| 1. |
- Almqvist, Henrik, et al.
(författare)
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Muconic Acid Production Using Engineered Pseudomonas putida KT2440 and a Guaiacol-Rich Fraction Derived from Kraft Lignin
- 2021
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Ingår i: ACS Sustainable Chemistry and Engineering. - : American Chemical Society (ACS). - 2168-0485. ; 9:24, s. 8097-8106
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Tidskriftsartikel (refereegranskat)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.
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| 2. |
- Carlquist, Magnus
(författare)
-
Enzymatic Reduction of Ketones
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Methods have been studied for the production of chiral alcohols, which are of importance in organic synthesis of pharmaceuticals, agro-chemicals and fragrances, etc., through the enzymatic or microbial reduction of ketones. Classical reaction engineering was combined with genetic engineering to improve the production of chiral alcohols and kinetic resolution of racemic ketones with high enantiomeric and diastereomeric purity, productivity, product yield and co-substrate yield. Reductases originating from baker’s yeast, Saccharomyces cerevisiae, were over-expressed in either S. cerevisiae or in the Gram-negative bacterium Escherichia coli. This increased reaction rates and selectivity towards the substrates for whole-cell reduction. Also, several mutants of S. cerevisiae and E. coli with altered metabolic carbon fluxes were constructed and compared regarding their ability to supply the aldo-keto reductase YPR1 with the essential coenzyme NADPH at a non-limiting rate. In order not to limit the reductase, S. cerevisiae required a higher NADPH regeneration rate, which was achieved by directing the carbon flux through the main NADPH-generating pathway, the pentose–phosphate pathway. However, this created a disturbance in the redox balance, which led to the need for fine-tuning of the glucose concentration during biomass production. The same modifications of the central carbon metabolism in E. coli did not increase the reaction rate. In fact, the highest initial reaction rate, productivity and co-substrate yield were obtained with the strain only over-expressing YPR1. This was even higher than for the best S. cerevisiae strain. However, the highest degree of conversion was obtained with S. cerevisiae, because of its significantly greater robustness. In fact, S. cerevisiae has potential to be recycled for sequential bioreductions. The second part of this work involved the study of the inhibition of human reductases, with the aim of reducing the side-effects of anthracycline antibiotics, a group of drugs commonly used to treat a wide variety of cancer diseases. Anthracyclines are reduced in the body to less potent and cardiotoxic alcohol metabolites. To reduce the side-effects of anthracyclines, reductase inhibitors could be administered concomitantly to prevent metabolite formation. Important functionalities for binding of flavonoid compounds to human carbonyl reductase 1 (CBR1) were identified by determining IC50 values for 11 different flavonoids and performing computational docking experiments with four of the best inhibitors. The knowledge acquired on the binding of different flavonoids to the catalytic site of CBR1 is of importance for the design of an optimal inhibitor.
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| 3. |
- Carlquist, Magnus, et al.
(författare)
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Flavonoids as inhibitors of human carbonyl reductase 1
- 2008
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Ingår i: Chemico-Biological Interactions. - : Elsevier BV. - 1872-7786 .- 0009-2797. ; 174:2, s. 98-108
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Tidskriftsartikel (refereegranskat)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 +/- 26.7 mu M and V-max =3490 +/- 220 mu mol/(min g protein). 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 +/- 1.2 mu M and K-iu = 2.8 +/- 1.6 mu M. 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 I 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 IIe140. (C) 2008 Elsevier Ireland Ltd. All rights reserved.
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| 4. |
- Carlquist, Magnus, et al.
(författare)
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Genetically engineered Saccharomyces cerevisiae for kinetic resolution of racemic bicyclo[3.3.1]nonane-2,6-dione
- 2008
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Ingår i: Tetrahedron: Asymmetry. - : Elsevier BV. - 0957-4166. ; 19:19, s. 2293-2295
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Tidskriftsartikel (refereegranskat)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.
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| 5. |
- Carlquist, Magnus, et al.
(författare)
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Kinetic resolution of racemic 5,6-epoxy-bicyclo[2.2.1]heptane-2-one using genetically engineered Saccharomyces cerevisiae
- 2009
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Ingår i: Journal of Molecular Catalysis B: Enzymatic. - : Elsevier BV. - 1873-3158 .- 1381-1177. ; 58:2, s. 98-102
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Tidskriftsartikel (refereegranskat)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.
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| 6. |
- Carlquist, Magnus, et al.
