REVIEW SUMMARY◥

MICROBIOTA

Chemical transformation ofxenobiotics by the humangut microbiotaNitzan Koppel, Vayu Maini Rekdal, Emily P. Balskus*

BACKGROUND: Humans ingest a multitudeof small molecules that are foreign to the body(xenobiotics), including dietary components,environmental chemicals, and pharmaceuti-cals. The trillions of microorganisms that in-habit our gastrointestinal tract (the human gutmicrobiota) can directly alter the chemicalstructures of such compounds, thusmodifyingtheir lifetimes, bioavailabilities, and biologicaleffects. Our knowledge of how gut microbialtransformations of xenobiotics affect humanhealth is in its infancy, which is surprising giventhe importance of the gut microbiota. We cur-rently lack an understanding of the extent towhich this metabolism varies between individ-uals, the mechanisms by which these microbialactivities influence humanbiology, and howwemight rationally manipulate these reactions.This deficiency stems largely from the difficultyof connecting this microbial chemistry to spe-cific organisms, genes, and enzymes.

ADVANCES: Over the past several decades,studies of gut microbiota–mediated modifica-

tion of xenobiotics have revealed that these or-ganisms collectively have a larger metabolicrepertoire than human cells. The chemical dif-ferences between human andmicrobial trans-formations of ingested compounds arise notonly from the increased diversity of enzymespresent in this complex and variable communitybut also from the distinct selection pressuresthat have shaped these activities. For example,whereas host metabolism evolved to facilitateexcretion of many xenobiotics from the body,microbial modifications of these compoundsand their human metabolites often support mi-crobial growth through provision of nutrients orproduction of energy. Notably, the chemistry ofmicrobial transformationsoftenopposesorreversesthat of host metabolism, altering the pharmaco-kinetic and pharmacodynamic properties ofxenobiotics and associated metabolites.The range of xenobiotics subject to gut micro-

bial metabolism is impressive and expanding.Gut microbes modify many classes of dietarycompounds, including complex polysaccha-rides, lipids, proteins, and phytochemicals. These

metabolic reactions are linked to a variety ofhealth benefits, aswell as disease susceptibilities.Gut microbes are also able to transform indus-trial chemicals and pollutants, altering theirtoxicities and lifetimes in the body. Similarly,

microbial transformationsof drugs can change theirpharmacokinetic proper-ties, be critical for prodrugactivation, and lead to un-desirable side effects orloss of efficacy. In the vast

majority of cases, the individual microbes andenzymes that mediate these reactions areunknown.Fueled by findings underscoring the relevance

of microbial xenobiotic metabolism to humanhealth, scientists are increasingly seeking todiscover and manipulate the enzymatic chem-istry involved in these transformations. Recentwork exploring how gut microbes metabolizethe drugs digoxin and irinotecan, as well asthe dietary nutrient choline, provides guidancefor such investigations. These studies, whichcombine traditional methods with modernapproaches, illustrate how a molecular under-standing of gut microbial xenobiotic metabo-lism can guide hypothesis-driven research intothe roles these reactions play in both micro-biota and host biology.

OUTLOOK: We still face a myriad of chal-lenges in understanding the gut microbiota’scontribution to xenobiotic metabolism. It isimperative that we connect the many knownmicrobial transformations with the genes andenzymes responsible for these activities, andknowledge of enzyme mechanism and bio-chemical logic will facilitate this objective.There also remains a great need to uncovercurrently unappreciated activities associatedwith this community. Revealing the full scopeof microbially mediated transformations inthe gutmay give us new insights into themanyvariable and contradictory studies regardingthe effects of diet, pollutants, and drugs onhuman health. Microbial genes and enzymeswill provide both specific targets for manipu-lation and diagnostic markers that can beincorporated into clinical studies and prac-tice. Ultimately, a molecular understandingof gut microbial xenobiotic metabolism willinform personalized nutrition, toxicology riskassessment, precision medicine, and drugdevelopment.▪

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The list of author affiliations is available in the full article online.*Corresponding author. Email: balskus@chemistry.harvard.eduThis is an open-access article distributed under the termsof the Creative Commons Attribution license(http://creativecommons.org/licenses/by/4.0/), whichpermits unrestricted use, distribution, and reproduction in anymedium, provided the original work is properly cited.Cite this article as N. Koppel et al., Science 356, eaag2770(2017). DOI: 10.1126/science.aag2770

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Chemicalmodifications

Human gut microbes metabolize xenobiotics. Themicroorganisms that inhabit the humangutalter the chemical structures of ingested compounds, includingdietarycomponents, industrialchemicals, anddrugs.Thesechangesaffect xenobiotic toxicity, biological activity, andbioavailability.Thegutmicrobialenzymesresponsibleformanyofthesetransformationsarepoorlyunderstood.Me,methyl.

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REVIEW◥

MICROBIOTA

Chemical transformation ofxenobiotics by the humangut microbiotaNitzan Koppel,1 Vayu Maini Rekdal,1 Emily P. Balskus1,2*

The human gut microbiota makes key contributions to the metabolism ofingested compounds (xenobiotics), transforming hundreds of dietary components,industrial chemicals, and pharmaceuticals into metabolites with altered activities,toxicities, and lifetimes within the body. The chemistry of gut microbial xenobioticmetabolism is often distinct from that of host enzymes. Despite their importantconsequences for human biology, the gut microbes, genes, and enzymes involvedin xenobiotic metabolism are poorly understood. Linking these microbialtransformations to enzymes and elucidating their biological effects is undoubtedlychallenging. However, recent studies demonstrate that integrating traditional andemerging technologies can enable progress toward this goal. Ultimately, a molecularunderstanding of gut microbial xenobiotic metabolism will guide personalized medicineand nutrition, inform toxicology risk assessment, and improve drug discoveryand development.

The human gut microbiota is a diverse andcomplex community of microorganisms thathas evolved with its host (1) and is deeplyintertwined with human biology. The esti-mated 1013 microbes that inhabit the hu-

man gastrointestinal (GI) tract play a central rolein many processes, including colonization resist-ance, immune system modulation, synthesis ofessential vitamins and nutrients, and digestionof polysaccharides (2–4). Gut microbes also modi-fy the chemical structures of numerous ingested,foreign compounds (xenobiotics), including die-tary components, environmental pollutants, andpharmaceuticals. Such transformations were iden-tified as early as the 1950s in humans, animalmodels, fecal samples, and individual microbes.In these studies, changes in metabolism in theabsence of microbes [i.e., germ-free (GF) animals]or upon microbial perturbation (i.e., antibiotictreatment or dietarymodulation) indicated thegut microbiota’s involvement in xenobiotic pro-cessing (5, 6).The human gut microbiota encodes a broad

diversity of enzymes, many of which are exclu-sivelymicrobial, thus expanding the repertoire ofmetabolic reactions occurring within the humanbody. Although clinical studies have revealedmarked interindividual variability in these micro-bial transformations, most of these reactions haveconsequences for the host that are not completelyunderstood. Gut microbial xenobiotic metabolitesare known to have altered bioactivity, bioavail-

ability, and toxicity and can interfere with theactivities of human xenobiotic-metabolizing en-zymes to affect the fates of other ingested mol-ecules. Despite the diverse and physiologicallyimportant consequences of these modifications,relatively little is known about the specific gutmicrobial strains, genes, and enzymes that me-diate xenobiotic metabolism.Here we review our current knowledge of how

the gutmicrobiota directlymodifies dietary com-pounds, environmental pollutants, and pharma-ceuticals. We discuss critical differences betweenthe chemistry of microbial and host xenobioticmetabolism and highlight opportunities for en-zyme discovery and characterization. By exam-ining several recent studies, we showhow gaininga molecular understanding of microbial xeno-biotic modifications can illuminate their con-nections to human health. Finally, we discussstrategies for uncovering the genetic and bio-chemical bases of these microbial activities andprovide an outlook on how understanding gutmicrobial xenobiotic metabolism will influencepersonalized nutrition, toxicology, andmedicine.

Gut microbial interactionswith xenobiotics

The majority of human microbiota-xenobioticinteractions occur within the GI tract. The dif-ferent regions of this organ system vary in epi-thelial cell physiology, pH, oxygen levels, andnutrient content, thus providing distinct habitatsfor microorganisms and influencing the types ofmetabolic processes that occur (7, 8). Hundredsof distinct microbial species colonize the humangut. Although obligate anaerobes such as theFirmicutes andBacteroidetes phyla typically dom-

inate, large variability in community compositionis observed among individuals (9, 10).Microbial metabolism of xenobiotics must be

understood in the context of the concurrent andoften competing metabolic processes occurringin the human host. Orally ingested compoundspass through the upper GI tract to the small in-testine where they can be modified by digestiveenzymes and absorbed by host tissues (11). Read-ily absorbed xenobiotics pass between or throughintestinal epithelial cells, where they may be pro-cessed by host enzymes before transport to theliver via the portal vein. Following exposure to theliver’s rich collection of metabolic enzymes, xeno-biotics and their metabolites enter systemic circu-lation, distributing into tissues and potentiallyaffecting distal organs. By contrast, intravenous-ly administered compounds circumvent this “first-pass”metabolism and are immediately introducedinto systemic circulation. Compounds in the circu-latory system are eventually further metabolizedand/or excreted, which generally occurs either viathe biliary duct back into the gut lumen (biliaryexcretion) or through the kidneys into the urine.Metabolites returned to the gut lumen can eithercontinue on to the large intestine, where they willeventually be excreted in the feces, or they canpotentially be reabsorbed by host cells in the smallintestine through a process known as enterohe-patic circulation.Xenobiotics can therefore encounter gutmicrobes

via multiple routes. In contrast to compoundsthat are absorbed in the small intestine, poorlyabsorbed xenobiotics continue through the smallintestine into the large intestine and may betransformed by gut microbes. Readily absorbedcompounds and compounds administered viaother routes (e.g., intravenous injection) can alsoreach gut microbes through biliary excretion.The products of gutmicrobialmetabolism can beabsorbed by the host and circulated systemicallyor interact locally with the epithelial cells liningthe GI tract. Ultimately, these microbial metab-olites are excreted in feces or filtered by thekidneys and eliminated in the urine. Overall,human andmicrobial transformations generatea complex intertwined metabolic network thataffects both the host and the members of themicrobiota.

