Phylogenetic diversity of the carbon monoxide-utilizing prokaryotes and their divergent carbon monoxide metabolisms in the human gut microbiome

Although the production of toxic CO within the human body has been detected, only a few CO-utilizing prokaryotes (CO utilizers) have been reported in the human gut, and their phylogenetic and physiological diversity remains unclear. Here, we unveiled more than thousand representative genomes originating from previously unexplored potential CO-utilizing prokaryotes, which contain CO dehydrogenase (CODH) genes. More than half of CODH-bearing prokaryotes possess genes for the autotrophic Wood–Ljungdahl pathway (WLP). However, 79% of these prokaryotes commonly lack a key gene for WLP, which encodes enzyme that synthesizes formate from CO2 and reductants such as H2, suggesting that they share a degenerated WLP. Instead, many were predicted to possess an alternative way of synthesizing formate from pyruvate, which is a product of glycolysis. In addition to degenerated WLP, seven genes neighboring the CODH gene were found, which may reflect diverse utilization of CO in the human gut. Our findings reveal the unique and diverse nature of CO metabolism in the human gut microbiome, suggesting its potential contribution to CO consumption and gut homeostasis. Impact statement Carbon monoxide (CO)-utilizing prokaryotes mitigate the toxic impact of CO by consuming it as energy and/or carbon sources. In addition to various environments, CO is also produced via multiple routes, such as heme degradation, in the human body and accumulates in the gut. Revealing CO-utilizing prokaryotes and their CO metabolisms in the human gut would contribute to gaining insight into how microbial community functions are involved in maintaining human gut homeostasis. Nevertheless, the limited number of CO utilizers in the human gut microbiome have been reported. In our study, a significant proportion of human gut microbial genomes belonging to diverse phyla were revealed to be of potential CO-utilizing prokaryotes. Additionally, the majority of CO-utilizing prokaryote genomes in the human gut have potentially remodeled the Wood– Ljungdahl pathway (WLP), one of the most well-known autotrophic pathways, to the degenerated, heterotrophic form. Moreover, there were seven other genes neighboring CODH in the human gut CO-utilizers, suggesting various CO utilization. Our findings would pave the way for future explorations into microbial metabolic adaptations and their implications for human health. Data summary The human gut prokaryote genomes were downloaded from HumGut database (Hiseni et al. 2021; https://arken.nmbu.no/~larssn/humgut/). The accession numbers of CODH/ACS-bearing genomes from environments without host-association (Inoue et al., 2022) are listed in Table S1. Metatranscriptomic datasets were downloaded from the NCBI Sequence Read Archive (SRA) under the Bioproject accession numbers PRJNA354235 and PRJNA707065 and their accession IDs are listed in Table S2.


Introduction
Carbon monoxide (CO) is a colorless, odorless, and tasteless gas that is toxic to many organisms (Ernst and Zibrak 1998).CO has a 200-250 times higher affinity for hemoproteins, such as hemoglobin and cytochrome c, than oxygen.Therefore, exposure to >100 ppm of CO inhibits oxygen transport and respiration, resulting in severe effects on human health (Ernst and Zibrak 1998;Alonso et al. 2003;Prockop and Chichkova 2007).Nevertheless, the human body produces 16.4-160 μmol, which correspond to 5-45 ppm, of CO per hour on average, primarily through heme degradation using heme oxygenase-1 (Morse and Choi 2005;Onyiah et al. 2014;Hopper et al. 2020).
From the human body and exogenous sources, CO accumulates in the gastrointestinal tract, potentially influencing the composition of intestinal microbiota (Hopper et al. 2020).
Through reactions catalyzed by the abovementioned CODHs, CO utilizers utilize CO as a carbon source and/or as the low redox potential (E 0 ′= −520 mV) for carbon fixation and/or energy conservation in various CO metabolizing pathways and respiratory chains.For instance, through the reduction and oxidation of the ferredoxin-like protein CooF and flavin adenine dinucleotidedependent NAD(P) oxidoreductase (FNOR), the reducing power of CO is supplied to NAD + , thereby generating NADH, which is available for various respiration and biosynthesis reactions (Tian et al. 2016;Inoue et al. 2019;Slobodkin et al. 2019).The reducing power of CO is also supplied to various terminal electron acceptors, including proton, nitrate, and oxygen, in the electron transport chains to generate an electrochemical gradient for ATP synthesis (Oelgeschläger and Rother 2008;Slobodkin et al. 2019;Adachi et al. 2020).
