丹参酮生物合成途径中C20位氧化酶改造研究  

Modification of C20 oxidase in tanshinone biosynthesis pathway

作　　者：曹小青 马晓惠 程亚田 李琦爽 卜俊玲 马莹[2] 郭娟 CAO Xiao-qing;MA Xiao-hui;CHENG Ya-tian;LI Qi-shuang;BU Jun-ling;MA Ying;GUO Juan(Yunnan Key Laboratory of Sustainable Utilization of Southern Medicine,College of Traditional Chinese Medicine,Yunnan University of Chinese Medicine,Kunming 650500,China;State Key Laboratory Breeding Base of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]云南中医药大学中药学院、云南省南药可持续利用研究重点实验室,云南昆明650500 [2]中国中医科学院中药资源中心、道地药材国家重点实验室培育基地,北京100700

出　　处：《中国中药杂志》2023年第9期2298-2306,共9页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82003904,31961133007,82011530137);国家重点研发计划项目(2020YFA0908000);中央本级重大增减支项目(2060302);中国中医科学院科技创新工程项目(CI2021A04110)。

摘　　要：丹参酮类化合物是丹参主要有效成分之一,在心血管类疾病治疗中发挥重要作用,丹参酮类化合物的微生物异源生产能够为含丹参中药制剂生产提供大量原材料,降低提取成本,缓解临床用药压力。丹参酮生物合成途径包含多个P450酶,高效优势的催化元件是丹参酮微生物生产的基础,该研究以丹参酮途径关键P450-C20位羟化酶CYP76AK1为研究对象,使用SWISS-MODEL、Robetta和AlphaFold2这3种蛋白建模方法,对所得蛋白模型进行分析,获取可靠蛋白结构,以分子对接和同源比对进行突变体蛋白的半理性设计。筛选发现了影响CYP76AK1氧化活性的关键氨基酸位点,通过真核表达对所得突变体进行体外功能研究。研究获得了对11-羟基柳杉酚具有连续氧化功能的CYP76AK1突变体元件,解析了影响其氧化活性的4个关键氨基酸位点,并根据突变结果分析3种蛋白建模方法的可靠性。研究首次报道了丹参中CYP76AK1的有效蛋白改造位点,为丹参酮类化合物合成生物学研究提供了C20位不同氧化活性的催化元件,为解析C20位修饰P450蛋白的连续氧化机制奠定基础。Tanshinones are one of the main effective components of Salvia miltiorrhiza,which play important roles in the treatment of cardiovascular diseases.Microbial heterogony production of tanshinones can provide a large number of raw materials for the production of traditional Chinese medicine(TCM)preparations containing S.miltiorrhiza,reduce the extraction cost,and relieve the pressure of clinical medication.The biosynthetic pathway of tanshinones contains multiple P450 enzymes,and the catalytic element with high efficiency is the basis of microbial production of tanshinones.In this study,the protein modification of CYP76AK1,a key P450-C20 hydroxylase in tanshinone pathway,was researched.The protein modeling methods SWISS-MODEL,Robetta,and AlphaFold2 were used,and the protein model was analyzed to obtain the reliable protein structure.The semi-rational design of mutant protein was carried out by molecular docking and homologous alignment.The key amino acid sites affecting the oxidation activity of CYP76AK1 were identified by molecular docking.The function of the obtained mutations was studied with yeast expression system,and the CYP76AK1 mutations with continuous oxidation function to 11-hydroxysugiol were obtained.Four key amino acid sites that affected the oxidation activity were analyzed,and the reliability of three protein modeling methods was analyzed according to the mutation results.The effective protein modification sites of CYP76AK1 were reported for the first time in this study,which provides a catalytic element for different oxidation activities at C20 site for the study of the synthetic biology of tanshinones and lays a foundation for the analysis of the continuous oxidation mechanism of P450-C20 modification.

关 键 词：细胞色素P450 定向进化 突变体 连续氧化 11-羟基柳杉酚 

分 类 号：S567.53[农业科学—中草药栽培]