Elucidation of an anaerobic pathway for metabolism of L-carnitine-derived γ-butyrobetaine to trimethylamine in human gut bacteria

ABSTRACTTrimethylamine (TMA) is an important gut microbial metabolite strongly associated with human disease. There are prominent gaps in our understanding of how TMA is produced from the essential dietary nutrient L-carnitine, particularly in the anoxic environment of the human gut where oxygen-dependent L-carnitine-metabolizing enzymes are likely inactive. Here, we elucidate the chemical and genetic basis for anaerobic TMA generation from the L-carnitine-derived metabolite γ-butyrobetaine (γbb) by the human gut bacterium Emergencia timonensis. We identify a set of genes upregulated by γbb and demonstrate that the enzymes encoded by the induced γbb utilization (bbu) gene cluster convert γbb to TMA. The key TMA-generating step is catalyzed by a previously unknown type of TMA-lyase enzyme that utilizes a flavin cofactor to catalyze a redox neutral transformation. We identify additional cultured and uncultured host-associated bacteria that possess the bbu gene cluster, providing insights into the distribution of anaerobic γbb metabolism. Lastly, we present genetic, transcriptional, and metabolomic evidence that confirms the relevance of this metabolic pathway in the human gut microbiota. These analyses indicate that the anaerobic pathway is a more substantial contributor to TMA generation from L-carnitine in the human gut than the previously proposed aerobic pathway. The discovery and characterization of the bbu pathway provides the critical missing link in anaerobic metabolism of L-carnitine to TMA, enabling investigation into the connection between this microbial function and human disease.SIGNIFICANCETrimethylamine (TMA) is a disease-associated metabolite produced in the human body exclusively by microbes. Gut microbes generate TMA from essential nutrients consumed in the human diet, including L-carnitine. However, our understanding of the biochemical mechanisms involved in these transformations is incomplete. In this work, we define the biochemical pathway and genetic components in gut bacteria required for anaerobic production of TMA from γ-butyrobetaine, a metabolite derived from L-carnitine. This discovery identifies a new type of TMA-producing enzyme and fills a critical gap in our knowledge of L-carnitine metabolism to TMA in the anaerobic environment of the human gut. This knowledge will enable evaluation of the link between L-carnitine metabolism and human disease, and the design of potential therapeutics.

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Elucidation of an anaerobic pathway for metabolism of l-carnitine–derived γ-butyrobetaine to trimethylamine in human gut bacteria

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2101498118 ◽

2021 ◽

Vol 118 (32) ◽

pp. e2101498118

Author(s):

Lauren J. Rajakovich ◽

Beverly Fu ◽

Maud Bollenbach ◽

Emily P. Balskus

Keyword(s):

Gene Cluster ◽

Human Disease ◽

Genetic Basis ◽

Human Gut ◽

Metabolizing Enzymes ◽

Associated Bacteria ◽

Microbial Function ◽

Dietary Nutrient ◽

Anoxic Environment

Trimethylamine (TMA) is an important gut microbial metabolite strongly associated with human disease. There are prominent gaps in our understanding of how TMA is produced from the essential dietary nutrient l-carnitine, particularly in the anoxic environment of the human gut where oxygen-dependent l-carnitine–metabolizing enzymes are likely inactive. Here, we elucidate the chemical and genetic basis for anaerobic TMA generation from the l-carnitine–derived metabolite γ-butyrobetaine (γbb) by the human gut bacterium Emergencia timonensis. We identify a set of genes up-regulated by γbb and demonstrate that the enzymes encoded by the induced γbb utilization (bbu) gene cluster convert γbb to TMA. The key TMA-generating step is catalyzed by a previously unknown type of TMA-lyase enzyme that utilizes a putative flavin cofactor to catalyze a redox-neutral transformation. We identify additional cultured and uncultured host-associated bacteria that possess the bbu gene cluster, providing insights into the distribution of anaerobic γbb metabolism. Lastly, we present genetic, transcriptional, and metabolomic evidence that confirms the relevance of this metabolic pathway in the human gut microbiota. These analyses indicate that the anaerobic pathway is a more substantial contributor to TMA generation from l-carnitine in the human gut than the previously proposed aerobic pathway. The discovery and characterization of the bbu pathway provides the critical missing link in anaerobic metabolism of l-carnitine to TMA, enabling investigation into the connection between this microbial function and human disease.

