Deciphering Human Gut MicrobiotaâNutrient Interactions: A Role for

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Deciphering human gut microbiotanutrient interactions: a role for biochemistry Carina Chittim, Stephania M. Irwin, and Emily P Balskus Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01277 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

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Biochemistry

Title: Deciphering human gut microbiota-nutrient interactions: a role for biochemistry

Authors – Carina L. Chittim, Stephania M. Irwin, Emily P. Balskus* Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St, Cambridge, Massachusetts 02138, United States. *Email: [email protected]

Abstract: The human gut contains trillions of microorganisms that play a central role in many aspects of host biology, including the provision of key nutrients from the diet. However, our appreciation of how gut microbes and their extensive metabolic capabilities affect the nutritional status of the human host is in its infancy. In this perspective, we highlight how recent efforts to elucidate the biochemical basis for gut microbial metabolism of dietary components are reshaping our view of these organisms’ roles in host nutrition. Gaining a molecular understanding of gut microbenutrient interactions will enhance our knowledge of how diet affects host health and disease, ultimately enabling personalized nutrition and therapeutics.

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Introduction

We inhabit a microbial planet. Bacteria, archaea, microscopic eukaryotes, and viruses colonize essentially every available habitat and help to maintain homeostasis in a diverse range of ecosystems, including the human body. The trillions of microbes that live in and on the human body are collectively referred to as the human microbiota. Current estimates indicate the human body contains a 1:1 ratio of microbial to host cells.1 However, the collective number of genes present in the human microbiome surpasses that of the human genome by an estimated 150fold.1,2 The expanded genetic potential of the human microbiota encodes an expanded set of metabolic capabilities with poorly understood roles in host biology.

Most human-associated microbes inhabit the gastrointestinal tract, with the human colon harboring one of the densest microbial communities known.3 Though these organisms have been studied for many years, the past decade has seen a dramatic increase in efforts to characterize the human gut microbiota and its links to host health and disease. Advances in DNA sequencing have enabled large-scale application of taxonomic profiling, the use of genes that are markers of phylogeny (typically the gene encoding the 16S subunit of the bacterial ribosome), to identify the organisms present in human-associated microbial communities.4 These efforts have revealed that the healthy human gut contains hundreds of different bacterial species and is typically dominated by obligate anaerobes from the phyla Bacteroidetes and Firmicutes. However, there is tremendous inter-individual variability in gut community composition. Meta-omics approaches (metagenomics, metatranscriptomics, metaproteomics and metabolomics) can provide insight into the functional potential of microbial communities.5 Application of these methods to the human gut microbiota has revealed that, despite variation in community composition, there may be some conservation of core biological functions.6


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However, the broad categorization of functions in these analyses, coupled with our poor understanding of the roles of the majority of genes in microbiomes, makes it difficult to assess the degree to which specific biochemical activities are maintained. Sequencing various patient populations has also revealed correlations between both the composition and functions of the gut microbiota and numerous diseases.7 Simultaneously, studies with gnotobiotic models that involve transplant of microbes into germ-free hosts have revealed a causal role for gut microbes in health and disease.8 Overall, these recent efforts have reinforced the idea that gut microbes play a central role in the functioning of the human body and that inter-individual differences in the gut microbiota likely influence host biology.

One of the aspects of host physiology in which the importance of the gut microbiota is most readily apparent is nutrition. Studies using germ-free animals have revealed that organisms raised in the absence of microbes have deficits in nutrient acquisition and metabolism,9,10 and the development of supplemented diets was critical for successful maintenance of these model systems. Beginning in the 1940s, a large body of work has shown that gut microbes play integral roles in metabolizing key dietary nutrients, including breaking down otherwise indigestible compounds.11-13 With the advent of modern metabolomics approaches, the end products of this process can be observed directly.14 But despite the importance of gut microbial interactions with dietary components, our understanding of the mechanisms by which particular transformations of nutrients affect host biology is extremely limited.

This gap in knowledge arises in part from a poor understanding of the molecular basis for gut microbial metabolic activities. Most metabolic transformations performed by this community have not been linked to specific organisms, genes, or enzymes.15 This leaves us unable to accurately assess the functional capabilities of individual gut communities; indeed, 85% of the


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genes in the gut microbiomes of subjects sequenced as part of the Human Microbiome Project cannot be readily linked to known microbial metabolic processes. To understand how the gut microbiota’s metabolic capabilities affect host nutrition, it is essential that we address this knowledge gap. In this perspective, we highlight selected recent advances in connecting gut microbial nutrient metabolism and transport to specific microbial genes and enzymes, including examples that span multiple classes of essential nutrients. These studies illustrate how gaining a molecular understanding of gut microbiota-nutrient interactions is reshaping our views of this community’s role in host nutrition (Figure 1). Continued efforts to elucidate this biochemistry will not only enhance our fundamental knowledge of how diet affects host health and disease, but will also be critical for enabling personalized nutrition and therapeutics.

carbohydrates

human gut microbiota

protein and amino acids

microbial genes and proteins

altered nutrient availabiity

nutritional requirements altered disease risk

lipids

links between diet and disease vitamins essential nutrients

molecular basis for gut microbiotanutrient interactions?

altered community composition

effect of diet on microbiota

Figure 1. The central role of biochemistry in understanding gut microbiota-nutrient interactions.

