16 results on '"Milker, Sofia"'
Search Results
2. First time β-farnesene production by the versatile bacterium Cupriavidus necator
- Author
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Milker, Sofia and Holtmann, Dirk
- Published
- 2021
- Full Text
- View/download PDF
3. Identification of vitamin B12 producing bacteria based on the presence of bluB/cobT2 homologues.
- Author
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Dudko, Darya, Milker, Sofia, Holtmann, Dirk, and Buchhaupt, Markus
- Subjects
VITAMINS ,GENE fusion ,VITAMIN B12 ,LIQUID chromatography-mass spectrometry ,BACTERIA - Abstract
Objectives: The objective of the study was to develop a strategy for the identification of new vitamin B
12 -producing species and to characterize their production capability using a fast and sensitive LC–MS/MS method developed in this study. Results: Searching for homologues of the bluB/cobT2 fusion gene known to be responsible for the production of the active vitamin B12 form in P. freudenreichii was shown to be a successful strategy for the identification of new vitamin B12 -producing strains. The analysis of the identified strains via LC–MS/MS showed the ability of Terrabacter sp. DSM102553, Yimella lutea DSM19828 and Calidifontibacter indicus DSM22967 to produce the active form of vitamin B12 . Further analysis of vitamin B12 production capability of Terrabacter sp. DSM102553 in M9 minimal medium and peptone-based media revealed that the highest yield of 2.65 µg of vitamin B12 per g dry cell weight was obtained in M9 medium. Conclusions: The proposed strategy enabled identification of Terrabacter sp. DSM102553, whose relatively high yields obtained in the minimal medium open new perspectives for the possible application of the strain for biotechnological vitamin B12 production. [ABSTRACT FROM AUTHOR]- Published
- 2023
- Full Text
- View/download PDF
4. Gram‐scale production of the sesquiterpene α‐humulene with Cupriavidus necator.
- Author
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Milker, Sofia, Sydow, Anne, Torres‐Monroy, Ingrid, Jach, Guido, Faust, Frederik, Kranz, Lea, Tkatschuk, Ljubov, and Holtmann, Dirk
- Abstract
Terpenoids have an impressive structural diversity and provide valuable substances for a variety of industrial applications. Among terpenes, the sesquiterpenes (C15) are the largest subclass with bioactivities ranging from aroma to health promotion. In this article, we show a gram‐scale production of the sesquiterpene α‐humulene in final aqueous concentrations of 2 g L−1 with the recombinant strain Cupriavidus necator pKR‐hum in a fed‐batch mode on fructose as carbon source and n‐dodecane as an extracting organic phase for in situ product removal. Since C. necator is capable of both heterotrophic and autotrophic growth, we additionally modeled the theoretically possible yields of a heterotrophic versus an autotrophic process on CO2 in industrially relevant quantities. We compared the cost‐effectiveness of both processes based on a production of 10 t α‐humulene per year, with both processes performing equally with similar costs and gains. Furthermore, the expression and activity of 3‐hydroxymethylglutaryl‐CoA reductase (hmgR) from Myxococcus xanthus was identified as the main limitation of our constructed C. necator pKR‐hum strain. Thus, we outlined possible solutions for further improvement of our production strain, for example, the replacement of the hmgR from M. xanthus by a plant‐based variant to increase α‐humulene production titers in the future. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
5. Biomass pretreatment affects Ustilago maydis in producing itaconic acid
- Author
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Klement Tobias, Milker Sofia, Jäger Gernot, Grande Philipp M, Domínguez de María Pablo, and Büchs Jochen
- Subjects
Ustilago maydis ,Itaconic acid ,Lignocellulose ,Pretreatment ,Seawater ,RAMOS ,Microbiology ,QR1-502 - Abstract
Abstract Background In the last years, the biotechnological production of platform chemicals for fuel components has become a major focus of interest. Although ligno-cellulosic material is considered as suitable feedstock, the almost inevitable pretreatment of this recalcitrant material may interfere with the subsequent fermentation steps. In this study, the fungus Ustilago maydis was used to produce itaconic acid as platform chemical for the synthesis of potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have investigated how pretreatment of ligno-cellulosic biomass precisely influences the subsequent fermentation by U. maydis. Thus, this current study aims to first characterize U. maydis in shake flasks and then to evaluate the influence of three exemplary pretreatment methods on the cultivation and itaconic acid production of this fungus. Cellulose enzymatically hydrolysed in seawater and salt-assisted organic-acid catalysed cellulose were investigated as substrates. Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as substrate. Results U. maydis was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, U. maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood. Conclusion The fungus U. maydis is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, U. maydis can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although U. maydis is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.
