Metabolism of Daidzein and Genistein by Gut Bacteria of the Class Coriobacteriia

Abstract

The intake of isoflavones is presumed to be associated with health benefits in humans, but also potential adverse effects of isoflavones are controversially discussed. Isoflavones can be metabolized by gut bacteria leading to modulation of the bioactivity, such as estrogenic effects. Especially bacterial strains of the Eggerthellaceae, a well-known bacterial family of the human gut microbiota, are able to convert the isoflavone daidzein into equol. In addition, metabolization of genistein is also described for strains of the Eggerthellaceae. The aim of this study was to identify and investigate gut bacterial strains of the family Eggerthellaceae as well as the narrowly related family Coriobacteriaceae which are able to metabolize daidzein and genistein. This study provides a comprehensive, polyphasic approach comprising in silico analysis of the equol gene cluster, detection of genes associated with the daidzein, and genistein metabolism via PCR and fermentation of these isoflavones. The in silico search for protein sequences that are associated with daidzein metabolism identified sequences with high similarity values in already well-known equol-producing strains. Furthermore, protein sequences that are presumed to be associated with daidzein and genistein metabolism were detected in the two type strains 'Hugonella massiliensis' and Senegalimassilia faecalis which were not yet described to metabolize these isoflavones. An alignment of these protein sequences showed that the equol gene cluster is highly conserved. In addition, PCR amplification supported the presence of genes associated with daidzein and genistein metabolism. Furthermore, the metabolism of daidzein and genistein was investigated in fermentations of pure bacterial cultures under strictly anaerobic conditions and proofed the metabolism of daidzein and genistein by the strains 'Hugonella massiliensis' DSM 101782^T and Senegalimassilia faecalis KGMB04484^T.

Keywords: Coriobacteriaceae; Eggerthellaceae; anaerobic; daidzein; equol; genistein; isoflavones; metabolism; microbial.

Figure 1

Metabolism of daidzein and genistein by human gut bacteria. Modified based on [6,7]. Daidzein (DAI); genistein (GEN); dihydrodaidzein (DH-DAI); dihydrogenistein (DH-GEN), tetrahydrodaidzein (TH-DAI); tetrahydrogenistein (TH-GEN); 5-hydroxyl-equol (5-OH-equol); O-desmethylangolensin (O-DMA); 6′-hydroxy-O-desmethylangolensin (6′-OH-O-DMA); daidzein reductase (DZNR), dihydrodaidzein reductase (DHDR); tetrahydrodaidzein reductase (THDR).

Figure 2

Cluster analyses of amino acid sequences of daidzein reductase (DZNR), dihydrodaidzein reductase (DHDR), tetrahydrodaidzein reductase (THDR), and dihydrodaidzein racemase (DDRC) of strains of the Eggerthellaceae and Coriobacteriaceae. Clustering was performed by multiple-alignment and UPGMA (unweighted pair group method with arithmetic mean) in BioNumerics 8.0. Green labeled strains are not yet described in the literature to be associated with daidzein or genistein metabolism, to the best of our knowledge.

Figure 3

Schematic representation of the gene organization within the equol gene cluster (dihydrodaidzein racemase (DDRC), tetrahydrodaidzein reductase (THDR), dihydrodaidzein reductase (DHDR), and daidzein reductase (DZNR)) as well partial downstream and upstream genes based on amino acid sequences. The respective accession numbers were obtained from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/, (accessed on 14 August 2019)) and aligned using clustalW algorithm in the CLC Sequence Viewer software (version 8, Qiagen).

Figure 4

Phylogenetic tree based on 16S rRNA gene sequences of a total of 29 strains belonging to the Eggerthellaceae and Coriobacteriaceae. Clustering was performed based on multiple alignment and unweighted pair group method with arithmetic mean (UPGMA). Results of a PCR with primers amplifying for dzr, ddr and tdr [29] are shown next to the cluster.