Biology subjects

Emond, S.

Publications and source records attributed to Emond, S..

3 recordsLinked to original sources

bioRxiv2023

Versatile Product Detection via Coupled Assays for Ultra-high-throughput Screening of Carbohydrate-Active-Enzymes in Microfluidic Droplets

Enzyme discovery and directed evolution are the two major contemporary approaches for the improvement of industrial processes by biocatalysis in various fields. Customization of catalysts for improvement of single enzyme reactions or de novo reaction development is often complex and tedious. The success of screening campaigns relies on the fraction of sequence space that can be sampled, whether for evolving a particular enzyme or screening metagenomes. Ultrahigh-throughput screening (uHTS) based on in-vitro compartmentalization in water-in-oil emulsion of picolitre droplets generated in microfluidic systems allows screening rates >1 kHz (or >107 per day). Screening for Carbohydrate Active Enzymes (CAZymes) catalysing biotechnologically valuable reactions in this format presents an additional challenge, because the released carbohydrates are difficult to monitor in high throughput. Activated substrates with large optically active hydrophobic leaving groups provide a generic optical readout, but the molecular recognition properties of sugars will be altered by incorporation of such fluoro- or chromophores and their typically higher reactivity, as leaving groups with lowered pKa values compared to native substrates make observation of promiscuous reactions more likely. To overcome these issues, we designed microdroplet assays in which optically inactive carbohydrate products are made visible by specific cascades: the primary reaction of an unlabelled substrate leads to an optical signal downstream. Successfully implementing such assays at the picoliter droplet scale allowed us to detect glucose, xylose, glucuronic acid and arabinose as final products of complex oligosaccharide degradation by glycoside hydrolases by absorbance measurements. Enabling the use of uHTS for screening CAZyme reactions that have been thus far elusive will chart a route towards faster and easier development of specific and efficient biocatalysts for biovalorisation, directing enzyme discovery towards catalysts for their natural rather than model substrates. Graphical abstract / TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/534725v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@13063eborg.highwire.dtl.DTLVardef@1efdcfcorg.highwire.dtl.DTLVardef@9562eeorg.highwire.dtl.DTLVardef@1923207_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

In vitro Evolution of Antibody Affinity via Insertional Mutagenesis Scanning of an Entire Antibody Variable Region

We report the first systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD was used to generate large libraries with random in-frame InDels across the entire scFv gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (ISM). An overall 256-fold affinity improvement of an anti-IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach and suggests that the results of this InDel approach and conventional exploration of point mutations can synergize to generate antibodies with higher affinity. SignificanceInsertion/deletion (InDel) mutations play key roles in genome and protein evolution. Despite their prominence in evolutionary history, the potential of InDels for changing function in protein engineering by directed evolution remains unexplored. Instead point mutagenesis is widely used. Here we create antibody libraries containing InDels and demonstrate that affinity maturation can be achieved in this way, establishing an alternative to the point mutation strategies employed in all previous in vitro selections. These InDels mirror the observation of considerable length variation in loops of natural antibodies originating from the same germline genes and be combined with point mutations, making both natural sources of functional innovation available for artificial evolution in the test tube.

biochemistry↗

bioRxiv2019

Access to unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

Insertions and deletions (InDels) are frequently observed in natural protein evolution, yet their potential remains untapped in laboratory evolution. Here we introduce a transposon mutagenesis approach (TRIAD) to generate libraries of random variants with short in-frame InDels, and screen TRIAD libraries to evolve a promiscuous arylesterase activity in a phosphotriesterase. The evolution exhibits features that are distinct from previous point mutagenesis campaigns: while the average activity of TRIAD variants is more deleterious, a larger proportion has successfully adapted for the new activity, exhibiting different functional profiles: (i) both strong and weak trade-off in original vs promiscuous activity are observed; (ii) trade-off is more severe (10- to 20-fold increased kcat/KM in arylesterase with [~]100-fold decreases in the original phosphotriesterase activity) and (iii) improvements show up in kcat rather than KM, suggesting novel adaptive solution. These distinct features make TRIAD an alternative to widely used point mutagenesis, providing access to functional innovations and traversing unexplored fitness landscape regions.

biochemistry↗