Model-driven design of a minimal medium for Akkermansia muciniphila confirms mucus adaptation

Abstract

The abundance of the human intestinal symbiont Akkermansia muciniphila has found to be inversely correlated with several diseases, including metabolic syndrome and obesity. A. muciniphila is known to use mucin as sole carbon and nitrogen source. To study the physiology and the potential for therapeutic applications of this bacterium, we designed a defined minimal medium. The composition of the medium was based on the genome-scale metabolic model of A. muciniphila and the composition of mucin. Our results indicate that A. muciniphila does not code for GlmS, the enzyme that mediates the conversion of fructose-6-phosphate (Fru6P) to glucosamine-6-phosphate (GlcN6P), which is essential in peptidoglycan formation. The only annotated enzyme that could mediate this conversion is Amuc-NagB on locus Amuc_1822. We found that Amuc-NagB was unable to form GlcN6P from Fru6P at physiological conditions, while it efficiently catalyzed the reverse reaction. To overcome this inability, N-acetylglucosamine needs to be present in the medium for A. muciniphila growth. With these findings, the genome-scale metabolic model was updated and used to accurately predict growth of A. muciniphila on synthetic media. The finding that A. muciniphila has a necessity for GlcNAc, which is present in mucin further prompts the adaptation to its mucosal niche.

Figure 1

Growth of Akkermansia muciniphila on CP medium supplemented with l‐threonine and different sugars. All sugars were supplemented to a total of 25 mM. The negative control was supplemented with GlcNAc/glucose and not inoculated. The data shown are averages of three biological replicates and two technical duplicates for each. The experiment was performed twice in duplicate; bars indicate standard deviations.

Figure 2

SCFA production and degradation of sugars. The degradation of GlcNAc and glucose results in a 1:1 ratio of acetate and propionate (A). The degradation of GlcNAc and GalNAc results in similar acetate and propionate ratios of 1.5:1 to 2:1 (B, C). The production of acetate and propionate on glucose and GlcN is in a 1:2 ratio (D). GlcN is still degraded when not supplemented with glucose, but no SCFA production was observed (E). The experiment was performed twice in duplicate; bars indicate standard deviations.

Figure 3

Core biochemical network of Akkermansia muciniphila with the distribution of expected fluxes in the main catabolic pathways under optimal growth conditions described in the article. Arrow width indicates relative changes in the fluxes on different substrates GalNac (red), GlcNac (blue) and mixture of GlcNac and glucose (yellow). Reactions for which all conditions showed similar fluxes are represented by black arrows. Model simulations were performed with equimolar total amounts of sugar. Figure has been adapted from (Ottman et al., 2017a).