Polysaccharide metabolism: a surprising origin for gut bacterial enzymes

Complex polysaccharides are perhaps the most extensively studied dietary substrates in the context of the human gut microbiota. Most of these molecules are indigestible by host enzymes, but undergo extensive metabolism by gut microbes.16,17 The end products of polysaccharide


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fermentation in the gut are short-chain fatty acids (SCFAs).18 SCFAs, including propionate, butyrate, and acetate, play a central role in host nutrition by providing a key energy source for colonocytes. This contribution can represent up to 10% of daily caloric requirements.19 In addition to SCFAs’ role as an energy source, they also interact with specific G protein-coupled receptors (GPRs) on host cells that are involved in regulation of lipid and glucose metabolism. Butyrate also binds to histone deacetylases (HDACs) in a manner that inhibits activity, subsequently impacting gene expression in human cells.20 The gut bacterial pathways and enzymes responsible for SCFA generation have been extensively reviewed.21-23

Accessing simple sugars from complex dietary carbohydrates requires that gut microbes hydrolyze a vast array of different glycosidic linkages.17 The group of enzymes that catalyzes these transformations has been designated carbohydrate-active enzymes (CAZymes). The expression of these CAZymes can be influenced by the availability of specific substrates and the presence of other gut microbes.24 Understanding these enzymes, their specificities, their regulation, and their distribution in the gut can help us assess which polysaccharides are likely to be processed by individual gut communities and perhaps predict responses to specific dietary interventions or prebiotics, complex substrates (typically polysaccharides) intended to stimulate activities of beneficial gut microbes.

The discovery of new CAZymes has also provided unexpected insights into the role of the human diet in shaping the metabolic capabilities of gut microbes. In 2010, Hehemann et al. used a rational bioinformatic approach to search the genome of the marine bacterium Zobellia galactanivorans for enzymes involved in metabolizing polysaccharides present in marine algae.25 They identified two new glycoside hydrolases (PorA and PorB) that were capable of cleaving the b-1,4-glycosidic linkages in porphyran, a sulfated polysaccharide found in the


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edible red alga Porphyra, in vitro (Figure 2A). These enzymes are the first b-porphyranases to be described and were unable to catalyze the cleavage of other complex polysaccharides, including agarose and k-carrageenan. Crystallization of each enzyme bound to a porphyran tetrasaccharide revealed the key recognition elements responsible for this substrate selectivity (Figure 2B). Specifically, the a-L-galactopyranose-6-sulfate at the –2 position is positioned in a cleft with its negatively charged C6 sulfate group bound within a pocket containing positively charged histidine and arginine residues. This binding pocket is not present in glycoside hydrolases that accept alternate substrates such as agarose, which do not contain negatively charged substituents (Figure 2B).

Figure 2: The discovery of b-porphyranase enzymes uncovers new interactions between food- and human-associated bacteria. A) PorA and PorB are glycoside hydrolases that cleave the b-1,4-glycosidic linkages in the sulfated seaweed polysaccharide porphyran. B) The crystal structure of PorA bound to a porphyran tetrasaccharide reveals amino acids involved in substrate recognition (PDB = 3ILF) as well as a key pocket for substrate binding. C) b-


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porphyranases were detected solely in gut bacterial species and gut metagenomes from Japanese subjects. The prominence of seaweed in the Japanese diet raises the intriguing possibility that gut bacteria acquired b-porphyranases via horizontal gene transfer from marine bacteria present in seaweed-based foods.

Gaining a molecular understanding of substrate recognition by PorA and PorB enabled bioinformatic searches for additional b-porphyranases in sequenced microbial genomes. In their analysis, Hehemann et al. identified six additional putative glycoside hydrolases that contained the residues involved in porphyran binding.25 These enzymes were located in other marine bacteria, with the exception of one b-porphyranase that was present in the genome of a human gut bacterium Bacteroides plebeius, a species that had only been isolated from Japanese individuals. Analysis of gut metagenomes from Japanese and North American individuals revealed b-porphyranase genes only in the Japanese subjects, suggesting this metabolic activity could be absent in the Western gut microbiota (Figure 2C). The high similarity and close phylogenetic relationship between the b-porphyranases found in B. plebeius and marine bacteria, along with the importance of seaweed in the Japanese diet, led the authors to propose that consumption of raw seaweed led to the transfer of the genes encoding porphyrandegrading enzymes from seaweed-associated marine bacteria into strains of B. plebeius residing in the gut, providing this organism with the ability to process a previously inaccessible dietary polysaccharide.

These findings not only illustrate the metabolic diversity present in the gut microbiotas of different populations, but also alter our views of how diet affects this microbial community. Specifically, this work suggests that interactions between food-associated microbes and species


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present in the human gut can actually change the metabolic capabilities of the gut microbiota. The discovery of new CAZymes in human gut microbes continues to be a dynamic area of biochemistry that will advance our knowledge of diet-microbe interactions, with notable recent achievements including the identification and characterization of mannan-26,27 and xylandegrading28,29 enzymes.