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A Bacterial Microcompartment Is Used for Choline Fermentation byEscherichia coli536

Journal of Bacteriology ◽

10.1128/jb.00764-17 ◽

2018 ◽

Vol 200 (10) ◽

Cited By ~ 12

Author(s):

Taylor I. Herring ◽

Tiffany N. Harris ◽

Chiranjit Chowdhury ◽

Sujit Kumar Mohanty ◽

Thomas A. Bobik

Keyword(s):

Electron Microscopy ◽

Heart Disease ◽

Gene Cluster ◽

Transcriptional Regulator ◽

Human Gut ◽

Content Type ◽

E Coli ◽

Bacterial Microcompartment ◽

New Type ◽

Insight Into

ABSTRACTBacterial choline degradation in the human gut has been associated with cancer and heart disease. In addition, recent studies found that a bacterial microcompartment is involved in choline utilization byProteusandDesulfovibriospecies. However, many aspects of this process have not been fully defined. Here, we investigate choline degradation by the uropathogenEscherichia coli536. Growth studies indicatedE. coli536 degrades choline primarily by fermentation. Electron microscopy indicated that a bacterial microcompartment was used for this process. Bioinformatic analyses suggested that the choline utilization (cut) gene cluster ofE. coli536 includes two operons, one containing three genes and a main operon of 13 genes. Regulatory studies indicate that thecutXgene encodes a positive transcriptional regulator required for induction of the maincutoperon in response to choline supplementation. Each of the 16 genes in thecutcluster was individually deleted, and phenotypes were examined. ThecutX,cutY,cutF,cutO,cutC,cutD,cutU, andcutVgenes were required for choline degradation, but the remaining genes of thecutcluster were not essential under the conditions used. The reasons for these varied phenotypes are discussed.IMPORTANCEHere, we investigate choline degradation inE. coli536. These studies provide a basis for understanding a new type of bacterial microcompartment and may provide deeper insight into the link between choline degradation in the human gut and cancer and heart disease. These are also the first studies of choline degradation inE. coli536, an organism for which sophisticated genetic analysis methods are available. In addition, thecutgene cluster ofE. coli536 is located in pathogenicity island II (PAI-II536) and hence might contribute to pathogenesis.

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Characterization and Detection of a Widely Distributed Gene Cluster That Predicts Anaerobic Choline Utilization by Human Gut Bacteria

mBio ◽

10.1128/mbio.00042-15 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 77

Author(s):

Ana Martínez-del Campo ◽

Smaranda Bodea ◽

Hilary A. Hamer ◽

Jonathan A. Marks ◽

Henry J. Haiser ◽

...

Keyword(s):

Gut Microbiota ◽

Gene Cluster ◽

Gut Bacteria ◽

Functional Gene ◽

Metagenomic Data ◽

Human Gut ◽

Bacterial Gene ◽

Human Gut Microbiota ◽

Choline Metabolism

ABSTRACTElucidation of the molecular mechanisms underlying the human gut microbiota's effects on health and disease has been complicated by difficulties in linking metabolic functions associated with the gut community as a whole to individual microorganisms and activities. Anaerobic microbial choline metabolism, a disease-associated metabolic pathway, exemplifies this challenge, as the specific human gut microorganisms responsible for this transformation have not yet been clearly identified. In this study, we established the link between a bacterial gene cluster, the choline utilization (cut) cluster, and anaerobic choline metabolism in human gut isolates by combining transcriptional, biochemical, bioinformatic, and cultivation-based approaches. Quantitative reverse transcription-PCR analysis andin vitrobiochemical characterization of twocutgene products linked the entire cluster to growth on choline and supported a model for this pathway. Analyses of sequenced bacterial genomes revealed that thecutcluster is present in many human gut bacteria, is predictive of choline utilization in sequenced isolates, and is widely but discontinuously distributed across multiple bacterial phyla. Given that bacterial phylogeny is a poor marker for choline utilization, we were prompted to develop a degenerate PCR-based method for detecting the key functional gene choline TMA-lyase (cutC) in genomic and metagenomic DNA. Using this tool, we found that new choline-metabolizing gut isolates universally possessedcutC. We also demonstrated that this gene is widespread in stool metagenomic data sets. Overall, this work represents a crucial step toward understanding anaerobic choline metabolism in the human gut microbiota and underscores the importance of examining this microbial community from a function-oriented perspective.IMPORTANCEAnaerobic choline utilization is a bacterial metabolic activity that occurs in the human gut and is linked to multiple diseases. While bacterial genes responsible for choline fermentation (thecutgene cluster) have been recently identified, there has been no characterization of these genes in human gut isolates and microbial communities. In this work, we use multiple approaches to demonstrate that the pathway encoded by thecutgenes is present and functional in a diverse range of human gut bacteria and is also widespread in stool metagenomes. We also developed a PCR-based strategy to detect a key functional gene (cutC) involved in this pathway and applied it to characterize newly isolated choline-utilizing strains. Both our analyses of thecutgene cluster and this molecular tool will aid efforts to further understand the role of choline metabolism in the human gut microbiota and its link to disease.

