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.

Keywords: Dynamic in vitro gastrointestinal model; Long-read DNA sequencing; Metabolomics; Metagenomics; Metatranscriptomics; UPLC-QqQ-MS/MS.

Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.

肠道微生物群在人类健康中扮演着重要角色，通过将膳食成分代谢为生物活性分子（包括短链脂肪酸和三甲胺）发挥作用。了解饮食干预如何调节微生物代谢对于开发减少有害代谢物（如三甲胺的前体——促动脉粥样硬化的三甲胺氧化物）的策略至关重要。在这项研究中，我们结合了动态体外胃肠道模型（simgi®）和纳米孔测序技术及代谢组学来探讨红迷迭香提取物对从左旋肉碱生成三甲胺的微生物代谢的影响。进行了元条形码、宏基因组和宏转录组分析，并伴随目标代谢物定量。我们的结果表明，微生物三甲胺生产主要发生在横结肠和降结肠部分，在与三甲胺产生相关的gbu簇所含分类群的转录活性增加时亦如此。红迷迭香提取物的施用暂时减少了左旋肉碱利用但对总体三甲胺水平的影响有限。同时，短链脂肪酸分析揭示了微生物发酵模式的变化，其中酸氨杆菌成为主要丁酸生成者。糖基水解酶谱分析确定了在模拟条件下拟杆菌和副拟杆菌属为关键的黏蛋白使用者。这些发现突显了肠道微生物群对左旋肉碱存在及其复杂碳水化合物可用性降低时表现出代谢可塑性的能力，并提供了有关针对三甲胺代谢的饮食干预措施的新的洞见。此外，本研究代表了首次将基于纳米孔的宏基因组学和以基因组为中心的宏转录组学与目标代谢组学结合应用于动态体外胃肠道模型中的整合方法。这种多组学方法能够详细重构涉及左旋肉碱利用和三甲胺形成的微生物代谢网络，为研究肠道微生物群-饮食相互作用机制提供了一个强大的工具。

关键词：动态体外胃肠道模型；长读DNA测序；代谢组学；宏基因组学；宏转录组学；UPLC-QqQ-MS/MS。