# Gut microbiota role in dietary protein metabolism and health-related outcomes: The two sides of the coin

Kevin J. Portune, Martin Beaumont, Anne-Marie Davila, Daniel Tomé, Francois Blachier, Yolanda Sanz

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## To cite this version:

Kevin J. Portune, Martin Beaumont, Anne-Marie Davila, Daniel Tomé, Francois Blachier, et al.. Gut microbiota role in dietary protein metabolism and health-related outcomes: The two sides of the coin. Trends in Food Science and Technology, 2016, 57 (B, SI), pp.213-232. 10.1016/j.tifs.2016.08.011. hal-01488443

HAL Id: hal-01488443

https://agroparistech.hal.science/hal-01488443

Submitted on 4 Apr 2024

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# Gut microbiota role in dietary protein metabolism and health-related outcomes: The two sides of the coin

Kevin J. Portune, Martin Beaumont, Anne-Marie Davila, Daniel Tomé, Francois Blachier, Yolanda Sanz

► To cite this version:

Kevin J. Portune, Martin Beaumont, Anne-Marie Davila, Daniel Tomé, Francois Blachier, et al.. Gut microbiota role in dietary protein metabolism and health-related outcomes: The two sides of the coin. Trends in Food Science and Technology, 2016, 57 (B, SI), pp.213-232. 10.1016/j.tifs.2016.08.011. hal-01488443

HAL Id: hal-01488443

https://agroparistech.hal.science/hal-01488443v1

Submitted on 4 Apr 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
1 Gut microbiota role in dietary protein metabolism and health-related outcomes:

2 the two sides of the coin

3

4 Kevin J. Portuneª, Martin Beaumontᵇ, Anne-Marie Davilaᵇ, Daniel Toméᵇ, François

5 Blachierᵇ, Yolanda Sanzª

6

7

8

9

10

11

12

13

14 Affiliations

15 ªMicrobial Ecology, Nutrition & Health Research Unit. Institute of Agrochemistry and

16 Food Technology, Spanish National Research Council, Valencia (IATA-CSIC), C/

17 Catedràtic Agustín Escardino Benlloch, 7, 46980, Paterna, Valencia, Spain

18

19 ᵇUMR Nutrition Physiology and Ingestive Behavior, AgroParisTech, INRA, Université

20 Paris-Saclay, 75005, Paris, France

21

22

23 Corresponding author: Kevin J. Portune

24 Email: kportune@iata.csic.es

25 Phone: +34 963 90 00 22
1 Gut microbiota role in dietary protein metabolism and health-related outcomes:

2 the two sides of the coin

3

4 Kevin J. Portuneª, Martin Beaumontᵇ, Anne-Marie Davilaᵇ, Daniel Toméᵇ, François

5 Blachierᵇ, Yolanda Sanzª

6

7

8

9

10

11

12

13

14 Affiliations

15 ªMicrobial Ecology, Nutrition & Health Research Unit. Institute of Agrochemistry and

16 Food Technology, Spanish National Research Council, Valencia (IATA-CSIC), C/

17 Catedràtic Agustín Escardino Benlloch, 7, 46980, Paterna, Valencia, Spain

18

19 ᵇUMR Nutrition Physiology and Ingestive Behavior, AgroParisTech, INRA, Université

20 Paris-Saclay, 75005, Paris, France

21

22

23 Corresponding author: Kevin J. Portune

24 Email: kportune@iata.csic.es

25 Phone: +34 963 90 00 22
26 **Abstract**

27 **Background**

28 Human gut bacteria can synthesize proteinogenic amino acids and produce a range of
29 metabolites via protein fermentation, some known to exert beneficial or harmful
30 physiological effects on the host. However, the effects of the type and amount of dietary
31 protein consumed on these metabolic processes, as well as the effects of the microbiota-
32 derived amino acids and related metabolites on the host health are still predominantly
33 unknown.

34 **Scope and Approach**

35 This review provides an up-to-date description of the dominant pathways/genes
36 involved in amino acid metabolism in gut bacteria, and provides an inventory of
37 metabolic intermediates derived from bacterial protein fermentation that may affect
38 human health. Advances in understanding bacterial protein fermentation pathways and
39 metabolites generated at a global level via the implementation of ‘omics’ technologies
40 are reviewed. Finally, the impact of dietary protein intake and high-protein diets on
41 human health is discussed.

42 **Key Findings and Conclusions**

43 The intestinal microbiota is able to synthesize amino acids, but the net result of
44 amino acid production and utilization, according to dietary patterns still needs to be
45 determined. The amount of ingested dietary protein appears to modify both the diversity
46 and composition of the intestinal microbiota as well as the luminal environment of the
47 intestinal epithelium and peripheral tissues. The understanding of the consequences of
48 such changes on the host physiology and pathophysiology is still in an early stage but
49 major progress is expected in the near future with the investigation of host-microbe
50 omics profiles from well-controlled human intervention studies.
26 **Abstract**

27 **Background**

28 Human gut bacteria can synthesize proteinogenic amino acids and produce a range of
29 metabolites via protein fermentation, some known to exert beneficial or harmful
30 physiological effects on the host. However, the effects of the type and amount of dietary
31 protein consumed on these metabolic processes, as well as the effects of the microbiota-
32 derived amino acids and related metabolites on the host health are still predominantly
33 unknown.

34 **Scope and Approach**

35 This review provides an up-to-date description of the dominant pathways/genes
36 involved in amino acid metabolism in gut bacteria, and provides an inventory of
37 metabolic intermediates derived from bacterial protein fermentation that may affect
38 human health. Advances in understanding bacterial protein fermentation pathways and
39 metabolites generated at a global level via the implementation of ‘omics’ technologies
40 are reviewed. Finally, the impact of dietary protein intake and high-protein diets on
41 human health is discussed.

42 **Key Findings and Conclusions**

43 The intestinal microbiota is able to synthesize amino acids, but the net result of
44 amino acid production and utilization, according to dietary patterns still needs to be
45 determined. The amount of ingested dietary protein appears to modify both the diversity
46 and composition of the intestinal microbiota as well as the luminal environment of the
47 intestinal epithelium and peripheral tissues. The understanding of the consequences of
48 such changes on the host physiology and pathophysiology is still in an early stage but
49 major progress is expected in the near future with the investigation of host-microbe
50 omics profiles from well-controlled human intervention studies.
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53

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**Key words:** Microbiota, microbiome, protein metabolism, high-protein diet

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**Key words:** Microbiota, microbiome, protein metabolism, high-protein diet

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## 1. Introduction

The dietary protein consumption level in humans is vastly different according to food availability and cultural dietary habits (Wu, et al., 2014). Although insufficient protein consumption remains a persistent problem in the developing world, the average daily protein intake in countries from Western Europe and the United States of America is generally higher than the recommended dietary intake of 0.83 g protein•kg⁻¹•day⁻¹ for adults (EFSA Panel on Dietetic Products, 2012; Rand, Pellett, & Young, 2003). In individuals consuming a high-protein (HP) diet as a way to reduce their body weight, the protein consumption generally consists of approximately two to three times the recommended dietary intake; and can even represent five times this latter value (Pesta & Samuel, 2014). Such diets have been shown to increase satiety, modify lipid metabolism, and facilitate short- and medium-term weight reduction (Westerterp-Plantenga, Nieuwenhuizen, Tome, Soenen, & Westerterp, 2009). Although a reduction of body weight in overweight and obese individuals is obviously associated with favorable outcomes in terms of health (Papillard-Marechal, et al., 2012), such dietary modification are also associated with potentially deleterious effects in both healthy situations in the long-term and in some pathological situations, notably in kidney diseases (Juraschek, Appel, Anderson, & Miller, 2013) and in inflammatory bowel diseases (Jowett, et al., 2004).

Besides host physiological factors, recent evidence demonstrates that human gut microbiota in the small and large intestine also plays a role in host dietary protein metabolism. The interplay between host and gut microbial metabolism is complex, with microbes utilizing and even competing for dietary and endogenous proteins.

Fermentation of amino acids by gut bacteria produces metabolites that can affect host protein/amino acid uptake (transport) and metabolism, as well as affect host cell
## 1. Introduction

The dietary protein consumption level in humans is vastly different according to food availability and cultural dietary habits (Wu, et al., 2014). Although insufficient protein consumption remains a persistent problem in the developing world, the average daily protein intake in countries from Western Europe and the United States of America is generally higher than the recommended dietary intake of 0.83 g protein·kg⁻¹·day⁻¹ for adults (EFSA Panel on Dietetic Products, 2012; Rand, Pellett, & Young, 2003). In individuals consuming a high-protein (HP) diet as a way to reduce their body weight, the protein consumption generally consists of approximately two to three times the recommended dietary intake; and can even represent five times this latter value (Pesta & Samuel, 2014). Such diets have been shown to increase satiety, modify lipid metabolism, and facilitate short- and medium-term weight reduction (Westerterp-Plantenga, Nieuwenhuizen, Tome, Soenen, & Westerterp, 2009). Although a reduction of body weight in overweight and obese individuals is obviously associated with favorable outcomes in terms of health (Papillard-Marechal, et al., 2012), such dietary modification are also associated with potentially deleterious effects in both healthy situations in the long-term and in some pathological situations, notably in kidney diseases (Juraschek, Appel, Anderson, & Miller, 2013) and in inflammatory bowel diseases (Jowett, et al., 2004).

Besides host physiological factors, recent evidence demonstrates that human gut microbiota in the small and large intestine also plays a role in host dietary protein metabolism. The interplay between host and gut microbial metabolism is complex, with microbes utilizing and even competing for dietary and endogenous proteins.

Fermentation of amino acids by gut bacteria produces metabolites that can affect host protein/amino acid uptake (transport) and metabolism, as well as affect host cell
101 physiology (Davila, et al., 2013). Bacteria can also synthesize amino acids, which can
102 be provided to the host (Metges, 2000). However, the net result of amino acid synthesis
103 and degradation remains largely to be determined along with the role of the gut
104 microbiota for the management of whole body nitrogen metabolism (Neis, Dejong, &
105 Rensen, 2015). Such knowledge is important since it will yield information regarding
106 the role of the microbiota in the utilization of amino acids from dietary origin in
107 different physiological and pathological situations, as well as the role of the microbiota
108 in the production of metabolites that could be available for the host and impact host
109 metabolism and other physiological functions.

110 Despite the relatively rapid transit of the luminal content in the small intestine, part
111 of the amino acid pool released from proteins through the action of pancreatic enzymes
112 can be used by the host enterocytes (Davila, et al., 2013) as well as by the small
113 intestinal microbiota (Dai, Zhang, Wu, & Zhu, 2010). Protein digestion in the
114 mammalian digestive tract is a very efficient process, being generally equal to or even
115 higher than 90% (Bos, et al., 2005). In the large intestine, where the microbiota
116 concentration is much higher and the transit time is longer than in the small intestine,
117 the remaining protein is broken down to peptides and amino acids via extracellular
118 bacterial proteases and peptidases (Macfarlane, Cummings, & Allison, 1986). In
119 contrast to the small intestine, however, the amino acids generated cannot be absorbed
120 to any significant extent by the large intestine epithelium, except during the neonatal
121 period in mammals (Darragh, Cranwell, & Moughan, 1994). Gut bacterial fermentation
122 of amino acids thus results in an accumulation of various metabolic end-products in the
123 luminal content, some of these metabolites being largely absorbed through the large
124 intestinal epithelial cells, while others are released in feces in large amounts (Davila, et
125 al., 2013). Several bacterial metabolites have also been shown to be active on colonic
101 physiology (Davila, et al., 2013). Bacteria can also synthesize amino acids, which can
102 be provided to the host (Metges, 2000). However, the net result of amino acid synthesis
103 and degradation remains largely to be determined along with the role of the gut
104 microbiota for the management of whole body nitrogen metabolism (Neis, Dejong, &
105 Rensen, 2015). Such knowledge is important since it will yield information regarding
106 the role of the microbiota in the utilization of amino acids from dietary origin in
107 different physiological and pathological situations, as well as the role of the microbiota
108 in the production of metabolites that could be available for the host and impact host
109 metabolism and other physiological functions.

110 Despite the relatively rapid transit of the luminal content in the small intestine, part
111 of the amino acid pool released from proteins through the action of pancreatic enzymes
112 can be used by the host enterocytes (Davila, et al., 2013) as well as by the small
113 intestinal microbiota (Dai, Zhang, Wu, & Zhu, 2010). Protein digestion in the
114 mammalian digestive tract is a very efficient process, being generally equal to or even
115 higher than 90% (Bos, et al., 2005). In the large intestine, where the microbiota
116 concentration is much higher and the transit time is longer than in the small intestine,
117 the remaining protein is broken down to peptides and amino acids via extracellular
118 bacterial proteases and peptidases (Macfarlane, Cummings, & Allison, 1986). In
119 contrast to the small intestine, however, the amino acids generated cannot be absorbed
120 to any significant extent by the large intestine epithelium, except during the neonatal
121 period in mammals (Darragh, Cranwell, & Moughan, 1994). Gut bacterial fermentation
122 of amino acids thus results in an accumulation of various metabolic end-products in the
123 luminal content, some of these metabolites being largely absorbed through the large
124 intestinal epithelial cells, while others are released in feces in large amounts (Davila, et
125 al., 2013). Several bacterial metabolites have also been shown to be active on colonic
epithelial cells, which, as detailed below, depending on their luminal concentrations, can exert beneficial or deleterious effects.

Bacterial metabolites which are not fully metabolized/detoxified by the colonic epithelial cells during their transcellular journey from the intestinal lumen to the bloodstream may reach the liver through the portal vein and then peripheral tissues where they can exert some biological effects, notably on kidney functions.

Investigations into the effects of microbially-derived metabolites on human health and the interaction of the microbiota with the human host have previously been limited due to the complexity of interactions between these two systems. The rapid advance of 'omics' technologies are beginning to expand our understanding of the relationships between the human host and gut microbiota by allowing a global analysis of the flow of host- and microbially-produced metabolites and genes involved in specific biochemical pathways (Qin, et al., 2010; Sridharan, et al., 2014). A thorough characterization and understanding of the bacterial pathways involved in amino acid metabolism and their derivatives is required for precise interpretation and prediction of dietary protein effects on the host health. Currently, a comprehensive review of those bacterial genes and metabolic routes is lacking.

This review merges up-to-date genomic information regarding amino acid-related metabolism in gut bacteria with their potential effects on human health. A description of the dominant pathways for bacterial amino acid biosynthesis as well as for amino acid degradation into metabolites that may play different roles in human health is provided. Moreover, information on the enzymes and homologous genes involved in these pathways as deduced from the KEGG database (Kanehisa, et al., 2014) is given. We then discuss the recent advances in understanding the effects of different dietary strategies (i.e. high-fat diet and HP diet) on the human gut microbiome and its role in
epithelial cells, which, as detailed below, depending on their luminal concentrations, can exert beneficial or deleterious effects.

Bacterial metabolites which are not fully metabolized/detoxified by the colonic epithelial cells during their transcellular journey from the intestinal lumen to the bloodstream may reach the liver through the portal vein and then peripheral tissues where they can exert some biological effects, notably on kidney functions.

Investigations into the effects of microbially-derived metabolites on human health and the interaction of the microbiota with the human host have previously been limited due to the complexity of interactions between these two systems. The rapid advance of 'omics' technologies are beginning to expand our understanding of the relationships between the human host and gut microbiota by allowing a global analysis of the flow of host- and microbially-produced metabolites and genes involved in specific biochemical pathways (Qin, et al., 2010; Sridharan, et al., 2014). A thorough characterization and understanding of the bacterial pathways involved in amino acid metabolism and their derivatives is required for precise interpretation and prediction of dietary protein effects on the host health. Currently, a comprehensive review of those bacterial genes and metabolic routes is lacking.

This review merges up-to-date genomic information regarding amino acid-related metabolism in gut bacteria with their potential effects on human health. A description of the dominant pathways for bacterial amino acid biosynthesis as well as for amino acid degradation into metabolites that may play different roles in human health is provided. Moreover, information on the enzymes and homologous genes involved in these pathways as deduced from the KEGG database (Kanehisa, et al., 2014) is given. We then discuss the recent advances in understanding the effects of different dietary strategies (i.e. high-fat diet and HP diet) on the human gut microbiome and its role in
151 protein/amino acid metabolism based on metagenomic and metabolomic studies.

152 Finally, we analyzed how this metabolic activity, notably in terms of bacterial
153 metabolite production, may be responsible for the effects of dietary protein intake levels
154 on health-related outcomes in both physiological and pathological situations as well as
155 underline research areas that need new developments.

156

157

158 **2. Bacterial synthesis of amino acids**

159 The effects of *de novo* production of amino acids by microbes on whole-body fluxes
160 and human health are still not clearly understood. Bacterial production of amino acids
161 that are accessible to the host may be useful to compensate indispensable amino acid
162 deficiency in low quality protein diets. However, bacterially-produced amino acids
163 could also have detrimental consequences in conditions such as insulin resistance in
164 type 2 diabetes where systemic concentrations of amino acids such as aromatic and
165 branched-chain amino acids are elevated (Neis, et al., 2015). A deeper understanding of
166 the effects of microbially-produced amino acids on host health is warranted, as well as a
167 revision of the biosynthetic pathways of amino acids in bacteria which is provided here.

168

169 **2.1. Pathways/genes involved in de novo biosynthesis of amino acids**

170 Due to the high metabolic cost of synthesizing amino acids, the carbon backbone of
171 all amino acids originates from common metabolic intermediates involved in processes
172 such as the tricarboxylic acid cycle, the pentose phosphate pathway and glycolysis
173 (Berg, Tymoczko, & Stryer, 2002). Among these intermediates, α-ketoglutarate plays a
174 central role in amino acid biosynthesis through its conversion to glutamate, as well as
175 its participation in the biosynthetic pathways of other amino acids. Amino acids can be
151 protein/amino acid metabolism based on metagenomic and metabolomic studies.

152 Finally, we analyzed how this metabolic activity, notably in terms of bacterial
153 metabolite production, may be responsible for the effects of dietary protein intake levels
154 on health-related outcomes in both physiological and pathological situations as well as
155 underline research areas that need new developments.

156

157

158 **2. Bacterial synthesis of amino acids**

159 The effects of *de novo* production of amino acids by microbes on whole-body fluxes
160 and human health are still not clearly understood. Bacterial production of amino acids
161 that are accessible to the host may be useful to compensate indispensable amino acid
162 deficiency in low quality protein diets. However, bacterially-produced amino acids
163 could also have detrimental consequences in conditions such as insulin resistance in
164 type 2 diabetes where systemic concentrations of amino acids such as aromatic and
165 branched-chain amino acids are elevated (Neis, et al., 2015). A deeper understanding of
166 the effects of microbially-produced amino acids on host health is warranted, as well as a
167 revision of the biosynthetic pathways of amino acids in bacteria which is provided here.

168

169 **2.1. Pathways/genes involved in de novo biosynthesis of amino acids**

170 Due to the high metabolic cost of synthesizing amino acids, the carbon backbone of
171 all amino acids originates from common metabolic intermediates involved in processes
172 such as the tricarboxylic acid cycle, the pentose phosphate pathway and glycolysis
173 (Berg, Tymoczko, & Stryer, 2002). Among these intermediates, α-ketoglutarate plays a
174 central role in amino acid biosynthesis through its conversion to glutamate, as well as
175 its participation in the biosynthetic pathways of other amino acids. Amino acids can be
grouped into families according to common starting products or use of common
enzymes for synthesis. These consist of the following families: glutamate, serine,
aspartate, pyruvate, and aromatic amino acid families, as well as several unique
pathways for individual amino acids (Umbarger, 1978). It is important to note that an
overwhelming amount of the literature on bacterial metabolism has historically been
focused on a few bacterial taxa, namely *Escherichia coli* and *Salmonella typhimurium*,
and to a lesser extent *Bacillus subtilis* and recently *Corynebacterium glutamicum*, thus
creating a potential bias towards mechanisms found in these organisms. Although many
of these pathways are conserved across bacterial lineages including those inhabiting the
intestine, diversity is found among different bacterial species at both the species and
strain level. For example, whole genome analysis has revealed that the common gut
bacterium *Clostridium perfringens* lacks numerous amino acid biosynthetic genes for
glutamate, arginine, histidine, lysine, methionine, serine, threonine, aromatic and
branched-chain amino acids (Shimizu, et al., 2002), while other *Clostridium* spp., such
as *Clostridium acetobutylicum*, has a complete set of genes for amino acid biosynthesis
(Nolling, et al., 2001). The gut bacterium *Lactobacillus johnsonii* also appears
incapable of carrying out *de novo* biosynthesis of almost all amino acids due to a lack of
complete biosynthetic pathways, and exhibits an apparent dependence on exogenous
host amino acids/peptides for protein synthesis (Pridmore, et al., 2004). Other animal
and human intestinal bacteria, including *Campylobacter jejuni*, *Helicobacter pylori*,
*Enterococcus faecalis* and *Streptococcus agalactiae* have also lost certain amino acid
biosynthetic pathways (Yu, Walker, Liu, & Zhang, 2009), suggesting a dependence on
availability of exogenous peptides/amino acid sources. Furthermore, the sole presence
of genes within a genome does not confirm their functionality. Genes for the
biosynthesis of all 20 standard amino acids have been identified in the common gut
grouped into families according to common starting products or use of common
enzymes for synthesis. These consist of the following families: glutamate, serine,
aspartate, pyruvate, and aromatic amino acid families, as well as several unique
pathways for individual amino acids (Umbarger, 1978). It is important to note that an
overwhelming amount of the literature on bacterial metabolism has historically been
focused on a few bacterial taxa, namely *Escherichia coli* and *Salmonella typhimurium*,
and to a lesser extent *Bacillus subtilis* and recently *Corynebacterium glutamicum*, thus
creating a potential bias towards mechanisms found in these organisms. Although many
of these pathways are conserved across bacterial lineages including those inhabiting the
intestine, diversity is found among different bacterial species at both the species and
strain level. For example, whole genome analysis has revealed that the common gut
bacterium *Clostridium perfringens* lacks numerous amino acid biosynthetic genes for
glutamate, arginine, histidine, lysine, methionine, serine, threonine, aromatic and
branched-chain amino acids (Shimizu, et al., 2002), while other *Clostridium* spp., such
as *Clostridium acetobutylicum*, has a complete set of genes for amino acid biosynthesis
(Nolling, et al., 2001). The gut bacterium *Lactobacillus johnsonii* also appears
incapable of carrying out *de novo* biosynthesis of almost all amino acids due to a lack of
complete biosynthetic pathways, and exhibits an apparent dependence on exogenous
host amino acids/peptides for protein synthesis (Pridmore, et al., 2004). Other animal
and human intestinal bacteria, including *Campylobacter jejuni*, *Helicobacter pylori*,
*Enterococcus faecalis* and *Streptococcus agalactiae* have also lost certain amino acid
biosynthetic pathways (Yu, Walker, Liu, & Zhang, 2009), suggesting a dependence on
availability of exogenous peptides/amino acid sources. Furthermore, the sole presence
of genes within a genome does not confirm their functionality. Genes for the
biosynthesis of all 20 standard amino acids have been identified in the common gut
201 bacterium *Lactococcus lactis*, although supplemented sources of certain amino acids
202 (isoleucine, valine, leucine, histidine, methionine, and glutamic acid) are also required
203 for growth since genes in these biosynthetic pathways have been demonstrated to be
204 non-functional due to point mutations (Bolotin, et al., 2001; Godon, et al., 1993). This
205 may also be the case with another gut bacterium *Staphylococcus aureus*, as two distinct
206 strains exhibited auxotrophy for different amino acids, despite the presence of complete
207 sets of genes for biosynthetic pathways of these essential amino acids in both strains
208 (Kuroda, et al., 2001). Given these different scenarios, it is likely that alternative
209 intestinal bacterial co-metabolic pathways and regulation strategies remain to be
210 discovered.

