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Page 6 of 15 Geerlings et al. Microbiome Res Rep 2024;3:36 https://dx.doi.org/10.20517/mrr.2024.06

Figure 1. Exponential growth of A. muciniphila as a function of glucose to GlcNAc ratios. Bioreactors at pH 7.0 were run with glucose to
GlcNAc carbon source ratios of 3:1 (Condition A), 10:1 (Condition B), and 20:1 (Condition C) or on mucin (Condition D). Purple arrows
indicate mid-log sampling, turquoise arrows indicate end-of-growth sampling, and the brown arrow indicates sampling of condition D.
The growth rate is indicated in the graph for conditions A-C. Asterisks at the arrows indicate either glucose and GlcNAc were depleted
(**) or only GlcNAc was depleted (*). Observed viscosity in the cultures is indicated with (++) meaning high viscosity, (+)
meaning medium viscosity, or (-) no viscosity observed. A. muciniphila: Akkermansia muciniphila; GlcNAc: N-acetylglucosamine.

Transcriptome response in exponential and stationary phase
The growth curve of condition A showed two different growing phases, the first one until approximately
27 h with a high growth rate (0.18 h ) and the second phase from 27 to 48 h (0.03 h ), the time at which the
-1
-1
glucose and GlcNAc were depleted [Figure 1]. This observation was also supported by the transcriptome
data that showed dozens of genes to be significantly higher expressed in the first compared to the second
growth phase [Figure 2, Supplementary Table 2, Supplementary Figure 4 and Supplementary File 1]. The
genes with the highest upregulation (> 5-fold) at the end of the first phase included several gene clusters
with two or more juxtaposed genes, such as the metabolic gene cluster encoding a glutaminase and a likely
glutamine-GABA antiporter (Amuc_0037-0038), as well as several stress proteins (Amuc_1406-1408, coding
for DnaK, GroES and GroEL). In addition, the expression of single genes was upregulated for other stress
proteins such as Skp (Amuc_0405) and HtpG (Amuc_2002), as well as genes involved in oxygen and other
stress responses, such as catalase (Amuc_2070), glutamate decarboxylase (Amuc_0372), and NAD(P)-
dependent oxidoreductase (Amuc_0777). Many of the genes observed to be upregulated in the end-growth
phase of this condition had no known function and were annotated as hypothetical. Genes that were
upregulated and annotated with a function included genes involved in transport systems for iron
(Amuc_1929-1931), potassium (Amuc_0830-0831 and Amuc_1151-1153), and phosphate (Amuc_1302-
1307), as well as phage production (such as Amuc_1355, Amuc_1936, and Amuc_1335). A notable
exception was the highly (14.8-fold) upregulated gene for a predicted lactoylglutathione lyase (Amuc_1878)
that has shown in Salmonella to be involved in the detoxification of methylglyoxal, known to be produced
in the gut but also in bioreactors with peptones . The transcriptome data are also supported by proteome
[45]
data, which showed that the global proteome is more conserved than the global transcriptome during