The complementary chemistry ofmicrobial xenobiotic metabolism

Within the distinctive and complex ecology of thehuman gut, microorganisms transform ingestedsubstrates via a broad range of enzymatic reac-tions. Gut microbes primarily use hydrolytic andreductive reactions to metabolize xenobiotics (5),many of which are distinct to these organisms.This is in stark contrast to host enzymes, whichtypically use oxidative and conjugative chemis-try. These differences are partially due to physio-logical context, but they also reflect distinctevolutionary pressures. Additionally, it is likelythat certain gut microbial enzymes were not evo-lutionarily selected to process specific xenobio-tics, but rather that metabolism arises fromrelaxed substrate specificity. Thus, the combined

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1Department of Chemistry and Chemical Biology, HarvardUniversity, 12 Oxford Street, Cambridge, MA 02138, USA.2Broad Institute, Cambridge, MA 02139, USA.*Corresponding author. Email: balskus@chemistry.harvard.edu

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metabolisms of host and microbiota generatemetabolites that would not be synthesized bythe host alone and can substantially alter thebioactivities and lifetimes of xenobiotics withinthe human body.Many of the enzyme classes associated with

xenobiotic metabolism (hydrolases, lyases, oxi-doreductases, and transferases) and highlightedhere are widely distributed among sequencedgut microorganisms (12–16). Metagenomic anal-yses have also revealed them tobe among themost

prevalent protein families in this environment(17, 18). It is therefore likely that many importanttransformations of xenobioticsmay be performedby multiple different phylogenetic groups of gutmicrobes. However, it is critical to note that broadannotations are not predictive of substrate spec-ificity, as enzymes with high sequence similaritycan catalyze distinct chemical reactions.Metabol-ic activities can also be discontinuously distrib-uted across closely related strains and acquiredvia horizontal gene transfer, making it problem-

atic to infer gut microbial metabolic capabilitiesfrom phylogenetic analyses alone. This issue high-lights the value of culture-based experimentsand rigorous biochemical characterization of gutmicrobial enzymes in understanding xenobioticmetabolism.

Host xenobiotic metabolism

Any discussion of microbial xenobiotic trans-formations must also consider host chemical ca-pabilities.Human xenobioticmetabolismgenerally

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Box 1. The chemistry of gut microbial and host xenobiotic metabolism. (A) Chemical logic of host xenobiotic metabolism. Commonly usedchemical strategies for microbial xenobiotic metabolism include (B) hydrolytic transformations, (C) lyase reactions, (D) reductive transformations,(E) functional group transfer reactions, and (F) transformations mediated by radical enzymes. Enz, enzyme; PLP, pyridoxal 5-phosphate; NAD(P)H, NADHor NADPH; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; Me, methyl; CoA, coenzyme A; SAM, S-adenosylmethionine.

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transforms nonpolar compounds into hydrophilic,higher–molecular weight metabolites that aremorereadily excreted (Box 1A). This process occurs in twostages: installation or exposure of polar func-tional groups (“phase I”) and conjugation of thesegroups to more-polar metabolites (“phase II”).Phase I enzymes perform oxidative, reductive, orhydrolytic reactions to generate hydroxyl groups,epoxides, thiols, and amines. The largest class ofphase I enzymes is the cytochrome P-450s, butcarboxylesterases and flavin monooxygenases(FMOs) are also important in xenobiotic process-ing (11). Transferase enzymes predominate inphase IImetabolism, appending glucuronyl,methyl,acetyl, sulfonyl, and glutathionyl groups ontoxenobiotics or phase Imetabolites (19). Polymor-phisms in xenobiotic-metabolizing genes influ-ence how individuals respond to both dietary andpharmaceutical interventions.

Hydrolytic reactions

Both the host and gut microbiota use hydrolyticchemistry to break down large ingested com-pounds into smaller products that may be fur-ther metabolized. Hydrolase enzymes catalyzethe addition of a water molecule to a substrate,followed by bond cleavage (Box 1B). The mostabundant and relevant hydrolases in the GI tractare proteases, glycosidases, and sulfatases, withthe microbiota contributing a broader range ofactivities than host enzymes. Proteases cleave thepeptide bonds linking the amino acids in poly-peptide chains. Whereas the small intestine isdominated by pancreatic serine proteases, thecolon contains many microbial cysteine- andmetalloproteases (20) with different substratespecificities and potentially different clinical con-sequences (21). Glycosidases hydrolyze glycosidicbonds using a dyad of carboxylic acid residuesand a water molecule, releasing free sugars (22).These enzymes process a huge diversity of glyco-conjugates and oligosaccharides and are broadlydistributed across gut microbes (15, 23). Sulfa-tases, which are also widespread, hydrolyze sul-fate esters generated by phase II host metabolismusing the unusual amino acid formylglycine (24).The hydrate form of this residue is thought toundergo transesterification with a sulfate estersubstrate to generate a tetrahedral intermediatethat breaks down to release sulfate and reformthe aldehyde (25).Hydrolytic reactions alter both the physical

properties and activities of xenobiotics and theirmetabolites. For example, removal of a glucuro-nide in the gut lumen is generally accompaniedby a decrease in polarity that can allow reab-sorption by host cells and thereby extend thelifetime of a molecule within the body, as seenwith glucuronide conjugates of nonsteroidal anti-inflammatory drugs and the cancer therapy iri-notecan (26, 27). Hydrolysis can also alter thebiological activity or toxicity of xenobiotics, asobserved for plant-derived glycosides like amyg-dalin and the artificial sweetener cyclamate(28, 29). Moreover, hydrolysis is often a prereq-uisite for further transformations, such as thefermentation of sugars released from indiges-

tible polysaccharides (30), and the products ofhydrolytic reactions (sugars, amino acids, andsulfate) often support microbial growth andsurvival in the gut.

Lyases

Lyase enzymes break C–C or C–X bonds (whereX = O, N, S, P, or halides) without relying onoxidation or the addition of water. Microbialpolysaccharide lyases (PLs) modify polysacchar-ides that contain a glycosidic bond at the b posi-tion relative to a carboxylic acid (e.g., alginate,pectin, chondroitin, and heparan) (Box 1C). Thepresence of the carboxylate enables removal ofthe a proton and subsequent b-elimination to yieldan a,b-unsaturated sugar and a hemiacetal (12).El Kaoutari et al. found that a single human gutmicrobiome encoded >5000 PLs (15), suggestingan enormous diversity of transformations thatcould support microbial growth.Microbial C–S b-lyases cleave C–S bonds found

inbothdietarycompoundsandcysteine-S-conjugatesof xenobiotics, which are formed by liver enzymes(Box 1C). These enzymes generate an aldiminelinkage between pyridoxal 5-phosphate (PLP) andthe a-amino group of the cysteine-derived substit-uent, acidifying the adjacent proton. b-eliminationreleases a thiol-containing metabolite and amino-acrylate, the latter of which spontaneously breaksdown to formammonia andpyruvate (31).Microbescan further metabolize these thiols, altering theirphysical properties and localization within thebody. For example, gut bacterial C–S b-lyasescleave cysteine-S-conjugates of polychlorinatedbiphenyls to produce thiol metabolites that arefurther methylated and accumulate in lipophilichost tissues (32). The consequences of C–S lyasechemistry for the microbiota are not well under-stood. This activitymayderive frompromiscuousPLP-dependent enzymes involved in “house-keeping” functions (31); however, ammonia gene-rated by C–S b-lyases can serve as a sole nitrogensource (33), pointing to a potential role in nutrientacquisition.

Reductive transformations

Gut microbes can reduce a wide range of func-tional groups, includingalkenesanda,b-unsaturatedcarboxylic acid derivatives, nitro, N-oxide, azo,and sulfoxide groups (Box 1D). Reductase enzymesmake use of various cofactors [e.g., NAD(P)H (i.e.,NADH or NADPH), flavin, Fe–S clusters, (siro)heme, molybdenum cofactor, and other metallo-cofactors] tomediate the transfer of electrons orhydride equivalents (H+, 2e–) to substrates (34–36).Biochemical and structural characterization ofgut microbial reductases has uncovered individ-ual enzymes that display broad substrate scopeand can transform multiple functional groups;consequently, their endogenous substrates andin vivo relevance are often unclear.Reduction typically decreases the polarity of

compounds and can alter charge, hybridization,and electrophilicity, which can affect the life-times and activities of metabolites in the body(37–39). Notably, electron transfer to xenobioticsmay enable anaerobic respiration in the human

gut, where oxygen is largely unavailable to serveas a terminal electron acceptor. Although reduc-tase enzymes are found in humans, many re-ductive transformations are exclusively microbial(6, 37, 40) and have not yet been linked to knownenzymes or organisms.

Functional group transfer reactions

Transferase enzymes move functional groupsbetween two substrates via nucleophilic substi-tution reactions. The gut microbiota transfersmethyl and acyl groups to or from xenobioticscaffolds (Box 1E). Addition reactions requirechemically activated cosubstrates, such as acetylcoenzyme A, adenosine triphosphate (ATP), orS-adenosylmethionine (SAM). Whereas enzymesthat remove acyl groups generally rely on hy-drolysis, demethylating enzymes use cofactors ca-pable of nucleophilic catalysis [e.g., cob(I)alaminand tetrahydrofolate] (41, 42).Installation and removal of these functional

groups can affect the lifetimes and bioactivitiesof xenobiotics in various ways. For instance, ace-tylation can serve as a detoxification mechanismby decreasing polarity and facilitating excretionfrom microbial cells. A notable example is theN-acetylation of the anti-inflammatory compound5-aminosalicylic acid (5-ASA) by microbial N-acetyltransferases (43), which yields a therapeuti-cally inactive metabolite. Host demethylation ofxenobiotics liberates polar groups for further con-jugation and excretion from the body (44), but inmicroorganisms, demethylation can provide acarbon source for growth (41).

Radical chemistry

Radical enzymes generate high-energy interme-diates containing an unpaired electron. Suchprocesses are often oxygen-sensitive and ener-getically demanding, but enable microbes to per-form chemically challenging reactions that areinaccessible by other modes of catalysis, includ-ing bond cleavage and formation (both C–C andC–X, where X = N, O, or halides) and skeletalrearrangements (45). Many radical enzymes usedin anaerobic metabolism share a common chem-ical logic. By using an enzyme- or cofactor-basedradical species, these enzymes typically generatea substrate-based radical intermediate throughsingle-electron transfer or homolytic bond cleav-age (Box 1F). This initial substrate-based radicalis then converted to a product-based radical.Formation of the final product often regeneratesthe initial enzyme- or cofactor-based radical tocomplete the catalytic cycle.Key classes of gut microbial radical enzymes

include radical SAM enzymes, cobalamin (B12)–dependent enzymes, and glycyl radical enzymes(GREs). These enzymes often mediate primarymetabolism in anaerobic microbes and can di-rectly or indirectly influence the fate of xenobio-tics in the human body. Reactions catalyzed bygut microbial GREs include generation of tri-methylamine (TMA) by choline trimethylamine-lyase (CutC) (46) and decarboxylation of thetyrosine-derivedmetabolitep-hydroxyphenylacetateby p-hydroxyphenylacetate decarboxylase (47).

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This latter reaction produces p-cresol, amoleculethat competes with xenobiotics for O-sulfationand detoxification by host enzymes (48).

Uncharacterized xenobiotic metabolism

The vast majority of gut microbial xenobiotictransformations cannot be linked to specific en-zymes and organisms. Whereas certain reactionscan, with reasonable confidence, be associatedwith one of the enzyme classes highlighted above,other uncharacterized metabolic activities can-not be readily explained with known biochem-istry. Below we discuss prominent gaps in ourknowledge of gut microbial xenobiotic metab-olism, focusing on transformations with in-triguing but poorly understood links to humanhealth. Although not comprehensive, this over-view highlights particularly notable opportu-nities for gut microbial enzyme discovery andcharacterization.

Metabolism of dietary compounds

Gut microbes process an enormous variety ofdietary compounds to extract nutrients and en-ergy (49). The types and extent of these mod-ifications vary substantially among individuals,presumably due to differences in the presenceand abundance of gut microbial enzymes, andthe bioactivities of the resulting metabolitesrange from beneficial to acutely toxic. As muchattention has been focused on microbial metab-olism of complex, plant-derived polysaccharides(4, 50, 51), we have chosen to highlight transfor-mations of noncarbohydrate dietary components.