211

212 **Glutamate Family (glutamate, glutamine, proline, arginine)**

213 Glutamate and glutamine are both key nitrogen/amino group donors for amino acid
214 synthesis and provide the major entry points of ammonia into bacterial metabolism (H.
215 Shimizu & Hirasawa, 2007). These enzyme pathways are particularly important for
216 some gut bacteria with a reliance on ammonia as a nitrogen source, such as many
217 *Bacteroides* spp., which cannot replace ammonia with other nitrogen sources such as
218 amino acids, peptides, urea or nitrate (reviewed in Fischbach & Sonnenburg, 2011).
219 Glutamate is one of the most important central metabolites in all bacteria providing a
220 link between carbon and nitrogen metabolism. Most enteric bacteria have two primary
221 pathways for the synthesis of glutamate (Fig. 1a) involving either the enzyme glutamate
222 dehydrogenase (GDH) or glutamine oxoglutarate aminotransferase (also called GOGAT
223 or glutamate synthase) (Reitzer & Magasanik, 1987; H. Shimizu & Hirasawa, 2007).
224 Preferential use of either pathway depends on both the energy state and intracellular
225 ammonium concentrations in the cells. GDH is preferentially utilized for glutamate
201 bacterium *Lactococcus lactis*, although supplemented sources of certain amino acids
202 (isoleucine, valine, leucine, histidine, methionine, and glutamic acid) are also required
203 for growth since genes in these biosynthetic pathways have been demonstrated to be
204 non-functional due to point mutations (Bolotin, et al., 2001; Godon, et al., 1993). This
205 may also be the case with another gut bacterium *Staphylococcus aureus*, as two distinct
206 strains exhibited auxotrophy for different amino acids, despite the presence of complete
207 sets of genes for biosynthetic pathways of these essential amino acids in both strains
208 (Kuroda, et al., 2001). Given these different scenarios, it is likely that alternative
209 intestinal bacterial co-metabolic pathways and regulation strategies remain to be
210 discovered.

211

212 **Glutamate Family (glutamate, glutamine, proline, arginine)**

213 Glutamate and glutamine are both key nitrogen/amino group donors for amino acid
214 synthesis and provide the major entry points of ammonia into bacterial metabolism (H.
215 Shimizu & Hirasawa, 2007). These enzyme pathways are particularly important for
216 some gut bacteria with a reliance on ammonia as a nitrogen source, such as many
217 *Bacteroides* spp., which cannot replace ammonia with other nitrogen sources such as
218 amino acids, peptides, urea or nitrate (reviewed in Fischbach & Sonnenburg, 2011).
219 Glutamate is one of the most important central metabolites in all bacteria providing a
220 link between carbon and nitrogen metabolism. Most enteric bacteria have two primary
221 pathways for the synthesis of glutamate (Fig. 1a) involving either the enzyme glutamate
222 dehydrogenase (GDH) or glutamine oxoglutarate aminotransferase (also called GOGAT
223 or glutamate synthase) (Reitzer & Magasanik, 1987; H. Shimizu & Hirasawa, 2007).
224 Preferential use of either pathway depends on both the energy state and intracellular
225 ammonium concentrations in the cells. GDH is preferentially utilized for glutamate
synthesis when both energy and carbon sources are limited for the cell but ammonium and phosphate are present in excess, while the GOGAT pathway is preferred when the cell is not under energy limitation since this pathway requires the expenditure of ATP (Helling, 1994). However, exceptions to this strategy are seen in the common gut bacterium *Bacteroides fragilis* which contains 2 distinct glutamate dehydrogenase enzymes: a dual cofactor NAD(P)H-dependent (gene = *gdhB*) enzyme whose activity depends on high ammonia concentrations, and a NADH-specific enzyme whose activity depends on high peptide concentrations in which ammonia has no effect on activity (Abrahams & Abratt, 1998; Yamamoto, Saito, & Ishimoto, 1987). In the Gram-positive bacterium *Bacillus subtilis*, glutamate is exclusively synthesized by reductive amination of α-ketoglutarate by the enzyme glutamate synthase encoded by the *gltAB* operon (Belitsky, 2002).

Glutamine is synthesized by the single reaction of glutamine synthetase (GS; EC 6.3.1.2) in which ammonia is added to glutamate through ATP hydrolysis (Fig. 1a).
Three distinct types of glutamine synthetases have been identified in bacteria: GSI is found in eubacteria and archaea including the ruminal *R. albus*, GSII is present only in eukaryotes and several soil bacteria: *Rhizobium*, *Frankia*, and *Streptomyces*, while GSIII has only been found in several unrelated bacteria *Bacteroides fragilis*, *Rhizobium leguminosarum*, and *Butyrivibrio fibrisolvens*, and *R. albus* (Brown, Masuchi, Robb, & Doolittle, 1994; Kim, Henriksen, Cann, & Mackie, 2014).

Several pathways exist for bacterial proline biosynthesis, however the most common pathway found in many groups involves a four-step process starting with the phosphorylation of glutamate (Fig. 1a) (reviewed in (Fichman, et al., 2014). Genomic analysis of proline biosynthesis has revealed that many eubacterial and archael species contain only one recognizable gene from this pathway (i.e. Δ1-pyrroline-5-carboxylate
synthesis when both energy and carbon sources are limited for the cell but ammonium and phosphate are present in excess, while the GOGAT pathway is preferred when the cell is not under energy limitation since this pathway requires the expenditure of ATP (Helling, 1994). However, exceptions to this strategy are seen in the common gut bacterium *Bacteroides fragilis* which contains 2 distinct glutamate dehydrogenase enzymes: a dual cofactor NAD(P)H-dependent (gene = *gdhB*) enzyme whose activity depends on high ammonia concentrations, and a NADH-specific enzyme whose activity depends on high peptide concentrations in which ammonia has no effect on activity (Abrahams & Abratt, 1998; Yamamoto, Saito, & Ishimoto, 1987). In the Gram-positive bacterium *Bacillus subtilis*, glutamate is exclusively synthesized by reductive amination of α-ketoglutarate by the enzyme glutamate synthase encoded by the *gltAB* operon (Belitsky, 2002).

Glutamine is synthesized by the single reaction of glutamine synthetase (GS; EC 6.3.1.2) in which ammonia is added to glutamate through ATP hydrolysis (Fig. 1a). Three distinct types of glutamine synthetases have been identified in bacteria: GSI is found in eubacteria and archaea including the ruminal *R. albus*, GSII is present only in eukaryotes and several soil bacteria: *Rhizobium*, *Frankia*, and *Streptomyces*, while GSIII has only been found in several unrelated bacteria *Bacteroides fragilis*, *Rhizobium leguminosarum*, and *Butyrivibrio fibrisolvens*, and *R. albus* (Brown, Masuchi, Robb, & Doolittle, 1994; Kim, Henriksen, Cann, & Mackie, 2014).

Several pathways exist for bacterial proline biosynthesis, however the most common pathway found in many groups involves a four-step process starting with the phosphorylation of glutamate (Fig. 1a) (reviewed in (Fichman, et al., 2014). Genomic analysis of proline biosynthesis has revealed that many eubacterial and archael species contain only one recognizable gene from this pathway (i.e. Δ1-pyrroline-5-carboxylate
251 reductase) while other species lack all genes from this pathway, suggesting that either
252 these bacteria are dependent on exogenous proline sources for growth or that alternative
253 pathways may exist that may not yet be elucidated (Fichman, et al., 2014).

254 Biosynthesis of arginine involves an eight-step process starting with the acetylation
255 of the glutamate (Fig. 1a). This step occurs through the so-called “classical” pathway
256 initially described for *E. coli* via the enzyme N-acetylglutamate synthase (NAGS) and
257 encoded by the gene *argA* (reviewed in (Xu, Labedan, & Glansdorff, 2007). In many
258 bacterial species, this step can also be achieved by an ornithine *N*-acetyltransferase
259 (OAT) with dual functional activities (E.C. 2.3.1.35 / 2.3.1.1) that can also synthesize
260 acetylglutamate *de novo* from acetyl-CoA and glutamate (Marc, et al., 2000; Xu, et al.,
261 2007).

262

263 **Serine Family (serine, glycine, cysteine)**

264 Serine is an important metabolite and precursor to multiple amino acids in bacteria
265 (glycine, cysteine, tryptophan). It is also a precursor for the metabolism of
266 sphingolipids, folate, methane, sulfur, cyanoamino acid, and pyruvate, and also
267 participates in the biosynthesis of purines and pyrimidines (Stolz, et al., 2007;
268 Umbarger, 1978). Furthermore, L-serine plays a fundamental role in stabilizing blood
269 sugar concentration in the liver (Remesy, Fafournoux, & Demigne, 1983). Synthesis of
270 serine is a three-step process starting with the glycolytic intermediate 3-
271 phosphoglycerate (Fig. 1b) (Peters-Wendisch, et al., 2005). Glycine is generated from
272 serine by the single enzyme serine hydroxymethyltransferase (Fig. 1b) that catalyzes the
273 reversible conversion of serine and tetrahydrofolate to glycine and 5,10-methylene
274 tetrahydrofolate, which is a key intermediate in the biosynthesis of purine, thymidine,
275 choline, glutathione and methionine (Trivedi, et al., 2002). In enterobacteria and
251 reductase) while other species lack all genes from this pathway, suggesting that either
252 these bacteria are dependent on exogenous proline sources for growth or that alternative
253 pathways may exist that may not yet be elucidated (Fichman, et al., 2014).

254 Biosynthesis of arginine involves an eight-step process starting with the acetylation
255 of the glutamate (Fig. 1a). This step occurs through the so-called “classical” pathway
256 initially described for *E. coli* via the enzyme N-acetylglutamate synthase (NAGS) and
257 encoded by the gene *argA* (reviewed in (Xu, Labedan, & Glansdorff, 2007). In many
258 bacterial species, this step can also be achieved by an ornithine *N*-acetyltransferase
259 (OAT) with dual functional activities (E.C. 2.3.1.35 / 2.3.1.1) that can also synthesize
260 acetylglutamate *de novo* from acetyl-CoA and glutamate (Marc, et al., 2000; Xu, et al.,
261 2007).

262

263 **Serine Family (serine, glycine, cysteine)**

264 Serine is an important metabolite and precursor to multiple amino acids in bacteria
265 (glycine, cysteine, tryptophan). It is also a precursor for the metabolism of
266 sphingolipids, folate, methane, sulfur, cyanoamino acid, and pyruvate, and also
267 participates in the biosynthesis of purines and pyrimidines (Stolz, et al., 2007;
268 Umbarger, 1978). Furthermore, L-serine plays a fundamental role in stabilizing blood
269 sugar concentration in the liver (Remesy, Fafournoux, & Demigne, 1983). Synthesis of
270 serine is a three-step process starting with the glycolytic intermediate 3-
271 phosphoglycerate (Fig. 1b) (Peters-Wendisch, et al., 2005). Glycine is generated from
272 serine by the single enzyme serine hydroxymethyltransferase (Fig. 1b) that catalyzes the
273 reversible conversion of serine and tetrahydrofolate to glycine and 5,10-methylene
274 tetrahydrofolate, which is a key intermediate in the biosynthesis of purine, thymidine,
275 choline, glutathione and methionine (Trivedi, et al., 2002). In enterobacteria and
276 *Bacillus subtilis*, the synthesis of cysteine is a two-step reaction (Fig. 1b) that involves
277 the substrates coenzyme A and hydrogen sulfide ($H_2S$) (Kredich, 1996).

278 *Bifidobacterium longum* lacks the genes for the final step with cysteine synthase and
279 may use alternative pathways with several enzymes such as succinylhomoserine and the
280 sulfur-containing compounds $H_2S$ or methanethiol produced by other colonic bacteria
281 (Schell, et al., 2002). Several strains of *Bifidobacterium bifidum* have further displayed
282 cysteine auxotrophy (Ferrario, et al., 2015), indicating a dependence of exogenous
283 sources of this amino acid.

284

285 **Aspartate Family (aspartate, asparagine, lysine, threonine, methionine)**

286 Aspartate is an important precursor for the biosynthesis of numerous amino acids. It
287 is synthesized from the transfer of an amino group from glutamate to oxaloacetate via
288 the enzyme aspartate transaminase (Fig. 2). Asparagine is commonly made by one of
289 two distinct asparagine synthetases in bacteria in which one enzyme utilizes ammonia
290 while the other synthetase carries out a transamination reaction from glutamine to
291 aspartate (Fig. 2) (Min, Pelaschier, Graham, Tumbula-Hansen, & Soll, 2002). Alternate
292 pathways exist in some gut bacteria, such as *B. longum*, which lack both types of
293 asparagine synthetases and likely use an asparaginyl-tRNA-dependent route (Schell, et
294 al., 2002).

295 Two main pathways exist for lysine biosynthesis in bacteria, the diaminopimelic acid
296 (DAP) and aminoadipic acid (AAA) pathways. The DAP pathway is used by most
297 bacteria and some archaea and utilizes aspartate and pyruvate as starting material with
298 meso-2-6-diaminopimelic acid as an intermediate (Fig. 2) (Patte, 1996). Four variations
299 of this pathway have been identified in bacteria: the succinylase (most common) (Fig.
300 2), acetylase, aminotransferase, and dehydrogenase pathways (Liu, White, & Whitman,
276 *Bacillus subtilis*, the synthesis of cysteine is a two-step reaction (Fig. 1b) that involves
277 the substrates coenzyme A and hydrogen sulfide ($H_2S$) (Kredich, 1996).

278 *Bifidobacterium longum* lacks the genes for the final step with cysteine synthase and
279 may use alternative pathways with several enzymes such as succinylhomoserine and the
280 sulfur-containing compounds $H_2S$ or methanethiol produced by other colonic bacteria
281 (Schell, et al., 2002). Several strains of *Bifidobacterium bifidum* have further displayed
282 cysteine auxotrophy (Ferrario, et al., 2015), indicating a dependence of exogenous
283 sources of this amino acid.

284

285 **Aspartate Family (aspartate, asparagine, lysine, threonine, methionine)**

286 Aspartate is an important precursor for the biosynthesis of numerous amino acids. It
287 is synthesized from the transfer of an amino group from glutamate to oxaloacetate via
288 the enzyme aspartate transaminase (Fig. 2). Asparagine is commonly made by one of
289 two distinct asparagine synthetases in bacteria in which one enzyme utilizes ammonia
290 while the other synthetase carries out a transamination reaction from glutamine to
291 aspartate (Fig. 2) (Min, Pelaschier, Graham, Tumbula-Hansen, & Soll, 2002). Alternate
292 pathways exist in some gut bacteria, such as *B. longum*, which lack both types of
293 asparagine synthetases and likely use an asparaginyl-tRNA-dependent route (Schell, et
294 al., 2002).

295 Two main pathways exist for lysine biosynthesis in bacteria, the diaminopimelic acid
296 (DAP) and aminoadipic acid (AAA) pathways. The DAP pathway is used by most
297 bacteria and some archaea and utilizes aspartate and pyruvate as starting material with
298 meso-2-6-diaminopimelic acid as an intermediate (Fig. 2) (Patte, 1996). Four variations
299 of this pathway have been identified in bacteria: the succinylase (most common) (Fig.
300 2), acetylase, aminotransferase, and dehydrogenase pathways (Liu, White, & Whitman,
301 2010). No organism is known to possess both DAP and AAA pathways (Liu, et al.,
302 2010). In *E. coli*, the first step in the DAP pathway employs three distinct aspartate
303 kinase isozymes (ThrA, MetL, and LysC), each specific to one of three different
304 biochemical pathways under regulation from lysine, methionine, and threonine
305 (Vitreschak, Lyubetskaya, Shirshin, Gelfand, & Lyubetsky, 2004).

306 Synthesis of threonine shares the first two steps of the biosynthetic pathway of lysine
307 (Fig. 2). In addition to the conversion of aspartate to 4-phospho-L-aspartate, ThrA and
308 MetL isozymes also contain a homoserine dehydrogenase (Hom) domain that carries
309 out the third step in the biosynthetic pathway in which aspartate 4-semialdehyde is
310 converted to homoserine (Fig. 2) (Vitreschak, et al., 2004). Methionine shares the first
311 three steps of the biosynthetic pathway of threonine (Fig. 2); after which several
312 different pathways are utilized by different bacteria to attach a sulfur group to O-
313 Succinyl-L-homoserine via either L-cysteine (Fig. 2) or incorporate inorganic sulfur
314 with O-acetylhomoserine to form homocysteine (Rodionov, Vitreschak, Mironov, &
315 Gelfand, 2004).

316

317 **Pyruvate Family (isoleucine, valine, leucine, alanine)**

318 The branched-chain amino acids (BCAA), valine, leucine and isoleucine share
319 common biosynthetic pathways all stemming from intermediates from pyruvate
320 metabolism. These pathways are so similar that many of the same enzymes are shared
321 for biosynthesis of all these amino acids (Fig. 3). In *E. coli* and *S. enterica* serovar
322 *Typhimurium*, three different isoenzymes of the enzyme acetolactate synthase (AHAS;
323 EC 2.2.1.6;) are made up of a large and small subunit encoded by the respective genes
324 *ilvIH*, *ilvBN*, and *ilvGM* (Umbarger, 1996). Isoleucine biosynthesis starts with the
325 conversion of threonine to 2-oxobutanoate via the enzyme threonine deaminase and
301 2010). No organism is known to possess both DAP and AAA pathways (Liu, et al.,

302 2010). In *E. coli*, the first step in the DAP pathway employs three distinct aspartate

303 kinase isozymes (ThrA, MetL, and LysC), each specific to one of three different

304 biochemical pathways under regulation from lysine, methionine, and threonine

305 (Vitreschak, Lyubetskaya, Shirshin, Gelfand, & Lyubetsky, 2004).

306 Synthesis of threonine shares the first two steps of the biosynthetic pathway of lysine

307 (Fig. 2). In addition to the conversion of aspartate to 4-phospho-L-aspartate, ThrA and

308 MetL isozymes also contain a homoserine dehydrogenase (Hom) domain that carries

309 out the third step in the biosynthetic pathway in which aspartate 4-semialdehyde is

310 converted to homoserine (Fig. 2) (Vitreschak, et al., 2004). Methionine shares the first

311 three steps of the biosynthetic pathway of threonine (Fig. 2); after which several

312 different pathways are utilized by different bacteria to attach a sulfur group to O-

313 Succinyl-L-homoserine via either L-cysteine (Fig. 2) or incorporate inorganic sulfur

314 with O-acetylhomoserine to form homocysteine (Rodionov, Vitreschak, Mironov, &

315 Gelfand, 2004).

316

317 **Pyruvate Family (isoleucine, valine, leucine, alanine)**

318 The branched-chain amino acids (BCAA), valine, leucine and isoleucine share

319 common biosynthetic pathways all stemming from intermediates from pyruvate

320 metabolism. These pathways are so similar that many of the same enzymes are shared

321 for biosynthesis of all these amino acids (Fig. 3). In *E. coli* and *S. enterica* serovar

322 *Typhimurium*, three different isoenzymes of the enzyme acetolactate synthase (AHAS;

323 EC 2.2.1.6;) are made up of a large and small subunit encoded by the respective genes

324 *ilvIH*, *ilvBN*, and *ilvGM* (Umbarger, 1996). Isoleucine biosynthesis starts with the

325 conversion of threonine to 2-oxobutanoate via the enzyme threonine deaminase and
326 then uses the same enzymes as those described for valine biosynthesis (Fig. 3). Leucine
327 biosynthesis uses the last intermediate from the valine pathway, 2-oxoisovalerate, to
328 carry out the initial reaction (Fig. 3) (Pátek, 2007). Leucine has also been demonstrated
329 to be biosynthesized via the precursor SCFA isovalerate via carboxylation in the gut
330 bacteria *Bacteroides fragilis* and *Prevotella ruminicola* (previously *Bacteroides ruminicola*) (Allison, Baetz, & Wiegel, 1984). Both of these bacteria also preferentially
331 utilize 2-methylbutyrate as a precursor for isoleucine biosynthesis instead of carrying
332 out *de novo* synthesis starting from glucose (Allison, et al., 1984). Several pathways
333 exist for alanine biosynthesis, with starting precursors such as pyruvate and aspartate
334 among the most common (Fig. 3). L-alanine is mainly synthesized from pyruvate and
335 glutamate via an alanine transaminase (Oikawa, 2007).
336

337

338 Aromatic Family (phenylalanine, tyrosine, tryptophan)

339 Biosynthesis of aromatic amino acids typically follows the shikimate pathway and
340 starts with the condensation of the glycolytic intermediate phosphoenolpyruvate and the
341 pentose phosphate pathway intermediate erythrose 4-phosphate, via the enzyme 3-
342 Deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase (Fig. 4) (Sprenger,
343 2007). In *E. coli*, three distinct isoenzymes (called AroF, AroG and AroH) are involved
344 in this step and each is specifically feed-back inhibited by the terminal end products
345 tyrosine, phenylalanine and tryptophan, respectively (Sprenger, 2007). A single non-
346 homologous DAHP synthase (AroA) is found in the Gram-positive *B. subtilis* (Panina,
347 Vitreschak, Mironov, & Gelfand, 2003). The pathway proceeds for 6 steps to the
348 common intermediate chorismate, which serves as a branchpoint to the individual
349 pathways for phenylalanine, tryptophan and tyrosine (Fig. 4).
326 then uses the same enzymes as those described for valine biosynthesis (Fig. 3). Leucine
327 biosynthesis uses the last intermediate from the valine pathway, 2-oxoisovalerate, to
328 carry out the initial reaction (Fig. 3) (Pátek, 2007). Leucine has also been demonstrated
329 to be biosynthesized via the precursor SCFA isovalerate via carboxylation in the gut
330 bacteria *Bacteroides fragilis* and *Prevotella ruminicola* (previously *Bacteroides ruminicola*) (Allison, Baetz, & Wiegel, 1984). Both of these bacteria also preferentially
331 utilize 2-methylbutyrate as a precursor for isoleucine biosynthesis instead of carrying
332 out *de novo* synthesis starting from glucose (Allison, et al., 1984). Several pathways
333 exist for alanine biosynthesis, with starting precursors such as pyruvate and aspartate
334 among the most common (Fig. 3). L-alanine is mainly synthesized from pyruvate and
335 glutamate via an alanine transaminase (Oikawa, 2007).
336

337

338 Aromatic Family (phenylalanine, tyrosine, tryptophan)

339 Biosynthesis of aromatic amino acids typically follows the shikimate pathway and
340 starts with the condensation of the glycolytic intermediate phosphoenolpyruvate and the
341 pentose phosphate pathway intermediate erythrose 4-phosphate, via the enzyme 3-
342 Deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase (Fig. 4) (Sprenger,
343 2007). In *E. coli*, three distinct isoenzymes (called AroF, AroG and AroH) are involved
344 in this step and each is specifically feed-back inhibited by the terminal end products
345 tyrosine, phenylalanine and tryptophan, respectively (Sprenger, 2007). A single non-
346 homologous DAHP synthase (AroA) is found in the Gram-positive *B. subtilis* (Panina,
347 Vitreschak, Mironov, & Gelfand, 2003). The pathway proceeds for 6 steps to the
348 common intermediate chorismate, which serves as a branchpoint to the individual
349 pathways for phenylalanine, tryptophan and tyrosine (Fig. 4).
Alternative pathways exist for synthesis of each respective aromatic amino acid in different bacterial species, but commonalities within the pathways remain. The first reaction for both phenylalanine and tyrosine uses the enzyme chorismate mutase to convert chorismate to prephenate. In *E. coli*, chorismate mutases are bifunctional enzymes which can participate in the first two steps of both phenylalanine and tyrosine biosynthesis from chorismate (Fig. 4) (Sprenger, 2007). In *E. coli*, both pathways then use either the enzyme aromatic amino acid aminotransferase or aspartate aminotransferase (EC 2.6.1.1) to catalyze the transamination reaction into each respective α-keto acid using glutamate as the amino group donor (Pittard, 1996) (Fig. 4). Aromatic amino acid aminotransferase can also participate in an alternative pathway that catalyzes the conversion of prephenate to L-arogenate which can be further converted to either phenylalanine or tyrosine using prephenate dehydratase or cyclohexadieny/prephenate dehydrogenase, respectively. Instead of *de novo* synthesis of phenylalanine, several gut bacteria (*B. fragilis* and *P. ruminicola*) preferentially use phenylacetate as a precursor for this amino acid (Allison, et al., 1984).

