Bioconversion of Pinoresinol into Matairesinol by Use of Recombinant Escherichia coli

Abstract

Lignans, a class of dimeric phenylpropanoid derivative found in plants, such as whole grains and sesame and flax seeds, have anticancer activity and can act as phytoestrogens. The lignans secoisolariciresinol and matairesinol can be converted in the mammalian proximal colon into enterolactone and enterodiol, respectively, which reduce the risk of breast and colon cancer. To establish an efficient bioconversion system to generate matairesinol from pinoresinol, the genes encoding pinoresinol-lariciresinol reductase (PLR) and secoisolariciresinol dehydrogenase (SDH) were cloned from Podophyllum pleianthum Hance, an endangered herb in Taiwan, and the recombinant proteins, rPLR and rSDH, were expressed in Escherichia coli and purified. The two genes, termed plr-PpH and sdh-PpH, were also linked to form two bifunctional fusion genes, plr-sdh and sdh-plr, which were also expressed in E. coli and purified. Bioconversion in vitro at 22°C for 60 min showed that the conversion efficiency of fusion protein PLR-SDH was higher than that of the mixture of rPLR and rSDH. The percent conversion of (؉)-pinoresinol to matairesinol was 49.8% using PLR-SDH and only 17.7% using a mixture of rPLR and rSDH. However, conversion of (؉)-pinoresinol by fusion protein SDH-PLR stopped at the intermediate product, secoisolariciresinol. In vivo, (؉)-pinoresinol was completely converted to matairesinol by living recombinant E. coli expressing PLR-SDH without addition of cofactors.

Lignans and Gut Microbiota: An Interplay Revealing Potential Health Implications

“…However, exactly defining the effectiveness of such conversion, according to the type of lignan molecule, is a difficult task due to the inconsistency of results. Nonetheless, both in vitro [19,22] and in vivo [23] studies reported that the catabolic transformation of SECO and MAT was observed to be greater than LAR, SYR, PINO, and sesamin and, in turn, that aglycones are more efficiently metabolized than diglucosylated derivatives. Except for MAT that generates EL but not ED, the contribution of each plant lignan to the whole pool of ED and EL is not entirely clear [19].…”

“…In another study, after the daily intake of 0.3 g flaxseed/kg body weight by healthy man for 1 week, the mean serum concentration of ED and EL increased 10-fold compared to baseline until reach 20 and 60 nM, respectively, but not all the participant responded to the same form since ED increased in all participants while EL remained unchanged in two subjects [26]. Many dietary interventions including pharmacokinetic studies in human [11] and animals [23,24] and prolonger plant lignan supplementation in human [13] have presented the mean values of ED and EL or only EL in biofluids before and after the intervention. Still, they have not discriminated among categories of different metabolic responses losing the opportunity to gather data to define how individuals respond to plant lignans supplementation as proposed by other authors [15].…”

Investigation and Expression of the Secoisolariciresinol Dehydrogenase Gene Involved in Podophyllotoxin Biosynthesis

“…Each bioconversion reaction was prepared with a total volume of 250 ll that contained 20 mM Tris buffer (pH 8.8), 5 mM DTT, 4 mM NAD, and 165 lM (-)-secoisolariciresinol [29]. The reaction was incubated at 30°C for 2 h. Each sample was extracted 39 with 300 ll ethyl acetate (EtOAc), the combined EtOAc extract was dried under N 2 (g), and the precipitate reconstituted in 200 ll methanol (MeOH) for HPLC and LC-MS analyses.…”

Assembly of Plant Enzymes in E. coli for the Production of the Valuable (−)-Podophyllotoxin Precursor (−)-Pluviatolide

“…Generally, the expression temperature and duration substantially influenced CYP719A23 expression (Table S13). Although the highest expression of CYP719A23 was achieved at 20 °C, 25 °C was chosen as a compromise for establishing the reaction cascade with regard to the optimal expression conditions for FiPLR and PpSDH reported elsewhere and confirmed in our previous study …”