(författare)
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Physiological heterogeneities in microbial populations and implications for physical stress tolerance
- 2012
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Background: Traditionally average values of the whole population are considered when analysing microbial cell cultivations. However, a typical microbial population in a bioreactor is heterogeneous in most phenotypes measurable at a single-cell level. There are indications that such heterogeneity may be unfavourable on the one hand (reduces yields and productivities), but also beneficial on the other hand (facilitates quick adaptation to new conditions - i.e. increases the robustness of the fermentation process). Understanding and control of microbial population heterogeneity is thus of major importance for improving microbial cell factory processes. Results: In this work, a dual reporter system was developed and applied to map growth and cell fitness heterogeneities within budding yeast populations during aerobic cultivation in well-mixed bioreactors. The reporter strain, which was based on the expression of green fluorescent protein (GFP) under the control of the ribosomal protein RPL22a promoter, made it possible to distinguish cell growth phases by the level of fluorescence intensity. Furthermore, by exploiting the strong correlation of intracellular GFP level and cell membrane integrity it was possible to distinguish subpopulations with high and low cell membrane robustness and hence ability to withstand freeze-thaw stress. A strong inverse correlation between growth and cell membrane robustness was observed, which further supports the hypothesis that cellular resources are limited and need to be distributed as a trade-off between two functions: growth and robustness. In addition, the trade-off was shown to vary within the population, and the occurrence of two distinct subpopulations shifting between these two antagonistic modes of cell operation could be distinguished. Conclusions: The reporter strain enabled mapping of population heterogeneities in growth and cell membrane robustness towards freeze-thaw stress at different phases of cell cultivation. The described reporter system is a valuable tool for understanding the effect of environmental conditions on population heterogeneity of microbial cells and thereby to understand cell responses during industrial process-like conditions. It may be applied to identify more robust subpopulations, and for developing novel strategies for strain improvement and process design for more effective bioprocessing.
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| 7. |
- Carlquist, Magnus, et al.
(författare)
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Process engineering for bioflavour production with metabolically active yeast - a minireview.
- 2015
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Ingår i: Yeast. - 1097-0061. ; 32:1, s. 123-143
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Forskningsöversikt (refereegranskat)abstract
- Flavours are biologically active molecules of large commercial interest in the food, cosmetics, detergent and pharmaceutical industry. The production of flavours can take place by either extraction from plant materials, chemical synthesis, through biological conversion of precursor molecules or through 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. One potential production host for bioflavour is yeast. In this mini-review, we give an overview of bioflavour production in yeast from the process engineering perspective. Two specific examples - production of 2-phenylethanol and vanillin - are used to illustrate process challenges and strategies used.
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| 8. |
- Carlquist, Magnus, et al.
(författare)
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Rationalisation of the substrate concentration dependent diastereoselectivity of a Saccharomyces cerevisiae short-chain dehydrogenase
- 2007
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Ingår i: Tetrahedron: Asymmetry. - : Elsevier BV. - 0957-4166. ; 18:21, s. 2554-2556
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Tidskriftsartikel (refereegranskat)abstract
- The diastereoselectivity of the carbonyl reduction of bicyclo[2.2.2]octane-2,6-dione, catalysed by the purified yeast cytosolic short-chain dehydrogenase Ymr226cp, was shown to be substrate concentration dependent. The changing selectivity was attributed to two distinct binding configurations of the substrate in the active site, each yielding a distinct hydroxy ketone diastereomer. By applying individual KM and Vmax values for each binding configuration, the concentration dependence could be modelled with Michaelis–Menten kinetics and the apparent KM and Vmax values for the generation of each diastereomer determined. This is to the best of our knowledge the first rationalisation of a substrate dependent stereoselectivity for a pro-chiral substrate with an isolated enzyme.
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| 9. |
- Dines Knudsen, Jan, et al.
(författare)
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NADH-dependent biosensor in Saccharomyces cerevisiae: principle and validation at the single cell level.
- 2014
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Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 4
-
Tidskriftsartikel (refereegranskat)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 the GPD2 promoter 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 the gpd1Δgpd2Δ strain 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.
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| 10. |
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| 11. |
- Fernandes, Rita Lencastre, et al.
(författare)
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Applying Mechanistic Models in Bioprocess Development
- 2013
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Ingår i: Advances in Biochemical Engineering, Biotechnology. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 0724-6145. ; 132, s. 137-166
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Tidskriftsartikel (refereegranskat)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.
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| 12. |
- Fernandes, Rita Lencastre, et al.
(författare)
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Cell mass and cell cycle dynamics of an asynchronous budding yeast population: Experimental observations, flow cytometry data analysis, and multi-scale modeling
- 2013
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 110:3, s. 812-826
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Tidskriftsartikel (refereegranskat)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. Biotechnol. Bioeng. 2013; 110: 812826. (c) 2012 Wiley Periodicals, Inc.
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| 13. |
- Gonçalves, Carolyne Caetano, et al.