Tryptophan biosynthesis is five-step pathway from chorismate (Fig. 4) in which glutamine or ammonia is used in the first step with anthranilate synthase (Nichols, 1996; Pittard, 1996). In the final step, serine is used as the amino group donor for tryptophan synthase (Sprenger, 2007). The tryptophan biosynthesis pathway is quite conserved in different bacteria, although differences in gene order and the enzymatic reactions carried out by separate or fused enzyme units exist in different bacteria (reviewed in Xie, Keyhani, Bonner, & Jensen, 2003). For example, all of the genes in the biosynthetic pathway for tryptophan have been identified in the gut bacterium *B. longum* except for TrpF (phosphoribosylanthranilate isomerase), indicating a replacement of this gene with an unidentified homolog (Schell, et al., 2002).
Alternative pathways exist for synthesis of each respective aromatic amino acid in different bacterial species, but commonalities within the pathways remain. The first reaction for both phenylalanine and tyrosine uses the enzyme chorismate mutase to convert chorismate to prephenate. In *E. coli*, chorismate mutases are bifunctional enzymes which can participate in the first two steps of both phenylalanine and tyrosine biosynthesis from chorismate (Fig. 4) (Sprenger, 2007). In *E. coli*, both pathways then use either the enzyme aromatic amino acid aminotransferase or aspartate aminotransferase (EC 2.6.1.1) to catalyze the transamination reaction into each respective α-keto acid using glutamate as the amino group donor (Pittard, 1996) (Fig. 4). Aromatic amino acid aminotransferase can also participate in an alternative pathway that catalyzes the conversion of prephenate to L-arogenate which can be further converted to either phenylalanine or tyrosine using prephenate dehydratase or cyclohexadieny/prephenate dehydrogenase, respectively. Instead of *de novo* synthesis of phenylalanine, several gut bacteria (*B. fragilis* and *P. ruminicola*) preferentially use phenylacetate as a precursor for this amino acid (Allison, et al., 1984).

Tryptophan biosynthesis is five-step pathway from chorismate (Fig. 4) in which glutamine or ammonia is used in the first step with anthranilate synthase (Nichols, 1996; Pittard, 1996). In the final step, serine is used as the amino group donor for tryptophan synthase (Sprenger, 2007). The tryptophan biosynthesis pathway is quite conserved in different bacteria, although differences in gene order and the enzymatic reactions carried out by separate or fused enzyme units exist in different bacteria (reviewed in Xie, Keyhani, Bonner, & Jensen, 2003). For example, all of the genes in the biosynthetic pathway for tryptophan have been identified in the gut bacterium *B. longum* except for TrpF (phosphoribosylanthranilate isomerase), indicating a replacement of this gene with an unidentified homolog (Schell, et al., 2002).
375

376 **Histidine**

377 Histidine biosynthesis is a complex ten-step enzymatic pathway (Fig. 5) encoded by
378 eight different genes, three of which (*hisD*, *hisB*, and *hisI*) encode bifunctional enzymes
379 (reviewed in (Alifano, et al., 1996). Most of the work on the histidine biosynthetic
380 pathway has mainly been studied in *E. coli* and *Salmonella typhimurium* and more
381 recently in the industrially important *Corynebacterium glutamicum*, demonstrating large
382 conservation along with some differences in the biochemical pathways between the
383 different species (Kulis-Horn, Persicke, & Kalinowski, 2014). Whole genome studies in
384 bacteria identified in the gut such as *L. lactis* confirm the presence of all the genes of
385 this pathway (Bolotin, et al., 2001).
386

386

387

388 **3. Overview of uptake systems and catabolism of proteins/amino acids in gut**
389 **bacteria**

390

391 **3.1. Protein hydrolysis and peptide/amino acid transporters**

392 Initial steps of bacterial protein catabolism include the extracellular hydrolysis of
393 proteins via different bacterial proteases. According to the MEROPS peptidase database
394 (Rawlings, Waller, Barrett, & Bateman, 2014), bacteria contain a highly diverse number
395 of different proteases, present in many common gut microbiota species such as
396 *Clostridium* spp. *Bacteroides* spp., *Lactobacillus* spp., etc. containing up to hundreds of
397 different identified proteases. Some bacteria, such as lactic acid bacteria, have
398 developed sophisticated proteolytic systems to compensate their reduced capabilities for
399 amino acid biosynthesis (Pessione, 2012). Proteolytic systems in lactic acid bacteria
375

376 **Histidine**

377 Histidine biosynthesis is a complex ten-step enzymatic pathway (Fig. 5) encoded by
378 eight different genes, three of which (*hisD*, *hisB*, and *hisI*) encode bifunctional enzymes
379 (reviewed in (Alifano, et al., 1996). Most of the work on the histidine biosynthetic
380 pathway has mainly been studied in *E. coli* and *Salmonella typhimurium* and more
381 recently in the industrially important *Corynebacterium glutamicum*, demonstrating large
382 conservation along with some differences in the biochemical pathways between the
383 different species (Kulis-Horn, Persicke, & Kalinowski, 2014). Whole genome studies in
384 bacteria identified in the gut such as *L. lactis* confirm the presence of all the genes of
385 this pathway (Bolotin, et al., 2001).
386

386

387

388 **3. Overview of uptake systems and catabolism of proteins/amino acids in gut**
389 **bacteria**

390

391 **3.1. Protein hydrolysis and peptide/amino acid transporters**

392 Initial steps of bacterial protein catabolism include the extracellular hydrolysis of
393 proteins via different bacterial proteases. According to the MEROPS peptidase database
394 (Rawlings, Waller, Barrett, & Bateman, 2014), bacteria contain a highly diverse number
395 of different proteases, present in many common gut microbiota species such as
396 *Clostridium* spp. *Bacteroides* spp., *Lactobacillus* spp., etc. containing up to hundreds of
397 different identified proteases. Some bacteria, such as lactic acid bacteria, have
398 developed sophisticated proteolytic systems to compensate their reduced capabilities for
399 amino acid biosynthesis (Pessione, 2012). Proteolytic systems in lactic acid bacteria
400 consist of either extracellular or membrane-bound proteases (principally PrtP and CEP)
401 that degrade proteins into oligopeptides, followed by their incorporation into the cell via
402 peptide transporters (Opp, Dpp, Dtp, for oligopeptide, dipeptide, and di- and tripeptide,
403 respectively) and finally numerous intracellular peptidases that degrade the peptides
404 into short peptides and amino acids (reviewed in Liu, Bayjanov, Renckens, Nauta, &
405 Siezen, 2010; Steiner, Naider, & Becker, 1995).

406 Amino acids and their derivatives are imported and exported from the bacterial cell
407 via transmembrane proteins comprising ATP-dependent ABC transporters, several
408 families of channel proteins, or secondary carriers relying on proton-motive force,
409 sodium-ion motive force, solute-solute exchange, or uniport (Saier, 2000). Efflux
410 systems for some amino acids such as lysine, arginine, threonine, cysteine, leucine,
411 isoleucine, and valine are well-known in the bacteria *E. coli* and *Clostridium*
412 glutamicum (Eggeling & Sahm, 2003), whereas to date no export systems for histidine
413 have been described in any organism (Kulis-Horn, et al., 2014).

414

3.2. Transamination, deamination and decarboxylation
---

416 Bacteria may directly incorporate available amino acids as substrates for protein
417 biosynthesis or may carry out catabolic reactions to use them as energy sources or to
418 produce other metabolites. Under aerobic conditions, bacteria typically convert
419 proteinogenic α-amino acids to α-oxo acids (aka. α-ketoacids) or saturated fatty acids
420 via transamination or deamination, which are further oxidized as energy sources in the
421 tricarboxylic acid (TCA) cycle. However, in the absence of oxygen or other suitable
422 electron acceptors, only strict or facultative anaerobic bacteria in the gut, such as
423 Clostridia and Fusobacteria, are capable of utilizing amino acids as energy sources, thus
424 fermenting amino acids to short-chain fatty acids (SCFAs), molecular hydrogen (H₂),
400 consist of either extracellular or membrane-bound proteases (principally PrtP and CEP)
401 that degrade proteins into oligopeptides, followed by their incorporation into the cell via
402 peptide transporters (Opp, Dpp, Dtp, for oligopeptide, dipeptide, and di- and tripeptide,
403 respectively) and finally numerous intracellular peptidases that degrade the peptides
404 into short peptides and amino acids (reviewed in Liu, Bayjanov, Renckens, Nauta, &
405 Siezen, 2010; Steiner, Naider, & Becker, 1995).

406 Amino acids and their derivatives are imported and exported from the bacterial cell
407 via transmembrane proteins comprising ATP-dependent ABC transporters, several
408 families of channel proteins, or secondary carriers relying on proton-motive force,
409 sodium-ion motive force, solute-solute exchange, or uniport (Saier, 2000). Efflux
410 systems for some amino acids such as lysine, arginine, threonine, cysteine, leucine,
411 isoleucine, and valine are well-known in the bacteria *E. coli* and *Clostridium*
412 glutamicum (Eggeling & Sahm, 2003), whereas to date no export systems for histidine
413 have been described in any organism (Kulis-Horn, et al., 2014).

414

415 **3.2. Transamination, deamination and decarboxylation**

416 Bacteria may directly incorporate available amino acids as substrates for protein
417 biosynthesis or may carry out catabolic reactions to use them as energy sources or to
418 produce other metabolites. Under aerobic conditions, bacteria typically convert
419 proteinogenic α-amino acids to α-oxo acids (aka. α-ketoacids) or saturated fatty acids
420 via transamination or deamination, which are further oxidized as energy sources in the
421 tricarboxylic acid (TCA) cycle. However, in the absence of oxygen or other suitable
422 electron acceptors, only strict or facultative anaerobic bacteria in the gut, such as
423 Clostridia and Fusobacteria, are capable of utilizing amino acids as energy sources, thus
424 fermenting amino acids to short-chain fatty acids (SCFAs), molecular hydrogen (H₂),
425 carbon dioxide (CO₂), and ammonia, with minor products of H₂S, methylmercaptane,
426 phenols, alcohols and organic acids (Davila, et al., 2013; J. Kim, Hetzel, Boiangiu, &
427 Buckel, 2004). Several mechanisms for α-amino acid degradation exist for anaerobic
428 bacteria, which includes the well-known Stickland reaction found in many proteolytic
429 Clostridia which involves the coupled oxidation and reduction of two respective amino
430 acids to organic acids. Other fermentation pathways found in various Clostridia as well
431 as Fusobacterium spp. and Acidaminococcus spp. involve single amino acids that act as
electron donors as well as acceptors (Fischbach & Sonnenburg, 2011; J. Kim, et al.,
432 2004). The genus Clostridium contains unique amino acid degradation pathways, such
433 as B12-dependent aminomutases, selenium containing oxidoreductases and oxygen-
434 sensitive 2-hydroxyacyl-CoA dehydratases (Fonknechten, et al., 2010).
435

436 Amino acids can also be metabolized through decarboxylation reactions ultimately
437 yielding amines and polyamines as products. Factors such as pH can influence the
438 activity of deaminases and decarboxylases, ultimately affecting the accumulation of
439 specific end products. Furthermore, many complex amino acids can undergo a series of
440 metabolic reactions that produce a large variety of structurally-related metabolic end-
441 products (Davila, et al., 2013).

442 Amino acid utilization may be achieved in a preferential manner, as Fonknechten et
443 al. (2010) demonstrated that *Clostridium sticklandii* preferentially used threonine,
444 arginine and serine for carbon and energy sources, but hardly utilized glutamate,
445 aspartate and aromatic amino acids, even though these catabolic pathways are found in
446 this organism. Furthermore, lysine degradation, which is a process that provides a major
447 energy source, was only observed in stationary growth phases (Fonknechten, et al.,
448 2010).

449
425 carbon dioxide (CO₂), and ammonia, with minor products of H₂S, methylmercaptane,
426 phenols, alcohols and organic acids (Davila, et al., 2013; J. Kim, Hetzel, Boiangiu, &
427 Buckel, 2004). Several mechanisms for α-amino acid degradation exist for anaerobic
428 bacteria, which includes the well-known Stickland reaction found in many proteolytic
429 Clostridia which involves the coupled oxidation and reduction of two respective amino
430 acids to organic acids. Other fermentation pathways found in various Clostridia as well
431 as Fusobacterium spp. and Acidaminococcus spp. involve single amino acids that act as
electron donors as well as acceptors (Fischbach & Sonnenburg, 2011; J. Kim, et al.,
432 2004). The genus Clostridium contains unique amino acid degradation pathways, such
433 as B12-dependent aminomutases, selenium containing oxidoreductases and oxygen-
434 sensitive 2-hydroxyacyl-CoA dehydratases (Fonknechten, et al., 2010).
435

436 Amino acids can also be metabolized through decarboxylation reactions ultimately
437 yielding amines and polyamines as products. Factors such as pH can influence the
438 activity of deaminases and decarboxylases, ultimately affecting the accumulation of
439 specific end products. Furthermore, many complex amino acids can undergo a series of
440 metabolic reactions that produce a large variety of structurally-related metabolic end-
441 products (Davila, et al., 2013).
442

442 Amino acid utilization may be achieved in a preferential manner, as Fonknechten et
443 al. (2010) demonstrated that *Clostridium sticklandii* preferentially used threonine,
444 arginine and serine for carbon and energy sources, but hardly utilized glutamate,
445 aspartate and aromatic amino acids, even though these catabolic pathways are found in
446 this organism. Furthermore, lysine degradation, which is a process that provides a major
447 energy source, was only observed in stationary growth phases (Fonknechten, et al.,
448 2010).
449
### 3.3. Factors influencing protein fermentation:

Many different factors may influence protein fermentation in the gut, such as substrate availability, transit time, pH and osmolarity. The ratio of available carbohydrates:protein determines substrate utilization by the gut microbiota (Smith & Macfarlane, 1996), and in humans, it has been shown that availability of complex carbohydrates lowers protein fermentation (Birkett, Muir, Phillips, Jones, & O'Dea, 1996; De Preter, et al., 2008; Geboes, et al., 2006). When the main energy sources (i.e. fermentable carbohydrates) for microbiota are abundant, nitrogenous substrates can be used for biosynthetic (anabolic) process and bacterial growth. On the contrary, proteins are catabolized by bacteria when energy is scarce, leading to the production of amino acid-derived end products. Due to high carbohydrate fermentation in the proximal colon, there is a progressive decrease of carbohydrate availability in the distal colon, resulting in higher protein fermentation (Macfarlane, Gibson, & Cummings, 1992).
Long transit time and elevated pH are also associated with high levels of protein fermentation (Cummings, Hill, Bone, Branch, & Jenkins, 1979; Macfarlane, Cummings, Macfarlane, & Gibson, 1989). Therefore, dietary carbohydrate and protein ratios may strongly influence the metabolic pathways activated in the large intestine and flow of metabolites generated.

### 4. Key intermediate products from bacterial protein/amino acid catabolism and effects on host physiology/health

Fermentation of amino acids derived from endogenous luminal and dietary protein by intestinal microbiota produces numerous metabolites with suspected or established
### 3.3. Factors influencing protein fermentation:

Many different factors may influence protein fermentation in the gut, such as substrate availability, transit time, pH and osmolarity. The ratio of available carbohydrates:protein determines substrate utilization by the gut microbiota (Smith & Macfarlane, 1996), and in humans, it has been shown that availability of complex carbohydrates lowers protein fermentation (Birkett, Muir, Phillips, Jones, & O'Dea, 1996; De Preter, et al., 2008; Geboes, et al., 2006). When the main energy sources (i.e. fermentable carbohydrates) for microbiota are abundant, nitrogenous substrates can be used for biosynthetic (anabolic) process and bacterial growth. On the contrary, proteins are catabolized by bacteria when energy is scarce, leading to the production of amino acid-derived end products. Due to high carbohydrate fermentation in the proximal colon, there is a progressive decrease of carbohydrate availability in the distal colon, resulting in higher protein fermentation (Macfarlane, Gibson, & Cummings, 1992).
Long transit time and elevated pH are also associated with high levels of protein fermentation (Cummings, Hill, Bone, Branch, & Jenkins, 1979; Macfarlane, Cummings, Macfarlane, & Gibson, 1989). Therefore, dietary carbohydrate and protein ratios may strongly influence the metabolic pathways activated in the large intestine and flow of metabolites generated.

### 4. Key intermediate products from bacterial protein/amino acid catabolism and effects on host physiology/health

Fermentation of amino acids derived from endogenous luminal and dietary protein by intestinal microbiota produces numerous metabolites with suspected or established
effects on host intestinal physiology, liver and peripheral tissues. Relevant examples of such metabolites are described below.

476

477 **4.1. Microbially-produced compounds with neuroactive properties**

Recent evidence has shown that bacteria isolated from the mammalian gut have the capacity to synthesize neuroactive compounds including neurotransmitters, many of which result from the catabolism of amino acids. These compounds include GABA (produced by *Lactobacillus* spp., *Bifidobacterium* spp., and *Lactococcus lactis*); norepinephrine (produced by *Escherichia* spp. and *Bacillus* spp.); dopamine (produced by *Bacillus* spp.); histamine (produced by numerous bacterial genera); and serotonin (produced by *Streptococcus* spp., *Escherichia* spp. and *Enterococcus* spp.) (Sanders, et al., 1998; Wall, Ross, & Stanton, 2014). In addition, bacteria have been demonstrated to produce other neuroactive compounds from amino acid degradation such as nitric oxide and the biogenic amines tryptamine and phenethylamine. It is worth noting that for most of these bacterial metabolites, the understanding of their precise effects on the intestine and peripheral tissues remains in its infancy.

γ-aminobutyrate (GABA), known as an inhibitory neurotransmitter, is microbially-produced by the decarboxylation of glutamate via the enzyme glutamate decarboxylase (Table 1). This enzyme makes up part of the glutamate decarboxylase (GAD) system found in several bacterial genera, which is implicated in acid tolerance by maintaining intracellular pH homeostasis through proton consumption (Feehily & Karatzas, 2013). Other factors in addition to acidic stress have been demonstrated to activate the GAD system, including sodium, polyamines and hypoxia (Feehily & Karatzas, 2013). GABA is subsequently exported from the cell via antiporters that import glutamate, or it remains in the cells and is metabolized to succinate via the GABA shunt pathway
effects on host intestinal physiology, liver and peripheral tissues. Relevant examples of such metabolites are described below.

476

477 **4.1. Microbially-produced compounds with neuroactive properties**

Recent evidence has shown that bacteria isolated from the mammalian gut have the capacity to synthesize neuroactive compounds including neurotransmitters, many of which result from the catabolism of amino acids. These compounds include GABA (produced by *Lactobacillus* spp., *Bifidobacterium* spp., and *Lactococcus lactis*); norepinephrine (produced by *Escherichia* spp. and *Bacillus* spp.); dopamine (produced by *Bacillus* spp.); histamine (produced by numerous bacterial genera); and serotonin (produced by *Streptococcus* spp., *Escherichia* spp. and *Enterococcus* spp.) (Sanders, et al., 1998; Wall, Ross, & Stanton, 2014). In addition, bacteria have been demonstrated to produce other neuroactive compounds from amino acid degradation such as nitric oxide and the biogenic amines tryptamine and phenethylamine. It is worth noting that for most of these bacterial metabolites, the understanding of their precise effects on the intestine and peripheral tissues remains in its infancy.

γ-aminobutyrate (GABA), known as an inhibitory neurotransmitter, is microbially-produced by the decarboxylation of glutamate via the enzyme glutamate decarboxylase (Table 1). This enzyme makes up part of the glutamate decarboxylase (GAD) system found in several bacterial genera, which is implicated in acid tolerance by maintaining intracellular pH homeostasis through proton consumption (Feehily & Karatzas, 2013). Other factors in addition to acidic stress have been demonstrated to activate the GAD system, including sodium, polyamines and hypoxia (Feehily & Karatzas, 2013). GABA is subsequently exported from the cell via antiporters that import glutamate, or it remains in the cells and is metabolized to succinate via the GABA shunt pathway
499 (Feehily & Karatzas, 2013; Karatzas, Brennan, Heavin, Morrissey, & O'Byrne, 2010).

500 Glutamate/GABA antiporters are found in numerous Gram-negative (Escherichia, Shigella, Brucella) and Gram-positive genera (Listeria, Lactobacillus, Lactococcus, Clostridium, Bifidobacterium) (Feehily & Karatzas, 2013). An in vivo study in mice 501 also revealed that chronic administration of Lactobacillus rhamnosus (JB-1) induced 502 changes in mRNA of GABA receptors B1b and A2, as well as reduced anxiety- and 503 depression-related behavior (Bravo, et al., 2011), although the direct production of 504 GABA by gut bacteria was not investigated. In vivo studies are required to evaluate 505 whether gut microbiota derived GABA is active on the host. 506

507
508 Serotonin, a neurotransmitter involved in numerous processes including behavior,
509 learning, appetite and glucose homeostasis, is produced in the human brain but also
510 notably in intestinal enteroendocrine cells (El-Merahbi, Loffler, Mayer, & Sumara,
511 2015). The key role of serotonin in the gut-brain axis has been extensively reviewed,
512 indicating that peripherally it is involved in modulation of the gut immune system,
513 gastrointestinal secretions, motility and visceral sensitivity and centrally in mood and
514 cognition (O'Mahony, Clarke, Borre, Dinan, & Cryan, 2015). In humans, serotonin is
515 produced from tryptophan degradation in a two-step process via the enzymes tryptophan
516 5-monooxygenase (tryptophan hyroxylase 1) and aromatic-L-amino-acid decarboxylase
517 (Table 1). It has been experimentally demonstrated in vitro that serotonin can also be
518 produced by several bacterial genera isolated from the human gut, including
519 Streptococcus, Escherichia, Enterococcus, Lactococcus and Lactobacillus (Wall, et al.,
520 2014). However, the full mechanism of direct serotonin production by bacteria has not
521 been clearly established. Analysis of the serotonin biosynthetic pathway in bacteria via
522 the KEGG database reveals that several bacterial genera contain homologous genes for
523 encoding aromatic-L-amino-acid decarboxylase used in the second step of this pathway
499 (Feehily & Karatzas, 2013; Karatzas, Brennan, Heavin, Morrissey, & O'Byrne, 2010).