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- 2012
- Full Text
- View/download PDF
6. Optimization of biotransformations in Escherichia coli: from biocatalyst to the process level
- Author
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Milker, Sofia
- Subjects
modelling ,whole-cell systems ,Prozessoptimierung ,biocatalysis ,process optimization ,Kaskaden ,cascades ,Modellierung ,Ganz-Zell-Systeme ,Biokatalyse - Abstract
Diese Dissertation behandelt Methoden, die f��r eine Optimierung biokatalytischer Transformationen n��tzlich sind. Optimierungen von biokatalytischen Prozessen sollten in verschiedenen Projektphasen durchgef��hrt werden: in der Phase der Katalysatorentwicklung, der Fermentations-/Kultivierungsentwicklung und der Bioprozess- sowie der Produktaufarbeitungsentwicklung. Verbesserungen in allen genannten Phasen tragen zur Produktivit��tssteigerung bei. Beispiele f��r solche Optimierungen sind in dieser Dissertation aufgef��hrt. Im ersten Kapitel wurde die Biotransformation in allen vier im letzten Abschnitt genannten Phasen optimiert. Ein biokatalytischer Prozess zur Synthese von Nylon-9, einem Spezialpolymer, wurde in der Gr��ssenordung von mehreren Dutzend Gramm etabliert. Die Nachfrage nach Spezialpolymeren wie Nylon-9 w��chst; jedoch ist die chemische Synthese der entsprechenden Monomere oft kompliziert. Diese Tatsache erleichtert die Etablierung biokatalytischer Syntheseverfahren. In dieser Arbeit wurde Cyclopendadecanone Monooxygenase (CPDMO) in Escherichia coli als Ganzzellkatalysator verwendet. Das Expressionssystem wurde angepasst, um stabile Expression des Enzyms zu garantieren (Phase der Katalysatorentwicklung). Die Expression wurde in einem definierten und optimierten Medium etabliert (Phase der Kultivierungsentwicklung). Die Implementierung des "Substrat-Feed-Product-Removal" (SFPR) Konzepts mit der entsprechenden Produktaufarbeitung rundete die Optimierung des Prozesses ab (Phase der Bioprozess-/Produktaufarbeitungsentwicklung). In dem Prozess wurde eine vorher beschriebene, gef��hrliche Pers��ure-katalysierte Oxidation durch einen sicheren und skalierbaren biokatalytischen Prozess mit Sauerstoff als Oxidant und Wasser als L��sungsmittel ersetzt. Der verbesserte Prozess setzte 42 g (0.28 mol) Keton zum entsprechenden Lakton um. Die isolierte Ausbeute nach einer sehr effizienten Produktaufarbeitung mit 95 % Wiederfindung der umgesetzten Stoffmenge betrug dabei 70 % (33 g), was einer volumetrischen Ausbeute von 8 g pures Lakton pro Liter Kulturvolumen entsprach. Im Folgenden wurde die M��glichkeit der Optimierung eines Ganzzellkatalysators untersucht (Phase der Katalysatorentwicklung). Ein kinetisches Modell f��r die Simulation und Optimierung einer enzymatischen in vivo Redox-Kaskade in Escherichia coli (E. coli) wurde erstellt, bestehend aus der Kombination einer Alkoholdehydrogenase (ADH), einer Enoatreduktase (ERED) und einer Baeyer-Villiger Monooxygenase (BVMO, speziell Cyclohexanone Monooxygenase (CHMO)). Die Kaskade diente zur Sythese von Laktonen. Das Modell wurde verwendet, um die aktiven intrazellul��ren Enzymkonzentrationen in den sequenziellen Biotransformationen zu bestimmen und um daraufhin den Engpass in der Laktonproduktion zu identifizieren sowie Optimierungsstrategien zu erstellen. Die in in vitro Experimenten mit isolierten Enzymen ermittelten Michaelis-Menten Parameter wurden verwendet, um die Verl��ufe der Intermediat- und Produktkonzentrationen in den in vivo Kaskadenreaktionen zu simulieren. Das Modell zeigte, dass das schnellste Enzym, die CHMO, der geschwindigkeitsbestimmende Schritt der Kaskade war. Der Grund daf��r war die geringe Konzentration der aktiven Form des Enzyms, die die Bildung von reversiblen Nebenreaktionen erm��glichte. Es wurde ein wesentlicher experimenteller