Dietary protein

Dietary protein is necessary for supplying hu-mans with essential amino acids, but the sourceand amount of protein can vary substantiallybetween different diets. The gut lumen is rich inboth host and microbial proteases, and studiesincreasingly indicate that differential microbialproteolytic activity may directly contribute tohuman disease. For example, the gut microbiotais associated with celiac disease (CD), a commonautoimmune disorder characterized by an in-flammatory response to dietary gluten found inwheat-based foods. This proline-rich proteinevades complete digestion by host proteases, re-sulting in the generationofhigh–molecularweight,immunogenic peptides. The gutmicrobiotamayaffect CD by altering gluten proteolysis. Fecal sus-pensions from healthy individuals and CD pa-tients process gluten proteins and immunogenicpeptidesdifferently (52). For instance, gluten-derivedpeptides generated by Pseudomonas aeruginosa,an opportunistic pathogen in CD patients, areprone to translocation across themouse intestineand elicit an enhanced gluten-specific immuneresponse in comparison with peptides producedby Lactobacillus spp. from healthy individuals(53). Identification of specific proteases respon-sible for gut microbial gluten processing couldnot only enable a better understanding of CDbut also inform therapeutic interventions forthis disease1, including enzymatic or probiotictreatments.

Gut microbes can also metabolize amino ac-ids obtained from dietary protein, including L-phenylalanine, L-tyrosine, and L-tryptophan, intoa range of bioactive products (54). For example,gut bacteria can metabolize L-tryptophan intomany products, including the antioxidant indole-3-propionic acid, the neurotransmitter tryptamine,and indole, the latter of which can undergo hy-droxylation and sulfation by hepatic enzymesto generate the uremic toxin indoxyl sulfate(55–57).

Dietary lipids

Variable gut microbial metabolism of lipids andlipid-derived compounds is associated with avariety of human diseases (58, 59). One notableexample involves dietary cholesterol, a majorcomponent of Western diets that is associatedwith increased risk of cardiovascular disease (60).While ingested cholesterol is absorbed in thesmall intestine and subsequently undergoes bil-iary excretion and enterohepatic circulation, gutmicrobial reduction of cholesterol generates co-prostanol, which cannot be reabsorbed and is ex-creted. This transformation therefore effectivelyremoves cholesterol from circulation. Coprostanolcomprises up to 50% of the steroids in humanfeces (61), and GF mice colonized with microbesfrom high- and low-cholesterol–reducing patientsproduce distinct amounts of coprostanol (62).Animal experiments also suggest that cholesterol-reducing bacteriamay decrease serum cholesterol(61). Studies of the cholesterol-reducing gut bac-teria Eubacterium coprostanoligenes indicate thatcoprostanol synthesis may involve oxidation to 5-cholesten-3-one followed by alkene isomerizationto 4-cholesten-3-one, conjugate reduction, andketone reduction (63). Identifying the enzymesresponsible for these transformations and char-acterizing their abundance in patients may beparticularly interesting, given that inhibition ofcholesterol reabsorption is a clinically validatedstrategy for lowering cholesterol (64).

Dietary phytochemicals

Identifying and characterizing gut microbial en-zymes may also help us to better understanddietary compounds that are associated with healthbenefits. For example, numerous studies impli-cate the gut microbiota in metabolizing poorlyabsorbed, polyphenolic compounds from plant-derived foods (65) including soy isoflavones,lignans from flaxseed and sesame seeds, flavo-noids like the catechins and gallate esters foundin tea, and ellagic acid from nuts and berries(66–69). These molecules are processed using arange of transformations, including ring cleav-age, demethylation, and dehydroxylation, whichgenerally produce metabolites with higher oralbioavailability, increased bioactivity, and a cor-relation with lowered disease risk (67, 68). Poly-phenolmetabolismvarieswidely among individuals(67), and further research is needed to elucidatewhether these microbial products can directlyaffect host biology or are biomarkers for di-sease susceptibility. From a chemical standpoint,studying polyphenolmetabolismwill also address

fundamental gaps in our understanding of gutmicrobial enzymes.

Artificial sweeteners

The microbiome may also interact with compo-nents of our diets that are added in the process offood manufacturing (e.g., artificial sweeteners,emulsifiers, and preservatives). For instance, al-though many artificial sweeteners are poorlymetabolized by humans, studies demonstrate thatthey are susceptible to microbial transformation.Gut microbes convert the artificial sweetener cy-clamate into cyclohexylamine via hydrolytic clea-vage of its sulfamate linkage. Cyclamate wasbanned in the United States after studies sug-gested that cyclohexylamine was carcinogenic,and both this finding and the continued use ofthis sweetener remain controversial (70). A cy-clamate hydrolyzing enzyme has been partiallypurified from a guinea pig–associated strain, buthuman gut microbial hydrolases with this acti-vity have not been identified (29). Gut microbescan also metabolize the artificial sweeteners ste-vioside and xylitol using unknown enzymes(71, 72). The gutmicrobiota acquires the ability totransform xylitol and cyclohexamate after pro-longed exposure, suggesting that long-term in-gestion of dietary components can select forparticular microbial metabolic functions (73).Understanding the microbiota’s role in metab-olizing components of processed foods couldbe important for assessing food safety and thelong-term effects of food additives on humanhealth.

Heterocyclic amines

Finally, an understanding of gut microbial meta-bolic activities could provide new insights intothe biological consequences of cooking practicesand food preparation. The mutagenic potentialof heterocyclic amines, poorly absorbed mole-cules produced during charring of meat andfish, can be altered by gut microbial metabo-lism. For example, gut microbes convert 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) into thepotential mutagen 7-hydroxy IQ (74) and can hy-drolyze IQ-glucuronide conjugates, prolongingthe lifetime of IQ in the body (75). Gnotobioticrats monoassociated with an Escherichia colistrain encoding a b-glucuronidase (uidA) havehigher levels of unconjugated IQ and increasedcolonic DNA damage compared with rats colon-ized with an isogenic uidA mutant (75). Theseresults appear to implicate the gut microbiotain the known link between charred meat andcancer (76).

Metabolism of industrial chemicalsand pollutants

Although there is an emerging appreciation forthe role of the gut microbiota in metabolizingpollutants and industrial chemicals, our knowl-edge of the specific transformations, strains, andenzymes involved lags far behind that of envi-ronmental microbes. However, it is clear thatmicrobial activities can alter the toxicity and bio-availability of these compounds, as well as extend

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host exposure to harmful substances. When eval-uating the safety of these compounds, it is thuscrucial to consider the consequences of gut mi-crobial metabolism. Here we discuss several typesof chemicals that have been implicated in humandisease risk and for which there is evidencethat gut microbial metabolism affects toxicity.

Chemicals used inindustrial manufacturing

Gut microbes reductively metabolize azo com-pounds, some of the first industrially importantsynthetic chemicals (40). Despite their use for>150 years as textile dyes, food colorings, andpharmaceuticals, we have an incomplete under-standing of the organisms and enzymes thatprocess these molecules. The reductive cleavageof azo linkages yields aniline products, and thisreaction can be performed by flavin- or NAD(P)H-dependent enzymes found in many eukaryotesand bacteria (13). Azoreductases have not yet beenextensively characterized from many human gutbacterial strains in which this activity has beenobserved, and isolates can vary in their ability toreduce different dyes (77). The biological conse-quences of azo reduction vary depending on thesubstrate. For example, although microbial trans-formation of azo food dyes generates metabolitesthat are considered to be nontoxic (34, 78), workerswith long-term exposure to textile dyes have anincreased risk of bladder cancers (79). Feeding azotextile dyes to conventional, but not GF,mice leadsto the accumulation of the mutagenic bis-anilinebenzidine in urine, which implicates microbialmetabolism in increasing carcinogen exposure(80). Thus, the toxic effects of azo compoundsmay depend on both the individual dye backboneand the presence of specificmetabolizing organisms.Gut microbes also metabolize the s-triazine

compoundmelamine, an industrial chemical usedin the production of various plastics, enhancingits toxicity in humans. Melamine added to infantformula in China caused kidney stones in 300,000children and led to at least six deaths (81). Sub-sequent studies inmice revealed that gut microbesdeaminate melamine to generate ammonia andcyanuric acid (82), the latter of which forms aninsoluble complex with melamine in vivo, leadingto renal toxicity (83). Klebsiella species are asso-ciated with cyanuric acid production in mice andgenerate this metabolite in vitro (82), but it re-mains unclear whether the gut microbiota or thisorganism contribute to melamine toxicity in hu-mans. As environmental bacteria use similar hy-drolytic chemistry to metabolize other industriallyrelevant s-triazines, including the herbicide atra-zine, these studies also raise the possibility that thegut microbiota may transform additional com-pounds in this class.

Heavy metals

In addition to organic pollutants, human gutmicrobes modify the structures and alter thetoxicities of various heavy metals, including bis-muth, arsenic, and mercury. Mercury bioaccu-mulates in living organisms, posing a threat tohuman health, and gut microbial metabolism

may affect mercury toxicity and lifetime in thebody. Rat fecal samples reduce methylmercury(CH3Hg+) to the less toxic inorganic mercury,thereby facilitating mercury excretion from thehost (84). Depletion of the gut flora in rats andmice can result in the accumulation of methyl-mercury, thereby causing neurological symptoms(85). The enzymes responsible for this protectiveactivity could include homologs of the demethyl-ating, organomercuric lyase (MerB) and mercuricreductase (MerA), which have been identifiedin human isolates (86). However, the abun-dance of mer genes did not correlate with fecalmethylmercury levels in a recent clinical study,raising the possibility of additional enzymes orindirect effects (87). Notably, incubation of a mix-ture of 16 metal(oid)s with suspensions from anin vitro simulator of the GI tract resulted in vol-atilization of many metal species and the produc-tion of As/S compounds not previously observedin biological systems (88). These studies indicatethat there aremajor gaps in our knowledge of thegut microbiota’s interactions with heavy metalsand the resulting toxicological implications.

Metabolism of pharmaceuticals

Apart from antibiotics, the human gut microbiotais known to transform >50 pharmaceuticals, span-ning many indications and host targets, intometabolites with altered pharmacological prop-erties (5, 89). In some cases, the teratogenic (90),toxic (26, 27), and even lethal (91) effects of thesemicrobial metabolic activities were not recog-nized until drugs were on the market. Ongoingstudies have illuminated a complex interplay be-tween drugs and gut microbes (92, 93), but wefocus here on examples of direct microbialmodifications.