500 Glutamate/GABA antiporters are found in numerous Gram-negative (Escherichia, Shigella, Brucella) and Gram-positive genera (Listeria, Lactobacillus, Lactococcus, Clostridium, Bifidobacterium) (Feehily & Karatzas, 2013). An in vivo study in mice 501 also revealed that chronic administration of Lactobacillus rhamnosus (JB-1) induced 502 changes in mRNA of GABA receptors B1b and A2, as well as reduced anxiety- and 503 depression-related behavior (Bravo, et al., 2011), although the direct production of 504 GABA by gut bacteria was not investigated. In vivo studies are required to evaluate 505 whether gut microbiota derived GABA is active on the host. 506

507
508 Serotonin, a neurotransmitter involved in numerous processes including behavior,
509 learning, appetite and glucose homeostasis, is produced in the human brain but also
510 notably in intestinal enteroendocrine cells (El-Merahbi, Loffler, Mayer, & Sumara,
511 2015). The key role of serotonin in the gut-brain axis has been extensively reviewed,
512 indicating that peripherally it is involved in modulation of the gut immune system,
513 gastrointestinal secretions, motility and visceral sensitivity and centrally in mood and
514 cognition (O'Mahony, Clarke, Borre, Dinan, & Cryan, 2015). In humans, serotonin is
515 produced from tryptophan degradation in a two-step process via the enzymes tryptophan
516 5-monooxygenase (tryptophan hyroxylase 1) and aromatic-L-amino-acid decarboxylase
517 (Table 1). It has been experimentally demonstrated in vitro that serotonin can also be
518 produced by several bacterial genera isolated from the human gut, including
519 Streptococcus, Escherichia, Enterococcus, Lactococcus and Lactobacillus (Wall, et al.,
520 2014). However, the full mechanism of direct serotonin production by bacteria has not
521 been clearly established. Analysis of the serotonin biosynthetic pathway in bacteria via
522 the KEGG database reveals that several bacterial genera contain homologous genes for
523 encoding aromatic-L-amino-acid decarboxylase used in the second step of this pathway
524 (Kanehisa, et al., 2014), but no homologous genes have presently been found for
525 tryptophan 5-monooxygenase. Bacteria can also directly interact with the host to induce
526 the production of serotonin by the host, with experimentally demonstrated effects on
527 host physiology (Yano, et al., 2015). A comparison between germ-free mice and
528 colonized mice (with either mouse or human gut microbiota) has shown that microbiota
529 increased colonic expression of tryptophan hydroxylase 1, the rate limiting enzyme for
530 mucosal serotonin synthesis, and thereby serotonin production, likely acting through
531 SCFAs (Reigstad, et al., 2015).

532 Histamine and its receptors were first described as part of the immune and
533 gastrointestinal systems, but their presence in the central nervous system and
534 implication in behavior and energy homeostasis is gaining increasing attention
535 (Baronio, et al., 2014). Production of histamine from the decarboxylation of the amino
536 acid histidine has been well demonstrated in numerous Gram-positive and Gram-
537 negative bacterial strains (Landete, De las Rivas, Marcobal, & Munoz, 2008). Gram-
538 positive bacteria, such as lactic acid bacteria, use a pyruvoyl-dependent histidine
539 decarboxylase (EC 4.1.1.22) (Table 1), whereas Gram-negative bacteria use a pyridoxal
540 phosphate-dependent histidine decarboxylase (Landete, De las Rivas, et al., 2008). In
541 Lactobacillus spp. histamine is readily exported from the cell using electrogenic
542 histidine/histamine antiporters (Molenaar, Bosscher, ten Brink, Driessen, & Konings,
543 1993). Bacterial histamine production can exert both positive and negative effects on
544 human health. Recently, Thomas et al. (2012) demonstrated that histamine derived from
545 a human gut isolate of the species Lactobacillus reuteri suppresses pro-inflammatory
546 TNF-α production in isolated Toll-like receptor 2-activated human monocytes cells,
547 indicating a potential beneficial effect of microbially-derived compounds for
548 inflammatory conditions. However, histamine production by bacteria has also been
524 (Kanehisa, et al., 2014), but no homologous genes have presently been found for
525 tryptophan 5-monooxygenase. Bacteria can also directly interact with the host to induce
526 the production of serotonin by the host, with experimentally demonstrated effects on
527 host physiology (Yano, et al., 2015). A comparison between germ-free mice and
528 colonized mice (with either mouse or human gut microbiota) has shown that microbiota
529 increased colonic expression of tryptophan hydroxylase 1, the rate limiting enzyme for
530 mucosal serotonin synthesis, and thereby serotonin production, likely acting through
531 SCFAs (Reigstad, et al., 2015).

532 Histamine and its receptors were first described as part of the immune and
533 gastrointestinal systems, but their presence in the central nervous system and
534 implication in behavior and energy homeostasis is gaining increasing attention
535 (Baronio, et al., 2014). Production of histamine from the decarboxylation of the amino
536 acid histidine has been well demonstrated in numerous Gram-positive and Gram-
537 negative bacterial strains (Landete, De las Rivas, Marcobal, & Munoz, 2008). Gram-
538 positive bacteria, such as lactic acid bacteria, use a pyruvoyl-dependent histidine
539 decarboxylase (EC 4.1.1.22) (Table 1), whereas Gram-negative bacteria use a pyridoxal
540 phosphate-dependent histidine decarboxylase (Landete, De las Rivas, et al., 2008). In
541 *Lactobacillus* spp. histamine is readily exported from the cell using electrogenic
542 histidine/histamine antiporters (Molenaar, Bosscher, ten Brink, Driessen, & Konings,
543 1993). Bacterial histamine production can exert both positive and negative effects on
544 human health. Recently, Thomas et al. (2012) demonstrated that histamine derived from
545 a human gut isolate of the species *Lactobacillus reuteri* suppresses pro-inflammatory
546 TNF-α production in isolated Toll-like receptor 2-activated human monocytes cells,
547 indicating a potential beneficial effect of microbially-derived compounds for
548 inflammatory conditions. However, histamine production by bacteria has also been
implicated in food poisoning related to the ingestion of fish fermented by histamine-producing bacteria (scombrotoxin) (Bjornsdottir-Butler, Green, Bolton, & McClellan-Green, 2015).

Tyrosine is a precursor for several hormones including the thyroid hormones thyroxine (3,5,3',5'-tetraiodothyronine) and triiodothyronine, as well as a precursor to L-dihydroxyphenylalanine (L-DOPA) which is in itself a precursor to the catecholamine neurotransmitters dopamine, norepinephrine and epinephrine. Bacterial tyrosinases (EC 1.14.18.1) (Table 1), which catalyze the conversion of tyrosine to L-DOPA, are widely found in many bacterial genera (Claus & Decker, 2006). Although *Escherichia* spp. and *Bacillus* spp. have been reported to produce norepinephrine (Wall, et al., 2014), enzymes necessary for production of the catecholamines norepinephrine and epinephrine have only been identified to date in animals, suggesting some mode of cooperation between host and gut microbes in the catecholamine biosynthetic process.
However, catecholamine stress hormones have been demonstrated to promote both bacterial growth and virulence (Freestone, 2013). Some opportunistic pathogens inhabiting the intestinal tract (*Escherichia coli*, *Staphylococcus aureus*, etc.) are equipped with catechol siderophore uptake systems that facilitate the import Fe(III)-catecholamine complexes and, thereby, their growth under iron-restricted conditions (Beasley, Marolda, Cheung, Buac, & Heinrichs, 2011).

Tyramine is a biogenic amine capable of causing food poisoning if consumed at high levels and is involved in hypertension and migraine syndromes (Marcobal, De las Rivas, Landete, Tabera, & Munoz, 2012; Millichap & Yee, 2003). Tyramine is produced by the decarboxylation of tyrosine by the bacterial enzyme tyrosine decarboxylase (Table 1), which has been identified in Gram-positive bacteria such as lactic acid bacteria, but putative tyrosine decarboxylases in Gram-negative bacteria
implicated in food poisoning related to the ingestion of fish fermented by histamine-producing bacteria (scombrotoxin) (Bjornsdottir-Butler, Green, Bolton, & McClellan-Green, 2015).

Tyrosine is a precursor for several hormones including the thyroid hormones thyroxine (3,5,3',5'-tetraiodothyronine) and triiodothyronine, as well as a precursor to L-dihydroxyphenylalanine (L-DOPA) which is in itself a precursor to the catecholamine neurotransmitters dopamine, norepinephrine and epinephrine. Bacterial tyrosinases (EC 1.14.18.1) (Table 1), which catalyze the conversion of tyrosine to L-DOPA, are widely found in many bacterial genera (Claus & Decker, 2006). Although *Escherichia* spp. and *Bacillus* spp. have been reported to produce norepinephrine (Wall, et al., 2014), enzymes necessary for production of the catecholamines norepinephrine and epinephrine have only been identified to date in animals, suggesting some mode of cooperation between host and gut microbes in the catecholamine biosynthetic process.
However, catecholamine stress hormones have been demonstrated to promote both bacterial growth and virulence (Freestone, 2013). Some opportunistic pathogens inhabiting the intestinal tract (*Escherichia coli*, *Staphylococcus aureus*, etc.) are equipped with catechol siderophore uptake systems that facilitate the import Fe(III)-catecholamine complexes and, thereby, their growth under iron-restricted conditions (Beasley, Marolda, Cheung, Buac, & Heinrichs, 2011).

Tyramine is a biogenic amine capable of causing food poisoning if consumed at high levels and is involved in hypertension and migraine syndromes (Marcobal, De las Rivas, Landete, Tabera, & Munoz, 2012; Millichap & Yee, 2003). Tyramine is produced by the decarboxylation of tyrosine by the bacterial enzyme tyrosine decarboxylase (Table 1), which has been identified in Gram-positive bacteria such as lactic acid bacteria, but putative tyrosine decarboxylases in Gram-negative bacteria
574 remain to be characterized (Marcobal, et al., 2012). Tyramine is exported from bacterial
575 cells via a tyrosine-tyramine antiporter (Linares, Fernandez, Martin, & Alvarez, 2009).
576 In Gram-positive bacteria, high concentrations of tyrosine and acidic pH conditions
577 have been demonstrated to increase the transcription of tyrosine decarboxylase and the
578 tyrosine-tyramine antiporter (Linares, et al., 2009; Marcobal, et al., 2012). Gut
579 microbiota derived tyramine has been shown to increase circulating serotonin levels by
580 elevating its synthesis by enteroendocrine cells (Yano, et al., 2015) although this
581 mechanism has not been defined.

582 Nitric oxide (NO) is produced from the amino acid arginine by many bacterial
583 species via the enzyme nitric oxide synthase (NOS) (Table 1), although only several
584 bacterial homologs of mammalian NOSs have been identified to date (Sudhamsu &
585 Crane, 2009). Little is known about the extent that microbially-produced NO impacts
586 the human host, notably at the intestinal mucosa level, although it is known that NO is
587 involved in immune defense mechanisms and exerts cytotoxic effects at high
588 concentrations.

589 Tryptamine is a β-arylamine neurotransmitter that has numerous biological roles
590 including a ligand for the sigma-2 receptor, a trace amine-associated receptor that
591 increases the inhibitory responses of cells to serotonin, and an inducer of serotonin
592 release from enteroendocrine cells which can modulate gastrointestinal motility and
593 may play a role in the pathology of inflammatory bowel diseases (Williams, et al., 2014
594 and references therein). Recently it was demonstrated that tryptamine is produced by
595 *Clostridium sporogenes* by the decarboxylation of tryptophan via a Trp decarboxylase
596 (Table 1) (Williams, et al., 2014). Williams et al. (2014) also used a phylogenetic-
597 informed screening of other decarboxylases from the gut microbiota to show that a
574 remain to be characterized (Marcobal, et al., 2012). Tyramine is exported from bacterial
575 cells via a tyrosine-tyramine antiporter (Linares, Fernandez, Martin, & Alvarez, 2009).
576 In Gram-positive bacteria, high concentrations of tyrosine and acidic pH conditions
577 have been demonstrated to increase the transcription of tyrosine decarboxylase and the
578 tyrosine-tyramine antiporter (Linares, et al., 2009; Marcobal, et al., 2012). Gut
579 microbiota derived tyramine has been shown to increase circulating serotonin levels by
580 elevating its synthesis by enteroendocrine cells (Yano, et al., 2015) although this
581 mechanism has not been defined.

582 Nitric oxide (NO) is produced from the amino acid arginine by many bacterial
583 species via the enzyme nitric oxide synthase (NOS) (Table 1), although only several
584 bacterial homologs of mammalian NOSs have been identified to date (Sudhamsu &
585 Crane, 2009). Little is known about the extent that microbially-produced NO impacts
586 the human host, notably at the intestinal mucosa level, although it is known that NO is
587 involved in immune defense mechanisms and exerts cytotoxic effects at high
588 concentrations.

589 Tryptamine is a β-arylamine neurotransmitter that has numerous biological roles
590 including a ligand for the sigma-2 receptor, a trace amine-associated receptor that
591 increases the inhibitory responses of cells to serotonin, and an inducer of serotonin
592 release from enteroendocrine cells which can modulate gastrointestinal motility and
593 may play a role in the pathology of inflammatory bowel diseases (Williams, et al., 2014
594 and references therein). Recently it was demonstrated that tryptamine is produced by
595 *Clostridium sporogenes* by the decarboxylation of tryptophan via a Trp decarboxylase
596 (Table 1) (Williams, et al., 2014). Williams et al. (2014) also used a phylogenetic-
597 informed screening of other decarboxylases from the gut microbiota to show that a
second Trp decarboxylase appears in the Firmicute *Ruminococcus gnavus*, and similar enzymes were found in at least 10% of the human population.

Phenethylamine is a trace amine that acts as a neurotransmitter and, depending on the concentrations, is associated with specific psychological disorders (Irsfeld, Spadafore, & Pruss, 2013) and can affect satiety and mood (Pessione, 2012). Phenethylamine is produced in bacteria by the decarboxylation of phenylalanine (Diaz, Fernandez, Prieto, & Garcia, 2001) via the enzyme aromatic-L-amino-acid decarboxylase (Table 1).

Potential physiological effects of microbially-derived phenethylamine on host health have yet to be elucidated.

## 4.2. Short- and branched-chain fatty acids

The main short-chain fatty acids (SCFAs) produced in the large intestine by the mammalian gut microbiota are acetate, butyrate and propionate (Rechkemmer, Ronnau, & von Engelhardt, 1988). Although it is well known that dietary substrates for SCFA production are mainly fibers and resistant starches (Laparra & Sanz, 2010), isolated colonic bacteria growing *in vitro* on proteins as the only available carbon source have been demonstrated to produce SCFAs as well as branched-chain fatty acids (BCFAs) (Neis, et al., 2015). Bacterial fermentation of glycine, alanine, threonine, glutamate, lysine and aspartate can produce acetate; threonine, glutamate and lysine can produce butyrate, and alanine and threonine can produce propionate (Davila, et al., 2013).

Nonetheless, the molecular pathways involved in production of these SCFAs are not well-defined.

Branched-chain fatty acids (BCFAs), namely isobutyrate, 2-methylbutyrate and isovalerate, are derived from branched-chain amino acids and are present at much lower concentrations in the large intestine luminal content (Liu, et al., 2014). Although a full
second Trp decarboxylase appears in the Firmicute *Ruminococcus gnavus*, and similar enzymes were found in at least 10% of the human population.

Phenethylamine is a trace amine that acts as a neurotransmitter and, depending on the concentrations, is associated with specific psychological disorders (Irsfeld, Spadafore, & Pruss, 2013) and can affect satiety and mood (Pessione, 2012). Phenethylamine is produced in bacteria by the decarboxylation of phenylalanine (Diaz, Fernandez, Prieto, & Garcia, 2001) via the enzyme aromatic-L-amino-acid decarboxylase (Table 1).

Potential physiological effects of microbially-derived phenethylamine on host health have yet to be elucidated.

## 4.2. Short- and branched-chain fatty acids

The main short-chain fatty acids (SCFAs) produced in the large intestine by the mammalian gut microbiota are acetate, butyrate and propionate (Rechkemmer, Ronnau, & von Engelhardt, 1988). Although it is well known that dietary substrates for SCFA production are mainly fibers and resistant starches (Laparra & Sanz, 2010), isolated colonic bacteria growing *in vitro* on proteins as the only available carbon source have been demonstrated to produce SCFAs as well as branched-chain fatty acids (BCFAs) (Neis, et al., 2015). Bacterial fermentation of glycine, alanine, threonine, glutamate, lysine and aspartate can produce acetate; threonine, glutamate and lysine can produce butyrate, and alanine and threonine can produce propionate (Davila, et al., 2013).

Nonetheless, the molecular pathways involved in production of these SCFAs are not well-defined.

Branched-chain fatty acids (BCFAs), namely isobutyrate, 2-methylbutyrate and isovalerate, are derived from branched-chain amino acids and are present at much lower concentrations in the large intestine luminal content (Liu, et al., 2014). Although a full
623 description of bacterial synthesis of BCFA from amino acids is beyond the scope of this
624 article, many Gram-positive bacteria use the primers isovaleryl-CoA, isobutyryl-CoA,
625 and 2-methylbutyryl-CoA derived from valine, isoleucine, and leucine to produce
626 BCFA using a specialized branched-chain-keto acid dehydrogenase complex (Cronan &
627 Thomas, 2009). The relative proportions of SCFAs and BCFAs depend on numerous
628 factors including the type of protein degraded as well as the available concentrations of
629 carbohydrates (Macfarlane & Macfarlane, 2012).

630 The SCFAs acetate, propionate and butyrate produced via bacterial fermentation of
631 carbohydrates and/or amino acids have been proposed to exert various physiological
632 effects, which have been recently reviewed (Hamer, et al., 2008). Briefly, for instance,
633 butyrate provides energy to colonocytes and regulates cell proliferation and
634 differentiation as well as the transcription of numerous genes involved in mucin
635 production and hormone secretion (i.e. PYY, GLP-1, GLP-2) that influence gut
636 integrity and transit, appetite and glucose metabolism (Daly, Cuff, Fung, & Shirazi-
637 Beechey, 2005). Butyrate also plays an anti-inflammatory role via different
638 mechanisms, including inhibition of LPS-mediated inflammatory cytokine secretion by
639 intestinal epithelial cells and other immune cells and via induction of colonic regulatory
640 T cells partly by an epigenetic modification of the forkhead box-P3 promoter
641 (Furusawa, et al., 2013). Propionate and acetate undergo partial oxidation in
642 colonocytes or can travel to the liver via the portal vein where they serve as substrates
643 for gluconeogenesis and lipogenesis (Tremaroli & Backhed, 2012). Acetate is also a
644 substrate for cholesterol synthesis, whereas propionate can decrease cholesterol
645 synthesis (Demigne, et al., 1995). In addition, SCFAs are ligands for G protein-coupled
646 receptors, namely Gpr41 and Gpr43, which are expressed in enteroendocrine L-cells of
647 the distal small intestine and colon (Reigstad, et al., 2015). Gpr41 deficiency has been
623 description of bacterial synthesis of BCFA from amino acids is beyond the scope of this
624 article, many Gram-positive bacteria use the primers isovaleryl-CoA, isobutyryl-CoA,
625 and 2-methylbutyryl-CoA derived from valine, isoleucine, and leucine to produce
626 BCFA using a specialized branched-chain-keto acid dehydrogenase complex (Cronan &
627 Thomas, 2009). The relative proportions of SCFAs and BCFAs depend on numerous
628 factors including the type of protein degraded as well as the available concentrations of
629 carbohydrates (Macfarlane & Macfarlane, 2012).

630 The SCFAs acetate, propionate and butyrate produced via bacterial fermentation of
631 carbohydrates and/or amino acids have been proposed to exert various physiological
632 effects, which have been recently reviewed (Hamer, et al., 2008). Briefly, for instance,
633 butyrate provides energy to colonocytes and regulates cell proliferation and
634 differentiation as well as the transcription of numerous genes involved in mucin
635 production and hormone secretion (i.e. PYY, GLP-1, GLP-2) that influence gut
636 integrity and transit, appetite and glucose metabolism (Daly, Cuff, Fung, & Shirazi-
637 Beechey, 2005). Butyrate also plays an anti-inflammatory role via different
638 mechanisms, including inhibition of LPS-mediated inflammatory cytokine secretion by
639 intestinal epithelial cells and other immune cells and via induction of colonic regulatory
640 T cells partly by an epigenetic modification of the forkhead box-P3 promoter
641 (Furusawa, et al., 2013). Propionate and acetate undergo partial oxidation in
642 colonocytes or can travel to the liver via the portal vein where they serve as substrates
643 for gluconeogenesis and lipogenesis (Tremaroli & Backhed, 2012). Acetate is also a
644 substrate for cholesterol synthesis, whereas propionate can decrease cholesterol
645 synthesis (Demigne, et al., 1995). In addition, SCFAs are ligands for G protein-coupled
646 receptors, namely Gpr41 and Gpr43, which are expressed in enteroendocrine L-cells of
647 the distal small intestine and colon (Reigstad, et al., 2015). Gpr41 deficiency has been
648 shown to be associated with reduced expression of peptide YY (PYY), a gut hormone
649 involved in satiety and gut motility (Samuel, et al., 2008). Direct administration of
650 SCFAs in the colon increase PYY plasma level, and *in vitro* administration on primary
651 colonic crypts also increase the release of PYY, suggesting a direct effect of SCFA on
652 secretion (Chambers, et al., 2014; Cherbut, et al., 1998). Butyrate can also bind the
653 GPR109a receptor expressed by intestinal macrophages and dendritic cells, thus
654 activating production of the anti-inflammatory cytokine Il-10 (Pabst & Bernhardt,
655 2010). BCFAs have also been shown to regulate electrolyte absorption and secretion
656 (Musch, Bookstein, Xie, Sellin, & Chang, 2001). It has been proposed that SCFAs
657 could exert effects beyond the gut via their interactions with Gpr expressed in different
658 tissues and cellular types (e.g. adipocytes, pancreatic cells, neuronal cells) and thereby
659 regulate metabolism by inducing energy expenditure and mitochondrial function (Gao,
660 et al., 2009). Whether these effects could be mediated by the SCFAs generated by the
661 gut microbiota requires further investigation.
662

662

663 **4.3. Sulfur-containing bacterial metabolites**

664 Fecal S-containing substances are a complex mixture of bacterial metabolites
665 including methanethiol and the well-studied H₂S, which is found at millimolar
666 concentrations in the human colonic luminal contents and in feces (Macfarlane, et al.,
667 1992). In addition to production of H₂S via reduction of inorganic sulfate by intestinal
668 sulfate reducing bacteria or microbial catabolism of intestinal sulfomucins, sulfide is
669 produced by intestinal microbiota activity through fermentation of several S-containing
670 amino acids of both dietary and endogenous origins (Blachier, et al., 2010). H₂S is
671 notably produced as a byproduct by the bacterial fermentation of cysteine via cysteine
672 desulfhydrases, cystathione-B-lyase and tryptophanase (Table 1) (Awano, Wada, Mori,
648 shown to be associated with reduced expression of peptide YY (PYY), a gut hormone
649 involved in satiety and gut motility (Samuel, et al., 2008). Direct administration of
650 SCFAs in the colon increase PYY plasma level, and *in vitro* administration on primary
651 colonic crypts also increase the release of PYY, suggesting a direct effect of SCFA on
652 secretion (Chambers, et al., 2014; Cherbut, et al., 1998). Butyrate can also bind the
653 GPR109a receptor expressed by intestinal macrophages and dendritic cells, thus
654 activating production of the anti-inflammatory cytokine Il-10 (Pabst & Bernhardt,
655 2010). BCFAs have also been shown to regulate electrolyte absorption and secretion
656 (Musch, Bookstein, Xie, Sellin, & Chang, 2001). It has been proposed that SCFAs
657 could exert effects beyond the gut via their interactions with Gpr expressed in different
658 tissues and cellular types (e.g. adipocytes, pancreatic cells, neuronal cells) and thereby
659 regulate metabolism by inducing energy expenditure and mitochondrial function (Gao,
660 et al., 2009). Whether these effects could be mediated by the SCFAs generated by the
661 gut microbiota requires further investigation.
662

662

663 **4.3. Sulfur-containing bacterial metabolites**

664 Fecal S-containing substances are a complex mixture of bacterial metabolites
665 including methanethiol and the well-studied H₂S, which is found at millimolar
666 concentrations in the human colonic luminal contents and in feces (Macfarlane, et al.,
667 1992). In addition to production of H₂S via reduction of inorganic sulfate by intestinal
668 sulfate reducing bacteria or microbial catabolism of intestinal sulfomucins, sulfide is
669 produced by intestinal microbiota activity through fermentation of several S-containing
670 amino acids of both dietary and endogenous origins (Blachier, et al., 2010). H₂S is
671 notably produced as a byproduct by the bacterial fermentation of cysteine via cysteine
672 desulfhydrases, cystathione-B-lyase and tryptophanase (Table 1) (Awano, Wada, Mori,
673 Nakamori, & Takagi, 2005). Fermentation of methionine yields the sulfur-containing
674 compound methanethiol (Davila, et al., 2013). Due to its lipophilic activity, H₂S
675 penetrates biological membranes (Reiffenstein, Hulbert, & Roth, 1992) and, when
676 present in excess, inhibits mitochondrial cytochrome c oxidase activity reversibly with a
677 binding constant similar to cyanide (Leschelle, et al., 2005). At lower concentrations,
678 H₂S is able to increase cellular respiration and ATP production by means of the
679 mitochondrial sulfide-oxidizing unit (Bouillaud & Blachier, 2011). Although several
680 lines of evidence suggest that excessive concentrations of sulfide in the intestinal
681 luminal content may be implicated in the etiology of ulcerative colitis (Pitcher &
682 Cummings, 1996) and in the risk of relapse after an inflammatory episode (Jowett, et
683 al., 2004), there is also evidence that endogenously-formed low levels of H₂S in
684 intestinal mucosa may participate in the resolution of mucosal inflammation (Flannigan,
685 et al., 2015). Experiments from colonocyte incubation and intra-colonic instillation
686 with millimolar concentrations of NaHS, used as a H₂S donor, indicate that hydrogen
687 sulfide reversibly inhibits colonocyte oxygen consumption and increases the expression
688 of hypoxia-inducible factor 1 alpha (HIF-1alpha) together with several inflammation-
689 related genes, namely inducible nitric oxide synthase (iNOS) and interleukin 6 (Il-6)
690 (Beaumont, et al., 2016). Importantly, it has been demonstrated that endogenously
691 produced H₂S maintains colon cancer cellular bioenergetics supporting colonic tumor
692 growth (Szabo, et al., 2013).

