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PUBLICATIONS
2024
Runda, Michael E.; Schmidt, Sandy
Light-driven bioprocesses Book Chapter
In: Holtmann, Dirk (Ed.): Bioprocess Intensification, Chapter 8, pp. 193–226, De Gruyter, Berlin, Boston, 2024, ISBN: 9783110760330.
@inbook{RundaSchmidt+2024+193+226,
title = {Light-driven bioprocesses},
author = {Michael E. Runda and Sandy Schmidt},
editor = {Dirk Holtmann},
url = {https://doi.org/10.1515/9783110760330-008},
doi = {doi:10.1515/9783110760330-008},
isbn = {9783110760330},
year = {2024},
date = {2024-07-01},
urldate = {2024-07-01},
booktitle = {Bioprocess Intensification},
pages = {193–226},
publisher = {De Gruyter},
address = {Berlin, Boston},
chapter = {8},
abstract = {Enzyme catalysis and photocatalysis are two research areas that have become of major interest in organic synthesis. This is mainly because both represent attractive strategies for making chemical synthesis more efficient and sustainable. Because enzyme catalysis offers several inherent advantages, such as high substrate specificity, regio-, and stereoselectivity, and activity under environmentally benign reaction conditions, biocatalysts are increasingly being adopted by the pharmaceutical and chemical industries. In addition, photocatalysis has proven to be a powerful approach for accessing unique reactivities upon light irradiation and performing reactions with an extended substrate range under milder conditions compared to light-independent alternatives. It is therefore not surprising that bio-and photocatalytic approaches are now often combined to exploit the exquisite selectivity of enzymes and the unique chemical transformations accessible to photocatalysis. In this chapter, we provide an overview of the wide variety of lightdriven bioprocesses, ranging from photochemical delivery of reducing equivalents to redox enzymes, photochemical cofactor regeneration, to direct photoactivation of enzymes. We also highlight the possibility of catalyzing non-natural reactions via photoinduced enzyme promiscuity and the combination of photo-and biocatalytic reactions used to create new synthetic methodologies.},
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pubstate = {published},
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}
Enzyme catalysis and photocatalysis are two research areas that have become of major interest in organic synthesis. This is mainly because both represent attractive strategies for making chemical synthesis more efficient and sustainable. Because enzyme catalysis offers several inherent advantages, such as high substrate specificity, regio-, and stereoselectivity, and activity under environmentally benign reaction conditions, biocatalysts are increasingly being adopted by the pharmaceutical and chemical industries. In addition, photocatalysis has proven to be a powerful approach for accessing unique reactivities upon light irradiation and performing reactions with an extended substrate range under milder conditions compared to light-independent alternatives. It is therefore not surprising that bio-and photocatalytic approaches are now often combined to exploit the exquisite selectivity of enzymes and the unique chemical transformations accessible to photocatalysis. In this chapter, we provide an overview of the wide variety of lightdriven bioprocesses, ranging from photochemical delivery of reducing equivalents to redox enzymes, photochemical cofactor regeneration, to direct photoactivation of enzymes. We also highlight the possibility of catalyzing non-natural reactions via photoinduced enzyme promiscuity and the combination of photo-and biocatalytic reactions used to create new synthetic methodologies.
Kok, Niels A. W.; Miao, Hui; Schmidt, Sandy
In vitro analysis of the three-component Rieske oxygenase cumene dioxygenase from Pseudomonas fluorescens IP01 Book Section
In: Academic Press, 2024, ISSN: 0076-6879.
@incollection{DEKOK2024,
title = {In vitro analysis of the three-component Rieske oxygenase cumene dioxygenase from Pseudomonas fluorescens IP01},
author = {Niels A. W. Kok and Hui Miao and Sandy Schmidt},
url = {https://www.sciencedirect.com/science/article/pii/S0076687924002246},
doi = {https://doi.org/10.1016/bs.mie.2024.05.013},
issn = {0076-6879},
year = {2024},
date = {2024-06-12},
urldate = {2024-01-01},
publisher = {Academic Press},
series = {Methods in Enzymology},
abstract = {Rieske non-heme iron-dependent oxygenases (ROs) are a versatile group of enzymes traditionally associated with the degradation of aromatic xenobiotics. In addition, ROs have been found to play key roles in natural product biosynthesis, displaying a wide catalytic diversity with typically high regio- and stereo- selectivity. However, the detailed characterization of ROs presents formidable challenges due to their complex structural and functional properties, including their multi-component composition, cofactor dependence, and susceptibility to reactive oxygen species. In addition, the substrate availability of natural product biosynthetic intermediates, the limited solubility of aromatic hydrocarbons, and the radical-mediated reaction mechanism can further complicate functional assays. Despite these challenges, ROs hold immense potential as biocatalysts for pharmaceutical applications and bioremediation. Using cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 as a model enzyme, this chapter details techniques for characterizing ROs that oxyfunctionalize aromatic hydrocarbons. Moreover, potential pitfalls, anticipated complications, and proposed solutions for the characterization of novel ROs are described, providing a framework for future RO research and strategies for studying this enzyme class. In particular, we describe the methods used to obtain CDO, from construct design to expression conditions, followed by a purification procedure, and ultimately activity determination through various activity assays.},
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}
Rieske non-heme iron-dependent oxygenases (ROs) are a versatile group of enzymes traditionally associated with the degradation of aromatic xenobiotics. In addition, ROs have been found to play key roles in natural product biosynthesis, displaying a wide catalytic diversity with typically high regio- and stereo- selectivity. However, the detailed characterization of ROs presents formidable challenges due to their complex structural and functional properties, including their multi-component composition, cofactor dependence, and susceptibility to reactive oxygen species. In addition, the substrate availability of natural product biosynthetic intermediates, the limited solubility of aromatic hydrocarbons, and the radical-mediated reaction mechanism can further complicate functional assays. Despite these challenges, ROs hold immense potential as biocatalysts for pharmaceutical applications and bioremediation. Using cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 as a model enzyme, this chapter details techniques for characterizing ROs that oxyfunctionalize aromatic hydrocarbons. Moreover, potential pitfalls, anticipated complications, and proposed solutions for the characterization of novel ROs are described, providing a framework for future RO research and strategies for studying this enzyme class. In particular, we describe the methods used to obtain CDO, from construct design to expression conditions, followed by a purification procedure, and ultimately activity determination through various activity assays.
Runda, Michael E.; Miao, Hui; Schmidt, Sandy
Protein fusion strategies for a multi-component Rieske oxygenase Journal Article
In: bioRxiv, vol. 2024.06.09.598105, 2024.
@article{Runda2024.06.09.598105,
title = {Protein fusion strategies for a multi-component Rieske oxygenase},
author = {Michael E. Runda and Hui Miao and Sandy Schmidt},
url = {https://www.biorxiv.org/content/early/2024/06/09/2024.06.09.598105},
doi = {10.1101/2024.06.09.598105},
year = {2024},
date = {2024-06-09},
urldate = {2024-06-09},
journal = {bioRxiv},
volume = {2024.06.09.598105},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Rieske oxygenases (ROs) are enzyme systems involved in microbial biodegradation or late-stage modifications during natural product biosynthesis. A major obstacle to working with ROs is their dependence on multi-component electron transfer chains (ETCs). Thereby, electrons from NAD(P)H are shuttled directly via a reductase (Red) or indirectly via an additional ferredoxin (Fd) to a terminal oxygenase (Oxy) for oxygen activation and subsequent substrate conversion. The present work evaluates potential fusion strategies to simplify the ETC of the three-component cumene dioxygenase (CDO) from Pseudomonas fluorescence. In in vitro reactions, the fusion of CDO-Red to CDO-Fd is the most suitable for activation of CDO-Oxy with product formation of approximately 22 mM (72 % conversion). Furthermore, protein fusion to CDO-Oxy was found to be feasible, highlighting the versatility of the redox partner fusion approach. Overall, this study aims to contribute to the research field of ROs by providing a promising strategy to simplify their multi-component nature.Competing Interest StatementThe authors have declared no competing interest.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Rieske oxygenases (ROs) are enzyme systems involved in microbial biodegradation or late-stage modifications during natural product biosynthesis. A major obstacle to working with ROs is their dependence on multi-component electron transfer chains (ETCs). Thereby, electrons from NAD(P)H are shuttled directly via a reductase (Red) or indirectly via an additional ferredoxin (Fd) to a terminal oxygenase (Oxy) for oxygen activation and subsequent substrate conversion. The present work evaluates potential fusion strategies to simplify the ETC of the three-component cumene dioxygenase (CDO) from Pseudomonas fluorescence. In in vitro reactions, the fusion of CDO-Red to CDO-Fd is the most suitable for activation of CDO-Oxy with product formation of approximately 22 mM (72 % conversion). Furthermore, protein fusion to CDO-Oxy was found to be feasible, highlighting the versatility of the redox partner fusion approach. Overall, this study aims to contribute to the research field of ROs by providing a promising strategy to simplify their multi-component nature.Competing Interest StatementThe authors have declared no competing interest.
Vajente, Matteo; Clerici, Riccardo; Ballerstedt, Hendrik; Blank, Lars M; Schmidt, Sandy
Using Cupriavidus necator H16 to provide a roadmap for increasing electroporation efficiency in non-model bacteria Journal Article
In: bioRxiv, vol. 2024.05.27.596136, 2024.
@article{Vajente2024.05.27.596136,
title = {Using Cupriavidus necator H16 to provide a roadmap for increasing electroporation efficiency in non-model bacteria},
author = {Matteo Vajente and Riccardo Clerici and Hendrik Ballerstedt and Lars M Blank and Sandy Schmidt},
url = {https://www.biorxiv.org/content/early/2024/05/29/2024.05.27.596136},
doi = {10.1101/2024.05.27.596136},
year = {2024},
date = {2024-05-29},
urldate = {2024-01-01},
journal = {bioRxiv},
volume = {2024.05.27.596136},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Bacteria are a treasure trove of metabolic reactions, but most industrial biotechnology applications rely on a limited set of established host organisms. In contrast, adopting non-model bacteria for the production of various chemicals of interest is often hampered by their limited genetic amenability coupled with their low transformation efficiency. In this study, we propose a series of steps that can be taken to increase electroporation efficiency in non-model bacteria. As a test strain, we use Cupriavidus necator H16, a lithoautotrophic bacterium that has been engineered to produce a wide range of products from CO2 and hydrogen. However, its low electroporation efficiency hinders the high-throughput genetic modifications required to develop C. necator into an industrially relevant host organism. First, we propose a species-independent technique based on natively methylated DNA and Golden Gate assembly to increase one-pot cloning and electroporation efficiency by 70-fold. Second, bioinformatic tools were used to predict defense systems and develop a restriction avoidance strategy that was used to introduce suicide plasmids by electroporation to obtain a domesticated strain. The results are discussed in the context of metabolic engineering of non-model bacteria. Competing Interest StatementThe authors have declared no competing interest.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Bacteria are a treasure trove of metabolic reactions, but most industrial biotechnology applications rely on a limited set of established host organisms. In contrast, adopting non-model bacteria for the production of various chemicals of interest is often hampered by their limited genetic amenability coupled with their low transformation efficiency. In this study, we propose a series of steps that can be taken to increase electroporation efficiency in non-model bacteria. As a test strain, we use Cupriavidus necator H16, a lithoautotrophic bacterium that has been engineered to produce a wide range of products from CO2 and hydrogen. However, its low electroporation efficiency hinders the high-throughput genetic modifications required to develop C. necator into an industrially relevant host organism. First, we propose a species-independent technique based on natively methylated DNA and Golden Gate assembly to increase one-pot cloning and electroporation efficiency by 70-fold. Second, bioinformatic tools were used to predict defense systems and develop a restriction avoidance strategy that was used to introduce suicide plasmids by electroporation to obtain a domesticated strain. The results are discussed in the context of metabolic engineering of non-model bacteria. Competing Interest StatementThe authors have declared no competing interest.
Runda, Michael E.; Miao, Hui; de Kok, Niels A. W.; Schmidt, Sandy
Developing Hybrid Systems to Address O2 Uncoupling in Multi-Component Rieske Oxygenases Journal Article
In: Journal of Biotechnology, vol. 389, pp. 22–29, 2024, ISSN: 0168-1656.
@article{Runda2024.02.16.580709b,
title = {Developing Hybrid Systems to Address O_{2} Uncoupling in Multi-Component Rieske Oxygenases},
author = {Michael E. Runda and Hui Miao and Niels A. W. de Kok and Sandy Schmidt},
url = {https://www.sciencedirect.com/science/article/pii/S0168165624001275},
doi = {doi.org/10.1016/j.jbiotec.2024.04.019},
issn = {0168-1656},
year = {2024},
date = {2024-04-30},
urldate = {2024-04-30},
journal = {Journal of Biotechnology},
volume = {389},
pages = {22–29},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Rieske non-heme iron oxygenases (ROs) are redox enzymes essential for microbial biodegradation and natural product synthesis. These enzymes utilize molecular oxygen for oxygenation reactions, making them very useful biocatalysts due to their broad reaction scope and high selectivities. The mechanism of oxygen activation in ROs involves electron transfers between redox centers of associated protein components, forming an electron transfer chain (ETC). Although the ETC is essential for electron replenishment, it carries the risk of reactive oxygen species (ROS) formation due to electron loss during oxygen activation. Our previous study linked ROS formation to O2 uncoupling in the flavin-dependent reductase of the three-component cumene dioxygenase (CDO). In the present study, we extend this finding by investigating the effects of ROS formation on the multi-component CDO system in a cell-free environment. In particular, we focus on the effects of hydrogen peroxide (H2O2) formation in the presence of a NADH cofactor regeneration system on the catalytic efficiency of CDO in vitro. Based on this, we propose the implementation of hybrid systems with alternative (non-native) redox partners for CDO, which are highly advantageous in terms of reduced H2O2 formation and increased product formation. The hybrid system consisting of the RO-reductase from phthalate dioxygenase (PDR) and CDO proved to be the most promising for the oxyfunctionalization of indene, showing a 4-fold increase in product formation (20 mM) over 24 h (TTN of 1515) at a 3-fold increase in production rate.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Rieske non-heme iron oxygenases (ROs) are redox enzymes essential for microbial biodegradation and natural product synthesis. These enzymes utilize molecular oxygen for oxygenation reactions, making them very useful biocatalysts due to their broad reaction scope and high selectivities. The mechanism of oxygen activation in ROs involves electron transfers between redox centers of associated protein components, forming an electron transfer chain (ETC). Although the ETC is essential for electron replenishment, it carries the risk of reactive oxygen species (ROS) formation due to electron loss during oxygen activation. Our previous study linked ROS formation to O2 uncoupling in the flavin-dependent reductase of the three-component cumene dioxygenase (CDO). In the present study, we extend this finding by investigating the effects of ROS formation on the multi-component CDO system in a cell-free environment. In particular, we focus on the effects of hydrogen peroxide (H2O2) formation in the presence of a NADH cofactor regeneration system on the catalytic efficiency of CDO in vitro. Based on this, we propose the implementation of hybrid systems with alternative (non-native) redox partners for CDO, which are highly advantageous in terms of reduced H2O2 formation and increased product formation. The hybrid system consisting of the RO-reductase from phthalate dioxygenase (PDR) and CDO proved to be the most promising for the oxyfunctionalization of indene, showing a 4-fold increase in product formation (20 mM) over 24 h (TTN of 1515) at a 3-fold increase in production rate.
Terholsen, Henrik; Schmidt, Sandy
Cell-free chemoenzymatic cascades with bio-based molecules Journal Article
In: Current Opinion in Biotechnology, vol. 85, pp. 103058, 2024, ISSN: 0958-1669.
@article{Terholsen2024,
title = {Cell-free chemoenzymatic cascades with bio-based molecules},
author = {Henrik Terholsen and Sandy Schmidt},
url = {https://doi.org/10.1016/j.copbio.2023.103058},
doi = {10.1016/j.copbio.2023.103058},
issn = {0958-1669},
year = {2024},
date = {2024-02-01},
urldate = {2023-12-27},
journal = {Current Opinion in Biotechnology},
volume = {85},
pages = {103058},
publisher = {Elsevier},
abstract = {For the valorization of various bio-based feedstocks, the combination of different catalytic systems with biocatalysis in chemoenzymatic cascades has been shown to have high potential. However, the development of such integrated catalytic systems is often limited by catalyst incompatibility. Therefore, incorporating novel catalytic concepts into the chemoenzymatic valorization of bio-based feedstocks is currently of great interest. This article provides an overview of the methods/approaches used to advance the development of chemoenzymatic cascades for the catalytic upgrading of bio-based feedstocks. It specifically focuses on recent developments in the combination of enzymes with organo- and chemocatalysis. Furthermore, current applications and future perspectives of integrating novel catalytic systems such as photo- and electrocatalysis toward new synthetic routes for the utilization of the often highly functionalized bio-based compounds are reviewed.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
For the valorization of various bio-based feedstocks, the combination of different catalytic systems with biocatalysis in chemoenzymatic cascades has been shown to have high potential. However, the development of such integrated catalytic systems is often limited by catalyst incompatibility. Therefore, incorporating novel catalytic concepts into the chemoenzymatic valorization of bio-based feedstocks is currently of great interest. This article provides an overview of the methods/approaches used to advance the development of chemoenzymatic cascades for the catalytic upgrading of bio-based feedstocks. It specifically focuses on recent developments in the combination of enzymes with organo- and chemocatalysis. Furthermore, current applications and future perspectives of integrating novel catalytic systems such as photo- and electrocatalysis toward new synthetic routes for the utilization of the often highly functionalized bio-based compounds are reviewed.