(författare)
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Bioprospecting Microbial Diversity for Lignin Valorization : Dry and Wet Screening Methods
- 2020
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 11
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Forskningsöversikt (refereegranskat)abstract
- Lignin is an abundant cell wall component, and it has been used mainly for generating steam and electricity. Nevertheless, lignin valorization, i.e. the conversion of lignin into high value-added fuels, chemicals, or materials, is crucial for the full implementation of cost-effective lignocellulosic biorefineries. From this perspective, rapid screening methods are crucial for time- and resource-efficient development of novel microbial strains and enzymes with applications in the lignin biorefinery. The present review gives an overview of recent developments and applications of a vast arsenal of activity and sequence-based methodologies for uncovering novel microbial strains with ligninolytic potential, novel enzymes for lignin depolymerization and for unraveling the main metabolic routes during growth on lignin. Finally, perspectives on the use of each of the presented methods and their respective advantages and disadvantages are discussed.
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| 14. |
- Gong, Haiyue, et al.
(författare)
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Boronic Acid Modified Polymer Nanoparticles for Enhanced Bacterial Deactivation
- 2019
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 20:24, s. 2991-2995
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Tidskriftsartikel (refereegranskat)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.
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| 15. |
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| 16. |
- Heins, Anna Lena, et al.
(författare)
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Quantitative flow cytometry to understand population heterogeneity in response to changes in substrate availability in escherichia coli and saccharomyces cerevisiae chemostats
- 2019
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Ingår i: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media SA. - 2296-4185. ; 7:AUG
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Tidskriftsartikel (refereegranskat)abstract
- Microbial cells in bioprocesses are usually described with averaged parameters. But in fact, single cells within populations vary greatly in characteristics such as stress resistance, especially in response to carbon source gradients. Our aim was to introduce tools to quantify population heterogeneity in bioprocesses using a combination of reporter strains, flow cytometry, and easily comprehensible parameters. We calculated mean, mode, peak width, and coefficient of variance to describe distribution characteristics and temporal shifts in fluorescence intensity. The skewness and the slope of cumulative distribution function plots illustrated differences in distribution shape. These parameters are person-independent and precise. We demonstrated this by quantifying growth-related population heterogeneity of Saccharomyces cerevisiae and Escherichia coli reporter strains in steady-state of aerobic glucose-limited chemostat cultures at different dilution rates and in response to glucose pulses. Generally, slow-growing cells showed stronger responses to glucose excess than fast-growing cells. Cell robustness, measured as membrane integrity after exposure to freeze-thaw treatment, of fast-growing cells was strongly affected in subpopulations of low membrane robustness. Glucose pulses protected subpopulations of fast-growing but not slower-growing yeast cells against membrane damage. Our parameters could successfully describe population heterogeneity, thereby revealing physiological characteristics that might have been overlooked during traditional averaged analysis.
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| 17. |
- Heitor Colombelli Manfrão-Netto, João, et al.
(författare)
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Metabolic engineering of Pseudomonas putida for production of vanillylamine from lignin-derived substrates
- 2021
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Ingår i: Microbial Biotechnology. - : Wiley. - 1751-7907 .- 1751-7915. ; 14:6, s. 2448-2462
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Tidskriftsartikel (refereegranskat)abstract
- Whole-cell bioconversion of technical lignins using Pseudomonas putida strains overexpressing amine transaminases (ATAs) has the potential to become an eco-efficient route to produce phenolic amines. Here, a novel cell growth-based screening method to evaluate the in vivo activity of recombinant ATAs towards vanillylamine in P. putida KT2440 was developed. It allowed the identification of the native enzyme Pp-SpuC-II and ATA from Chromobacterium violaceum (Cv-ATA) as highly active towards vanillylamine in vivo. Overexpression of Pp-SpuC-II and Cv-ATA in the strain GN442ΔPP_2426, previously engineered for reduced vanillin assimilation, resulted in 94- and 92-fold increased specific transaminase activity, respectively. Whole-cell bioconversion of vanillin yielded 0.70 ± 0.20 mM and 0.92 ± 0.30 mM vanillylamine, for Pp-SpuC-II and Cv-ATA, respectively. Still, amine production was limited by a substantial re-assimilation of the product and formation of the by-products vanillic acid and vanillyl alcohol. Concomitant overexpression of Cv-ATA and alanine dehydrogenase from Bacillus subtilis increased the production of vanillylamine with ammonium as the only nitrogen source and a reduction in the amount of amine product re-assimilation. Identification and deletion of additional native genes encoding oxidoreductases acting on vanillin are crucial engineering targets for further improvement.
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| 18. |
- Hirschberg, Daniel, et al.
(författare)
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Detection of phosphorylated peptides in proteomic analyses using microfluidic compact disk technology.
- 2004
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 76:19
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Tidskriftsartikel (refereegranskat)abstract
- A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.
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| 19. |
- Johanson, Ted, et al.
(författare)
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Reaction and strain engineering for improved stereo-selective whole-cell reduction of a bicyclic diketone
- 2008
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 77:5, s. 1111-1118
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Tidskriftsartikel (refereegranskat)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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| 20. |
- Knudsen, Jan Dines, et al.
