Rutin ,Flavonoid ,Gene Expression ,Mixed inhibition ,Saccharomyces cerevisiae ,Transfection ,Toxicology ,Polymerase Chain Reaction ,Substrate Specificity ,Inhibitory Concentration 50 ,chemistry.chemical_compound ,Humans ,Enzyme Inhibitors ,Binding site ,Flavonoids ,chemistry.chemical_classification ,Antibiotics, Antineoplastic ,Binding Sites ,Plant Extracts ,Chemistry ,Daunorubicin ,General Medicine ,Protein Structure, Tertiary ,Alcohol Oxidoreductases ,Kinetics ,Enzyme ,Biochemistry ,Docking (molecular) ,Quercetin - Abstract
Human carbonyl reductase 1 (CBR1), that is one of the enzymes responsible for the reduced efficiency of treatments by the antineoplastic agents anthracyclines, was functionally expressed in Saccharomyces cerevisiae. CBR1 was purified and kinetically characterised using daunorubicin as substrate. CBR1-catalysed reduction of daunorubicin followed an apparent Michaelis-Menten kinetics with K(M)=85.2+/-26.7microM and V(max)=3490+/-220micromol/(mingprotein). The type of inhibition for the flavonoid compound rutin was determined by studying initial reaction rates in the presence of rutin. The inhibition kinetics was found to follow an apparent mixed inhibition with K(ic)=1.8+/-1.2microM and K(iu)=2.8+/-1.6microM. IC50-values were also determined for a set of flavonoids in order to identify essential structure for inhibition activity. Computational docking experiments of the four best inhibitors to the catalytic site of CBR1 showed that the flavonoid skeleton structure was the binding part of the molecule. The presence of a sugar moiety in 1 and 2, or a sugar mimicking part in 9, directed the orientation of the flavonoid so that the sugars were pointing outwards, giving rise to a stabilising effect to the binding. Finally, additional binding epitopes that interacted with various parts of the flavonoid ligand were identified and could potentially be targeted for further improvement of inhibition activity. These included; hydrogen-binding sites surrounding Ser139 and Cys226, Met234 and Tyr193 or Trp229; aromatic-aromatic interaction with Tyr193, Trp229 or NADPH; van der Waals interactions with Ile140.
- Published
- 2008
17. NADH-dependent biosensor in Saccharomyces cerevisiae: principle and validation at the single cell level
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Jan Knudsen, Magnus Carlquist, and Marie F. Gorwa-Grauslund
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Single cell analysis ,Acetoin ,Saccharomyces cerevisiae ,Biophysics ,Heterologous ,Dehydrogenase ,Biology ,biology.organism_classification ,Applied Microbiology and Biotechnology ,Yeast ,Green fluorescent protein ,Redox balance ,chemistry.chemical_compound ,Biochemistry ,chemistry ,Single-cell analysis ,Original Article ,NAD+ kinase ,NADH biosensor - Abstract
A reporter system was constructed to measure perturbations in the NADH/NAD+co-factor balance in yeast, by using the green fluorescent protein gene under the control of theGPD2promoter that is induced under conditions of excess of NADH. High fluorescence levels were obtained in a glycerol 3-phosphate dehydrogenase double deletion strain (gpd1Δgpd2Δ), which is deficient in the ability to regenerate NAD+via glycerol formation. The responsiveness of the reporter system to externally induced perturbations in NADH oxidation was also evaluated in thegpd1Δgpd2Δstrain background by addition of acetoin, as well as by introduction of a set of heterologous xylose reductases (XRs) having different selectivities for NADH. Addition of acetoin during cell proliferation under oxygen-limited conditions resulted in a more than 2-fold decrease in mean fluorescence intensity as compared to the control experiment. Strains carrying XRs with different selectivities for NADH could be distinguished at the single cell level, so that the XR with the highest selectivity for NADH displayed the lowest fluorescence. In conclusion, the designed system successfully allowed for monitoring perturbations in the cellular redox metabolism caused by environmental changes, or by heterologous gene expression. The reporter system displayed high resolution in distinguishing cytosolic NADH oxidation capacity and hence has potential to be used for high-throughput screening based on the fluorescence of single cells.