Anti-inflammatory and GI agents

Multiple drugs that target the GI tract are affectedby gutmicrobes, either by direct chemicalmodi-fication or indirectly through the many inter-actions these organisms have with host cells inthis environment. Notably, many of these agentsrely onmicrobial metabolism for converting inac-tive precursors (prodrugs) to pharmaceuticallyactive compounds. Prominent examples are anti-inflammatory drugs that contain azo linkages,including the inflammatory bowel disease (IBD)medication sulfasalazine (38). Gutmicrobes reducesulfasalazine into sulfapyridine and the active anti-inflammatory agent 5-ASA, and various intestinalbacteria can further metabolize 5-ASA intoN-acetyl5-ASA, a metabolite that lacks anti-inflammatoryactivity. Considerable variability in acetylation rateshas been observed in human fecal samples (94). To-gether with differences in azo reduction, this ob-servation could potentially explain variabletherapeutic efficacy of sulfasalazine in patients.N-acetyl ASA inhibits the growth of anaerobes,including Clostridium difficile (43), which sug-gests that this activity could affect gut micro-biome composition. This observation is particularlynoteworthy given our increasing appreciationof the participation of the gut microbiota inIBD pathogenesis. Other gut microbial activities

that are responsible for prodrug activation in-clude reduction of the sulfoxide found in theanti-inflammatory compound sulindac (95) andreduction of theN-oxide of the anti-diarrheal drugloperamide (39). Gaining a better understandingof the specific organisms and enzymes responsi-ble for these activities and their presence inpatients could aid in drug selection and dosing.

Cancer chemotherapy

Patient response to chemotherapy can differ dra-matically between individuals, in terms of effi-cacy as well as the severity of side effects, andemerging studies suggest that differences in thegut microbiota may contribute to this phenom-enon. In addition to modulating the host im-mune system, gut microbes can directly alterthe structures of cancer therapies and their me-tabolites, affecting their interactions with hostcells. Recent work indicates that microbes mayhave a high potential for modifying the chemicalstructures of commonly used chemotherapeutics(96). Co-incubation with either E. coli or Listeriawelshimeri either increased or decreased the ef-ficacy of half of a panel of 30 anticancer drugstoward cancer cell lines. Assays with E. coli and asubset of these drugs (gemcitabine, fludarabine,and CB1954) revealed evidence for direct chem-ical modification by the bacteria. Finally, thepresence of E. coli altered the efficacy of che-motherapy in vivo in a manner consistent withthe observed metabolic activities. These prelim-inary observations suggest that structural mod-ification of drugs by gut or tumor-associatedmicrobes could contribute to interindividual var-iation in cancer therapy, but examination oflarger panels of microbes and detailed charac-terization of drug metabolites are needed to ex-tend these findings.

Central nervous system (CNS) drugs

In addition to affecting drugs that act locally, gutmicrobial metabolism can also influence the ef-ficacy of therapeutics that target distant organsystems. Many prominent examples can be foundamong CNS drugs. For example, oral levodopa(L-dopa) is used to treat Parkinson’s disease, acondition characterizedbydopaminergic-neuronaldeath. L-dopa crosses the blood-brain barrier,whereit is decarboxylated by host enzymes to restoredopamine levels (97). However, extensive metab-olism within the gut by both host and micro-bial enzymes affects the concentration of drugreaching the brain. Microbial decarboxylation(98) and p-dehydroxylation convert L-dopa tom-tyramine, which can be further oxidized to m-hydroxyphenylacetic acid (99). Differences in theseactivities may contribute to the substantial varia-tion observed in patient response to L-dopa (100).Although a tyrosine decarboxylase from a food-associated strain of Lactobacillus brevis acceptsL-dopa in vitro (101), the human gut microbesand enzymes responsible for L-dopa metabolismare unknown. As studies continue to reveal con-nections between the gutmicrobiota and variousneurological diseases (102), it will become in-creasingly important to identify and characterize

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additional microbial interactions with CNS-targeted drugs.

Herbal supplements andtraditional medicines

Similar to dietary phytochemicals, gut microbescan also transform poorly absorbed constituentsof herbal and traditional remedies, which canlead to potential health benefits or harmful sideeffects. There are many examples of the variableefficacy of traditional medicines, which may bepartly due to the complexity of active ingredients,as well as to differences in gut microbial metabo-lism of these treatments. For example, amygdalinis amandelonitrile glycoside found in almonds andfruit pits that was used as an alternative cancertreatment in the 1960s (103), although clinicaltrials demonstrated no improvements in cancersurvival or symptoms (104). In fact, subsequentanimal experiments showed that gut microbeshydrolyze the glycosidic linkage of amygdalin torelease mandelonitrile, which spontaneouslybreaks down to produce benzaldehyde and toxiccyanide (28, 105). Gut microbes also metabolizethe plant-derived benzoisoquinoline alkaloid ber-berine in a way that allows intestinal absorption(106) and convert ginsenosides, themajor bioactivecomponents of ginseng, into metabolites that in-hibit cytochromeP-450s and affect host xenobioticmetabolism (107). Because these remedies are notregulated to the same extent as pharmaceuticals,their modes of action are typically less well char-acterized. Elucidating how gut microbes processthese compounds may contribute extensively toour fundamental understanding of their effects.

Gut microbial biotransformation comesof age: Recent case studies

Attempts to decipher the biological consequencesof gutmicrobial xenobioticmetabolism have beenhinderedby ourpoorunderstandingof these trans-formations. Although many associations exist be-

tween ingestion of xenobiotics processed by gutmicrobes and health status, we have limited in-formation about the distribution of these ac-tivities in patient populations.Metabolic functionsrarely correlate directly withmicrobial phylogeny,and considerable strain-level variation exists evenwithin the same species (108), which limits theinformation gained from assessing the compo-sition of the gut microbiota alone. Large-scalemetagenomic analyses that claim to detect dif-ferences in putative xenobiotic metabolism path-ways (109, 110) have not provided experimentalevidence supporting predicted changes in activ-ities. To elucidate how gut microbial metabolisminfluences human health, it is critical to connectfunctions of interest with genes and enzymes. Re-cent studies show how the combination of tradi-tional methods with emerging technologies canenable functional analysis. These examples alsoshow how a molecular understanding of gut mi-crobial xenobiotic metabolism can aid in manip-ulating these activities to benefit health outcomes.

Digoxin: Identifying a predictivebiomarker for drug metabolism

Digitalis purpurea (foxglove) plant extracts werefirst used to treat “dropsy” (congestive heart fail-ure) more than 230 years ago (111). The activeconstituent of foxglove is the cardiac glycosidedigoxin, which inhibits Na+/K+ ATPases incardiac myocytes, causing an influx of calciumand enhanced muscular contraction. Digoxin hasa very narrow therapeutic window, requiring care-ful monitoring to avoid toxicity. More than 10%of patients taking digoxin excrete high levels ofdihydrodigoxin, an inactivemetabolite derived fromreduction of an a,b-unsaturated lactone (Fig. 1A).Early studies implicated the gut microbiota indrug inactivation, as coadministering digoxin withantibiotics decreased or abolished dihydrodi-goxin production (112). Moreover, fecal samplesfrom dihydrodigoxin-excreting individuals were

found to completely metabolize digoxin. Subse-quent isolation of digoxin-metabolizing microbesrevealed that a single organism, Eubacteriumlentum (renamed Eggerthella lenta), was respon-sible (113). However, E. lenta was also found inpatients who did not excrete dihydrodigoxin,illustrating that the presence of this species alonewas not predictive of activity.Although E. lenta’s role in digoxin metabolism

has been appreciated for decades, challenges ingrowing the organism and a lack of genetic toolshampered efforts to understand this transfor-mation. More recently, Turnbaugh and co-workersused RNA sequencing (RNA-seq) and comparativegenomics to identify a digoxin-inducible gene clus-ter present only in digoxin-metabolizing E. lentastrains (37) (Fig. 1B). The cardiac glycoside re-ductase (cgr) operon from this organism encodestwo proteins that resemble bacterial reductasesinvolved in anaerobic respiration. Bioinformaticanalyses suggest that a membrane-associated cy-tochrome (Cgr1) transfers electrons through aseries of hemes to a predicted flavin-dependentreductase (Cgr2) that converts digoxin to dihy-drodigoxin. The presence of cgr genes correlateswith digoxin reduction by E. lenta strains. Fur-thermore, experiments in GF mice monoassoci-ated with E. lenta strains and incubations withhuman fecal samples indicate that these genescould be useful biomarkers for digoxin inacti-vation (37). Consequently, knowledge of the con-ditions influencing cgr gene expression successfullyinformed the design of a dietary intervention thatreduces digoxin metabolism in vivo (37). Overall,this work delineated an approach that may begeneralized to study additional inducible micro-bial metabolic activities.

Choline: Uncovering a broadlydistributed disease-associated activity

Gut microbes anaerobically convert choline, anessential nutrient found in meat, eggs, and milk,

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Fig. 1. Identifying gut microbial genes that predict cardiac drug metabolism. (A) E. lenta reductive metabolism leads to cardiac drug inactivation.(B) A combination of culture-based studies, sequencing, and bioinformatics helped to identify microbial genes associated with digoxin metabolism in humans.

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into TMA, which is further transformed intotrimethylamineN-oxide (TMAO) by FMOs in theliver (Fig. 2A). This co-metabolic pathway is im-plicated in several human diseases, includingcardiovascular disease (58, 59). A chemically guided,rational genome-mining effort ultimately identifiedthe genes and enzymes thatmediate this process(Fig. 2B) (114). Choline fermentation begins witha C–N bond cleavage reaction that resembles thefirst step in ethanolamine utilization, a transforma-tion catalyzed by a B12-dependent radical enzyme(ethanolamine ammonia-lyase). Hypothesizing thatthese pathways might share certain reactions, wesearched the genome of the choline-metabolizing,animal-associatedstrainDesulfovibriodesulfuricansfor homologs of known ethanolamine-degradingenzymes from the pathogen Salmonella enterica.This analysis revealed the choline utilization (cut)gene cluster and CutC, a GRE that converts cholineinto TMA and acetaldehyde (46, 114, 115).Biochemical and structural studies have re-

vealed CutC active-site residues that interact withthe trimethylammonium group of choline andmediate C–N bond cleavage (46, 115). These con-served amino acids are specific to this particularGREandhaveenabledaccurate identificationof thecut pathway in numerous, phylogenetically di-verse human gut bacteria (14). The discovery ofCutC has aided interventions to modulate cholinemetabolism in vivo, including the design of gutcommunities with decreased TMA production(116) and small-molecule inhibitors targeting thispathway (117). Such inhibitors could be potentialtherapeutics to treat diseases linked to TMA andTMAO production.

Irinotecan: Inhibitingmicrobiota-mediated toxicity

Irinotecan (CPT-11) is a prodrug of SN-38, a topo-isomerase inhibitor used for treating cancer(Fig. 3A). SN-38 is glucuronidated by host liverenzymes into an inactive conjugate (SN-38G),which enters the gut via biliary excretion. Gut

bacterial b-glucuronidase enzymes hydrolyzeSN-38G in the large intestine to regeneratethe active chemotherapeutic agent (26). SN-38then enters colonic epithelial cells, causing in-testinal damage and severe diarrhea, side ef-fects that limit the use of this otherwise effectivedrug.Inhibition of gut bacterial b-glucuronidases is

an intriguing approach for preventing drug re-activation. As these enzymes are broadly distrib-uted in commensal bacteria and are present inhumans, inhibitors need to be selective for bac-terial b-glucuronidases and nontoxic to both hostcells and other gut microbes. Redinbo and co-workers have used an in vitro high-throughputscreen to successfully identify potent and selec-tive inhibitors of gut bacterial b-glucuronidases(Fig. 3B) (26). These inhibitors were effectiveagainst multiple, distantly related gut bacteriabut did not target the human b-glucuronidase.Structural studies revealed that these compoundsinteract with an active-site loop distinct to bac-terial b-glucuronidases, which explains their se-lectivity. Administration of one of these inhibitorsto mice prevented reactivation of SN-38 in thegut and concomitant toxicity. Further work ex-amining the crystal structures and activities ofadditional gut bacterial b-glucuronidases identi-fied a conserved Asn-Lys motif that interacts withthe carboxylic acid of the glucuronic acid sugar.This motif is absent from glycosidases that ac-cept different substrates, which will help toidentify these enzymes in sequencing data setsand elucidate the effects of inhibitors on differ-ent types of gut organisms (23). Because bacterialb-glucuronidases can deconjugate glucuronidesderived frommany dietary compounds and drugs,inhibitors of these enzymes may be useful in othertherapeutic contexts (27). By modulating specificmetabolic activities, additional small-moleculeinhibitors of microbial gut enzymes may helpto uncover the roles of particular transforma-tions in this complex habitat and could be

excellent starting points for developingmicrobiota-targeted drugs.

