On synergy between ultrahigh throughput screening and machine learning in biocatalyst engineering

Faraday Discussions, Journal Year: 2024, Volume and Issue: 252, P. 89 - 114

Published: Jan. 1, 2024

Protein design and directed evolution have separately contributed enormously to protein engineering. Without being mutually exclusive, the former relies on computation from first principles, while latter is a combinatorial approach based chance. Advances in ultrahigh throughput (uHT) screening, next generation sequencing machine learning may create alternative routes engineered proteins, where functional information linked specific sequences interpreted extrapolated

Language: Английский

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Versatile Product Detection via Coupled Assays for Ultrahigh-Throughput Screening of Carbohydrate-Active Enzymes in Microfluidic Droplets

ACS Catalysis, Journal Year: 2023, Volume and Issue: 13(15), P. 10232 - 10243

Published: July 21, 2023

Enzyme discovery and directed evolution are the two major contemporary approaches for improvement of industrial processes by biocatalysis in various fields. Customization catalysts single enzyme reactions or de novo reaction development is often complex tedious. The success screening campaigns relies on fraction sequence space that can be sampled, whether evolving a particular metagenomes. Ultrahigh-throughput (uHTS) based vitro compartmentalization water-in-oil emulsion picoliter droplets generated microfluidic systems allows rates >1 kHz (or >107 per day). Screening carbohydrate-active enzymes (CAZymes) catalyzing biotechnologically valuable this format presents an additional challenge because released carbohydrates difficult to monitor high throughput. Activated substrates with large optically active hydrophobic leaving groups provide generic optical readout, but molecular recognition properties sugars will altered incorporation such fluoro- chromophores their typically higher reactivity, as lowered pKa values compared native make observation promiscuous more likely. To overcome these issues, we designed microdroplet assays which inactive carbohydrate products made visible specific cascades: primary unlabeled substrate leads signal downstream. Successfully implementing at droplet scale allowed us detect glucose, xylose, glucuronic acid, arabinose final oligosaccharide degradation glycoside hydrolases absorbance measurements. Enabling use uHTS CAZyme have been thus far elusive chart route toward faster easier efficient biocatalysts biovalorization, directing challenging natural rather than model substrates.

Language: Английский

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In Vitro Enzyme Self-Selection Using Molecular Programs

ACS Synthetic Biology, Journal Year: 2024, Volume and Issue: 13(2), P. 474 - 484

Published: Jan. 11, 2024

Directed evolution provides a powerful route for in vitro enzyme engineering. State-of-the-art techniques functionally screen up to millions of variants using high throughput microfluidic sorters, whose operation remains technically challenging. Alternatively, self-selection methods, analogous vivo complementation strategies, open the way even higher throughputs, but have been demonstrated only few specific activities. Here, we leverage synthetic molecular networks generalize compartmentalized processes. We introduce programmable circuit architecture that can link an arbitrary target enzymatic activity replication its encoding gene. Microencapsulation bacterial expression library with this autonomous selection results single-step and screening-free enrichment genetic sequences coding programmed phenotypes. demonstrate potential approach nicking Nt.BstNBI (NBI). applied conditions enrich thermostability or catalytic efficiency, manipulating 107 microcompartments 5 × 105 at once. Full gene reads libraries nanopore sequencing revealed detailed mutational landscapes, suggesting key role electrostatic interactions DNA enzyme's turnover. The most beneficial mutations, identified after single round self-selection, provided with, respectively, 20 times 3 °C increased thermostability. Based on modular programming architecture, does not require complex instrumentation be repurposed other enzymes, including those are related chemistry.

Language: Английский

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Revolutionizing Molecular Design for Innovative Therapeutic Applications through Artificial Intelligence

Molecules, Journal Year: 2024, Volume and Issue: 29(19), P. 4626 - 4626

Published: Sept. 29, 2024

The field of computational protein engineering has been transformed by recent advancements in machine learning, artificial intelligence, and molecular modeling, enabling the design proteins with unprecedented precision functionality. Computational methods now play a crucial role enhancing stability, activity, specificity for diverse applications biotechnology medicine. Techniques such as deep reinforcement transfer learning have dramatically improved structure prediction, optimization binding affinities, enzyme design. These innovations streamlined process allowing rapid generation targeted libraries, reducing experimental sampling, rational tailored properties. Furthermore, integration approaches high-throughput techniques facilitated development multifunctional novel therapeutics. However, challenges remain bridging gap between predictions validation addressing ethical concerns related to AI-driven This review provides comprehensive overview current state future directions engineering, emphasizing their transformative potential creating next-generation biologics advancing synthetic biology.

Language: Английский

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Polysaccharide degradation in an Antarctic bacterium: Discovery of glycoside hydrolases from remote regions of the sequence space

International Journal of Biological Macromolecules, Journal Year: 2025, Volume and Issue: unknown, P. 140113 - 140113

Published: Jan. 1, 2025

Language: Английский

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Droplet microfluidic screening to engineer angiotensin-converting enzyme 2 (ACE2) catalytic activity

Journal of Biological Engineering, Journal Year: 2025, Volume and Issue: 19(1)

Published: Feb. 3, 2025

Angiotensin-Converting Enzyme 2 (ACE2) is a crucial peptidase in human peptide hormone signaling, catalyzing the conversion of Angiotensin-II to Angiotensin-(1–7), which activates Mas receptor and elicits vasodilation, increased blood flow, reduced inflammation, decreased pathological tissue remodeling. This study leverages protein engineering enhance ACE2's therapeutic potential for treating conditions such as respiratory viral infections, acute distress syndrome, diabetes. Surrogate substrates used traditional high-throughput screening methods peptidases often fail accurately mimic native substrates, leading less effective enzyme variants. Here, we developed an ultra-high-throughput droplet microfluidic platform screen on substrates. Our assay detects substrate cleavage via free amino acid release, providing precise measurement biologically relevant activity. Using this new platform, screened large library ACE2 variants, identifying position 187 hotspot enhancing Further focused revealed K187T variant, exhibited fourfold increase catalytic efficiency (kcat/KM) over wild-type ACE2. work demonstrates microfluidics engineering, offering robust accessible method optimize properties clinical applications.