- Published
- 2014
18. Process engineering for bioflavour production with metabolically active yeasts - a mini-review
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Magnus, Carlquist, Brian, Gibson, Yonca, Karagul Yuceer, Adamantini, Paraskevopoulou, Mari, Sandell, Angel I, Angelov, Velitchka, Gotcheva, Angel D, Angelov, Marlene, Etschmann, Gustavo M, de Billerbeck, and Gunnar, Lidén
- Subjects
Flavoring Agents ,Fungal Proteins ,Metabolic Engineering ,Yeasts - Abstract
Flavours are biologically active molecules of large commercial interest in the food, cosmetics, detergent and pharmaceutical industries. The production of flavours can take place by either extraction from plant materials, chemical synthesis, biological conversion of precursor molecules or de novo biosynthesis. The latter alternatives are gaining importance through the rapidly growing fields of systems biology and metabolic engineering, giving efficient production hosts for the so-called 'bioflavours', which are natural flavour and/or fragrance compounds obtained with cell factories or enzymatic systems. Yeasts are potential production hosts for bioflavours. In this mini-review, we give an overview of bioflavour production in yeasts from the process-engineering perspective. Two specific examples, production of 2-phenylethanol and vanillin, are used to illustrate the process challenges and strategies used.
- Published
- 2014
19. Biocatalytic potential of vanillin aminotransferase from Capsicum chinense
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Abdel Rahman Ismail, Magnus Carlquist, Marie-Francoise Gorwa-Grauslund, and Nora Weber
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Benzylamines ,Transamination ,Transaminase ,chemistry.chemical_compound ,Putative aminotransferase ,PAMT ,Escherichia coli ,Citrate synthase ,Capsaicinoids ,Transaminases ,Plant Proteins ,chemistry.chemical_classification ,VAMT ,Acetophenone ,Whole-cell biocatalysis ,biology ,Vanillin ,Vanillylamine ,Enzyme ,chemistry ,Biochemistry ,Biocatalysis ,1-phenylethylamine ,Benzaldehydes ,biology.protein ,Stereoselectivity ,Capsicum ,Research Article ,Biotechnology - Abstract
Background The conversion of vanillin to vanillylamine is a key step in the biosynthetic route towards capsaicinoids in pungent cultivars of Capsicum sp. The reaction has previously been annotated to be catalysed by PAMT (putative aminotransferase; [GenBank: AAC78480.1, Swiss-Prot: O82521]), however, the enzyme has previously not been biochemically characterised in vitro. Results The biochemical activity of the transaminase was confirmed by direct measurement of the reaction with purified recombinant enzyme. The enzyme accepted pyruvate, and oxaloacetate but not 2-oxoglutarate as co-substrate, which is in accordance with other characterised transaminases from the plant kingdom. The enzyme was also able to convert (S)-1-phenylethylamine into acetophenone with high stereo-selectivity. Additionally, it was shown to be active at a broad pH range. Conclusions We suggest PAMT to be renamed to VAMT (vanillin aminotransferase, abbreviation used in this study) as formation of vanillin from vanillylamine could be demonstrated. Furthermore, due to high stereoselectivity and activity at physiological pH, VAMT is a suitable candidate for biocatalytic transamination in a recombinant whole-cell system.
- Published
- 2014
20. Genetically engineered Saccharomyces cerevisiae for kinetic resolution of racemic bicyclo[3.3.1]nonane-2,6-dione
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Kenneth Wärnmark, Magnus Carlquist, Carl-Johan Wallentin, and Marie-Francoise Gorwa-Grauslund
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biology ,Stereochemistry ,Chemistry ,Organic Chemistry ,Saccharomyces cerevisiae ,Dehydrogenase ,Reductase ,biology.organism_classification ,Catalysis ,Yeast ,Kinetic resolution ,Inorganic Chemistry ,Biocatalysis ,Physical and Theoretical Chemistry ,Enantiomeric excess ,Selectivity - Abstract
Whole cells of the genetically engineered Saccharomyces cerevisiae strain TMB4100 (1% PGI, YMR226c) were Used as the biocatalyst for the kinetic resolution of racemic bicyclo[3.3.1]nonane-2,6-dione rac-1. The yeast's phosphoglucose isomerase activity was decreased, and the short-chain dehydrogenase/reductase encoded by YMR226c was overexpressed. This reduced the demand for the glucose to regenerate NADPH, while at the same time the reaction rate and selectivity towards (-)-1 became higher. The demand for yeast biomass also decreased, facilitating down-stream processing, which is of considerable importance oil a large scale. With 15 g dry weight/L of the genetically engineered yeast TMB4100 (1% PGI, YMR226c), 40 g/L rac-1 was kinetically resolved within 24 h producing pure (+)-1 with all enantiomeric excess (ee) of 100% after 75% conversion. This corresponds to a biochemical selectivity constant of E = 10.3 +/- 2.2. Thus, compared with conventional methods which use commercial baker's yeast as a biocatalyst, the reaction system was significantly improved, and Would be superior in a large-scale process. (C) 2008 Elsevier Ltd. All rights reserved. (Less)
- Published
- 2008
21. Applying mechanistic models in bioprocess development
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Rita, Lencastre Fernandes, Vijaya Krishna, Bodla, Magnus, Carlquist, Anna-Lena, Heins, Anna, Eliasson Lantz, Gürkan, Sin, and Krist V, Gernaey
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Research Design ,Fermentation ,Biological Assay ,Saccharomyces cerevisiae ,Models, Theoretical - Abstract