693

694 **4.4. Aromatic compounds**

695 In addition to neuroactive compounds, the catabolism of aromatic amino acids
696 (phenylalanine, tyrosine and tryptophan) by the microbiota also produces phenolic and
697 indolic compounds (Nyangale, Mottram, & Gibson, 2012). Recent support for a role of
673 Nakamori, & Takagi, 2005). Fermentation of methionine yields the sulfur-containing
674 compound methanethiol (Davila, et al., 2013). Due to its lipophilic activity, H₂S
675 penetrates biological membranes (Reiffenstein, Hulbert, & Roth, 1992) and, when
676 present in excess, inhibits mitochondrial cytochrome c oxidase activity reversibly with a
677 binding constant similar to cyanide (Leschelle, et al., 2005). At lower concentrations,
678 H₂S is able to increase cellular respiration and ATP production by means of the
679 mitochondrial sulfide-oxidizing unit (Bouillaud & Blachier, 2011). Although several
680 lines of evidence suggest that excessive concentrations of sulfide in the intestinal
681 luminal content may be implicated in the etiology of ulcerative colitis (Pitcher &
682 Cummings, 1996) and in the risk of relapse after an inflammatory episode (Jowett, et
683 al., 2004), there is also evidence that endogenously-formed low levels of H₂S in
684 intestinal mucosa may participate in the resolution of mucosal inflammation (Flannigan,
685 et al., 2015). Experiments from colonocyte incubation and intra-colonic instillation
686 with millimolar concentrations of NaHS, used as a H₂S donor, indicate that hydrogen
687 sulfide reversibly inhibits colonocyte oxygen consumption and increases the expression
688 of hypoxia-inducible factor 1 alpha (HIF-1alpha) together with several inflammation-
689 related genes, namely inducible nitric oxide synthase (iNOS) and interleukin 6 (Il-6)
690 (Beaumont, et al., 2016). Importantly, it has been demonstrated that endogenously
691 produced H₂S maintains colon cancer cellular bioenergetics supporting colonic tumor
692 growth (Szabo, et al., 2013).

693

694 **4.4. Aromatic compounds**

695 In addition to neuroactive compounds, the catabolism of aromatic amino acids
696 (phenylalanine, tyrosine and tryptophan) by the microbiota also produces phenolic and
697 indolic compounds (Nyangale, Mottram, & Gibson, 2012). Recent support for a role of
698 gut microbiota in the production of various aromatic amino acid metabolites has
699 recently been demonstrated by comparing metabolites between specific pathogen-free
700 (SPF) mice and germ-free (GF) mice (Sridharan, et al., 2014). However, bacterial
701 metabolism of aromatic compounds largely depends on the type of carbon availability,
702 as fermentable carbohydrates largely inhibit aromatic amino acid fermentation (Smith &
703 Macfarlane, 1996).

704 Phenol is produced from the conversion of tyrosine via the enzyme tyrosine phenol-
705 lyase (Table 1). Optimal conditions for production of phenol by anaerobic bacteria in
706 the human large intestine include a near-neutral pH of 6.8 and availability of free amino
707 acids as opposed to peptides (Smith & Macfarlane, 1997). The types of intestinal
708 bacteria may also play a significant role in production of phenol as aerobic bacteria
709 tended to produce phenol from tyrosine degradation *in vitro* while anaerobic bacteria
710 produced *p*-cresol (Bone, Tamm, & Hill, 1976). Phenol has been shown to decrease the
711 integrity of the barrier function of colonocytes *in vitro* (Hughes, Kurth, McGilligan,
712 McGlynn, & Rowland, 2008) and impaired the viability of human colonic epithelial
713 cells at concentrations higher than 1.25 mM (Pedersen, Brynskov, & Saermark, 2002).

714 Formation of *p*-cresol (or 4 methylphenol) by microbes begins with the two-step
715 conversion of tyrosine to 4-hydroxyphenylacetate (Meyer & Hostetter, 2012), although
716 the genes involved in this conversion are unknown (Table 1) (Dawson, et al., 2011). In
717 the final step, the bacterium *Clostridium difficile* uses the enzyme *p*-
718 hydroxyphenylacetate decarboxylase (genes *hpdB*, *hpaC*, *hpdA*) to convert 4-
719 hydroxyphenylacetate to *p*-cresol (Selmer & Andrei, 2001; Yu, Blaser, Andrei, Pierik,
720 & Selmer, 2006). The *hpdBCA* operon is rarely found in gut microflora other than
721 *Clostridium* spp. (Dawson, et al., 2011), and therefore it is unknown to what extent, if
722 any, other gut genera play a role in *p*-cresol formation. Production of *p*-cresol is
698 gut microbiota in the production of various aromatic amino acid metabolites has
699 recently been demonstrated by comparing metabolites between specific pathogen-free
700 (SPF) mice and germ-free (GF) mice (Sridharan, et al., 2014). However, bacterial
701 metabolism of aromatic compounds largely depends on the type of carbon availability,
702 as fermentable carbohydrates largely inhibit aromatic amino acid fermentation (Smith &
703 Macfarlane, 1996).

704 Phenol is produced from the conversion of tyrosine via the enzyme tyrosine phenol-
705 lyase (Table 1). Optimal conditions for production of phenol by anaerobic bacteria in
706 the human large intestine include a near-neutral pH of 6.8 and availability of free amino
707 acids as opposed to peptides (Smith & Macfarlane, 1997). The types of intestinal
708 bacteria may also play a significant role in production of phenol as aerobic bacteria
709 tended to produce phenol from tyrosine degradation *in vitro* while anaerobic bacteria
710 produced *p*-cresol (Bone, Tamm, & Hill, 1976). Phenol has been shown to decrease the
711 integrity of the barrier function of colonocytes *in vitro* (Hughes, Kurth, McGilligan,
712 McGlynn, & Rowland, 2008) and impaired the viability of human colonic epithelial
713 cells at concentrations higher than 1.25 mM (Pedersen, Brynskov, & Saermark, 2002).

714 Formation of *p*-cresol (or 4 methylphenol) by microbes begins with the two-step
715 conversion of tyrosine to 4-hydroxyphenylacetate (Meyer & Hostetter, 2012), although
716 the genes involved in this conversion are unknown (Table 1) (Dawson, et al., 2011). In
717 the final step, the bacterium *Clostridium difficile* uses the enzyme *p*-hydroxyphenylacetate decarboxylase (genes *hpdB*, *hpaC*, *hpdA*) to convert 4-hydroxyphenylacetate to *p*-cresol (Selmer & Andrei, 2001; Yu, Blaser, Andrei, Pierik, & Selmer, 2006). The *hpdBCA* operon is rarely found in gut microflora other than *Clostridium* spp. (Dawson, et al., 2011), and therefore it is unknown to what extent, if any, other gut genera play a role in *p*-cresol formation. Production of *p*-cresol is
723 stimulated by Fe(III) and competitive growth conditions in *Clostridium* spp. (Doerner,
724 Mason, Kridelbaugh, & Loughrin, 2009; Selmer & Andrei, 2001). The *p*-cresol
725 concentration in human feces averages approximately 0.4 mM (Gostner, et al., 2006;
726 Lecerf, et al., 2012). These compounds are absorbed from the intestinal lumen to the
727 portal bloodstream through colonocytes, metabolized in the liver and finally excreted by
728 the kidneys with more than 90% of urinary phenolic compounds being recovered as *p*-
729 cresol (Hughes, Magee, & Bingham, 2000). At millimolar concentrations, *p*-cresol
730 inhibits human colonocyte proliferation and cell respiration and increases superoxide
731 production (Andriamihaja, et al., 2015). Most importantly, in this latter study, *p*-cresol
732 was found to be genotoxic towards human colonocytes. *p*-cresol can be conjugated in
733 the colonic epithelium and in the liver, generating *p*-glucuronide and cresyl sulfate,
734 (Evenepoel, Meijers, Bammens, & Verbeke, 2009). Phenolic compound sulphation has
735 been shown to be impaired in the mucosa of ulcerative colitis patients (Ramakrishna,
736 Roberts-Thomson, Pannall, & Roediger, 1991). Interestingly, *p*-cresyl sulfate promotes
737 insulin resistance in chronic kidney disease (CKD) patients (Koppe, et al., 2013) and is
738 found at elevated concentrations in the urine of autism patients (Heinken & Thiele,
739 2015). It can also cause damage in renal tubular cells by induction of oxidative stress
740 through activation of NADPH oxidase (Watanabe, et al., 2013).

741 Indole is produced from tryptophan via the enzyme tryptophanase (Table 1), which is
742 found only in microbes (Meyer & Hostetter, 2012). After absorption through the
743 intestinal epithelium, indole is transported to the liver where it undergoes hydroxylation
744 by the host to 3-hydroxy-indole and finally sulfonated to indoxyl sulfate, a uremic
745 toxin, via a sulfotransferase (Meijers & Evenepoel, 2011; Wikoff, et al., 2009). In
746 contrast, indole also has a potential beneficial effect on host intestinal epithelial cells, as
747 treatment of human enterocytes with indole was found to result in increased expression
723 stimulated by Fe(III) and competitive growth conditions in *Clostridium* spp. (Doerner,
724 Mason, Kridelbaugh, & Loughrin, 2009; Selmer & Andrei, 2001). The *p*-cresol
725 concentration in human feces averages approximately 0.4 mM (Gostner, et al., 2006;
726 Lecerf, et al., 2012). These compounds are absorbed from the intestinal lumen to the
727 portal bloodstream through colonocytes, metabolized in the liver and finally excreted by
728 the kidneys with more than 90% of urinary phenolic compounds being recovered as *p*-
729 cresol (Hughes, Magee, & Bingham, 2000). At millimolar concentrations, *p*-cresol
730 inhibits human colonocyte proliferation and cell respiration and increases superoxide
731 production (Andriamihaja, et al., 2015). Most importantly, in this latter study, *p*-cresol
732 was found to be genotoxic towards human colonocytes. *p*-cresol can be conjugated in
733 the colonic epithelium and in the liver, generating *p*-glucuronide and cresyl sulfate,
734 (Evenepoel, Meijers, Bammens, & Verbeke, 2009). Phenolic compound sulphation has
735 been shown to be impaired in the mucosa of ulcerative colitis patients (Ramakrishna,
736 Roberts-Thomson, Pannall, & Roediger, 1991). Interestingly, *p*-cresyl sulfate promotes
737 insulin resistance in chronic kidney disease (CKD) patients (Koppe, et al., 2013) and is
738 found at elevated concentrations in the urine of autism patients (Heinken & Thiele,
739 2015). It can also cause damage in renal tubular cells by induction of oxidative stress
740 through activation of NADPH oxidase (Watanabe, et al., 2013).

741 Indole is produced from tryptophan via the enzyme tryptophanase (Table 1), which is
742 found only in microbes (Meyer & Hostetter, 2012). After absorption through the
743 intestinal epithelium, indole is transported to the liver where it undergoes hydroxylation
744 by the host to 3-hydroxy-indole and finally sulfonated to indoxyl sulfate, a uremic
745 toxin, via a sulfotransferase (Meijers & Evenepoel, 2011; Wikoff, et al., 2009). In
746 contrast, indole also has a potential beneficial effect on host intestinal epithelial cells, as
747 treatment of human enterocytes with indole was found to result in increased expression
748 of genes involved in the mucosal barrier functions and was associated with a reduction
749 of inflammatory parameters (Bansal, Alaniz, Wood, & Jayaraman, 2010). Secretion of
750 the incretin GLP-1 from enteroendocrine cells is increased during short exposures to
751 indole via interference of voltage-gated K⁺ channels, but reduced over long periods of
752 exposure to indole via slowing ATP production by blocking NADH dehydrogenase
753 (Chimerel, et al., 2014). Besides its role in glucose metabolism through increasing
754 insulin secretion, GLP-1 is involved in inhibiting gastric secretion and motility; these
755 phenomenons being associated with increased satiety (Steinert, Beglinger, & Langhans,
756 2015). The indole derivative indole-3-aldehyde was also shown to regulate gut mucosal
757 immune response through aryl hydrocarbon receptor as well as intestinal barrier
758 function and inflammation through its sensing by pregnane X receptor (Venkatesh, et
759 al., 2014; Zelante, et al., 2013).

760 Serum levels of the amino acid tryptophan itself also have a demonstrated role in
761 host health, as decreases in this amino acid have been associated with increased immune
762 activation, inflammatory diseases such as Crohn's disease, as well as cognitive deficit
763 (Gupta, et al., 2012; Widner, et al., 1999). This may be particularly detrimental in
764 elderly populations, as increased proportions of genes from gut microbiota involved in
765 aromatic amino acid metabolism (including tryptophan), as well as decreased levels of
766 tryptophan have been reported in elderly patients (Collino, et al., 2013; Rampelli, et al.,
767 2013).

768

769 **4.5. Polyamines**

770 Intestinal luminal polyamines (i.e. agmatine, putrescine, spermidine, cadaverine,
771 etc.) can originate from dietary components, microbiota metabolism (Davila, et al.,
772 2013), and endogenous origin, notably released from desquamated intestinal epithelial
748 of genes involved in the mucosal barrier functions and was associated with a reduction
749 of inflammatory parameters (Bansal, Alaniz, Wood, & Jayaraman, 2010). Secretion of
750 the incretin GLP-1 from enteroendocrine cells is increased during short exposures to
751 indole via interference of voltage-gated K⁺ channels, but reduced over long periods of
752 exposure to indole via slowing ATP production by blocking NADH dehydrogenase
753 (Chimerel, et al., 2014). Besides its role in glucose metabolism through increasing
754 insulin secretion, GLP-1 is involved in inhibiting gastric secretion and motility; these
755 phenomenons being associated with increased satiety (Steinert, Beglinger, & Langhans,
756 2015). The indole derivative indole-3-aldehyde was also shown to regulate gut mucosal
757 immune response through aryl hydrocarbon receptor as well as intestinal barrier
758 function and inflammation through its sensing by pregnane X receptor (Venkatesh, et
759 al., 2014; Zelante, et al., 2013).

760 Serum levels of the amino acid tryptophan itself also have a demonstrated role in
761 host health, as decreases in this amino acid have been associated with increased immune
762 activation, inflammatory diseases such as Crohn's disease, as well as cognitive deficit
763 (Gupta, et al., 2012; Widner, et al., 1999). This may be particularly detrimental in
764 elderly populations, as increased proportions of genes from gut microbiota involved in
765 aromatic amino acid metabolism (including tryptophan), as well as decreased levels of
766 tryptophan have been reported in elderly patients (Collino, et al., 2013; Rampelli, et al.,
767 2013).

768

769 **4.5. Polyamines**

770 Intestinal luminal polyamines (i.e. agmatine, putrescine, spermidine, cadaverine,
771 etc.) can originate from dietary components, microbiota metabolism (Davila, et al.,
772 2013), and endogenous origin, notably released from desquamated intestinal epithelial
773 cells. Gram-negative bacteria such as *E. coli* produce high concentrations of putrescine
774 and spermidine in minimal media (Tabor & Tabor, 1985). Numerous amines (including
775 polyamines) have been measured in the intestinal luminal contents at concentrations
776 ranging from micro- to millimolar (Osborne & Seidel, 1990). Intestinal epithelial cells
777 have the capacity to take up polyamines from the intestinal luminal contents (Blachier,
778 et al., 1992).

779 Agmatine is formed by the decarboxylation of the amino acid arginine via the
780 enzyme arginine decarboxylase (Table 1). Agmatine, which is produced and released
781 by colonic bacteria as well as by desquamated intestinal epithelial cells and ingested in
782 food, exerts inhibitory effects on colonocyte proliferation (Mayeur, et al., 2005).

783 Putrescine can be synthesized by bacteria from the amino acid arginine either through
784 the intermediate ornithine or through agmatine (Nakada & Itoh, 2003). Agmatine is
785 either directly converted to putrescine via the enzyme arginine decarboxylase, or goes
786 through a two-step process which uses the enzymes agmatine deiminase and N-
787 carbamoylputrescine amidase (Table 1). The agmatine deiminase pathway has been
788 found in several bacterial genera such as *Pseudomonas*, *Enterococcus*, *Bacillus* and
789 *Lactobacillus* (Landete, Arena, Pardo, Manca de Nadra, & Ferrer, 2008). Agmatine-
790 putrescine antiporters have been identified in bacterial genera and function by importing
791 agmatine and exporting putrescine (Polo, Gil-Ortiz, Cantin, & Rubio, 2012). Putrescine
792 synthesis has been shown to be strictly necessary for colonic epithelial cell proliferation
793 (Gamet, Cazenave, Trocheris, Denis-Pouxviel, & Murat, 1991; Mouille, Delpal,
794 Mayeur, & Blachier, 2003).

795 In many bacteria including *E. coli*, spermidine is synthesized from putrescine and
796 decarboxylated S-adenosylmethionine (SAM) via spermidine synthase (Tabor & Tabor,
797 1985). Recently, bacteria lacking spermidine synthase orthologues were discovered to
773 cells. Gram-negative bacteria such as *E. coli* produce high concentrations of putrescine
774 and spermidine in minimal media (Tabor & Tabor, 1985). Numerous amines (including
775 polyamines) have been measured in the intestinal luminal contents at concentrations
776 ranging from micro- to millimolar (Osborne & Seidel, 1990). Intestinal epithelial cells
777 have the capacity to take up polyamines from the intestinal luminal contents (Blachier,
778 et al., 1992).

779 Agmatine is formed by the decarboxylation of the amino acid arginine via the
780 enzyme arginine decarboxylase (Table 1). Agmatine, which is produced and released
781 by colonic bacteria as well as by desquamated intestinal epithelial cells and ingested in
782 food, exerts inhibitory effects on colonocyte proliferation (Mayeur, et al., 2005).

783 Putrescine can be synthesized by bacteria from the amino acid arginine either through
784 the intermediate ornithine or through agmatine (Nakada & Itoh, 2003). Agmatine is
785 either directly converted to putrescine via the enzyme arginine decarboxylase, or goes
786 through a two-step process which uses the enzymes agmatine deiminase and N-
787 carbamoylputrescine amidase (Table 1). The agmatine deiminase pathway has been
788 found in several bacterial genera such as *Pseudomonas*, *Enterococcus*, *Bacillus* and
789 *Lactobacillus* (Landete, Arena, Pardo, Manca de Nadra, & Ferrer, 2008). Agmatine-
790 putrescine antiporters have been identified in bacterial genera and function by importing
791 agmatine and exporting putrescine (Polo, Gil-Ortiz, Cantin, & Rubio, 2012). Putrescine
792 synthesis has been shown to be strictly necessary for colonic epithelial cell proliferation
793 (Gamet, Cazenave, Trocheris, Denis-Pouxviel, & Murat, 1991; Mouille, Delpal,
794 Mayeur, & Blachier, 2003).

795 In many bacteria including *E. coli*, spermidine is synthesized from putrescine and
796 decarboxylated S-adenosylmethionine (SAM) via spermidine synthase (Tabor & Tabor,
797 1985). Recently, bacteria lacking spermidine synthase orthologues were discovered to
798 have an alternate pathway in which spermidine is formed by the enzymes
799 carboxynorspermidine dehydrogenase (aka carboxynorspermidine synthase) and
800 carboxynorspermidine decarboxylase via the intermediate carboxyspermidine (Lee, et
801 al., 2009). This alternative pathway of spermidine synthesis has been identified as the
802 dominant pathway in the human gut microbiota as well as diverse human pathogens and
803 is critical for growth in selected species (Hanfrey, et al., 2011). In host cells, SAM can
804 react with spermidine via another enzyme, spermine synthase, to produce spermine.
805 Prokaryotic cells do not appear to contain spermine synthase, but they can produce a
806 different tetra-amine compound, thermospermine, that has been detected in lower
807 eukaryotes and plants (Minguet, Vera-Sirera, Marina, Carbonell, & Blazquez, 2008).
808 Spermidine and spermine are polycationic amines that are involved in numerous
809 processes such as mitigating oxidative stress, and induction of autophagy to stimulate
810 cellular longevity (Eisenberg, et al., 2009; Yamamoto, et al., 2012).