Among the CO-mediated CO 2 fixation pathways, the Wood-Lijungdahl pathway (WLP; reductive acetyl-CoA pathway) has been best studied.The WLP is recognized as the most ancient autotrophic pathway, which was possessed by the last universal common ancestor (LUCA) (Weiss et al. 2016;Adam et al. 2018).This pathway consists of two branches, carbonyl and methyl branches (Ragsdale and Pierce 2008;Adam et al. 2018).In the carbonyl branch, CO 2 is reduced to CO by Ni-CODH and incorporated into the carbonyl moiety of acetyl-CoA.Alternatively, CO can be obtained directly from the surrounding environment and incorporated into the carbonyl branch (Ragsdale 2008).In the methyl branch, formate is synthesized from CO 2 and H 2 is synthesized by formate dehydrogenase (Fdh), which are then reduced stepwise to a methyl moiety (CH 3 -) by formyltetrahydrofolate synthase (Fhs), methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD), and methylene-tetrahydrofolate reductase (MetF).The generated carbonyl and methyl moieties are incorporated into acetyl-CoA along with coenzyme A (CoA) by acetyl-CoA synthase (ACS), which forms a complex with Ni-CODH (CODH/ACS).Acetyl-CoA is then converted into various compounds such as short-chain fatty acids (SCFA), including acetate, by CO utilizers (Miller and Wolin 1996;Trischler et al. 2022).In many of these acetogenic CO utilizers, WLP is linked to energy conservation using Rnf complexes and/or energy-converting hydrogenase (Ech), where the oxidation of reduced ferredoxin is coupled with the reduction of NAD + and protons, respectively (Diender et al. 2015;Schoelmerich and Müller 2019).
Although the CO-utilizing abilities of human gut prokaryotes have not yet been fully elucidated, accumulating evidence suggests that CO utilizers are present in the human gut microbiome.Recently, two human gut bacteria, Blautia luti and B. wexlerae, were found to consume CO via the WLP (Trischler et al. 2022).Furthermore, several gastrointestinal prokaryotes such as Clostridioides and Marvinbryantia possess genes for WLP (Wolin et al. 2003;Yao et al. 2023).
However, the diversity of CO utilizers and their metabolic pathways in the human gut remain poorly understood.
With the advancement of sequencing technologies and bioinformatic tools, extensive databases of human gut prokaryotic genomes have been established.Almeida et al. (2021) created a Unified Human Gastrointestinal Genome (UHGG) collection comprising 204,938 non-redundant genomes.Hiseni et al. (2021) screened more than 5,700 healthy human gut metagenomes and constructed HumGut, a genome collection containing over 381,000 genomes.Analyzing these databases would deepen our understanding of human intestinal prokaryotes, including CO utilizers and uncultured prokaryotes, which are dominant in the human gut (Almeida et al. 2019(Almeida et al. , 2021;;Omae et al. 2019).In this study, we used the HumGut database to investigate CO utilizers and their CO-utilizing pathways in the human gut microbiome.

Detection of cooS and coxL in human gut prokaryote genomes
CODH-bearing genomes were searched from 30,691 representative human gut prokaryotic genomes, using cooS and coxL as genetic markers for Ni-and Mo-CODHs, respectively.The homology search was performed using NCBI blast+ 2.13.0 with cutoffs of an E-value of 10 −10 , a sequence length of 200 aa, and a sequence identity of 30%, using the following amino acid Thermosinus carboxydivorans mini CooS (WP_007288589.1),respectively (Inoue et al., 2019(Inoue et al., , 2022;;Techtmann et al., 2012).The Ni-CODH clades of the detected CooS were determined by their amino acid sequence identity with representative CooS sequences.The detected CooS/CoxL sequences were aligned using MAFFT v7.487 with the E-INS-i application (Katoh and Standley 2013).To examine the motifs of CooS, previously used criteria (Inoue et al. 2019) were adopted: no amino acid substitutions in two C-clusters comprising Ni, Fe, and S; a B-cluster comprising cubane-type 4Fe-4S; and a D-cluster comprising an additional 4Fe-4S at the subunit interface (Dobbek et al., 2001;Doukov et al. 2002;Inoue et al. 2019).A similar analysis was performed for CoxL with slight modifications.A homology search was performed using Oligotropha carboxidovorans CoxL (WP_013913730.1) as the query.The sequences that conserved the Form I CoxL active site (AYRCSFR) (King 2003) alone were selected.The detected Ni-CODHs are listed in Table S1.All genomes containing Ni-CODHs are listed in Table S2.

