Biology subjects

Charnock, S.

Publications and source records attributed to Charnock, S..

3 recordsLinked to original sources

bioRxiv2026

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

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

microbiology↗

bioRxiv2026

Bi-level diversity optimisation for representative protein panel selection

Selecting representative subsets from large protein sequence datasets is a common challenge in enzyme discovery and related tasks under limited screening capacity. In practice, candidate panels are often constructed using clustering-based redundancy reduction or manual selection guided by phylogenetic or similarity-network analyses, which do not directly optimise subset diversity and require threshold tuning or expert interpretation. Here, we present a bi-level diversity-optimisation framework for representative protein panel selection implemented using a local search heuristic that iteratively updates panel composition to improve diversity. The method formulates panel design as a combinatorial optimisation problem over pairwise distance matrices, combining a MaxMin objective to enforce minimum separation between selected sequences with a MaxSum objective to increase global dispersion. This formulation enables the direct construction of fixed-cardinality panels while remaining independent of the similarity representation used to compute pairwise distances. Benchmarking across four Pfam families shows that the bi-level formulation consistently reduces redundancy among selected sequences, lowering maximum pairwise identity by 43-46% relative to the previous MaxSum-based formulation, while maintaining comparable or improved EC-label coverage. The framework can incorporate sequence- or structure-based similarity measures, providing a flexible strategy for constructing diverse representative panels across homologous protein families.

bioinformatics↗

bioRxiv2024

Thermostable in vitro transcription-translation for enzyme screening in microdroplets

BackgroundIn vitro expression involves the utilization of the transcription and translation machinery derived from the cell to produce one or more proteins of interest and has found widespread application in the optimization of gene circuits or metabolic pathways in synthetic biology but also in pharmaceutical manufacturing. Most in vitro expression systems available are active at moderate temperatures but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. Moreover, given the fact that the main barrier toward the general use of in vitro expression is its high price compared with host-based recombinant expression, there is a need to develop alternative in vitro expression systems operating at high temperatures and compatible with technologies that enable ultrahigh-throughput screening in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. ResultsTo this end, we produced high-expression cell-free extracts from Thermus thermophilus for in vitro translation and supplemented them with thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable to other thermostable in vitro expression systems, while the preparation procedure is simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. Although the composition of these extracts showed a high background in carboxyl esterase assays, {beta}-glucosidase and cellobiose hydrolase activities could be measured with minimal background. ConclusionsCell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.

microbiology↗