Outlook and challenges

Although our appreciation of the human gutmicrobiota’s role in transforming xenobiotics hasincreased, our understanding of the biologicalimplications of these reactions is limited. Thefuture challenges lie in identifying the organisms,genes, and enzymes involved in knownmetabolicprocesses; uncovering important but currentlyunappreciated activities; andelucidating the effectsof this chemistry on both host and microbiota.

Connecting xenobiotic metabolism toorganisms, genes, and enzymes

As we have discussed above, most examples ofgut microbial xenobiotic metabolism are asso-ciated with the whole gut community. Surveys ofhuman gut isolates, such as HumanMicrobiomeProject (HMP) reference strains (118), or cul-turing from human fecal samples can identifyindividual organisms with particular metaboliccapabilities. Examining intact communities viaapproaches like stable isotope probing, fluores-cence in situ hybridization, and imaging massspectrometry can also be used to detect individ-ual cells that metabolize particular compounds(119). Coupled with single-cell genomics, thesemethods may aid investigations of activities as-sociated with organisms that are challenging tocultivate (120).Both traditional approaches (chemical or trans-

poson mutagenesis and activity-guided proteinpurification) andmore recent strategies (rationalgenome mining, comparative genomics, RNA-seq,and functional metagenomics)may be used to linkmetabolic activities with genes (37, 114, 121). How-ever, a lack of genetic tools for many gut microbesand the narrow range of heterologous hosts avail-able for functional characterization still limit prog-ress toward this objective. An appreciation ofthe chemical reactivity required for xenobiotic

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Fig. 2. Uncovering gut microbial enzymes that convert dietary choline to disease-associated metabolites. (A) Choline is metabolized by a gutmicrobial-human co-metabolic pathway into the disease-associated metabolites trimethylamine (TMA) and trimethylamine N-oxide (TMAO).(B) A chemically guided, rational genome-mining effort enabled the identification and characterization of enzymes involved in gut microbial anaerobiccholine metabolism.

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degradation is vital for informing genome andmetagenome mining, as well as interpreting andrationalizing results fromothermethods.Microbesin which enzymes and metabolic pathways arebetter studied, including pathogens and environ-mental strains involved in bioremediation, mayalso offer clues to support microbiome-focuseddiscovery efforts.The examples of xenobiotic metabolism sur-

veyed above likely represent a small fractionof the transformations taking place in the gutcommunity. Metabolomics in human subjectswill play a key role in illuminating currently un-appreciated gut microbial metabolic processes,particularly for transformations that depend onadditional microbial and host activities or inter-actions (122).

Linking uncharacterized genes inmicrobiomes to xenobiotic metabolism

We should expect to uncover interesting gut mi-crobial activities by investigating the vast num-ber of uncharacterized or misannotated genespresent in human gut microbiomes. Notably,86% of the genes from HMP stool metagenomescannot be assigned to known metabolic path-ways, and half cannot be annotated (10). Manymembers of large enzyme superfamilies also haveunknown functions and are typically misanno-tated. Collaborative efforts such as the EnzymeFunction Initiative have advanced bioinformaticand experimental approaches for functionallycharacterizing such enzymes (123), including pro-tein sequence similarity and genome neighbor-hood network analyses, high-throughput liganddocking, and structural genomics. These methodshighlight divergent sequences that likely possessdistinct functions and provide context for linkinguncharacterized enzymeswithmetabolic pathways

and substrates. Conversely, an understanding ofthe chemistry involved in known xenobiotic metab-olism may pinpoint particular enzyme super-families as startingpoints to prospect for additionaltransformations.Comprehensively characterizing proteins that

lack homology to known enzymes presents amoresubstantial challenge and will likely require novelapproaches that incorporate automation and high-throughput assay formats. Meanwhile, it is criticalto prioritize uncharacterized proteins for furtherstudy by considering both ecological context (i.e.,abundance and distribution in the gut micro-biome) and host health status, including expo-sure to xenobiotics (16, 124).

Harnessing knowledge of microbialmetabolism to improve human health

Deciphering how gut microbial transformationof xenobiotics affects host health will require theintegration of clinical studies with mechanisticexperiments in model systems and organisms(Fig. 4A). These efforts will necessitate identi-fying microbial genes and/or metabolites thatare reliable, diagnostic markers for activities ofinterest. Obtaining data from patients is partic-ularly important, and existing epidemiologicalstudies linking diet or xenobiotic exposure tohealth outcomes should be reexamined with gutmicrobial participation in mind.Diet is clearly a cornerstone of human health.

Though many epidemiological studies linkingdietary patterns with health outcomes yield con-flicting results, few have taken gut microbialmetabolic capabilities into account. A comprehen-sivemolecular understanding of how gutmicrobesprocess dietary components is essential for therational use of “functional foods” or prebiotics totreat conditions such as metabolic disease and

malnutrition. This knowledge can also informpersonalized nutrition, in which diets are indi-vidually customized for patients’ metabolic pro-files and gut microbiotas (Fig. 4B) (125).Similarly to dietary studies, attempts to cor-

relate pollutant exposure with health outcomesoften yield conflicting results that could, in part,arise from variations in microbial metabolic acti-vities (126). Gut microbial enzymes that alter thetoxicity of industrial chemicals and environmentalpollutants could serve as biomarkers to inform riskassessments among populations exposed to thesecompounds (Fig. 4C). It may also become possibleto use gut microbial metabolism to help removeharmful compounds from the body and preventdisease, analogous to bioremediation of pollutedenvironments (127).Finally, knowledge of how gut microbes trans-

form pharmaceuticals should be further integratedinto drug development, clinical trial design, andclinical practice. Understanding which functionalgroups are prone to microbial metabolism allowsmedicinal chemists to either avoid these structuralfeatures or incorporate them into prodrugs to en-able selective activation in the GI tract (38, 39). Gutmicrobial enzymes thatmediateharmfulmetabolicactivities may also represent a new class of drugtargets. Finally, knowledge of gutmicrobial metab-olism could shape clinical trial design and clinicalpractice by allowing physicians to screen for det-rimental or beneficial activities before prescribingdrugs (Fig. 4D). Ultimately, personalized medicinewill require a better understanding of the distri-bution of specific microbial metabolic functionswithin human populations.

Conclusions

A major challenge facing human gut microbiotaresearch is the need to move beyond cataloging

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Fig. 3. Preventing drug reactivation and toxicity by inhibiting gut microbial enzymes. (A) Microbial cleavage of the glucuronidated drug conjugate ofthe cancer chemotherapeutic SN-38 leads to drug reactivation and toxicity within the gut. UDP, uridine diphosphate. (B) High-throughput screening identifiedspecific inhibitors of bacterial b-glucuronidases. These compounds alleviated the GI toxicity associated with irinotecan metabolism. Et, ethyl.

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the organisms and genes in this community toelucidating the mechanisms underlying theirinfluence onhost health. By altering the chemicalstructures of ingested compounds, gut microbescan mediate the effects of diet, pollutants, anddrugs on host physiology. Individual variationremains a major challenge, and although manysuch metabolic activities have been identified,few have been connected to organisms, genes,and enzymes. Moving forward, it is essential thatwe incorporate enzyme discovery and character-ization efforts into investigations of gut micro-bial xenobiotic metabolism. Only by gaining amolecular understanding of these processes canwe leverage the remarkable chemical abilities ofthis community to improve human health.

REFERENCES AND NOTES

1. A. H. Moeller et al., Cospeciation of gut microbiota withhominids. Science 353, 380–382 (2016). doi: 10.1126/science.aaf3951; pmid: 27463672

2. C. G. Buffie et al., Precision microbiome reconstitutionrestores bile acid mediated resistance to Clostridium difficile.Nature 517, 205–208 (2015). doi: 10.1038/nature13828;pmid: 25337874

3. G. Sharon et al., Specialized metabolites from themicrobiome in health and disease. Cell Metab. 20, 719–730(2014). doi: 10.1016/j.cmet.2014.10.016; pmid: 25440054

4. A. Koh, F. De Vadder, P. Kovatcheva-Datchary, F. Bäckhed,From dietary fiber to host physiology: Short-chain fatty acidsas key bacterial metabolites. Cell 165, 1332–1345 (2016).doi: 10.1016/j.cell.2016.05.041; pmid: 27259147

5. T. Sousa et al., The gastrointestinal microbiota as a site forthe biotransformation of drugs. Int. J. Pharm. 363, 1–25(2008). doi: 10.1016/j.ijpharm.2008.07.009; pmid: 18682282

6. H. Danielsson, B. Gustafsson, On serum-cholesterol levelsand neutral fecal sterols in germ-free rats; bile acids andsteroids 59. Arch. Biochem. Biophys. 83, 482–485 (1959).doi: 10.1016/0003-9861(59)90056-6; pmid: 13813997

7. R. Sender, S. Fuchs, R. Milo, Revised estimates for thenumber of human and bacteria cells in the body. PLOS Biol.14, e1002533 (2016). doi: 10.1371/journal.pbio.1002533;pmid: 27541692

8. J. Aron-Wisnewsky, J. Doré, K. Clement, The importance ofthe gut microbiota after bariatric surgery. Nat. Rev.Gastroenterol. Hepatol. 9, 590–598 (2012). doi: 10.1038/nrgastro.2012.161; pmid: 22926153

9. P. B. Eckburg et al., Diversity of the human intestinalmicrobial flora. Science 308, 1635–1638 (2005).doi: 10.1126/science.1110591; pmid: 15831718

10. The Human Microbiome Project Consortium, Structure,function and diversity of the healthy human microbiome.Nature 486, 207–214 (2012). doi: 10.1038/nature11234;pmid: 22699609

11. B. Wang, L. Hu, T. Siahaan, Drug Delivery: Principles andApplications (Wiley, ed. 2, 2016).

12. R. J. Linhardt, P. M. Galliher, C. L. Cooney, Polysaccharidelyases. Appl. Biochem. Biotechnol. 12, 135–176 (1987).doi: 10.1007/BF02798420; pmid: 3521491

13. A. Ryan et al., Identification of NAD(P)H quinoneoxidoreductase activity in azoreductases from P. aeruginosa:Azoreductases and NAD(P)H quinone oxidoreductasesbelong to the same FMN-dependent superfamily of enzymes.PLOS ONE 9, e98551 (2014). doi: 10.1371/journal.pone.0098551; pmid: 24915188