Phylogenetic tree construction
The taxonomic assignment of 30,691 prokaryotic genomes was performed using GTDB-tk v2.1.1,with the reference data version R207 (Chaumeil et al. 2019).The names of the phyla were corrected according to the method described by Oren and Garrity (2021).Genome distances among the cooS-containing genomes were calculated using Mashtree v1.2.0 with 1,000 replicates and a minimum depth value of 0 (Ondov et al. 2016;Katz et al. 2019).The obtained tree was then visualized using the R package, ggtree v3.2.1 (Yu et al. 2017).Of the 3,534 human gut metagenome samples (Hiseni et al. 2021), the number of metagenome samples, from which bacterial genomes with sequence identity of 95% or higher to a particular genome were detected, was displayed as a heatmap on the outer layer of the phylogenetic tree.

Genome-based prediction of metabolic functions
To characterize the metabolic functions of the CO utilizer in the human gut, three groups of genomes were analyzed using METABOLIC v4.0 (Zhou et al. 2022).The first group comprised the CODH-bearing genomes detected in the HumGut database.The second group comprised the non-CODH-bearing genomes of human gut prokaryotes belonging to a family or order with CODHbearing genomes, that were detected in the HumGut database.The third group comprised the CODH/ACS-bearing bacteria found in environments other than the intestine.The proportions of genomes encoding functions in each genus were calculated for each group.From the HumGut database, we selected Ni-CODH-lacking bacterial genomes belonging to orders or families containing Ni-CODH-bearing bacteria, resulting in 194 genomes (the second group).We also retrieved the Ni-CODH/ACS-possessing genomes of bacteria isolated from environments other than the human gut, including soil, sludge, hot springs, reactors, lakes, and the deep sea, from the NCBI reference sequence (RefSeq) database, resulting in 37 genomes (the third group).The RefSeq ID and isolation sites of the host bacteria are listed in Table S3.

Genomic-based predictions of the degenerated WLP
In the present study, the gene composition of the CODH/ACS-bearing genomes was analyzed.
All the KOs annotated to bacterial genomes in the human gut and other environments were listed, and the presence of each KO was checked in each genome to calculate the proportion of genomes carrying a given gene in each genome group.When two or more KOs were annotated to a single gene, all annotated KOs were considered for calculating the proportion.Since K00656 (PflD) contains members of PFL-like proteins without PFL activity, proteins annotated as K00656 were aligned using MAFFT v7.487 with the E-INS-i application, and only the sequences that conserved the Cys-Cys active sites (Sawers and Watson 1998) were regarded as potential PFL-coding sequences.The proportions of the genomes carrying the given genes are listed in Table S6.
To and mini CODH (1) (Fig. 1c).Although a similar survey was conducted for Mo-CODH using CoxL as a marker, Mo-CODH, which possesses a conserved active site for form I CoxL, was not found in any human gut genome.
To assess the prevalence of potential CO utilizers in humans, we checked for the presence of CODH-bearing genomes among the available 3,534 datasets of healthy human gut metagenomes (Hiseni et al., 2021) (Fig. 1e).The CODH-bearing genomes derived from the Lachnospirales genera Fusicatenibacter and Blautia were present in 48% and 26% of the metagenome datasets, respectively.
The Veillonelalles genera Veillonella was detected in 8.3% of the metagenome datasets (Fig. 1e, Table S2).The Oscillospirales genera Faecousia and CAG-170 were detected in 13% and 18% of the metagenome datasets, respectively.The above CODH-bearing genomes were appeared more prevalent than or as prevalent as the two prominent CODH-lacking Bacteroidales genera, Phocaeicola and Bacteroides, that were detected in several human gut microbiomes (Hiseni et al. 2021), as Phocaeicola and Bacteroides were detected in the 18% and 31% metagenome datasets on average, respectively.In contrast, some CODH-bearing genomes were present in only a limited number of the human gut metagenome datasets (Fig. 1e, Table S2).For instance, the CODH-bearing genomes from the genus Eubacterium in the order Eubacteriales were found in only 0.2% of metagenomes on average.and F and mini CODH from the inner layer, respectively.The outermost layer shows the prevalence of each genome reported in the previous study (Hiseni et al. 2021).The number indicates the metagenome samples where each genome was analyzed in 3,534 metagenome datasets.

