Ultrahigh-throughput screening in microfluidic droplets: a faster route to new enzymes

Gantz, Maximilian; Aleku, Godwin A.; Hollfelder, Florian

doi:10.1016/j.tibs.2021.11.001

Cited by 13 publications

(12 citation statements)

References 10 publications

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“…We sampled all these variables combinatorially in an ultrahigh-throughput manner by using microfluidic droplets, which allow screening of the entire library in a single experiment. In this microfluidic assay, individual bacterial cells expressing a single library sequence were co-compartmentalized with the mixture of fluorogenic substrates and metals, and then lysed in the droplet ( Figure 1b, Figure S1 ) 18,19 . After incubation, the droplets were dielectrophoretically sorted at ultrahigh throughput by fluorescence-activated droplet sorting (FADS) on a microfluidic chip 20 .…”

Section: Resultsmentioning

confidence: 99%

Selection of a Promiscuous Minimalist cAMP Phosphodiesterase from a Library ofDe NovoDesigned Proteins

Fernández

Wang

Gantz

et al. 2023

Preprint

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The ability of unevolved amino acid sequences to become biological catalysts was key to the emergence of life on Earth. However, billions of years of evolution separate complex modern enzymes from their simpler early ancestors. To study how unevolved sequences can develop new functions, we screened for enzymatic activity in a collection of > 1 million novel sequences based on a de novo 4-helix bundle library of semi-random sequences. To mirror evolutionary selection for biological function, we screened the collection using ultrahigh-throughput droplet microfluidics to identify features that yield phosphoesterase activity. Characterization of active hits demonstrated that acquiring new function required a large jump in sequence space: screening enriched for truncations that removed > 40% of the protein chain and introduced a catalytically important cysteine. The truncated protein dimerized into a dynamic alpha-helical structure, consistent with the idea that gain of function was accompanied by an increase in structural dynamics relative to the parental 4-helix bundle. The purified protein catalyzes the hydrolysis of a range of phosphodiesters, with the greatest activity toward the biological second messenger cyclic AMP (cAMP). The novel cAMPase is a manganese-dependent metalloenzyme and catalyzes cAMP hydrolysis with a rate acceleration on the order of 10e9 and catalytic proficiency on the order of 10e14 M-1, comparable to large enzymes shaped by billions of years of evolution. These findings suggest that fragmentation to modular primordial peptides can be a fertile avenue for introducing structural and functional diversity into proteins.

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Section: Resultsmentioning

confidence: 99%

Selection of a Promiscuous Minimalist cAMP Phosphodiesterase from a Library ofDe NovoDesigned Proteins

Fernández

Wang

Gantz

et al. 2023

Preprint

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“…We envisage that this system can inspire future cascades for the preparation of allylic amines from simple starting materials. For example, coupling the CAR-RedAm system to an upstream biosynthetic pathway for the de novo synthesis of cinnamic acids or to a CO 2 -fixing enzyme. , Although the present study only focused on the synthesis of linear secondary and tertiary allylic amines, our future interests include identifying and developing suitable biocatalysts for the asymmetric version of this transformation using ultra-high-throughput enzyme screening technologies …”

Section: Discussionmentioning

confidence: 99%

“…73,74 Although the present study only focused on the synthesis of linear secondary and tertiary allylic amines, our future interests include identifying and developing suitable biocatalysts for the asymmetric version of this transformation using ultra-high-throughput enzyme screening technologies. 75 ■ ASSOCIATED CONTENT * sı Supporting Information…”

Section: ■ Conclusionmentioning

confidence: 99%

Enzymatic N-Allylation of Primary and Secondary Amines Using Renewable Cinnamic Acids Enabled by Bacterial Reductive Aminases

Aleku

Titchiner

Roberts

et al. 2022

ACS Sustainable Chem. Eng.

