Identification and Expression of Genes Involved in the Conversion of Daidzein and Genistein by the Equol-Forming Bacterium Slackia isoflavoniconvertens

Schröder, Christine; Matthies, Anastasia; Engst, Wolfram; Blaut, Michaël; Braune, Annett

doi:10.1128/aem.03693-12

Cited by 104 publications

(140 citation statements)

References 30 publications

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“…Wild-type DHDR has a catalytic efficiency that is higher by a factor of about 23.5 for (S)-DHD than for (R)-DHD in terms of the k cat /K m ratio. We confirmed that DHDR prefers (S)-DHD to (R)-DHD as its substrate (9,13).…”

Section: Resultssupporting

confidence: 70%

“…Although DHDRs from S. isoflavoniconvertens (13) and Lactococcus sp. strain 20-82 (10) were previously characterized based on their kinetic parameters, cofactor specificity, and preference for (S)-DHD, the analyses were restricted only to neutral pH, which prohibited a complete understanding of DHDR's characteristics.…”

Section: Discussionmentioning

confidence: 99%

“…Similar identifications of equol metabolism in Slackia sp. have also been reported (12,13). According to these reports, daidzein is converted to (S)-equol by four enzymes ( Fig.…”

mentioning

confidence: 99%

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P212A Mutant of Dihydrodaidzein Reductase Enhances ( S )-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System

Lee

Kim

et al. 2016

Appl Environ Microbiol

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d(S)-Equol, a gut bacterial isoflavone derivative, has drawn great attention because of its potent use for relieving female postmenopausal symptoms and preventing prostate cancer. Previous studies have reported on the dietary isoflavone metabolism of several human gut bacteria and the involved enzymes for conversion of daidzein to (S)-equol. However, the anaerobic growth conditions required by the gut bacteria and the low productivity and yield of (S)-equol limit its efficient production using only natural gut bacteria. In this study, the low (S)-equol biosynthesis of gut microorganisms was overcome by cloning the four enzymes involved in the biosynthesis from Slackia isoflavoniconvertens into Escherichia coli BL21(DE3). The reaction conditions were optimized for (S)-equol production from the recombinant strain, and this recombinant system enabled the efficient conversion of 200 M and 1 mM daidzein to (S)-equol under aerobic conditions, achieving yields of 95% and 85%, respectively. Since the biosynthesis of trans-tetrahydrodaidzein was found to be a rate-determining step for (S)-equol production, dihydrodaidzein reductase (DHDR) was subjected to rational site-directed mutagenesis. The introduction of the DHDR P212A mutation increased the (S)-equol productivity from 59.0 mg/liter/h to 69.8 mg/liter/h in the whole-cell reaction. The P212A mutation caused an increase in the (S)-dihydrodaidzein enantioselectivity by decreasing the overall activity of DHDR, resulting in undetectable activity for (R)-dihydrodaidzein, such that a combination of the DHDR P212A mutant with dihydrodaidzein racemase enabled the production of (3S,4R)-tetrahydrodaidzein with an enantioselectivity of >99%.

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Section: Resultssupporting

confidence: 70%

Section: Discussionmentioning

confidence: 99%

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P212A Mutant of Dihydrodaidzein Reductase Enhances ( S )-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System

Lee

Kim

et al. 2016

Appl Environ Microbiol

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“…The gene cloned in pPSG-IBA 3 or pPSG-IBA 5 was expressed in E. coli KRX following induction by rhamnose (0.1% [wt/vol]) and cultivation overnight at 25°C. Purification of tagged proteins from cell extracts was done using a 1-ml Strep-Tactin Sepharose column (IBA) as described previously (21).…”

