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Page 10 of 15 Geerlings et al. Microbiome Res Rep 2024;3:36 https://dx.doi.org/10.20517/mrr.2024.06

Table 1. Differential expression of genes involved in cell division comparing high-GlcNAc to the mucin condition
High-GlcNAc vs. mucin
Genes Description
Fold change P value
Amuc_1176 Cell division inhibitor 2.62 6.57E-48
Amuc_0348 Cell division protein FtsH 1.50 4.32E-36 Upregulated in
high GlcNAc
Amuc_1052 Cell division trigger factor 1.43 7.41E-31
Amuc_1558 RIP metalloprotease RseP 0.22 1.63E-06
Amuc_0662 Polypeptide-transport-associated domain-containing protein FtsQ-type 0.31 4.58E-08
*
Amuc_0540 Cell shape-determining protein MreB 0.45 6.6E-11
Amuc_0652 Peptidoglycan glycosyltransferase 0.59 4.79E-19
Amuc_0649 Transcriptional regulator MraZ 0.62 3.34E-17
Amuc_0153 Cell division protein FtsA 0.64 1.97E-18
Upregulated
Amuc_0152 Tubulin/FtsZ GTPase 0.88 1.24E-26 in mucin
Amuc_2076 Cell division FtsK 1.16 3.66E-34
Amuc_0658 Cell cycle protein 1.50 3.38E-41
Amuc_0514 Peptidoglycan glycosyltransferase 1.67 1.26E-41
Amuc_1317 Integral membrane protein CcmA involved in cell shape determination 2.03 2.88E-46
*
Genes that were also upregulated in the preliminary transcriptome data comparing soy medium to mucin medium. GlcNAc: N-
acetylglucosamine; RIP: regulated intramembrane proteolysis.

Amuc_2096), and an additional significantly upregulated glycosyltransferase cluster (Amuc_1139-1142), a
gene cluster containing multiple aldo/keto reductases (Amuc_1796-1809), and the gene for anaerobic
ribonucleoside-triphosphate reductase activating protein (Amuc_0860). Moreover, additional complete
gene clusters related to stress responses were found to be significantly upregulated in both high-GlcNAc
and low-GlcNAc conditions, as compared to mucin, including a gene cluster encoding ribosomal proteins
(Amuc_0294-0308), an iron transport cluster (Amuc_1930 until Amuc_1934), and a potential flavin
biosynthesis gene cluster (Amuc_0421-0426) [Supplementary File 1]. Other genes that may be involved in
stress response but not part of a gene cluster were also identified to be upregulated in both GlcNAc
conditions. This includes genes for rubrerythrin (Amuc_2055-2056), catalase (Amuc_2070),
oxidoreductases (Amuc_0116, Amuc_0777, Amuc_1072, Amuc_1176 and Amuc_1389), ribonucleoside-
triphosphate reductase (Amuc_0862), and glutamate decarboxylase (Amuc_0372).

Next to the transcriptomic stress response, elongated cells were observed in the bioreactors containing
glucose and GlcNAc in comparison to the small oval-shaped cells visible when A. muciniphila was
cultivated on mucin [Supplementary Figure 3]. Elongated cells were also observed in the previously
mentioned soy medium cultures [Supplementary Figure 1], together with an increased expression of
Amuc_0540 encoding cell shape-determining protein MreB in mucin as compared to soy medium.
Following this observation, we performed a more in-depth analysis of the expression of genes involved in
cell elongation and division [Table 1 and Supplementary File 1]. Overall, the transcriptomic stress response
and elongated cells observed in the cultures grown in synthetic medium without mucin indicate that a stress
response is triggered in the absence of mucin in this medium.

DISCUSSION
In this study, we assessed the cultivation of A. muciniphila food-grade pea-peptone medium with different
concentrations of carbon sources to produce cells suitable for therapeutic applications. The use of food-
grade synthetic and non-allergenic medium supplemented with glucose and GlcNAc resulted in high cell
yields and fast growth of A. muciniphila. Furthermore, we gained detailed insight into physiology by a
combination of biochemical analysis as well as transcriptional and proteomic analysis. In addition, we