Transcriptional Regulation of the Equol Biosynthesis Gene Cluster in Adlercreutzia equolifaciens DSM19450T

Abstract

Given the emerging evidence of equol's benefit to human health, understanding its synthesis and regulation in equol-producing bacteria is of paramount importance. <i>Adlercreutzia</i> <i>equolifaciens</i> DSM19450^T is a human intestinal bacterium -for which the whole genome sequence is publicly available- that produces equol from the daidzein isoflavone. In the present work, daidzein (between 50 to 200 μM) was completely metabolized by cultures of <i>A.</i> <i>equolifaciens</i> DSM19450^T after 10 h of incubation. However, only about one third of the added isoflavone was transformed into dihydrodaidzein and then into equol. Transcriptional analysis of the ORFs and intergenic regions of the bacterium's equol gene cluster was therefore undertaken using RT-PCR and RT-qPCR techniques with the aim of identifying the genetic elements of equol biosynthesis and its regulation mechanisms. Compared to controls cultured without daidzein, the expression of all 13 contiguous genes in the equol cluster was enhanced in the presence of the isoflavone. Depending on the gene and the amount of daidzein in the medium, overexpression varied from 0.5- to about 4-log₁₀ units. Four expression patterns of transcription were identified involving genes within the cluster. The genes <i>dzr</i>, <i>ddr</i> and <i>tdr</i>, which code for daidzein reductase, dihydrodaidzein reductase and tetrahydrodaidzein reductase respectively, and which have been shown involved in equol biosynthesis, were among the most strongly expressed genes in the cluster. These expression patterns correlated with the location of four putative ρ-independent terminator sequences in the cluster. All the intergenic regions were amplified by RT-PCR, indicating the operon to be transcribed as a single RNA molecule. These findings provide new knowledge on the metabolic transformation of daidzein into equol by <i>A.</i> <i>equolifaciens</i> DSM19450^T, which might help in efforts to increase the endogenous formation of this compound and/or its biotechnological production.