811 The polyamine cadaverine is synthesized from lysine in a one-step reaction with
812 lysine decarboxylase (Table 1) and has been shown to provide an acid resistance
813 mechanism in *E. coli* (Le Gall, et al., 2011). However, the effects of cadaverine on the
814 colonic epithelial cells remain unknown.

815

816 **4.6. Ammonia**

817 Ammonia provides the source of nitrogen for all amino acids and is primarily
818 assimilated through either glutamate dehydrogenase or the glutamine
819 synthetase/GOGAT cycle. It is also used directly in the biosynthesis of various amino
820 acids including glutamate, glutamine, asparagine, valine, isoleucine, leucine,
821 phenylalanine, tyrosine and tryptophan (Figs. 1-4). Ammonia can be produced by the
822 microbial degradation of numerous amino acids to specific metabolites (i.e. arginine to
798 have an alternate pathway in which spermidine is formed by the enzymes
799 carboxynorspermidine dehydrogenase (aka carboxynorspermidine synthase) and
800 carboxynorspermidine decarboxylase via the intermediate carboxyspermidine (Lee, et
801 al., 2009). This alternative pathway of spermidine synthesis has been identified as the
802 dominant pathway in the human gut microbiota as well as diverse human pathogens and
803 is critical for growth in selected species (Hanfrey, et al., 2011). In host cells, SAM can
804 react with spermidine via another enzyme, spermine synthase, to produce spermine.
805 Prokaryotic cells do not appear to contain spermine synthase, but they can produce a
806 different tetra-amine compound, thermospermine, that has been detected in lower
807 eukaryotes and plants (Minguet, Vera-Sirera, Marina, Carbonell, & Blazquez, 2008).
808 Spermidine and spermine are polycationic amines that are involved in numerous
809 processes such as mitigating oxidative stress, and induction of autophagy to stimulate
810 cellular longevity (Eisenberg, et al., 2009; Yamamoto, et al., 2012).

811 The polyamine cadaverine is synthesized from lysine in a one-step reaction with
812 lysine decarboxylase (Table 1) and has been shown to provide an acid resistance
813 mechanism in *E. coli* (Le Gall, et al., 2011). However, the effects of cadaverine on the
814 colonic epithelial cells remain unknown.

815

816 **4.6. Ammonia**

817 Ammonia provides the source of nitrogen for all amino acids and is primarily
818 assimilated through either glutamate dehydrogenase or the glutamine
819 synthetase/GOGAT cycle. It is also used directly in the biosynthesis of various amino
820 acids including glutamate, glutamine, asparagine, valine, isoleucine, leucine,
821 phenylalanine, tyrosine and tryptophan (Figs. 1-4). Ammonia can be produced by the
822 microbial degradation of numerous amino acids to specific metabolites (i.e. arginine to
putrescine, tyrosine to phenol, tryptophan to indole, Table 1). Ammonia (taken as the sum of NH₃ and NH₄⁺) is found at millimolar concentrations in the large intestine luminal content of mammals including humans (Mouille, Robert, & Blachier, 2004). In humans, the luminal ammonia concentration progressively increases from the ascending to the descending colon (Macfarlane, et al., 1992), in accordance with a higher rate of protein fermentation in the distal colon. The luminal ammonia concentration in the large intestine is primarily the net result of microbiota utilization and production through amino acid deamination, urea hydrolysis, and absorption from the luminal content to the portal blood, with the unabsorbed/unmetabolized ammonia being excreted in feces (Eklou-Lawson, et al., 2009). Although relatively large amounts of ammonia can be transferred from the intestinal lumen to the bloodstream, a part of this ammonia can be metabolized by colonocytes into citrulline and glutamine (Eklou-Lawson, et al., 2009; Mouille, et al., 2004), allowing control of the intracellular ammonia concentration in colonocytes during its transfer from the luminal content to the portal bloodstream.

Ammonia inhibits mitochondrial oxygen consumption in a dose-dependent manner, leading to the concept that excessive luminal ammonia concentration behaves as a metabolic troublemaker towards colonocyte energy metabolism (Andriamihaja, et al., 2010). Accordingly, high millimolar concentrations of ammonia have been shown to markedly inhibit short-chain fatty acid oxidation in isolated colonocytes (Cremin, Fitch, & Fleming, 2003).

5. Gut microbiome features related to amino acid metabolism as a function of the subject's metabolic phenotype and the diet
putrescine, tyrosine to phenol, tryptophan to indole, Table 1). Ammonia (taken as the sum of NH₃ and NH₄⁺) is found at millimolar concentrations in the large intestine luminal content of mammals including humans (Mouille, Robert, & Blachier, 2004). In humans, the luminal ammonia concentration progressively increases from the ascending to the descending colon (Macfarlane, et al., 1992), in accordance with a higher rate of protein fermentation in the distal colon. The luminal ammonia concentration in the large intestine is primarily the net result of microbiota utilization and production through amino acid deamination, urea hydrolysis, and absorption from the luminal content to the portal blood, with the unabsorbed/unmetabolized ammonia being excreted in feces (Eklou-Lawson, et al., 2009). Although relatively large amounts of ammonia can be transferred from the intestinal lumen to the bloodstream, a part of this ammonia can be metabolized by colonocytes into citrulline and glutamine (Eklou-Lawson, et al., 2009; Mouille, et al., 2004), allowing control of the intracellular ammonia concentration in colonocytes during its transfer from the luminal content to the portal bloodstream.

Ammonia inhibits mitochondrial oxygen consumption in a dose-dependent manner, leading to the concept that excessive luminal ammonia concentration behaves as a metabolic troublemaker towards colonocyte energy metabolism (Andriamihaja, et al., 2010). Accordingly, high millimolar concentrations of ammonia have been shown to markedly inhibit short-chain fatty acid oxidation in isolated colonocytes (Cremin, Fitch, & Fleming, 2003).

5. Gut microbiome features related to amino acid metabolism as a function of the subject's metabolic phenotype and the diet
Recent metagenomic studies have allowed a detailed examination of the metabolic capacity of the mammalian gut microbiome in metabolizing nitrogenous components, particularly amino acid-related compounds. An important insight from these studies revealed that the gut microbiome contains a large enrichment of genes involved in amino acid metabolism compared to the human genome (Gill, et al., 2006), thus expanding the human metabolic capacity to form a more diverse number of metabolites.

In particular, the human microbiome had large enrichments of genes involved in the biosynthesis of lysine, phenylalanine, tyrosine, tryptophan, valine, leucine and isoleucine as well as enrichment in genes associated with the metabolism of alanine, aspartate, glutamate, histidine, methionine, glycine, serine, threonine and the urea cycle, with only slight to moderate enrichments of genes for the metabolism of other amino acids and non-protein amino acids (Gill, et al., 2006). Qin et al. (2010) also found an enrichment of genes involved in pathways such as the biosynthesis of lysine, phenylalanine, tyrosine, tryptophan, valine, leucine and isoleucine in the human gut microbiome compared to the host genome. Since biosynthetic pathways for essential amino acids do not exist in humans, it is not surprising that gut microbiota has developed a specialized set of genes and metabolic pathways for synthesizing these essential nutrients to ensure its survival.

Metagenomic sequencing analysis has shown that the metabolic phenotype (obese versus lean subjects) and the diet (e.g. high-fat diets, prebiotic intake, etc.) are associated with shifts in both specific microbiota taxonomic groups and functions of the mammalian gut microbiome, such as those involved in the metabolism of amino acids.

Recent studies comparing the short-term effects of animal- and plant-based diets on the human gut microbiota and expression of metabolic-related genes using RNA-Seq revealed an increased expression of several catabolic amino acid genes for glutamine
Recent metagenomic studies have allowed a detailed examination of the metabolic capacity of the mammalian gut microbiome in metabolizing nitrogenous components, particularly amino acid-related compounds. An important insight from these studies revealed that the gut microbiome contains a large enrichment of genes involved in amino acid metabolism compared to the human genome (Gill, et al., 2006), thus expanding the human metabolic capacity to form a more diverse number of metabolites.

In particular, the human microbiome had large enrichments of genes involved in the biosynthesis of lysine, phenylalanine, tyrosine, tryptophan, valine, leucine and isoleucine as well as enrichment in genes associated with the metabolism of alanine, aspartate, glutamate, histidine, methionine, glycine, serine, threonine and the urea cycle, with only slight to moderate enrichments of genes for the metabolism of other amino acids and non-protein amino acids (Gill, et al., 2006). Qin et al. (2010) also found an enrichment of genes involved in pathways such as the biosynthesis of lysine, phenylalanine, tyrosine, tryptophan, valine, leucine and isoleucine in the human gut microbiome compared to the host genome. Since biosynthetic pathways for essential amino acids do not exist in humans, it is not surprising that gut microbiota has developed a specialized set of genes and metabolic pathways for synthesizing these essential nutrients to ensure its survival.

Metagenomic sequencing analysis has shown that the metabolic phenotype (obese versus lean subjects) and the diet (e.g. high-fat diets, prebiotic intake, etc.) are associated with shifts in both specific microbiota taxonomic groups and functions of the mammalian gut microbiome, such as those involved in the metabolism of amino acids.

Recent studies comparing the short-term effects of animal- and plant-based diets on the human gut microbiota and expression of metabolic-related genes using RNA-Seq revealed an increased expression of several catabolic amino acid genes for glutamine
and glutamate in animal-based diets, while increased expression of biosynthetic pathways for these amino acids was observed for plant diets (David, et al., 2014). David et al. (2014) also found that KEGG modules and pathways involved in methionine and leucine biosynthesis and cysteine metabolism were significantly associated with animal diets, while histidine biosynthesis and lysine and branched-chain amino acid degradation were significantly associated with plant diets.

In studies in rodents, Turnbaugh et al. (2006) found that KEGG pathways related to lysine biosynthesis and D-alanine metabolism were significantly enriched in the pooled cecal microbiome of *ob/ob* obese mice relative to the pooled lean cecal microbiome, while the KEGG pathways involved in the metabolism of glutamate, glycine, serine, threonine, cysteine, arginine and proline and the biosynthesis of phenylalanine, tyrosine and tryptophan were depleted in the *ob/ob* mouse microbiome relative to the lean one.

Comparison of obese/lean sibling pairs of mice revealed that KEGG pathways involved in phenylalanine, tyrosine and tryptophan biosynthesis were also depleted in the cecal microbiomes of the obese mice (Turnbaugh, et al., 2006). Furthermore, using fecal samples from obese and lean human twin pairs, Turnbaugh et al. (2009) revealed a depletion of the KEGG pathway involved in tryptophan metabolism in obese twins compared to their lean counterparts. Recent studies by Everard et al. (2014) revealed that the clusters of orthologous group (COG) for amino acid transport and metabolism was enriched in both high-fat diet-fed (HFD) mice and high-fat diet-fed mice treated with the prebiotic oligofructose (HFD-Pre) compared to controls with and without prebiotic treatment, with the HFD-Pre mice yielding the highest enrichment of all groups. Nevertheless, the physiological meaning of those changes in gut microbiome amino acid metabolic pathways associated with genetically (*ob/ob*) or diet-induced (HFD) obesity remains unknown. Furthermore, to our knowledge there have not been
and glutamate in animal-based diets, while increased expression of biosynthetic pathways for these amino acids was observed for plant diets (David, et al., 2014). David et al. (2014) also found that KEGG modules and pathways involved in methionine and leucine biosynthesis and cysteine metabolism were significantly associated with animal diets, while histidine biosynthesis and lysine and branched-chain amino acid degradation were significantly associated with plant diets.

In studies in rodents, Turnbaugh et al. (2006) found that KEGG pathways related to lysine biosynthesis and D-alanine metabolism were significantly enriched in the pooled cecal microbiome of *ob/ob* obese mice relative to the pooled lean cecal microbiome, while the KEGG pathways involved in the metabolism of glutamate, glycine, serine, threonine, cysteine, arginine and proline and the biosynthesis of phenylalanine, tyrosine and tryptophan were depleted in the *ob/ob* mouse microbiome relative to the lean one.

Comparison of obese/lean sibling pairs of mice revealed that KEGG pathways involved in phenylalanine, tyrosine and tryptophan biosynthesis were also depleted in the cecal microbiomes of the obese mice (Turnbaugh, et al., 2006). Furthermore, using fecal samples from obese and lean human twin pairs, Turnbaugh et al. (2009) revealed a depletion of the KEGG pathway involved in tryptophan metabolism in obese twins compared to their lean counterparts. Recent studies by Everard et al. (2014) revealed that the clusters of orthologous group (COG) for amino acid transport and metabolism was enriched in both high-fat diet-fed (HFD) mice and high-fat diet-fed mice treated with the prebiotic oligofructose (HFD-Pre) compared to controls with and without prebiotic treatment, with the HFD-Pre mice yielding the highest enrichment of all groups. Nevertheless, the physiological meaning of those changes in gut microbiome amino acid metabolic pathways associated with genetically (*ob/ob*) or diet-induced (HFD) obesity remains unknown. Furthermore, to our knowledge there have not been
897 detailed metagenomic studies examining the effect of a high-protein (HP) diet in either
898 murine models or humans.

899 Recent metabolomic studies have provided further evidence of the role of gut
900 microbiota in amino acid metabolism. Zheng et al. (2011) compared metabolites
901 produced by the host (rat) and microbiota during antibiotic administration and also after
902 a 14-day recovery period and showed that the recovered mammalian gut microbiota
903 alters the host's systemic metabolism in terms of production of short chain fatty acids,
904 tryptophan and tyrosine and possibly indole-melatonin. Metabolomic analyses have
905 also recently suggested that individual amino acids may play key roles in diet-related
906 diseases. For example, high concentrations of branched-chain amino acids (BCAA) and
907 their respective metabolites have been linked with obesity and type 2 diabetes, while
908 aromatic amino acids and high ratios of glutamate/glutamine have also been associated
909 with type 2 diabetes (reviewed in Heinken & Thiele, 2015). Although the association of
910 BCAA and other amino acids with insulin resistance and type 2 diabetes has been well-
911 known for decades, Newgard et al. (2009) recently performed metabolomic profiling of
912 obese and lean humans to show that the addition of branched-chain amino acids (valine,
913 leucine, isoleucine) and two aromatic acids (phenylalanine and tyrosine) to a high-fat
914 diet contributes to the development of obesity-associated insulin resistance. Wang et al.
915 (2011) also carried out metabolic profiling to investigate whether specific metabolites
916 could predict diabetes development and discovered that the same five branched-chain
917 and aromatic acids were strongly associated with the onset of diabetes, thus suggesting
918 a potential role of amino acid metabolism in the pathogenesis of diet-related diabetes.
919 Although the direct role of the gut microbiota in production of these amino acids has not
920 yet been clearly defined in vivo, theoretically the gut bacteria have the capacity to
897 detailed metagenomic studies examining the effect of a high-protein (HP) diet in either
898 murine models or humans.

899 Recent metabolomic studies have provided further evidence of the role of gut
900 microbiota in amino acid metabolism. Zheng et al. (2011) compared metabolites
901 produced by the host (rat) and microbiota during antibiotic administration and also after
902 a 14-day recovery period and showed that the recovered mammalian gut microbiota
903 alters the host's systemic metabolism in terms of production of short chain fatty acids,
904 tryptophan and tyrosine and possibly indole-melatonin. Metabolomic analyses have
905 also recently suggested that individual amino acids may play key roles in diet-related
906 diseases. For example, high concentrations of branched-chain amino acids (BCAA) and
907 their respective metabolites have been linked with obesity and type 2 diabetes, while
908 aromatic amino acids and high ratios of glutamate/glutamine have also been associated
909 with type 2 diabetes (reviewed in Heinken & Thiele, 2015). Although the association of
910 BCAA and other amino acids with insulin resistance and type 2 diabetes has been well-
911 known for decades, Newgard et al. (2009) recently performed metabolomic profiling of
912 obese and lean humans to show that the addition of branched-chain amino acids (valine,
913 leucine, isoleucine) and two aromatic acids (phenylalanine and tyrosine) to a high-fat
914 diet contributes to the development of obesity-associated insulin resistance. Wang et al.
915 (2011) also carried out metabolic profiling to investigate whether specific metabolites
916 could predict diabetes development and discovered that the same five branched-chain
917 and aromatic acids were strongly associated with the onset of diabetes, thus suggesting
918 a potential role of amino acid metabolism in the pathogenesis of diet-related diabetes.
919 Although the direct role of the gut microbiota in production of these amino acids has not
920 yet been clearly defined in vivo, theoretically the gut bacteria have the capacity to
921 synthesize all of these essential amino acids and, thereby, play a role in those
922 conditions. However, more work is needed to elucidate this connection.

923 In the case of increased dietary protein intake, the amount of undigested protein that
924 is transferred to the large intestine markedly increases (Chacko & Cummings, 1988).
925 Consequently, more substrate is available for bacterial amino acid catabolism. However,
926 the consequences of this increased availability is not well described at a metabolic level
927 as only a few studies have only partially examined microbial-derived metabolite profiles
928 in HP diets by a non-targeted analytical approach in which all potential metabolites are
929 analyzed. Russell et al. (2011) demonstrated that HP diets with low and moderate
930 carbohydrate intake in humans displayed increased branched-chain fatty acids,
931 phenylacetic acid and N-nitroso compounds compared to a weight-maintenance diet
932 with moderate protein levels at the beginning of the intervention study. Reduced
933 proportions of butyrate and antioxidant phenolic acids were also detected in the HP low-
934 carbohydrate diets (Russell, et al., 2011) likely as a result of the low carbohydrate
935 intake. Those changes in fecal metabolic profiles were associated with a decrease in
936 proportions of the known butyrate-producing bacteria *Roseburia/Eubacterium rectale*
937 (Russell, et al., 2011). Other studies in humans found that H₂S fecal concentration was
938 increased in humans fed a meat-rich diet (Magee, Richardson, Hughes, & Cummings,
939 2000), and increased levels of phenol, *p*-cresol and phenylacetate have been detected in
940 urine and feces of individuals receiving HP diets (Cummings, et al., 1979; Geypens, et
941 al., 1997; Russell, et al., 2011). Increasing the amount of alimentary protein also results
942 in an increase of the luminal and fecal ammonia concentration in humans (Geypens, et
943 al., 1997). HP diets in humans also caused elevations in plasma levels and urinary
944 excretion of indoxyl sulfate and urinary excretion of indoxyl glucuronide, kynurenic
945 acid and quinolinic acid (Poesen, et al., 2015). Although several of these compounds are
921 synthesize all of these essential amino acids and, thereby, play a role in those
922 conditions. However, more work is needed to elucidate this connection.

923 In the case of increased dietary protein intake, the amount of undigested protein that
924 is transferred to the large intestine markedly increases (Chacko & Cummings, 1988).
925 Consequently, more substrate is available for bacterial amino acid catabolism. However,
926 the consequences of this increased availability is not well described at a metabolic level
927 as only a few studies have only partially examined microbial-derived metabolite profiles
928 in HP diets by a non-targeted analytical approach in which all potential metabolites are
929 analyzed. Russell et al. (2011) demonstrated that HP diets with low and moderate
930 carbohydrate intake in humans displayed increased branched-chain fatty acids,
931 phenylacetic acid and N-nitroso compounds compared to a weight-maintenance diet
932 with moderate protein levels at the beginning of the intervention study. Reduced
933 proportions of butyrate and antioxidant phenolic acids were also detected in the HP low-
934 carbohydrate diets (Russell, et al., 2011) likely as a result of the low carbohydrate
935 intake. Those changes in fecal metabolic profiles were associated with a decrease in
936 proportions of the known butyrate-producing bacteria *Roseburia/Eubacterium rectale*
937 (Russell, et al., 2011). Other studies in humans found that H₂S fecal concentration was
938 increased in humans fed a meat-rich diet (Magee, Richardson, Hughes, & Cummings,
939 2000), and increased levels of phenol, *p*-cresol and phenylacetate have been detected in
940 urine and feces of individuals receiving HP diets (Cummings, et al., 1979; Geypens, et
941 al., 1997; Russell, et al., 2011). Increasing the amount of alimentary protein also results
942 in an increase of the luminal and fecal ammonia concentration in humans (Geypens, et
943 al., 1997). HP diets in humans also caused elevations in plasma levels and urinary
944 excretion of indoxyl sulfate and urinary excretion of indoxyl glucuronide, kynurenic
945 acid and quinolinic acid (Poesen, et al., 2015). Although several of these compounds are
strictly produced only by gut microbiota, no study has effectively examined the direct role of the gut microbiota in the production of these metabolites.

In murine models, HP diets have been shown to modify the composition and the diversity of the colonic microbiota in rats, with associated changes in the total amount of ammonia, SCFAs, H₂S, branched-chain fatty acids, ethanol and several organic acids (L- and D-lactate, succinate) in the colonic luminal content (Liu, et al., 2014). Although this experiment did not determine the direct role of microbiota composition and increased substrate availability in the changes observed after HP diet ingestion, it appears that both parameters are likely to be involved in such changes. A metabolomic analysis comparing a HP, low-carbohydrate (HPLC) diet to a moderate protein/carbohydrate (MPMC) diet in a rat model revealed that the HPLC diet induced weight loss and reduced adipose weight, and the plasma metabolites glucose, insulin, triglyceride, linoleate, palmitate, α-glycerophosphate and pyroglutamic acid and caused a significant increase in several plasma metabolites (i.e. urea, pyruvate, α-tocopherol, 2-oxoisocaproate, and β-hydroxybutyrate) (Mu, Yang, Luo, & Zhu, 2015). In the plasma of mice, increases in tryptophan and other aromatic acid-derived metabolites such as indole-3-acetic acid, p-cresyl glucuronide, phenyl sulfate and phenylacetic acid were detected in HP diets (Poesen, et al., 2015). In the urinary metabolite profile, the HPLC urinary metabolite profile showed an increase in the fatty acids palmitate and stearate and a reduction of pantothenate and the TCA cycle intermediates citrate, 2-ketoglutarate and malate (Mu, et al., 2015). In addition to murine models, study designs in humans still need to be refined to integrate data from complementary functional omics-technologies to progress in the understanding of the flow of metabolites between the microbiota and the host derived from dietary protein and their physiological consequences on the metabolic phenotype.
strictly produced only by gut microbiota, no study has effectively examined the direct role of the gut microbiota in the production of these metabolites.

In murine models, HP diets have been shown to modify the composition and the diversity of the colonic microbiota in rats, with associated changes in the total amount of ammonia, SCFAs, H₂S, branched-chain fatty acids, ethanol and several organic acids (L- and D-lactate, succinate) in the colonic luminal content (Liu, et al., 2014). Although this experiment did not determine the direct role of microbiota composition and increased substrate availability in the changes observed after HP diet ingestion, it appears that both parameters are likely to be involved in such changes. A metabolomic analysis comparing a HP, low-carbohydrate (HPLC) diet to a moderate protein/carbohydrate (MPMC) diet in a rat model revealed that the HPLC diet induced weight loss and reduced adipose weight, and the plasma metabolites glucose, insulin, triglyceride, linoleate, palmitate, α-glycerophosphate and pyroglutamic acid and caused a significant increase in several plasma metabolites (i.e. urea, pyruvate, α-tocopherol, 2-oxoisocaproate, and β-hydroxybutyrate) (Mu, Yang, Luo, & Zhu, 2015). In the plasma of mice, increases in tryptophan and other aromatic acid-derived metabolites such as indole-3-acetic acid, p-cresyl glucuronide, phenyl sulfate and phenylacetic acid were detected in HP diets (Poesen, et al., 2015). In the urinary metabolite profile, the HPLC urinary metabolite profile showed an increase in the fatty acids palmitate and stearate and a reduction of pantothenate and the TCA cycle intermediates citrate, 2-ketoglutarate and malate (Mu, et al., 2015). In addition to murine models, study designs in humans still need to be refined to integrate data from complementary functional omics-technologies to progress in the understanding of the flow of metabolites between the microbiota and the host derived from dietary protein and their physiological consequences on the metabolic phenotype.
971

972 **6. Impact of protein intake levels on health-related outcomes: possible**

973 **contribution of gut microbiota metabolic pathways**

974 Some of the metabolites produced by the microbiota from amino acids have been
975 shown to be active on numerous host functions as illustrated above. These results
976 suggest that some of the effects of HP diets on host metabolism and physiology may
977 involve some of these metabolites and, therefore, microbiota-mediated metabolic
978 pathways. In the following sections, the effects of high dietary protein intake observed
979 in human and animal studies are summarized and suggest mechanisms that might
980 implicate the gut bacteria-derived metabolites (Fig. 6).

