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Page 12 of 15 Geerlings et al. Microbiome Res Rep 2024;3:36 https://dx.doi.org/10.20517/mrr.2024.06

[46]
the trigger factor was found to cause defective cell division in E. coli . Therefore, the upregulation of this
gene Amuc_1052 may also be involved in the observed cell elongation of A. muciniphila. The upregulation
of cell division genes in the mucin condition supports the differences observed in cell size between mucin
and glucose/GlcNAc conditions.

The protein Amuc_1100 is gaining increasing interest in several studies for its positive effect on host
health [35,47] . The gene cluster associated with pili production, including Amuc_1100, showed significant
upregulation in the condition supplemented with mucin compared to the GlcNAc conditions. The
proteome data support this observation, but the protein abundance ratio observed was less than 10.
However, as the proteome data sets relate to relative amounts of proteins, absolute amounts of proteins
could be higher in the GlcNAc conditions as the OD600 value was approximately 8-fold higher in the high-
GlcNAc condition than the mucus condition.

The upregulation of multiple stress-related genes and gene clusters was identified in fermentations on
GlcNAc compared to mucin. First, the upregulation of the phosphate ABC transporter system may indicate
a phosphate limitation in these cultures . To overcome this limitation, additional phosphate sources could
[48]
be added to the food-grade medium. Furthermore, several genes previously found to be involved in the
oxygen stress response of A. muciniphila were found to be upregulated in conditions without mucin as
[32]
well . However, these fermentations were run anaerobically simultaneously, as was the case with the mucin
condition. Therefore, it is likely that this stress response was not specific for oxygen, but rather a form of
cross-protection against other stresses . Another stress response that was activated in conditions
[49]
containing glucose and GlcNAc was the production of EPS (Amuc_2077-2096). Interestingly, the expression
of the genes involved in EPS production decreased along with the decreasing concentration of GlcNAc, as
well as the glycosyltransferase cluster (Amuc_1139 until Amuc_1142). The production of EPS in bacteria is
often a mechanism to cope with harsh environmental conditions, as extensively studied for lactic acid
[50]
bacteria . For A. muciniphila, the high-GlcNAc condition, with limiting medium components, may be
sub-optimal. Additionally, within condition A, it was visible that during the mid-growth phase, stress-
related genes were upregulated as compared to the end phase; this included 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). However, the upregulation of stress-related genes
does not limit growth rate and biomass production as observed in these fermentations. We showed that pea
peptone-based food-grade medium supplemented with glucose and GlcNAc instead of mucin results in
high biomass formation of A. muciniphila, which may be used for its production for therapeutic purposes.

Other comparisons of the growth of A. muciniphila on mucin versus specific carbon sources have been
made [24,25] . A transcriptome comparison was conducted between cultivations supplemented with mucin and
those supplemented with glucose . Despite the differences in cultivation, cultures with glucose as a carbon
[24]
[24]
source also showed upregulation of stress-related genes in comparison to mucin . When comparing the
[24]
data of the previous study and the present one, we noted that the expression of many genes was similar.
Furthermore, similar differences were also documented for genes and proteins involved in mucin
degradation in both studies. In the present proteome analysis, we found that on the protein level, it was
clearly visible that the mucin culture had an upregulation of proteins involved in mucin degradation, since
these proteins were located in the top protein abundance ratios. Similar findings were documented
previously . In contrast, there were also differences between the previous study and the present one,
[24]
[24]
notably including differentially expressed stress-related genes. Moreover, the differences noted here in the
expression of genes involved in cell division were not found in the earlier study. This may be due to the low
growth rates and final biomass that were observed in the cultures with mucin only, while in our studies with