Schröder, Simon; Maier, Artur; Schmidt, Sandy; Mügge, Carolin; Tischler, Dirk
Enhancing biocatalytical N-N bond formation with the actinobacterial piperazate synthase KtzT Journal Article
In: Molecular Catalysis, vol. 553, no. 113733, pp. 11, 2024, ISSN: 2468-8231.
@article{Maier2024,
title = {Enhancing biocatalytical N-N bond formation with the actinobacterial piperazate synthase KtzT},
author = {Simon Schröder and Artur Maier and Sandy Schmidt and Carolin Mügge and Dirk Tischler},
url = {https://www.sciencedirect.com/science/article/pii/S2468823123008155},
doi = {https://doi.org/10.1016/j.mcat.2023.113733},
issn = {2468-8231},
year = {2024},
date = {2024-01-15},
urldate = {2024-01-15},
journal = {Molecular Catalysis},
volume = {553},
number = {113733},
pages = {11},
abstract = {Natural compounds with nitrogen-nitrogen bonds are diverse and have applications in medicine and agriculture. l-Piperazic acid (Piz), an α-hydrazino acid, is one of few naturally occurring compounds of its kind. Yet, Piz and its derivatives are valuable building blocks for bioactive compounds. Few NNzymes, enzymes capable of forming NN bonds, have been identified thus far. The hemoenzyme KtzT from Kutzneria sp. 744 catalyzes the intramolecular NN bond formation of N5‑hydroxy-l-ornithine (OH-Orn) to form Piz, a natural building block of kutznerides. The latter has antifungal and antibiotic properties. In our study, we established an improved expression method, with significantly improved yields (ca. 35-fold) of heme-loaded enzyme, making the enzyme much more accessible for laboratory studies. In vitro biochemical characterization under conditions for NN bond formation indicated a considerable thermo- and pH-flexibility, with optimal reaction conditions at 30 °C and 10 mM Tris buffer at pH 9 together with low salinity, paving the way for more complex applications involving KtzT. We have also identified two homologous enzymes from extremophilic organisms to exhibit piperazate-forming activity. In silico structural studies, combined with phylogenetic analysis, resulted in a heme- and substrate-binding model, suggesting target enzyme residues that we propose are critical for the structural integrity and catalytic activity of KtzT. Following this approach, we investigated the potential role of a cysteine residue in a dimer-stabilizing disulfide bridge. The interplay of in vitro and in silico data therefore provides crucial functional information on this enzyme class.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Natural compounds with nitrogen-nitrogen bonds are diverse and have applications in medicine and agriculture. l-Piperazic acid (Piz), an α-hydrazino acid, is one of few naturally occurring compounds of its kind. Yet, Piz and its derivatives are valuable building blocks for bioactive compounds. Few NNzymes, enzymes capable of forming NN bonds, have been identified thus far. The hemoenzyme KtzT from Kutzneria sp. 744 catalyzes the intramolecular NN bond formation of N5‑hydroxy-l-ornithine (OH-Orn) to form Piz, a natural building block of kutznerides. The latter has antifungal and antibiotic properties. In our study, we established an improved expression method, with significantly improved yields (ca. 35-fold) of heme-loaded enzyme, making the enzyme much more accessible for laboratory studies. In vitro biochemical characterization under conditions for NN bond formation indicated a considerable thermo- and pH-flexibility, with optimal reaction conditions at 30 °C and 10 mM Tris buffer at pH 9 together with low salinity, paving the way for more complex applications involving KtzT. We have also identified two homologous enzymes from extremophilic organisms to exhibit piperazate-forming activity. In silico structural studies, combined with phylogenetic analysis, resulted in a heme- and substrate-binding model, suggesting target enzyme residues that we propose are critical for the structural integrity and catalytic activity of KtzT. Following this approach, we investigated the potential role of a cysteine residue in a dimer-stabilizing disulfide bridge. The interplay of in vitro and in silico data therefore provides crucial functional information on this enzyme class.
2023
Grandi, Eleonora; Özgen, Fatma Feyza; Schmidt, Sandy; Poelarends, Gerrit J
Enzymatic Oxy- and Amino-Functionalization in Biocatalytic Cascade Synthesis: Recent Advances and Future Perspectives Journal Article
In: Angewandte Chemie International Edition, vol. n/a, no. n/a, pp. e202309012, 2023, ISSN: 1433-7851.
@article{Edition2023,
title = {Enzymatic Oxy- and Amino-Functionalization in Biocatalytic Cascade Synthesis: Recent Advances and Future Perspectives},
author = {Eleonora Grandi and Fatma Feyza Özgen and Sandy Schmidt and Gerrit J Poelarends},
url = {https://doi.org/10.1002/anie.202309012},
doi = {https://doi.org/10.1002/anie.202309012},
issn = {1433-7851},
year = {2023},
date = {2023-08-01},
urldate = {2023-08-01},
journal = {Angewandte Chemie International Edition},
volume = {n/a},
number = {n/a},
pages = {e202309012},
publisher = {John Wiley & Sons, Ltd},
abstract = {Biocatalytic cascades are a powerful tool for building complex molecules containing oxygen and nitrogen functionalities. Moreover, the combination of multiple enzymes in one pot offers the possibility to minimize downstream processing and waste production. In this review, we illustrate various recent efforts in the development of multi-step syntheses involving C-O and C-N bond-forming enzymes to produce high value-added compounds, such as pharmaceuticals and polymer precursors. Both in vitro and in vivo examples are discussed, revealing the respective advantages and drawbacks. The use of engineered enzymes to boost the cascades outcome is also addressed and current co-substrate and cofactor recycling strategies are presented, highlighting the importance of atom economy. Finally, tools to overcome current challenges for multi-enzymatic oxy- and amino-functionalization reactions are discussed, including flow systems with immobilized biocatalysts and cascades in confined nanomaterials.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Biocatalytic cascades are a powerful tool for building complex molecules containing oxygen and nitrogen functionalities. Moreover, the combination of multiple enzymes in one pot offers the possibility to minimize downstream processing and waste production. In this review, we illustrate various recent efforts in the development of multi-step syntheses involving C-O and C-N bond-forming enzymes to produce high value-added compounds, such as pharmaceuticals and polymer precursors. Both in vitro and in vivo examples are discussed, revealing the respective advantages and drawbacks. The use of engineered enzymes to boost the cascades outcome is also addressed and current co-substrate and cofactor recycling strategies are presented, highlighting the importance of atom economy. Finally, tools to overcome current challenges for multi-enzymatic oxy- and amino-functionalization reactions are discussed, including flow systems with immobilized biocatalysts and cascades in confined nanomaterials.
Runda, Michael E.; Kok, Niels A. W.; Schmidt, Sandy
Cover Feature - Rieske Oxygenases and Other Ferredoxin-Dependent Enzymes: Electron Transfer Principles and Catalytic Capabilities Journal Article
In: ChemBioChem, vol. e202300466, 2023, ISSN: 14397633.
@article{Runda2023d,
title = {Cover Feature - Rieske Oxygenases and Other Ferredoxin-Dependent Enzymes: Electron Transfer Principles and Catalytic Capabilities},
author = {Michael E. Runda and Niels A. W. Kok and Sandy Schmidt},
url = {https://doi.org/10.1002/cbic.202300466},
doi = {10.1002/cbic.202300078},
issn = {14397633},
year = {2023},
date = {2023-07-04},
urldate = {2023-07-04},
journal = {ChemBioChem},
volume = {e202300466},
abstract = {Enzymes that depend on sophisticated electron transfer via ferredoxins (Fds) exhibit outstanding catalytic capabilities, but despite decades of research, many of them are still not well understood or exploited for synthetic applications. This review aims to provide a general overview of the most important Fd-dependent enzymes and the electron transfer processes involved. While several examples are discussed, we focus in particular on the family of Rieske non-heme iron-dependent oxygenases (ROs). In addition to illustrating their electron transfer principles and catalytic potential, the current state of knowledge on structure–function relationships and the mode of interaction between the redox partner proteins is reviewed. Moreover, we highlight several key catalyzed transformations, but also take a deeper dive into their engineerability for biocatalytic applications. The overall findings from these case studies highlight the catalytic capabilities of these biocatalysts and could stimulate future interest in developing additional Fd-dependent enzyme classes for synthetic applications.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Enzymes that depend on sophisticated electron transfer via ferredoxins (Fds) exhibit outstanding catalytic capabilities, but despite decades of research, many of them are still not well understood or exploited for synthetic applications. This review aims to provide a general overview of the most important Fd-dependent enzymes and the electron transfer processes involved. While several examples are discussed, we focus in particular on the family of Rieske non-heme iron-dependent oxygenases (ROs). In addition to illustrating their electron transfer principles and catalytic potential, the current state of knowledge on structure–function relationships and the mode of interaction between the redox partner proteins is reviewed. Moreover, we highlight several key catalyzed transformations, but also take a deeper dive into their engineerability for biocatalytic applications. The overall findings from these case studies highlight the catalytic capabilities of these biocatalysts and could stimulate future interest in developing additional Fd-dependent enzyme classes for synthetic applications.
Runda, Michael Ernst; Kremser, Bastian; Özgen, Feyza; Schmidt, Sandy
An Optimized System for the Study of Rieske Oxygenase-catalyzed Hydroxylation Reactions In vitro Journal Article
In: ChemCatChem, vol. e202300371, 2023, ISBN: 2013206534.
@article{Runda2023c,
title = {An Optimized System for the Study of Rieske Oxygenase-catalyzed Hydroxylation Reactions In vitro},
author = {Michael Ernst Runda and Bastian Kremser and Feyza Özgen and Sandy Schmidt},
url = {https://doi.org/10.1002/cctc.202300371},
doi = {10.1002/cctc.202300371},
isbn = {2013206534},
year = {2023},
date = {2023-06-06},
urldate = {2023-06-06},
journal = {ChemCatChem},
volume = {e202300371},
abstract = {Rieske non-heme iron oxygenases (ROs) are primarily known for their ability to catalyze the stereoselective formation of vicinal cis-diols in a single step, endowing valuable products for pharmaceutical and chemical applications. In addition, ROs can catalyze several other oxidation reactions with high regio- and stereoselectivity and typically broad substrate scope. Owing to their dependence on multicomponent electron transfer, the majority of synthetic applications of ROs relies on recombinant whole-cell catalysts. In this context, important properties of the multicomponent system that determine the catalytic efficiency, including electron transfer via redox partner proteins, stability and uncoupling, have been investigated to a lesser extent in recent years. Here, we show for one of the most prominent ROs, the cumene dioxygenase from Pseudomonas fluorescens IP01 (CDO) that by developing and optimizing an efficient in vitro system, high catalytic activities can be achieved. In addition, we highlight that an efficient and continuous supplementation of electrons to the oxygenase is required to sustain their catalytic activity, while uncoupling can be a major limitation in CDO efficiency and stability.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Rieske non-heme iron oxygenases (ROs) are primarily known for their ability to catalyze the stereoselective formation of vicinal cis-diols in a single step, endowing valuable products for pharmaceutical and chemical applications. In addition, ROs can catalyze several other oxidation reactions with high regio- and stereoselectivity and typically broad substrate scope. Owing to their dependence on multicomponent electron transfer, the majority of synthetic applications of ROs relies on recombinant whole-cell catalysts. In this context, important properties of the multicomponent system that determine the catalytic efficiency, including electron transfer via redox partner proteins, stability and uncoupling, have been investigated to a lesser extent in recent years. Here, we show for one of the most prominent ROs, the cumene dioxygenase from Pseudomonas fluorescens IP01 (CDO) that by developing and optimizing an efficient in vitro system, high catalytic activities can be achieved. In addition, we highlight that an efficient and continuous supplementation of electrons to the oxygenase is required to sustain their catalytic activity, while uncoupling can be a major limitation in CDO efficiency and stability.
Alphand, Véronique; Berkel, Willem J H Van; Jurkaš, Valentina; Kara, Selin; Kourist, Robert; Kroutil, Wolfgang; Mascia, Francesco; Nowaczyk, Marc M; Paul, Caroline E; Schmidt, Sandy; Spasic, Jelena; Tamagnini, Paula; Winkler, Christoph K.
Exciting Enzymes: Current State and Future Perspective of Photobiocatalysis Journal Article
In: ChemPhotoChem, vol. 202200325, 2023.
@article{Alphand2023,
title = {Exciting Enzymes: Current State and Future Perspective of Photobiocatalysis},
author = {Véronique Alphand and Willem J H Van Berkel and Valentina Jurkaš and Selin Kara and Robert Kourist and Wolfgang Kroutil and Francesco Mascia and Marc M Nowaczyk and Caroline E Paul and Sandy Schmidt and Jelena Spasic and Paula Tamagnini and Christoph K. Winkler},
url = {https://chemistry-europe.onlinelibrary.wiley.com/doi/full/10.1002/cptc.202200325},
doi = {10.1002/cptc.202200325},
year = {2023},
date = {2023-05-02},
urldate = {2023-05-02},
journal = {ChemPhotoChem},
volume = {202200325},
abstract = {The recent increase of interest in photocatalysis spread to biocatalysis and triggered a rush for the development of light-dependent enzyme-mediated or enzyme-coupled processes. After several years of intense research on photobiocatalysis, it is time to evaluate the state of the field in a structured manner. In this Perspective, we suggest to group photobiocatalysis into distinct disciplines and provide principal guidelines and standards for the reporting of photobiocatalytic research results as well as advice on performing photobiocatalytic reactions. Overall, we assess that the field contributes to the diversity of biocatalytic reactions while offering the selectivity of enzymes to photocatalysis. We foresee that the ongoing excitement for light-dependent enzymatic processes will lead to the discovery of novel photobiocatalytic mechanisms to complement biocatalysis with new bond-forming reactions and will provide additional innovative strategies to utilize light as a possible benign energy source.},
keywords = {},
pubstate = {published},
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The recent increase of interest in photocatalysis spread to biocatalysis and triggered a rush for the development of light-dependent enzyme-mediated or enzyme-coupled processes. After several years of intense research on photobiocatalysis, it is time to evaluate the state of the field in a structured manner. In this Perspective, we suggest to group photobiocatalysis into distinct disciplines and provide principal guidelines and standards for the reporting of photobiocatalytic research results as well as advice on performing photobiocatalytic reactions. Overall, we assess that the field contributes to the diversity of biocatalytic reactions while offering the selectivity of enzymes to photocatalysis. We foresee that the ongoing excitement for light-dependent enzymatic processes will lead to the discovery of novel photobiocatalytic mechanisms to complement biocatalysis with new bond-forming reactions and will provide additional innovative strategies to utilize light as a possible benign energy source.
Runda, Michael E; Schmidt, Sandy
Light-driven bioprocesses Journal Article
In: Physical Sciences Reviews, pp. 1–34, 2023.
