Development of a Universal NADH Detection Assay for High Throughput Enzyme Evolution Using Fluorescence Activated Droplet Sorting

Directed evolution is an enzyme engineering approach based on the generation and screening of large mutagenesis libraries, with a view to discovering enzymes with improved properties such as activity, specificity or stability. Recently, droplet-based microfluidics has emerged as a powerful technology enabling ultra-high throughput screening of enzyme libraries and the effective identification and isolation of novel, improved enzyme variants, outperforming conventional enzyme screening platforms by several orders of magnitude in terms of speed and chemical consumption. When using droplet-based platforms fluorescence remains the predominant choice for detection of enzymatic activity due to its high sensitivity and low limits of detection. However, this approach often requires the use of labeled, non-natural substrates, which are typically not commercially available. In addition, fluorescence detection is only suitable for a few enzyme classes such as hydrolases or oxidases, whose reactions can often lead to a fluorescent signal. Herein, we describe an assay that enables fluorescence detection of enzymatic activity through a reaction cascade for the industrially important enzyme subclass of dehydrogenases. By applying a hydrogen peroxide-forming NADH oxidase coupled with peroxidase-catalyzed fluorescence generation, quantification of NADH and dehydrogenase activity becomes possible. We explored the utility of this assay in the evolution of a low performing alcohol dehydrogenase from Sphingomonas species A1 (SpsADH). A fluorescence-activated droplet sorting (FADS) platform was utilized for the screening of a 50,000 variant SpsADH library towards the non-native substrate L-guluronate, a primary component of macroalgae, with the potential to serve as raw material for the bio-based production of chemicals. Significantly, we found an enzyme variant with a 2.6-fold improvement in catalytic efficiency kcat/Km towards the non-native substrate, with only a single round of mutagenesis. The screening of SpsADH libraries confirms the ability of the developed method to enrich active enzyme variants.