松香烷型三环二萜关键C14位CYP450的挖掘与功能研究

Oxidation is one of the most common post-modification reactions in the terpenoid biosynthesis pathways. It is mainly catalyzed by cytochrome P450-dependent monooxygenase (CYP450). Due to the low sequence homology and high substrate specificity, the identification of CYP450 has been one of the toughest obstacles in the elucidation of terepnoid biosynthesis pathway. C-14 oxidation is widely distributed in the abietane-type diterpenoids. However, the responsible CYP450 remains elusive. Isodon lophanthoides var. gerardiana is a traditional Chinese herbal medicine rich in C14-oxidative abietane-type diterpenoids. Using a combination of transcriptomics, homologous comparison and tissue-specific expression pattern analysis, 10 CYP450 candidates were isolated from I. lophanthoides after two rounds of screening. A third round of screening will be conducted base on the methyl jasmonate (MJ)-induced expression pattern analysis; the full-length of the candidate genes will be obtained using RACE-PCR. The functions of the resulting final CYP450 candidates will be studied using heterologous expression systems in both Saccharomyces cerevisiae and plants by co-transformation of high-yield yeast chassis cells, in vitro enzymatic reaction, transient transformation of tobacco leaves, GC-MS analysis, etc. The aim of this study is to reveal the function of the C-14 oxidase in order to elucidate the biosynthetic pathway of abietane-type diterpenes and provide key genetic resources for the further metabolite engineering of abietane-type diterpenes.

氧化是萜类后修饰中最常见的方式，主要由CYP450催化完成。由于较低的序列同源性和较高的底物特异性，CYP450的研究一直是次生代谢领域的难点之一。C14位氧化修饰广泛存在于许多具有重要药理活性的松香烷型三环二萜中，而负责的关键CYP450基因至今未被克隆。本项目以富含松香烷型三环二萜的药用植物狭基线纹香茶菜为研究对象，通过叶片转录组测序，结合同源比对和组织特异性表达模式进行两轮筛选，获得了10个C14位CYP450候选基因。我们将在此基础上根据茉莉酸甲酯(MJ)诱导表达模式开展第三轮筛选；通过RACE-PCR技术获得候选基因全长。随后拟借助酵母和植物表达系统开展功能研究，通过共转化高产酵母底盘细胞、体外酶促反应、烟草叶片瞬时表达、气质联用等技术和方法，揭示松香烷型三环二萜C14位CYP450功能，完善松香烷型三环二萜生物合成途径，并为这类化合物的代谢工程研究提供关键的基因资源。

岭南特色中草药狭基线纹香茶菜中富含高度氧化的松香烷型三环二萜，具有多种重要的药理活性。本项目旨在分离狭基线纹香茶菜中参与松香烷型二萜生物合成的关键P450，并对它们的功能进行研究，取得研究成果如下：1.首先我们系统分离和鉴定了狭基线纹香茶菜中二萜合酶基因家族，为下游P450的研究提供了底物保证；2.利用分离得到的二萜合酶基因，我们构建并优化了松香烷型二萜直接前体miltiradiene高产的酵母底盘细胞；3.利用转录组和代谢组数据，结合上游二萜合酶组织表达特异和诱导特性，我们建立了一套筛选P450基因的方法并最终得到8个候选基因；4.利用优化的酵母底盘和传统的烟草瞬时表达系统，我们成功从8个候选基因中得到5个具有活性的P450基因，其中3个为铁锈醇合酶/11-羟基铁锈醇合酶，2 个P450产生了新产物，由于新产物量相对较低，目前正在进行优化，以提高产量用于结构鉴定。本项目相关成果发表于Plant and cell physiology杂志1篇，目前投稿Planta杂志正在按照审稿意见修改1篇，新功能P450部分待完成产物结构鉴定后，可撰写论文进行公开发表。通过该项目培养硕士研究生和科研助理各一名。