Beweis erbracht, dass die geringen Konzentration an Flavin- und Nicotinamidkofaktoren der Grund f��r die drastisch verminderte Leistung der in vivo Kaskade verantworlich waren. Als N��chstes wurde das Verhalten von CHMO im Ganzzellsystem untersucht, um Gr��nde f��r die geringe Leistung in vivo zu finden sowie um Optimierungsstrategien zu erarbeiten (Phase der Katalysatorentwicklung). Die Messungen der in vivo Aktivit��t und Stabilit��t der CHMO erfolgten im E. coli Ganzzellsystem. Die CHMO wurde oft als in vitro instabil beschrieben. Vor Kurzem wurde postuliert, dass sich das Enzym in Abwesenheit von notwendigen Kofaktoren und Antioxidantien sehr schnell deaktiviert. Zudem wurde das Enzym im letzten Kapitel als der geschwindigkeitsbestimmende Schritt einer im Ganzzellsystem exprimierten enzymatischen Kaskade identifiziert. Jedoch wurde die in vivo Stabilit��t dieses Enzyms soweit kaum erforscht. Die Aktivit��t und Stabilit��t der CHMO wurde unter g��ngigen E. coli Expressionsbedingungen gemessen sowie die F��higkeit des Wirtsorganismus diese Eigenschaften der CHMO aufrechtzuerhalten mittels Metabolomics analysiert. Es zeigte sich, dass E. coli nicht in der Lage war, die notwendigen Kofaktorkonzentrationen bereitzustellen, die notwendig sind, um CHMO zu stabilisieren. Dennoch wurde die CHMO in ausreichenden Konzentrationen im E. coli produziert und die Konzentrationen waren nach der Produktion f��r den Zeitraum der Messungen bis zum Experimentende unver��ndert. Biotechnologische Anwendungen in diesem Wirtsorganismus mussten somit bisher mit der Restaktivit��t der CHMO von 5 % auskommen. Deswegen k��nnten andere Mikroorganismen effizientere Produktionsplattformen f��r die Produktion der CHMO und verwandter Enzyme bieten. Als letzter Optimierungsschritt in der Phasen der Katalysator- und Kultivierungsentwicklung wurde die in vivo Charakterisierung der Dihydroxyacetonkinase (DhaK), produziert in E. coli, pr��sentiert. Der Schwerpunkt lag dabei auf den physiologischen und metabolomischen Ver��nderungen im Expressionssystem im Vergleich mit dem E. coli Wildstamm. Die DhaK kann zur Synthese von Dihydroxyacetonphosphat (DHAP) aus Dihydroxyaceton (DHA) unter ATP Verbrauch eingesetzt werden und k��nnte somit zur Erleichterung von Aldolreaktionen in vivo dienen, da DHAP f��r viele Aldolasen ein Donormolek��l darstellt. Trotz der metabolischen Last der Enzymsynthese und Plasmiderhaltung, zeigte der DhaK Stamm ��hnliche physiologische Charakteristika wie der Wildtyp. Im Gegensatz zum Wildtyp, zeigte die DhaK Mutante Aufnahme von DHA sowie erh��hte intrazellul��re DHAP Konzentrationen w��hrend des exponentiellen Wachstums. Die Metabolomanalyse zeigte, dass der ATP Pool, der f��r die enzymatische Phosphorylierung von DHA ben��tigt wird, w��hrend der nicht-wachsenden Bedingungen zu gering war und diese somit nur unter wachsenden Bedingungen stattfinden konnte. Die Modellierung beider St��mme zeigte die M��glichkeit eines Bypasses der oberen Glykolyse durch die Expression der DhaK und die Zugabe von DHA. Diese Ans��tze bed��rfen weiterer Untersuchungen, vor Allem mit 13CMetabolischer Flussanalyse. Abschliessend wurde das vorgestellte System mit einer Aldolproduzierenden enzymatischen Kaskade in E. coli modelliert und in silico als f��rderlich f��r DHAP-abh��ngige Aldolreaktionen befunden. Die Optimierungen der verschiedenen Phasen einer biokatalytischen Transformation wurden in dieser Arbeit kombiniert angewendet und verschiedene Methoden wurden unterst��tzend herangezogen - Modellierung, Metabolomanalyse und Optimierungen im Reaktionsdesign. Diese wurden erfolgreich angewendet, um Biotransformationen in lebenden Ganzzellsystemen zu erm��glichen., This thesis deals with tools for optimization of biocatalytical transformations. The optimization of biocatalysis