Majority of CODH-bearing bacteria were acetate-producing prokaryotes in the human gut
As most of the potential CO utilizers detected in this study have been uncultured and thereby uncharacterized, we reconstructed the genome-based metabolic functions of the potential CO utilizers bearing Ni-CODH genes in the human gut microbiome using METABOLIC v4.0 (Zhou et al. 2022).Twenty-three metabolic functions were detected in the potential human gut CO utilizers, and the acetate production function was conserved among them (Fig. 2).In particular, the analysis revealed that 1,162/1,302 Ni-CODH-bearing human gut genomes, which correspond to 73/82 genera, contained genes for acetate production (acdA, ack, and pta) for fermentation, suggesting that most potential CO utilizers produce acetate in the human gut (Fig. 2).Exceptions were the Selenomonadales genera (Centipeda, Mitsuokella, and Selenomonas), whose all 27 genomes lacked the abovementioned genes for acetate production.
To evaluate whether these functions were specific to potential CO utilizers, we compared the metabolic functions of 1,302 Ni-CODH-bearing human gut microbial genomes to those of 194 Ni-CODH-lacking genomes that were closely related to the Ni-CODH-bearing ones.All functions other than CO metabolism were observed in both types of genomes; thus, we could not find any specific metabolic functions in the potential CO utilizers (Fig. 2).This suggests that CO metabolism may be engaged in accessory functions that support the existing functions conserved in both Ni-CODHbearing and Ni-CODH-lacking human gut microbiomes.

Eight cooS genomic contexts were identified in the human gut prokaryote genomes
Genes that encode proteins involved in CO metabolism are often located close to cooS in the genome, enabling the prediction of their physiological roles based on the genomic context (Inoue et al. 2019;Matson et al. 2011;Techtmann et al. 2012).To gain insight into the physiological roles of CO metabolism in the human gut microbiome, we analyzed 15 genes located in the upstream and downstream regions of cooS in the 1,302 human gut microbial genomes (Fig. 3, Table S4, S5).
Totally, 1,150 KOs and 1,285 COGs were annotated for genes within the 1,380 cooS contexts in the 1,302 genomes (Tables S4 and S5).Genomic contexts were manually classified into eight types: WLP, PEPCK, FNOR, ABC transporter, Fe-only hydrogenase, MFS transporter, uncharacterized dehydrogenase, and Cysteine synthase.Below, we describe the estimated physiological functions and detailed phylogenetic distributions of genomic context types (Fig. 3).
The WLP type, characterized by the presence of the gene for ACS β subunit (AcsB; K14138, COG1614), was the most prevalent in the human gut microbial genomes, being detected from 593 of the 1,380 cooS contexts.The WLP context was observed in Oscillospirales (8/13 genera, that is, eight CODH-bearing genera within the total genera of the order; 323/411 CODH-bearing genomes, that is, 323 CODH genomes within the total genomes of the order), Lachnospirales (18/38 genera, 198/379 genomes), Peptostreptococcales (4/9 genera, 13/41 genomes), and Eubacterales Eubacterium (3/3 genomes).All genera conserved the consecutive gene arrangement of cooS-cooC-acsB, except for the Peptostreptococcales genera, which had the gene arrangement of cooS-cooC-fhs and acsB at 10 genes downstream of cooS (Tables S4 and S5).acsCDE was found in the cooS contexts of the Lachnospirales and Peptostreptococcus genera but not in seven out of eight Oscillospirales genera (Fig. 3, Table S4, S5).
The second type is PEPCK, which has been unpreceded in cooS genomic contexts.PEPCK (pckA; K01610, COG1866) is a phosphoenolpyruvate carboxykinase that catalyzes the conversion of phosphoenolpyruvate (PEP), CO 2 , and ADP to oxaloacetate (OAA) and ATP, and vice versa.The PEPCK type was found among the genomes of Veillonelales genus Veillonella (189/342 genomes).
