Flavonoid-Modifying Capabilities of the Human Gut Microbiome-An In Silico Study

Abstract

Flavonoids are a major group of dietary plant polyphenols and have a positive health impact, but their modification and degradation in the human gut is still widely unknown. Due to the rise of metagenome data of the human gut microbiome and the assembly of hundreds of thousands of bacterial metagenome-assembled genomes (MAGs), large-scale screening for potential flavonoid-modifying enzymes of human gut bacteria is now feasible. With sequences of characterized flavonoid-transforming enzymes as queries, the Unified Human Gastrointestinal Protein catalog was analyzed and genes encoding putative flavonoid-modifying enzymes were quantified. The results revealed that flavonoid-modifying enzymes are often encoded in gut bacteria hitherto not considered to modify flavonoids. The enzymes for the physiologically important daidzein-to-equol conversion, well studied in Slackiaisoflavoniconvertens, were encoded only to a minor extent in Slackia MAGs, but were more abundant in Adlercreutzia equolifaciens and an uncharacterized Eggerthellaceae species. In addition, enzymes with a sequence identity of about 35% were encoded in highly abundant MAGs of uncultivated Collinsella species, which suggests a hitherto uncharacterized daidzein-to-equol potential in these bacteria. Of all potential flavonoid modification steps, O-deglycosylation (including derhamnosylation) was by far the most abundant in this analysis. In contrast, enzymes putatively involved in C-deglycosylation were detected less often in human gut bacteria and mainly found in Agathobacter faecis (formerly Roseburia faecis). Homologs to phloretin hydrolase, flavanonol/flavanone-cleaving reductase and flavone reductase were of intermediate abundance (several hundred MAGs) and mainly prevalent in Flavonifractor plautii. This first comprehensive insight into the black box of flavonoid modification in the human gut highlights many hitherto overlooked and uncultured bacterial genera and species as potential key organisms in flavonoid modification. This could lead to a significant contribution to future biochemical-microbiological investigations on gut bacterial flavonoid transformation. In addition, our results are important for individual nutritional recommendations and for biotechnological applications that rely on novel enzymes catalyzing potentially useful flavonoid modification reactions.

Keywords: human gut microbiome; isoflavones; metagenomes; personalized nutrition; phytohormones; plant metabolites; polyphenols.

Figure 1

Flavonoid O-glycosidase homologs in human gut MAGs. The PID (percent amino acid sequence identity) threshold to the queries (see color code) was set to 50 (For abbreviations and details, see Table 1). Hits were filtered for at least 50 occurrences, so that each bubble represents a number of redundant sequences ranging from 50 to 772.

Figure 2

Rhamnosidase homologs in human gut MAGs. A PID threshold of 65 was chosen for the queries shown in the color code (for abbreviations and details, see Table 1; hits with a lower PID are shown in Supplementary Figure S4). Hits were filtered for at least 20 occurrences, so that each bubble represents a number of redundant sequences ranging from 20 to 2176.

Figure 3

Two characterized C-deglycosylation gene clusters. (A) Eubacterium cellulosolvens with five genes involved in C-deglycosylation [17] and (B) strain PUE [25,26], with dgpA encoding an oxidoreductase and dgpBC as oxo-puerarin-deglycosylating enzymes, of which all three are involved in C-deglycosylation. Genes encoding deglycosylating enzymes are shown in turquoise, accessory genes in violet. The full GenBank accession number is given for the first gene and only variable digits are given for the downstream genes.

Figure 4

Homologs to enzymes involved in flavonoid C-degylcosylation using sequences of strain PUE. Hits were filtered for the co-occurrence of all three genes required for C-deglycosylation (dgpABC) in the same MAG. Hits were filtered for at least 2 occurrences, so that each bubble represents a number of redundant sequences ranging from 2 to 820.

Figure 5

Homologs to enzymes involved in daidzein-to-equol transformation in human gut MAGs. Hits were filtered for the co-occurrence of all three genes required for daidzein-to-equol conversion (dzr, ddr, tdr) in a single MAG. For a plot showing the hits to individually occurring genes (mainly dzr), see Supplementary Figure S7.

Figure 6

Reductive degradation of flavonoids depictured with apigenin as a flavone example.

Figure 7

Flr sequence hits of human gut MAGs. A PID threshold of 40 was chosen to the queries shown in the color code (for abbreviations and details see Table 1). Hits were filtered for at least 10 occurrences, so that each bubble represents a number of redundant sequences ranging from 10 to 245.

Figure 8

Fcr-like enzymes in human gut MAGs. Hits were filtered for at least 10 occurrences, so that each bubble represents a number of redundant sequences ranging from 10 to 804.

Figure 9

Distribution of Phy homologs in human gut MAGs. Depicted are MAGs with at least five identical amino acid sequences. Hits were filtered for at least ten occurrences, so that each bubble represents a number of redundant sequences ranging from 10 to 578.

Figure 10

Overview of the flavonoid conversion pathways in the human gut based on the MAGs study. Thickness of arrows reflects the abundance of observed hits to the sequences of the characterized flavonoid-transforming enzymes. Color code: blue, flavonoid trivial names; orange, flavonoid-converting enzymes; green, most abundant bacterial species carrying out the corresponding reaction in this analysis. For abbreviations, see text.