Amino acid metabolism: a new link between gut microbes and the nervous system

Humans obtain essential amino acids from dietary protein and potentially also from de novo synthesis by the gut microbiota.30 Both host and gut microbial enzymes can hydrolyze ingested proteins to release these critical building blocks, with early work demonstrating the proteolytic capabilities of both gut contents and fecal samples.31 Genomic analyses have also shown that human gut microbes encode numerous proteases, peptidases, and amino acid peptide transporters that likely affect amino acid metabolism within the host.32,33 A subset of these proteases and peptidases have been discussed elsewhere, especially in connection to bacteria found predominantly in fermented foods.32,34

In addition to contributing to protein synthesis by both host and microbes, free amino acids are also subject to further transformations by the gut microbiota. Many of these pathways are uniquely microbial. For example, Stickland fermentation involves the coupled oxidation and reduction of pairs of amino acids and is a major route for energy production in human gut commensal and pathogenic Clostridium, with this metabolic process playing a particularly important role in colonization of the human gut by Clostridium difficile.35,36 The production of indole from tryptophan is carried out by multiple species within the gut, and the resulting host metabolite indoxyl sulfate is a suspected uremic toxin.37-39 Likewise, the gut microbial


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conversion of tyrosine to p-cresol has been linked to autism, and excess production of this metabolite can interfere with host metabolism of drugs such as acetaminophen.40,41 Gut microbial metabolism of aromatic amino acids can also produce immunomodulatory metabolites, as is seen with the conversion of histidine to histamine and tryptophan to indoleproprionic acid.42,43 Overall, it is becoming clear that the biological consequences of dietary protein-gut microbiota interactions extend far beyond simply accessing free amino acids.

One particularly intriguing area of host biology that may be affected by gut microbial amino acid metabolism is nervous system function. Monoamine neurotransmitters, including serotonin and catecholamines, are derived from aromatic amino acids.44 While it was originally thought that monoamine neurotransmitters only signaled in the central nervous system, their role in other systems is now appreciated.44 While it has been known for almost 100 years that bacteria from the human gut can produce neurotransmitters and other neuroactive metabolites, the biological consequences of this metabolism are not yet well defined.11 At the same time, it is becoming increasingly clear that the gut microbiota influences nervous system function. Germ-free mice exhibit strikingly altered behavioral tendencies compared to conventional animals,45 and have decreased excitability of a subset of neurons in the enteric nervous system.46 Studies in various patient cohorts have revealed strong links between neurological disorders and gastrointestinal dysfunction, and work with gnotobiotic animal models has shown that the gut microbiota can contribute to conditions such as autism and Parkinson’s disease.47,48 Though accumulating evidence clearly supports the existence of a ‘gut microbiota-brain axis’, the molecular mechanisms underlying these effects are not well understood. One intriguing possibility is that gut microbial transformation of dietary amino acids to neuroactive metabolites may play a role in this process.


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Recent work uncovering enzymes involved in the unexpected production of the neurotransmitter tryptamine by human gut bacteria highlights the prominent gaps that still remain in our appreciation of gut microbiota-amino acid interactions. Tryptamine is a trace amine neurotransmitter that has been previously thought to modulate the serotonin response in myenteric neurons.49 However, recent work has suggested tryptamine does not activate serotonin release from enterochromaffin cells.50 As tryptamine and serotonin share tryptophan as a common precursor, increased tryptamine levels may reflect a global change in tryptophan metabolism that also affects serotonin production. The precise biological function of microbiallyproduced tryptamine is still not fully understood.

Tryptamine is formed from decarboxylation of the amino acid tryptophan, and the tryptophan decarboxylase enzymes that catalyze this transformation are widespread in eurkaryotes.51 Though tryptamine had been observed previously in human stool,52 tryptophan decarboxylase activity had been reported only in a few bacteria that were not members of the human gut microbiota.53-55 Williams et al. serendipitously discovered that the human gut bacterium Clostridium sporogenes could convert tryptophan to tryptamine (Figure 3A).56 They identified candidate enzyme(s) for this transformation by searching the C. sporogenes genome for genes encoding pyridoxal phosphate (PLP)-dependent decarboxylase enzymes. This targeted search revealed one gene (CLOSPO_02085) that was annotated as a tyrosine decarboxylase. In vitro biochemical characterization confirmed that the encoded enzyme was indeed a tryptophan decarboxylase (Figure 3A).

Searches of additional gut bacterial genomes and heterologous expression of putative tryptophan decarboxylases in E. coli identified one additional tryptamine-generating enzyme in Ruminococcus gnavus. Interestingly, this enzyme (RUMGA_01526) shared only low sequence


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Biochemistry

similarity with the decarboxylase from C. sporogenes. A crystal structure of the R. gnavus tryptophan decarboxylase enzyme bound to the PLP adduct derived from the mechanism-based inhibitor (S)-a-fluoromethyltryptophan revealed the active site residues involved in recognizing the indole side chain of tryptophan (Figure 3B). This molecular information helped Williams et al. to identify tryptophan decarboxylase-encoding genes in assembled gut metagenomes from 86 healthy subjects. Intriguingly, this enzyme could be detected in only ~10% of these samples, suggesting the potential for a high degree of inter-individual variability in tryptamine production by the human gut microbiota.