981

982 **6.1. HP diet-induced satiety**

983 Two recent meta-analyses of clinical trials concluded that, when compared to
984 normoproteic (NP) diet, a HP diet modestly reduces body weight, blood pressure, fat
985 mass and triglyceride levels while sparing fat-free mass (Santesso, et al., 2012;
986 Wycherley, Moran, Clifton, Noakes, & Brinkworth, 2012). Those effects are mainly
987 attributed to dietary protein induced satiety (Westerterp-Plantenga, et al., 2009). In
988 humans, HP diet-induced satiety is associated with an increase of PYY plasma levels
989 (Batterham, et al., 2006). Implication of gut microbiota in those mechanisms, however,
990 has not been investigated yet. Nevertheless, SCFAs, indole, tryptamine and tyramine are
991 compounds produced by the gut microbiota from amino acid precursors and are
992 candidates for mediating the effects of HP diet on enteroendocrine cell hormone
993 secretion with an impact on satiety (Figure 6).

994

995 **6.2. HP diet effects on the intestinal mucosa**
971

972 **6. Impact of protein intake levels on health-related outcomes: possible**

973 **contribution of gut microbiota metabolic pathways**

974 Some of the metabolites produced by the microbiota from amino acids have been
975 shown to be active on numerous host functions as illustrated above. These results
976 suggest that some of the effects of HP diets on host metabolism and physiology may
977 involve some of these metabolites and, therefore, microbiota-mediated metabolic
978 pathways. In the following sections, the effects of high dietary protein intake observed
979 in human and animal studies are summarized and suggest mechanisms that might
980 implicate the gut bacteria-derived metabolites (Fig. 6).

981

982 **6.1. HP diet-induced satiety**

983 Two recent meta-analyses of clinical trials concluded that, when compared to
984 normoproteic (NP) diet, a HP diet modestly reduces body weight, blood pressure, fat
985 mass and triglyceride levels while sparing fat-free mass (Santesso, et al., 2012;
986 Wycherley, Moran, Clifton, Noakes, & Brinkworth, 2012). Those effects are mainly
987 attributed to dietary protein induced satiety (Westerterp-Plantenga, et al., 2009). In
988 humans, HP diet-induced satiety is associated with an increase of PYY plasma levels
989 (Batterham, et al., 2006). Implication of gut microbiota in those mechanisms, however,
990 has not been investigated yet. Nevertheless, SCFAs, indole, tryptamine and tyramine are
991 compounds produced by the gut microbiota from amino acid precursors and are
992 candidates for mediating the effects of HP diet on enteroendocrine cell hormone
993 secretion with an impact on satiety (Figure 6).

994

995 **6.2. HP diet effects on the intestinal mucosa**
996 In the rat model, HP diet consumption for 15 days resulted in a marked reduction in
997 the height of the colonocyte brush-border when compared with control animals
998 receiving a NP/hyperglucidic isocaloric diet (Andriamihaja, et al., 2010). This
999 morphological change could be related to lower energy efficiency in HP rat colonocytes
1000 due to proton leaks in the mitochondrial inner membrane (Andriamihaja, et al., 2010). A
1001 reduction of the mucosal myeloperoxidase activity (representative of neutrophil
1002 infiltration) together with a down-regulation of mucosal $T_{helper}$ cytokines was measured
1003 in the ileum of rats receiving a HP diet (Lan, et al., 2015). This decreased inflammatory
1004 status was associated with a hyperplasia of mucus-producing cells concomitant with an
1005 increased expression of Muc2 at both the gene and protein levels (Lan, et al., 2015). A
1006 HP diet also induced DNA damages in rat colonocytes, in association with an increase
1007 in the bacterially-derived genotoxic metabolite *p*-cresol (Toden, Bird, Topping, &
1008 Conlon, 2005).

1009 In an intervention study comparing volunteers receiving either HP diets or low
1010 protein isocaloric diet for two weeks, no evidence was found for a role of protein
1011 fermentation in gut toxicity in healthy human subjects despite identification of several
1012 metabolites in fecal water with presumably cytotoxic and genotoxic effects towards
1013 colonic epithelial cells (Windey, et al., 2012). SCFA, $H_2S$, *p*-cresol and ammonia are
1014 metabolites produced from amino acids that could interfere with colonocyte energy
1015 metabolism and mucin secretion and may mediate the effects of HP diet on the
1016 intestinal mucosa. These metabolites could partly result from the activity of gut
1017 microbiota (Figure 6). However, the consequences of a HP intake on large intestine
1018 mucosal health have never been directly investigated in humans.

1019

1020 **6.2.3. HP diet effects in inflammatory bowel diseases**
996 In the rat model, HP diet consumption for 15 days resulted in a marked reduction in
997 the height of the colonocyte brush-border when compared with control animals
998 receiving a NP/hyperglucidic isocaloric diet (Andriamihaja, et al., 2010). This
999 morphological change could be related to lower energy efficiency in HP rat colonocytes
1000 due to proton leaks in the mitochondrial inner membrane (Andriamihaja, et al., 2010). A
1001 reduction of the mucosal myeloperoxidase activity (representative of neutrophil
1002 infiltration) together with a down-regulation of mucosal $T_{helper}$ cytokines was measured
1003 in the ileum of rats receiving a HP diet (Lan, et al., 2015). This decreased inflammatory
1004 status was associated with a hyperplasia of mucus-producing cells concomitant with an
1005 increased expression of Muc2 at both the gene and protein levels (Lan, et al., 2015). A
1006 HP diet also induced DNA damages in rat colonocytes, in association with an increase
1007 in the bacterially-derived genotoxic metabolite *p*-cresol (Toden, Bird, Topping, &
1008 Conlon, 2005).

1009 In an intervention study comparing volunteers receiving either HP diets or low
1010 protein isocaloric diet for two weeks, no evidence was found for a role of protein
1011 fermentation in gut toxicity in healthy human subjects despite identification of several
1012 metabolites in fecal water with presumably cytotoxic and genotoxic effects towards
1013 colonic epithelial cells (Windey, et al., 2012). SCFA, $H_2S$, *p*-cresol and ammonia are
1014 metabolites produced from amino acids that could interfere with colonocyte energy
1015 metabolism and mucin secretion and may mediate the effects of HP diet on the
1016 intestinal mucosa. These metabolites could partly result from the activity of gut
1017 microbiota (Figure 6). However, the consequences of a HP intake on large intestine
1018 mucosal health have never been directly investigated in humans.

1019

1020 **6.2.3. HP diet effects in inflammatory bowel diseases**
1021 SCFAs, H₂S, indolic compounds, serotonin and histamine are all amino acid-derived
1022 bacterial metabolites which are likely to interfere with the process of mucosal
1023 inflammation due to their effects on the mucosal immune response and on the
1024 maintaining of the epithelial barrier function (Figure 6). As reviewed by Halmos and
1025 Gibson (2015), only a few studies have examined the impact of the level of protein
1026 consumption on the risk of inflammatory bowel disease or risk of relapse in diagnosed
1027 patients. Jowett et al. (2004) reported an increased risk of relapse over 1 year in
1028 ulcerative colitis patients with the highest meat and protein consumption. A HP diet was
1029 also associated with an increased risk of incident inflammatory bowel diseases
1030 (Jantchou, Morois, Clavel-Chapelon, Boutron-Ruault, & Carbonnel, 2010), while Shoda
1031 et al. (1996) demonstrated that increased consumption of animal protein was related to
1032 increased incidence of Crohn's disease. However, another study did not find any
1033 association between elevated protein intake and development of ulcerative colitis (Hart,
1034 et al., 2008).

1035

1036 **6.2.4. HP diet effects in colorectal cancer**

1037 Experimental studies with animal models of chemically-induced colonic preneoplastic
1038 and neoplastic lesions have shown that dietary protein can influence the colonic
carcinogenesis process, depending on their quantity and quality (McIntosh & Le Leu,
1039 2001). The production of amino-acid derived genotoxic metabolites produced by the
1040 microbiota (e.g. *p*-cresol) could be implicated in this process. However, the complexity
1041 of the Western diet makes the identification of alimentary compounds that impact the
1042 risk of colorectal cancer a difficult task. In this context, it has been proposed that the
1043 positive association between high consumption of red and processed meat and
1044 colorectal cancer found in some epidemiological studies, may result from both the
1045
1021 SCFAs, H₂S, indolic compounds, serotonin and histamine are all amino acid-derived
1022 bacterial metabolites which are likely to interfere with the process of mucosal
1023 inflammation due to their effects on the mucosal immune response and on the
1024 maintaining of the epithelial barrier function (Figure 6). As reviewed by Halmos and
1025 Gibson (2015), only a few studies have examined the impact of the level of protein
1026 consumption on the risk of inflammatory bowel disease or risk of relapse in diagnosed
1027 patients. Jowett et al. (2004) reported an increased risk of relapse over 1 year in
1028 ulcerative colitis patients with the highest meat and protein consumption. A HP diet was
1029 also associated with an increased risk of incident inflammatory bowel diseases
1030 (Jantchou, Morois, Clavel-Chapelon, Boutron-Ruault, & Carbonnel, 2010), while Shoda
1031 et al. (1996) demonstrated that increased consumption of animal protein was related to
1032 increased incidence of Crohn's disease. However, another study did not find any
1033 association between elevated protein intake and development of ulcerative colitis (Hart,
1034 et al., 2008).

1035

1036 **6.2.4. HP diet effects in colorectal cancer**

1037 Experimental studies with animal models of chemically-induced colonic preneoplastic
1038 and neoplastic lesions have shown that dietary protein can influence the colonic
carcinogenesis process, depending on their quantity and quality (McIntosh & Le Leu,
1039 2001). The production of amino-acid derived genotoxic metabolites produced by the
1040 microbiota (e.g. *p*-cresol) could be implicated in this process. However, the complexity
1041 of the Western diet makes the identification of alimentary compounds that impact the
1042 risk of colorectal cancer a difficult task. In this context, it has been proposed that the
1043 positive association between high consumption of red and processed meat and
1044 colorectal cancer found in some epidemiological studies, may result from both the
1045
1046 composition of meat (e.g. heme and protein) and from compounds generated by the
1047 cooking process (Kim, Coelho, & Blachier, 2013). In support of this hypothesis, a
1048 recent study has shown that hydrogen sulfide, which is produced by the gut microbiota
1049 from L-cysteine released from dietary and endogenous protein, can drive mucin
1050 denaturation and possibly increase the access of heme (a cytotoxic and genotoxic
1051 compound) to colonic epithelial cells (Ijssennagger, et al., 2015).

1052

1053 **6.3. HP diet effects on kidney function**

1054 It was concluded from a systematic review that indoxyl sulfate and *p*-cresyl sulfate
1055 are toxic for kidneys notably through reactive-oxygen species (ROS) generation
1056 (Vanholder, Schepers, Pletinck, Nagler, & Glorieux, 2014). Moreover, it has been
1057 proposed that *p*-cresol level in the blood is an indicator of CKD severity (Bammens,
1058 Evenepoel, Keuleers, Verbeke, & Vanrenterghem, 2006). Recently, it has been shown
1059 that CKD alters gut microbiota composition (Vaziri, Wong, et al., 2013) together with
1060 an increased bacterial metabolic capacity for nitrogen utilization, as evidenced by the
1061 increased abundance of urease, indole- and *p*-cresol-forming enzymes in CKD patients
1062 (Wong, et al., 2014). Indeed, *p*-cresyl sulfate (or glucuronide) and indoxyl sulfate are
1063 known conjugates of the gut microbiota-produced metabolites *p*-cresol and indole,
1064 respectively, and are elevated in the serum of chronic kidney diseases (CKD) patients
1065 (Vanholder & Glorieux, 2015). Furthermore, the elevated uremia in CKD induces an
1066 important diffusion of urea into the intestinal lumen, and it has been shown that elevated
1067 urea concentration impairs intestinal barrier function and induces inflammation in the
1068 digestive tract, most likely through excessive ammonia production by bacterial ureases
1069 (Vaziri, Yuan, & Norris, 2013), raising the view that CKD may impact gut health.
1070 Those events may play a causative role in the establishment of CKD-associated
1046 composition of meat (e.g. heme and protein) and from compounds generated by the
1047 cooking process (Kim, Coelho, & Blachier, 2013). In support of this hypothesis, a
1048 recent study has shown that hydrogen sulfide, which is produced by the gut microbiota
1049 from L-cysteine released from dietary and endogenous protein, can drive mucin
1050 denaturation and possibly increase the access of heme (a cytotoxic and genotoxic
1051 compound) to colonic epithelial cells (Ijssennagger, et al., 2015).

1052

1053 **6.3. HP diet effects on kidney function**

1054 It was concluded from a systematic review that indoxyl sulfate and *p*-cresyl sulfate
1055 are toxic for kidneys notably through reactive-oxygen species (ROS) generation
1056 (Vanholder, Schepers, Pletinck, Nagler, & Glorieux, 2014). Moreover, it has been
1057 proposed that *p*-cresol level in the blood is an indicator of CKD severity (Bammens,
1058 Evenepoel, Keuleers, Verbeke, & Vanrenterghem, 2006). Recently, it has been shown
1059 that CKD alters gut microbiota composition (Vaziri, Wong, et al., 2013) together with
1060 an increased bacterial metabolic capacity for nitrogen utilization, as evidenced by the
1061 increased abundance of urease, indole- and *p*-cresol-forming enzymes in CKD patients
1062 (Wong, et al., 2014). Indeed, *p*-cresyl sulfate (or glucuronide) and indoxyl sulfate are
1063 known conjugates of the gut microbiota-produced metabolites *p*-cresol and indole,
1064 respectively, and are elevated in the serum of chronic kidney diseases (CKD) patients
1065 (Vanholder & Glorieux, 2015). Furthermore, the elevated uremia in CKD induces an
1066 important diffusion of urea into the intestinal lumen, and it has been shown that elevated
1067 urea concentration impairs intestinal barrier function and induces inflammation in the
1068 digestive tract, most likely through excessive ammonia production by bacterial ureases
1069 (Vaziri, Yuan, & Norris, 2013), raising the view that CKD may impact gut health.
1070 Those events may play a causative role in the establishment of CKD-associated
1071 dysbiosis. Moreover, CKD patients generally have a lower fiber intake, prolonged transit time and protein malabsorption, all of which favor protein fermentation in the gut (Evenepoel, et al., 2009).

1074 Moderate protein intake (0.6-0.8 g protein/kg/day) has been demonstrated to be beneficial for CKD patients (Fouque, Pelletier, Mafra, & Chauveau, 2011). Reduced renal exposure to gut microbiota amino acid-derived metabolites through control of protein intake may contribute to better health outcomes. In healthy subjects, HP diets increase glomerular filtration rate, but the long-term consequences for kidney health remains unclear (Marckmann, Osther, Pedersen, & Jespersen, 2015). Thus, renal consequences of increased colonic protein fermentation should be further studied in healthy subjects.

1082

## 6.4. Strategies to limit toxicity associated with the control of protein fermentation

1083 Increased fermentable carbohydrate intake is one of the nutritional strategies that may help to limit adverse effects of protein fermentation, as demonstrated in an intervention study which has shown that dietary resistant starch lowers excretion of ammonia and phenols (Birkett, et al., 1996). In healthy subjects, *p*-cresyl sulfate and indoxyl sulfate levels are markedly lower in vegetarians than in omnivores, likely because of a higher fiber intake and lower protein intake (Patel, Luo, Plummer, Hostetter, & Meyer, 2012). In rats, supplementation with resistant starch protects against HP diet-induced DNA damages in colonocytes in association with decreased excretion of *p*-cresol (Toden, Belobrajdic, Bird, Topping, & Conlon, 2010). Resistant carbohydrate supplementation also attenuates renal injury possibly through a decreased production of amino acid-derived metabolites by the microbiota (Vaziri, et al., 2014). In CKD patients, improved disease markers (including lower *p*-cresyl sulfate) were
1071 dysbiosis. Moreover, CKD patients generally have a lower fiber intake, prolonged transit time and protein malabsorption, all of which favor protein fermentation in the gut (Evenepoel, et al., 2009).

1074 Moderate protein intake (0.6-0.8 g protein/kg/day) has been demonstrated to be beneficial for CKD patients (Fouque, Pelletier, Mafra, & Chauveau, 2011). Reduced renal exposure to gut microbiota amino acid-derived metabolites through control of protein intake may contribute to better health outcomes. In healthy subjects, HP diets increase glomerular filtration rate, but the long-term consequences for kidney health remains unclear (Marckmann, Osther, Pedersen, & Jespersen, 2015). Thus, renal consequences of increased colonic protein fermentation should be further studied in healthy subjects.

1082

## 6.4. Strategies to limit toxicity associated with the control of protein fermentation

1084 Increased fermentable carbohydrate intake is one of the nutritional strategies that may help to limit adverse effects of protein fermentation, as demonstrated in an intervention study which has shown that dietary resistant starch lowers excretion of ammonia and phenols (Birkett, et al., 1996). In healthy subjects, *p*-cresyl sulfate and indoxyl sulfate levels are markedly lower in vegetarians than in omnivores, likely because of a higher fiber intake and lower protein intake (Patel, Luo, Plummer, Hostetter, & Meyer, 2012). In rats, supplementation with resistant starch protects against HP diet-induced DNA damages in colonocytes in association with decreased excretion of *p*-cresol (Toden, Belobrajdic, Bird, Topping, & Conlon, 2010). Resistant carbohydrate supplementation also attenuates renal injury possibly through a decreased production of amino acid-derived metabolites by the microbiota (Vaziri, et al., 2014). In CKD patients, improved disease markers (including lower *p*-cresyl sulfate) were
1096 observed after resistant starch supplementation (Vanholder & Glorieux, 2015).

1097 Unfortunately, CKD patients are advised to restrict their fruit and vegetable intake to
1098 avoid potassium overload, thus excluding one of the main fiber sources. In that context,
1099 the use of fibers may be an efficient tool to reduce protein fermentation (De Preter,
1100 Hamer, Windey, & Verbeke, 2011). This has been demonstrated in human studies with
1101 isotope-labeled biomarkers showing that fiber (lactulose) efficiently lowered ammonia
1102 and *p*-cresol production by the gut microbiota (De Preter, et al., 2004).

1103 Consumption of specific probiotic bacteria belonging to the genera *Lactobacillus* and
1104 *Bifidobacterium* has also been shown to decrease urinary *p*-cresol although to a lesser
1105 extent than with prebiotics, and with no effect on ammonia (De Preter, et al., 2004;
1106 Wutzke, Lotz, & Zipprich, 2010). In CKD patients, some probiotic bacteria used to
1107 reduce the formation of gut-derived toxins have been investigated, but the level of
1108 evidence remains low (Rossi, et al., 2014). Combining pre- and probiotics (known as
1109 “synbiotics”) has also been considered an attractive approach, but requires further
1110 clinical investigation (De Preter, et al., 2007).

1111 Other strategies such as elementary nutrition (free amino acid supplementation) or
1112 the use of substrates that bind deleterious compounds derived from amino-acid
1113 fermentation (e.g. zinc that binds H₂S) have been proposed to minimize their effects in
1114 medical conditions (e.g. CKD patients) (Mimoun, et al., 2012), but are out of the scope
1115 of this review.

1116

1117

1118 **7. Conclusion and perspectives.**

1119 Dietary protein metabolism is the result of the interplay between host and gut
1120 bacterial metabolic pathways but their respective roles and contributions to host
1096 observed after resistant starch supplementation (Vanholder & Glorieux, 2015).

1097 Unfortunately, CKD patients are advised to restrict their fruit and vegetable intake to
1098 avoid potassium overload, thus excluding one of the main fiber sources. In that context,
1099 the use of fibers may be an efficient tool to reduce protein fermentation (De Preter,
1100 Hamer, Windey, & Verbeke, 2011). This has been demonstrated in human studies with
1101 isotope-labeled biomarkers showing that fiber (lactulose) efficiently lowered ammonia
1102 and *p*-cresol production by the gut microbiota (De Preter, et al., 2004).

1103 Consumption of specific probiotic bacteria belonging to the genera *Lactobacillus* and
1104 *Bifidobacterium* has also been shown to decrease urinary *p*-cresol although to a lesser
1105 extent than with prebiotics, and with no effect on ammonia (De Preter, et al., 2004;
1106 Wutzke, Lotz, & Zipprich, 2010). In CKD patients, some probiotic bacteria used to
1107 reduce the formation of gut-derived toxins have been investigated, but the level of
1108 evidence remains low (Rossi, et al., 2014). Combining pre- and probiotics (known as
1109 “synbiotics”) has also been considered an attractive approach, but requires further
1110 clinical investigation (De Preter, et al., 2007).

1111 Other strategies such as elementary nutrition (free amino acid supplementation) or
1112 the use of substrates that bind deleterious compounds derived from amino-acid
1113 fermentation (e.g. zinc that binds H₂S) have been proposed to minimize their effects in
1114 medical conditions (e.g. CKD patients) (Mimoun, et al., 2012), but are out of the scope
1115 of this review.

1116

1117

1118 **7. Conclusion and perspectives.**

1119 Dietary protein metabolism is the result of the interplay between host and gut
1120 bacterial metabolic pathways but their respective roles and contributions to host
1121 physiology and metabolic health remain undefined. Several gut microbiota-derived
1122 amino acid metabolites may theoretically have both beneficial and deleterious
1123 physiological consequences on host cells and tissues, but so far their effects have only
1124 been tested individually (i.e. in un-physiological conditions). The use of global-scale
1125 omics technologies in tightly-controlled intervention studies will help to identify the
1126 specific gut microbial metabolic pathways activated in response to dietary protein and
1127 to disentangle the flow of metabolites between the host and the gut microbiota and their
1128 contribution to host physiology. Moreover, since human diets consist of a wide range of
1129 different protein sources (plant and animal) with different characteristics (digestibility,
1130 amino acid composition); these differences also need to be taken into consideration for
1131 interpretation of metadata obtained in the context of a varied diet. These future studies
1132 will represent an important and necessary step for a better understanding of the complex
1133 interplay between diet, microbiota and host metabolism and physiology and will
1134 contribute to informing microbiome-based dietary recommendations.

1135

1136

1137 Acknowledgements

1138 This work is supported by the European Union's Seventh Framework Program under
1139 the grant agreement no 613979 (MyNewGut).

