Sub-single-turnover quantification of enzyme catalysis at ultrahigh throughput via a versatile NAD(P)H coupled assay in microdroplets

Abstract

Enzyme engineering and discovery are crucial for a future sustainable bioeconomy, and harvesting new biocatalysts from large libraries through directed evolution or functional metagenomics requires accessible, rapid assays. Ultra-high throughput screening can often require an optical readout, leading to the use of model substrates that may not accurately report on activity for the target reaction and may require bespoke synthesis. In contrast, coupled assays represent a modular ‘plug-and-play’ system, where any pairing of enzyme/substrate may be investigated, if the reaction can produce a common intermediate which links the catalytic reaction to a detection cascade readout. Here we establish a detection cascade, producing a fluorescent readout in response to NAD(P)H via glutathione reductase and a subsequent thiol-mediated uncaging reaction, with a 30 nM detection limit. We demonstrate its utility for the glycosidaseAxyAgu115A (producing monosaccharides from a natural biofuel feedstock) and report a three orders of magnitude improved sensitivity compared to absorbance-based systems, so that less than one catalytic turnover per enzyme molecule expressed from a single cell is detectable. These advantages are brought to bear in plate formats, but also in picoliter emulsion droplets, where enrichments of 950-fold suggest that large libraries can be interrogated against a specific query substrate.

Carbohydrate-active enzyme (CAZyme) discovery and engineering via (Ultra)high-throughput screening

“…85 More recent work by the Hollfelder laboratory has expanded this approach by using sugar dehydrogenases to enable measurement of the release of free sugars such as glucuronic acid, xylose, and galactose. 78,86 This method uses the reduction of NAD(P)H by these enzymes during sugar oxidation to initiate an enzymatic cascade that results in reduction of a tetrazolium derivative (WST-1) to form a UV/Vis absorbent formazan. 78 Ladeveze et al show in their proof of concept that enzymes active on beechwood xylan, wheat arabinoxylan and xylobiose can be detected and that absorbance-activated droplet sorting can be used to sort active clones.…”

“…The creativity and ingenuity displayed within the field, particularly in the use of coupled assays and highly relevant substrates (or ideally the desired substrate) to detect activity stand as highlights and portend future developments. In moving this forward there is likely to be synergy between the engineering of CAZymes and the engineering of the coupling enzymes used (whether they be oxidases, 79,81 dehydrogenases, 78,86 O-glycopeptidases 173 or whatever).…”

“…In brief, the production of NAD(P)H by the sugar dehydrogenase is then coupled to the reduction of glutathione disulfide to glutathione by glutathione reductase. 86 This free reduced glutathione can then release a quenched carboxy-coumarin fluorophore through nucleophilic cleavage of a sulfonic ester. 86 The resulting fluorescent signal offers a much lower limit of detection compared to the absorbance based assay.…”

“… 86 This free reduced glutathione can then release a quenched carboxy-coumarin fluorophore through nucleophilic cleavage of a sulfonic ester. 86 The resulting fluorescent signal offers a much lower limit of detection compared to the absorbance based assay. 86 This approach should prove useful in enzyme discovery campaigns via methods such as functional metagenomics where low enzyme expression levels are common.…”

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

“…When single emulsions are converted into double emulsions, commercial flow cytometers can be used for detection of fluorophores 14 . Further, lab-on-a-chip devices miniaturise more complex liquid handling operations and coupled assays enlarge the range of reactions that can be assayed 11,15,16,17 . Indeed, functional assays for all E.C.…”