非天然人参皂苷12β-O-Glc-PPD代谢途径在酿酒酵母中的构建与优化

Ginsenosides are usually produced through glycosylation at C3-OH, C6-OH and C20-OH of ginsenoside aglycones, whereas C12-OH glycosylated ginsenosides have never been discovered in Panax ginseng. It is known that the unnatural ginsenoside 12β-O-Glc-PPD chemically synthesized from protopanoxadiol (PPD) exhibits more effective anti-tumor activity than natural ones. In this project, a gene of glycosyltransferase was cloned from Bacillus subtilis which catalyzes the glycosylation at C3-OH and C12-OH of PPD, while DS and CYP450 genes involved in the ginsenoside biosynthetic pathway were cloned from Panax ginseng, the gene of CYP450 reductase was cloned from Arabidopsis thaliana, and the gene of glycosyl hydrolase which catalyzes the specific hydrolysis of C3-O-glucosyl was cloned from Lentinula edodes. All the above-mentioned genes are used as primary elements to construct metabolic pathway of 12β-O-Glc-PPD in Saccharomyces cerevisiae. The biosynthesis of 12β-O-Glc-PPD is further enhanced by the strategies of “broaden sources of income and reduce expenditure”, which mainly include up-regulation of the key enzyme expression, overexpression of molecular chaperones and transcription factors, and reduction of the branch metabolic flow. By this project, we explore a new pathway leading to mass production of unnatural ginsenoside 12β-O-Glc-PPD by the strategies of synthetic biology, which will provide raw materials for the research of new anti-tumor drugs and therefore have important theoretical significance and good application prospects.

天然人参皂苷由人参皂苷苷元C3-OH、C6-OH或C20-OH糖基化生成，在人参中从未发现C12-OH糖基化皂苷，而以原人参二醇（PPD）为前体化学合成的C12-OH糖基化皂苷12β-O-Glc-PPD比天然人参皂苷具有更强的抗肿瘤活性。本项目从枯草芽孢杆菌中克隆到一个能同时催化原人参二醇C3-OH和C12-OH糖基化的糖基转移酶基因，结合从人参中克隆的达玛烯二醇-II合酶和 CYP450基因、从拟南芥中克隆的CYP450还原酶基因、从真菌香菇中克隆的可专一水解C3位糖基的糖基水解酶基因，在酿酒酵母中构建12β-O-Glc-PPD代谢途径，并通过上调关键酶表达、过表达分子伴侣及转录因子、下调支路代谢等“开源节流”措施，提高工程菌中12β-O-Glc-PPD的产量。本项目通过合成生物学策略开发能产生非天然人参皂苷的新资源，为抗肿瘤创新药物研究提供原料，具有重要的理论意义和良好的应用前景。

天然人参皂苷由人参皂苷苷元C3-OH、C6-OH或C20-OH糖基化生成，在人参中从未发现C12-OH糖基化皂苷。本项目从枯草芽孢杆菌中克隆到一个能同时催化原人参二醇C3-OH和C12-OH糖基化的糖基转移酶UGT109A1基因，从人参中克隆到达玛烯二醇-II合酶DS基因和原人参二醇合酶PPDS基因，从拟南芥中克隆到CYP450还原酶ATR1基因，利用上述基因在酿酒酵母中构建了非天然人参皂苷3β,12β-Di-O-Glc-PPD代谢途径，通过底盘细胞优化、CRISPR/Cas9系统介导外源基因多拷贝整合、上调关键酶表达、下调支路代谢途径、过表达转录因子HAC1等多重优化策略，使工程菌在3 L发酵罐中3β,12β-Di-O-Glc-PPD的产量达到152.9 mg/L。将从真菌香菇中克隆的可专一水解C3位糖基的糖基水解酶LXYL-P1-2基因转入上述产3β,12β-Di-O-Glc-PPD的工程菌，在酿酒酵母中构建了12β-O-Glc-PPD代谢途径，获得了产12β-O-Glc-PPD的工程菌。通过体内药理活性评价证明，3β,12β-Di-O-Glc-PPD和12β-O-Glc-PPD均具有显著的抗肺癌活性，且12β-O-Glc-PPD的活性优于3β,12β-Di-O-Glc-PPD。本项目基于合成生物学技术构建了能产生非天然人参皂苷3β,12β-Di-O-Glc-PPD和12β-O-Glc-PPD的微生物细胞工厂，获得抗肺癌药物候选物，为创新药物研发奠定了基础。