should be performed on different levels: on the catalyst level, on the fermentation/cultivation level, and on the process level as well as the downstream processing (DSP) level. All levels contribute to the enhancement of productivity and examples for their optimization are given in the following theses. In the first chapter, the optimization was performed on all the levels mentioned above. The development of a biocatalytic process on the multi-dozen gram scale for the synthesis of a precursor to Nylon-9, a specialty polyamide, was established. Such materials are growing in demand, but their corresponding monomers are often difficult to synthesize, giving rise to biocatalytic approaches. Here,the cyclopentadecanone monooxygenase as an Escherichia coli (E. coli) whole-cell biocatalyst was chosen as a catalytic entity and produced in a stable expressing system (biocatalyst level) in a defined medium, which was optimized prior to upscaling (cultivation level). Together with the implementation of a substrate feeding-product removal concept, an DSP was established (process/DSP level). A previously described hazardous peracid-mediated oxidation was thus replaced with a safe and scalable protocol, using aerial oxygen as oxidant and water as reaction solvent. The engineered process converted 42 g (0.28 mol) starting material ketone to the corresponding lactone with an isolated yield of 70 % (33 g), after highly efficient DSP with 95 % recovery of the converted material, translating to a volumetric yield of 8 g pure product per liter. Subsequently, the possibility of optimization on whole-cell biocatalyst level was investigated. A kinetic model for the simulation and optimization of an in vivo redox cascade in E. coli, using a combination of an alcohol dehydrogenase, an enoate reductase, and a Baeyer-Villiger monooxygenase (CHMO) for the synthesis of lactones was developed. The model was used to estimate the concentrations of active enzyme in the sequential biotransformations to identify bottlenecks together with their reasons and how to overcome them. The adapted Michaelis-Menten parameters from in vitro experiments with isolated enzymes were estimated, and these values were used to simulate the change in concentrations of intermediates and products during the in vivo cascade reactions. The model indicated the CHMO, the fastest enzyme, to be rate-determining due to the low concentration of the active form, opening up reversible reaction channels towards side products. Substantial experimental evidence was provided, that a low intracellular concentration of flavin and nicotinamide cofactors drastically throttled the performance of the in vivo cascade. As a next step, the performance of the CHMO in a whole-cell catalyst was investigated to obtain knowledge about its poor performance in vivo and to propose optimization strategies (catalyst level). The measurement of in vivo activity and stability of the CHMO in the recombinant host E. coli was performed. This enzyme was often described as poorly stable in vitro, and has recently been found to deactivate rapidly in the absence of its essential cofactors and of anti-oxidants. Additionally, it was identified as the rate limiting step of an enzymatic cascade in the previous chapter. Its stability in vivo was scarcely studied, so far. The activity and stability of CHMO in E. coli during common conditions for over-expression was measured, and the ability of the host to support these properties by metabolomics analyses was investigated. The results showed that E. coli failed to provide the intracellular levels of cofactors required to functionally stabilize CHMO, although the biocatalyst was produced in high concentration, and was invariably detectable after protein synthesis had stopped. Biotechnological applications in this host possibly relied on a residual activity of approx. 