The available knowledge on the mechanisms of a bioprocess system is central to process analytical technology. In this respect, mechanistic modeling has gained renewed attention, since a mechanistic model can provide an excellent summary of available process knowledge. Such a model therefore incorporates process-relevant input (critical process variables)-output (product concentration and product quality attributes) relations. The model therefore has great value in planning experiments, or in determining which critical process variables need to be monitored and controlled tightly. Mechanistic models should be combined with proper model analysis tools, such as uncertainty and sensitivity analysis. When assuming distributed inputs, the resulting uncertainty in the model outputs can be decomposed using sensitivity analysis to determine which input parameters are responsible for the major part of the output uncertainty. Such information can be used as guidance for experimental work; i.e., only parameters with a significant influence on model outputs need to be determined experimentally. The use of mechanistic models and model analysis tools is demonstrated in this chapter. As a practical case study, experimental data from Saccharomyces cerevisiae fermentations are used. The data are described with the well-known model of Sonnleitner and Käppeli (Biotechnol Bioeng 28:927-937, 1986) and the model is analyzed further. The methods used are generic, and can be transferred easily to other, more complex case studies as well.
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- 2013
22. Applying Mechanistic Models in Bioprocess Development
- Author
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Krist V. Gernaey, Anna Eliasson Lantz, Vijaya Krishna Bodla, Anna-Lena Heins, Guerkan Sin, Rita Lencastre Fernandes, and Magnus Carlquist
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Process (engineering) ,Computer science ,media_common.quotation_subject ,Process analytical technology ,Experimental data ,Identifiability ,Value (computer science) ,Quality (business) ,Sensitivity (control systems) ,Biochemical engineering ,Bioprocess ,media_common - Abstract
The available knowledge on the mechanisms of a bioprocess system is central to process analytical technology. In this respect, mechanistic modeling has gained renewed attention, since a mechanistic model can provide an excellent summary of available process knowledge. Such a model therefore incorporates process-relevant input (critical process variables)-output (product concentration and product quality attributes) relations. The model therefore has great value in planning experiments, or in determining which critical process variables need to be monitored and controlled tightly. Mechanistic models should be combined with proper model analysis tools, such as uncertainty and sensitivity analysis. When assuming distributed inputs, the resulting uncertainty in the model outputs can be decomposed using sensitivity analysis to determine which input parameters are responsible for the major part of the output uncertainty. Such information can be used as guidance for experimental work; i.e., only parameters with a significant influence on model outputs need to be determined experimentally. The use of mechanistic models and model analysis tools is demonstrated in this chapter. As a practical case study, experimental data from Saccharomyces cerevisiae fermentations are used. The data are described with the well-known model of Sonnleitner and Kappeli (Biotechnol Bioeng 28: 927-937, 1986) and the model is analyzed further. The methods used are generic, and can be transferred easily to other, more complex case studies as well. (Less)
- Published
- 2012
23. Bioreduction
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Nádia Skorupa Parachin, Magnus Carlquist, and Marie-F Gorwa-Grauslund
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- 2010
24. Comparison of engineered Saccharomyces cerevisiae and engineered Escherichia coli for the production of an optically pure keto alcohol
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Magnus Carlquist, Nádia Skorupa Parachin, and Marie-Francoise Gorwa-Grauslund
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Saccharomyces cerevisiae ,Aldo-Keto Reductases ,Reductase ,Pentose phosphate pathway ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Bridged Bicyclo Compounds ,Aldehyde Reductase ,medicine ,Escherichia coli ,NADPH regeneration ,chemistry.chemical_classification ,Aldo-keto reductase ,biology ,Organisms, Genetically Modified ,Stereoisomerism ,General Medicine ,Ketones ,biology.organism_classification ,Yeast ,Alcohol Oxidoreductases ,Enzyme ,Biochemistry ,chemistry ,Alcohols ,Oxidation-Reduction ,NADP ,Biotechnology - Abstract
In this study, the production of enantiomerically pure (1R,4S,6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one ((−)-2) through stereoselective bioreduction was used as a model reaction for the comparison of engineered Saccharomyces cerevisiae and engineered Escherichia coli as biocatalysts. For both microorganisms, over-expression of the gene encoding the NADPH-dependent aldo-keto reductase YPR1 resulted in high purity of the keto alcohol (−)-2 (>99% ee, 97–98% de). E. coli had three times higher initial reduction rate but S. cerevisiae continued the reduction reaction for a longer time period, thus reaching a higher conversion of the substrate (95%). S. cerevisiae was also more robust than E. coli, as demonstrated by higher viability during bioreduction. It was also investigated whether the NADPH regeneration rate was sufficient to supply the over-expressed reductase with NADPH. Five strains of each microorganism with varied carbon flux through the NADPH regenerating pentose phosphate pathway were genetically constructed and compared. S. cerevisiae required an increased NADPH regeneration rate to supply YPR1 with co-enzyme while the native NADPH regeneration rate was sufficient for E. coli.