Gene composition varied among PEPCK-type genomic contexts.Of the 189 PEPCK-type genomic contexts of Veillonella, 144 included a putative oxyR, which encodes a transcriptional regulator of LysR family (K04761, COG0583) and responds to oxidative stress (Sen and Imlay 2021) in the upstream region adjacent to cooS.Genes for nitrate reduction (narGHIJK) were also observed in 111 Veillonella PEPCK-type genomic contexts, of which 78 genomes also included oxyR (Fig. 3).
The third is FNOR type that has been predicted to be associated with CO utilization in the previous studies (Geelhoed et al. 2016;Inoue et al. 2019Inoue et al. , 2022)).FNOR (COG1251) is often present in the CO utilizers and is involved in energy conservation through CO oxidation associated with the reduction of NAD(P) + (Geelhoed et al. 2016).In human gut microbial genomes, FNOR genes were found in 138 cooS contexts of bacteria, including Lachnospirales (15/38 genera, 92/379 genomes) and Peptostreptococcales (5/9 genera, 18/41 genomes) (Fig. 3).
In 17 of these, pflAB (K04069 and K00656) was found within the genomic context of cooS (Fig. 3).
In addition to the Fe-hydrogenase-type context, COG4624 was also found in the WLP-type context in 27 genomes.
"MFS transporter," "functionally uncharacterized dehydrogenase," and "Cysteine synthase" were relatively minor types, as they occupied fewer than 2% of the CODH-bearing genomes and were observed in specific taxa.The MFS transporter is responsible for importing or exporting a wide range of substrates across the membrane using a substrate concentration gradient.The MFS transporters encoded in this context were annotated as COG2223 (NarK, nitrate transporter) and K08177 (OxlT, oxalate/formate antiporter) in Selenomonadales Mitsuokella (16/20 genomes) (Fig. 3).

Fdh-lacking WLP is common in the human gut microbiome
As the WLP-type genomic context was the most prevalent among the 1,302 potential CO utilizers in the human gut, we further investigated whether human gut prokaryotes bearing the WLP context were capable of acetyl-CoA synthesis through the WLP.Especially, since WLP could be functional even when cooS and acsB are not located in the same genomic context (Gencic and Grahame 2020), we here referred to the 667 genomes bearing both cooS and acsB as CODH/ACSbearing genomes regardless of whether to retain them as the WLP type context.The presence of other WLP genes such as cooCF, fdh, fhs, folD, metF, and acsCDE was surveyed in all the genomes containing genes for CODH/ACS (667 genomes) (Fig. 4).
More than 88% of CODH/ACS-bearing genomes retained almost the full set of WLP genes, including acsCD, cooC, fhs, folD, and metF (Fig. 4, Fig. S1), suggesting that most of the potential human gut CO utilizers bearing CODH/ACS are capable of CO-mediated acetyl-CoA synthesis.
Interestingly, acsE, a bacteria-specific gene for the methyltransferase subunits of ACS, was not detected in 78% of the Oscillospirales genomes (7/9 genera) (Fig. 4), whereas acsE was conserved in other CODH/ACS-bearing genomes.Although a nearly full set of WLP genes was detected, we found that the Fdh genes for formate synthesis (fdhA, fdhF, and fdoG) were not detected in 95%, 96%, and 79% of the CODH/ACS-bearing genomes, respectively (Fig. 4), and none of the Fdh genes were observed in 79% of the genomes.WLP lacking the Fdh gene is not unprecedented, but some bacterial isolates appear to be capable of CO metabolism with it (Trischler et al. 2022).Compared to non-human gut microbial genomes with WLP genes, Fdh genes (fdhA, fdhF, and fdoG) were missing in a smaller proportion of bacteria isolated from other environments (22%, 79%, and 8%, respectively) (Fig. S1), and none of the Fdh genes was observed in 2.7% of the genomes.Thus, the lack of formate synthesis might be a feature prevalent in the human gut potential CO utilizer.
The above analyses indicate that there may be CO 2 /H 2 -independent sources of formate for human gut CODH/ACS-bearing bacteria.To estimate how formate is obtained, we surveyed the genes involved in alternative formate synthesis or import by CODH/ACS-bearing bacteria (Fig. 4).