(författare)
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Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae
- 2015
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Ingår i: Biotechnology Reports. - : Elsevier BV. - 2215-017X. ; 7, s. 107-119
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Tidskriftsartikel (refereegranskat)abstract
- A control point for keeping redox homeostasis in Saccharomyces cerevisiae during fermentative growth is the dynamic regulation of transcription for the glycerol-3-phosphate dehydrogenase 2 (GPD2) gene. In this study, the possibility to steer the activity of the GPD2 promoter was investigated by placing it in strains with different ability to reoxidise NADH, and applying different environmental conditions. Flow cytometric analysis of reporter strains expressing green fluorescent protein (GFP) under the control of the GPD2 promoter was used to determine the promoter activity at the single-cell level. When placed in a gpd1Δgpd2Δ strain background, the GPD2 promoter displayed a 2-fold higher activity as compared to the strong constitutive glyceraldehyde-3-phosphate dehydrogenase (TDH3). In contrast, the GPD2 promoter was found to be inactive when cells were cultivated in continuous mode at a growth rate of 0.3 h-1 and in conditions with excess oxygen (i.e. with an aeration of 2.5 vvm, and a stirring of 800 rpm). In addition, a clear window of operation where the gpd1Δgpd2Δ strain can be grown with the same efficiency as wild type yeast was identified. In conclusion, the flow cytometry mapping revealed conditions where the GPD2 promoter was either completely inactive or hyperactive, which has implications for its implementation in future biotechnological applications such as for process control of heterologous gene expression.
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| 21. |
- Knudsen, Jan, et al.
(författare)
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Increased availability of NADH in metabolically engineered baker's yeast improves transaminase-oxidoreductase coupled asymmetric whole-cell bioconversion.
- 2016
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 22. |
- Lindh, Tova, et al.
(författare)
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Expression of the Bacterial Enzyme IdeS Using a GFP Fusion in the Yeast Saccharomyces cerevisiae
- 2023. - 2nd
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Ingår i: Bacterial pathogenesis : Methods and protocols - Methods and protocols. - 1940-6029. - 9781071632437 ; , s. 131-146
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Bokkapitel (refereegranskat)abstract
- Bacterial proteases are important enzymes used in several technical applications where controlled cleavage of proteins is needed. They are challenging enzymes to express recombinantly as parts of the proteome can be hydrolyzed by their activity. The eukaryotic model organism Saccharomyces cerevisiae is potentially a good expression host as it tolerates several stress conditions and is known to better express insoluble proteins compared to bacterial systems. In this chapter we describe how the protease IdeS from Streptococcus pyogenes can be expressed in S. cerevisiae. The expression of IdeS was followed by constructing a fused protein with GFP and measuring the fluorescence with flow cytometry. The protease presence was confirmed with a Western blot assay and activity was measured with an in vitro assay. To reduce potentially toxic effect on the host cell, the growth and production phases were separated by using the inducible promoter GAL1p to control recombinant gene expression. The protocol provided may be adopted for other bacterial proteases through minor modifications of the fused protein.
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| 23. |
- Muratovska, Nina, et al.
(författare)
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Engineering Saccharomyces cerevisiae for production of the capsaicinoid nonivamide
- 2022
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 21
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundCapsaicinoids 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.ConclusionsBaker’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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| 24. |
- Muratovska, Nina, et al.
(författare)
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Recombinant yeast for production of the pain receptor modulator nonivamide from vanillin
- 2023
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Ingår i: Frontiers in Chemical Engineering. - : Frontiers Media SA. - 2673-2718. ; 4
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Tidskriftsartikel (refereegranskat)abstract
- We report on the development of a method based on recombinant yeast Saccharomyces cerevisiae to produce nonivamide, a capsaicinoid and potent agonist of the pain receptor TRPV1. Nonivamide was produced in a two-step batch process where yeast was i) grown aerobically on glucose and ii) used to produce nonivamide from vanillin and non-anoic acid by bioconversion. The yeast was engineered to express multiple copies of an amine transaminase from Chromobacterium violaceum (CvTA), along with an NADH-dependent alanine dehydrogenase from Bacillus subtilis (BsAlaDH) to enable efficient reductive amination of vanillin. Oxygen-limited conditions and the use of ethanol as a co-substrate to regenerate NADH were identified to favour amination over the formation of the by-products vanillic alcohol and vanillic acid. The native alcohol dehydrogenase ADH6 was deleted to further reduce the formation of vanillic alcohol. A two-enzyme system consisting of an N-acyltransferase from Capsicum annuum (CaAT), and a CoA ligase from Sphingomonas sp. Ibu-2 (IpfF) was co-expressed to produce the amide. This study provides proof of concept for yeast-based production of non-ivamide by combined transamination and amidation of vanillin.
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| 25. |
- Muratovska, Nina, et al.