@article{Runda2023b,
title = {Light-driven bioprocesses},
author = {Michael E Runda and Sandy Schmidt},
url = {https://doi.org/10.1515/psr-2022-0109},
doi = {10.1515/psr-2022-0109},
year = {2023},
date = {2023-04-17},
urldate = {2023-01-01},
journal = {Physical Sciences Reviews},
pages = {1--34},
abstract = {Enzyme catalysis and photocatalysis are two research areas that have become of major interest in organic synthesis. This is mainly because both represent attractive strategies for making chemical synthesis more efficient and sustainable. Because enzyme catalysis offers several inherent advantages, such as high substrate specificity, regio-, and stereoselectivity, and activity under environmentally benign reaction conditions, biocatalysts are increasingly being adopted by the pharmaceutical and chemical industries. In addition, photocatalysis has proven to be a powerful approach for accessing unique reactivities upon light irradiation and performing reactions with an extended substrate range under milder conditions compared to light-independent alternatives. It is therefore not surprising that bio- and photocatalytic approaches are now often combined to exploit the exquisite selectivity of enzymes and the unique chemical transformations accessible to photocatalysis. In this chapter, we provide an overview of the wide variety of light-driven bioprocesses, ranging from photochemical delivery of reducing equivalents to redox enzymes, photochemical cofactor regeneration, to direct photoactivation of enzymes. We also highlight the possibility of catalyzing non-natural reactions via photoinduced enzyme promiscuity and the combination of photo- and biocatalytic reactions used to create new synthetic methodologies.},
keywords = {},
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Enzyme catalysis and photocatalysis are two research areas that have become of major interest in organic synthesis. This is mainly because both represent attractive strategies for making chemical synthesis more efficient and sustainable. Because enzyme catalysis offers several inherent advantages, such as high substrate specificity, regio-, and stereoselectivity, and activity under environmentally benign reaction conditions, biocatalysts are increasingly being adopted by the pharmaceutical and chemical industries. In addition, photocatalysis has proven to be a powerful approach for accessing unique reactivities upon light irradiation and performing reactions with an extended substrate range under milder conditions compared to light-independent alternatives. It is therefore not surprising that bio- and photocatalytic approaches are now often combined to exploit the exquisite selectivity of enzymes and the unique chemical transformations accessible to photocatalysis. In this chapter, we provide an overview of the wide variety of light-driven bioprocesses, ranging from photochemical delivery of reducing equivalents to redox enzymes, photochemical cofactor regeneration, to direct photoactivation of enzymes. We also highlight the possibility of catalyzing non-natural reactions via photoinduced enzyme promiscuity and the combination of photo- and biocatalytic reactions used to create new synthetic methodologies.
Runda, Michael Ernst; Kok, Niels A W; Schmidt, Sandy
Rieske Oxygenases and other Ferredoxin-dependent Enzymes: Electron Transfer Principles and Catalytic Capabilities Journal Article
In: ChemBioChem, pp. e202300078, 2023, ISSN: 1439-4227.
@article{Runda2023,
title = {Rieske Oxygenases and other Ferredoxin-dependent Enzymes: Electron Transfer Principles and Catalytic Capabilities},
author = {Michael Ernst Runda and Niels A W Kok and Sandy Schmidt},
url = {https://doi.org/10.1002/cbic.202300078},
doi = {https://doi.org/10.1002/cbic.202300078},
issn = {1439-4227},
year = {2023},
date = {2023-03-25},
urldate = {2023-03-01},
journal = {ChemBioChem},
pages = {e202300078},
publisher = {John Wiley & Sons, Ltd},
abstract = {Enzymes that depend on sophisticated electron transfer via ferredoxins (Fds) exhibit outstanding catalytic capabilities, but despite decades of research, many of them are still not well understood nor exploited for synthetic applications. This review aims to provide a general overview of the most important Fd-dependent enzymes and the electron transfer processes involved. While several examples are discussed, we focus in particular on the family of Rieske non-heme iron-dependent oxygenases (ROs). In addition to illustrating their electron transfer principles and catalytic potential, the current state of knowledge on structure-function relationships and the mode of interaction between the redox partner proteins is reviewed. Moreover, we highlight several key catalyzed transformations, but also take a deeper dive into their engineerability for biocatalytic applications. The overall findings from these case studies highlight the catalytic capabilities of these biocatalysts and may stimulate future interest in developing additional Fd-dependent enzyme classes for synthetic applications.},
keywords = {},
pubstate = {published},
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Enzymes that depend on sophisticated electron transfer via ferredoxins (Fds) exhibit outstanding catalytic capabilities, but despite decades of research, many of them are still not well understood nor exploited for synthetic applications. This review aims to provide a general overview of the most important Fd-dependent enzymes and the electron transfer processes involved. While several examples are discussed, we focus in particular on the family of Rieske non-heme iron-dependent oxygenases (ROs). In addition to illustrating their electron transfer principles and catalytic potential, the current state of knowledge on structure-function relationships and the mode of interaction between the redox partner proteins is reviewed. Moreover, we highlight several key catalyzed transformations, but also take a deeper dive into their engineerability for biocatalytic applications. The overall findings from these case studies highlight the catalytic capabilities of these biocatalysts and may stimulate future interest in developing additional Fd-dependent enzyme classes for synthetic applications.
Kok, Niels A W De; Schmidt, Sandy
Tapping into abiological reaction chemistries in biocatalysis Journal Article
In: Chem Catalysis, no. 2022, pp. 100493, 2023, ISSN: 2667-1093.
@article{https://doi.org/10.1016/j.checat.2022.100493,
title = {Tapping into abiological reaction chemistries in biocatalysis},
author = {Niels A W De Kok and Sandy Schmidt},
url = {https://doi.org/10.1016/j.checat.2022.100493},
doi = {10.1016/j.checat.2022.100493},
issn = {2667-1093},
year = {2023},
date = {2023-01-18},
urldate = {2023-01-18},
journal = {Chem Catalysis},
number = {2022},
pages = {100493},
publisher = {Elsevier Inc.},
abstract = {Biocatalysis has established itself as a vital tool in the chemical and pharmaceutical industries. However, challenged by the sheer number of chemical reactions available to small-molecule catalysts, the quest to harness biocatalysts for novel transformations has become a veritable hotbed of research. Herein, we discuss previous and actual developments in this area ranging from enzyme discovery for novel function to directed evolution, computational enzyme design, and the construction of artificial (metallo)enzymes, as well as exciting new developments in the field of photobiocatalysis for tapping into abiological reaction chemistries in enzyme catalysis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Biocatalysis has established itself as a vital tool in the chemical and pharmaceutical industries. However, challenged by the sheer number of chemical reactions available to small-molecule catalysts, the quest to harness biocatalysts for novel transformations has become a veritable hotbed of research. Herein, we discuss previous and actual developments in this area ranging from enzyme discovery for novel function to directed evolution, computational enzyme design, and the construction of artificial (metallo)enzymes, as well as exciting new developments in the field of photobiocatalysis for tapping into abiological reaction chemistries in enzyme catalysis.
2022
Schmidt, Sandy
Photoexcited enzymes for asymmetric Csp3-Csp3 cross-electrophile couplings Journal Article
In: Angewandte Chemie International Edition, vol. n/a, no. n/a, 2022.
@article{https://doi.org/10.1002/anie.202214313,
title = {Photoexcited enzymes for asymmetric Csp3-Csp3 cross-electrophile couplings},
author = {Sandy Schmidt },
url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.202214313},
doi = {https://doi.org/10.1002/anie.202214313},
year = {2022},
date = {2022-10-14},
urldate = {2022-01-01},
journal = {Angewandte Chemie International Edition},
volume = {n/a},
number = {n/a},
abstract = {Enzymes have several advantages over conventional catalysts for organic synthesis. Over the last two decades, much effort has been made to further extend the scope of biocatalytic reactions available to synthetic chemists, particularly by expanding the repertoire of enzymes for abiological transformations. In this regard, exciting new developments in the area of photobiocatalysis enable now the introduction of non-natural reactivity in enzymes to solve long-standing synthetic challenges. A recently described example from the Hyster group demonstrates in an unprecedented way how the combination of photochemistry with enzyme catalysis empowers the catalytic asymmetric construction of Csp 3–Csp 3 bonds with high chemo- and enantioselectivity.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Enzymes have several advantages over conventional catalysts for organic synthesis. Over the last two decades, much effort has been made to further extend the scope of biocatalytic reactions available to synthetic chemists, particularly by expanding the repertoire of enzymes for abiological transformations. In this regard, exciting new developments in the area of photobiocatalysis enable now the introduction of non-natural reactivity in enzymes to solve long-standing synthetic challenges. A recently described example from the Hyster group demonstrates in an unprecedented way how the combination of photochemistry with enzyme catalysis empowers the catalytic asymmetric construction of Csp 3–Csp 3 bonds with high chemo- and enantioselectivity.
Özgen, Fatma Feyza; Jorea, Alexandra; Capaldo, Luca; Kourist, Robert; Ravelli, Davide; Schmidt, Sandy
Cover Picture - The Synthesis of Chiral gamma-Lactones by Merging Decatungstate Photocatalysis with Biocatalysis Journal Article
In: ChemCatChem, 2022, ISSN: 18673899.
@article{Ozgen2022b,
title = {Cover Picture - The Synthesis of Chiral gamma-Lactones by Merging Decatungstate Photocatalysis with Biocatalysis},
author = {Fatma Feyza Özgen and Alexandra Jorea and Luca Capaldo and Robert Kourist and Davide Ravelli and Sandy Schmidt},
url = {https://doi.org/10.1002/cctc.202201064},
doi = {https://doi.org/10.1002/cctc.202201064},
issn = {18673899},
year = {2022},
date = {2022-09-16},
urldate = {2022-09-16},
journal = {ChemCatChem},
abstract = {The implementation of light-driven catalytic processes in biocatalysis opens a golden window of opportunities. We hereby report the merging of photocatalytic C−C bond formation with enzymatic asymmetric reduction for the direct conversion of simple aldehydes and acrylates or unsaturated carboxylic acids into chiral $gamma$-lactones. Tetrabutylammonium decatungstate (TBADT) is employed as the photocatalyst to trigger the hydroacylation of the starting olefins, yielding the corresponding keto esters/acids. Subsequently, an alcohol dehydrogenase converts the intermediate to the chiral alcohol, which undergoes lactonization to the desired $gamma$-lactone. The photochemoenzymatic synthesis of aliphatic and aromatic $gamma$-lactones was thereby achieved with up to >99 % ee and >99 % yield. This synthesis highlights the power of building molecular complexity by merging photocatalysis with biocatalysis to access high-value added chiral compounds from simple, cheap and largely available starting materials.},
keywords = {},
pubstate = {published},
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}
The implementation of light-driven catalytic processes in biocatalysis opens a golden window of opportunities. We hereby report the merging of photocatalytic C−C bond formation with enzymatic asymmetric reduction for the direct conversion of simple aldehydes and acrylates or unsaturated carboxylic acids into chiral $gamma$-lactones. Tetrabutylammonium decatungstate (TBADT) is employed as the photocatalyst to trigger the hydroacylation of the starting olefins, yielding the corresponding keto esters/acids. Subsequently, an alcohol dehydrogenase converts the intermediate to the chiral alcohol, which undergoes lactonization to the desired $gamma$-lactone. The photochemoenzymatic synthesis of aliphatic and aromatic $gamma$-lactones was thereby achieved with up to >99 % ee and >99 % yield. This synthesis highlights the power of building molecular complexity by merging photocatalysis with biocatalysis to access high-value added chiral compounds from simple, cheap and largely available starting materials.
Schmidt, Sandy
Expanding the repertoire of Rieske oxygenases for O-demethylations Journal Article
In: Chem Catalysis, vol. 2, no. 8, pp. 1843–1845, 2022, ISSN: 2667-1093.
@article{Schmidt2022,
title = {Expanding the repertoire of Rieske oxygenases for O-demethylations},
author = {Sandy Schmidt},
url = {https://doi.org/10.1016/j.checat.2022.07.005},
doi = {https://doi.org/10.1016/j.checat.2022.07.005},
issn = {2667-1093},
year = {2022},
date = {2022-08-18},
urldate = {2022-08-18},
journal = {Chem Catalysis},
volume = {2},
number = {8},
pages = {1843--1845},
publisher = {Elsevier Inc.},
abstract = {Lignin is presently regarded as both highly underutilized and promising feedstock for diverse applications. Enzymes for aromatic O-demethylations are of particular interest in this context. In this issue of Chem Catalysis, Michener and co-workers expand the enzyme universe with Rieske-type aryl-O-demethylases acting on lignin-derived substrates.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Lignin is presently regarded as both highly underutilized and promising feedstock for diverse applications. Enzymes for aromatic O-demethylations are of particular interest in this context. In this issue of Chem Catalysis, Michener and co-workers expand the enzyme universe with Rieske-type aryl-O-demethylases acting on lignin-derived substrates.
Özgen, Fatma Feyza; Jorea, Alexandra; Capaldo, Luca; Kourist, Robert; Ravelli, Davide; Schmidt, Sandy
The Synthesis of Chiral gamma-Lactones by Merging Decatungstate Photocatalysis with Biocatalysis Journal Article
In: ChemCatChem, vol. n/a, no. n/a, 2022, ISSN: 1867-3880.
@article{Ozgen2022,
title = {The Synthesis of Chiral gamma-Lactones by Merging Decatungstate Photocatalysis with Biocatalysis},
author = {Fatma Feyza Özgen and Alexandra Jorea and Luca Capaldo and Robert Kourist and Davide Ravelli and Sandy Schmidt},
url = {https://doi.org/10.1002/cctc.202200855},
doi = {https://doi.org/10.1002/cctc.202200855},
issn = {1867-3880},
year = {2022},
date = {2022-07-01},
urldate = {2022-07-01},
journal = {ChemCatChem},
volume = {n/a},
number = {n/a},
publisher = {John Wiley & Sons, Ltd},
abstract = {The implementation of light-driven catalytic processes in biocatalysis opens a golden window of opportunities. We hereby report the merging of photocatalytic C-C bond formation with enzymatic asymmetric reduction for the direct conversion of simple aldehydes and acrylates or unsaturated carboxylic acids into chiral ?-lactones. Tetrabutylammonium decatungstate (TBADT) is employed as the photocatalyst to trigger the hydroacylation of the starting olefins, yielding the corresponding keto esters/acids. Subsequently, an alcohol dehydrogenase converts the intermediate to the chiral alcohol, which undergoes lactonization to the desired ?-lactone. The photochemoenzymatic synthesis of aliphatic and aromatic ?-lactones was thereby achieved with up to >99 % ee and >99 % yield. This synthesis highlights the power of building molecular complexity by merging photocatalysis with biocatalysis to access high-value added chiral compounds from simple, cheap and largely available starting materials.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The implementation of light-driven catalytic processes in biocatalysis opens a golden window of opportunities. We hereby report the merging of photocatalytic C-C bond formation with enzymatic asymmetric reduction for the direct conversion of simple aldehydes and acrylates or unsaturated carboxylic acids into chiral ?-lactones. Tetrabutylammonium decatungstate (TBADT) is employed as the photocatalyst to trigger the hydroacylation of the starting olefins, yielding the corresponding keto esters/acids. Subsequently, an alcohol dehydrogenase converts the intermediate to the chiral alcohol, which undergoes lactonization to the desired ?-lactone. The photochemoenzymatic synthesis of aliphatic and aromatic ?-lactones was thereby achieved with up to >99 % ee and >99 % yield. This synthesis highlights the power of building molecular complexity by merging photocatalysis with biocatalysis to access high-value added chiral compounds from simple, cheap and largely available starting materials.
Kristina, Haslinger; Sandy, Schmidt
Späte Funktionalisierung mit Biokatalysatoren aus Naturstoffsynthesen Journal Article
In: Nachrichten aus der Chemie, no. August, pp. 62-65, 2022.
@article{Enzymkandidaten2022,
title = {Späte Funktionalisierung mit Biokatalysatoren aus Naturstoffsynthesen},
author = {Haslinger Kristina and Schmidt Sandy},
url = {https://doi.org/10.1002/nadc.20224127080},
doi = {10.1002/nadc.20224127080},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Nachrichten aus der Chemie},
number = {August},
pages = {62-65},
abstract = {Der Abschnitt „Späte Funktionalisierung mit Biokatalysatoren aus Naturstoffsynthesen“ beschäftigt sich mit der Frage, wie sich funktionelle Gruppen gezielt in Moleküle einführen lassen, ohne auf langwierige Schutzgruppenstrategien rückgreifen zu müssen. Dabei können spezialisierte Enzyme helfen, die vielfach in natürlichen Synthesewegen von Naturstof-fen vorkommen. Es gibt einige Beispiele, wie sich diese Enzyme in der chemischen Synthese nutzen lassen.},
keywords = {},
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Der Abschnitt „Späte Funktionalisierung mit Biokatalysatoren aus Naturstoffsynthesen“ beschäftigt sich mit der Frage, wie sich funktionelle Gruppen gezielt in Moleküle einführen lassen, ohne auf langwierige Schutzgruppenstrategien rückgreifen zu müssen. Dabei können spezialisierte Enzyme helfen, die vielfach in natürlichen Synthesewegen von Naturstof-fen vorkommen. Es gibt einige Beispiele, wie sich diese Enzyme in der chemischen Synthese nutzen lassen.
2021
Özgen, Fatma Feyza; Runda, Michael E.; Schmidt, Sandy
Photo-biocatalytic Cascades: Combining Chemical and Enzymatic Transformations Fueled by Light Journal Article
In: ChemBioChem, vol. 22, no. 5, pp. 790–806, 2021, ISSN: 14397633.