To gain deeper insights into the functions specifically encoded in the human gut microbial genomes with WLP, we compared all the genes in the genomes of human gut bacteria and other environmental bacteria that contain WLP genes (Fig. 2, Table S6).We observed that genes involved in carbohydrate and saccharide degradation were more prevalent in the human gut microbiome containing WLP genes than in bacteria isolated from other environments.For example, the gene for beta-galactosidase (lacZ) was identified in 69% of the human gut bacterial genomes with WLP genes, whereas it was only present in 8% of the genomes of CO utilizers with WLP isolated from other environments (Fig. 2, Table S6).These results suggest that potential human gut CO utilizers with WLP are capable of utilizing more diverse carbohydrates and/or polysaccharides than the CO utilizers of other environments, producing more pyruvate through glycolysis.For pyruvate metabolism, 53% of the human gut CODH/ACS-bearing genomes possessed pflB.PFOR (por) is also involved in WLP by supplying reduced ferredoxin from pyruvate oxidation (Ragsdale 2004) and is present in 90% of the human gut CODH/ACS-bearing genomes.These genes were less frequently observed in CODH/ACS-bearing bacteria in other environments: por and pflB were present in 73% and 22% of the bacteria in other environments, respectively (Fig. S1).
Not only genes for carbohydrate/saccharide degradation (above), but also the genes for Rnf complex (rnfABCDEG) were detected in a high proportion of WLP-bearing CO utilizers in the human gut (>73%).Group b FeFe-hydrogenase, which is involved in ferredoxin-coupled H 2 production (Benoit et al. 2020), was relatively enriched in human gut WLP-bearing CO utilizers (64%) than in bacteria from other environments (24%) (Fig. S1).
Fig. 4 The presence rates of the WLP genes and other related genes in the human gut genomes.
Genomes possessing cooS and acsB were analyzed to reveal the presence of WLP-related and other genes.The presence rates are displayed for each genus on a heatmap.

Discussion
Given the continuous production of CO through heme degradation in the human body (Hopper et al. 2020), it was predicted that a significant number of CO-utilizing prokaryotes exist in the human gastrointestinal tract, contributing to the consumption of the accumulating harmful CO.Previous studies indeed demonstrated the rapid consumption of CO by fresh human fecal samples (Levine et al. 1982) and by two Blautia strains isolated from human feces (Trischler et al. 2022).However, the mechanism through which taxonomically and phylogenetically diverse human gut prokaryotes become potential CO utilizers in the human gut microbiome remains unclear.In the present study, we analyzed CODH-bearing genomes derived from potential CO utilizers in the human gut microbiome and uncovered various Ni-CODH-bearing genomes belonging to 248 species and 82 genera across 8 phyla (Table S1).To date, only two human gut prokaryotes have been experimentally verified as CO utilizers (Trischler et al. 2022).Therefore, our findings extensively expand the catalogue of potential human gut CO utilizers.
Our findings are not limited to phylogenetic diversity but include the functional diversity of CO utilizers.The previously identified CO-mediated metabolic pathway in human gut-derived prokaryotes is the WLP.However, as their CODH genomic contexts can be divided into eight distinct types presumably involved in distinct physiological roles, the diverse taxa of the human gut microbiome are potentially involved in CO utilization through various pathways.