A

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L-tryptophan CLOSPO_02085 or RUMGA_01526

human gut bacteria Clostridium sporogenes Ruminococcus gnavus

tryptophan decarboxylase PLP

neurologically active metabolite

+

CO2

NH 2

HN

tryptamine

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(S)-α-fluromethyltyrosine (FMT)

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Figure 3: The identification of a gut bacterial enzyme involved in tryptamine production connects metabolism of dietary amino acids to nervous system function. (A) Tryptophan decarboxylases from Clostridium sporogenes (CLOSPO_02085) and Ruminococcus gnavus (RUMGA_01526) convert tryptophan to the neuroactive metabolite tryptamine. (B) A crystal structure of the R. gnavus tryptophan decarboxylase bound to the PLP-adduct derived from exposure of the enzyme to the mechanism-based inhibitor (S)-a-fluoromethyltryptophan (PDB = 4OBV) reveals active site residues critical for substrate recognition.

This final observation has important implications for gut microbe-nervous system interactions, suggesting that the presence of both specific dietary precursors and specific metabolic activities may influence the effects this community has on the host nervous system. Indeed, Williams et al. showed that tryptamine stimulates ion secretion by colonic epithelial cells ex vivo, indicating a potential role for this gut microbial metabolite in GI mobility.56 Overall, this work should inspire further investigations of gut microbial protein and amino acid metabolism, with an eye toward elucidating how differences in dietary protein intake combine with gut microbial metabolic capabilities to shape the production of neuroactive small molecules.

Lipid metabolism

The larger nutrient class of lipids includes fat soluble vitamins, mono-, di-, and tri-glycerides, phospholipids, sterols, and fatty acids. These molecules are present in the diet and interact with the gut microbiota. Many of the lipids present in the colon come from bile released into the gastrointestinal tract postprandially.57 Bile salts are synthesized in the liver with sterol cores that are conjugated to either taurine or glycine. Microbial deconjugation of bile salts by bile salt hydrolases (BSH) is the key step in regulating the circulation and excretion pathways of bile


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acids. BSH enzymes from many gut bacteria, including Bacteroides fragilis, Clostridium perfrigens, Bifidobacterium longum, Lactobacillus plantarum, have been biochemically characterized.12,58,59 In 2014, a study showed BSH activity could alter host expression profiles of lipid metabolizing, cholesterol metabolizing, and homeostasis regulating genes.60 In addition to BSHs, gut isolates can generate secondary bile acids through addition modification to the steroid scaffold including 7a-dehydroxylation.58 The genes responsible for many of these transformation and have been characterized in a variety of gut isolates.61-63 However, much less known about the specific microbial enzymes responsible for metabolizing additional classes of lipids. Developing our biochemical knowledge of gut microbial lipid-interactions may provide us with new insights into in obesity, cardiovascular disease, and other diseases associated with altered lipid metabolism.

Essential nutrient metabolism: uncovering the consequences of gut microbial nutrient consumption

It has been long appreciated that gut microbiota can positively affect the availability of critical nutrients for the host. This can occur via microbial modulation of host nutrient uptake pathways, release of nutrients from dietary sources, and de novo synthesis of nutrients such as essential vitamins.64,65 Daily recommended vitamin intake may not always be met by diet alone, particularly in the case of undernutrition. Gut bacterial synthesis of folic acid, biotin, riboflavin, and Vitamin K, may help to support unmet nutritional requirements.64 Beyond these essential vitamins, studies in germ-free animals have revealed the gut microbiota can enhance the availability of glycine-conjugated aromatic amino acids,66 iron,67 and additional nutrients through poorly defined mechanisms.


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Recent efforts to understand a disease-associated gut microbial metabolic pathway have begun to reshape our view of how gut microbes affect nutrient availability for the host. Choline is an essential dietary nutrient that plays multiple roles in the human body.68 Choline is a critical component of host cell membrane lipids such as phosphatidylcholine and sphingomyelin, is metabolized to the neurotransmitter acetylcholine, and is a precursor to glycine betaine, which is a methyl donor for the synthesis of S-adenosylmethionine via the enzyme betaine-homocysteine methyltransferase.69

Over 100 years ago, it was noted that certain bacteria convert choline into trimethylamine (TMA) under anaerobic conditions.70 This metabolic activity was subsequently identified in many anaerobic microbial habitats, including the human gut. TMA is a uniquely microbial metabolite in the human body and is further processed to trimethylamine N-oxide (TMAO) by a flavin monooxygenase 3 in the liver (Figure 4A).71 Both TMA and TMAO are linked to human disease. In the last decade, metabolomics studies have uncovered correlations between elevated levels of TMAO in plasma and multiple diseases including atherosclerosis, colon cancer, diabetes, non-alcoholic fatty liver disease, and kidney disease.72 However, this association has not been consistently found across all disease cohorts examined; a recent study observed no correlation between elevated levels of TMAO and cardiovascular outcomes for patients with renal disease.73 TMA also plays a central role in the inherited metabolic disease trimethylaminuria or ‘fish malodor syndrome’, in which patients have mutations in the gene encoding FMO3, resulting in deficiencies in TMA oxidation and excretion of this odorous microbial metabolite.74

Despite its strong connections to disease, the genetic and biochemical basis for anaerobic microbial choline metabolism was unknown until 2012 when we reported the discovery of a choline utilization (cut) gene cluster in the sheep gut microbe Desulfovibrio desulfuricans. The


