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D’Aimmo et al. Microbiome Res Rep 2024;3:11 https://dx.doi.org/10.20517/mrr.2023.59 Page 11 of 22

materials. Furthermore, folate-producing probiotics must meet several other requirements, such as fulfilling
[11]
the EFSA criteria and actively producing folate in the gut ecosystem.
The idea of folate trophic probiotics seems feasible and should be explored further. It is conceivable that
bifidobacteria adapted to the human colon have been selected for folate production because of the mutual
benefits it provides.

BIOSYNTHESIS OF FOLATE BY BIFIDOBACTERIA: FROM GENES TO PHENOTYPE
In silico studies for folate production
The complete biochemical pathway of folate biosynthesis in bifidobacteria is still being investigated. By
comparing identified folate biosynthesis genes present in other organisms with bifidobacterial genomes,
genome information can be used to identify genes and corresponding proteins that are likely involved in
folate biosynthesis .
[65]
Several databases and web resources are available for studying microbial genomes. The US Department of
Energy Joint Genome Institute (DOE JGI) established the online Integrated Microbial Genome System
(IMG) as a genome search and annotation platform that supports distribution and genome analyses. With
[83]
databases such as IMG and MicrobesOnline , it is possible to study and compare microorganisms’
[84]
genomes. For example, these databases allow researchers to check whether a microorganism has examples
of all the necessary genes to produce a vitamin such as folate. However, the mere presence of genes does not
guarantee that the corresponding proteins are functional.

All known type strains of the species of bifidobacteria have been sequenced to date. At the time of writing,
the databases contained genome information about the 111 currently described species of bifidobacteria
(https://site.unibo.it/subcommittee-lactobacillus-bifidobacterium/en). In silico analysis of the folate
biosynthesis genes for all 111 bifidobacterial species was performed. The species genomes were examined
for the presence of genes involved in the biosynthesis of DHPPP, THF-polyglutamate, chorismate, and
pABA [Figure 2]. As B. adolescentis is known to be capable of de novo folate production, the genes from
B. adolescentis were taken as a reference for BlastP searches. Based on the identity values, strains in Figure 2
are marked as being involved in folate biosynthesis or not. The identity value of 50% is used as a threshold
to define the presence or absence of a gene in a particular strain.All 111 bifidobacterial species have been
found to possess the required genes to produce chorismate by yielding shikimate [Figures 2 and 4].

In general, de novo folate biosynthesis requires the precursors of both DHPPP and pABA. Upon analyzing
the genomes, it was found that most species carry the pabA gene, which encodes aminodeoxychorismate
synthase for pABA biosynthesis. However, only B. adolescentis, B. angulatum, B. breve and B. dentium that
are typically found in humans, B. aesculapii, B. callithricos, B. callithrichidarum, B. goeldii, B. myosotis,
B. moukalabense, B. parmae and B. stellenboschense from non-human primates and B. ruminantium,
B. porcinum, B. rousetti and B. subtile from other animals have the pabC gene, that encodes the 4-amino-4-
deoxychorismate lyase and are considered able to complete the pABA biosynthesis process [Figure 4 and
Supplementary Table 2]. The bifidobacterial phylogenetic tree of the 111 species tested showed that folate-
producing species cluster in the same branches [Figure 5].

All human-derived species, except B. gallicum, are prototrophic or require pABA for folate production. For
bifidobacterial species in non-human primates [85-87] , about one-third are prototrophic, one-third can
produce folate in the presence of pABA, and one-third are auxotrophic for the folate; moreover, most
bifidobacterial “animal species” were found to lack crucial genes, namely folE and folBK, which encode the