Section: Chemicals (ϯ)-Taxifolin and (ϯ)mentioning

confidence: 99%

Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols

et al. 2016

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The enzyme catalyzing the ring-contracting conversion of the flavanonol taxifolin to the auronol alphitonin in the course of flavonoid degradation by the human intestinal anaerobe Eubacterium ramulus was purified and characterized. It stereospecifically catalyzed the isomerization of (؉)-taxifolin but not that of (؊)-taxifolin. The K m for (؉)-taxifolin was 6.4 ؎ 0.8 M, and the V max was 108 ؎ 4 mol min ؊1 (mg protein) ؊1 . The enzyme also isomerized (؉)-dihydrokaempferol, another flavanonol, to maesopsin. Inspection of the encoding gene revealed its complete identity to that of the gene encoding chalcone isomerase (CHI) from E. ramulus. Based on the reported X-ray crystal structure of CHI (M. Gall et al., Angew Chem Int Ed 53:1439 -1442, 2014, http://dx.doi.org/10.1002/anie.201306952), docking experiments suggest the substrate binding mode of flavanonols and their stereospecific conversion. Mutation of the active-site histidine (His33) to alanine led to a complete loss of flavanonol isomerization by CHI, which indicates that His33 is also essential for this activity. His33 is proposed to mediate the stereospecific abstraction of a proton from the hydroxymethylene carbon of the flavanonol C-ring followed by ring opening and recyclization. A flavanonol-isomerizing enzyme was also identified in the flavonoid-converting bacterium Flavonifractor plautii based on its 50% sequence identity to the CHI from E. ramulus. IMPORTANCE Chalcone isomerase was known to be involved in flavone/flavanone conversion by the human intestinal bacterium E. ramulus.Here we demonstrate that this enzyme moreover catalyzes a key step in the breakdown of flavonols/flavanonols. Thus, a single isomerase plays a dual role in the bacterial conversion of dietary bioactive flavonoids. The identification of a corresponding enzyme in the human intestinal bacterium F. plautii suggests a more widespread occurrence of this isomerase in flavonoid-degrading bacteria. F lavonoids represent a major group of plant-derived polyphenolic compounds and have been implicated in beneficial effects on human health (1-4). The flavonoids are classified into several subgroups, which include, among others, flavonols, flavanonols, flavones, flavanones, and chalcones. Quercetin (Fig. 1) is a highly abundant dietary flavonoid that shows a broad range of biological activities. Thus, this flavonol has been intensively studied with respect to its role in disease prevention and use in therapy (5-7). The effects of quercetin and other flavonoids depend on how they are metabolized in the human body, including their conversion by intestinal bacteria (8). Beside deglycosylation and deconjugation, intestinal bacteria are also able to further transform the resulting flavonoid aglycones. However, knowledge about the corresponding bacterial species and, in particular, the enzymes involved is still limited.Eubacterium ramulus and Flavonifractor plautii (formerly Clostridium orbiscindens) are human intestinal bacteria that have been demonstrated to cleave the central heterocyc...

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“…The reported isoflavone metabolites were utilized for various metabolomic studies to investigate correlations among host physiology, host metabolism, and gut microbiota changes (Lee et al 2012;Kobayashi et al 2013). The genes involved in the biotransformation of daidzein and the metabolites were also cloned from the isolated intestinal bacteria for the biochemical research (Shimada et al 2010;Tsuji et al 2012;Schröder et al 2013). Therefore, the isolation of new intestinal bacteria and characterization of the catalytic properties are of great significant.…”

Section: Discussionmentioning

confidence: 99%

Deglycosylation of flavonoid O-glucosides by human intestinal bacteria Enterococcus sp. MRG-2 and Lactococcus sp. MRG-IF-4

Kim

Han

2016

Appl Biol Chem

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Daidzin, daidzein 7-O-glucoside, is a major isoflavone in soybean and acts as a phytoestrogen. By intestinal bacteria dietary, daidzin is hydrolyzed to the aglycone daidzein and further converted to the more reduced metabolites, such as dihydrodaidzein, tetrahydrodaidzein, and equol. Human intestinal bacteria Enterococcus sp. MRG-2 and Lactococcus sp. MRG-IF-4, which convert daidzin to daidzein, were isolated under anaerobic condition, and identified by 16S rRNA gene sequence analysis. Changes of OD 600 and pH were measured during the anaerobic growth, and deglycosylation kinetics of daidzin and genistin were measured by HPLC. Both bacteria also converted other isoflavone 7-O-glucosides, glycitin, ononin, and sissotrin, to their aglycones, glycitein, formononetin, and biochanin A, respectively. Apigetrin was deglycosylated to apigenin by these bacteria too. However, rutin, hesperidin, and naringin were not converted to the aglycones. Phylogeny analysis of the isolated strains also found that bacterial species identified by 16S rRNA gene sequence was not correlated with its metabolic ability of flavonoid biotransformation.

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Identification and Expression of Genes Involved in the Conversion of Daidzein and Genistein by the Equol-Forming Bacterium Slackia isoflavoniconvertens

Cited by 104 publications

References 30 publications

P212A Mutant of Dihydrodaidzein Reductase Enhances ( S )-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System

P212A Mutant of Dihydrodaidzein Reductase Enhances ( S )-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System

Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols

Deglycosylation of flavonoid O-glucosides by human intestinal bacteria Enterococcus sp. MRG-2 and Lactococcus sp. MRG-IF-4

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