Ginsenoside Ro (Ro), the only identified oleanane-type pentacyclic ginsenoside, has the ability to provide antitumor, anticoagulant and other functions and has the potential to be applied in pharmaceuticals and tonics. Currently, Ro is mainly derived by direct extraction from ginseng; however, this method is not sustainable and cannot meet the increasing demand due to its low content and long culture period. In this study, we selected 29 enzymes from 15 different species to screen for key enzymes for Ro synthesis. Through both in vitro and in vivo characterization, seven enzymes from five species with high catalytic efficiency and substrate specificity were selected to construct an artificial dual glycosylation pathway for the de novo biosynthesis of Ro in Saccharomyces cerevisiae. The main route of Ro synthesis was identified through the in vitro enzymatic kinetics. Several metabolic engineering strategies were employed to strengthen Ro synthesis. Finally, the Ro titer reached 528.0 ± 18.0 mg L⁻¹ in 5 L fed-batch fermentation, which was more than 374 000-fold higher than the reported data.

中文翻译：

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在酿酒酵母中通过人工糖基化途径可持续生产稀有齐墩果型人参皂甙

人参皂甙Ro（Ro）是唯一鉴定的齐墩果烷型五环人参皂甙，具有抗肿瘤、抗凝血等功能，具有在药物和滋补品中的应用潜力。目前，Ro主要是从人参中直接提取得到的；但这种方法不可持续，由于含量低、培养周期长，无法满足日益增长的需求。在这项研究中，我们从 15 个不同物种中选择了 29 种酶来筛选用于 Ro 合成的关键酶。通过体外和体内表征，从5个物种中选择了7种具有高催化效率和底物特异性的酶，构建了Ro从头生物合成的人工双糖基化途径。酿酒酵母。通过体外酶动力学确定了 Ro 合成的主要途径。采用了几种代谢工程策略来加强 Ro 合成。最后，在 5 L 补料分批发酵中，Ro 滴度达到 528.0 ± 18.0 mg L ^-1，比报道的数据高出 374 000 倍以上。