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Allylic amines are a versatile class of synthetic precursors of many valuable nitrogen-containing organic compounds, including pharmaceuticals. Enzymatic allylic amination methods provide a sustainable route to these compounds but are often restricted to allylic primary amines. We report a biocatalytic system for the reductive N -allylation of primary and secondary amines, using biomass-derivable cinnamic acids. The two-step one-pot system comprises an initial carboxylate reduction step catalyzed by a carboxylic acid reductase to generate the corresponding α,β-unsaturated aldehyde in situ . This is followed by reductive amination of the aldehyde catalyzed by a bacterial reductive aminase pIR23 or BacRedAm to yield the corresponding allylic amine. We exploited pIR23, a prototype bacterial reductive aminase, self-sufficient in catalyzing formal reductive amination of α,β-unsaturated aldehydes with various amines, generating a broad range of secondary and tertiary amines accessed in up to 94% conversion under mild reaction conditions. Analysis of products isolated from preparative reactions demonstrated that only selective hydrogenation of the C=N bond had occurred, preserving the adjacent alkene moiety. This process represents an environmentally benign and sustainable approach for the synthesis of secondary and tertiary allylic amine frameworks, using renewable allylating reagents and avoiding harsh reaction conditions. The selectivity of the system ensures that bis-allylation of the alkylamines and (over)reduction of the alkene moiety are avoided.

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“…Droplet-based high-throughput screening methods facilitate analysis of extraordinary sample sizes with throughputs in the tens of kHz range implying a capacity of up to over 100 million independent reactions in a working day. [1][2][4][5][6][7][8][9] This type of screening is advantageous in particular for diagnostic applications, [10][11][12][13] or to identify efficient catalytic systems, 9, 14-15 e.g. by interrogating protein libraries in directed evolution, [16][17][18][19][20][21] and functional metagenomics, [22][23][24] and in strain selections [25][26] or bioprospecting.…”

Section: Introductionmentioning

confidence: 99%

Rapid quantitative assessment of small molecule leakage from microdroplets by flow cytometry and improvement of fluorophore retention in biochemical assays

Zinchenko

Devenish

Hollfelder

2023

Preprint

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Microdroplets are compartments made in the laboratory that allow the miniaturisation of chemical and biological experiments to the femto- to picolitre scale, replacing the classical test tube with a droplet. Ideally containment of the contents of individual droplets would be perfect, but in reality this situation rarely occurs. Instead the leaking of molecules even from intact droplets presents a challenge to the success of miniaturisation and must be assessed on a case-by-case basis. We now present a new method for quantitative determination of leakage: a sheath fluid-free flow cytometer (Guava EasyCyte) is used to directly determine the fluorescence of water-in-oil droplets as a function of time. We validate this method by demonstrating that this assessment of leakage provides a framework for experimental improvements that reduce the leakage of two widely used fluorophores. A 40-fold better retention compared to current protocols is achieved for resorufin with an optimized mix (oil: FC-70, surfactant: 0.1% w/w AZ900C, additive: 1% BSA) to maintain useful retention for up to 130 hours. Likewise leakage of the fluorophore methylumbelliferone is reduced by 75-fold. The availability of a method to quantitate leakage quickly for a variety of experimental conditions will facilitate future applications of droplet-based experiments (e.g. in directed evolution or diagnostics), aid miniaturisation of lab-scale assays into this format, and improve the degrees of freedom in setting up such ultrahigh-throughput experiments.

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Ultrahigh-throughput screening in microfluidic droplets: a faster route to new enzymes

Cited by 13 publications

References 10 publications

Selection of a Promiscuous Minimalist cAMP Phosphodiesterase from a Library ofDe NovoDesigned Proteins

Selection of a Promiscuous Minimalist cAMP Phosphodiesterase from a Library ofDe NovoDesigned Proteins

Enzymatic N-Allylation of Primary and Secondary Amines Using Renewable Cinnamic Acids Enabled by Bacterial Reductive Aminases

Rapid quantitative assessment of small molecule leakage from microdroplets by flow cytometry and improvement of fluorophore retention in biochemical assays

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