黄连中四氢小檗碱氧化酶基因CcTHBO的功能表征
Functional characterization of tetrahydroberberine oxidase gene encoding CcTHBO in Coptis chinensis
为探究黄连中四氢小檗碱氧化酶((S)-tetrahydroberberine oxidase,THBO)的功能,对黄连的转录组数据进行序列比对和结构域分析,筛选得到一条编码THBO的基因CcTHBO,进而将该基因构建至毕赤酵母表达载体上,在毕赤酵母中进行过量表达,并通过亲和层析的方法纯化得到活性蛋白。纯化得到的CcTHBO在体外与(S)-四氢小檗碱和(S)-四氢非洲防己碱反应,经液相色谱-串联质谱法(Liquid Chromatography-Tandem Mass spectrometry,LC-MS/MS)对反应产物进行分析,证明CcTHBO可以催化(S)-四氢小檗碱和(S)-四氢非洲防己碱氧化脱氢分别生成小檗碱和非洲防己碱。黄连THBO基因在毕赤酵母中的成功表达及其功能表征,为进一步通过合成生物学技术大量合成小檗碱等黄连生物碱打下了基础。
o explore the function of (S)-tetrahydroberberine oxidase (THBO) of Coptis chinensis, sequence alignment and conserveddomain analysis were performed on the transcriptome data. One gene CcTHBO encoding THBO was obtained that was then constructed into the Pichia pastoris expression vector. CcTHBO was overexpressed in Pichia pastoris and purified by affinity chromatography whose activity was tested in vitro using (S)-tetrahydroberberine and (S)-tetrahydrocolumbamine as substrates. The reaction products were analyzed by Liquid Chromatography-Tandem Mass spectrometry (LC-MS/MS), whichdemonstrated that CcTHBO could catalyze the oxidative dehydrogenation of (S)-tetrahydroberberine and (S)-tetrahydrocolumbamine to form berberine and columbamine, respectively. Successful expression of CcTHBO in Pichia pastoris and its functional characterization thus lay the foundation for further large-scale biosynthesis of alkaloids in Coptis chinensis such as berberine through synthetic biology technology.
赵彬鑫、虞沂
生物科学研究方法、生物科学研究技术生物化学生物工程学
生物化学黄连(S)-四氢小檗碱氧化酶小檗碱非洲防己碱
BiochemistryCoptis chinensis(S)-tetrahydroberberine oxidaseBerberineColumbamine
赵彬鑫,虞沂.黄连中四氢小檗碱氧化酶基因CcTHBO的功能表征[EB/OL].(2022-05-11)[2025-08-23].http://www.paper.edu.cn/releasepaper/content/202205-51.点此复制
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