1140

1141

1142

1143

1144

1145
1121 physiology and metabolic health remain undefined. Several gut microbiota-derived
1122 amino acid metabolites may theoretically have both beneficial and deleterious
1123 physiological consequences on host cells and tissues, but so far their effects have only
1124 been tested individually (i.e. in un-physiological conditions). The use of global-scale
1125 omics technologies in tightly-controlled intervention studies will help to identify the
1126 specific gut microbial metabolic pathways activated in response to dietary protein and
1127 to disentangle the flow of metabolites between the host and the gut microbiota and their
1128 contribution to host physiology. Moreover, since human diets consist of a wide range of
1129 different protein sources (plant and animal) with different characteristics (digestibility,
1130 amino acid composition); these differences also need to be taken into consideration for
1131 interpretation of metadata obtained in the context of a varied diet. These future studies
1132 will represent an important and necessary step for a better understanding of the complex
1133 interplay between diet, microbiota and host metabolism and physiology and will
1134 contribute to informing microbiome-based dietary recommendations.

1135

1136

1137 Acknowledgements

1138 This work is supported by the European Union's Seventh Framework Program under
1139 the grant agreement no 613979 (MyNewGut).

1140

1141

1142

1143

1144

1145
1146 References

1147 Abrahams, G. L., & Abratt, V. R. (1998). The NADH-dependent glutamate

1148 dehydrogenase enzyme of *Bacteroides fragilis* Bfl is induced by peptides in the

1149 growth medium. *Microbiology*, **144** ( Pt 6 ), 1659-1667.

1150 Alifano, P., Fani, R., Lio, P., Lazcano, A., Bazzicalupo, M., Carlomagno, M. S., &

1151 Bruni, C. B. (1996). Histidine biosynthetic pathway and genes: structure,

1152 regulation, and evolution. *Microbiol Rev*, **60**, 44-69.

1153 Allison, M. J., Baetz, A. L., & Wiegel, J. (1984). Alternative pathways for biosynthesis

1154 of leucine and other amino acids in *Bacteroides ruminicola* and *Bacteroides*

1155 *fragilis*. *Appl Environ Microbiol*, **48**, 1111-1117.

1156 Andriamihaja, M., Davila, A. M., Eklou-Lawson, M., Petit, N., Delpal, S., Allek, F.,

1157 Blais, A., Delteil, C., Tome, D., & Blachier, F. (2010). Colon luminal content

1158 and epithelial cell morphology are markedly modified in rats fed with a high-

1159 protein diet. *Am J Physiol Gastrointest Liver Physiol*, **299**, G1030-1037.

1160 Andriamihaja, M., Lan, A., Beaumont, M., Audebert, M., Wong, X., Yamada, K., Yin,

1161 Y., Tome, D., Carrasco-Pozo, C., Gotteland, M., Kong, X., & Blachier, F.

1162 (2015). The deleterious metabolic and genotoxic effects of the bacterial

1163 metabolite *p*-cresol on colonic epithelial cells. *Free Radic Biol Med*, **85**, 219-

1164 227.

1165 Awano, N., Wada, M., Mori, H., Nakamori, S., & Takagi, H. (2005). Identification and

1166 functional analysis of *Escherichia coli* cysteine desulfhydrases. *Appl Environ*

1167 *Microbiol*, **71**, 4149-4152.

1168 Bammens, B., Evenepoel, P., Keuleers, H., Verbeke, K., & Vanrenterghem, Y. (2006).

1169 Free serum concentrations of the protein-bound retention solute *p*-cresol predict

1170 mortality in hemodialysis patients. *Kidney Int*, **69**, 1081-1087.
1146 **References**

1147 Abrahams, G. L., & Abratt, V. R. (1998). The NADH-dependent glutamate

1148 dehydrogenase enzyme of *Bacteroides fragilis* Bfl is induced by peptides in the

1149 growth medium. *Microbiology*, 144 ( Pt 6 ), 1659-1667.

1150 Alifano, P., Fani, R., Lio, P., Lazcano, A., Bazzicalupo, M., Carlomagno, M. S., &

1151 Bruni, C. B. (1996). Histidine biosynthetic pathway and genes: structure,

1152 regulation, and evolution. *Microbiol Rev*, *60*, 44-69.

1153 Allison, M. J., Baetz, A. L., & Wiegel, J. (1984). Alternative pathways for biosynthesis

1154 of leucine and other amino acids in *Bacteroides ruminicola* and *Bacteroides*

1155 *fragilis*. *Appl Environ Microbiol*, *48*, 1111-1117.

1156 Andriamihaja, M., Davila, A. M., Eklou-Lawson, M., Petit, N., Delpal, S., Allek, F.,

1157 Blais, A., Delteil, C., Tome, D., & Blachier, F. (2010). Colon luminal content

1158 and epithelial cell morphology are markedly modified in rats fed with a high-

1159 protein diet. *Am J Physiol Gastrointest Liver Physiol*, *299*, G1030-1037.

1160 Andriamihaja, M., Lan, A., Beaumont, M., Audebert, M., Wong, X., Yamada, K., Yin,

1161 Y., Tome, D., Carrasco-Pozo, C., Gotteland, M., Kong, X., & Blachier, F.

1162 (2015). The deleterious metabolic and genotoxic effects of the bacterial

1163 metabolite *p*-cresol on colonic epithelial cells. *Free Radic Biol Med*, *85*, 219-

1164 227.

1165 Awano, N., Wada, M., Mori, H., Nakamori, S., & Takagi, H. (2005). Identification and

1166 functional analysis of *Escherichia coli* cysteine desulfhydrases. *Appl Environ*

1167 *Microbiol*, *71*, 4149-4152.

1168 Bammens, B., Evenepoel, P., Keuleers, H., Verbeke, K., & Vanrenterghem, Y. (2006).

1169 Free serum concentrations of the protein-bound retention solute *p*-cresol predict

1170 mortality in hemodialysis patients. *Kidney Int*, *69*, 1081-1087.
1171 Bansal, T., Alaniz, R. C., Wood, T. K., & Jayaraman, A. (2010). The bacterial signal
1172 indole increases epithelial-cell tight-junction resistance and attenuates indicators
1173 of inflammation. *Proc Natl Acad Sci U S A*, *107*, 228-233.

1174 Baronio, D., Gonchoroski, T., Castro, K., Zanatta, G., Gottfried, C., & Riesgo, R.
1175 (2014). Histaminergic system in brain disorders: lessons from the translational
1176 approach and future perspectives. *Ann Gen Psychiatry*, *13*, 34.

1177 Batterham, R. L., Heffron, H., Kapoor, S., Chivers, J. E., Chandarana, K., Herzog, H.,
1178 Le Roux, C. W., Thomas, E. L., Bell, J. D., & Withers, D. J. (2006). Critical role
1179 for peptide YY in protein-mediated satiation and body-weight regulation. *Cell
1180 Metab*, *4*, 223-233.

1181 Beasley, F. C., Marolda, C. L., Cheung, J., Buac, S., & Heinrichs, D. E. (2011).
1182 *Staphylococcus aureus* transporters Hts, Sir, and Sst capture iron liberated from
1183 human transferrin by Staphyloferrin A, Staphyloferrin B, and catecholamine
1184 stress hormones, respectively, and contribute to virulence. *Infect Immun*, *79*,
1185 2345-2355.

1186 Beaumont, M., Andriamihaja, M., Lan, A., Khodorova, N., Audebert, M., Blouin, J. M.,
1187 Grauso, M., Lancha, L., Benetti, P. H., Benamouzig, R., Tome, D., Bouillaud,
1188 F., Davila, A. M., & Blachier, F. (2016). Detrimental effects for colonocytes of
1189 an increased exposure to luminal hydrogen sulfide: The adaptive response. *Free
1190 Radic Biol Med*, *93*, 155-164.

1191 Belitsky, B. R. (2002). Chapter 16: Biosynthesis of amino acids of the glutamate and
1192 aspartate families, alanine, and polyamines. In A. Sonenshein, R. Losick & J.
1193 Hoch (Eds.), *Bacillus subtilis and Its Closest Relatives*. Washington, D.C.: ASM
1194 Press.
1171 Bansal, T., Alaniz, R. C., Wood, T. K., & Jayaraman, A. (2010). The bacterial signal
1172 indole increases epithelial-cell tight-junction resistance and attenuates indicators
1173 of inflammation. *Proc Natl Acad Sci U S A*, **107**, 228-233.

1174 Baronio, D., Gonchoroski, T., Castro, K., Zanatta, G., Gottfried, C., & Riesgo, R.
1175 (2014). Histaminergic system in brain disorders: lessons from the translational
1176 approach and future perspectives. *Ann Gen Psychiatry*, **13**, 34.

1177 Batterham, R. L., Heffron, H., Kapoor, S., Chivers, J. E., Chandarana, K., Herzog, H.,
1178 Le Roux, C. W., Thomas, E. L., Bell, J. D., & Withers, D. J. (2006). Critical role
1179 for peptide YY in protein-mediated satiation and body-weight regulation. *Cell
1180 Metab*, **4**, 223-233.

1181 Beasley, F. C., Marolda, C. L., Cheung, J., Buac, S., & Heinrichs, D. E. (2011).
1182 *Staphylococcus aureus* transporters Hts, Sir, and Sst capture iron liberated from
1183 human transferrin by Staphyloferrin A, Staphyloferrin B, and catecholamine
1184 stress hormones, respectively, and contribute to virulence. *Infect Immun*, **79**,
1185 2345-2355.

1186 Beaumont, M., Andriamihaja, M., Lan, A., Khodorova, N., Audebert, M., Blouin, J. M.,
1187 Grauso, M., Lancha, L., Benetti, P. H., Benamouzig, R., Tome, D., Bouillaud,
1188 F., Davila, A. M., & Blachier, F. (2016). Detrimental effects for colonocytes of
1189 an increased exposure to luminal hydrogen sulfide: The adaptive response. *Free
1190 Radic Biol Med*, **93**, 155-164.

1191 Belitsky, B. R. (2002). Chapter 16: Biosynthesis of amino acids of the glutamate and
1192 aspartate families, alanine, and polyamines. In A. Sonenshein, R. Losick & J.
1193 Hoch (Eds.), *Bacillus subtilis and Its Closest Relatives*. Washington, D.C.: ASM
1194 Press.
1195 Berg, J. M., Tymoczko, J. L., & Stryer, L. (2002). *Biochemistry, Fifth Edition*. New York: W. H. Freeman.

1197 Birkett, A., Muir, J., Phillips, J., Jones, G., & O'Dea, K. (1996). Resistant starch lowers fecal concentrations of ammonia and phenols in humans. *Am J Clin Nutr*, 63, 766-772.

1200 Bjornsdottir-Butler, K., Green, D. P., Bolton, G. E., & McClellan-Green, P. D. (2015). Control of Histamine-Producing Bacteria and Histamine Formation in Fish Muscle by Trisodium Phosphate. *J Food Sci*, 80, M1253-1258.

1203 Blachier, F., Davila, A. M., Mimoun, S., Benetti, P. H., Atanasiu, C., Andriamihaja, M., Benamouzig, R., Bouillaud, F., & Tome, D. (2010). Luminal sulfide and large intestine mucosa: friend or foe? *Amino Acids*, 39, 335-347.

1206 Blachier, F., M'Rabet-Touil, H., Posho, L., Morel, M. T., Bernard, F., Darcy-Vrillon, B., & Duee, P. H. (1992). Polyamine metabolism in enterocytes isolated from newborn pigs. *Biochim Biophys Acta*, 1175, 21-26.

1209 Bolotin, A., Wincker, P., Mauger, S., Jaillon, O., Malarme, K., Weissenbach, J., Ehrlich, S. D., & Sorokin, A. (2001). The complete genome sequence of the lactic acid bacterium *Lactococcus lactis* ssp. lactis IL1403. *Genome Res*, 11, 731-753.

1213 Bone, E., Tamm, A., & Hill, M. (1976). The production of urinary phenols by gut bacteria and their possible role in the causation of large bowel cancer. *Am J Clin Nutr*, 29, 1448-1454.

1216 Bos, C., Juillet, B., Fouillet, H., Turlan, L., Dare, S., Luengo, C., N'Tounda, R., Benamouzig, R., Gausseres, N., Tome, D., & Gaudichon, C. (2005).

1218 Postprandial metabolic utilization of wheat protein in humans. *Am J Clin Nutr*, 81, 87-94.
1195 Berg, J. M., Tymoczko, J. L., & Stryer, L. (2002). *Biochemistry, Fifth Edition*. New York: W. H. Freeman.

1197 Birkett, A., Muir, J., Phillips, J., Jones, G., & O'Dea, K. (1996). Resistant starch lowers fecal concentrations of ammonia and phenols in humans. *Am J Clin Nutr*, 63, 766-772.

1200 Bjornsdottir-Butler, K., Green, D. P., Bolton, G. E., & McClellan-Green, P. D. (2015). Control of Histamine-Producing Bacteria and Histamine Formation in Fish Muscle by Trisodium Phosphate. *J Food Sci*, 80, M1253-1258.

1203 Blachier, F., Davila, A. M., Mimoun, S., Benetti, P. H., Atanasiu, C., Andriamihaja, M., Benamouzig, R., Bouillaud, F., & Tome, D. (2010). Luminal sulfide and large intestine mucosa: friend or foe? *Amino Acids*, 39, 335-347.

1206 Blachier, F., M'Rabet-Touil, H., Posho, L., Morel, M. T., Bernard, F., Darcy-Vrillon, B., & Duee, P. H. (1992). Polyamine metabolism in enterocytes isolated from newborn pigs. *Biochim Biophys Acta*, 1175, 21-26.

1209 Bolotin, A., Wincker, P., Mauger, S., Jaillon, O., Malarme, K., Weissenbach, J., Ehrlich, S. D., & Sorokin, A. (2001). The complete genome sequence of the lactic acid bacterium *Lactococcus lactis* ssp. lactis IL1403. *Genome Res*, 11, 731-753.

1213 Bone, E., Tamm, A., & Hill, M. (1976). The production of urinary phenols by gut bacteria and their possible role in the causation of large bowel cancer. *Am J Clin Nutr*, 29, 1448-1454.

1216 Bos, C., Juillet, B., Fouillet, H., Turlan, L., Dare, S., Luengo, C., N'Tounda, R., Benamouzig, R., Gausseres, N., Tome, D., & Gaudichon, C. (2005).

1218 Postprandial metabolic utilization of wheat protein in humans. *Am J Clin Nutr*, 81, 87-94.
1220 Bouillaud, F., & Blachier, F. (2011). Mitochondria and sulfide: a very old story of poisoning, feeding, and signaling? *Antioxid Redox Signal*, 15, 379-391.
1221
1222 Bravo, J. A., Forsythe, P., Chew, M. V., Escaravage, E., Savignac, H. M., Dinan, T. G., Bienenstock, J., & Cryan, J. F. (2011). Ingestion of *Lactobacillus* strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. *Proc Natl Acad Sci U S A*, 108, 16050-16055.
1223
1224
1225
1226 Brown, J. R., Masuchi, Y., Robb, F. T., & Doolittle, W. F. (1994). Evolutionary relationships of bacterial and archaeal glutamine synthetase genes. *J Mol Evol*, 38, 566-576.
1227
1228
1229 Chacko, A., & Cummings, J. H. (1988). Nitrogen losses from the human small bowel: obligatory losses and the effect of physical form of food. *Gut*, 29, 809-815.
1230
1231 Chambers, E. S., Viardot, A., Psichas, A., Morrison, D. J., Murphy, K. G., Zac-Varghese, S. E., MacDougall, K., Preston, T., Tedford, C., Finlayson, G. S., Blundell, J. E., Bell, J. D., Thomas, E. L., Mt-Isa, S., Ashby, D., Gibson, G. R., Kolida, S., Dhillo, W. S., Bloom, S. R., Morley, W., Clegg, S., & Frost, G. (2014). Effects of targeted delivery of propionate to the human colon on appetite regulation, body weight maintenance and adiposity in overweight adults. *Gut*.
1232
1233
1234
1235
1236
1237 Cherbut, C., Ferrier, L., Roze, C., Anini, Y., Blottiere, H., Lecannu, G., & Galmiche, J. P. (1998). Short-chain fatty acids modify colonic motility through nerves and polypeptide YY release in the rat. *Am J Physiol*, 275, G1415-1422.
1238
1239
1240 Chimerel, C., Emery, E., Summers, D. K., Keyser, U., Gribble, F. M., & Reimann, F. (2014). Bacterial metabolite indole modulates incretin secretion from intestinal enteroendocrine L cells. *Cell Rep*, 9, 1202-1208.
1241
1242
1243 Claus, H., & Decker, H. (2006). Bacterial tyrosinases. *Syst Appl Microbiol*, 29, 3-14.
1220 Bouillaud, F., & Blachier, F. (2011). Mitochondria and sulfide: a very old story of poisoning, feeding, and signaling? *Antioxid Redox Signal*, 15, 379-391.
1221
1222 Bravo, J. A., Forsythe, P., Chew, M. V., Escaravage, E., Savignac, H. M., Dinan, T. G., Bienenstock, J., & Cryan, J. F. (2011). Ingestion of *Lactobacillus* strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. *Proc Natl Acad Sci U S A*, 108, 16050-16055.
1223
1224
1225
1226 Brown, J. R., Masuchi, Y., Robb, F. T., & Doolittle, W. F. (1994). Evolutionary relationships of bacterial and archaeal glutamine synthetase genes. *J Mol Evol*, 38, 566-576.
1227
1228
1229 Chacko, A., & Cummings, J. H. (1988). Nitrogen losses from the human small bowel: obligatory losses and the effect of physical form of food. *Gut*, 29, 809-815.
1230
1231 Chambers, E. S., Viardot, A., Psichas, A., Morrison, D. J., Murphy, K. G., Zac-Varghese, S. E., MacDougall, K., Preston, T., Tedford, C., Finlayson, G. S., Blundell, J. E., Bell, J. D., Thomas, E. L., Mt-Isa, S., Ashby, D., Gibson, G. R., Kolida, S., Dhillo, W. S., Bloom, S. R., Morley, W., Clegg, S., & Frost, G. (2014). Effects of targeted delivery of propionate to the human colon on appetite regulation, body weight maintenance and adiposity in overweight adults. *Gut*.
1232
1233
1234
1235
1236
1237 Cherbut, C., Ferrier, L., Roze, C., Anini, Y., Blottiere, H., Lecannu, G., & Galmiche, J. P. (1998). Short-chain fatty acids modify colonic motility through nerves and polypeptide YY release in the rat. *Am J Physiol*, 275, G1415-1422.
1238
1239
1240 Chimerel, C., Emery, E., Summers, D. K., Keyser, U., Gribble, F. M., & Reimann, F. (2014). Bacterial metabolite indole modulates incretin secretion from intestinal enteroendocrine L cells. *Cell Rep*, 9, 1202-1208.
1241
1242
1243 Claus, H., & Decker, H. (2006). Bacterial tyrosinases. *Syst Appl Microbiol*, 29, 3-14.
1244 Collino, S., Montoliu, I., Martin, F. P., Scherer, M., Mari, D., Salvioli, S., Bucci, L.,
1245 Ostan, R., Monti, D., Biagi, E., Brigidi, P., Franceschi, C., & Rezzi, S. (2013).
1246 Metabolic signatures of extreme longevity in northern Italian centenarians reveal
1247 a complex remodeling of lipids, amino acids, and gut microbiota metabolism.
1248 *PLoS One*, 8, e56564.
1249 Cremin, J. D., Jr., Fitch, M. D., & Fleming, S. E. (2003). Glucose alleviates ammonia-
1250 induced inhibition of short-chain fatty acid metabolism in rat colonic epithelial
1251 cells. *Am J Physiol Gastrointest Liver Physiol*, 285, G105-114.
1252 Cronan, J. E., & Thomas, J. (2009). Bacterial fatty acid synthesis and its relationships
1253 with polyketide synthetic pathways. *Methods Enzymol*, 459, 395-433.
1254 Cummings, J. H., Hill, M. J., Bone, E. S., Branch, W. J., & Jenkins, D. J. (1979). The
1255 effect of meat protein and dietary fiber on colonic function and metabolism. II.
1256 Bacterial metabolites in feces and urine. *Am J Clin Nutr*, 32, 2094-2101.
1257 Dai, Z. L., Zhang, J., Wu, G., & Zhu, W. Y. (2010). Utilization of amino acids by
1258 bacteria from the pig small intestine. *Amino Acids*, 39, 1201-1215.
1259 Daly, K., Cuff, M. A., Fung, F., & Shirazi-Beechey, S. P. (2005). The importance of
1260 colonic butyrate transport to the regulation of genes associated with colonic
1261 tissue homeostasis. *Biochem Soc Trans*, 33, 733-735.
1262 Darragh, A. J., Cranwell, P. D., & Moughan, P. J. (1994). Absorption of lysine and
1263 methionine from the proximal colon of the piglet. *Br J Nutr*, 71, 739-752.
1264 David, L. A., Maurice, C. F., Carmody, R. N., Gootenberg, D. B., Button, J. E., Wolfe,
1265 B. E., Ling, A. V., Devlin, A. S., Varma, Y., Fischbach, M. A., Biddinger, S. B.,
1266 Dutton, R. J., & Turnbaugh, P. J. (2014). Diet rapidly and reproducibly alters the
1267 human gut microbiome. *Nature*, 505, 559-563.
1244 Collino, S., Montoliu, I., Martin, F. P., Scherer, M., Mari, D., Salvioli, S., Bucci, L.,
1245 Ostan, R., Monti, D., Biagi, E., Brigidi, P., Franceschi, C., & Rezzi, S. (2013).
1246 Metabolic signatures of extreme longevity in northern Italian centenarians reveal
1247 a complex remodeling of lipids, amino acids, and gut microbiota metabolism.
1248 *PLoS One*, 8, e56564.
1249 Cremin, J. D., Jr., Fitch, M. D., & Fleming, S. E. (2003). Glucose alleviates ammonia-
1250 induced inhibition of short-chain fatty acid metabolism in rat colonic epithelial
1251 cells. *Am J Physiol Gastrointest Liver Physiol*, 285, G105-114.
1252 Cronan, J. E., & Thomas, J. (2009). Bacterial fatty acid synthesis and its relationships
1253 with polyketide synthetic pathways. *Methods Enzymol*, 459, 395-433.
1254 Cummings, J. H., Hill, M. J., Bone, E. S., Branch, W. J., & Jenkins, D. J. (1979). The
1255 effect of meat protein and dietary fiber on colonic function and metabolism. II.
1256 Bacterial metabolites in feces and urine. *Am J Clin Nutr*, 32, 2094-2101.
1257 Dai, Z. L., Zhang, J., Wu, G., & Zhu, W. Y. (2010). Utilization of amino acids by
1258 bacteria from the pig small intestine. *Amino Acids*, 39, 1201-1215.
1259 Daly, K., Cuff, M. A., Fung, F., & Shirazi-Beechey, S. P. (2005). The importance of
1260 colonic butyrate transport to the regulation of genes associated with colonic
1261 tissue homeostasis. *Biochem Soc Trans*, 33, 733-735.
1262 Darragh, A. J., Cranwell, P. D., & Moughan, P. J. (1994). Absorption of lysine and
1263 methionine from the proximal colon of the piglet. *Br J Nutr*, 71, 739-752.
1264 David, L. A., Maurice, C. F., Carmody, R. N., Gootenberg, D. B., Button, J. E., Wolfe,
1265 B. E., Ling, A. V., Devlin, A. S., Varma, Y., Fischbach, M. A., Biddinger, S. B.,
1266 Dutton, R. J., & Turnbaugh, P. J. (2014). Diet rapidly and reproducibly alters the
1267 human gut microbiome. *Nature*, 505, 559-563.