4-Coumaroyl-CoA ligase (4CL) is a key enzyme in the phenylpropanoid pathway, which is involved in the biosynthesis of various specialized metabolites such as flavonoids, coumarins, lignans, and lignin. Plants have several 4CLs showing divergence in sequence: class I 4CLs involved in lignin metabolism, class II 4CLs associated with flavonoid metabolism, and atypical 4CLs and 4CL-like proteins of unknown function. Shikonin, a Boraginaceae-specific specialized metabolite in red gromwell (Lithospermum erythrorhizon), is biosynthesized from p-hydroxybenzoic acid, and the involvement of 4CL in its biosynthesis has long been debated. In this study, we demonstrated the requirement of 4CL for shikonin biosynthesis using a 4CL-specific inhibitor. In silico analysis of the L. erythrorhizon genome revealed the presence of at least eight 4CL genes, among which the expression of three (Le4CL3, Le4CL4, and Le4CL5) showed a positive association with shikonin production. Phylogenetic analysis indicated that Le4CL5 belongs to class I 4CLs, while Le4CL3 and Le4CL4 belong to clades that are distant from class I and class II. Interestingly, both Le4CL3 and Le4CL4 have peroxisome targeting signal 1 in their C-terminal region, and subcellular localization analysis revealed that both localize to the peroxisome. We targeted each of the three Le4CL genes by CRISPR/Cas9-mediated mutagenesis and observed remarkably lower shikonin production in Le4CL3-ge and Le4CL4-ge genome-edited lines compared with the vector control. We therefore conclude that peroxisomal Le4CL3 and Le4CL4 are responsible for shikonin production and propose a model for metabolite-specific 4CL distribution in L. erythrorhizon.

中文翻译：

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过氧化物酶体4-香豆酰辅酶A连接酶参与紫草紫草素的生产


4-香豆酰辅酶A连接酶（4CL）是苯丙素途径中的关键酶，参与黄酮类、香豆素、木脂素和木质素等各种专门代谢物的生物合成。植物有几种 4CL 在序列上表现出差异：I 类 4CL 涉及木质素代谢，II 类 4CL 与类黄酮代谢相关，以及非典型 4CL 和功能未知的 4CL 样蛋白。紫草素是红紫草（紫草）中紫草科特有的特殊代谢物，由对羟基苯甲酸生物合成，4CL 参与其生物合成长期以来一直存在争议。在这项研究中，我们使用 4CL 特异性抑制剂证明了紫草素生物合成需要 4CL。对红根草基因组的计算机分析揭示了至少八个 4CL 基因的存在，其中三个（Le4CL3、Le4CL4 和 Le4CL5）的表达与紫草素的产生呈正相关。系统发育分析表明，Le4CL5属于I类4CL，而Le4CL3和Le4CL4属于远离I类和II类的进化枝。有趣的是，Le4CL3 和 Le4CL4 在其 C 末端区域都有过氧化物酶体靶向信号 1，亚细胞定位分析表明两者都定位于过氧化物酶体。我们通过 CRISPR/Cas9 介导的诱变分别针对三个 Le4CL 基因，并观察到与载体对照相比，Le4CL3-ge 和 Le4CL4-ge 基因组编辑品系中紫草素的产量显着降低。因此，我们得出结论，过氧化物酶体 Le4CL3 和 Le4CL4 负责紫草素的产生，并提出了红根草中代谢物特异性 4CL 分布的模型。