(författare)
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Towards engineered yeast as production platform for capsaicinoids
- 2022
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Ingår i: Biotechnology Advances. - : Elsevier BV. - 0734-9750. ; 59
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Forskningsöversikt (refereegranskat)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.
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| 26. |
- Narayanan, Venkatachalam, et al.
(författare)
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Increased lignocellulosic inhibitor tolerance of Saccharomyces cerevisiae cell populations in early stationary phase
- 2017
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Background: Production of second-generation bioethanol and other bulk chemicals by yeast fermentation requirescells that tolerate inhibitory lignocellulosic compounds at low pH. Saccharomyces cerevisiae displays high plasticitywith regard to inhibitor tolerance, and adaptation of cell populations to process conditions is essential for reachingefficient and robust fermentations.Results: In this study, we assessed responses of isogenic yeast cell populations in different physiological states tocombinations of acetic acid, vanillin and furfural at low pH. We found that cells in early stationary phase (ESP) exhibitedsignificantly increased tolerance compared to cells in logarithmic phase, and had a similar ability to initiategrowth in the presence of inhibitors as pre-adapted cells. The ESP cultures consisted of subpopulations with differentbuoyant cell densities which were isolated with flotation and analysed separately. These so-called quiescent (Q) andnon-quiescent (NQ) cells were found to possess similar abilities to initiate growth in the presence of lignocellulosicinhibitors at pH 3.7, and had similar viabilities under static conditions. Therefore, differentiation into Q-cells was notthe cause for increased tolerance of ESP cultures. Flow cytometry analysis of cell viability, intracellular pH and reactiveoxygen 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 highfrequency of cells with low pHiand low ROS levels. Furthermore, only a subpopulation of ESP cultures was able totolerate lignocellulosic inhibitors at low pH, while pre-adapted cell populations displayed an almost uniform high toleranceto the adverse condition. This was in stark contrast to cell populations growing exponentially in non-inhibitorymedium 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 environmentswhich may be beneficial from a process perspective for fermentation of non-detoxified lignocellulosic substrates atlow pH. Furthermore, flow cytometry analysis of pHiand ROS level distributions in ESP cultures revealed responsesthat were characteristic for populations with high tolerance to lignocellulosic inhibitors. Measurement of populationdistribution responses as described herein may be applied to predict the outcome of environmental perturbationsand thus can function as feedback for process control of yeast fitness during lignocellulosic fermentation.
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| 27. |
- Ofuji Osiro, Karen, et al.
(författare)
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Assessing the effect of d-xylose on the sugar signaling pathways of Saccharomyces cerevisiae in strains engineered for xylose transport and assimilation
- 2018
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1364. ; 18:1
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Tidskriftsartikel (refereegranskat)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.
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| 28. |
- Perruca-Foncillas, Raquel, et al.
(författare)
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Assessment of fluorescent protein candidates for multi-color flow cytometry analysis of Saccharomyces cerevisiae
- 2022
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Ingår i: Biotechnology Reports. - : Elsevier BV. - 2215-017X. ; 34
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Tidskriftsartikel (refereegranskat)abstract
- Transcription factor-based biosensors represent promising tools in the construction and evaluation of efficient cell factories for the sustainable production of fuels, chemicals and pharmaceuticals. They can notably be designed to follow the production of a target compound or to monitor key cellular properties, such as stress or starvation. In most cases, the biosensors are built with fluorescent protein (FP) genes as reporter genes because of the direct correlation between promoter activity and fluorescence level that can be measured using, for instance, flow cytometry or fluorometry. The expansion of available FPs offers the possibility of using several FPs - and biosensors – in parallel in one host, with simultaneous detection using multicolor flow cytometry. However, the technique is currently limited by the unavailability of combinations of FP whose genes can be successfully expressed in the host and whose fluorescence can be efficiently distinguished from each other.In the present study, the broad collection of available FPs was explored and four different FPs were successfully expressed in the yeast Saccharomyces cerevisiae: yEGFP, mEGFP, CyOFP1opt and mBeRFPopt. After studying their fluorescence signals, population heterogeneity and possible interactions, we recommend two original combinations of FPs for bi-color flow cytometry: mEGFP together with either CyOFP1opt or mBeRFPopt, as well as the combination of all three FPs mEGFP, CyOFP1opt and mBeRFPopt for tri-color flow cytometry. These combinations will allow to perform different types of bi-color or possibly tri-color flow cytometry and FACS experiments with yeast, such as phenotype evaluation, screening or sorting, by single-laser excitation with a standard 488 nm blue laser.
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| 29. |
- Perruca Foncillas, Raquel, et al.
(författare)
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Assessment of the TRX2p-yEGFP Biosensor to Monitor the Redox Response of an Industrial Xylose-Fermenting Saccharomyces cerevisiae Strain during Propagation and Fermentation
- 2023
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Ingår i: Journal of Fungi. - 2309-608X. ; 9:6
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Tidskriftsartikel (refereegranskat)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.
