Picoliter Cell Lysate Assays in Microfluidic Droplet Compartments for Directed Enzyme Evolution

Kintses, Bálint; Hein, Christopher; Mohamed, Mark F.; Fischlechner, Martin; Courtois, Fabienne; Lainé, Céline; Hollfelder, Florian

doi:10.1016/j.chembiol.2012.06.009

Cited by 210 publications

(245 citation statements)

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“…This is also in line with the sorter error rate being on the order of 10 −4 indicating that it should not significantly affect the enrichment, which is more than ten times lower. A good agreement between η max and the actual enrichment was also reported by Fallah-Araghi et al 28 In contrast, Kintses et al (2012) 30 found an enrichment of ca. 1/20 of η max , likely due to the fact that their sorting experiment involved a library rather than a binary mixture of two strains, the library being a more difficult model system.…”

Section: Model Selection For α-Amylase Expressionsupporting

confidence: 84%

High-throughput screening for industrial enzyme production hosts by droplet microfluidics

et al. 2014

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A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its α-amylase production, close to the theoretical maximum enrichment. Furthermore, a 10 5 member yeast cell library is screened yielding a clone with a more than 2-fold increase in α-amylase production. The increase in enzyme production results from an improvement of the cellular functions of the production host in contrast to previous droplet-based directed evolution that has focused on improving enzyme protein structure. In the workflow presented, enzyme producing single cells are encapsulated in 20 pL droplets with a fluorogenic reporter substrate. The coupling of a desired phenotype (secreted enzyme concentration) with the genotype (contained in the cell) inside a droplet enables selection of single cells with improved enzyme production capacity by droplet sorting. The platform has a throughput over 300 times higher than that of the current industry standard, an automated microtiter plate screening system. At the same time, reagent consumption for a screening experiment is decreased a million fold, greatly reducing the costs of evolutionary engineering of production strains.

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Section: Model Selection For α-Amylase Expressionsupporting

confidence: 84%

High-throughput screening for industrial enzyme production hosts by droplet microfluidics

et al. 2014

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“…Droplet-based microfluidic screening provides a flexible platform for assaying enzyme activity over a broad range of reaction conditions (10)(11)(12)(13). We adapted our screening protocol to include a heat challenge and enriched for mutations that increase the enzyme's thermostability.…”

Section: Discussionmentioning

confidence: 99%

“…Through comprehensive mutagenesis and functional characterization of this enzyme, we were able, with minimal bias, to discover residues crucial to function and identify mutations that enhance its activity at elevated temperatures. This approach can be applied to any enzyme whose chemical activity can be measured with a fluorogenic assay in microfluidic droplets (10)(11)(12)(13). Our method extends the applicability of deep mutational scanning to a wide range of protein functions and reaction conditions not accessible by other high-throughput methods.…”

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confidence: 98%

Dissecting enzyme function with microfluidic-based deep mutational scanning

Romero

Tran

Abate

2015

Proc. Natl. Acad. Sci. U.S.A.

212

183

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Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme's sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence-function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based deep mutational scanning provides a comprehensive and unbiased view of the enzyme function landscape. The mapping displays expected patterns of mutational tolerance and a strong correspondence to sequence variation within the enzyme family, but also reveals previously unreported sites that are crucial for glycosidase function. We modified the screening protocol to include a hightemperature incubation step, and the resulting thermotolerance landscape allowed the discovery of mutations that enhance enzyme thermostability. Droplet microfluidics provides a general platform for enzyme screening that, when combined with DNAsequencing technologies, enables high-throughput mapping of enzyme sequence space.protein engineering | droplet-based microfluidics | high-throughput DNA sequencing E nzymes are powerful biological catalysts capable of remarkably accelerating the rates of chemical transformations (1). The molecular bases of these rate accelerations are often complex, using multiple steps, multiple catalytic mechanisms, and relying on numerous molecular interactions, in addition to those provided by the main catalytic groups. This complexity imposes a significant barrier to understanding how an enzyme's sequence impacts its function and, thus, on our ability to rationally design biocatalysts with new or enhanced functions (2-4).Comprehensive mappings of sequence-function relationships can be used to dissect the molecular basis of protein function in an unbiased manner (5). Growth selections or in vitro binding screens can be combined with next-generation DNA sequencing to generate detailed mappings between a protein's sequence and its biochemical properties, such as binding affinity, enzymatic activity, and stability (6-9). This deep mutational scanning approach has been used to study the structure of the protein fitness landscape, discover new functional sites, improve molecular energy functions, and identify beneficial combinations of mutations for protein engineering. However, these methods rely on functional assays coupled to cell growth or protein binding, severely limiting the types of proteins that can be analyzed. For example, most enzymes of biological or industrial relevance cannot be analyzed using existing methods because they do not catalyze a reaction that can be directly coupled to cell growth. Experimental advances are needed to broaden the applicability of deep mutational scanning to the diverse palette of functions performed by enzymes.In this paper, we present a general method for mapping protein sequence-func...

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“…Single occupancy of cells in droplets can be controlled by the cell concentration and follows a Poisson distribution 24 . In the droplet, the two aqueous phases from the separate inlets are mixed, the cells are lysed 25 and the enzyme catalyst is liberated and thus able to react with substrate. The reaction is conducted in emulsion droplets at 30 °C with agarose in its liquid (sol) form (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, in contrast to transient, fluid droplets in microfluidic devices, the compartmentalization is robustly 'immortalized' in GSBs. The stability of GSBs allows them to be handled and analyzed in aqueous solution by standard FACS equipment (rather than custom-made microfluidic on-chip emulsion sorters 25,[31][32][33][34] ). FACS can also be employed for cell-based selections, but they either rely on in vivo live/dead assays 35 or require that the reaction product must be captured in or on cells 36,37 .…”

Section: Discussionmentioning

confidence: 99%

Evolution of enzyme catalysts caged in biomimetic gel-shell beads

et al. 2014

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Natural evolution relies on the improvement of biological entities by rounds of diversification and selection. In the laboratory, directed evolution has emerged as a powerful tool for the development of new and improved biomolecules, but it is limited by the enormous workload and cost of screening sufficiently large combinatorial libraries. Here we describe the production of gel-shell beads (GSBs) with the help of a microfluidic device. These hydrogel beads are surrounded with a polyelectrolyte shell that encloses an enzyme, its encoding DNA and the fluorescent reaction product. Active clones in these manmade compartments can be identified readily by fluorescence-activated sorting at rates >107 GSBs per hour. We use this system to perform the directed evolution of a phosphotriesterase (a bioremediation catalyst) caged in GSBs and isolate a 20-fold faster mutant in less than one hour. We thus establish a practically undemanding method for ultrahigh-throughput screening that results in functional hybrid composites endowed with evolvable protein components. Evolution of Catalysts Caged in Biomimetic Gel-Shell Beads AbstractNatural evolution relies on improvement of biological entities by rounds of

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Picoliter Cell Lysate Assays in Microfluidic Droplet Compartments for Directed Enzyme Evolution

Cited by 210 publications

References 50 publications

High-throughput screening for industrial enzyme production hosts by droplet microfluidics

High-throughput screening for industrial enzyme production hosts by droplet microfluidics

Dissecting enzyme function with microfluidic-based deep mutational scanning

Evolution of enzyme catalysts caged in biomimetic gel-shell beads

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