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first step of this metabolic process is a C–N bond cleavage event that converts choline to TMA and acetaldehyde. Recognizing potential parallels between this reaction and the well-studied bacterial pathway for ethanolamine utilization, we were able to identify the cut gene cluster by searching the D. desulfuricans genome for homologs of genes involved in ethanolamine metabolism. Encoded within this gene cluster was the choline TMA-lyase (CutC), a glycyl radical enzyme (GRE) that catalyzes the critical C–N bond cleavage reaction (Figure 4A).75 GREs use glycine- and cysteine-centered radical intermediates to perform challenging radicalbased reactions, with the key glycine centered radical generated posttranslationally by an activating enzyme partner that is a member of the radical SAM enzyme family. Biochemical and structural characterization of CutC confirmed its activity, revealed a strict specificity for choline as a substrate, and identified conserved active site residues that are important for catalysis but are not found in GREs that perform different transformations (Figure 4B).75,76

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CutC choline TMA-lyase

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CC– choline metabolism absent from gut microbiota

atherosclerosis diabetes liver disease kidney disease trimethylaminuria

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CC– CC+ decreased flux

choline depleted in CC+ mice

SAM

40

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*

0

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DNA methylation epigenetic changes

Figure 4: The discovery and characterization of choline trimethylamine-lyase (CutC) reveals the consequences of gut microbial nutrient consumption. A) CutC is a glycyl radical enzyme that converts choline to acetaldehyde and the disease-associated metabolite


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trimethylamine (TMA). B) Structural characterization of CutC (PDB = 5FAU) reveals unique, conserved active site residues that provide a bioinformatic signature of this activity and guide the discovery of choline-metabolizing human gut microbes. C) Deletion of cutC in the genetically tractable choline utilizing strain E. coli MS 200-1 provides a strategy for removing this microbial activity from a model gut microbiota and assessing its role in host biology. (D) Mice with a choline consuming microbiota (CC+) have decreased levels of serum choline compared to mice lacking this activity. (E) CC+ mice display altered one-carbon metabolism and epigenetic changes.

Gaining a molecular understanding of CutC has facilitated efforts to decipher its distribution in gut microbes and roles in host biology. Further genome analyses revealed the cut gene cluster in multiple bacterial phyla present the human gut microbiota.77 However, this metabolic activity is discontinuously distributed across individual gut microbial species, complicating efforts to use community composition as a predictor of choline metabolism. Additional experiments demonstrated that the cut gene cluster was predictive of choline metabolism across phylogenetically distant organisms and was present in both newly isolated choline utilizing gut microbes as well as human gut metagenomes.77

Another important outcome of this study was the identification of a genetically tractable choline utilizing gut bacterium, E. coli MS 200-1. The availability of this strain allowed us to devise an approach for manipulating gut microbial choline metabolism in a host setting: ‘deleting’ this metabolic activity from the gut microbiota. We colonized two groups of germ-free mice with defined, model gut communities that consisted of six strains of non-choline metabolizing human gut bacteria and either wild-type E. coli MS 200-1 or a cutC deletion mutant (Figure 4C).78 While removing choline metabolism from the gut microbiota didn’t have a large effect on gut


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microbiota composition, it dramatically altered metabolite levels (Figure 4D). As anticipated, TMAO production was abolished in the mice colonized with the community containing the cutC mutant. Strikingly, we also observed differences in serum choline, with the mice possessing choline-consuming gut microbiota having dramatically reduced levels of this nutrient. 78

We hypothesized that this decrease in choline availability was due to excess consumption of this nutrient by the gut microbiota. Subsequent analyses of additional host metabolites and biological phenotypes support with this proposal.78 In addition to choline, mice with a cholineconsuming gut microbiota had decreased levels of other molecules that linked to one-carbon metabolism (Figure 4E). Both adult mice and pups in utero also displayed alterations levels of in DNA methylation in various tissues. Similar metabolic and epigenetic changes have been previously observed in mice fed choline-deficient diets.68,79 Additionally, the presence of the CutC enzyme in the gut microbiota led to pronounced phenotypes in two mouse models where one-carbon metabolism is known to be important.78 Mice with a choline consuming gut microbiota displayed alterations in lipid metabolism and fat pad mass in a model of diet-induced metabolic disease, including a dramatically increased accumulation of liver trigylcerides. Genetically predisposed adults and offspring also had increased anxiety behavior. Overall, this work suggests that negative effects of gut microbial choline metabolism on host health may arise from the combined consequences of TMA/TMAO production and choline depletion.

More broadly, these findings are helping to reshape our view of the microbiota’s role in host nutrition. In uncovering these negative consequences of gut microbial choline consumption on host biology, we have highlighted the potential for gut microbes to compete with the host for critical nutrients. Variations in gut microbial nutrient and vitamin consumption may be an important but underappreciated factor in determining host nutrient requirements, and this


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phenomenon may be particularly important in the context of choline. Most individuals do not consume enough dietary choline to meet recommended daily requirements (425 mg/day for women and 550 mg/day for men), and inadequate choline intake has been linked to multiple health problems, including fatty liver, muscle damage, and even neurological disorders.68,80 Moreover, alterations in choline metabolism, including decreased levels of choline and increased TMAO, are found in children with acute malnutrition.81 Future research should explore how differences in this gut microbial metabolic activity affect choline requirements across different populations. Finally, it will be important to explore whether gut microbiota-host nutrient competition extends to additional vitamins and nutrients beyond choline.

