Biotransformation of an exogenously supplied isoflavonoid by transgenic tobacco cells expressing alfalfa isoflavone reductase

Isoflavone reductase (IFR) from alfalfa catalyzes the NADPH-dependent reduction of 2'-hydroxy isoflavones to 2'-hydroxyisoflavanones such as vestitone, thereby performing a key step in isoflavonoid phytoalexin biosynthesis. Tobacco (Nicotiana tabacum L. cv. Xanthi) was transformed with an alfalfa cDNA encoding IFR regulated by an enhanced cauliflower mosaic virus 35S promoter and used to generate cell suspension cultures. Transformed cells expressed high levels of IFR activity and could take up 2'-hydroxyformononetin (2'OHF; IFR substrate) and convert it to vestitone. Both 2'OHF and vestitone were each converted to two sugar conjugates. The structure of the most abundant vestitone conjugate was determined using NMR spectroscopic analysis to be a novel vestitone 2'-O-glucoside. Liquid chromatography-mass spectroscopy (LC-MS) indicated that the molecular weights of the three less abundant conjugates were consistent with hexose conjugates of vestitone and 2'OHF. The IFR reduction and conjugation reactions were complete within 4 h, with the net percentage conversion of 2'OHF to vestitone ranging as high as 60%.