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Equol is the isoflavone-derived metabolite with the greatest estrogenic and antioxidant activity. It is produced from daidzein by fastidious and oxygen-susceptible intestinal bacteria, which hinders their use at an industrial scale. Therefore, expressing the equol production machinery into easily-cultivable hosts would expedite the heterologous production of this compound. In this work, four genes (racemase, tdr, ddr and dzr) coding for key enzymes involved in equol production in Adlercreutzia equolifaciens DSM19450T were synthesized and cloned in a pUC-derived vector (pUC57-equol) that was introduced in Escherichia coli. Recombinant clones of E. coli produced equol in cultures supplemented with daidzein (equol precursor) and dihydrodaidzein (intermediate compound). To check whether equol genes were expressed in Gram-positive bacteria, the pUC57-equol construct was cloned into the low-copy-number vector pIL252, and the new construct (pIL252-pUC57-equol) introduced into model strains of Lacticaseibacillus casei and Lactococcus lactis. L. casei clones carrying pIL252-pUC57-equol produced a small amount of equol from dihydrodaidzein but not from daidzein, while L. lactis recombinant clones produced no equol from either of the substrates. This is the first time that A. equolifaciens equol genes have been cloned and expressed in heterologous hosts. E. coli clones harboring pUC57-equol could be used for biotechnological production of equol. [ABSTRACT FROM AUTHOR]

Isoflavones are transformed in the gut into more estrogen-like compounds or into inactive molecules. However, neither the intestinal microbes nor the pathways leading to the synthesis of isoflavone-derived metabolites are fully known. In the present work, 73 fecal isolates from three women with an equol-producing phenotype were considered to harbor equol-related genes by qPCR. After typing, 57 different strains of different taxa were tested for their ability to act on the isoflavones daidzein and genistein. Strains producing small to moderate amounts of dihydrodaidzein and/or O-desmethylangolensin (O-DMA) from daidzein and dihydrogenistein from genistein were recorded. However, either alone or in several strain combinations, equol producers were not found, even though one of the strains, W18.34a (also known as IPLA37004), was identified as Adlercreutzia equolifaciens, a well-described equol-producing species. Analysis and comparison of A. equolifaciens W18.34a and A. equolifaciens DSM19450T (an equol producer bacterium) genome sequences suggested a deletion in the former involving a large part of the equol operon. Furthermore, genome comparison of A. equolifaciens and Asaccharobacter celatus (other equol-producing species) strains from databases indicated many of these also showed deletions within the equol operon. The present results contribute to our knowledge to the activity of gut bacteria on soy isoflavones.

Two intestinal bacterial strains MT4s-5 and MT42 involved in the degradation of (−)-epigallocatechin (EGC) were isolated from rat feces. Strain MT4s-5 was tentatively identified as Adlercreutzia equolifaciens. This strain converted EGC into not only 1-(3, 4, 5-trihydroxyphenyl)-3-(2, 4, 6-trihydroxyphenyl)propan-2-ol ( 1), but also 1-(3, 5-dihydroxyphenyl)-3-(2, 4, 6-trihydroxyphenyl)propan-2-ol ( 2), and 4′-dehydroxylated EGC ( 7). Type strain (JCM 9979) of Eggerthella lenta was also found to convert EGC into 1. Strain MT42 was identified as Flavonifractor plautii and converted 1 into 4-hydroxy-5-(3, 4, 5-trihydroxyphenyl)valeric acid ( 3) and 5-(3, 4, 5-trihydroxyphenyl)-γ-valerolactone ( 4) simultaneously. Strain MT42 also converted 2 into 4-hydroxy-5-(3, 5-dihydroxyphenyl)valeric acid ( 5), and 5-(3, 5-dihydroxyphenyl)-γ-valerolactone ( 6). Furthermore, F. plautii strains ATCC 29863 and ATCC 49531 were found to catalyze the same reactions as strain MT42. Interestingly, formation of 2 from EGC by strain MT4s-5 occurred rapidly in the presence of hydrogen supplied by syntrophic bacteria. Strain JCM 9979 also formed 2 in the presence of the hydrogen or formate. Strain MT4s-5 converted 1, 3, and 4 to 2, 5, and 6, respectively, and the conversion was stimulated by hydrogen, whereas strain JCM 9979 could catalyze the conversion only in the presence of hydrogen or formate. On the basis of the above results together with previous reports, the principal metabolic pathway of EGC and EGCg by catechin-degrading bacteria in gut tract is proposed. [ABSTRACT FROM AUTHOR]

Isoflavones are transformed in the gut into more estrogen-like compounds or into inactive molecules. However, neither the intestinal microbes nor the pathways leading to the synthesis of isoflavone-derived metabolites are fully known. In the present work, 73 fecal isolates from three women with an equol-producing phenotype were considered to harbor equol-related genes by qPCR. After typing, 57 different strains of different taxa were tested for their ability to act on the isoflavones daidzein and genistein. Strains producing small to moderate amounts of dihydrodaidzein and/or O-desmethylangolensin (O-DMA) from daidzein and dihydrogenistein from genistein were recorded. However, either alone or in several strain combinations, equol producers were not found, even though one of the strains, W18.34a (also known as IPLA37004), was identified as Adlercreutzia equolifaciens, a well-described equol-producing species. Analysis and comparison of A. equolifaciens W18.34a and A. equolifaciens DSM19450T (an equol producer bacterium) genome sequences suggested a deletion in the former involving a large part of the equol operon. Furthermore, genome comparison of A. equolifaciens and Asaccharobacter celatus (other equol-producing species) strains from databases indicated many of these also showed deletions within the equol operon. The present results contribute to our knowledge to the activity of gut bacteria on soy isoflavones. [ABSTRACT FROM AUTHOR]

Given the emerging evidence of equol's benefit to human health, understanding its synthesis and regulation in equol-producing bacteria is of paramount importance. Adlercreutzia equolifaciens DSM19450T 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 A. equolifaciens DSM19450T 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 dzr, ddr and tdr, 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 A. equolifaciens DSM19450T, which might help in efforts to increase the endogenous formation of this compound and/or its biotechnological production. [ABSTRACT FROM AUTHOR]