Vitamin biosynthesis, metabolism, and transport: key factors in shaping the gut microbiota?

Vitamins are important for enabling critical enzymatic reactions in both the host and gut microbiota. As highlighted earlier, one of the critical factors that differs between germ-free and conventional mice is an increased requirement for dietary vitamins in the absence of microbes, suggesting that the gut microbiota may help to supply these molecules to the host via de novo synthesis or enhancing their uptake by host cells.82,83 Characterizing the pathways that different gut microbes use to synthesize, metabolize, and transport vitamins, as well as assessing their presence in individual organisms and patients, represents a fascinating area of human microbiota research. Though the gut microbiota’s role in increasing vitamin availability for the host is appreciated, less is known about how dietary- and gut microbe-derived vitamins influence this microbial community. Recent efforts to study corrinoid transport and metabolism in anaerobic microbes, including members of the human gut microbiota, have revealed new


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enzymatic chemistry and point towards the availability of vitamins and their exchange within communities as a potential contributor to the stability of these ecosystems.

Corrinoids are essential cofactors that consist of a cobalt ion bound within a heterocyclic corrin ring system and coordinated by both an upper ligand and a lower ligand that is connected to the corrin scaffold via a nucleotide loop (Figure 5A).84 Unlike many essential vitamins, only certain bacteria and archaea can synthesize corrinoids.84,85 While humans require vitamin B12 (cobalamin), microbes can make and use a much larger array of these cofactors that can differ in the identities of both the upper and lower ligands.85-87 While some microbes can make corrinoids de novo, most are predicted to require an exogenous source of corrinoids. Within the human gut, microbes are thought to primarily use B12 derived from the host diet and can remodel this cofactor to access a suite of corrinoids that contain a variety of different lower ligands.88 The availability of distinct corrinoids may play a key role in the ecology of microbial communities, as individual organisms typically use only a subset of these cofactors.85,89 Intriguingly, variations in the presence and abundance of different corrinoids have been observed in fecal samples from different human subjects.90 However, the range of corrinoids synthesized and used by gut microbes, as well as many of their precise roles in microbial metabolism in this environment remain largely uncharacterized.

In 2014, Degnan and co-workers assessed the distribution of genes involved in corrinoid biosynthesis, acquisition, and use across 313 sequenced human gut microbial genomes sequenced as part of the Human Microbiome Project.85 This survey indicates that while the majority (83%) of these strains likely use corrinoids, only a fraction (25%) are predicted to synthesize these vitamins, including members of the Firmicutes and Actinobacteria phyla. Unexpectedly, their analysis also revealed the presence of multiple predicted B12 transporters


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encoded in the genomes of single strains, with the majority of species from the dominant gut phylum Bacteroidetes having at least two homologs of the BtuBFCD transporter. This system consists of a TonB-dependent outer membrane transporter (BtuB), which is present only in Gram-negative bacteria, a periplasmic binding protein (BtuF), and an ABC transporter (BtuCD). Three predicted BtuB homologs and two sets of BtuFCD proteins are transporters are encoded in the genome of the model gut microbe Bacteroides thetaiotaomicron. Competition experiments using a series of B. thetaiotaomicron mutants containing single, pairwise, and complete deletions of the three btuB genes revealed that each of these systems is functional for B12 transport and displays a different relative preference for corrinoids containing alternate lower ligands (Figure 5B).85 This observation may indicate that B. thetaiotaomicron has adapted to an environment that contains a dynamic pool of corrinoids. Competing the wild type and mutant strains in a gnotobiotic mouse model revealed a striking 4,000-fold loss in fitness in the DbtuB2 mutant that was exacerbated when mice were fed a B12-defecient diet, highlighting the importance of B12-dependent transport and metabolism in this environment.85

Another recent study uncovered the genes and enzymes that enable anaerobic microbes to construct both 5,6-dimethylbenzimidazole (DMB), the lower ligand found in B12, as well as additional benzimidazole-based lower ligands.91 By searching the genome of the B12-producing acetogen Eubacterium limosum for genes located downstream of cobalamin riboswitches, Hazra et al. identified a six-gene operon encoding a homolog of CobT, the enzyme responsible for lower ligand attachment, and five additional enzymes (Figure 5C). Expression of this putative benzimidazole (bza) gene cluster in an E. coli host resulted in the conversion of the cobalamin precursor cobinamide, which is missing lower ligand, to vitamin B12. This result confirmed the role of the bza genes in DMB biosynthesis. Introducing subsets of this gene cluster into E. coli revealed the functional roles of each enzyme and delineated a biosynthetic pathway that begins


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with the conversion of 5-aminoimidazole ribotide to 5-hydroxybenzimidazole by two radical SAM enzymes (BzaA and BzaB) (Figure 5D). These enzymes are both homologs of ThiC, a radical SAM enzyme that participates in thiamin biosynthesis. Hazra et al. also showed that in some anaerobes, a single radical SAM enzyme (BzaF) accomplishes this transformation. Next, SAMdependent

methyltransferase

BzaC

converts

5-hydroxybenzimidazole

to

5-

methyoxybenzimidazole. Finally, the predicted B12-binding-Fe–S/SAM superfamily proteins BzaD and BzaE are responsible for installing two methyl groups on the benzimidazole scaffold to generate DMB.