5 %. Other microorganisms might thus offer a more efficient solution for recombinant production of CHMO, and related enzymes. As the last optimization step on the catalyst and cultivation level, an in vivo characterization of dihydroxyacetone kinase (DhaK) expressed in E. coli was presented with regard to physiological and metabolical changes compared to E. coli wt strain. This enzyme can facilitate aldol reactions in vivo by increasing intracellular dihydroxyacetone phosphate (DHAP, aldol donor molecule) concentrations. The DhaK phosphorylates dihydroxyacetone (DHA) to DHAP by consuming ATP. Overall, despite the metabolic burden of plasmid maintenance and protein expression, the DhaK strain showed similar physiological behavior compared to the wt strain. In contrast to E. coli wt, the DhaK strain took up DHA during the exponential growth phase and showed higher intracellular concentrations of DHAP. Metabolomics analysis also indicated that the pool of ATP, which is needed for the enzymatic phosphorylation of DHA, was too low under non-growing conditions, so that the reaction could only proceed during exponential growth. Modeling both strains revealed the possibility of bypassing the upper glycolysis by expressing DhaK and feeding DHA, which needs to be investigated further with carbon source 13C labeling by dynamic metabolomics approaches. In summary, the presented system was modeled together with an aldol producing enzymatic cascade in E. coli and was found to be of benefit for DHAP-dependent aldol reactions in silico. The combination of the different levels of optimization applied in this theses - accompanied by the methods modeling, metabolomics and optimizational tools for reaction design - were successfully applied to facilitate biotransformations in living systems.
- Published
- 2017
- Full Text
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7. Cell Factory Design and Optimization for the Stereoselective Synthesis of Polyhydroxylated Compounds.
- Author
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Wiesinger, Thomas, Bayer, Thomas, Milker, Sofia, Mihovilovic, Marko D., and Rudroff, Florian
- Published
- 2018
- Full Text
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8. Escherichia coli Fails to Efficiently Maintain the Activity of an Important Flavin Monooxygenase in Recombinant Overexpression.
- Author
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Milker, Sofia, Goncalves, Leticia C. P., Fink, Michael J., and Rudroff, Florian
- Subjects
ESCHERICHIA coli ,MONOOXYGENASES ,GENETIC overexpression - Abstract
This paper describes the measurement and analysis of in vivo activity and stability of cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMO), a model Baeyer-Villiger monooxygenase, in the recombinant host Escherichia coli. This enzyme was often described as poorly stable in vitro, and has recently been found to deactivate rapidly in the absence of its essential cofactors and antioxidants. Its stability in vivo was scarcely studied, so far. Under conditions common for the overexpression of CHMO we investigated the ability of the host to support these properties using metabolomics. Our results showed that E. coli failed to provide the intracellular levels of cofactors required to functionally stabilize the enzyme, although the biocatalyst was produced in high concentration, and was invariably detected after protein synthesis had stopped. We thus infer that biotechnological applications of CHMO with this host relied on a residual activity of approximately 5-10%. Other microorganisms might offer a more efficient solution for recombinant production of CHMO and related enzymes. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