14. A. Martínez-del Campo et al., Characterization and detectionof a widely distributed gene cluster that predicts anaerobiccholine utilization by human gut bacteria. mBio 6, e00042-15(2015). doi: 10.1128/mBio.00042-15; pmid: 25873372

15. A. El Kaoutari, F. Armougom, J. I. Gordon, D. Raoult,B. Henrissat, The abundance and variety of carbohydrate-active enzymes in the human gut microbiota. Nat. Rev.Microbiol. 11, 497–504 (2013). doi: 10.1038/nrmicro3050;pmid: 23748339

16. B. J. Levin et al., A prominent glycyl radical enzyme in humangut microbiomes metabolizes trans-4-hydroxy-L-proline.Science 355, eaai8386 (2017). doi: 10.1126/science.aai8386;pmid: 28183913

17. K. Kurokawa et al., Comparative metagenomics revealedcommonly enriched gene sets in human gut microbiomes.DNA Res. 14, 169–181 (2007). doi: 10.1093/dnares/dsm018;pmid: 17916580

18. K. Ellrott, L. Jaroszewski, W. Li, J. C. Wooley,A. Godzik, Expansion of the protein repertoire innewly explored environments: Human gut microbiomespecific protein families. PLOS Comput. Biol. 6,e1000798 (2010). doi: 10.1371/journal.pcbi.1000798;pmid: 20532204

19. P. Jancova, P. Anzenbacher, E. Anzenbacherova, Phase IIdrug metabolizing enzymes. Biomed. Pap. Med. Fac. Univ.Palacky Olomouc Czech Repub. 154, 103–116 (2010).doi: 10.5507/bp.2010.017; pmid: 20668491

20. J. Wang, V. Yadav, A. L. Smart, S. Tajiri, A. W. Basit,Stability of peptide drugs in the colon. Eur. J. Pharm. Sci.78, 31–36 (2015). doi: 10.1016/j.ejps.2015.06.018;pmid: 26111980

21. H. Tozaki et al., Degradation of insulin and calcitonin andtheir protection by various protease inhibitors in rat caecalcontents: Implications in peptide delivery to the colon.J. Pharm. Pharmacol. 49, 164–168 (1997). doi: 10.1111/j.2042-7158.1997.tb06773.x; pmid: 9055189

22. W. W. Kallemeijn, M. D. Witte, T. Wennekes, J. M. Aerts,Mechanism-based inhibitors of glycosidases: Design andapplications. Adv. Carbohydr. Chem. Biochem. 71, 297–338(2014). doi: 10.1016/B978-0-12-800128-8.00004-2;pmid: 25480507

Koppel et al., Science 356, eaag2770 (2017) 23 June 2017 9 of 11

Fig. 4. Potential implications of understanding gut microbial xenobiotic metabolism. (A) Interfacing clinical studies and hypothesis-driven researchin model systems is essential for elucidating the biological consequences of gut microbial xenobiotic metabolism. Incorporating a mechanisticunderstanding of microbial transformations, along with knowledge of host genetics and metabolism, could (B) inform personalized nutrition, (C) improvetoxicological risk assessment, and (D) enable personalized medicine.

RESEARCH | REVIEWon June 7, 2018

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Dow

nloaded from

23. B. D. Wallace et al., Structure and inhibition of microbiomeb-glucuronidases essential to the alleviation of cancer drugtoxicity. Chem. Biol. 22, 1238–1249 (2015). doi: 10.1016/j.chembiol.2015.08.005; pmid: 26364932

24. J. E. Ulmer et al., Characterization of glycosaminoglycan(GAG) sulfatases from the human gut symbiont Bacteroidesthetaiotaomicron reveals the first GAG-specific bacterialendosulfatase. J. Biol. Chem. 289, 24289–24303 (2014).doi: 10.1074/jbc.M114.573303; pmid: 25002587

25. G. Lukatela et al., Crystal structure of human arylsulfatase A:The aldehyde function and the metal ion at the active sitesuggest a novel mechanism for sulfate ester hydrolysis.Biochemistry 37, 3654–3664 (1998). doi: 10.1021/bi9714924;pmid: 9521684

26. B. D. Wallace et al., Alleviating cancer drug toxicity byinhibiting a bacterial enzyme. Science 330, 831–835 (2010).doi: 10.1126/science.1191175; pmid: 21051639

27. K. S. Saitta et al., Bacterial b-glucuronidase inhibitionprotects mice against enteropathy induced by indomethacin,ketoprofen or diclofenac: Mode of action andpharmacokinetics. Xenobiotica 44, 28–35 (2014).doi: 10.3109/00498254.2013.811314; pmid: 23829165

28. J. H. Carter, M. A. McLafferty, P. Goldman, Role of thegastrointestinal microflora in amygdalin (laetrile)-inducedcyanide toxicity. Biochem. Pharmacol. 29, 301–304 (1980).doi: 10.1016/0006-2952(80)90504-3; pmid: 7362642

29. T. Nimura, T. Tokieda, T. Yamaha, Partial purification andsome properties of cyclamate sulfamatase. J. Biochem. 75,407–417 (1974). doi: 10.1093/oxfordjournals.jbchem.a130407; pmid: 4209783

30. D. R. Donohoe et al., The microbiome and butyrate regulateenergy metabolism and autophagy in the mammaliancolon. Cell Metab. 13, 517–526 (2011). doi: 10.1016/j.cmet.2011.02.018; pmid: 21531334

31. A. J. Cooper et al., Cysteine S-conjugate b-lyases: Importantroles in the metabolism of naturally occurring sulfur andselenium-containing compounds, xenobiotics and anticanceragents. Amino Acids 41, 7–27 (2011). doi: 10.1007/s00726-010-0552-0; pmid: 20306345

32. S. P. Claus, H. Guillou, S. Ellero-Simatos, The gut microbiota:A major player in the toxicity of environmental pollutants?NPJ Biofilms Microbiomes 2, 16003 (2016). doi: 10.1038/npjbiofilms.2016.3

33. I. Rossol, A. Pühler, The Corynebacterium glutamicumaecD gene encodes a C-S lyase with a,b-elimination activitythat degrades aminoethylcysteine. J. Bacteriol. 174,2968–2977 (1992). doi: 10.1128/jb.174.9.2968-2977.1992;pmid: 1569026

34. F. Rafii, J. D. Hall, C. E. Cerniglia, Mutagenicity of azo dyesused in foods, drugs and cosmetics before and afterreduction by Clostridium species from the human intestinaltract. Food Chem. Toxicol. 35, 897–901 (1997). doi: 10.1016/S0278-6915(97)00060-4; pmid: 9409630

35. S. C. Lee, A. G. Renwick, Sulphoxide reduction by ratintestinal flora and by Escherichia coli in vitro. Biochem.Pharmacol. 49, 1567–1576 (1995). doi: 10.1016/0006-2952(95)00093-F; pmid: 7786297

36. H. Laue, M. Friedrich, J. Ruff, A. M. Cook, Dissimilatory sulfitereductase (desulfoviridin) of the taurine-degrading, non-sulfate-reducing bacterium Bilophila wadsworthia RZATAUcontains a fused DsrB-DsrD subunit. J. Bacteriol. 183,1727–1733 (2001). doi: 10.1128/JB.183.5.1727-1733.2001;pmid: 11160104

37. H. J. Haiser et al., Predicting and manipulating cardiac druginactivation by the human gut bacterium Eggerthella lenta.Science 341, 295–298 (2013). doi: 10.1126/science.1235872;pmid: 23869020

38. M. A. Peppercorn, P. Goldman, The role of intestinal bacteriain the metabolism of salicylazosulfapyridine. J. Pharmacol.Exp. Ther. 181, 555–562 (1972). pmid: 4402374

39. K. Lavrijsen et al., Reduction of the prodrug loperamide oxideto its active drug loperamide in the gut of rats, dogs, andhumans. Drug Metab. Dispos. 23, 354–362 (1995).pmid: 7628301

40. F. Rafii, C. E. Cerniglia, Reduction of azo dyes andnitroaromatic compounds by bacterial enzymes from thehuman intestinal tract. Environ. Health Perspect. 103(suppl. 5), 17–19 (1995). doi: 10.1289/ehp.95103s417;pmid: 8565901

41. T. Kumano, E. Fujiki, Y. Hashimoto, M. Kobayashi, Discoveryof a sesamin-metabolizing microorganism and a new enzyme.Proc. Natl. Acad. Sci. U.S.A. 113, 9087–9092 (2016).doi: 10.1073/pnas.1605050113; pmid: 27444012

42. T. Ticak, D. J. Kountz, K. E. Girosky, J. A. Krzycki,D. J. Ferguson Jr., A nonpyrrolysine member of the widelydistributed trimethylamine methyltransferase family is aglycine betaine methyltransferase. Proc. Natl. Acad. Sci. U.S.A.111, E4668–E4676 (2014). doi: 10.1073/pnas.1409642111;pmid: 25313086

43. C. Deloménie et al., Identification and functionalcharacterization of arylamine N-acetyltransferases ineubacteria: Evidence for highly selective acetylation of 5-aminosalicylic acid. J. Bacteriol. 183, 3417–3427 (2001).doi: 10.1128/JB.183.11.3417-3427.2001; pmid: 11344150

44. D. Sutton, A. M. Butler, L. Nadin, M. Murray, Role of CYP3A4in human hepatic diltiazem N-demethylation: Inhibition ofCYP3A4 activity by oxidized diltiazem metabolites.J. Pharmacol. Exp. Ther. 282, 294–300 (1997). pmid: 9223567

45. W. Buckel, B. T. Golding, Radical enzymes in anaerobes.Annu. Rev. Microbiol. 60, 27–49 (2006). doi: 10.1146/annurev.micro.60.080805.142216; pmid: 16704345

46. S. Bodea, M. A. Funk, E. P. Balskus, C. L. Drennan, Molecularbasis of C–N bond cleavage by the glycyl radicalenzyme choline trimethylamine-lyase. Cell Chem. Biol. 23,1206–1216 (2016). doi: 10.1016/j.chembiol.2016.07.020;pmid: 27642068

47. T. Selmer, P. I. Andrei, p-Hydroxyphenylacetatedecarboxylase from Clostridium difficile. Eur. J. Biochem. 268,1363–1372 (2001). doi: 10.1046/j.1432-1327.2001.02001.x;pmid: 11231288

48. T. A. Clayton, D. Baker, J. C. Lindon, J. R. Everett,J. K. Nicholson, Pharmacometabonomic identification of asignificant host-microbiome metabolic interaction affectinghuman drug metabolism. Proc. Natl. Acad. Sci. U.S.A. 106,14728–14733 (2009). doi: 10.1073/pnas.0904489106;pmid: 19667173

49. X. He, M. L. Marco, C. M. Slupsky, Emerging aspects of foodand nutrition on gut microbiota. J. Agric. Food Chem. 61,9559–9574 (2013). doi: 10.1021/jf4029046; pmid: 24028159

50. H. J. Flint, E. A. Bayer, M. T. Rincon, R. Lamed, B. A. White,Polysaccharide utilization by gut bacteria: Potential for newinsights from genomic analysis. Nat. Rev. Microbiol. 6,121–131 (2008). doi: 10.1038/nrmicro1817; pmid: 18180751

