首页|超表达HnCYP82M3和DsTRI基因对颠茄托品烷生物碱合成的影响

超表达HnCYP82M3和DsTRI基因对颠茄托品烷生物碱合成的影响

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托品烷生物碱(TAs)是一类临床上广泛应用的抗胆碱药物，颠茄是提取TAs的主要药源植物，利用代谢工程技术大量获取TAs具有重要的产业价值。本研究基于开源引流代谢工程策略，在颠茄发根中超表达TAs合成途径分支处的细胞色素氧化酶(cytochrome oxidase，CYP82M3)HnCYP82M3(来源于天仙子)和托品酮还原酶Ⅰ(tropinone reductase Ⅰ，TRI)DsTRI(来源于曼陀罗)两个酶基因，以期通过CYP82M3催化合成托品酮(开源)，TRI进而将托品酮引入TAs合成的代谢方向(引流)，大量促进3种TAs的积累。从天仙子根中克隆了HnCYP82M3基因，其编码氨基酸与颠茄AbCYP82M3序列一致性高达91。7％。单独超表达HnCYP82M3不影响颠茄发根TAs的含量，说明CYP82M3不是TAs合成的关键酶。同时超表达HnCYP82M3和DsTRI极大促进了 3种TAs的积累，莨菪碱、山莨菪和东莨菪碱含量分别是对照的4。97倍、2。83倍和2。19倍，并且提高幅度大于DsTRI单表达。本研究证明分支点处酶基因共表达的开源引流策略是有效提高颠茄TAs合成的代谢工程新方法，为工业化大量获取TAs奠定了理论和技术基础。
Enhancement of tropane alkaloids biosynthesisinAtropabelladonna hariy root by overexpression of HnCYP82M3 and DsTRI genes
Tropane alkaloids(TAs)are a class of anticholinergic drugs widely used in clinical practice and mainly extracted from plant,among which Atopa belladonna is the main commercial drug source.It is of great industrial value to obtain TAs in large quantities by plant metabolic engineering.In TAs pathway,cytochrome oxidase CYP82M3 catalyze the synthesis of tropinone and then tropinone reductase I(TRI)compete with TRⅡ for tropinone to form tropine leading to the TAs synthesis(drainage).In this study,based on the"increasing flow and drainage"metabolic engineering strategy,two genes,namely HnCYP82M3 and DsTRI from Hyoscyamus niger and Datura stramonium,respectively,were overexpressed in the hair roots ofA.belladonna,with a view to promote the TAs accumulation.The HnCYP82M3 gene was cloned from the root of H.niger,and it encoded amino acid with 91.7％sequence identity with AbCYP82M3 from A.belladonna.Overexpression of HnCYP82M3 alone did not affect the content of TAs in hair roots of A.belladonna,indicating that CYP82M3 was not a key enzyme in TAs biosynthesis.Simultaneous overexpression of HnCYP82M3 and DsTRI greatly promoted the accumulation of the three TAs,and the contents of hyoscyamine,anisodamine and scopolamine were 4.97 times,2.83 times and 2.19 times that of the control,respectively,and the increase amplitude was greater than that of single overexpression of DsTRI.This study showed that the"increasing flow and drainage"strategy of enzyme genes co-expression at branch points was a promising metabolic engineering method to effectively improve the biosynthesis of TAs in A.belladonna,and laid a theoretical and technical foundation for the large-scale industrial acquisition of TAs.

CYP82M3TRItropane alkaloidsAtropa belladonna L.metabolic engineering

穆德会、刘艳红、陈漂漂、谭艾娟、马炳南、潘航、张明生、强玮

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贵州大学生命科学学院/农业生物工程研究院,山地植物资源保护与种质创新教育部重点实验室,贵州贵阳 550025

CYP82M3 TRI·托品烷生物碱 颠茄 代谢工程

国家自然科学基金贵州省省级科技计划贵州大学培育项目贵州大学引进人才科研项目贵州省中药材产业技术体系建设项目贵州大学大学生创新创业训练计划剑河县省级科技特派员试验示范项目

32200225黔科合基础-ZK[2022]一般096贵大培育[2019]51号贵大人基合字[2017]58GZCYTX-02贵大028号剑科特派合[2022]10号

2024

10.16438/j.0513-4870.2023-0939

药学学报
中国药学会 中国医学科学院药物研究所

药学学报

CSTPCD北大核心
影响因子：1.274
ISSN：0513-4870
年,卷(期)：2024.59(3)
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