Mechanisms for biochemical and genetic control in lignin and lignan biosyntheses

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First, systems for chiral HPLC separation and chiral LC-MS analysis of several lignans were established, which provide important basis for tracer experiments with stable isotopes in studies of lignan biosynthesis. The chiral LC-MS system was successfully applied to the assay of enzyme activity for enantioselective formation of (-)-secoisolariciresinol.Second, callus was successfully induced from explants of Forsythia koreana and the production systems for the cultured cells producing both lignans and lignan-synthesizing enzymes have been established. The calli have shown to produce several lignan glycosides. Many of the calli produced significant amounts of matairesinol (aglycones), exhibiting enzymatic activity forming secoisolariciresinol, a precursor of matairesinol biosynthesis, from coniferyl alcohol; comparable to those of field growing F.koreana plants. The results indicate potential use of F.koreana tissue cultures as a source of lignan synthesizing enzyme(s).Cell-free extracts from F.koreana catalyzed enantioselective formation of (-)-, but not (+)-,[^2H_<10>]secoisolariciresinol from [^2H_<10>]- coniferyl alcohol in the presence of NADPH and H_2O_2. Surprisingly, (-)- pinoresinol and (-)-lariciresinol were also formed in the incubation although they are not natural enantiomers isolated from F.koreana. In addition, the enzyme system catalyzed enantioselective reduction of (+)- lariciresinol to afford (-)-secoisolariciresinol in the presence of NADPH. Mechanisms for the enantioselective formation of (-)-secoisolariciresinol from coniferyl alcohol were proposed. The optical activity of lignans is formed probably by some of peroxidases and NADPH:quinone oxidoreductases. There are multiple forms of those enzymes in the young shoots from F. koreana. We also discussed strategies on the molecular cloning for the enzymes involved in lignan biosynthesis.