51. N. M. Koropatkin, E. A. Cameron, E. C. Martens, How glycanmetabolism shapes the human gut microbiota. Nat. Rev.Microbiol. 10, 323–335 (2012). doi: 10.1038/nrmicro2746;pmid: 22491358

52. A. Caminero et al., Differences in gluten metabolism amonghealthy volunteers, coeliac disease patients and first-degreerelatives. Br. J. Nutr. 114, 1157–1167 (2015). doi: 10.1017/S0007114515002767; pmid: 26428276

53. A. Caminero et al., Duodenal bacteria from patients with celiacdisease and healthy subjects distinctly affect gluten breakdownand immunogenicity. Gastroenterology 151, 670–683 (2016).doi: 10.1053/j.gastro.2016.06.041; pmid: 27373514

54. F. Blachier, F. Mariotti, J. F. Huneau, D. Tomé, Effects ofamino acid-derived luminal metabolites on the colonicepithelium and physiopathological consequences. AminoAcids 33, 547–562 (2007). doi: 10.1007/s00726-006-0477-9; pmid: 17146590

55. W. R. Wikoff et al., Metabolomics analysis reveals largeeffects of gut microflora on mammalian blood metabolites.Proc. Natl. Acad. Sci. U.S.A. 106, 3698–3703 (2009).doi: 10.1073/pnas.0812874106; pmid: 19234110

56. B. B. Williams et al., Discovery and characterization of gutmicrobiota decarboxylases that can produce theneurotransmitter tryptamine. Cell Host Microbe 16, 495–503(2014). doi: 10.1016/j.chom.2014.09.001; pmid: 25263219

57. A. S. Devlin et al., Modulation of a circulating uremic Solutevia rational genetic manipulation of the gut microbiota.Cell Host Microbe 20, 709–715 (2016). doi: 10.1016/j.chom.2016.10.021; pmid: 27916477

58. C. E. Cho, M. A. Caudill, Trimethylamine-N-oxide: Friend, foe,or simply caught in the cross-fire? Trends Endocrinol. Metab.28, 121–130 (2017). doi: 10.1016/j.tem.2016.10.005;pmid: 27825547

59. D. Fennema, I. R. Phillips, E. A. Shephard, Trimethylamineand trimethylamine N-oxide, a flavin-containingmonooxygenase 3 (FMO3)-mediated host-microbiomemetabolic axis implicated in health and disease. Drug Metab.Dispos. 44, 1839–1850 (2016). doi: 10.1124/dmd.116.070615;pmid: 27190056

60. J. C. Escolà-Gil et al., The cholesterol content of Westerndiets plays a major role in the paradoxical increase in high-density lipoprotein cholesterol and upregulates themacrophage reverse cholesterol transport pathway.

Arterioscler. Thromb. Vasc. Biol. 31, 2493–2499 (2011).doi: 10.1161/ATVBAHA.111.236075; pmid: 21885848

61. I. A. Macdonald, V. D. Bokkenheuser, J. Winter,A. M. McLernon, E. H. Mosbach, Degradation of steroids inthe human gut. J. Lipid Res. 24, 675–700 (1983).pmid: 6350517

62. P. Gérard et al., Gnotobiotic rats harboring human intestinalmicrobiota as a model for studying cholesterol-to-coprostanol conversion. FEMS Microbiol. Ecol. 47, 337–343(2004). doi: 10.1016/S0168-6496(03)00285-X;pmid: 19712322

63. D. Ren, L. Li, A. W. Schwabacher, J. W. Young, D. C. Beitz,Mechanism of cholesterol reduction to coprostanol byEubacterium coprostanoligenes ATCC 51222. Steroids 61,33–40 (1996). doi: 10.1016/0039-128X(95)00173-N;pmid: 8789734

64. C. A. Dujovne et al., Efficacy and safety of a potent newselective cholesterol absorption inhibitor, ezetimibe, inpatients with primary hypercholesterolemia. Am. J. Cardiol.90, 1092–1097 (2002). doi: 10.1016/S0002-9149(02)02798-4;pmid: 12423709

65. L. Valdés et al., The relationship between phenoliccompounds from diet and microbiota: Impact on humanhealth. Food Funct. 6, 2424–2439 (2015). doi: 10.1039/C5FO00322A; pmid: 26068710

66. R. García-Villalba, D. Beltrán, J. C. Espín, M. V. Selma,F. A. Tomás-Barberán, Time course production of urolithinsfrom ellagic acid by human gut microbiota. J. Agric. FoodChem. 61, 8797–8806 (2013). doi: 10.1021/jf402498b;pmid: 23984796

67. C. Atkinson, C. L. Frankenfeld, J. W. Lampe, Gut bacterialmetabolism of the soy isoflavone daidzein: Exploring therelevance to human health. Exp. Biol. Med. 230, 155–170(2005). pmid: 15734719

68. J. Tang et al., Tea consumption and mortality of all cancers,CVD and all causes: A meta-analysis of eighteen prospectivecohort studies. Br. J. Nutr. 114, 673–683 (2015). doi: 10.1017/S0007114515002329; pmid: 26202661

69. T. Clavel, G. Henderson, W. Engst, J. Doré, M. Blaut,Phylogeny of human intestinal bacteria that activate thedietary lignan secoisolariciresinol diglucoside.FEMS Microbiol. Ecol. 55, 471–478 (2006). doi: 10.1111/j.1574-6941.2005.00057.x; pmid: 16466386

70. B. A. Bopp, R. C. Sonders, J. W. Kesterson, A. G. Renwick,Toxicological aspects of cyclamate and cyclohexylamine.Crit. Rev. Toxicol. 16, 213–306 (1986). doi: 10.3109/10408448609037465; pmid: 2420530

71. A. G. Renwick, S. M. Tarka, Microbial hydrolysis of steviolglycosides. Food Chem. Toxicol. 46 (suppl. 7), S70–S74(2008). doi: 10.1016/j.fct.2008.05.008; pmid: 18550247

72. R. Krishnan et al., The effect of dietary xylitol on the abilityof rat caecal flora to metabolise xylitol. Aust. J. Exp. Biol.Med. Sci. 58, 639–652 (1980). doi: 10.1038/icb.1980.66;pmid: 6791623

73. A. G. Renwick, The metabolism of intense sweeteners.Xenobiotica 16, 1057–1071 (1986). doi: 10.3109/00498258609038983; pmid: 3541395

74. F. Kassie et al., Intestinal microflora plays a crucial role in thegenotoxicity of the cooked food mutagen 2-amino-3-methylimidazo [4,5-f]quinoline. Carcinogenesis 22, 1721–1725(2001). doi: 10.1093/carcin/22.10.1721; pmid: 11577015

75. C. Humblot et al., b-glucuronidase in human intestinalmicrobiota is necessary for the colonic genotoxicity of thefood-borne carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline in rats. Carcinogenesis 28, 2419–2425 (2007).doi: 10.1093/carcin/bgm170; pmid: 17660508

76. A. J. Cross et al., A large prospective study of meatconsumption and colorectal cancer risk: An investigation ofpotential mechanisms underlying this association. CancerRes. 70, 2406–2414 (2010). doi: 10.1158/0008-5472.CAN-09-3929; pmid: 20215514

77. F. Rafii, W. Franklin, C. E. Cerniglia, Azoreductase activity ofanaerobic bacteria isolated from human intestinal microflora.Appl. Environ. Microbiol. 56, 2146–2151 (1990).pmid: 2202258

78. J. F. Borzelleca, K. Depukat, J. B. Hallagan, Lifetimetoxicity/carcinogenicity studies of FD & C blue no. 1(brilliant blue FCF) in rats and mice. Food Chem. Toxicol.28, 221–234 (1990). doi: 10.1016/0278-6915(90)90034-K;pmid: 2358248

79. Z. Singh, P. Chadha, Textile industry and occupational cancer.J. Occup. Med. Toxicol. 11, 39 (2016). doi: 10.1186/s12995-016-0128-3; pmid: 27532013

Koppel et al., Science 356, eaag2770 (2017) 23 June 2017 10 of 11

RESEARCH | REVIEWon June 7, 2018

http://science.sciencemag.org/

Dow

nloaded from

80. R. P. Bos et al., Internal exposure of rats to benzidine derivedfrom orally administered benzidine-based dyes afterintestinal azo reduction. Toxicology 40, 207–213 (1986).doi: 10.1016/0300-483X(86)90080-6; pmid: 3726894

81. J. R. Ingelfinger, Melamine and the global implications of foodcontamination. N. Engl. J. Med. 359, 2745–2748 (2008).doi: 10.1056/NEJMp0808410; pmid: 19109571

82. X. Zheng et al., Melamine-induced renal toxicity ismediated by the gut microbiota. Sci. Transl. Med. 5,172ra22 (2013). doi: 10.1126/scitranslmed.3005114;pmid: 23408055

83. H. Wang, C. Geng, J. Li, A. Hu, C. P. Yu, Characterization of anovel melamine-degrading bacterium isolated from amelamine-manufacturing factory in China. Appl. Microbiol.Biotechnol. 98, 3287–3293 (2014). doi: 10.1007/s00253-013-5363-2; pmid: 24297478

84. I. R. Rowland, M. J. Davies, P. Grasso, Metabolism ofmethylmercuric chloride by the gastro-intestinal flora of therat. Xenobiotica 8, 37–43 (1978). doi: 10.3109/00498257809060381; pmid: 626001

85. I. R. Rowland, M. J. Davies, J. G. Evans, The effect of thegastrointestinal flora on tissue content of mercury andorganomercurial neurotoxicity in rats given methylmercuricchloride. Dev. Toxicol. Environ. Sci. 8, 79–82 (1980).pmid: 7308081

86. C. A. Liebert, J. Wireman, T. Smith, A. O. Summers,Phylogeny of mercury resistance (mer) operons of gram-negative bacteria isolated from the fecal flora of primates.Appl. Environ. Microbiol. 63, 1066–1076 (1997).pmid: 9055422

87. S. E. Rothenberg et al., The role of gut microbiota in fetalmethylmercury exposure: Insights from a pilot study. Toxicol.Lett. 242, 60–67 (2016). doi: 10.1016/j.toxlet.2015.11.022;pmid: 26626101

88. R. A. Diaz-Bone, T. R. van de Wiele, Biovolatilization ofmetal(loid)s by intestinal microorganisms in the simulatorof the human intestinal microbial ecosystem. Environ. Sci.Technol. 43, 5249–5256 (2009). doi: 10.1021/es900544c;pmid: 19708349

89. P. Spanogiannopoulos, E. N. Bess, R. N. Carmody,P. J. Turnbaugh, The microbial pharmacists within us: Ametagenomic view of xenobiotic metabolism. Nat. Rev.Microbiol. 14, 273–287 (2016). doi: 10.1038/nrmicro.2016.17;pmid: 26972811

90. S. Takeno, Y. Hirano, A. Kitamura, T. Sakai, Comparativedevelopmental toxicity and metabolism of nitrazepam in ratsand mice. Toxicol. Appl. Pharmacol. 121, 233–238 (1993).doi: 10.1006/taap.1993.1150; pmid: 8346540

91. H. Okuda et al., Lethal drug interactions of sorivudine, a newantiviral drug, with oral 5-fluorouracil prodrugs. Drug Metab.Dispos. 25, 270–273 (1997). pmid: 9152608

