CAS-Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, 200032, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
CAS-Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.
Metab Eng. 2021 Jul;66:87-97. doi: 10.1016/j.ymben.2021.04.006. Epub 2021 Apr 15.
The Chinese medicinal plant Panax notoginseng has been traditionally used to activate blood flow and circulation, and to prevent blood stasis. P. notoginseng contains protopanaxatriol (PPT)-type saponins as its main active compounds, thus distinguishing it from the other two famous Panax species, P. ginseng and P. quinquefolius. Ginsenoside Rg1 (Rg1), notoginsenoside R1 (NgR1), and notoginsenoside R2 (NgR2) are three major PPT-type saponins in P. notoginseng and possess potential cardiovascular protection activities. However, their use in medical applications has long been hampered by the lack of sustainable and low-cost industrial-scale preparation methods. In this study, a PPT-producing yeast chassis strain was designed and constructed based on a previously constructed and optimized protopanaxadiol (PPD)-producing Saccharomyces cerevisiae strain, and further optimized by systemically engineering and optimizing the expression level of its key P450 biopart. Rg1-producing yeast strains were constructed by introducing PgUGT71A53 and PgUGT71A54 into the PPT chassis strain. The fermentation titer of Rg1 reached 1.95 g/L. A group of UDP-glycosyltransferases (UGT) from P. notoginseng and P. ginseng were characterized, and were found to generate NgR1 and NgR2 by catalyzing the C-O-Glc xylosylation of Rg1 and Rh1, respectively. Using one of these UGTs, PgUGT94Q13, and the previously identified PgUGT71A53 and PgUGT71A54, the biosynthetic pathway to produce saponins NgR1 and NgR2 from PPT could be available. The NgR1 cell factory was further developed by introducing PgUGT94Q13 and a heterologous UDP-xylose biosynthetic pathway from Arabidopsis thaliana into the highest Rg1-producing cell factory. The NgR2-producing cell factory was constructed by introducing PgUGT71A54, PgUGT94Q13, and the UDP-xylose biosynthetic pathway into the PPT chassis. De novo production of NgR1 and NgR2 reached 1.62 g/L and 1.25 g/L, respectively. Beyond the realization of artificial production of the three valuable saponins Rg1, NgR1, and NgR2 from glucose, our work provides a green and sustainable platform for the efficient production of other PPT-type saponins in engineered yeast strains, and promotes the industrial application of PPT-type saponins as medicine and functional foods.
中文草药三七历来被用于活血化淤和防止血淤。三七含有达玛烷型皂甙作为其主要活性化合物，因此与其他两种著名的人参属植物，人参和西洋参相区别。人参皂甙 Rg1（Rg1）、三七皂甙 R1（NgR1）和三七皂甙 R2（NgR2）是三七中的三种主要达玛烷型皂甙，具有潜在的心血管保护活性。然而，它们在医学应用中的使用长期以来一直受到缺乏可持续和低成本的工业规模制备方法的阻碍。在这项研究中，基于先前构建和优化的产生原人参二醇的酿酒酵母菌株，设计并构建了一个产生达玛烷二醇（PPD）的酵母底盘菌株，并通过系统地工程改造和优化其关键 P450 生物部件的表达水平进行了进一步优化。通过将 PgUGT71A53 和 PgUGT71A54 引入 PPT 底盘菌株，构建了产生 Rg1 的酵母菌株。Rg1 的发酵产量达到 1.95g/L。对一组来自三七和人参的尿苷二磷酸-糖基转移酶（UGT）进行了表征，发现它们通过催化 Rg1 和 Rh1 的 C-O-Glc 木糖基化，分别生成 NgR1 和 NgR2。利用其中一种 UGT，PgUGT94Q13，以及之前鉴定的 PgUGT71A53 和 PgUGT71A54，可以构建从 PPT 生物合成 NgR1 和 NgR2 皂甙的生物合成途径。通过将 PgUGT94Q13 和来自拟南芥的异源 UDP-木糖生物合成途径引入到最高产 Rg1 的细胞工厂中，进一步开发了 NgR1 细胞工厂。通过将 PgUGT71A54、PgUGT94Q13 和 UDP-木糖生物合成途径引入 PPT 底盘，构建了 NgR2 产生细胞工厂。从头合成 NgR1 和 NgR2 的产量分别达到 1.62g/L 和 1.25g/L。除了从葡萄糖中实现三种有价值的皂甙 Rg1、NgR1 和 NgR2 的人工生产外，我们的工作还为在工程酵母菌株中高效生产其他达玛烷型皂甙提供了一个绿色可持续的平台，并促进了达玛烷型皂甙作为药物和功能性食品的工业应用。