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| 30. |
- Pozdniakova, Tatiana A., et al.
(författare)
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Optimization of a hybrid bacterial/Arabidopsis thaliana fatty acid synthase system II in Saccharomyces cerevisiae
- 2023
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Ingår i: Metabolic Engineering Communications. - 2214-0301. ; 17
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Tidskriftsartikel (refereegranskat)abstract
- Fatty acids are produced by eukaryotes like baker's yeast Saccharomyces cerevisiae mainly using a large multifunctional type I fatty acid synthase (FASI) where seven catalytic steps and a carrier domain are shared between one or two protein subunits. While this system may offer efficiency in catalysis, only a narrow range of fatty acids are produced. Prokaryotes, chloroplasts and mitochondria rely instead on a FAS type II (FASII) where each catalytic step is carried out by a monofunctional enzyme encoded by a separate gene. FASII is more flexible and capable of producing a wider range of fatty acid structures, such as the direct production of unsaturated fatty acids. An efficient FASII in the preferred industrial organism S. cerevisiae could provide a platform for developing sustainable production of specialized fatty acids. We functionally replaced either yeast FASI genes (FAS1 or FAS2) with a FASII consisting of nine genes from Escherichia coli (acpP, acpS and fab -A, -B, -D, -F, -G, -H, -Z) as well as three from Arabidopsis thaliana (MOD1, FATA1 and FATB). The genes were expressed from an autonomously replicating multicopy vector assembled using the Yeast Pathway Kit for in-vivo assembly in yeast. Two rounds of adaptation led to a strain with a maximum growth rate (μmax) of 0.19 h−1 without exogenous fatty acids, twice the growth rate previously reported for a comparable strain. Additional copies of the MOD1 or fabH genes resulted in cultures with higher final cell densities and three times higher lipid content compared to the control.
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| 31. |
- Seshagiri Rao, Nikhil, et al.
(författare)
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Flow cytometric analysis reveals culture condition dependent variations in phenotypic heterogeneity of Limosilactobacillus reuteri
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11
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Tidskriftsartikel (refereegranskat)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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| 32. |
- Seshagiri Rao, Nikhil, et al.
(författare)
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Non-inhibitory levels of oxygen during cultivation increase freeze-drying stress tolerance in Limosilactobacillus reuteri DSM 17938
- 2023
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Ingår i: Frontiers in Microbiology. - 1664-302X. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- The physiological effects of oxygen on Limosilactobacillus reuteri DSM 17938 during cultivation and the ensuing properties of the freeze-dried probiotic product was investigated. On-line flow cytometry and k-means clustering gating was used to follow growth and viability in real time during cultivation. The bacterium tolerated aeration at 500 ml/min, with a growth rate of 0.74 ± 0.13 h-1 which demonstrated that low levels of oxygen did not influence the growth kinetics of the bacterium. Modulation of the redox metabolism was, however, seen already at non-inhibitory oxygen levels by 1.5-fold higher production of acetate and 1.5-fold lower ethanol production. A significantly higher survival rate in the freeze-dried product was observed for cells cultivated in presence of oxygen compared to absence of oxygen (61.8 ± 2.4 % vs 11.5 ± 4.3 %), coinciding with a higher degree of unsaturated fatty acids (UFA:SFA ratio of 10 for air sparged vs 3.59 for N2 sparged conditions.). Oxygen also resulted in improved bile tolerance and boosted 5’nucleotidase activity (370 U/L vs 240 U/L in N2 sparged conditions) but lower tolerance to acidic conditions compared bacteria grown under complete anaerobic conditions which survived up to 90 min of exposure at pH 2. Overall, our results indicate the controlled supply of oxygen during production may be used as means for probiotic activity optimisation of L. reuteri DSM 17938.
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| 33. |
- Skorupa Parachin, Nádia, et al.
(författare)
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Bioreduction
- 2010
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Ingår i: Enclyclopedia of Industrial Biotechnology : Bioprocess, Bioseparation and Cell Technology - Bioprocess, Bioseparation and Cell Technology. - Hoboken, NJ, USA : John Wiley & Sons, Inc.. - 9780471799306 - 9780470054581
-
Bokkapitel (refereegranskat)abstract
- Bioreduction has emerged over the years as an alternative method to organic synthesis for the generation of chiral precursors of commercial interest. Bioreductions operate under mild conditions of pH and temperature with the help of highly regio- and enantio-selective oxidoreductase enzymes.In this contribution, the different oxidoreductase families involved in bioreductions are exemplified and their main characteristics are presented. The wide spectrum of oxidoreductase substrates (including ketones, diketones, ketoesters, aldehydes, alkenes, and keto acids) is discussed and both preparative and industrial scale examples are reported. The advantages and disadvantages of using isolated enzymatic systems versus whole-cell systems for bioreduction are discussed in terms of cost, specificity, stereoselectivity, and cofactor regeneration. The contribution is also reviewing strategies for improving the biocatalyst at the cell or enzyme level, which include process engineering, metabolic engineering as well as structure-based and nonstructure-based enzyme engineering. Finally, the potential role of metagenomics for isolating novel biocatalysts from different environments is discussed.
