Application of nanopore long-read sequencing and metabolomics in an in vitro dynamic intestinal digestion model: A genome-centric metatranscriptomic approach to investigating microbial TMA and SCFA metabolism.

The gut microbiota plays a relevant role in human health by metabolizing dietary components into bioactive molecules, including short-chain fatty acids and trimethylamine. Understanding how dietary interventions modulate microbial metabolism is key to developing strategies for reducing harmful metabolites such as TMA, a precursor of the pro-atherogenic trimethylamine-N-oxide. In this study, we integrated a dynamic in vitro gastrointestinal model (simgi®) with nanopore sequencing technology and metabolomics to investigate the impact of red thyme extract on microbial trimethylamine metabolism from L-carnitine. Metabarcoding, metagenomic, and metatranscriptomic analyses were performed alongside targeted metabolite quantification. Our results showed that microbial trimethylamine production primarily occurred in the transverse and descending colon compartments, coinciding with increased transcriptional activity of taxa harboring gbu cluster, associated with trimethylamine production. The administration of red thyme extract transiently reduced L-carnitine utilization but had a limited effect on overall trimethylamine levels. In parallel, short-chain fatty acids analysis revealed a shift in microbial fermentation patterns, with Acidaminococcus emerging as a dominant butyrate producer. Carbohydrate-active enzyme profiling identified Bacteroides and Parabacteroides genera as key mucin utilizers under the simulation conditions. These findings highlight the metabolic plasticity of the gut microbiota in response to the presence of L-carnitine and reduced complex carbohydrates availability, and provide new insights into microbial functional responses to dietary interventions targeting trimethylamine metabolism. Additionally, this study represents the first integration of nanopore-based metagenomics and genome-centric metatranscriptomics with targeted metabolomics in a dynamic in vitro gastrointestinal model. This multi-omics approach enabled a detailed reconstruction of the microbial metabolic network involved in L-carnitine utilization and trimethylamine formation, offering a powerful tool for mechanistic studies of gut microbiota-diet interactions.