Production of butyrate from lysine and the Amadori product fructoselysine by a human gut commensal

Abstract

Human intestinal bacteria produce butyrate, which has signalling properties and can be used as energy source by enterocytes thus influencing colonic health. However, the pathways and the identity of bacteria involved in this process remain unclear. Here we describe the isolation from the human intestine of Intestinimonas strain AF211, a bacterium that can convert lysine stoichiometrically into butyrate and acetate when grown in a synthetic medium. Intestinimonas AF211 also converts the Amadori product fructoselysine, which is abundantly formed in heated foods via the Maillard reaction, into butyrate. The butyrogenic pathway includes a specific CoA transferase that is overproduced during growth on lysine. Bacteria related to Intestinimonas AF211 as well as the genetic coding capacity for fructoselysine conversion are abundantly present in colonic samples from some healthy human subjects. Our results indicate that protein can serve as a source of butyrate in the human colon, and its conversion by Intestinimonas AF211 and related butyrogens may protect the host from the undesired side effects of Amadori reaction products.

Figure 1. L-lysine and fructoselysine conversion by strain Intestinimonas AF211 throughout time.

Carbon recovery was 87% and 101% for the lysine (a) and fructoselysine (b) utilization, respectively. Exact concentrations of substrates and products and optical density values are included in Supplementary Table 1. Values are mean of biological duplicates. Error bars indicate s.d.

Figure 2. Model for fructoselysine metabolism in Intestinimonas AF211.

The locus tag and fold induction of the proteins based on the proteomic data are indicated in the brackets. Fd, ferredoxin; ND, not detected; Ppase, pyrophosphatase; Rnf, proton pumping Rnf cluster. According to our model, fructoselysine is taken up by ABC transporter (AF_00952-00955), which is phosphorylated by fructoselysine kinase (AF_00949) to form fructoselysine-6-phosphate that is subsequently cleaved by fructoseamine deglycase (AF_00951) into lysine and glucose-6-phosphate (indicated by the purple arrows). Lysine is then degraded via the lysine pathway (black arrows) while glucose-6-phosphate is metabolized via glycolysis and the acetyl-CoA pathway (blue arrows) as described in the text (Supplementary Fig. 4). There are various links between the lysine and acetyl-CoA pathways and the one at the level of acetoacetyl-CoA, involving acetate to generate acetoacetate and acetyl-CoA, is indicated (dashed black arrows). For simplicity, only the key reactions are shown and other links or the conversion of acetyl-CoA formed via acetyl-CoA pathway to acetate have been omitted. Fructoselysine, lysine, butyrate, acetate, lactate and CO₂ are indicated in different colours highlighting their distinctive positions in the pathways. ATP is in red to indicate reactions that either require or generate energy via substrate-level phosphorylation and electron transport chain. Lactate formation is dependent on the amount of exogenous acetate and redox state (hence both lactate formation and acetate uptake are indicated by dashed green arrows). Therefore, the overall fructoselysine stoichiometry depends on the presence or absence of exogenous acetate as well as environmental conditions and activity of biosynthetic pathways. In the presence of acetate (such as in the human colon), fructoselysine is converted in approximately three butyrate (two butyrate are formed via acetyl-CoA pathway and one butyrate through the lysine pathway), while no lactate is produced; however, when no exogenous acetate is present, fructoselysine is converted into approximately two butyrate and one lactate (see Supplementary Table 2 for details).

Figure 3. Elucidation of lysine pathway via ¹H-decoupled ¹³C-NMR spectrum and 2D HMBC spectrum.

(a) High-resolution ¹H-decoupled ¹³C-NMR spectra showing L-[6-¹³C]lysine ¹³C-labelled fermentation products. [2-¹³C]butyrate, [2-¹³C]acetate and [4-¹³C]butyrate had a chemical shift of 42.33, 25.99 and 15.95 p.p.m., respectively. (b) 2D HMBC spectrum for [6-¹³C]lysine is shown. (c) Percentages of labelled butyrate fractions (see Supplementary Figs 2 and 3 for more details).

Figure 4. Phylogeny of CoA transferase and enzyme activity.

(a) Phylogenetic tree of predicted CoA transferases from Intestinimonas AF211 (bold) and other representative anaerobes. The tree was based on sequences from butyryl-CoA:acetate CoA transferase (But, in blue), butyryl-CoA:4-hydroxybutyrate CoA transferase (4Hbt, in purple), butyryl-CoA:acetoacetate CoA transferase (Ato) alpha subunit (AtoD, in orange), beta subunit (AtoA, in brown) and acetyl-CoA:acetoacetate CoA transferase (AtoC, in green), respectively. Green dots indicate non-intestinal isolates. Intestinimonas AF211 proteins induced during growth on lysine are indicated by the red arrows. (b) Butyryl-CoA:acetoacetate CoA transferase activity in crude cell extracts. Each measurement was performed with biological duplicates and 4–6 replicate measurements. Values represent mean of replicates. Error bars indicate s.d.'s. Green and red bars represent the enzyme activity of Intestinimonas AF211 grown in lysine and glucose plus acetate, respectively; blue bar is the negative control with A. rhamnosivorans DSM26241 grown on glucose, which is not capable of lysine fermentation.