@article{Ozgen2021b,
title = {Photo-biocatalytic Cascades: Combining Chemical and Enzymatic Transformations Fueled by Light},
author = {Fatma Feyza Özgen and Michael E. Runda and Sandy Schmidt},
url = {https://doi.org/10.1002/cbic.202000587},
doi = {10.1002/cbic.202000587},
issn = {14397633},
year = {2021},
date = {2021-03-01},
journal = {ChemBioChem},
volume = {22},
number = {5},
pages = {790--806},
publisher = {John Wiley & Sons, Ltd},
abstract = {In the field of green chemistry, light – an attractive natural agent – has received particular attention for driving biocatalytic reactions. Moreover, the implementation of light to drive (chemo)enzymatic cascade reactions opens up a golden window of opportunities. However, there are limitations to many current examples, mostly associated with incompatibility between the enzyme and the photocatalyst. Additionally, the formation of reactive radicals upon illumination and the loss of catalytic activities in the presence of required additives are common observations. As outlined in this review, the main question is how to overcome current challenges to the exploitation of light to drive (chemo)enzymatic transformations. First, we highlight general concepts in photo-biocatalysis, then give various examples of photo-chemoenzymatic (PCE) cascades, further summarize current synthetic examples of PCE cascades and discuss strategies to address the limitations.},
keywords = {},
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In the field of green chemistry, light – an attractive natural agent – has received particular attention for driving biocatalytic reactions. Moreover, the implementation of light to drive (chemo)enzymatic cascade reactions opens up a golden window of opportunities. However, there are limitations to many current examples, mostly associated with incompatibility between the enzyme and the photocatalyst. Additionally, the formation of reactive radicals upon illumination and the loss of catalytic activities in the presence of required additives are common observations. As outlined in this review, the main question is how to overcome current challenges to the exploitation of light to drive (chemo)enzymatic transformations. First, we highlight general concepts in photo-biocatalysis, then give various examples of photo-chemoenzymatic (PCE) cascades, further summarize current synthetic examples of PCE cascades and discuss strategies to address the limitations.
Schmidt, Sandy
Applications of mixed microbial cultures in industrial biotechnology Book Chapter
In: Kourist, Robert; Schmidt, Sandy (Ed.): The Autotrophic Biorefinery, pp. 353–384, De Gruyter, Berlin, Boston, 2021.
@inbook{Schmidt+2021+353+384b,
title = {Applications of mixed microbial cultures in industrial biotechnology},
author = {Sandy Schmidt},
editor = {Robert Kourist and Sandy Schmidt},
url = {https://doi.org/10.1515/9783110550603-013},
doi = {10.1515/9783110550603-013},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
booktitle = {The Autotrophic Biorefinery},
pages = {353--384},
publisher = {De Gruyter},
address = {Berlin, Boston},
abstract = {Microbial consortia are ubiquitous found in nature and are applied in biotechnological processes such as fermentation, waste treatment and agriculture for millennia. Those consortia consist of member organisms that are together more robust to environmental changes, exhibit reduced metabolic burden, possess expanded metabolic capabilities compared to monocultures and are communicating between species. Those unique strengths of microbial consortia make them an attractive production platform for many biotechnological applications. Moreover, mixed cultures of autotrophic and heterotrophic microorganisms open up the possibility to combine the strengths of both; the autotrophic metabolism promoting a sustainable and green bio-economy with the efficiency of high-performing heterotrophs to manufacture high-value added products. As a result, the field of mixed culture biotechnology has significantly developed and expanded towards the engineering of microbial consortia to expand the scope of possible applications. This chapter provides an outline of the recent developments in mixed culture biotechnology highlighting several examples of mixed cultures used for the production of diverse products. Furthermore, insights into the type of interactions observed in microbial consortia and current limitations associated to these bioproduction systems are discussed. },
keywords = {},
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Microbial consortia are ubiquitous found in nature and are applied in biotechnological processes such as fermentation, waste treatment and agriculture for millennia. Those consortia consist of member organisms that are together more robust to environmental changes, exhibit reduced metabolic burden, possess expanded metabolic capabilities compared to monocultures and are communicating between species. Those unique strengths of microbial consortia make them an attractive production platform for many biotechnological applications. Moreover, mixed cultures of autotrophic and heterotrophic microorganisms open up the possibility to combine the strengths of both; the autotrophic metabolism promoting a sustainable and green bio-economy with the efficiency of high-performing heterotrophs to manufacture high-value added products. As a result, the field of mixed culture biotechnology has significantly developed and expanded towards the engineering of microbial consortia to expand the scope of possible applications. This chapter provides an outline of the recent developments in mixed culture biotechnology highlighting several examples of mixed cultures used for the production of diverse products. Furthermore, insights into the type of interactions observed in microbial consortia and current limitations associated to these bioproduction systems are discussed.
Schmidt, Sandy; Schallmey, Anett; Kourist, Robert
Multi-Enzymatic Cascades In Vitro Book Section
In: Kara, Selin; Rudroff, Florian (Ed.): Enzyme Cascade Design and Modelling, pp. 31–48, Springer International Publishing, Cham, 2021, ISBN: 9783030657185.
@incollection{Schmidt2021b,
title = {Multi-Enzymatic Cascades In Vitro},
author = {Sandy Schmidt and Anett Schallmey and Robert Kourist},
editor = {Selin Kara and Florian Rudroff},
url = {https://doi.org/10.1007/978-3-030-65718-5_3},
doi = {10.1007/978-3-030-65718-5_3},
isbn = {9783030657185},
year = {2021},
date = {2021-01-01},
booktitle = {Enzyme Cascade Design and Modelling},
pages = {31--48},
publisher = {Springer International Publishing},
address = {Cham},
abstract = {The combination of enzymatic reactions in a simultaneous or sequential fashion by designing artificial synthetic cascades allows for the synthesis of complex compounds from simple precursors. Such multi-catalytic cascade reactions not only bear a great potential to minimize downstream processing steps but can also lead to a drastic reduction of the produced waste. With the growing toolbox of biocatalysts, alternative routes employing enzymatic transformations towards manifold and diverse target molecules become accessible. In vitro cascade reactions open up new possibilities for efficient regeneration of the required cofactors such as nicotinamide cofactors or nucleoside triphosphates. They are represented by a vast array of two-enzyme cascades that have been designed by coupling the activity of a cofactor regenerating enzyme to the product generating enzyme. However, the implementation of cascade reactions requires careful consideration, particularly with respect to whether the pathway is constructed concurrently or sequentially. In this regard, this chapter describes how biocatalytic cascades are classified, and how such cascade reactions can be employed in order to solve synthetic problems. Recent developments in the area of dynamic kinetic resolution or cofactor regeneration and showcases are presented. We also highlight the factors that influence the design and implementation of purely enzymatic cascades in one-pot or multi-step pathways in an industrial setting.},
keywords = {},
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}
The combination of enzymatic reactions in a simultaneous or sequential fashion by designing artificial synthetic cascades allows for the synthesis of complex compounds from simple precursors. Such multi-catalytic cascade reactions not only bear a great potential to minimize downstream processing steps but can also lead to a drastic reduction of the produced waste. With the growing toolbox of biocatalysts, alternative routes employing enzymatic transformations towards manifold and diverse target molecules become accessible. In vitro cascade reactions open up new possibilities for efficient regeneration of the required cofactors such as nicotinamide cofactors or nucleoside triphosphates. They are represented by a vast array of two-enzyme cascades that have been designed by coupling the activity of a cofactor regenerating enzyme to the product generating enzyme. However, the implementation of cascade reactions requires careful consideration, particularly with respect to whether the pathway is constructed concurrently or sequentially. In this regard, this chapter describes how biocatalytic cascades are classified, and how such cascade reactions can be employed in order to solve synthetic problems. Recent developments in the area of dynamic kinetic resolution or cofactor regeneration and showcases are presented. We also highlight the factors that influence the design and implementation of purely enzymatic cascades in one-pot or multi-step pathways in an industrial setting.
Kourist, Robert; Schmidt, Sandy (Ed.)
The Autotrophic Biorefinery: Raw Materials from Biotechnology Book
De Gruyter, 2021, ISBN: 9783110550603.
@book{+2021,
title = {The Autotrophic Biorefinery: Raw Materials from Biotechnology},
editor = {Robert Kourist and Sandy Schmidt},
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date = {2021-01-01},
urldate = {2021-01-01},
publisher = {De Gruyter},
abstract = {The depletion of fossil resources and an ever-growing human population create an increasing demand for the development of sustainable processes for the utilization of renewable resources. As autotrophic microorganisms offer numerous metabolic pathways for the fixation of carbon dioxide and the metabolic utilization of light, electricity and inorganic energy donors, they are expected to play a pivotal role in an emerging carbon neutral society.
This text-book presents the metabolic principles of autotrophy and current efforts for their utilization in biotechnology, including photoautotrophic, chemolithoautotrophic and electroautotrophic organisms. It outlines how modern molecular biology and process engineering create technologies that allow to use industrial off-gases and inorganic energy for the synthesis of bio-based plastics, materials and other chemical products.
The text-book is ideally suited for students in advanced graduate and master courses and offers a reference for PhD students, engineers, chemists, biologists and all with an interests in biotechnology and renewable resources.},
keywords = {},
pubstate = {published},
tppubtype = {book}
}
The depletion of fossil resources and an ever-growing human population create an increasing demand for the development of sustainable processes for the utilization of renewable resources. As autotrophic microorganisms offer numerous metabolic pathways for the fixation of carbon dioxide and the metabolic utilization of light, electricity and inorganic energy donors, they are expected to play a pivotal role in an emerging carbon neutral society.
This text-book presents the metabolic principles of autotrophy and current efforts for their utilization in biotechnology, including photoautotrophic, chemolithoautotrophic and electroautotrophic organisms. It outlines how modern molecular biology and process engineering create technologies that allow to use industrial off-gases and inorganic energy for the synthesis of bio-based plastics, materials and other chemical products.
The text-book is ideally suited for students in advanced graduate and master courses and offers a reference for PhD students, engineers, chemists, biologists and all with an interests in biotechnology and renewable resources.
This text-book presents the metabolic principles of autotrophy and current efforts for their utilization in biotechnology, including photoautotrophic, chemolithoautotrophic and electroautotrophic organisms. It outlines how modern molecular biology and process engineering create technologies that allow to use industrial off-gases and inorganic energy for the synthesis of bio-based plastics, materials and other chemical products.
The text-book is ideally suited for students in advanced graduate and master courses and offers a reference for PhD students, engineers, chemists, biologists and all with an interests in biotechnology and renewable resources.
2020
Schmidt, Sandy; Bornscheuer, Uwe T.
Baeyer-Villiger monooxygenases: From protein engineering to biocatalytic applications Book Section
In: Chaiyen, Pimchai; Tamanoi, Fuyuhiko B T - The Enzymes (Ed.): Enzymes, vol. 47, pp. 231–281, Academic Press, 2020, ISSN: 18746047.
@incollection{Schmidt2020b,
title = {Baeyer-Villiger monooxygenases: From protein engineering to biocatalytic applications},
author = {Sandy Schmidt and Uwe T. Bornscheuer},
editor = {Pimchai Chaiyen and Fuyuhiko B T - The Enzymes Tamanoi},
url = {https://www.sciencedirect.com/science/article/pii/S187460472030010X http://dx.doi.org/10.1016/bs.enz.2020.05.007},
doi = {10.1016/bs.enz.2020.05.007},
issn = {18746047},
year = {2020},
date = {2020-07-18},
urldate = {2020-01-01},
booktitle = {Enzymes},
volume = {47},
pages = {231--281},
publisher = {Academic Press},
edition = {1},
abstract = {Biocatalytic processes are well established for the synthesis of high-value fine chemicals, especially for chiral pharmaceutical intermediates, by using natural or engineered enzymes. In contrast, examples for the enzymatic synthesis of bulk chemicals are still rare. Especially for the synthesis of polymer precursors such as ɛ-caprolactone, that is still produced under harsh conditions by using peracetic acid, Baeyer-Villiger monooxygenases (BVMOs) represent promising alternative catalysts that can perform the reaction under mild conditions. However, industrial production of this bulk chemical using a biocatalyst such as a BVMO has not been achieved yet due to a number of reasons. In this book chapter, we are emphasizing the versatility of BVMOs and their catalyzed reactions, and address several examples where protein engineering was applied in order to overcome several limitations associated to the use of BVMOs. Finally, we highlight several examples of BVMO applications, either in single enzyme transformations, or BVMOs involved in cascade reactions. By mainly focusing on recent developments and achievements in the field, we outline different concepts that were developed in order to pave the way for an industrial application of BVMOs.},
keywords = {},
pubstate = {published},
tppubtype = {incollection}
}
Biocatalytic processes are well established for the synthesis of high-value fine chemicals, especially for chiral pharmaceutical intermediates, by using natural or engineered enzymes. In contrast, examples for the enzymatic synthesis of bulk chemicals are still rare. Especially for the synthesis of polymer precursors such as ɛ-caprolactone, that is still produced under harsh conditions by using peracetic acid, Baeyer-Villiger monooxygenases (BVMOs) represent promising alternative catalysts that can perform the reaction under mild conditions. However, industrial production of this bulk chemical using a biocatalyst such as a BVMO has not been achieved yet due to a number of reasons. In this book chapter, we are emphasizing the versatility of BVMOs and their catalyzed reactions, and address several examples where protein engineering was applied in order to overcome several limitations associated to the use of BVMOs. Finally, we highlight several examples of BVMO applications, either in single enzyme transformations, or BVMOs involved in cascade reactions. By mainly focusing on recent developments and achievements in the field, we outline different concepts that were developed in order to pave the way for an industrial application of BVMOs.
Büchsenschütz, Hanna C.; Vidimce-Risteski, Viktorija; Eggbauer, Bettina; Schmidt, Sandy; Winkler, Christoph K.; Schrittwieser, Joerg H.; Kroutil, Wolfgang; Kourist, Robert
Stereoselective Biotransformations of Cyclic Imines in Recombinant Cells of Synechocystis sp. PCC 6803 Journal Article
In: ChemCatChem, vol. 12, no. 3, pp. 726–730, 2020, ISSN: 18673899.
@article{Buchsenschutz2020a,
title = {Stereoselective Biotransformations of Cyclic Imines in Recombinant Cells of Synechocystis sp. PCC 6803},
author = {Hanna C. Büchsenschütz and Viktorija Vidimce-Risteski and Bettina Eggbauer and Sandy Schmidt and Christoph K. Winkler and Joerg H. Schrittwieser and Wolfgang Kroutil and Robert Kourist},
url = {https://doi.org/10.1002/cctc.201901592},
doi = {10.1002/cctc.201901592},
issn = {18673899},
year = {2020},
date = {2020-02-01},
journal = {ChemCatChem},
volume = {12},
number = {3},
pages = {726--730},
publisher = {John Wiley & Sons, Ltd},
abstract = {Light-driven biotransformations in recombinant cyanobacteria allow to employ photosynthetic water-splitting for cofactor-regeneration and thus, to save the use of organic electron donors. The genes of three recombinant imine reductases (IREDs) were expressed in the cyanobacterium Synechocystis sp. PCC 6803 and eight cyclic imine substrates were screened in whole-cell biotransformations. While initial reactions showed low to moderate rates, optimization of the reaction conditions in combination with promoter engineering allowed to alleviate toxicity effects and achieve full conversion of prochiral imines with initial rates of up to 6.3 mM h−1. The high specific activity of up to 22 U gCDW−1 demonstrates that recombinant cyanobacteria can provide large amounts of NADPH during whole cell reactions. The excellent optical purity of the products with up to >99 %ee underlines the usefulness of cyanobacteria for the stereoselective synthesis of amines.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Light-driven biotransformations in recombinant cyanobacteria allow to employ photosynthetic water-splitting for cofactor-regeneration and thus, to save the use of organic electron donors. The genes of three recombinant imine reductases (IREDs) were expressed in the cyanobacterium Synechocystis sp. PCC 6803 and eight cyclic imine substrates were screened in whole-cell biotransformations. While initial reactions showed low to moderate rates, optimization of the reaction conditions in combination with promoter engineering allowed to alleviate toxicity effects and achieve full conversion of prochiral imines with initial rates of up to 6.3 mM h−1. The high specific activity of up to 22 U gCDW−1 demonstrates that recombinant cyanobacteria can provide large amounts of NADPH during whole cell reactions. The excellent optical purity of the products with up to >99 %ee underlines the usefulness of cyanobacteria for the stereoselective synthesis of amines.
Özgen, F. Feyza; Runda, Michael E.; Burek, Bastien O.; Wied, Peter; Bloh, Jonathan Z.; Kourist, Robert; Schmidt, Sandy
Artifizielle Lichtsammelkomplexe ermöglichen Rieske‐Oxygenase‐ katalysierte Hydroxylierungen in nicht‐photosynthetischen Zellen Journal Article
In: Angewandte Chemie, vol. 132, no. 10, pp. 4010–4016, 2020, ISSN: 0044-8249.
