新疆紫草中紫草素生物合成途径关键酶HGC基因克隆及功能研究

Shikonins, the main active ingredients of the endangered medicinal plant Arnebia euchroma, have a variety of pharmacological effects and great economic value. Tissue culture has been used to replace direct extraction method in the production of shikonin and mitigate the shortage of natural resources. However, the yield of shikonin still failed to meet the needs of industry. Increased yield by culture cell would be achieved through the regulation of shikonin biosynthesis. 3''-hydroxy-geranylhydroquinone cyclase (HGC) is the key enzyme in shikonin biosynthesis and an important target in metabolic engineering, but it has not yet been characterized. After metabolomic and transcriptomic comparison of the shikonin proficient red line and deficient white cell line of A. euchroma, we will partially purify the crude enzyme from the red line and clarify the reaction mechanism of HGC using the active ingredient. Based on the above results, we are going to select the predicted HGC genes and construct yeast engineering strains to verify the function of predicted genes through enzymatic reaction in vitro. By means of gene overexpression and RNA interference, we are planning to not only clarify the role of HGC gene in shikonin biosynthesis but also aquire the shikonin proficient cell line with enhanced expression level of the gene. In this study, we intended to clone and analyze the function of HGC gene, which encode the key enzyme in shikonin biosynthesis of A. euchroma, through comprehensive bioinformatics, chemistry, and molecular biology methods. The expected outcomes will provide a new target for the regulation of shikonin biosynthesis and pave the way for the clarification of the further pathway, which may help to relief the stress of the wild A. euchroma resources.

紫草素类化合物是濒危药用植物新疆紫草的主要有效成分，具有广泛药理活性和显著经济价值。以组织培养代替直接提取缓解了紫草素天然来源短缺，但产量无法满足工业需求。应用代谢工程调控紫草素生物合成可提高产量。3''-羟基牻牛儿基对苯二酚环化酶（HGC）是紫草素生物合成途径关键酶和重要调控靶标，迄今国内外尚未克隆鉴定。本课题分析新疆紫草高产紫草素（红色）和低产（白色）细胞系之间代谢组和转录组差异后，再提取红色细胞系粗酶检测HGC活性并确认反应机制。据此克隆HGC基因，在酵母中外源表达验证功能。通过抑制和过表达组织培养细胞HGC基因，揭示其对紫草素生物合成的调控作用，获得HGC表达上调的高产紫草素细胞系。本课题综合运用生物信息学、化学和分子生物学方法挖掘HGC基因并研究其功能，为利用代谢工程手段调控紫草素生物合成提供全新靶标，并为解析紫草素后续生物合成途径奠定基础，对缓解新疆紫草野生资源压力有重要意义。

紫草素类化合物是濒危药用植物新疆紫草的主要有效成分，具有广泛药理活性和显著经济价值。以组织培养代替直接提取缓解了紫草素天然来源短缺，但产量无法满足工业需求。应用合成生物学技术异源生产紫草素可提高产量。GBA氧化脱羧酶（GBAD）和GHQ C-3''羟基化酶（GHQH）是紫草素生物合成途径关键酶和重要调控靶标，迄今国内外尚无相关报道。本课题分析新疆紫草高产紫草素（红色）和低产（白色）细胞系之间代谢组和转录组差异，据此克隆GHQH基因，在酵母中外源表达验证功能。通过抑制毛状根GHQH基因，揭示其对紫草素生物合成的调控作用。利用分子模拟技术对担子菌FAD依赖单氧化酶VibMO1进行蛋白结构改造，获得了可高效催化GBA脱羧羟基化反应的突变体。本课题综合运用生物信息学、化学、分子生物学和蛋白质工程技术获得GBAD及GHQH，为利用合成生物学手段实现紫草素生产提供新的生物元件，并为解析紫草素后续生物合成途径奠定基础，对缓解新疆紫草野生资源压力有重要意义。