Ultrahigh throughput evolution of tryptophan synthase in droplets via an aptamer-biosensor DOI Open Access
Remkes A. Scheele, Yanik Weber, Friederike E. H. Nintzel

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Oct. 12, 2023

Abstract Tryptophan synthase catalyzes the synthesis of a wide array non-canonical amino acids and is an attractive target for directed evolution. Droplet microfluidics offers ultrahigh throughput approach to evolution (>10 7 experiments per day), enabling search biocatalysts in wider regions sequence space with reagent consumption minimized picoliter volume (per library member). While majority screening campaigns this format on record relied optically active reaction product, new assay needed tryptophan synthase. not fluorogenic visible light spectrum thus falls outside scope conventional droplet microfluidic read-outs which are incompatible UV detection at high throughput. Here, we engineer DNA aptamer into biosensor quantitatively report production droplets. The utility was validated by identifying 5-fold improved synthases from ∼100,000 protein variants. More generally work establishes use DNA-aptamer sensors read-out widening

Language: Английский

Ultrahigh-Throughput Enzyme Engineering and Discovery in In Vitro Compartments DOI Creative Commons
Maximilian Gantz, Stefanie Neun,

Elliot J. Medcalf

et al.

Chemical Reviews, Journal Year: 2023, Volume and Issue: 123(9), P. 5571 - 5611

Published: May 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.

Language: Английский

Citations

52
Covalent Organic Frameworks for the Purification of Recombinant Enzymes and Heterogeneous Biocatalysis DOI
Satyadip Paul, Mani Gupta, Ashok Kumar Mahato

et al.

Journal of the American Chemical Society, Journal Year: 2023, Volume and Issue: 146(1), P. 858 - 867

Published: Dec. 30, 2023

Recombinant enzymes have gained prominence due to their diverse functionalities and specificity are often a greener alternative in biocatalysis. This context makes purifying recombinant from host cells other impurities crucial. The primary goal is isolate the pure enzyme of interest ensure its stability under ambient conditions. Covalent organic frameworks (COFs), renowned for well-ordered structure permeability, offer promising approach histidine-tagged (His-tagged) enzymes. Furthermore, immobilizing within COFs represents growing field heterogeneous In this study, we developed flow-based technology utilizing nickel-infused covalent framework (Ni-TpBpy COF) combine two distinct processes: purification His-tagged immobilization simultaneously. Our work primarily focuses on three β-glucosidase, cellobiohydrolase, endoglucanase as well proteins with varying molecular weights, namely, green fluorescent protein (27 kDa) BG Rho (88 kDa). We employed Ni-TpBpy column matrix showcase versatility our system. Additionally, successfully obtained COF immobilized enzymes, which can serve catalyst hydrolysis p-nitrophenyl-β-d-glucopyranoside carboxymethylcellulose. These demonstrated catalytic activity comparable that free counterparts, added advantages recyclability enhanced conditions an extended period, ranging 60 90 days. contrasts do not maintain effectively over time.

Language: Английский

Citations

26
Open microfluidics: droplet microarrays as next generation multiwell plates for high throughput screening DOI Creative Commons
Robert John Strutt, Bijing Xiong, Vanessa Fabienne Abegg

et al.

Lab on a Chip, Journal Year: 2024, Volume and Issue: 24(5), P. 1064 - 1075

Published: Jan. 1, 2024

Droplet microarrays underpin novel experimentation across the biological and chemical sciences. This perspective explores operations analysis with droplet microarrays, placing focus on a comparison to traditional multiwell plates.

Language: Английский

Citations

12
A review of the principles and biotechnological applications of glycoside hydrolases from extreme environments DOI Creative Commons

Ellie Ashcroft,

José Muñoz-Muñoz

International Journal of Biological Macromolecules, Journal Year: 2024, Volume and Issue: 259, P. 129227 - 129227

Published: Jan. 5, 2024

It is apparent that Biocatalysts are shaping the future by providing a more sustainable approach to established chemical processes. Industrial processes rely heavily on use of toxic compounds and high energy or pH reactions, factors both contributes worsening climate crisis. Enzymes found in bacterial systems other microorganisms, from glaciers Arctic sandy deserts Abu Dhabi, provide key tools understanding as how we can progress biotechnology sector. These extremophilic bacteria harness adaptive enzymes capable withstanding harsh reaction conditions terms stability reactivity. Carbohydrate-active enzymes, including glycoside hydrolases carbohydrate esterases, extremely beneficial for presence biocatalysis. Their involvement industry spans laundry detergents paper pulp treatment degrading oligo/polysaccharides into their monomeric products almost all detrimental environments. This includes exceedingly temperatures, pHs even absence water. In this review, discuss structure function different extremophiles, they be applied industrial-scale reactions replace chemicals, reduce waste, decrease consumption.

Language: Английский

Citations

10
Microfluidics-driven high-throughput phenotyping and screening in synthetic biology: from single cells to cell-free systems DOI
Tae-Ok Kim, Minji Ko,

Eugene Rha

et al.