9. Kinetic Modeling of an Enzymatic Redox Cascade In Vivo Reveals Bottlenecks Caused by Cofactors.
- Author
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Milker, Sofia, Fink, Michael J., Oberleitner, Nikolin, Ressmann, Anna K., Bornscheuer, Uwe T., Mihovilovic, Marko D., and Rudroff, Florian
- Subjects
- *
OXIDATION-reduction reaction kinetics , *BOTTLENECKS (Manufacturing) , *COFACTORS (Biochemistry) , *SIMULATION methods & models , *ALCOHOL dehydrogenase - Abstract
We describe the development of a kinetic model for the simulation and optimization of an in vivo redox cascade in E. coli in which a combination of an alcohol dehydrogenase, an enoate reductase, and a Baeyer-Villiger monooxygenase is used for the synthesis of lactones. The model was used to estimate the concentrations of active enzyme in the sequential biotransformations to identify bottlenecks together with their reasons and how to overcome them. We estimated adapted Michaelis-Menten parameters from in vitro experiments with isolated enzymes and used these values to simulate the change in the concentrations of intermediates and products during the in vivo cascade reactions. Remarkably, the model indicated that the fastest enzyme was rate-determining because of the unexpectedly low concentration of the active form, which opens up reversible reaction channels towards byproducts. We also provide substantial experimental evidence that a low intracellular concentration of flavin and nicotinamide cofactors drastically decreased the performance of the in vivo cascade drastically. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
10. In Vivo Synthesis of Polyhydroxylated Compounds from a 'Hidden Reservoir' of Toxic Aldehyde Species.
- Author
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Bayer, Thomas, Milker, Sofia, Wiesinger, Thomas, Winkler, Margit, Mihovilovic, Marko D., and Rudroff, Florian
- Subjects
- *
ALDEHYDES , *POLYPHENOLS , *ESCHERICHIA coli , *CARBOXYLIC acids , *PSEUDOMONAS putida - Abstract
Synthetic enzyme cascades in living cells often lack efficiency owing to the formation of byproducts by endogenous enzymes or toxicity of the cascade intermediates. Highly reactive aldehyde species can trigger a metabolic stress response, and this leads to undesired side reactions and decreased yields. Owing to the metabolic background of Escherichia coli ( E. coli), aldehydes may be irreversibly oxidized to carboxylic acids or reduced to the corresponding alcohols. Herein, we applied an approach to equilibrate the aldehyde concentration in vivo. We oxidized primary alcohols to the corresponding aldehydes by AlkJ, an alcohol dehydrogenase from Pseudomonas putida. Introduction of a carboxylic acid reductase from Nocardia iowensis allowed the target compound to be retrieved from the carboxylate sink. Further reduction of the aldehydes to alcohols by endogenous E. coli enzymes completed the equilibration between alcohols, aldehydes, and carboxylic acids. Thus, the aldehyde concentrations remained below nonviable concentrations. We demonstrated the concept on several primary alcohols, which reached the redox equilibrium within 6 h and persisted up to 24 h. Subsequent combination with a dihydroxyacetone-dependent aldolase (Fsa1-A129S, E. coli) demonstrated that the reactive aldehyde species were freely available and gave the aldol product, (3 S,4 R)-1,3,4-trihydroxy-5-phenylpentan-2-one, in 70 % yield within short reaction times. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
11. Non-hazardous biocatalytic oxidation in Nylon-9 monomer synthesis on a 40 g scale with efficient downstream processing.
- Author
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Milker, Sofia, Fink, Michael J., Rudroff, Florian, and Mihovilovic, Marko D.
- Abstract
ABSTRACT This paper describes the development of a biocatalytic process on the multi-dozen gram scale for the synthesis of a precursor to Nylon-9, a specialty polyamide. Such materials are growing in demand, but their corresponding monomers are often difficult to synthesize, giving rise to biocatalytic approaches. Here, we implemented cyclopentadecanone monooxygenase as an Escherichia coli whole-cell biocatalyst in a defined medium, together with a substrate feeding-product removal concept, and an optimized downstream processing (DSP). A previously described hazardous peracid-mediated oxidation was thus replaced with a safe and scalable protocol, using aerial oxygen as oxidant, and water as reaction solvent. The engineered process converted 42 g (0.28 mol) starting material ketone to the corresponding lactone with an isolated yield of 70% (33 g), after highly efficient DSP with 95% recovery of the converted material, translating to a volumetric yield of 8 g pure product per liter. Biotechnol. Bioeng. 2017;114: 1670-1678. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
12. Mutagenesis-Independent Stabilization of Class B Flavin Monooxygenases in Operation.
- Author
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Goncalves, Leticia C. P., Kracher, Daniel, Milker, Sofia, Fink, Michael J., Rudroff, Florian, Ludwig, Roland, Bommarius, Andreas S., and Mihovilovic, Marko D.
