Harnessing N-glycans as Next Generation Prebiotics

N-linked glycans are structurally diverse carbohydrate moieties that are covalently boundto dietary glycoproteins. Upon ingestion, N-glycans from dietary glycoproteins escape absorption in the small intestine and travel to the colon for fermentation. Ample evidence has indicated that certain gut-associated microbes can degrade N-glycans in vitro and in vivo. However, how N-glycans and subtle differences between N-glycan structures impact the gut microbiome has not been studied. This dissertation addresses these questions, and advances methods to study N-glycans as next-generation prebiotics. To begin to address this, a novel yeast-synthesized whey isolate was compared to traditional bovine whey isolate in their protein and N-glycome composition. The bovine whey contained predominantly complex type N-glycans while the yeast-synthesized whey contained exclusively oligomannose N-glycans. Next, an in vitro model using three taxonomically and functionally distinct fecal communities’ representative of gut microbiota were used to screen how differences in these whey protein isolates influenced the gut microbiota. Significant differences in microbial composition, such as alpha diversity, were seen across all three microbial communities between the protein isolates. The compositional differences across all three microbial communities between the protein isolates suggested that each of the proteins' respective N-glycomes drove these differences. Next, the four dietary protein isolates, bovine whey, egg, pea, and soy were investigated. The N-glycomes' of the four protein isolates were characterized, and in vitro fermentation was performed to assess how these N-glycoproteins influenced microbial ecology, short-chain fatty acid production, and N-glycan degradation strategies across the three fecal communities. Composition and short-chain fatty acid production significantly differed in ii response to the glycoprotein sources in a microbiota-dependent manner. Glycoside hydrolase profiles, which are indicative of carbohydrate degradation, were significantly different between all three microbial communities regardless of treatment. Additionally, the glycoproteins had a significant impact on the glycoside hydrolase and KEGG ortholog expression profiles across each microbial community, suggesting that the differences in N-glycomes drove distinct gene expression. Finally, predicted endo-beta-N- acetylglucosaminidases (ENGases), which cleave the chitobiose core of N-glycans, were identified from the metagenomic sequencing of fecal samples and cloned. Three predicted ENGases were then tested for their catalytic ability to remove N-glycans from proteins. An RNase B gel shift was seen when incubated with all three of the cloned ENGases, indicating that all three enzymes had catalytic activity. Furthermore, the enzymes were tested on proteins that contained a mix of complex and oligomannose, exclusively complex, or core alpha 1,3 fucosylation. All three enzymes were exclusively active on oligomannose but could not remove complex or alpha 1,3 fucosylated N- glycans. Together, these experiments demonstrate how subtle differences in N-glycan composition impact the gut microbiome in vitro. Furthermore, these experiments demonstrate a proof-of-concept that isolate N-glycans by harnessing the genetic capabilities of the gut microbiome.

Level thesis:degree_level

Doctorate Degree

Year dc:date.issued

2026

Author dc:creator

• Bolino, Matthew John

Advisor dc:contributor.advisor

• Frese, Steven A

Language dc:language.iso

en_US, English

Repository record dc:identifier.uri

https://scholarwolf.unr.edu/handle/11714/11820

OAI identifier oai:identifier

oai:scholarwolf.unr.edu:11714/11820