@article{FeyzaOzgen2020b,
title = {Artifizielle Lichtsammelkomplexe ermöglichen Rieske‐Oxygenase‐ katalysierte Hydroxylierungen in nicht‐photosynthetischen Zellen},
author = {F. Feyza Özgen and Michael E. Runda and Bastien O. Burek and Peter Wied and Jonathan Z. Bloh and Robert Kourist and Sandy Schmidt},
doi = {10.1002/ange.201914519},
issn = {0044-8249},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {Angewandte Chemie},
volume = {132},
number = {10},
pages = {4010--4016},
abstract = {In dieser Studie wurde ein auf E. coli basierendes Ganzzellsystem über Lichtsammelkomplexe an Rieske-Oxygenasen(RO)-katalysierte Hydroxylierungen in vivo angekoppelt. Obwohl diese Enzyme vielversprechende Biokatalysatoren darstellen, wird ihre praktische Anwendbarkeit durch ihre Abhängigkeit von NAD(P)H sowie ihre Mehrkomponentennatur und intrinsische Instabilität in zellfreien Systemen beeinträchtigt. Um die Grenzen von E. coli als Chassis für künstliche Photosynthese zu erforschen, sowie aufgrund der berichteten Instabilität von ROs, haben wir diese herausfordernden Enzyme als Modellsystem verwendet. Der Licht-getriebene Ansatz beruht auf Lichtsammelkomplexen wie beispielsweise Eosin Y, 5(6)-Carboxyeosin oder Rose Bengal und Elektronendonoren (EDTA, MOPS oder MES), die von den Zellen leicht aufgenommen werden können. Die erzielten Produktbildungen von bis zu 1.3 g L−1 und Raten von bis zu 1.6 mm h−1 zeigen, dass dies ein vergleichbarer Ansatz zu typischen Ganzzelltransformationen in E. coli ist. Die Anwendbarkeit dieser photokatalytischen Synthese wurde demonstriert und ist das erste Beispiel eines photoinduzierten RO-Systems.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In dieser Studie wurde ein auf E. coli basierendes Ganzzellsystem über Lichtsammelkomplexe an Rieske-Oxygenasen(RO)-katalysierte Hydroxylierungen in vivo angekoppelt. Obwohl diese Enzyme vielversprechende Biokatalysatoren darstellen, wird ihre praktische Anwendbarkeit durch ihre Abhängigkeit von NAD(P)H sowie ihre Mehrkomponentennatur und intrinsische Instabilität in zellfreien Systemen beeinträchtigt. Um die Grenzen von E. coli als Chassis für künstliche Photosynthese zu erforschen, sowie aufgrund der berichteten Instabilität von ROs, haben wir diese herausfordernden Enzyme als Modellsystem verwendet. Der Licht-getriebene Ansatz beruht auf Lichtsammelkomplexen wie beispielsweise Eosin Y, 5(6)-Carboxyeosin oder Rose Bengal und Elektronendonoren (EDTA, MOPS oder MES), die von den Zellen leicht aufgenommen werden können. Die erzielten Produktbildungen von bis zu 1.3 g L−1 und Raten von bis zu 1.6 mm h−1 zeigen, dass dies ein vergleichbarer Ansatz zu typischen Ganzzelltransformationen in E. coli ist. Die Anwendbarkeit dieser photokatalytischen Synthese wurde demonstriert und ist das erste Beispiel eines photoinduzierten RO-Systems.
Özgen, F. Feyza; Runda, Michael E.; Burek, Bastien O.; Wied, Peter; Bloh, Jonathan Z.; Kourist, Robert; Schmidt, Sandy
Artificial Light-Harvesting Complexes Enable Rieske Oxygenase Catalyzed Hydroxylations in Non-Photosynthetic cells Journal Article
In: Angewandte Chemie - International Edition, vol. 59, no. 10, pp. 3982–3987, 2020, ISSN: 15213773.
@article{Ozgen2019,
title = {Artificial Light-Harvesting Complexes Enable Rieske Oxygenase Catalyzed Hydroxylations in Non-Photosynthetic cells},
author = {F. Feyza Özgen and Michael E. Runda and Bastien O. Burek and Peter Wied and Jonathan Z. Bloh and Robert Kourist and Sandy Schmidt},
doi = {10.1002/anie.201914519},
issn = {15213773},
year = {2020},
date = {2020-01-01},
journal = {Angewandte Chemie - International Edition},
volume = {59},
number = {10},
pages = {3982--3987},
abstract = {In this study, we coupled a well-established whole-cell system based on E. coli via light-harvesting complexes to Rieske oxygenase (RO)-catalyzed hydroxylations in vivo. Although these enzymes represent very promising biocatalysts, their practical applicability is hampered by their dependency on NAD(P)H as well as their multicomponent nature and intrinsic instability in cell-free systems. In order to explore the boundaries of E. coli as chassis for artificial photosynthesis, and due to the reported instability of ROs, we used these challenging enzymes as a model system. The light-driven approach relies on light-harvesting complexes such as eosin Y, 5(6)-carboxyeosin, and rose bengal and sacrificial electron donors (EDTA, MOPS, and MES) that were easily taken up by the cells. The obtained product formations of up to 1.3 g L−1 and rates of up to 1.6 mm h−1 demonstrate that this is a comparable approach to typical whole-cell transformations in E. coli. The applicability of this photocatalytic synthesis has been demonstrated and represents the first example of a photoinduced RO system.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In this study, we coupled a well-established whole-cell system based on E. coli via light-harvesting complexes to Rieske oxygenase (RO)-catalyzed hydroxylations in vivo. Although these enzymes represent very promising biocatalysts, their practical applicability is hampered by their dependency on NAD(P)H as well as their multicomponent nature and intrinsic instability in cell-free systems. In order to explore the boundaries of E. coli as chassis for artificial photosynthesis, and due to the reported instability of ROs, we used these challenging enzymes as a model system. The light-driven approach relies on light-harvesting complexes such as eosin Y, 5(6)-carboxyeosin, and rose bengal and sacrificial electron donors (EDTA, MOPS, and MES) that were easily taken up by the cells. The obtained product formations of up to 1.3 g L−1 and rates of up to 1.6 mm h−1 demonstrate that this is a comparable approach to typical whole-cell transformations in E. coli. The applicability of this photocatalytic synthesis has been demonstrated and represents the first example of a photoinduced RO system.
2019
Schweiger, Anna K.; Ríos-Lombardía, Nicolás; Winkler, Christoph K.; Schmidt, Sandy; Morís, Francisco; Kroutil, Wolfgang; González-Sabín, Javier; Kourist, Robert
Using Deep Eutectic Solvents to Overcome Limited Substrate Solubility in the Enzymatic Decarboxylation of Bio-Based Phenolic Acids Journal Article
In: ACS Sustainable Chemistry and Engineering, vol. 7, no. 19, pp. 16364–16370, 2019, ISSN: 21680485.
@article{Schweiger2019,
title = {Using Deep Eutectic Solvents to Overcome Limited Substrate Solubility in the Enzymatic Decarboxylation of Bio-Based Phenolic Acids},
author = {Anna K. Schweiger and Nicolás Ríos-Lombardía and Christoph K. Winkler and Sandy Schmidt and Francisco Morís and Wolfgang Kroutil and Javier González-Sabín and Robert Kourist},
url = {https://doi.org/10.1021/acssuschemeng.9b03455},
doi = {10.1021/acssuschemeng.9b03455},
issn = {21680485},
year = {2019},
date = {2019-10-01},
journal = {ACS Sustainable Chemistry and Engineering},
volume = {7},
number = {19},
pages = {16364--16370},
publisher = {American Chemical Society},
abstract = {Phenolic acid decarboxylase from Bacillus subtilis (BsPAD) converts several p-hydroxycinnamic acid derivatives into the corresponding p-hydroxystyrenes, which are considered to be promising bio-based aromatic chemicals. Despite the enzyme's high activity and stability, the low solubility of its substrates presents severe limitations for the establishment of industrial processes. Accordingly, deep eutectic solvents (DESs) have emerged as interesting alternatives to aqueous or organic solvents and biphasic systems as they offer unique reaction conditions while remaining biocompatible and biodegradable. Herein, we show that BsPAD could tolerate choline chloride (ChCl)-based eutectic solvents with 0-50% water content, which allowed conversion to the corresponding p-hydroxystyrene derivatives (>99%) at substrate loadings of up to 300 mM due to the exceptional solubilizing properties of this solvent. As the enzyme showed some remarkable reactivity differences in DES and water, we further explored the substrate scope of the enzyme and a mutant with increased space in the active site. The comparison of substrates with different substituents on the aryl group indicated that the substrate preference is determined by steric, rather than electronic effects. Furthermore, we report that the choice of the solvent influences the acceptance of different substrates as evidenced by the fact that DES strongly favored the conversion of caffeic acid, which is only poorly converted in aqueous media.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Phenolic acid decarboxylase from Bacillus subtilis (BsPAD) converts several p-hydroxycinnamic acid derivatives into the corresponding p-hydroxystyrenes, which are considered to be promising bio-based aromatic chemicals. Despite the enzyme's high activity and stability, the low solubility of its substrates presents severe limitations for the establishment of industrial processes. Accordingly, deep eutectic solvents (DESs) have emerged as interesting alternatives to aqueous or organic solvents and biphasic systems as they offer unique reaction conditions while remaining biocompatible and biodegradable. Herein, we show that BsPAD could tolerate choline chloride (ChCl)-based eutectic solvents with 0-50% water content, which allowed conversion to the corresponding p-hydroxystyrene derivatives (>99%) at substrate loadings of up to 300 mM due to the exceptional solubilizing properties of this solvent. As the enzyme showed some remarkable reactivity differences in DES and water, we further explored the substrate scope of the enzyme and a mutant with increased space in the active site. The comparison of substrates with different substituents on the aryl group indicated that the substrate preference is determined by steric, rather than electronic effects. Furthermore, we report that the choice of the solvent influences the acceptance of different substrates as evidenced by the fact that DES strongly favored the conversion of caffeic acid, which is only poorly converted in aqueous media.
Assil-Companioni, Leen; Schmidt, Sandy; Heidinger, Petra; Schwab, Helmut; Kourist, Robert
Hydrogen-Driven Cofactor Regeneration for Stereoselective Whole-Cell C=C Bond Reduction in Cupriavidus necator Journal Article
In: ChemSusChem, vol. 12, no. 11, pp. 2361–2365, 2019, ISSN: 1864564X.
@article{Assil-Companioni2019b,
title = {Hydrogen-Driven Cofactor Regeneration for Stereoselective Whole-Cell C=C Bond Reduction in Cupriavidus necator},
author = {Leen Assil-Companioni and Sandy Schmidt and Petra Heidinger and Helmut Schwab and Robert Kourist},
url = {https://doi.org/10.1002/cssc.201900327},
doi = {10.1002/cssc.201900327},
issn = {1864564X},
year = {2019},
date = {2019-06-01},
journal = {ChemSusChem},
volume = {12},
number = {11},
pages = {2361--2365},
publisher = {John Wiley & Sons, Ltd},
abstract = {The coupling of recombinantly expressed oxidoreductases to endogenous hydrogenases for cofactor recycling permits the omission of organic cosubstrates as sacrificial electron donors in whole-cell biotransformations. This increases atom efficiency and simplifies the reaction. A recombinant ene-reductase was expressed in the hydrogen-oxidizing proteobacterium Cupriavidus necator H16. In hydrogen-driven biotransformations, whole cells catalyzed asymmetric C=C bond reduction of unsaturated cyclic ketones with stereoselectivities up to >99 % enantiomeric excess. The use of hydrogen as a substrate for growth and cofactor regeneration is particularly attractive because it represents a strategy for improving atom efficiency and reducing side product formation associated with the recycling of organic cofactors.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The coupling of recombinantly expressed oxidoreductases to endogenous hydrogenases for cofactor recycling permits the omission of organic cosubstrates as sacrificial electron donors in whole-cell biotransformations. This increases atom efficiency and simplifies the reaction. A recombinant ene-reductase was expressed in the hydrogen-oxidizing proteobacterium Cupriavidus necator H16. In hydrogen-driven biotransformations, whole cells catalyzed asymmetric C=C bond reduction of unsaturated cyclic ketones with stereoselectivities up to >99 % enantiomeric excess. The use of hydrogen as a substrate for growth and cofactor regeneration is particularly attractive because it represents a strategy for improving atom efficiency and reducing side product formation associated with the recycling of organic cofactors.
Schmermund, Luca; Jurkaš, Valentina; Özgen, F. Feyza; Barone, Giovanni D.; Büchsenschütz, Hanna C.; Winkler, Christoph K.; Schmidt, Sandy; Kourist, Robert; Kroutil, Wolfgang
Photo-Biocatalysis: Biotransformations in the Presence of Light Journal Article
In: ACS Catalysis, vol. 9, no. 5, pp. 4115–4144, 2019, ISSN: 21555435.
@article{Schmermund2019b,
title = {Photo-Biocatalysis: Biotransformations in the Presence of Light},
author = {Luca Schmermund and Valentina Jurkaš and F. Feyza Özgen and Giovanni D. Barone and Hanna C. Büchsenschütz and Christoph K. Winkler and Sandy Schmidt and Robert Kourist and Wolfgang Kroutil},
url = {https://doi.org/10.1021/acscatal.9b00656},
doi = {10.1021/acscatal.9b00656},
issn = {21555435},
year = {2019},
date = {2019-05-01},
journal = {ACS Catalysis},
volume = {9},
number = {5},
pages = {4115--4144},
publisher = {American Chemical Society},
abstract = {Light has received increased attention for various chemical reactions but also in combination with biocatalytic reactions. Because currently only a few enzymatic reactions are known, which per se require light, most transformations involving light and a biocatalyst exploit light either for providing the cosubstrate or cofactor in an appropriate redox state for the biotransformation. In selected cases, a promiscuous activity of known enzymes in the presence of light could be induced. In other approaches, light-induced chemical reactions have been combined with a biocatalytic step, or light-induced biocatalytic reactions were combined with chemical reactions in a linear cascade. Finally, enzymes with a light switchable moiety have been investigated to turn off/on or tune the actual reaction. This Review gives an overview of the various approaches for using light in biocatalysis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Light has received increased attention for various chemical reactions but also in combination with biocatalytic reactions. Because currently only a few enzymatic reactions are known, which per se require light, most transformations involving light and a biocatalyst exploit light either for providing the cosubstrate or cofactor in an appropriate redox state for the biotransformation. In selected cases, a promiscuous activity of known enzymes in the presence of light could be induced. In other approaches, light-induced chemical reactions have been combined with a biocatalytic step, or light-induced biocatalytic reactions were combined with chemical reactions in a linear cascade. Finally, enzymes with a light switchable moiety have been investigated to turn off/on or tune the actual reaction. This Review gives an overview of the various approaches for using light in biocatalysis.
Zhang, Wuyuan; Fueyo, Elena Fernandez; Hollmann, Frank; Martin, Laura Leemans; Pesic, Milja; Wardenga, Rainer; Höhne, Matthias; Schmidt, Sandy
Combining Photo-Organo Redox- and Enzyme Catalysis Facilitates Asymmetric C-H Bond Functionalization Journal Article
In: European Journal of Organic Chemistry, vol. 2019, no. 1, pp. 80–84, 2019, ISSN: 10990690.
@article{Zhang2019gb,
title = {Combining Photo-Organo Redox- and Enzyme Catalysis Facilitates Asymmetric C-H Bond Functionalization},
author = {Wuyuan Zhang and Elena Fernandez Fueyo and Frank Hollmann and Laura Leemans Martin and Milja Pesic and Rainer Wardenga and Matthias Höhne and Sandy Schmidt},
url = {https://doi.org/10.1002/ejoc.201801692},
doi = {10.1002/ejoc.201801692},
issn = {10990690},
year = {2019},
date = {2019-01-01},
journal = {European Journal of Organic Chemistry},
volume = {2019},
number = {1},
pages = {80--84},
publisher = {John Wiley & Sons, Ltd},
abstract = {In this study, we combined photo-organo redox catalysis and biocatalysis to achieve asymmetric C–H bond functionalization of simple alkane starting materials. The photo-organo catalyst anthraquinone sulfate (SAS) was employed to oxyfunctionalise alkanes to aldehydes and ketones. We coupled this light-driven reaction with asymmetric enzymatic functionalisations to yield chiral hydroxynitriles, amines, acyloins and $alpha$-chiral ketones with up to 99 % ee. In addition, we demonstrate functional group interconversion to alcohols, esters and carboxylic acids. The transformations can be performed as concurrent tandem reactions. We identified the degradation of substrates and inhibition of the biocatalysts as limiting factors affecting compatibility, due to reactive oxygen species generated in the photocatalytic step. These incompatibilities were addressed by reaction engineering, such as applying a two-phase system or temporal and spatial separation of the catalysts. Using a selection of eleven starting alkanes, one photo-organo catalyst and 8 diverse biocatalysts, we synthesized 26 products and report for the model compounds benzoin and mandelonitrile > 97 % ee at gram scale.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In this study, we combined photo-organo redox catalysis and biocatalysis to achieve asymmetric C–H bond functionalization of simple alkane starting materials. The photo-organo catalyst anthraquinone sulfate (SAS) was employed to oxyfunctionalise alkanes to aldehydes and ketones. We coupled this light-driven reaction with asymmetric enzymatic functionalisations to yield chiral hydroxynitriles, amines, acyloins and $alpha$-chiral ketones with up to 99 % ee. In addition, we demonstrate functional group interconversion to alcohols, esters and carboxylic acids. The transformations can be performed as concurrent tandem reactions. We identified the degradation of substrates and inhibition of the biocatalysts as limiting factors affecting compatibility, due to reactive oxygen species generated in the photocatalytic step. These incompatibilities were addressed by reaction engineering, such as applying a two-phase system or temporal and spatial separation of the catalysts. Using a selection of eleven starting alkanes, one photo-organo catalyst and 8 diverse biocatalysts, we synthesized 26 products and report for the model compounds benzoin and mandelonitrile > 97 % ee at gram scale.