92. M. Vétizou et al., Anticancer immunotherapy by CTLA-4blockade relies on the gut microbiota. Science 350,1079–1084 (2015). doi: 10.1126/science.aad1329;pmid: 26541610

93. N. R. Shin et al., An increase in the Akkermansia spp.population induced by metformin treatment improvesglucose homeostasis in diet-induced obese mice. Gut 63,727–735 (2014). doi: 10.1136/gutjnl-2012-303839;pmid: 23804561

94. R. A. van Hogezand et al., Bacterial acetylation of 5-aminosalicylic acid in faecal suspensions cultured underaerobic and anaerobic conditions. Eur. J. Clin. Pharmacol. 43,189–192 (1992). doi: 10.1007/BF01740669; pmid: 1425876

95. H. A. Strong, A. G. Renwick, C. F. George, Y. F. Liu, M. J. Hill,The reduction of sulphinpyrazone and sulindac by intestinalbacteria. Xenobiotica 17, 685–696 (1987). doi: 10.3109/00498258709043976; pmid: 3630204

96. P. Lehouritis et al., Local bacteria affect the efficacy ofchemotherapeutic drugs. Sci. Rep. 5, 14554 (2015).doi: 10.1038/srep14554; pmid: 26416623

97. D. B. Calne et al., Idiopathic Parkinsonism treated with anextracerebral decarboxylase inhibitor in combination withlevodopa. BMJ 3, 729–732 (1971). doi: 10.1136/bmj.3.5777.729; pmid: 4938431

98. J. Bergmark et al., Decarboxylation of orally administeredL-dopa in the human digestive tract. Naunyn SchmiedebergsArch. Pharmacol. 272, 437–440 (1972). doi: 10.1007/BF00501249; pmid: 4260263

99. B. R. Goldin, M. A. Peppercorn, P. Goldman, Contributions ofhost and intestinal microflora in the metabolism of L-dopa bythe rat. J. Pharmacol. Exp. Ther. 186, 160–166 (1973).pmid: 4723308

100. J. G. Nutt, N. H. Holford, The response to levodopa inParkinson’s disease: Imposing pharmacological law andorder. Ann. Neurol. 39, 561–573 (1996). doi: 10.1002/ana.410390504; pmid: 8619540

101. K. Zhang, Y. Ni, Tyrosine decarboxylase from Lactobacillusbrevis: Soluble expression and characterization. Protein Expr.Purif. 94, 33–39 (2014). doi: 10.1016/j.pep.2013.10.018;pmid: 24211777

102. G. Sharon, T. R. Sampson, D. H. Geschwind, S. K. Mazmanian,The central nervous system and the gut microbiome.Cell 167, 915–932 (2016). doi: 10.1016/j.cell.2016.10.027;pmid: 27814521

103. A. S. Relman, Closing the books on laetrile. N. Engl. J. Med.306, 236 (1982). doi: 10.1056/NEJM198201283060410;pmid: 7054687

104. C. G. Moertel et al., A clinical trial of amygdalin (laetrile) inthe treatment of human cancer. N. Engl. J. Med. 306,201–206 (1982). doi: 10.1056/NEJM198201283060403;pmid: 7033783

105. G. J. Strugala, A. G. Rauws, R. Elbers, Intestinal first passmetabolism of amygdalin in the rat in vitro. Biochem.Pharmacol. 35, 2123–2128 (1986). doi: 10.1016/0006-2952(86)90580-0; pmid: 3089225

106. R. Feng et al., Transforming berberine into its intestine-absorbable form by the gut microbiota. Sci. Rep. 5, 12155(2015). doi: 10.1038/srep12155; pmid: 26174047

107. Y. Liu et al., Ginsenoside metabolites, rather than naturallyoccurring ginsenosides, lead to inhibition of humancytochrome P450 enzymes. Toxicol. Sci. 91, 356–364(2006). doi: 10.1093/toxsci/kfj164; pmid: 16547074

108. K. T. Konstantinidis, J. M. Tiedje, Genomic insights thatadvance the species definition for prokaryotes. Proc. Natl.Acad. Sci. U.S.A. 102, 2567–2572 (2005). doi: 10.1073/pnas.0409727102; pmid: 15701695

109. J. Li et al., An integrated catalog of reference genes in thehuman gut microbiome. Nat. Biotechnol. 32, 834–841 (2014).doi: 10.1038/nbt.2942; pmid: 24997786

110. A. Das, M. Srinivasan, T. S. Ghosh, S. S. Mande,Xenobiotic metabolism and gut microbiomes. PLOS ONE11, e0163099 (2016). doi: 10.1371/journal.pone.0163099;pmid: 27695034

111. W. Withering, An Account of the Foxglove (printed byM. Swinney for G. G. J. and J. Robinson, London,Birmingham, 1785).

112. J. Lindenbaum, D. G. Rund, V. P. Butler Jr., D. Tse-Eng,J. R. Saha, Inactivation of digoxin by the gut flora:Reversal by antibiotic therapy. N. Engl. J. Med. 305,789–794 (1981). doi: 10.1056/NEJM198110013051403;pmid: 7266632

113. J. R. Saha, V. P. Butler Jr., H. C. Neu, J. Lindenbaum,Digoxin-inactivating bacteria: Identification in human gutflora. Science 220, 325–327 (1983). doi: 10.1126/science.6836275; pmid: 6836275

114. S. Craciun, E. P. Balskus, Microbial conversion of choline totrimethylamine requires a glycyl radical enzyme. Proc. Natl.Acad. Sci. U.S.A. 109, 21307–21312 (2012). doi: 10.1073/pnas.1215689109; pmid: 23151509

115. S. Craciun, J. A. Marks, E. P. Balskus, Characterization ofcholine trimethylamine-lyase expands the chemistry of glycylradical enzymes. ACS Chem. Biol. 9, 1408–1413 (2014).doi: 10.1021/cb500113p; pmid: 24854437

116. K. A. Romano, E. I. Vivas, D. Amador-Noguez, F. E. Rey,Intestinal microbiota composition modulates cholinebioavailability from diet and accumulation of theproatherogenic metabolite trimethylamine-N-oxide.mBio 6, e02481-14 (2015). doi: 10.1128/mBio.02481-14;pmid: 25784704

117. Z. Wang et al., Non-lethal inhibition of gut microbialtrimethylamine production for the treatment ofatherosclerosis. Cell 163, 1585–1595 (2015). doi: 10.1016/j.cell.2015.11.055; pmid: 26687352

118. The Human Microbiome Jumpstart Reference StrainsConsortium, A catalog of reference genomes from the humanmicrobiome. Science 328, 994–999 (2010). doi: 10.1126/science.1183605; pmid: 20489017

119. D. Berry et al., Host-compound foraging by intestinalmicrobiota revealed by single-cell stable isotopeprobing. Proc. Natl. Acad. Sci. U.S.A. 110, 4720–4725(2013). doi: 10.1073/pnas.1219247110; pmid: 23487774

120. R. S. Lasken, Genomic sequencing of unculturedmicroorganisms from single cells. Nat. Rev. Microbiol.10, 631–640 (2012). doi: 10.1038/nrmicro2857;pmid: 22890147

121. L. Tasse et al., Functional metagenomics to mine the humangut microbiome for dietary fiber catabolic enzymes. GenomeRes. 20, 1605–1612 (2010). doi: 10.1101/gr.108332.110;pmid: 20841432

122. M. Li et al., Symbiotic gut microbes modulate humanmetabolic phenotypes. Proc. Natl. Acad. Sci. U.S.A. 105,2117–2122 (2008). doi: 10.1073/pnas.0712038105;pmid: 18252821

123. J. A. Gerlt et al., Enzyme Function Initiative-EnzymeSimilarity Tool (EFI-EST): A web tool for generating proteinsequence similarity networks. Biochim. Biophys. Acta 1854,1019–1037 (2015). doi: 10.1016/j.bbapap.2015.04.015;pmid: 25900361

124. C. A. Kolmeder et al., Comparative metaproteomics anddiversity analysis of human intestinal microbiota testifiesfor its temporal stability and expression of core functions.PLOS ONE 7, e29913 (2012). doi: 10.1371/journal.pone.0029913; pmid: 22279554

125. D. Zeevi et al., Personalized nutrition by prediction ofglycemic responses. Cell 163, 1079–1094 (2015).doi: 10.1016/j.cell.2015.11.001; pmid: 26590418

126. K. S. Kim et al., Associations of organochlorinepesticides and polychlorinated biphenyls in visceral vs.subcutaneous adipose tissue with type 2 diabetesand insulin resistance. Chemosphere 94, 151–157(2014). doi: 10.1016/j.chemosphere.2013.09.066;pmid: 24161582

127. C. S. Karigar, S. S. Rao, Role of microbial enzymes in thebioremediation of pollutants: A review. Enzyme Res. 2011,805187 (2011). doi: 10.4061/2011/805187; pmid: 21912739

ACKNOWLEDGMENTS

Human gut microbiota research in the Balskus laboratory has beensupported by the Smith Family Award for Biomedical Research, theDamon Runyon-Rachleff Innovation Award, a George W. MerckFellowship, a Fellowship for Science and Engineering from theDavid and Lucille Packard Foundation, a Howard Hughes MedicalInstitute–Gates Faculty Scholar Award (OPP1158186), theMassachusetts Institute of Technology Center for MicrobiomeInformatics and Therapeutics, a Sloan Research Fellowship, aBlavatnik Biomedical Accelerator Award, Takeda, the DefenseAdvanced Research Projects Agency (HR0011-16-2-0013), the NIH(CA208834), and the NSF (MCB-1650086). N.K. acknowledges aGraduate Research Fellowship from the NSF (DGE1144152). V.M.R.is funded by an NIH training grant (5T32GM007598). E.P.B. hasreceived honoraria from Procter & Gamble, Cubist (now Merck),Boehringer Ingelheim, New England Biolabs, Eli Lilly, Pfizer,Novartis, and Merck. This work is licensed under a CreativeCommons Attribution 4.0 International (CC BY 4.0) license, whichpermits unrestricted use, distribution, and reproduction in anymedium, provided the original work is properly cited. To view a copyof this license, visit http://creativecommons.org/licenses/by/4.0/.This license does not apply to figures/photos/artwork orother content included in the article that is credited to a thirdparty; obtain authorization from the rights holder before usingsuch material.

10.1126/science.aag2770

Koppel et al., Science 356, eaag2770 (2017) 23 June 2017 11 of 11

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Chemical transformation of xenobiotics by the human gut microbiotaNitzan Koppel, Vayu Maini Rekdal and Emily P. Balskus

DOI: 10.1126/science.aag2770 (6344), eaag2770.356Science 

, this issue p. eaag2770Sciencehuman health and well-being.pharmaceuticals, or be toxic. All of these consequences of our companion microbes can have important impacts on composition of microorganisms in the gut, the subsequent products may have nutritionally beneficial effects, modifymetabolism, its interaction with somatic metabolism, and interindividual variation. Depending on the functional

review the distinguishing features of microbial xenobioticet al.whether food, drugs, or pollutants. Koppel −−we ingest The human gut is packed with actively metabolizing microorganisms. These have a transformative effect on what

One person's meat is another's poison

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REFERENCES

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