Ultrahigh-throughput screening for sialic acid-active enzymes in the microbial genetic diversity

Authors

Martinez-Salvador, J., Trujillo-Cubillo, S., Blas-Munoz, L., Conte, M., Fessner, W.-D., Charnock, S., Finnigan, J., Hidalgo, A.

Abstract

Sialic acids (Sias) and related nonulosonic acids are critical components of glycoconjugates involved in host-pathogen interactions, immune regulation, and cell signalling. Despite their biotechnological relevance, the diversity of enzymes involved in Sia biosynthesis remains largely underexplored due to limitations in culture-dependent methods and the lack of (ultra)high-throughput screening strategies. Here, we report the development of a highly sensitive droplet-based microfluidic screening platform enabling the functional discovery of sialic acid aldolases in environmental metagenomes. The method integrates a fluorescence-coupled enzymatic cascade compatible with fluorescence-activated droplet sorting (FADS), allowing the screening of >10 droplets per experiment, as well as a downstream validation strategy for the selected hits. Although some limitations were identified, the system demonstrated high sensitivity and was utilised for the screening of a metagenomic library from garden soil. During this campaign, a potential new sialic acid aldolase enzyme was identified. This work establishes a generalizable framework for measuring complex, multi-step enzymatic functions at ultrahigh throughput using coupled cascades in droplets

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 28 Aug 2026.

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