Özgen, Fatma Feyza; Schmidt, Sandy
Rieske non-heme iron dioxygenases: Applications and future perspectives Book Section
In: Husain, Qayyum; Ullah, Mohammad Fahad (Ed.): Biocatalysis: Enzymatic Basics and Applications, pp. 57–82, Springer International Publishing, Cham, 2019, ISBN: 9783030250232.
@incollection{Ozgen2019a,
title = {Rieske non-heme iron dioxygenases: Applications and future perspectives},
author = {Fatma Feyza Özgen and Sandy Schmidt},
editor = {Qayyum Husain and Mohammad Fahad Ullah},
url = {https://doi.org/10.1007/978-3-030-25023-2_4},
doi = {10.1007/978-3-030-25023-2_4},
isbn = {9783030250232},
year = {2019},
date = {2019-01-01},
booktitle = {Biocatalysis: Enzymatic Basics and Applications},
pages = {57--82},
publisher = {Springer International Publishing},
address = {Cham},
abstract = {The stereo- and regioselective oxidative functionalization of olefins is amongst the most challenging reactions in organic syntheses. In particular, the catalytic asymmetric dihydroxylation of alkenes has attracted considerable attention due to the facile further transformation of the chiral diol products into valuable derivatives, making them important building blocks for the pharmaceutical and chemical industry. Nature's creativity in developing solutions for C–H-bond functionalization reactions like hydroxylations at activated or non-activated C–H-bonds is remarkably shown by an impressive list of metal-dependent enzymes. These enzymes, like the Rieske non-heme iron oxygenases (ROs) are able to activate molecular oxygen in order to generate reactive oxygen species capable of hydroxylating alkyl-substrates and they also promote further oxidative transformations. For many of these reactions no ‘classical' chemical counterpart is known. ROs represent promising biocatalysts for these reactions since they are the only enzymes known to catalyze the stereoselective formation of vicinal cis-diols in one step. They are soluble multicomponent systems that harness the reductive power of NAD(P)H for oxygen activation. Due to their versatility, ROs are considered as the non-heme analogues of cytochrome P450 monooxygenases and, in addition to their relaxed substrate specificity, these enzymes can also catalyze various oxidation reactions, resulting in an enormous potential of these enzymes for manifold synthetically useful transformations. This chapter describes the current understanding of the structural determinants and the catalytic behavior of dioxygenase-catalyzed reactions, and highlights how in several cases this knowledge has been harnessed to design tailored catalysts for the synthesis of various natural products, polyfunctionalized metabolites and pharmaceutical intermediates. Moreover, the chapter also gives insights into recently characterized ROs catalyzing unusual reactions as well as applications in chemo-enzymatic cascade reactions for natural product synthesis.},
keywords = {},
pubstate = {published},
tppubtype = {incollection}
}
The stereo- and regioselective oxidative functionalization of olefins is amongst the most challenging reactions in organic syntheses. In particular, the catalytic asymmetric dihydroxylation of alkenes has attracted considerable attention due to the facile further transformation of the chiral diol products into valuable derivatives, making them important building blocks for the pharmaceutical and chemical industry. Nature's creativity in developing solutions for C–H-bond functionalization reactions like hydroxylations at activated or non-activated C–H-bonds is remarkably shown by an impressive list of metal-dependent enzymes. These enzymes, like the Rieske non-heme iron oxygenases (ROs) are able to activate molecular oxygen in order to generate reactive oxygen species capable of hydroxylating alkyl-substrates and they also promote further oxidative transformations. For many of these reactions no ‘classical' chemical counterpart is known. ROs represent promising biocatalysts for these reactions since they are the only enzymes known to catalyze the stereoselective formation of vicinal cis-diols in one step. They are soluble multicomponent systems that harness the reductive power of NAD(P)H for oxygen activation. Due to their versatility, ROs are considered as the non-heme analogues of cytochrome P450 monooxygenases and, in addition to their relaxed substrate specificity, these enzymes can also catalyze various oxidation reactions, resulting in an enormous potential of these enzymes for manifold synthetically useful transformations. This chapter describes the current understanding of the structural determinants and the catalytic behavior of dioxygenase-catalyzed reactions, and highlights how in several cases this knowledge has been harnessed to design tailored catalysts for the synthesis of various natural products, polyfunctionalized metabolites and pharmaceutical intermediates. Moreover, the chapter also gives insights into recently characterized ROs catalyzing unusual reactions as well as applications in chemo-enzymatic cascade reactions for natural product synthesis.
2018
Fiorentini, Filippo; Hatzl, Anna Maria; Schmidt, Sandy; Savino, Simone; Glieder, Anton; Mattevi, Andrea
The Extreme Structural Plasticity in the CYP153 Subfamily of P450s Directs Development of Designer Hydroxylases Journal Article
In: Biochemistry, vol. 57, no. 48, pp. 6701–6714, 2018, ISSN: 15204995.
@article{Fiorentini2018b,
title = {The Extreme Structural Plasticity in the CYP153 Subfamily of P450s Directs Development of Designer Hydroxylases},
author = {Filippo Fiorentini and Anna Maria Hatzl and Sandy Schmidt and Simone Savino and Anton Glieder and Andrea Mattevi},
url = {https://doi.org/10.1021/acs.biochem.8b01052},
doi = {10.1021/acs.biochem.8b01052},
issn = {15204995},
year = {2018},
date = {2018-12-01},
journal = {Biochemistry},
volume = {57},
number = {48},
pages = {6701--6714},
publisher = {American Chemical Society},
abstract = {CYP153s are bacterial class I P450 enzymes traditionally described as alkane hydroxylases with a high terminal regioselectivity. They have been more recently shown to also catalyze hydroxylations at nonactivated carbon atoms of small heterocycles. The aim of our work was to perform an extensive characterization of this subfamily in order to deliver a toolbox of CYP153 enzymes for further development as biocatalysts. Through the screening of recently sequenced bacterial genomes, 20 CYP153s were selected, comprising 17 single monooxygenase domains and three multidomain variants, where the monooxygenase domain is naturally fused to its redox partners in a single polypeptide chain. The 20 novel variants were heterologously expressed, and their activity was screened toward octane and small heterocycles. A more extended substrate characterization was then performed on three representative candidates, and their crystal structures were unveiled and compared with those of the known CYP153A7 and CYP153A33. The tested enzymes displayed a wide range of activities, ranging from $ømega$ and $ømega$-1 hydroxylations of lauric acid to indigo-generating indole modification. The comparative analysis highlighted a conserved architecture and amino acid composition of the catalytic core close to the heme, while showing a huge degree of structural plasticity and flexibility in those regions hosting the substrate recognition sites. Although dealing with this type of conformational variability adds a layer of complexity and difficulty to structure-based protein engineering, such diversity in substrate acceptance and recognition promotes the investigated CYP153s as a prime choice for tailoring designer hydroxylases.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
CYP153s are bacterial class I P450 enzymes traditionally described as alkane hydroxylases with a high terminal regioselectivity. They have been more recently shown to also catalyze hydroxylations at nonactivated carbon atoms of small heterocycles. The aim of our work was to perform an extensive characterization of this subfamily in order to deliver a toolbox of CYP153 enzymes for further development as biocatalysts. Through the screening of recently sequenced bacterial genomes, 20 CYP153s were selected, comprising 17 single monooxygenase domains and three multidomain variants, where the monooxygenase domain is naturally fused to its redox partners in a single polypeptide chain. The 20 novel variants were heterologously expressed, and their activity was screened toward octane and small heterocycles. A more extended substrate characterization was then performed on three representative candidates, and their crystal structures were unveiled and compared with those of the known CYP153A7 and CYP153A33. The tested enzymes displayed a wide range of activities, ranging from $ømega$ and $ømega$-1 hydroxylations of lauric acid to indigo-generating indole modification. The comparative analysis highlighted a conserved architecture and amino acid composition of the catalytic core close to the heme, while showing a huge degree of structural plasticity and flexibility in those regions hosting the substrate recognition sites. Although dealing with this type of conformational variability adds a layer of complexity and difficulty to structure-based protein engineering, such diversity in substrate acceptance and recognition promotes the investigated CYP153s as a prime choice for tailoring designer hydroxylases.
Dong, Jia Jia; Fernández-Fueyo, Elena; Hollmann, Frank; Paul, Caroline E.; Pesic, Milja; Schmidt, Sandy; Wang, Yonghua; Younes, Sabry; Zhang, Wuyuan
Biocatalytic Oxidation Reactions: A Chemist's Perspective Journal Article
In: Angewandte Chemie - International Edition, vol. 57, no. 30, pp. 9238–9261, 2018, ISSN: 15213773.
@article{Dong2018ab,
title = {Biocatalytic Oxidation Reactions: A Chemist's Perspective},
author = {Jia Jia Dong and Elena Fernández-Fueyo and Frank Hollmann and Caroline E. Paul and Milja Pesic and Sandy Schmidt and Yonghua Wang and Sabry Younes and Wuyuan Zhang},
url = {https://doi.org/10.1002/anie.201800343},
doi = {10.1002/anie.201800343},
issn = {15213773},
year = {2018},
date = {2018-07-01},
journal = {Angewandte Chemie - International Edition},
volume = {57},
number = {30},
pages = {9238--9261},
publisher = {John Wiley & Sons, Ltd},
abstract = {Oxidation chemistry using enzymes is approaching maturity and practical applicability in organic synthesis. Oxidoreductases (enzymes catalysing redox reactions) enable chemists to perform highly selective and efficient transformations ranging from simple alcohol oxidations to stereoselective halogenations of non-activated C−H bonds. For many of these reactions, no “classical” chemical counterpart is known. Hence oxidoreductases open up shorter synthesis routes based on a more direct access to the target products. The generally very mild reaction conditions may also reduce the environmental impact of biocatalytic reactions compared to classical counterparts. In this Review, we critically summarise the most important recent developments in the field of biocatalytic oxidation chemistry and identify the most pressing bottlenecks as well as promising solutions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Oxidation chemistry using enzymes is approaching maturity and practical applicability in organic synthesis. Oxidoreductases (enzymes catalysing redox reactions) enable chemists to perform highly selective and efficient transformations ranging from simple alcohol oxidations to stereoselective halogenations of non-activated C−H bonds. For many of these reactions, no “classical” chemical counterpart is known. Hence oxidoreductases open up shorter synthesis routes based on a more direct access to the target products. The generally very mild reaction conditions may also reduce the environmental impact of biocatalytic reactions compared to classical counterparts. In this Review, we critically summarise the most important recent developments in the field of biocatalytic oxidation chemistry and identify the most pressing bottlenecks as well as promising solutions.
Dong, JiaJia; Fernández-Fueyo, Elena; Hollmann, Frank; Paul, Caroline E.; Pesic, Milja; Schmidt, Sandy; Wang, Yonghua; Younes, Sabry; Zhang, Wuyuan
Biokatalytische Oxidationsreaktionen - aus der Sicht eines Chemikers Journal Article
In: Angewandte Chemie, vol. 130, no. 30, pp. 9380–9404, 2018, ISSN: 1521-3757.
@article{Dong2018,
title = {Biokatalytische Oxidationsreaktionen - aus der Sicht eines Chemikers},
author = {JiaJia Dong and Elena Fernández-Fueyo and Frank Hollmann and Caroline E. Paul and Milja Pesic and Sandy Schmidt and Yonghua Wang and Sabry Younes and Wuyuan Zhang},
doi = {10.1002/ange.201800343},
issn = {1521-3757},
year = {2018},
date = {2018-01-01},
journal = {Angewandte Chemie},
volume = {130},
number = {30},
pages = {9380--9404},
abstract = {Enzyme gewinnen immer mehr Bedeutung als Katalysatoren füror- ganische Oxidationsreaktionen. Mit Oxidoreduktasen stehen dem Chemiker vielseitige Werkzeuge fürselektive und effiziente Oxida- tionsreaktionen zur Verfügung. Ihr Spektrum reicht von einfachen Alkoholoxidationen bis hin zur stereoselektiven Halogenierung nicht- aktivierter C-H-Bindungen. Fürviele biokatalytischeOxidationen ist bisher kein chemisches Pendant bekannt. Daher ermçglichen Oxido- reduktasen kürzere Syntheserouten. Darüber hinaus sind die generell milderen Reaktionsbedingungen biokatalytischer Oxidationen at- traktiv.Indiesem Aufsatz fassen wir einige aktuelle Entwicklungen im Bereichder biokatalytischen Oxidation zusammen. Wirbeschreiben auchderzeitige Grenzen und vielversprechende Lçsungsansätze.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Enzyme gewinnen immer mehr Bedeutung als Katalysatoren füror- ganische Oxidationsreaktionen. Mit Oxidoreduktasen stehen dem Chemiker vielseitige Werkzeuge fürselektive und effiziente Oxida- tionsreaktionen zur Verfügung. Ihr Spektrum reicht von einfachen Alkoholoxidationen bis hin zur stereoselektiven Halogenierung nicht- aktivierter C-H-Bindungen. Fürviele biokatalytischeOxidationen ist bisher kein chemisches Pendant bekannt. Daher ermçglichen Oxido- reduktasen kürzere Syntheserouten. Darüber hinaus sind die generell milderen Reaktionsbedingungen biokatalytischer Oxidationen at- traktiv.Indiesem Aufsatz fassen wir einige aktuelle Entwicklungen im Bereichder biokatalytischen Oxidation zusammen. Wirbeschreiben auchderzeitige Grenzen und vielversprechende Lçsungsansätze.
Schmidt, Sandy; Castiglione, Kathrin; Kourist, Robert
Overcoming the Incompatibility Challenge in Chemoenzymatic and Multi-Catalytic Cascade Reactions Journal Article
In: Chemistry - A European Journal, vol. 24, no. 8, pp. 1755–1768, 2018, ISSN: 15213765.
@article{Schmidt2018eb,
title = {Overcoming the Incompatibility Challenge in Chemoenzymatic and Multi-Catalytic Cascade Reactions},
author = {Sandy Schmidt and Kathrin Castiglione and Robert Kourist},
doi = {10.1002/chem.201703353},
issn = {15213765},
year = {2018},
date = {2018-01-01},
journal = {Chemistry - A European Journal},
volume = {24},
number = {8},
pages = {1755--1768},
abstract = {Multi-catalytic cascade reactions bear a great potential to minimize downstream and purification steps, leading to a drastic reduction of the produced waste. In many examples, the compatibility of chemo- and biocatalytic steps could be easily achieved. Problems associated with the incompatibility of the catalysts and their reactions, however, are very frequent. Cascade-like reactions can hardly occur in this way. One possible solution to combine, in principle, incompatible chemo- and biocatalytic reactions is the defined control of the microenvironment by compartmentalization or scaffolding. Current methods for the control of the microenvironment of biocatalysts go far beyond classical enzyme immobilization and are thus believed to be very promising tools to overcome incompatibility issues and to facilitate the synthetic application of cascade reactions. In this Minireview, we will summarize recent synthetic examples of (chemo)enzymatic cascade reactions and outline promising methods for their spatial control either by using bio-derived or synthetic systems.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Multi-catalytic cascade reactions bear a great potential to minimize downstream and purification steps, leading to a drastic reduction of the produced waste. In many examples, the compatibility of chemo- and biocatalytic steps could be easily achieved. Problems associated with the incompatibility of the catalysts and their reactions, however, are very frequent. Cascade-like reactions can hardly occur in this way. One possible solution to combine, in principle, incompatible chemo- and biocatalytic reactions is the defined control of the microenvironment by compartmentalization or scaffolding. Current methods for the control of the microenvironment of biocatalysts go far beyond classical enzyme immobilization and are thus believed to be very promising tools to overcome incompatibility issues and to facilitate the synthetic application of cascade reactions. In this Minireview, we will summarize recent synthetic examples of (chemo)enzymatic cascade reactions and outline promising methods for their spatial control either by using bio-derived or synthetic systems.