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- 2009
25. Reaction and strain engineering for improved stereo-selective whole-cell reduction of a bicyclic diketone
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Torbjörn Frejd, Ted Johanson, Marie-Francoise Gorwa-Grauslund, Magnus Carlquist, Cecilia Olsson, and Andreas Rudolf
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Diketone ,Saccharomyces cerevisiae Proteins ,Strain (chemistry) ,Bicyclic molecule ,Chemistry ,Stereochemistry ,Substrate (chemistry) ,Stereoisomerism ,General Medicine ,Saccharomyces cerevisiae ,Reductase ,Hydrogen-Ion Concentration ,Applied Microbiology and Biotechnology ,Substrate Specificity ,Bridged Bicyclo Compounds ,Yield (chemistry) ,Stereoselectivity ,Enantiomeric excess ,Oxidation-Reduction ,Biotechnology - Abstract
Reduction of bicyclo[2.2.2]octane-2,6-dione to (1R, 4S, 6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one by whole cells of Saccharomyces cerevisiae was improved using an engineered recombinant strain and process design. The substrate inhibition followed a Han-Levenspiel model showing an effective concentration window between 12 and 22 g/l, in which the activity was kept above 95%. Yeast growth stage, substrate concentration and a stable pH were shown to be important parameters for effective conversion. The over-expression of the reductase gene YDR368w significantly improved diastereoselectivity compared to previously reported results. Using strain TMB4110 expressing YDR368w in batch reduction with pH control, complete conversion of 40 g/l (290 mM) substrate was achieved with 97% diastereomeric excess (de) and >99 enantiomeric excess (ee), allowing isolation of the optically pure ketoalcohol in 84% yield.
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- 2007
26. Dynamics in population heterogeneity during batch and continuous fermentation of Saccharomyces cerevisiae
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Rita Lencastre Fernandes, Anna-Lena Heins, Krist V. Gernaey, Anna Eliasson Lantz, Magnus Carlquist, Luisa Lundin, and Søren J. Sørensen
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Continuous fermentation ,biology ,Chemistry ,Dynamics (mechanics) ,Saccharomyces cerevisiae ,Population Heterogeneity ,ComputingMilieux_LEGALASPECTSOFCOMPUTING ,Bioengineering ,General Medicine ,Food science ,biology.organism_classification ,Molecular Biology ,Biotechnology - Abstract
Traditionally, microbial populations in optimization studies of fermentation processes have been considered homogeneous. However, research has shown that a typical microbial population in fermentation is heterogeneous. There are indications that this heterogeneity may be both beneficial (facilitates quick adaptation to new conditions) and harmful (reduces yields and productivities)[1,2]. Typically, gradients of e.g. dissolved oxygen, substrates, and pH are observed in industrial scale fermentation processes. Consequently, microbial cells circulating throughout a bioreactor experience rapid environmental changes, which might pose stress on the cells, affect their metabolism and consequently influence the level of heterogeneity of the population. To gain a deeper understanding of population heterogeneity and the triggering phenomena, a Saccharomyces cerevisiae growth reporter strain based on the expression of green fluorescent protein (GFP) was constructed which enable to perform single cell analysis, and thereby provides a tool to map population heterogeneity. A factorial design experiment followed by multivariate data analysis demonstrated a highly dynamic behavior with regard to subpopulation distribution during different growth stages. To further simulate which effect gradients have on population heterogeneity, glucose and ethanol perturbations during continuous cultivation were performed. Physiological changes were analyzed on single cell level by using flow cytometry followed by cell sorting of different subpopulations. Furthermore the expression of the reporter gene was examined by qPCR. It could be demonstrated that pulses had a clear influence on population distribution. In conclusion, we now have a tool to study the effect environmental gradients have on population heterogeneity.
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- 2012
27. Heterogeneous Microbial Populations: using flow cytometric data for building dynamic distributed models
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Rita Lencastre Fernandes, Magnus Carlquist, Luisa Lundin, Anna-Lena Heins, Abhishek Dutta, Ingmar Nopens, Anker Degn Jensen, Johansen, Søren J., Anna Eliasson Lantz, and Krist Gernaey
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