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为了研究人参达玛烯二醇-Ⅱ合酶(dammarenediol-Ⅱ synthase,DS)在酿酒酵母中的重组表达和定位,本研究克隆了人参中达玛烯二醇-Ⅱ合酶基因ds,并将其与绿色荧光蛋白基因gfp融合,构建了相应的重组表达质粒pESC-HIS-DS和pESC-HIS-DS-GFP,转化酿酒酵母INVSc1,获得了重组菌INVSc1-DS和INVSc1-DS-GFP。通过差速离心法制备重组菌微粒体,荧光显微镜下观察达玛烯二醇-Ⅱ合酶的表达及亚细胞定位,并通过酶促反应对该酶进行了功能鉴定,结果表明,DS为膜结合型蛋白,在体外能催化2,3-氧化鲨烯生成达玛烯二醇-Ⅱ。对重组菌发酵产物进行分析,结果表明,重组菌中产生了达玛烯二醇-Ⅱ,且通过将ds与gfp融合,重组菌中的达玛烯二醇-Ⅱ产量由7.53 mg·g~(-1)提高到12.24 mg·g~(-1),该结果为优化在酿酒酵母中构建的人参皂苷代谢途径提供了新思路。
In order to study the recombinant expression and localization of ginseng dammarenediol-Ⅱ synthase (DS) in Saccharomyces cerevisiae, dsDNA of darmacen-2-one synthase gene was cloned in this study The recombinant plasmid pESC-HIS-DS and pESC-HIS-DS-GFP were constructed by fusion with the green fluorescent protein gene gfp and transformed into Saccharomyces cerevisiae INVSc1 to obtain the recombinant vectors INVSc1-DS and INVSc1-DS-GFP. Recombinant bacterial microsomes were prepared by differential centrifugation. The expression and subcellular localization of damarane diol-Ⅱ synthase were observed under a fluorescence microscope. The enzyme was identified by enzymatic reaction. The results showed that DS was a membrane Binding type protein, in vitro can catalyze the 2,3-oxidation of squalene to generate darlenediol-Ⅱ. The results of the analysis of the fermentation products of the recombinant bacteria showed that there was a reaction of damahenediol-Ⅱ in the recombinant bacteria, and the yield of darhamide-Ⅱ in the recombinant bacteria increased from 7.53 mg · g ~ (-1) to 12.24 mg · g -1, which provided a new idea for optimizing the metabolic pathway of ginsenosides constructed in Saccharomyces cerevisiae.