Beneath the Sweet Surface

Abstract

The surface of microorganisms is often decorated with a wide variety of glycans and glycoconjugates. These biomolecules play key roles in structural support, cellular communication, immune recognition, and host–pathogen interactions. Despite their importance, glycan analysis still lags far behind that of DNA and proteins. This is mainly because glycans are not directly encoded in the genome but arise from the combined action of glycosyltransferases and glycosidases. Their characterization is further complicated by extensive structural diversity, as glycans consist of a large and expanding repertoire of monosaccharides. In addition, restricted access to these building blocks hinders functional studies and the exploitation of glycans as antimicrobial targets. This thesis aims to advance the discovery of microbial metabolites, glycomolecule precursors, and carbohydrate-active enzymes using mass spectrometry and bioinformatic approaches.

Chapter 2 reviews advanced mass spectrometry-based methods for the identification and quantification of monosaccharides and their nucleotide-activated forms. Key analytical challenges are highlighted, including the differentiation of stereoisomers and the limited availability of reference standards. Complementary strategies, such as high-performance separation techniques, advanced fragmentation methods, and isotope tracing, are discussed for improved structural characterization and pathway analysis.

Building on these analytical foundations, Chapter 3 presents SugarBase, a mass spectrometry and bioinformatic pipeline for untargeted exploration of microbial nucleotide sugar networks. SugarBase combines narrow-window DIA fragmentation with a theoretical sugar composition database for parent ion annotation. The platform revealed species-specific nucleotide sugar profiles, including distinct nonulosonic acid profiles in Campylobacter jejuni strains and previously unannotated compounds across diverse microbes, including a higher-carbon ulosonic acid in Magnetospirillum.

Extending this work to complex microbial systems, Chapter 4 includes a collaborative study adapting the Chapter 3 methodology into a targeted metabolomics workflow to quantify nucleotides in granular biofilms involved in enhanced biological phosphorus removal. Using fast quenching, boiling-water extraction, and high-resolution mass spectrometry with ¹³C-labeled internal standards, the approach enabled measurement of adenylate and uridylate energy charge dynamics. Applied to a lab-scale system, it revealed energy pool fluctuations during acetate uptake and polyphosphate degradation, demonstrating that energy imbalances can underlie metabolic switching in biofilms.

In Chapter 5, we demonstrate that microbial metabolite extracts can serve as effective and readily accessible substrate sources for glycosyltransferase assays, without further purification. Using a Campylobacter jejuni extract containing diverse nonulosonic acid derivatives, we show substrate promiscuity of the previously identified pseudaminyltransferase from Acinetobacter baumannii, currently the only confirmed enzyme of its kind. These findings support the production of glycans and glycoconjugates relevant for antimicrobial and vaccine development.

While glycans and glycoconjugates are central to many biological processes, carbohydrate-active enzymes are of similar interest due to their potential to replace harsh chemical processes and their ability to enable new products. A key challenge remains the targeted identification of enzymes with desired activities and characteristics. In Chapter 6, we combine enrichment cultures with metagenomics and metaproteomics to discover glycoside hydrolases. This strategy enables functional enrichment under customized conditions and yields a focused set of enzyme candidates. One of the candidates was subsequently confirmed as a pullulan-degrading enzyme using a mass spectrometry-based activity assay.

Finally, Chapter 7 summarizes some of the main findings, discusses main challenges and outlines future research directions.