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Combining LC-MS metabolomics and next generation sequencing to study the interactions between herbal medicines and human gut bacteria in-vitro

10.1055/s-0037-1608574 ◽

2017 ◽

Author(s):

EM Pferschy-Wenzig ◽

K Koskinen ◽

A Roßmann ◽

K Ardjomand-Woelkart ◽

C Moissl-Eichinger ◽

...

Keyword(s):

Next Generation Sequencing ◽

Herbal Medicines ◽

Gut Bacteria ◽

Next Generation ◽

Human Gut ◽

Generation Sequencing

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Faculty Opinions recommendation of Functional metagenomics reveals novel pathways of prebiotic breakdown by human gut bacteria.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718118305.793485814 ◽

2013 ◽

Author(s):

Jo Handelsman ◽

Heather Allen

Keyword(s):

Gut Bacteria ◽

Functional Metagenomics ◽

Human Gut

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Faculty Opinions recommendation of Human gut bacteria contain acquired interbacterial defence systems.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.736828921.793567257 ◽

2019 ◽

Author(s):

Michael Kurilla

Keyword(s):

Gut Bacteria ◽

Human Gut

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Flavonoid-Modifying Capabilities of the Human Gut Microbiome—An In Silico Study

Nutrients ◽

10.3390/nu13082688 ◽

2021 ◽

Vol 13 (8) ◽

pp. 2688

Author(s):

Tobias Goris ◽

Rafael R. C. Cuadrat ◽

Annett Braune

Keyword(s):

Gut Microbiome ◽

Large Scale ◽

Health Impact ◽

Gut Bacteria ◽

Human Gut ◽

Positive Health ◽

Human Gut Microbiome ◽

Nutritional Recommendations ◽

Genes Encoding ◽

A Minor

Flavonoids are a major group of dietary plant polyphenols and have a positive health impact, but their modification and degradation in the human gut is still widely unknown. Due to the rise of metagenome data of the human gut microbiome and the assembly of hundreds of thousands of bacterial metagenome-assembled genomes (MAGs), large-scale screening for potential flavonoid-modifying enzymes of human gut bacteria is now feasible. With sequences of characterized flavonoid-transforming enzymes as queries, the Unified Human Gastrointestinal Protein catalog was analyzed and genes encoding putative flavonoid-modifying enzymes were quantified. The results revealed that flavonoid-modifying enzymes are often encoded in gut bacteria hitherto not considered to modify flavonoids. The enzymes for the physiologically important daidzein-to-equol conversion, well studied in Slackiaisoflavoniconvertens, were encoded only to a minor extent in Slackia MAGs, but were more abundant in Adlercreutzia equolifaciens and an uncharacterized Eggerthellaceae species. In addition, enzymes with a sequence identity of about 35% were encoded in highly abundant MAGs of uncultivated Collinsella species, which suggests a hitherto uncharacterized daidzein-to-equol potential in these bacteria. Of all potential flavonoid modification steps, O-deglycosylation (including derhamnosylation) was by far the most abundant in this analysis. In contrast, enzymes putatively involved in C-deglycosylation were detected less often in human gut bacteria and mainly found in Agathobacter faecis (formerly Roseburia faecis). Homologs to phloretin hydrolase, flavanonol/flavanone-cleaving reductase and flavone reductase were of intermediate abundance (several hundred MAGs) and mainly prevalent in Flavonifractor plautii. This first comprehensive insight into the black box of flavonoid modification in the human gut highlights many hitherto overlooked and uncultured bacterial genera and species as potential key organisms in flavonoid modification. This could lead to a significant contribution to future biochemical-microbiological investigations on gut bacterial flavonoid transformation. In addition, our results are important for individual nutritional recommendations and for biotechnological applications that rely on novel enzymes catalyzing potentially useful flavonoid modification reactions.

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