A

O

NH 2 O

NH 2

Me Me

Alternative lower ligands

O

H 2N O

Me Me H

O

NH 2

N

N

Co

N

Me

NH Me

O O

HO

P O

H

Me

Me

N

Me

H

H

Me

N

Me

N H

5,6-dimethylbenzimidazole (DMB); found in B12

Me Me NH 2

N

O O

N H

N

H 2N O

N

N

O

O

N

N

N

N H

NH 2

Me

N

Me

N

N N

adenine

benzimidazole

HO

HO

N H

5-methoxybenzimidazole

N H

NH 2

Me

5-methylbenzimidazole

p-cresol

phenol

H OH

corrinoid

B

C OM

BtuB1

BtuB2

BtuB3

bzaA

IM

BtuF2

BtuF3

BtuC2

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BtuD2

BtuD3

bzaB

cobT

bzaC

bzaD

bzaE

cobalamin riboswitch

Import into cells

D O 2-O

Me

2-methyladenine

N H

variable lower ligand

N

3PO

HO

N N OH

NH 2

BzaA BzaB radical SAM

H N N

BzaC OH

O-methyltransferase

H N N

BzaD OMe B12-binding

Fe-S/SAM superfamily


H N

Me

N

OMe

BzaE B12-binding Fe-S/SAM superfamily

DMB

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Figure 5. The identification and characterization of proteins involved in the transport and biosynthesis of corrinoids highlights how interactions with vitamins may shape the gut microbiota. (A) General structure of cyanocobalamin (vitamin B12) and the lower ligands of other corrinoids. (B) Different transport systems in Bacteroides thetaiotaomicron handle distinct corrinoids. Lower ligands preferentially transported by different BtuB homologs are color-coded. (C) The bza operon in Eubacterium limosum that is responsible for dimethylbenzimidazole generation. (D) Proposed biosynthetic pathway for dimethylbenzimidazole in anaerobic microbes.

Identifying the bza genes allowed Hazra et al. to mine additional bacterial genomes to uncover additional anaerobic microbes capable of synthesizing B12.91 In addition to the full gene cluster, they found organisms that contained subsets of the bza genes. As all of the benzamidazole intermediates generated by the Bza enzymes are known to be used as alternative lower ligands, the authors hypothesized these strains would make corrinoids other than B12. A combination of culturing and heterologous expression of these smaller bza gene clusters confirmed the involvement of these genes in producing corrinoids with alternative lower ligands. In all cases examined, the set of bza genes encoded in a genome accurately predicted the structure of the corrinoid produced. This finding indicates that these gene clusters will be useful markers of corrinoid production by anaerobes, including human gut microbes.

Together with emerging knowledge of corrinoid transport in gut microbes, this work and additional biochemical studies of the enzyme(s) involved in lower ligand incorporation are helping to define the range of corrinoids made and exchanged in anaerobic microbial habitats


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like the human gut.86,87,91 This knowledge will not only provide new insights into the importance of vitamins in both host-microbe and microbe-microbe metabolic interactions, but may also inform strategies for manipulating the gut microbiota through altering dietary corrinoid availability. By highlighting how the vitamins we consume may contribute to the stability and health of this microbial community, this work is helping facilitate the move toward a ‘microbefocused’ view of host nutrition.

Conclusions and Future Directions

The past two decades have seen dramatic advances in our understanding of the composition of the human gut microbiota and our appreciation of its central role in host biology. To further advance this area of science and realize the enormous potential of these organisms to modulate human health, it is imperative that we gain a mechanistic understanding of this microbial community. The work discussed here represents a small fraction of gut microbe-nutrient interactions. Continued efforts to elucidate the molecular basis for gut microbial nutrient biosynthesis, metabolism, and transport will not only reveal new biochemistry but will also fundamentally change our understanding of human nutrition.

Gaining this knowledge will provide new strategies for enhancing human health and preventing disease. Elucidating the gut microbiota’s role in maintaining key nutrient levels and subsequent impact on host biology could lead to improved nutritional guidelines and recommendations. Personalized dietary recommendations could be guided by the presence or absence of enzymes involved in nutrient acquisition (such as PorA and PorB) or the production of diseaseassociated metabolites (such as CutC). This knowledge could also guide use of therapeutic foods and other diet-based interventions to treat human diseases linked to the gut microbiota,


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complementing traditional therapeutic strategies. Obesity and cardiometabolic diseases may present particularly exciting opportunities for mechanistic investigations. The connection between the gut microbiota and the development of obesity has been explored in both animal models and human cohorts.92 While gut microbiota composition may not strongly correlate with obesity in humans, studies in animal models have shown that diet can alter the gut microbiota.93 These changes can affect host gene expression, modulate metabolic and inflammatory pathways, and ultimately influence weight gain and adiposity.93

Elucidating the molecular mechanisms underlying gut microbiota-nutrient interactions could also enhance our understanding of undernutrition, potentially leading to new treatment and prevention strategies. Undernutrition is highly prevalent in the developing world and is thought to be the cause of over 55% of child deaths worldwide.94 Even with dietary interventions, this condition can cause life-long consequences. Recent studies performed in Bangladesh95 and Malawi96 have suggested that the gut microbiota likely plays a key role in the progression of undernutrition. Germ-free animals are nutrient deprived and exhibit similar characteristics to malnourished patients.9 These results have inspired the development of dietary interventions intended to reshape the gut microbiota to alleviate malnutrition.97,98 Gaining a better understanding of how the gut microbiota produces and metabolites key nutrients may provide new insights into the pathophysiology of undernutrition as well as explain the efficacy of these dietary interventions.