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| 34. |
- Skorupa Parachin, Nadia, et al.
(författare)
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Comparison of engineered Saccharomyces cerevisiae and engineered Escherichia coli for the production of an optically pure keto alcohol.
- 2009
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 84, s. 487-497
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Tidskriftsartikel (refereegranskat)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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| 35. |
- Sreenivas, Krishnan, et al.
(författare)
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Evaluation of Pyrophosphate‐Driven Proton Pumps in Saccharomyces cerevisiae under Stress Conditions
- 2024
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Ingår i: Microorganisms. - 2076-2607. ; 12:3
-
Tidskriftsartikel (refereegranskat)abstract
- In Saccharomyces cerevisiae, pH homeostasis is reliant on ATP due to the use of proton-translocating ATPase (H+-ATPase) which constitutes a major drain within cellular ATP supply. Here, an exogenous proton-translocating pyrophosphatase (H+-PPase) from Arabidopsis thaliana, which uses inorganic pyrophosphate (PPi) rather than ATP, was evaluated for its effect on reducing the ATP burden. The H+-Ppase was localized to the vacuolar membrane or to the cell membrane, and their impact was studied under acetate stress at a low pH. Biosensors (pHluorin and mQueen-2m) were used to observe changes in intracellular pH (pHi) and ATP levels during growth on either glucose or xylose. A significant improvement of 35% in the growth rate at a pH of 3.7 and 6 g·L−1 acetic acid stress was observed in the vacuolar membrane H+-PPase strain compared to the parent strain. ATP levels were elevated in the same strain during anaerobic glucose and xylose fermentations. During anaerobic xylose fermentations, co-expression of pHluorin and a vacuolar membrane H+-PPase improved the growth characteristics by means of an improved growth rate (11.4%) and elongated logarithmic growth duration. Our study identified a potential method for improving productivity in the use of S. cerevisiae as a cell factory under the harsh conditions present in industry.
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| 36. |
- Weber, Nora, et al.
(författare)
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Biocatalytic potential of vanillin aminotransferase from Capsicum chinense.
- 2014
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Ingår i: BMC Biotechnology. - : Springer Science and Business Media LLC. - 1472-6750. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 37. |
- Weber, Nora, et al.
(författare)
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Engineered baker's yeast as whole-cell biocatalyst for one-pot stereo-selective conversion of amines to alcohols
- 2014
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 13
-
Tidskriftsartikel (refereegranskat)abstract
- Background: One-pot multi-step biocatalysis is advantageous over step-by-step synthesis as it reduces the number of process operation units, leading to significant process intensification. Whole-cell biocatalysis with metabolically active cells is especially valuable since all enzymes can be co-expressed in the cell whose metabolism can be exploited for supply of co-substrates and co-factors. Results: In this study, a heterologous enzymatic system consisting of omega-transaminase and ketone reductase was introduced in Saccharomyces cerevisiae, and evaluated for one-pot stereo-selective conversion of amines to alcohols. The system was applied for simultaneous kinetic resolution of racemic 1-phenylethylamine to (R)-1-phenylethylamine and reduction of the ketone intermediate to (R)-1-phenylethanol. Glucose was used as sole co-substrate for both the supply of amine acceptor and the regeneration of NADPH in the reduction step. Conclusions: The whole-cell biocatalyst was shown to sustain transaminase-reductase-catalyzed enantioselective conversion of amines to alcohols with glucose as co-substrate. The transamination catalyzed by recombinant vanillin aminotransferase from Capsicum chinense proved to be the rate-limiting step as a three-fold increase in transaminase gene copy number led to a two-fold increased conversion. The (R)-selective NADPH-dependent alcohol dehydrogenase from Lactobacillus kefir proved to be efficient in catalyzing the reduction of the acetophenone generated in the transamination reaction.
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| 38. |
- Weber, Nora, et al.
(författare)
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Exploiting cell metabolism for biocatalytic whole-cell transamination by recombinant Saccharomyces cerevisiae.
- 2014
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 98:10, s. 4615-4624
-
Tidskriftsartikel (refereegranskat)abstract
- The potential of Saccharomyces cerevisiae for biocatalytic whole-cell transamination was investigated using the kinetic resolution of racemic 1-phenylethylamine (1-PEA) to (R)-1-PEA as a model reaction. As native yeast do not possess any ω-transaminase activity for the reaction, a recombinant yeast biocatalyst was constructed by overexpressing the gene coding for vanillin aminotransferase from Capsicum chinense. The yeast-based biocatalyst could use glucose as the sole co-substrate for the supply of amine acceptor via cell metabolism. In addition, the biocatalyst was functional without addition of the co-factor pyridoxal-5'-phosphate (PLP), which can be explained by a high inherent cellular capacity to sustain PLP-dependent reactions in living cells. In contrast, external PLP supplementation was required when cell viability was low, as it was the case when using pyruvate as a co-substrate. Overall, the results indicate a potential for engineered S. cerevisiae as a biocatalyst for whole-cell transamination and with glucose as the only co-substrate for the supply of amine acceptor and PLP.