We observed that WLP is most prevalent in the genomes of human gut microbes, such as those of Oscillospirales, Lachnospirales, and Peptostreptococcales.The WLP observed in this study likely synthesized a carbonyl group in a canonical manner using CO 2 or exogenous CO.However, WLP does not synthesize methyl groups with CO 2 -derived formate because Fdh genes were not detected in almost all of the WLP-bearing human gut microbial genomes (Fig. 5a).Instead, they are potentially capable of obtaining formate through alternative biosynthesis pathways or by import from extracellular fractions, as is evident from the retention of genes for PFL and/or formate transporters, suggesting variations in cellular functions associated with WLP.Especially in PFL-bearing FDHlacking species, considering that PFL synthesizes formate and acetyl-CoA from pyruvate, glycolysis would become one of the primary sources of carbon and reducing power for their Fdh-lacking WLP (Fig. 5a).Because glycolysis requires electron acceptors and WLP requires electron donors, which are often supplied by H 2 via CO 2 reduction, Fdh-deficient WLP may play a role in the electron and carbon sinks of glycolysis in these CO utilizers in the human gut (Fig. 5a).Importantly, many of the potential CO utilizers with such heterotrophic WLP also retain genes to degrade sugars and polysaccharides to greater extent than the CO utilizers of other environments (Table S6).The presence of these genes, coupled with the availability of carbohydrates in the human intestinal tract and Clostridiales (C.bovifaecis and Clostridioides difficile), lack fdh but retain WLP genes (Wolin et al. 2003;Yao et al. 2020Yao et al. , 2023)).This study proposes that the "heterotrophic WLP" lacking FDH are not limited to Blautia, Marvinbryantia, and Clostridium but more prevalently utilized in the human gut CO utilizers.The previous findings in Levine et al. (1982), which observed higher CO consumption activity of feces samples in the presence of glucose (0.7 mL/h, g feces) than in the absence of glucose (0.2 mL/h, g feces) (Levine et al. 1982), might at least partially be caused by the heterotrophic WLP-bearing human gut CO utilizers.In these heterotrophic WLP systems, the dynamics of carbon and electron fluxes may deviate from those observed in the conventional Fdhcontaining WLP system, because of their lack of reliance on CO 2 and H 2 .The coexistence of PFL and PFOR suggests that carbon and electron fluxes are balanced depending on the available carbohydrates and electron donors in the environment, because PFL supplies formate for WLP, whereas PFOR can provide reduced ferredoxin to Rnf and WLP (Fig. 5a).Furthermore, Rnf, FeFe hydrogenase, and the electron transport chain enzymes (such as NADH, quinone oxidoreductase, and succinate dehydrogenase) in B. wexlerae likely contribute to maintain redox equilibrium and facilitate energy conservation in the "heterotrophic WLP"-bearing CO utilizers (Fig. 5a).
A CO metabolic pathway, which has not been previously reported, was predicted in 190 genomes of the genus Veillonella (Fig. 5b).Veillonella mainly harbored clade B CODH, a phylogenetically separate variant from the predominant WLP-associated clade E CODH (Fig. 1).The cooS genome of Veillonella contained genes encoding PEPCK and NarKGHJI (Fig. 2).Because nitrate reduction requires electron donors, CO oxidation by Ni-CODH might work as an electron donor and be involved in energy conservation in Veillonella (Fig. 5b), and a biochemical link between CO oxidation and nitrate reduction was observed in Deferribacter autotrophicus (Slobodkin et al. 2019).PEPCK is involved in the reversible catalytic reactions between PEP, CO 2 , ADP, oxaloacetate, and ATP, although the direction of PEP-to-oxaloacetate is energetically unfavorable.
Nevertheless, the directions of the catalytic reactions by PEPCK vary among species and environmental conditions; in particular, PEPCK is used for oxaloacetate synthesis by fixing CO 2 using capnophilic bacteria (Zelle et al. 2010).If PEPCK in Veillonella also functions in an anaplerotic direction from PEP to oxaloacetate in the human gut environment, CO 2 derived from CO may be fixed with PEP, resulting in oxaloacetate.This catalytic mechanism might also support the viability and growth of Veillonella by supplying ATP.PEPCK and nitrate reduction appear to be functionally linked with lactate metabolism in some species of Veillonella (Inderlied and Delwiche 1973;Ng et al. 1982;Wicaksono et al. 2020).Lactate metabolism likely supplies reductants and substrates for nitrate reduction and CO 2 fixation by PEPCK for oxaloacetate synthesis (Wicaksono et al. 2020).Since our metabolic pathway analyses (Fig. 2) suggested that CO metabolism is involved in facilitating existing cellular functions, Ni-CODH, encoded in the same context as PEPCK and Nar, might support the supply of reductants and CO 2 during lactate metabolism.Thus, biochemical links between CO metabolism and nitrate reduction-and PEPCK-mediated catalytic reactions are plausible.However, as the reconstructed pathways described above are based only on metagenomeassembled genomes, the possibility of them functioning separately, at least in part, cannot be ruled out.
