Mechanisms for biochemical and genetic control in lignin and lignan biosyntheses

首先，建立了几种木脂素类化合物的手性HPLC分离和手性LC-MS分析体系，为木脂素类化合物生物合成研究中稳定同位素示踪实验提供了重要依据。将手性LC-MS系统成功地应用于（-）-开环异落叶松树脂醇对映体选择性合成酶活性的测定。第二，成功地从朝鲜连翘外植体中诱导出愈伤组织，建立了同时产生木脂素和木脂素合成酶的细胞培养体系。愈伤组织已显示出产生几种木脂素糖苷。许多愈伤组织产生显著量的matairesinol（糖苷配基），表现出从松柏醇形成开环异落叶松树脂醇（matairesinol生物合成的前体）的酶活性;与田间生长的朝鲜F.koreana植物的那些相当。在NADPH和H_2O_2存在下，朝鲜冷杉无细胞提取物催化[^2H_ ]-松柏醇对映选择性生成（-）-[^2H_ ]开环异落叶松树脂酚，而不催化（+）-[^2H<10>_]开环异落叶松树脂酚<10>。令人惊讶的是，（-）-松叶醇和（-）-落叶松树脂酚也形成了温育，虽然它们不是天然的对映异构体分离自F. koreana。此外，在NADPH存在下，该酶催化（+）-落叶松树脂酚对映选择性还原生成（-）-开环异落叶松树脂酚。提出了由松柏醇不对称合成（-）-开环异落叶松树脂酚的反应机理。木脂素类化合物的光学活性可能是由过氧化物酶和NADPH：醌氧化还原酶形成的。这些酶在F.朝鲜语。我们还讨论了木脂素生物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.