Language: Английский

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A High-Throughput Cell-Free Enzyme Screening System Using Redox-Responsive Hydrogel Beads as Artificial Compartments

ACS Synthetic Biology, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 13, 2025

We have developed a rapid, simple, and high-throughput screening system for recombinant enzymes using disulfide-bonded hydrogel beads (HBs) produced via microfluidic method. These redox-responsive HBs were compatible with the biosynthesis of enzyme mutants cell-free protein synthesis, fluorescent staining through an enzymatic reaction, genetic information recovery after fluorescence-activated droplet sorting (FADS). The expression microbial transglutaminase zymogen (MTGz) synthesis cross-linking-reactivity-based product validated. Next-generation sequencing (NGS) analysis genes recovered from highly identified novel mutation sites (N25 N27) in propeptide domain. introduction these mutations allowed design engineered active MTGz, demonstrating potential as artificial compartments FADS-based selection that catalyze peptide cross-linking reactions.

Language: Английский

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From specialization to broad adoption: Key trends in droplet microfluidic innovations enhancing accessibility to non-experts

Biomicrofluidics, Journal Year: 2025, Volume and Issue: 19(2)

Published: March 1, 2025

Droplet microfluidics has emerged as a versatile and powerful tool for various analytical applications, including single-cell studies, synthetic biology, directed evolution, diagnostics. Initially, access to droplet was predominantly limited specialized technology labs. However, the landscape is shifting with increasing availability of commercialized manipulation technologies, thereby expanding its use non-specialized Although these commercial solutions offer robust platforms, their adaptability often constrained compared in-house developed devices. Consequently, both within industry academia, significant efforts are being made further enhance robustness automation droplet-based not only facilitate transfer non-expert laboratories but also reduce experimental failures. This Perspective article provides an overview recent advancements aimed at accessibility systems enabling complex manipulations. The discussion encompasses diverse aspects such generation, reagent addition, splitting, washing, incubation, sorting, dispensing. Moreover, alternative techniques like double emulsions hydrogel capsules, minimizing or eliminating need microfluidic operations by end user, explored. These developments foreseen integration intricate manipulations users in workflows, fostering broader faster adoption across scientific domains.

Language: Английский

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Engineered T7 RNA polymerase reduces dsRNA formation by lowering terminal transferase and RNA‐dependent RNA polymerase activities

FEBS Journal, Journal Year: 2025, Volume and Issue: unknown

Published: March 3, 2025

T7 RNA polymerase (RNAP), the preferred tool for in vitro transcription (IVT), can generate double‐stranded (dsRNA) by‐products that elicit immune stress and pose safety concerns. By combining molecular beacon‐based fluorescence‐activated droplet sorting (FADS) utilized random library screening with site‐directed mutagenesis aimed at facilitating conformational changes RNAP, we successfully identified four mutants exhibit reduced dsRNA content: M1 (V214A), M7 (F162S/A247T), M11 (K180E) M14 (A70Q). Furthermore, combinatorial mutant M17 (A70Q/F162S/K180E) exhibited significantly production under various conditions. Cellular experiments confirm application potential of mutants, displaying mitigated responses enhanced protein translation compared to wild‐type protein. We then observed a close correlation between terminal transferase RNA‐dependent RNAP (RDRP) activities RNAP. The activity adds several nucleotides terminus RNAs, while RDRP extends complementary region formed by self‐pairing. In summary, developed novel approach engineering demonstrated its variants or improved product integrity.

Language: Английский

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NebulaPlate: a droplet microfluidic platform to analyze platelet aggregation

Journal of Nanobiotechnology, Journal Year: 2025, Volume and Issue: 23(1)

Published: March 5, 2025

The accurate assessment of platelet activity is crucial in clinical practice and scientific research owing to the pivotal role platelets progression cardiovascular conditions, such as arterial thrombotic diseases. However, conventional methods are currently limited by their requirement substantial blood samples inadequate high-throughput capabilities, therapeutic resistance induced antiplatelet agents impedes treatment efficacy. In this study, we developed a microdroplet-based function detection method, referred NebulaPlate, achieve miniaturized robust assessment, thereby overcoming current challenges. NebulaPlate supports merging with drugs confined picoliter microdroplets leverages an imaging-based analysis automatically identify platelets, evaluate aggregation, determine P-selectin expression within anchored microdroplets. We experimentally confirmed feasibility aggregation assays on using various representative drugs. Requiring only 0.3 mL whole blood/chip, which corresponds approximately 100 platelets/reaction, reduced consumption single assay. This represents reduction 10 times compared that techniques. Moreover, our experimental results validity reproducibility performed NebulaPlate. Our highlights important developments field provides fresh prospects for future therapies personalized medicine. it introduces new possibilities related

Language: Английский

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