Engineered enzymes for the synthesis of pharmaceuticals and other high-value products

Nature Synthesis, Год журнала: 2024, Номер 3(1), С. 19 - 32

Опубликована: Янв. 4, 2024

Язык: Английский

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Ultrahigh-Throughput Enzyme Engineering and Discovery in In Vitro Compartments

Chemical Reviews, Год журнала: 2023, Номер 123(9), С. 5571 - 5611

Опубликована: Май 1, 2023

Novel and improved biocatalysts are increasingly sourced from libraries via experimental screening. The success of such campaigns is crucially dependent on the number candidates tested. Water-in-oil emulsion droplets can replace classical test tube, to provide in vitro compartments as an alternative screening format, containing genotype phenotype enabling a readout function. scale-down micrometer droplet diameters picoliter volumes brings about >107-fold volume reduction compared 96-well-plate Droplets made automated microfluidic devices be integrated into modular workflows set up multistep protocols involving various detection modes sort >107 variants day with kHz frequencies. repertoire assays available for covers all seven enzyme commission (EC) classes, setting stage widespread use microfluidics everyday biochemical experiments. We review practicalities adapting discovery detailed kinetic characterization. These new ways working will not just accelerate experiments currently limited by capacity but profoundly change paradigms we probe. By interfacing results ultrahigh-throughput next-generation sequencing deep learning, strategies directed evolution implemented, examined, evaluated.

Язык: Английский

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Direct visible-light-excited flavoproteins for redox-neutral asymmetric radical hydroarylation

Nature Catalysis, Год журнала: 2023, Номер 6(11), С. 996 - 1004

Опубликована: Сен. 14, 2023

Язык: Английский

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Making Enzymes Suitable for Organic Chemistry by Rational Protein Design

ChemBioChem, Год журнала: 2022, Номер 23(14)

Опубликована: Апрель 7, 2022

This review outlines recent developments in protein engineering of stereo- and regioselective enzymes, which are prime interest organic pharmaceutical chemistry as well biotechnology. The widespread application enzymes was hampered for decades due to limited enantio-, diastereo- regioselectivity, the reason why most chemists were not interested biocatalysis. attitude began change with advent semi-rational directed evolution methods based on focused saturation mutagenesis at sites lining binding pocket. Screening constitutes labor-intensive step (bottleneck), is various research groups continuing develop techniques generation small smart mutant libraries. Rational enzyme design, traditionally an alternative evolution, provides collections mutants require minimal screening. approach first thermostabilization, did enter field stereoselectivity until later. Computational guides such Rosetta algorithms, HotSpot Wizard metric, machine learning (ML) contribute significantly decision making. newest advancements show that CAST/ISM rational design no longer separate tracks, instead, they have started merge. Indeed, researchers utilizing two approaches learned from each other. Today, toolbox includes primarily because possibility controlling by has ensured reliability when facing synthetic challenges. also written hope undergraduate graduate education will include more so than past.

Язык: Английский

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Focused rational iterative site-specific mutagenesis (FRISM): A powerful method for enzyme engineering

Molecular Catalysis, Год журнала: 2023, Номер 553, С. 113755 - 113755

Опубликована: Дек. 9, 2023

Язык: Английский

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Iron Heme Enzyme-Catalyzed Cyclopropanations with Diazirines as Carbene Precursors: Computational Explorations of Diazirine Activation and Cyclopropanation Mechanism

Journal of the American Chemical Society, Год журнала: 2024, Номер 146(5), С. 2959 - 2966

Опубликована: Янв. 25, 2024

The mechanism of cyclopropanations with diazirines as air-stable and user-friendly alternatives to commonly employed diazo compounds within iron heme enzyme-catalyzed carbene transfer reactions has been studied by means density functional theory (DFT) calculations model systems, quantum mechanics/molecular mechanics (QM/MM) calculations, molecular dynamics (MD) simulations the cyclopropanation transition state in enzyme active site. reaction is initiated a direct diazirine-diazo isomerization occurring site enzyme. In contrast, an proceeding via formation free intermediate lieu direct, one-step process was observed for systems. Subsequent benzyl acrylate takes place through stepwise C–C bond diradical intermediate, delivering cyclopropane product. origin diastereo- enantioselectivity investigated MD simulations, which indicate preferred cis-cyclopropane steric control.

Язык: Английский

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High-Throughput Screening Techniques for the Selection of Thermostable Enzymes

Journal of Agricultural and Food Chemistry, Год журнала: 2024, Номер 72(8), С. 3833 - 3845

Опубликована: Янв. 29, 2024

The acquisition of a thermostable enzyme is an indispensable prerequisite for its successful implementation in industrial applications and the development novel functionalities. Various protein engineering approaches, including rational design, semirational directed evolution, have been employed to enhance thermostability. However, all these approaches require sensitive reliable high-throughput screening (HTS) technologies efficiently rapidly identify variants with improved properties. While numerous reviews focus on modification strategies enhancing thermostability, there dearth literature reviewing HTS methods specifically aimed at this objective. Herein, we present comprehensive overview various utilized modifying thermostability across different platforms. Additionally, highlight significant recent examples that demonstrate application methods. Furthermore, address technical challenges associated used discuss valuable perspectives promote further advancements field. This review serves as authoritative reference source offering theoretical support selecting appropriate tailored specific enzymes aim improving their

Язык: Английский

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Asymmetric α-benzylation of cyclic ketones enabled by concurrent chemical aldol condensation and biocatalytic reduction

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Янв. 2, 2024

Abstract Chemoenzymatic cascade catalysis has emerged as a revolutionary tool for streamlining traditional retrosynthetic disconnections, creating new possibilities the asymmetric synthesis of valuable chiral compounds. Here we construct one-pot concurrent chemoenzymatic by integrating organobismuth-catalyzed aldol condensation with ene-reductase (ER)-catalyzed enantioselective reduction, enabling formal α-benzylation cyclic ketones. To achieve this, develop pair enantiocomplementary ERs capable reducing α-arylidene ketones, lactams, and lactones. Our engineered mutants exhibit significantly higher activity, up to 37-fold, broader substrate specificity compared parent enzyme. The key success is due well-tuned hydride attack distance/angle and, more importantly, synergistic proton-delivery triade Tyr28-Tyr69-Tyr169. Molecular docking density functional theory (DFT) studies provide important insights into bioreduction mechanisms. Furthermore, demonstrate synthetic utility best in several synthons.

Язык: Английский

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Efficient and feasible biocatalysts: Strategies for enzyme improvement. A review

International Journal of Biological Macromolecules, Год журнала: 2024, Номер 276, С. 133978 - 133978

Опубликована: Июль 20, 2024

Язык: Английский

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Enhancing enzymatic activity with nanoparticle display – an updated compendium and engineering outlook

Molecular Systems Design & Engineering, Год журнала: 2024, Номер 9(7), С. 679 - 704

Опубликована: Янв. 1, 2024

Schematic depicting enzyme kinetic enhancement when displayed on a nanoparticle surface. We provide state of the art review this phenomenon describing what is known about how it arises along with examples grouped by nanomaterials.

Язык: Английский

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