In conclusion, gut microbial metabolism of dietary components and essential nutrients has been evident for some time. However, a lack of knowledge of the biochemical basis for these transformations has made it challenging to study these activities and decipher their roles in host nutrition. The work discussed here showcases how elucidating the molecular basis for gut


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microbe-nutrient interactions can open the door to a mechanistic understanding of this microbial community, highlighting the tremendous opportunities for biochemists and enzymologists in this field.

Acknowledgements

The authors acknowledge Yolanda Huang, Li Zha, and Dr. Spencer Peck for helpful discussions and for their comments on this manuscript.

Funding

Work on gut microbial choline metabolism in the Balskus Lab has been supported by a Smith Family Award for Excellence in Biomedical Research, a Packard Fellowship for Science and Engineering (2013-39267), and a Blavatnik Biomedical Accelerator Award from Harvard University.


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Graphical abstract: carbohydrates


protein and amino acids lipids vitamins essential nutrients

molecular basis for gut microbiotanutrient interactions?


Biochemistry human gut microbiota

carbohydrates

1 protein and amino acids 2 3 lipids 4 5 vitamins 6 7 8 essential nutrients 9 10

Page 38altered of 43 microbial genes and proteins

nutrient availabiity

nutritional requirements altered disease risk

links between diet and disease

ACS

molecular basis for gut microbiotaParagon Plus Environment nutrient interactions?

altered community composition

effect of diet on microbiota

A 39 of 43OH Page

Biochemistry

OH

PorA and PorB glycoside hydrolases

1 2 3 4 5 6 7B 8 9 10 11 12 13 14 15 16 17 18

O

O HO

OH

O

O

O 3SO

+ H2 O

OH

O

O HO

algal-associated bacterium Zobellia galactanivorans

OH

HO

OH

OH

R59

lactose-6-sulfate (L6S)

C Origin of Metagenomic Data Set Japanese CAZymes specific for starches found in seaweed

L6S

Gal

control CAZyme

3.5 Å 2.9 Å

ACS Paragon Plus Environment R133

OH

O

OH

W56

pocket for porphyran recognition

O 3SO

galactose (Gal)

porphyran

H53

+

OH

HO

North American

β-porphyranase 1#()#+2)"$&% β-agarase 1"3")"&% α-amylase 1".24"&%

0 100% % metagenomes detected in

A

dietary protein Biochemistry

Page 40 of 43

1 O 2 3 OH 4 NH 2 HN 5 L-tryptophan 6 CLOSPO_02085 7 human gut bacteria or RUMGA_01526 8 Clostridium sporogenes tryptophan decarboxylase Ruminococcus gnavus 9 PLP 10 11 12 neurologically + CO 13 active metabolite NH 2 HN 14 tryptamine 15 16 B (S)-α-fluromethylL-tryptophan 17 tyrosine (FMT) side chain binding pocket 18 L339 19 L126 20 L336 H 21 N L355 22 F98 H120 23 24 O 25 (S)-α-FMT T356 26 OPO 3227 HO PLP 28 ACS Paragon PlusK306 Environment H305 N 29 Me 30 PLP-inhibitor adduct 31 2

A Page 41 of 43 Me

CutC choline TMA-lyase glycyl radical

N H

Biochemistry

H N

H

TMA/TMAO disease links:

FMO3 flavin monooxygenase

O

atherosclerosis

serum choline (μM)

N

serum TMAO (μM)

diabetes Me 1 N + Me Me liver disease N Me Me Me Me O Me Me 2HO kidney disease many human gut host liver trimethylaminuria trimethylamine-N-oxide trimethylamine 3 choline microbial species acetaldehyde (TMA) (TMAO) 4 5 60 D 80 B C germ-free mice 6 conserved interactions **** with trimethylammonium group 60 7 40 Y508 F395 Gly loop 8 40 9 * 20 “core community” CC+ choline 20 10 choline metabolism + choline metabolizer present in gut microbiota 11 0 0 G821 12 CC– CC+ CC– CC+ 13 Y208 decreased E C489 14 D218 flux DNA 15 ACS Paragon Plus Environment choline SAM “core community” methylation CC– 16 + cutC mutant choline metabolism Cys loop T502 depleted in epigenetic E491 absent from gut microbiota CC+ mice changes 17 O

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 42 of 43

Page 43 of 43 Biochemistry carbohydrates


protein and amino acids

1 2 3 4

lipids

ACS Paragon Plus Environment molecular basis

vitamins

essential nutrients

for gut microbiotanutrient interactions?

Deciphering Human Gut MicrobiotaâNutrient Interactions: A Role for

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