Schmidt, Sandy; Dörr, Mark; Bornscheuer, Uwe T.
Library growth and protein expression: Optimal and reproducible microtiter plate expression of recombinant enzymes in E. coli using MTP Shakers Journal Article
In: Methods in Molecular Biology, vol. 1685, pp. 145–156, 2018, ISSN: 10643745.
@article{Schmidt2018c,
title = {Library growth and protein expression: Optimal and reproducible microtiter plate expression of recombinant enzymes in E. coli using MTP Shakers},
author = {Sandy Schmidt and Mark Dörr and Uwe T. Bornscheuer},
doi = {10.1007/978-1-4939-7366-8_8},
issn = {10643745},
year = {2018},
date = {2018-01-01},
journal = {Methods in Molecular Biology},
volume = {1685},
pages = {145--156},
abstract = {Escherichia coli (E. coli) as heterologous host enables the recombinant expression of the desired protein in high amounts. Nevertheless, the expression in such a host, especially by utilizing a strong induction system, can result in insoluble and/or inactive protein fractions (inclusion bodies). Furthermore, the expression of different enzyme variants often leads to a diverse growth behavior of the E. coli clones resulting in the identification of false-positives when screening a mutant library. Thus, we developed a protocol for an optimal and reproducible protein expression in microtiter plates showcased for the expression of the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. By emerging this protocol, several parameters concerning the expression medium, the cultivation temperatures, shaking conditions as well as time and induction periods for CHMO were investigated. We employed a microtiter plate shaker with humidity and temperature control (Cytomat™) (integrated in a robotic platform) to obtain an even growth and expression over the plates. Our optimized protocol provides a comprehensive overview of the key factors influencing a reproducible protein expression and this should serve as basis for the adaptation to other enzyme classes.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Escherichia coli (E. coli) as heterologous host enables the recombinant expression of the desired protein in high amounts. Nevertheless, the expression in such a host, especially by utilizing a strong induction system, can result in insoluble and/or inactive protein fractions (inclusion bodies). Furthermore, the expression of different enzyme variants often leads to a diverse growth behavior of the E. coli clones resulting in the identification of false-positives when screening a mutant library. Thus, we developed a protocol for an optimal and reproducible protein expression in microtiter plates showcased for the expression of the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. By emerging this protocol, several parameters concerning the expression medium, the cultivation temperatures, shaking conditions as well as time and induction periods for CHMO were investigated. We employed a microtiter plate shaker with humidity and temperature control (Cytomat™) (integrated in a robotic platform) to obtain an even growth and expression over the plates. Our optimized protocol provides a comprehensive overview of the key factors influencing a reproducible protein expression and this should serve as basis for the adaptation to other enzyme classes.
2017
Böhmer, Stefanie; Köninger, Katharina; Gómez-Baraibar, Álvaro; Bojarra, Samiro; Mügge, Carolin; Schmidt, Sandy; Nowaczyk, Marc M.; Kourist, Robert
Enzymatic oxyfunctionalization driven by photosynthetic water-splitting in the cyanobacterium Synechocystis sp. PCC 6803 Miscellaneous
2017, ISSN: 20734344.
@misc{Bohmer2017,
title = {Enzymatic oxyfunctionalization driven by photosynthetic water-splitting in the cyanobacterium Synechocystis sp. PCC 6803},
author = {Stefanie Böhmer and Katharina Köninger and Álvaro Gómez-Baraibar and Samiro Bojarra and Carolin Mügge and Sandy Schmidt and Marc M. Nowaczyk and Robert Kourist},
doi = {10.3390/catal7080240},
issn = {20734344},
year = {2017},
date = {2017-01-01},
booktitle = {Catalysts},
volume = {7},
number = {8},
abstract = {Photosynthetic water-splitting is a powerful force to drive selective redox reactions. The need of highly expensive redox partners such as NADPH and their regeneration is one of the main bottlenecks for the application of biocatalysis at an industrial scale. Recently, the possibility of using the photosystem of cyanobacteria to supply high amounts of reduced nicotinamide to a recombinant enoate reductase opened a new strategy for overcoming this hurdle. This paper presents the expansion of the photosynthetic regeneration system to a Baeyer–Villiger monooxygenase. Despite the potential of this strategy, this work also presents some of the encountered challenges as well as possible solutions, which will require further investigation. The successful enzymatic oxygenation shows that cyanobacterial whole-cell biocatalysis is an applicable approach that allows fuelling selective oxyfunctionalisation reactions at the expense of light and water. Yet, several hurdles such as side-reactions and the cell-density limitation, probably due to self-shading of the cells, will have to be overcome on the way to synthetic applications.},
keywords = {},
pubstate = {published},
tppubtype = {misc}
}
Photosynthetic water-splitting is a powerful force to drive selective redox reactions. The need of highly expensive redox partners such as NADPH and their regeneration is one of the main bottlenecks for the application of biocatalysis at an industrial scale. Recently, the possibility of using the photosystem of cyanobacteria to supply high amounts of reduced nicotinamide to a recombinant enoate reductase opened a new strategy for overcoming this hurdle. This paper presents the expansion of the photosynthetic regeneration system to a Baeyer–Villiger monooxygenase. Despite the potential of this strategy, this work also presents some of the encountered challenges as well as possible solutions, which will require further investigation. The successful enzymatic oxygenation shows that cyanobacterial whole-cell biocatalysis is an applicable approach that allows fuelling selective oxyfunctionalisation reactions at the expense of light and water. Yet, several hurdles such as side-reactions and the cell-density limitation, probably due to self-shading of the cells, will have to be overcome on the way to synthetic applications.
Schmidt, Sandy; Almeida, Tiago Pedroso De; Rother, Dörte; Hollmann, Frank
Towards environmentally acceptable synthesis of chiral alpha-hydroxy ketones: Via oxidase-lyase cascades Journal Article
In: Green Chemistry, vol. 19, no. 5, pp. 1226–1229, 2017, ISSN: 14639270.
@article{Schmidt2017,
title = {Towards environmentally acceptable synthesis of chiral alpha-hydroxy ketones: Via oxidase-lyase cascades},
author = {Sandy Schmidt and Tiago Pedroso De Almeida and Dörte Rother and Frank Hollmann},
url = {http://dx.doi.org/10.1039/C7GC00020K},
doi = {10.1039/c7gc00020k},
issn = {14639270},
year = {2017},
date = {2017-01-01},
urldate = {2017-01-01},
journal = {Green Chemistry},
volume = {19},
number = {5},
pages = {1226--1229},
publisher = {The Royal Society of Chemistry},
abstract = {The one-pot multistep enzymatic oxidation of aliphatic and benzylic alcohols to the corresponding aldehydes combined with their subsequent carboligation to chiral $alpha$-hydroxy ketones has been exemplarily evaluated in terms of being a "green" biocatalytic approach. Besides the potential to start from bio-derived alcohols, this concept avoids the direct use of the reactive aldehyde intermediates, enables addition of high substrate concentrations in one liquid phase while maintaining enzyme activity and enables a simplified product isolation with diminished waste formation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The one-pot multistep enzymatic oxidation of aliphatic and benzylic alcohols to the corresponding aldehydes combined with their subsequent carboligation to chiral $alpha$-hydroxy ketones has been exemplarily evaluated in terms of being a "green" biocatalytic approach. Besides the potential to start from bio-derived alcohols, this concept avoids the direct use of the reactive aldehyde intermediates, enables addition of high substrate concentrations in one liquid phase while maintaining enzyme activity and enables a simplified product isolation with diminished waste formation.
Schmidt, Sandy; Gröger, Harald; Kourist, Robert
Durch Trennen verbinden Journal Article
In: Nachrichten aus der Chemie, vol. 65, no. 10, pp. 985–988, 2017, ISSN: 14399598.
@article{Schmidt2017c,
title = {Durch Trennen verbinden},
author = {Sandy Schmidt and Harald Gröger and Robert Kourist},
doi = {10.1002/nadc.20174058532},
issn = {14399598},
year = {2017},
date = {2017-01-01},
urldate = {2017-01-01},
journal = {Nachrichten aus der Chemie},
volume = {65},
number = {10},
pages = {985--988},
abstract = {Mehrstufensynthesen gelingen unter milden Reaktionsbedingungen, in hohen Raum-Zeit-Ausbeuten und mit hervorragender Stereoselektivität, wenn enzymatisch und metallisch katalysierte Reaktionen kombiniert werden. Allerdings sind beide Systeme schwierig zu verbinden, da Metalle Enzyme deaktivieren können. Funktionieren kann es, wenn beide Katalysatoren in getrennten Reaktionsräumen arbeiten.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Mehrstufensynthesen gelingen unter milden Reaktionsbedingungen, in hohen Raum-Zeit-Ausbeuten und mit hervorragender Stereoselektivität, wenn enzymatisch und metallisch katalysierte Reaktionen kombiniert werden. Allerdings sind beide Systeme schwierig zu verbinden, da Metalle Enzyme deaktivieren können. Funktionieren kann es, wenn beide Katalysatoren in getrennten Reaktionsräumen arbeiten.
Rauch, M.; Schmidt, S.; Arends, I. W. C. E.; Oppelt, K.; Kara, S.; Hollmann, F.
Photobiocatalytic alcohol oxidation using LED light sources Journal Article
In: Green Chemistry, vol. 19, no. 2, pp. 376–379, 2017, ISSN: 14639270.
@article{Rauch2017,
title = {Photobiocatalytic alcohol oxidation using LED light sources},
author = {M. Rauch and S. Schmidt and I. W. C. E. Arends and K. Oppelt and S. Kara and F. Hollmann},
doi = {10.1039/c6gc02008a},
issn = {14639270},
year = {2017},
date = {2017-01-01},
journal = {Green Chemistry},
volume = {19},
number = {2},
pages = {376--379},
abstract = {The photocatalytic oxidation of NADH using a flavin photocatalyst and a simple blue LED light source is reported. This in situ NAD+ regeneration system can be used to promote biocatalytic, enantioselective oxidation reactions. Compared to the traditional use of white light bulbs this method enables very significant reductions in energy consumption and CO2 emission.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The photocatalytic oxidation of NADH using a flavin photocatalyst and a simple blue LED light source is reported. This in situ NAD+ regeneration system can be used to promote biocatalytic, enantioselective oxidation reactions. Compared to the traditional use of white light bulbs this method enables very significant reductions in energy consumption and CO2 emission.
Reimer, Anna; Wedde, Severin; Staudt, Svenja; Schmidt, Sandy; Höffer, Diana; Hummel, Werner; Kragl, Udo; Bornscheuer, Uwe T.; Gröger, Harald
Process Development through Solvent Engineering in the Biocatalytic Synthesis of the Heterocyclic Bulk Chemical epsilon-Caprolactone Journal Article
In: Journal of Heterocyclic Chemistry, vol. 54, no. 1, pp. 391–396, 2017, ISSN: 19435193.
@article{Reimer2017,
title = {Process Development through Solvent Engineering in the Biocatalytic Synthesis of the Heterocyclic Bulk Chemical epsilon-Caprolactone},
author = {Anna Reimer and Severin Wedde and Svenja Staudt and Sandy Schmidt and Diana Höffer and Werner Hummel and Udo Kragl and Uwe T. Bornscheuer and Harald Gröger},
doi = {10.1002/jhet.2595},
issn = {19435193},
year = {2017},
date = {2017-01-01},
urldate = {2017-01-01},
journal = {Journal of Heterocyclic Chemistry},
volume = {54},
number = {1},
pages = {391--396},
abstract = {For an alternative synthetic approach toward the heterocyclic industrial chemical $epsilon$-caprolactone, which is based on a biocatalytic oxidation of readily available cyclohexanol with air in aqueous media (using an alcohol dehydrogenase and a Baeyer–Villiger monooxygenase as enzyme components), a solvent engineering has been carried out identifying isooctane as a suitable co-solvent. Biotransformations in an aqueous-isooctane biphasic solvent system were found to proceed faster at both investigated substrate concentrations of 40 and 80 mm, respectively, compared with the analogous enzymatic reactions in pure aqueous medium. In addition, in all cases quantitative conversions were observed after a reaction time of 23 h when using isolated enzymes. The achievements indicate a high compatibility of isooctane [10%(v/v)] with the enzymes as well as the potential for an in situ removal of the organic reaction components, thus decreasing inhibition and/or destabilization effects of these organic components on the enzymes used. In contrast, so far, the use of recombinant whole-cells gave less satisfactory results, which might be due to limitations of the permeation of, for example, the substrate through the cell membrane.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
For an alternative synthetic approach toward the heterocyclic industrial chemical $epsilon$-caprolactone, which is based on a biocatalytic oxidation of readily available cyclohexanol with air in aqueous media (using an alcohol dehydrogenase and a Baeyer–Villiger monooxygenase as enzyme components), a solvent engineering has been carried out identifying isooctane as a suitable co-solvent. Biotransformations in an aqueous-isooctane biphasic solvent system were found to proceed faster at both investigated substrate concentrations of 40 and 80 mm, respectively, compared with the analogous enzymatic reactions in pure aqueous medium. In addition, in all cases quantitative conversions were observed after a reaction time of 23 h when using isolated enzymes. The achievements indicate a high compatibility of isooctane [10%(v/v)] with the enzymes as well as the potential for an in situ removal of the organic reaction components, thus decreasing inhibition and/or destabilization effects of these organic components on the enzymes used. In contrast, so far, the use of recombinant whole-cells gave less satisfactory results, which might be due to limitations of the permeation of, for example, the substrate through the cell membrane.
Younes, Sabry H. H.; Ni, Yan; Schmidt, Sandy; Kroutil, Wolfgang; Hollmann, Frank
Alcohol Dehydrogenases Catalyze the Reduction of Thioesters Journal Article
In: ChemCatChem, vol. 9, no. 8, pp. 1389–1392, 2017, ISSN: 18673899.
@article{Younes2017b,
title = {Alcohol Dehydrogenases Catalyze the Reduction of Thioesters},
author = {Sabry H. H. Younes and Yan Ni and Sandy Schmidt and Wolfgang Kroutil and Frank Hollmann},
doi = {10.1002/cctc.201700165},
issn = {18673899},
year = {2017},
date = {2017-01-01},
journal = {ChemCatChem},
volume = {9},
number = {8},
pages = {1389--1392},
abstract = {Alcohol dehydrogenases are well-established catalysts for various reduction reactions. However, the reduction of carboxylic acid derivatives has not yet been reported with these enzymes. In this contribution, we demonstrated that carboxylic acid thioesters could be readily reduced by a range of alcohol dehydrogenases, albeit at significantly reduced rates relative to those observed for corresponding ketones. A molecular explanation, especially for the lower turnover rates for thioesters relative to those obtained for ketones, is presented, as is a preliminary substrate scope.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Alcohol dehydrogenases are well-established catalysts for various reduction reactions. However, the reduction of carboxylic acid derivatives has not yet been reported with these enzymes. In this contribution, we demonstrated that carboxylic acid thioesters could be readily reduced by a range of alcohol dehydrogenases, albeit at significantly reduced rates relative to those observed for corresponding ketones. A molecular explanation, especially for the lower turnover rates for thioesters relative to those obtained for ketones, is presented, as is a preliminary substrate scope.
Scherkus, Christian; Schmidt, Sandy; Bornscheuer, Uwe T.; Gröger, Harald; Kara, Selin; Liese, Andreas
Kinetic insights into ϵ-caprolactone synthesis: Improvement of an enzymatic cascade reaction Journal Article
In: Biotechnology and Bioengineering, vol. 114, no. 6, pp. 1215–1221, 2017, ISSN: 10970290.
