南欧大戟巨大戟醇当归酸酯类活性成分生物合成关键酶基因的鉴定和功能研究

Ingenol and its analogues from Euphorbia peplus are a type of Euphorbia diterpenes attracting the great interest due to their wide range of bioactivity, among which ingenol mebutate was approved by the FDA as a first-in-class treatment for actinic keratosis, a precancerous skin condition. Recently, our group also characterized 5β-O-angelate-20-deoxyingenol holding a promise for treatment of neurodegenerative disease. However, the extremely low content of these compounds in plant and low yield in chemical synthesis preclude their broader application in drug market. The synthetic biology is considered particularly promising as an alternative methodology to obtain these valuable terpenoids, however, their biosynthetic pathway remains to be elucidated. In our application, a Euphorbia peplus inbred plant producing higher level of these type compounds and showing low heterozygosity is chosen as subject. We will use genomic mining, transcriptome mining, bioinformatic analysis, and biochemical analysis to identify genes involved in the biosynthetic pathway. We plan to assay the enzymes activity using Nicotiana benthamiana transient expression system, in combination with RNAi, CRISPR-Cas9 and chemical synthesis methods, to finally elucidate the biosynthetic pathway. Moreover, the obtained intermediates will be evaluated for their bioactivity. Our research is significant of providing enough supply for ingenol mebutate and discover and develop more promising new drugs.

南欧大戟中的巨大戟醇酯是一类具有重要应用价值和前景的天然产物，如3-O-当归酸酯（商品名Picato®)已被FDA批准用于治疗光线性角化病，我们发现的20-脱氧-5-O-当归酸酯可作为治疗神经退行性疾病的先导化合物。但因在植物中含量低，并且化学合成工艺繁琐、收率低，无法满足药物开发的需要。合成生物学方法被认为是解决药源问题的有效途径，然而其生物合成途径尚不清楚。本项目拟以经多代自交纯合的巨大戟醇酯高产南欧大戟二倍体植株为研究对象，利用基因组、转录组、生物信息学、生物化学等分析手段，分离参与巨大戟醇酯类生物合成途径的酶基因，通过RNAi、CRISPR-Cas9、烟草细胞瞬时表达体系、以及化学合成等方法，验证酶功能，最终阐明其生物合成途径；开展生物合成中间体的生物活性研究。课题的实施对该类化合物发展为新型的、更具临床应用前景的药物具有重要的理论意义和应用前景。

南欧大戟中含有活性好但含量低的巨大戟醇酯二萜类化合物，如FDA批准用于治疗日光角化病的巨大戟醇甲基丁烯酸酯，及本课题组发现的可作为治疗神经退行性疾病的先导化合物20-去氧-5-O-当归酸酯等。本项目主要研究它们的生物合成途径，为最终通过合成生物学等方法生产该类化合物奠定基础。该类化合物多含有酰基取代，且为活性基团，克隆鉴定催化巨大戟醇酯酰基化酶具有重要的理论和实际意义。植物中负责酰基化反应的主要为以酰基辅酶A为供体的BAHD超家族基因，本项目通过基因组、转录组测序和生物信息学分析，在南欧大戟中挖掘到参与巨大戟醇酯酰基化的BAHD候选基因，通过植物化学和化学合成方法获得合适的底物，利用体外酶活分析、烟草瞬时表达等方法鉴定候选基因的功能，最终鉴定到了催化20-去氧巨大戟醇5位羟基乙酰化的BAHD酰基转移酶，它与20-去氧-5-O-当归酸酯竞争底物，在南欧大戟中敲除该基因，可以提高20-去氧-5-O-当归酸酯的含量。通过定点突变和标记底物探针，研究了相关萜类合酶的催化机制。此外，获得了15个新颖的巨大戟醇二萜类似物。其中7个新的二萜可促进溶酶体生物发生，包括一个新颖的二萜其溶酶体诱导活性与明星分子20-去氧巨大戟醇-5-当归酯相当，但二者化学结构差异巨大，暗示新的溶酶体生成机制。这些研究结果加深我们对真核生物的溶酶体生成和调节机制的认识。.在项目执行期间，发表了学术论文8篇，其中SCI收录7篇。培养了硕士研究生4名。项目主持人入选云南省中青年学术和技术后备人才。