This study revealed the phylogenetic diversity of CO utilizers and their divergent CO-utilizing pathways in the human gut microbiome.Because trace amounts of CO are constantly produced in the human body, CO utilizers may constantly consume CO in the human gut microbiota, avoiding the accumulation of high concentrations of toxic CO in the gut.Therefore, shedding light on CO utilizers would help elucidate the microbial mechanisms underpinning intestinal homeostasis in humans.

Fig. 1
Fig. 1 The CODH-bearing prokaryotic genomes in the human gut microbiome (a) Taxonomic classification of publicly available 30,691 human gut prokaryotic genomes.The genomes were the representatives of more than 0.38 million genomes, clustered with 97.5% nucleotide sequence identity (Hiseni et al., 2021).The numbers in parentheses show those of genomes.(b) The number of genomes that possess 1-3 CODHs.No genome possessed four or more CODHs in the used dataset.(c) The number of genomes that possess a CODH belonging to any of the six clades.(d) Taxonomic classification of the CODH-bearing genomes.(e) Phylogenetic tree of the CODH-bearing genomes.The tip color represents the genus level classifications.The heat maps around the phylogenetic tree exhibits order level taxonomic classifications, CODH clades B, C, D, E,

Fig. 2
Fig. 2 Metabolic function hits detected in the three groups of genomesThe metabolic functions of the three genome groups were analyzed using METABOLIC v4.0.The yaxis represents the genomes grouped by phylogeny (genera and orders) and origin.The first group consists of CODH-bearing genomes detected in the HumGut database (red).The second group comprises non-CODH-bearing genomes, which belong to the same family as the CODH-bearing genomes in the HumGut database (yellow).The third group comprised CO utilizers found in environments other than that of the intestines (blue).The x-axis represents the functions identified in each genome group.The functions were characterized by the presence of genes in METABOLIC.The following list represents the functions and the detected genes associated with each function, which are ordered from left to right in the columns: thermophilic-specific (reverse gyrase), amino acid utilization (4-aminobutyrate aminotransferase and related aminotransferases, aminotransferase class I and II, phosphoserine aminotransferase, ornithine/acetylornithine aminotransferase, branchedchain amino acid aminotransferase/4-amino-4-deoxychorismate lyase, aspartate/tyrosine/aromatic

Fig. 3
Fig. 3 The genomic context of CODH (cooS) in the CODH-bearing genomes in the human gut microbiome The heatmap on the left shows the number of genes detected within the 15 genes upstream and downstream of cooS in the genomes of each genus.Genera with more than nine CODH-bearing genomes were selected (red font), and the gene map around cooS of the genera is shown on the right.Each box represents protein-coding genes and their coded strands.The gray box represents the others and the hypothetical protein.cooS, carbon monoxide dehydrogenase catalytic subunit gene; cooC,

Fig. 5
Fig. 5 Genome-based metabolic reconstructions of the CO-utilizing bacteria in the human gut (a) A schematic of Fdh (formate dehydrogenase)-lacking WLP commonly identified in the human gut bacteria, including genera Blautia and Fusicatenibacter from Bacillota order Lachnospirales.The figure shows an example of metabolic pathways.Pyruvate which is mainly obtained via glycolysis is converted to acetyl-CoA and formate by pflB (pyruvate: formate lysase) (red lines).Formate is then progressively converted to methyl moiety in WLP (orange lines) using formyl-tetrahydrofolate synthase (Fhs), methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD), and methylenetetrahydrofolate reductase (MetF).CO and CH3 are then incorporated to acetyl-CoA with CO dehydrogenase/acetyl-CoA synthase (CODH/ACS).The WLP may contribute to toxic CO utilization, carbon acquisition, sink of electrons, and carbons obtained by glycolysis in the human gut prokaryotes.(b) Prediction of CO utilization in the Bacillota order Veillonellales, Veillonella.In Veillonella, CODH is present in the vicinity of genes for phosphoenolpyruvate carboxykinase (PEPCK) and nitrate reductase (NarGHIJK).The reducing power of CO may be used to reduce nitrate in the respiratory chain (green) and/or for the anaplerotic reaction by PEPCK, converting PEP and CO2 to oxaloacetate (OAA) and ATP (orange lines).The question marks represent the prediction of electron/energy flows obtained from CO. PC, pyruvate carboxylase.OAA, oxaloacetate.G6P, glucose-6-phosphate.