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| 39. |
- WEBER, NORA, et al.
(författare)
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Improvement of whole-cell transamination with Saccharomyces cerevisiae using metabolic engineering and cell pre-adaptation
- 2017
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 16:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Whole-cell biocatalysis based on metabolically active baker's yeast with engineered transamination activity can be used to generate molecules carrying a chiral amine moiety. A prerequisite is though to express efficient ω-transaminases and to reach sufficient intracellular precursor levels. Results: Herein, the efficiency of three different ω-transaminases originating from Capsicum chinense, Chromobacterium violaceum, and Ochrobactrum anthropi was compared for whole-cell catalyzed kinetic resolution of racemic 1-phenylethylamine to (R)-1-phenylethylamine. The gene from the most promising candidate, C. violaceum ω-transaminase (CV-TA), was expressed in a strain lacking pyruvate decarboxylase activity, which thereby accumulate the co-substrate pyruvate during glucose assimilation. However, the conversion increased only slightly under the applied reaction conditions. In parallel, the effect of increasing the intracellular pyridoxal-5'-phosphate (PLP) level by omission of thiamine during cultivation was investigated. It was found that without thiamine, PLP supplementation was redundant to keep high in vivo transamination activity. Furthermore, higher reaction rates were achieved using a strain containing several copies of CV-TA gene, highlighting the necessity to also increase the intracellular transaminase level. At last, this strain was also investigated for asymmetric whole-cell bioconversion of acetophenone to (S)-1-phenylethylamine using l-alanine as amine donor. Although functionality could be demonstrated, the activity was extremely low indicating that the native co-product removal system was unable to drive the reaction towards the amine under the applied reaction conditions. Conclusions: Altogether, our results demonstrate that (R)-1-phenylethylamine with >99% ee can be obtained via kinetic resolution at concentrations above 25 mM racemic substrate with glucose as sole co-substrate when combining appropriate genetic and process engineering approaches. Furthermore, the engineered yeast strain with highest transaminase activity was also shown to be operational as whole-cell catalyst for the production of (S)-1-phenylethylamine via asymmetric transamination of acetophenone, albeit with very low conversion.
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| 40. |
- Werin, Balder, et al.
(författare)
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Evaluation of heterologous expression in Pichia pastoris of Pine Weevil TRPA1 by GFP and flow cytometry
- 2024
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Ingår i: Microbial Cell Factories. - 1475-2859. ; 23
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The wasabi receptor, also known as the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel, is a potential target for development of repellents for insects, like the pine weevil (Hylobius abietis) feeding on conifer seedlings and causing damage in forestry. Heterologous expression of TRPA1 from pine weevil in the yeast Pichia pastoris can potentially provide protein for structural and functional studies. Here we take advantage of the Green Fluorescent Protein (GFP) tag to examine the various steps of heterologous expression, to get more insight in clone selection, expression and isolation of the intact purified protein.RESULTS: The sequence of HaTRPA1 is reported and GFP-tagged constructs were made of the full-length protein and a truncated version (Δ1-708 HaTRPA1), lacking the N-terminal ankyrin repeat domain. Clones were screened on GFP expression plates, induced in small liquid cultures and in fed-batch cultures, and evaluated by flow cytometry and fluorescence microscopy. The screening on plates successfully identifies low-expression clones, but fails to predict the ranking of the best performing clones in small-scale liquid cultures. The two constructs differ in their cellular localization. Δ1-708 HaTRPA1 is found in a ring at the perimeter of cell, whereas HaTRPA1 is forming highly fluorescent speckles in interior regions of the cell. The pattern is consistent in different clones of the same construct and persists in fed-batch culture. The expression of Δ1-708 HaTRPA1 decreases the viability more than HaTRPA1, and in fed-batch culture it is clear that intact cells first express Δ1-708 HaTRPA1 and then become damaged. Purifications show that both constructs suffer from degradation of the expressed protein, but especially the HaTRPA1 construct.CONCLUSIONS: The GFP tag makes it possible to follow expression by flow cytometry and fluorescence microscopy. Analyses of localization, cell viability and expression show that the former two parameters are specific for each of the two evaluated constructs, whereas the relative expression of the constructs varies with the cultivation method. High expression is not all that matters, so taking damaged cells into account, something that may be linked to protein degradation, is important when picking the most suitable construct, clone, and expression scheme.
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