Mechanisms for biochemical and genetic control in lignin and lignan biosyntheses

首先，建立了几种木脂素的手性高效液相色谱分离和手性LC-MS分析体系，为木脂素生物合成的稳定同位素示踪实验提供了重要依据。利用手性LC-MS系统成功地测定了不对称合成(-)-二异落叶松脂醇的酶活性。其次，从朝鲜连翘外植体中成功地诱导出愈伤组织，并建立了培养细胞同时产生木脂素和木脂素合成酶的生产体系。愈伤组织产生了几种木脂素糖苷。许多愈伤组织产生大量的松脂醇(苷元)，表现出酶活性，从松树醇生物合成松脂醇的前体--次异松脂醇；与田间种植的朝鲜铁杉相当。结果表明，在NADPH和H_2O_2存在下，朝鲜金针菇组织培养物有可能作为木脂素合成酶(S)的来源。朝鲜金针菇的无细胞提取物能催化(-)-，而不是()-，[^2H_&lt；10&gt；]-针叶松脂醇在NADPH和H_2O_2存在下选择性地生成(-)-，[^2H_&lt；10&gt；]-二异落叶松脂醇。令人惊讶的是，(-)-松脂醇和(-)-落叶松脂醇也在培养过程中生成，尽管它们不是从朝鲜金针菇中分离出来的天然对映体。此外，在NADPH存在下，该酶体系还催化()-落叶松香酚对映体选择性还原为(-)-二异落叶松香酚。提出了松柏醇对映体选择性生成(-)-二异落叶松脂醇的反应机理。木脂素的光学活性可能是由一些过氧化物酶和NADPH：苯醌氧化还原酶形成的。这些酶在朝鲜铁杉的幼芽中有多种形式。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.