Mechanisms for biochemical and genetic control in lignin and lignan biosyntheses

首先，建立了几种木脂素的手性HPLC分离体系和手性LC-MS分析体系，为木脂素生物合成研究中稳定同位素示踪实验提供了重要依据。手性LC-MS系统成功地应用于(-)-二异松脂醇对映选择性形成酶活性的测定。其次，以连翘为外植体成功诱导愈伤组织，并建立了同时产生木脂素和木脂素合成酶的细胞生产体系。愈伤组织可以产生几种木脂素苷。许多愈伤组织产生了大量的松柏醇（苷元），表现出酶促活性，形成了松柏醇生物合成松柏醇的前体——二异松柏醇；可与田间种植的韩属植物相媲美。研究结果表明，韩国假丝莲组织培养物可能作为木脂素合成酶的来源。在NADPH和H_2O_2存在下，韩国红的无细胞提取物可催化[^2H_<10>]-松柏醇（-)-而非(+)- [^2H_<10>]-异松柏醇对映选择性生成[-）-,[^2H_<10>]。令人惊讶的是，(-)-松脂醇和(-)-松脂醇和(-)-松脂醇和虽然它们不是从韩国真菌中分离出来的天然对映体，但也在孵育过程中形成。此外，该酶系统在NADPH存在下催化(+)-松脂醇的对映选择性还原生成(-)-二异松脂醇。提出了松柏醇对映选择性生成(-)-二异松脂醇的机理。木脂素的光学活性可能是由一些过氧化物酶和醌氧化还原酶形成的。这些酶有多种形式存在于韩国红毛菌的幼芽中。讨论了木脂素合成相关酶的分子克隆策略。

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.