@article{Scherkus2017b,
title = {Kinetic insights into ϵ-caprolactone synthesis: Improvement of an enzymatic cascade reaction},
author = {Christian Scherkus and Sandy Schmidt and Uwe T. Bornscheuer and Harald Gröger and Selin Kara and Andreas Liese},
doi = {10.1002/bit.26258},
issn = {10970290},
year = {2017},
date = {2017-01-01},
journal = {Biotechnology and Bioengineering},
volume = {114},
number = {6},
pages = {1215--1221},
abstract = {A computational approach for the simulation and prediction of a linear three-step enzymatic cascade for the synthesis of ϵ-caprolactone (ECL) coupling an alcohol dehydrogenase (ADH), a cyclohexanone monooxygenase (CHMO), and a lipase for the subsequent hydrolysis of ECL to 6-hydroxyhexanoic acid (6-HHA). A kinetic model was developed with an accuracy of prediction for a fed-batch mode of 37% for substrate cyclohexanol (CHL), 90% for ECL, and >99% for the final product 6-HHA. Due to a severe inhibition of the CHMO by CHL, a batch synthesis was shown to be less efficient than a fed-batch approach. In the fed-batch synthesis, full conversion of 100 mM CHL was 28% faster with an analytical yield of 98% compared to 49% in case of the batch synthesis. The lipase-catalyzed hydrolysis of ECL to 6-HHA circumvents the inhibition of the CHMO by ECL enabling a 24% higher product concentration of 6-HHA compared to ECL in case of the fed-batch synthesis without lipase. Biotechnol. Bioeng. 2017;114: 1215–1221. textcopyright 2017 Wiley Periodicals, Inc.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A computational approach for the simulation and prediction of a linear three-step enzymatic cascade for the synthesis of ϵ-caprolactone (ECL) coupling an alcohol dehydrogenase (ADH), a cyclohexanone monooxygenase (CHMO), and a lipase for the subsequent hydrolysis of ECL to 6-hydroxyhexanoic acid (6-HHA). A kinetic model was developed with an accuracy of prediction for a fed-batch mode of 37% for substrate cyclohexanol (CHL), 90% for ECL, and >99% for the final product 6-HHA. Due to a severe inhibition of the CHMO by CHL, a batch synthesis was shown to be less efficient than a fed-batch approach. In the fed-batch synthesis, full conversion of 100 mM CHL was 28% faster with an analytical yield of 98% compared to 49% in case of the batch synthesis. The lipase-catalyzed hydrolysis of ECL to 6-HHA circumvents the inhibition of the CHMO by ECL enabling a 24% higher product concentration of 6-HHA compared to ECL in case of the fed-batch synthesis without lipase. Biotechnol. Bioeng. 2017;114: 1215–1221. textcopyright 2017 Wiley Periodicals, Inc.
Wedde, Severin; Rommelmann, Philipp; Scherkus, Christian; Schmidt, Sandy; Bornscheuer, Uwe T.; Liese, Andreas; Gröger, Harald
In: Green Chemistry, vol. 19, no. 5, pp. 1286–1290, 2017, ISSN: 14639270.
@article{Wedde2017b,
title = {An alternative approach towards poly-epsilon-caprolactone through a chemoenzymatic synthesis: Combined hydrogenation, bio-oxidations and polymerization without the isolation of intermediates},
author = {Severin Wedde and Philipp Rommelmann and Christian Scherkus and Sandy Schmidt and Uwe T. Bornscheuer and Andreas Liese and Harald Gröger},
doi = {10.1039/c6gc02529c},
issn = {14639270},
year = {2017},
date = {2017-01-01},
urldate = {2017-01-01},
journal = {Green Chemistry},
volume = {19},
number = {5},
pages = {1286--1290},
abstract = {A novel synthetic route towards the polymer poly-$epsilon$-caprolactone based on a chemoenzymatic reaction sequence was developed. Initial hydrogenation of phenol to cyclohexanol gave a crude product, which was directly used without work-up for a subsequent biocatalytic double oxidation towards $epsilon$-caprolactone by means of an alcohol dehydrogenase and a monooxygenase. In order to overcome product inhibition effects, an in situ-product removal strategy via extraction of $epsilon$-caprolactone from an aqueous reaction medium with an organic solvent in the presence of a permeable polydimethylsiloxane membrane was applied. Furthermore, this in situ-product removal was combined with lipase-catalyzed polymerization in the organic phase at 25 °C. The obtained crude product contained a polymer fraction with a degree of polymerization comparable to commercial poly-$epsilon$-caprolactone.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A novel synthetic route towards the polymer poly-$epsilon$-caprolactone based on a chemoenzymatic reaction sequence was developed. Initial hydrogenation of phenol to cyclohexanol gave a crude product, which was directly used without work-up for a subsequent biocatalytic double oxidation towards $epsilon$-caprolactone by means of an alcohol dehydrogenase and a monooxygenase. In order to overcome product inhibition effects, an in situ-product removal strategy via extraction of $epsilon$-caprolactone from an aqueous reaction medium with an organic solvent in the presence of a permeable polydimethylsiloxane membrane was applied. Furthermore, this in situ-product removal was combined with lipase-catalyzed polymerization in the organic phase at 25 °C. The obtained crude product contained a polymer fraction with a degree of polymerization comparable to commercial poly-$epsilon$-caprolactone.
2016
Beier, Andy; Bordewick, Sven; Genz, Maika; Schmidt, Sandy; Bergh, Tom; Peters, Christin; Joosten, Henk Jan; Bornscheuer, Uwe T.
Switch in Cofactor Specificity of a Baeyer–Villiger Monooxygenase Journal Article
In: ChemBioChem, vol. 17, no. 24, pp. 2312–2315, 2016, ISSN: 14397633.
@article{Beier2016bb,
title = {Switch in Cofactor Specificity of a Baeyer–Villiger Monooxygenase},
author = {Andy Beier and Sven Bordewick and Maika Genz and Sandy Schmidt and Tom Bergh and Christin Peters and Henk Jan Joosten and Uwe T. Bornscheuer},
url = {https://doi.org/10.1002/cbic.201600484},
doi = {10.1002/cbic.201600484},
issn = {14397633},
year = {2016},
date = {2016-12-01},
journal = {ChemBioChem},
volume = {17},
number = {24},
pages = {2312--2315},
publisher = {John Wiley & Sons, Ltd},
abstract = {Baeyer–Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMOAcineto); this used NADH 4200-fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79 %) compared to NADPH (58 %) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen-bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Baeyer–Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMOAcineto); this used NADH 4200-fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79 %) compared to NADPH (58 %) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen-bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.
Dörr, Mark; Fibinger, Michael P. C.; Last, Daniel; Schmidt, Sandy; Santos-Aberturas, Javier; Böttcher, Dominique; Hummel, Anke; Vickers, Clare; Voss, Moritz; Bornscheuer, Uwe T.
Fully automatized high-throughput enzyme library screening using a robotic platform Journal Article
In: Biotechnology and Bioengineering, vol. 113, no. 7, pp. 1421–1432, 2016, ISSN: 10970290.
@article{Dorr2016,
title = {Fully automatized high-throughput enzyme library screening using a robotic platform},
author = {Mark Dörr and Michael P. C. Fibinger and Daniel Last and Sandy Schmidt and Javier Santos-Aberturas and Dominique Böttcher and Anke Hummel and Clare Vickers and Moritz Voss and Uwe T. Bornscheuer},
doi = {10.1002/bit.25925},
issn = {10970290},
year = {2016},
date = {2016-01-01},
journal = {Biotechnology and Bioengineering},
volume = {113},
number = {7},
pages = {1421--1432},
abstract = {A fully automatized robotic platform has been established to facilitate high-throughput screening for protein engineering purposes. This platform enables proper monitoring and control of growth conditions in the microtiter plate format to ensure precise enzyme production for the interrogation of enzyme mutant libraries, protein stability tests and multiple assay screenings. The performance of this system has been exemplified for four enzyme classes important for biocatalysis such as Baeyer-Villiger monooxygenase, transaminase, dehalogenase and acylase in the high-throughput screening of various mutant libraries. This allowed the identification of novel enzyme variants in a sophisticated and highly reliable manner. Furthermore, the detailed optimization protocols should enable other researchers to adapt and improve their methods.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A fully automatized robotic platform has been established to facilitate high-throughput screening for protein engineering purposes. This platform enables proper monitoring and control of growth conditions in the microtiter plate format to ensure precise enzyme production for the interrogation of enzyme mutant libraries, protein stability tests and multiple assay screenings. The performance of this system has been exemplified for four enzyme classes important for biocatalysis such as Baeyer-Villiger monooxygenase, transaminase, dehalogenase and acylase in the high-throughput screening of various mutant libraries. This allowed the identification of novel enzyme variants in a sophisticated and highly reliable manner. Furthermore, the detailed optimization protocols should enable other researchers to adapt and improve their methods.
Balke, Kathleen; Schmidt, Sandy; Genz, Maika; Bornscheuer, Uwe T.
Switching the Regioselectivity of a Cyclohexanone Monooxygenase toward (+)-trans-Dihydrocarvone by Rational Protein Design Journal Article
In: ACS Chemical Biology, vol. 11, no. 1, pp. 38–43, 2016, ISSN: 15548937.
@article{Balke2016b,
title = {Switching the Regioselectivity of a Cyclohexanone Monooxygenase toward (+)-trans-Dihydrocarvone by Rational Protein Design},
author = {Kathleen Balke and Sandy Schmidt and Maika Genz and Uwe T. Bornscheuer},
doi = {10.1021/acschembio.5b00723},
issn = {15548937},
year = {2016},
date = {2016-01-01},
journal = {ACS Chemical Biology},
volume = {11},
number = {1},
pages = {38--43},
abstract = {The regioselectivity of the Baeyer-Villiger monooxygenase-catalyzed oxidation is governed mostly by electronic effects leading to the migration of the higher substituted residue. However, in some cases, substrate binding occurs in a way that the less substituted residue lies in an antiperiplanar orientation to the peroxy bond in the Criegee intermediate yielding in the formation of the "abnormal" lactone product. We are the first to demonstrate a complete switch in the regioselectivity of the BVMO from Arthrobacter sp. (CHMOArthro) as exemplified for (+)-trans-dihydrocarvone by redesigning the active site of the enzyme. In the designed triple mutant, the substrate binds in an inverted orientation leading to a ratio of 99:1 in favor of the normal lactone instead of exclusive formation of the abnormal lactone in case of the wild type enzyme. In order to validate our computational study, the beneficial mutations were successfully transferred to the CHMO from Acinetobacter sp. (CHMOAcineto), again yielding in a complete switch of regioselectivity.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The regioselectivity of the Baeyer-Villiger monooxygenase-catalyzed oxidation is governed mostly by electronic effects leading to the migration of the higher substituted residue. However, in some cases, substrate binding occurs in a way that the less substituted residue lies in an antiperiplanar orientation to the peroxy bond in the Criegee intermediate yielding in the formation of the "abnormal" lactone product. We are the first to demonstrate a complete switch in the regioselectivity of the BVMO from Arthrobacter sp. (CHMOArthro) as exemplified for (+)-trans-dihydrocarvone by redesigning the active site of the enzyme. In the designed triple mutant, the substrate binds in an inverted orientation leading to a ratio of 99:1 in favor of the normal lactone instead of exclusive formation of the abnormal lactone in case of the wild type enzyme. In order to validate our computational study, the beneficial mutations were successfully transferred to the CHMO from Acinetobacter sp. (CHMOAcineto), again yielding in a complete switch of regioselectivity.
Genz, Maika; Melse, Okke; Schmidt, Sandy; Vickers, Clare; Dörr, Mark; Bergh, Tom; Joosten, Henk Jan; Bornscheuer, Uwe T.
Engineering the Amine Transaminase from Vibrio fluvialis towards Branched-Chain Substrates Journal Article
In: ChemCatChem, vol. 8, no. 20, pp. 3199–3202, 2016, ISSN: 18673899.
@article{Genz2016b,
title = {Engineering the Amine Transaminase from Vibrio fluvialis towards Branched-Chain Substrates},
author = {Maika Genz and Okke Melse and Sandy Schmidt and Clare Vickers and Mark Dörr and Tom Bergh and Henk Jan Joosten and Uwe T. Bornscheuer},
doi = {10.1002/cctc.201601007},
issn = {18673899},
year = {2016},
date = {2016-01-01},
journal = {ChemCatChem},
volume = {8},
number = {20},
pages = {3199--3202},
abstract = {Chiral amines are important building blocks, especially for the pharmaceutical industry. Although amine transaminases (ATAs) are versatile enzymes to synthesize chiral amines, the wildtype enzymes do not accept ketones with two large substituents next to the carbonyl functionality. Using bioinformatic tools to design a seven-site mutant library followed by high-throughput screening, we were able to identify variants of the enzyme from Vibrio fluvialis (VF-ATA) with a widened binding pocket, as exemplified for a range of ketones. Three variants allowed the asymmetric synthesis of 2,2-dimethyl-1-phenylpropan-1-amine—not accessible by any wildtype ATA described so far. The best variant containing four mutations (L56V, W57C, F85V, V153A) gave 100 % conversion of the ketone to yield the amine with an enantiomeric excess value >99 %, notably with preference for the (R)-enantiomer. In silico modeling enabled the reconstruction of the substrate binding mode to the newly evolved pocket and, hence, allowed explanation of the experimental results.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Chiral amines are important building blocks, especially for the pharmaceutical industry. Although amine transaminases (ATAs) are versatile enzymes to synthesize chiral amines, the wildtype enzymes do not accept ketones with two large substituents next to the carbonyl functionality. Using bioinformatic tools to design a seven-site mutant library followed by high-throughput screening, we were able to identify variants of the enzyme from Vibrio fluvialis (VF-ATA) with a widened binding pocket, as exemplified for a range of ketones. Three variants allowed the asymmetric synthesis of 2,2-dimethyl-1-phenylpropan-1-amine—not accessible by any wildtype ATA described so far. The best variant containing four mutations (L56V, W57C, F85V, V153A) gave 100 % conversion of the ketone to yield the amine with an enantiomeric excess value >99 %, notably with preference for the (R)-enantiomer. In silico modeling enabled the reconstruction of the substrate binding mode to the newly evolved pocket and, hence, allowed explanation of the experimental results.
Scherkus, Christian; Schmidt, Sandy; Bornscheuer, Uwe T.; Gröger, Harald; Kara, Selin; Liese, Andreas
A Fed-Batch Synthetic Strategy for a Three-Step Enzymatic Synthesis of Poly-ϵ-caprolactone Journal Article
In: ChemCatChem, vol. 8, no. 22, pp. 3446–3452, 2016, ISSN: 18673899.
@article{Scherkus2016b,
title = {A Fed-Batch Synthetic Strategy for a Three-Step Enzymatic Synthesis of Poly-ϵ-caprolactone},
author = {Christian Scherkus and Sandy Schmidt and Uwe T. Bornscheuer and Harald Gröger and Selin Kara and Andreas Liese},
doi = {10.1002/cctc.201600806},
issn = {18673899},
year = {2016},
date = {2016-01-01},
journal = {ChemCatChem},
volume = {8},
number = {22},
pages = {3446--3452},
abstract = {A three-step enzymatic reaction sequence for the synthesis of poly-ϵ-caprolactone (PCL) was designed running in a fed-batch operation. The first part of the cascade consisted of two oxidation steps starting with alcohol dehydrogenase catalyzed oxidation from cyclohexanol to cyclohexanone and further oxidation to ϵ-caprolactone (ECL) by means of a Baeyer–Villiger monooxygenase. As a third step, lipase-catalyzed hydrolysis of the lactone to 6-hydroxyhexanoic acid (6-HHA) was designed. With this biocatalytic multistep process reported herein, severe substrate surplus and product inhibition could be circumvented by the fed-batch operation by adding the cyclohexanol substrate and by in situ product removal of ECL by hydrolysis, respectively. Up to 283 mm product concentration of 6-HHA was reached in the fed-batch operated process without loss in productivity within 20 h. After extraction and subsequent polymerization catalyzed by Candida antarctica lipase B, analysis of the unfractionated polymer revealed a bimodal distribution of the polymer population, which reached a mass average molar mass (Mw) value of approximately 63 000 g mol−1 and a dispersity (Mw/Mn) of 1.1 for the higher molecular weight population, which thus revealed an alternative route to the conventional synthesis of PCL.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A three-step enzymatic reaction sequence for the synthesis of poly-ϵ-caprolactone (PCL) was designed running in a fed-batch operation. The first part of the cascade consisted of two oxidation steps starting with alcohol dehydrogenase catalyzed oxidation from cyclohexanol to cyclohexanone and further oxidation to ϵ-caprolactone (ECL) by means of a Baeyer–Villiger monooxygenase. As a third step, lipase-catalyzed hydrolysis of the lactone to 6-hydroxyhexanoic acid (6-HHA) was designed. With this biocatalytic multistep process reported herein, severe substrate surplus and product inhibition could be circumvented by the fed-batch operation by adding the cyclohexanol substrate and by in situ product removal of ECL by hydrolysis, respectively. Up to 283 mm product concentration of 6-HHA was reached in the fed-batch operated process without loss in productivity within 20 h. After extraction and subsequent polymerization catalyzed by Candida antarctica lipase B, analysis of the unfractionated polymer revealed a bimodal distribution of the polymer population, which reached a mass average molar mass (Mw) value of approximately 63 000 g mol−1 and a dispersity (Mw/Mn) of 1.1 for the higher molecular weight population, which thus revealed an alternative route to the conventional synthesis of PCL.