- Subjects
FLAVINS ,MUTAGENESIS ,MONOOXYGENASES ,PHARMACEUTICAL chemistry ,BIOTECHNOLOGY - Abstract
This paper describes the stabilization of flavin-dependent monooxygenases under reaction conditions, using an engineered formulation of additives (the natural cofactors NADPH and FAD, and superoxide dismutase and catalase as catalytic antioxidants). This way, a 10
3 - to 104 -fold increase of the half-life was reached without resource-intensive directed evolution or structure-dependent protein engineering methods. The stabilized enzymes are highly valued for their synthetic potential in biotechnology and medicinal chemistry (enantioselective sulfur, nitrogen and Baeyer-Villiger oxidations; oxidative human metabolism), but widespread application was so far hindered by their notorious fragility. Our technology immediately enables their use, does not require structural knowledge of the biocatalyst, and creates a strong basis for the targeted development of improved variants by mutagenesis. [ABSTRACT FROM AUTHOR]- Published
- 2017
- Full Text
- View/download PDF
13. Designer Microorganisms for Optimized Redox Cascade Reactions - Challenges and Future Perspectives.
- Author
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Bayer, Thomas, Milker, Sofia, Wiesinger, Thomas, Rudroff, Florian, and Mihovilovic, Marko. D.
- Subjects
- *
MICROORGANISMS , *OXIDATION-reduction reaction , *CHEMICAL reactions , *BIOCATALYSIS , *ESCHERICHIA coli - Abstract
An immense number of chemical reactions are carried out simultaneously in living cells. Nature's optimization approach encompasses the assembly of reactions in cascades and to embed them in finely tuned metabolic networks. With the vast progress in the field of biocatalysis, man-made cascades, especially redox cascades, have reached a degree of complexity that needs tools for improved control and optimization. Combined strategies from biocatalysis, metabolic engineering and synthetic biology lead to the establishment of artificial metabolic pathways with minimized interference with the cellular host environment. This review will focus on genetic and metabolic engineering tools for the assembly and introduction of de novo redox pathways into the host Escherichia coli and will present state of the art redox cascades performed by tailor-made microbial cell factories. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
14. Front Cover: Cell Factory Design and Optimization for the Stereoselective Synthesis of Polyhydroxylated Compounds (ChemBioChem 4/2018).
- Author
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Wiesinger, Thomas, Bayer, Thomas, Milker, Sofia, Mihovilovic, Marko D., and Rudroff, Florian
- Published
- 2018
- Full Text
- View/download PDF
15. Back Cover: In Vivo Synthesis of Polyhydroxylated Compounds from a 'Hidden Reservoir' of Toxic Aldehyde Species (ChemCatChem 15/2017).
- Author
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Bayer, Thomas, Milker, Sofia, Wiesinger, Thomas, Winkler, Margit, Mihovilovic, Marko D., and Rudroff, Florian
- Subjects
- *
POLYPHENOLS , *CHEMICAL synthesis - Abstract
The Cover shows a biochemical cell factory (Escherichia coli) for the synthesis of chiral polyhydroxylated compounds via an artificial enzyme cascade.In their Communication, T. Bayer et al. presented a rational approach for tuning the concentration of toxic aldehyde intermediates in living cells. By combining an alcohol dehydrogenase and a carboxylic acid reductase, the redox equilibrium between alcohol, aldehyde and carboxylic acid species can be adjusted, thus maintaining the aldehyde concentration in a ‘hidden reservoir’ and therefore below the toxicity level, yet freely available for subsequent aldolase mediated reaction. More information can be found in the Communication by T. Bayer et al. on page 2919 in Issue 15, 2017 (DOI: 10.1002/cctc.201700469). [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
16. ChemInform Abstract: Designer Microorganisms for Optimized Redox Cascade Reactions - Challenges and Future Perspectives.
- Author
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Bayer, Thomas, Milker, Sofia, Wiesinger, Thomas, Rudroff, Florian, and Mihovilovic, Marko D.
- Subjects
- *
OXIDATION-reduction reaction , *MICROORGANISMS - Abstract
Review: [217 refs. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
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