Biotechnology and Bioprocess Engineering, Journal Year: 2024, Volume and Issue: 29(1), P. 25 - 33

Published: Feb. 1, 2024

Language: Английский

Citations

6
Microdroplet screening rapidly profiles a biocatalyst to enable its AI-assisted engineering DOI Open Access
Maximilian Gantz, Simon V. Mathis, Friederike E. H. Nintzel

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: April 8, 2024

Abstract Engineering enzyme biocatalysts for higher efficiency is key to enabling sustainable, ‘green’ production processes the chemical and pharmaceutical industry. This challenge can be tackled from two angles: by directed evolution, based on labor-intensive experimental testing of variant libraries, or computational methods, where sequence-function data are used predict biocatalyst improvements. Here, we combine both approaches into a two-week workflow, ultra-high throughput screening library imine reductases (IREDs) in microfluidic devices provides not only selected ‘hits’, but also long-read sequence linked fitness scores >17 thousand variants. We demonstrate engineering an IRED chiral amine synthesis mapping functional information one go, ready interpretation extrapolation protein engineers with help machine learning (ML). calculate position-dependent mutability combinability mutations comprehensively illuminate complex interplay driven synergistic, often positively epistatic effects. Interpreted easy-to-use regression tree-based ML algorithms designed suit evaluation random whole-gene mutagenesis data, 3-fold improved ‘hits’ obtained extrapolated further give up 23-fold improvements catalytic rate after handful mutants. Our campaign paradigmatic future that will rely access large maps as profiles way responds mutation. These chart function exploiting synergy rapid combined extrapolation.

Language: Английский

Citations

6
On synergy between ultrahigh throughput screening and machine learning in biocatalyst engineering DOI Creative Commons
Maximilian Gantz, Simon V. Mathis, Friederike E. H. Nintzel

et al.

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

Citations

6
Layer-by-layer (LBL) self-assembly efficient immobilization of glucose oxidase onto PDMS microfluidic chip towards glucose biosensing DOI

Kemeng Zhou,

Yaoyao Yu,

Zhihua Wang

et al.

Journal of Polymer Research, Journal Year: 2025, Volume and Issue: 32(1)

Published: Jan. 1, 2025

Language: Английский

Citations

0
Ultrahigh Throughput Evolution of Tryptophan Synthase in Droplets via an Aptamer Sensor DOI Creative Commons
Remkes A. Scheele, Yanik Weber, Friederike E. H. Nintzel

et al.

ACS Catalysis, Journal Year: 2024, Volume and Issue: 14(8), P. 6259 - 6271

Published: April 10, 2024

Tryptophan synthase catalyzes the synthesis of a wide array noncanonical amino acids and is an attractive target for directed evolution. Droplet microfluidics offers ultrahigh throughput approach to evolution (up 107 experiments per day), enabling search biocatalysts in wider regions sequence space with reagent consumption minimized picoliter volume (per library member). While majority screening campaigns this format on record relied optically active reaction product, new assay needed tryptophan synthase. not fluorogenic visible light spectrum thus falls outside scope conventional droplet microfluidic readouts, which are incompatible UV detection at high throughput. Here, we engineer DNA aptamer into sensor quantitatively report production droplets. The utility was validated by identifying five-fold improved synthases from ∼100,000 protein variants. More generally, work establishes use DNA-aptamer sensors read-out widening

Language: Английский

Citations

3
Fluorogenic, Subsingle-Turnover Monitoring of Enzymatic Reactions Involving NAD(P)H Provides a Generalized Platform for Directed Ultrahigh-Throughput Evolution of Biocatalysts in Microdroplets DOI Creative Commons
Matthew Penner, Oskar James Klein, Maximilian Gantz

et al.

Journal of the American Chemical Society, Journal Year: 2025, Volume and Issue: unknown

Published: March 24, 2025

Enzyme engineering and discovery are crucial for a sustainable future bioeconomy. Harvesting new biocatalysts from large libraries through directed evolution or functional metagenomics requires accessible, rapid assays. Ultrahigh-throughput screening formats often require optical readouts, leading to the use of model substrates that may misreport target activity necessitate bespoke synthesis. This is particular challenge when glycosyl hydrolases, which leverage molecular recognition beyond glycosidic bond, so complex chemical synthesis would have be deployed build fluoro- chromogenic substrate. In contrast, coupled assays represent modular "plug-and-play" system: any enzyme–substrate pairing can investigated, provided reaction produce common intermediate links catalytic detection cascade readout. Here, we establish producing fluorescent readout in response NAD(P)H via glutathione reductase subsequent thiol-mediated uncaging reaction, with low nanomolar limit plates. Further scaling down microfluidic droplet possible: fluorophore leakage-free report 3 orders magnitude-improved sensitivity compared absorbance-based systems, resolution 361,000 product molecules per droplet. Our approach enables nonfluorogenic droplet-based enrichments, applicability hydrolases imine reductases (IREDs). To demonstrate assay's readiness combinatorial experiments, one round was performed select glycosidase processing natural substrate, beechwood xylan, improved kinetic parameters pool >106 mutagenized sequences.

Language: Английский

Citations

0