Deng Meijuan, Wang Binhao, Zhou Jieyu, Dong Jinjun, Ni Ye, Han Ruizhi
State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu 214122, China.
Key laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
J Agric Food Chem. 2025 Apr 2;73(13):7944-7953. doi: 10.1021/acs.jafc.5c00964. Epub 2025 Mar 19.
Rare ginsenoside Rh1, exhibiting great potential in the food industry, is limited by its natural scarcity. This constraint has driven the development of biocatalytic synthesis approaches, yet robust enzymes capable of efficient production remain elusive. Here, we employed the ancestral sequence reconstruction (ASR) approach to create a thermostable UDP-dependent glycosyltransferase (UGT227) for Rh1 synthesis from 20()-protopanaxatriol (PPT). UGT227 exhibited enhanced thermostability ( = 44.2 h at 60 °C) but initially yielded only 15% Rh1. Semirational engineering generated the I83A/F285 M variant, increasing the yield to 92%. For economic viability, the I83A/F285 M variant was coexpressed with sucrose synthase (AtSUS1), enabling the use of cost-effective sucrose for UDP-glucose regeneration. This integration achieved a 99.9% yield at a 1 mM PPT. Molecular dynamics simulations revealed that the enlarged binding pocket entrance of I83A/F285 M contributed to the enhanced Rh1 yield. Our findings offer strategies for efficient biosynthesis of Rh1 and pave the way for economically feasible production.
稀有皂苷Rh1在食品工业中具有巨大潜力，但其天然稀缺性限制了它的发展。这种限制推动了生物催化合成方法的发展，但能够高效生产的强大酶仍然难以获得。在这里，我们采用祖先序列重建（ASR）方法，创建了一种热稳定的UDP依赖性糖基转移酶（UGT227），用于从20（S）-原人参三醇（PPT）合成Rh1。UGT227表现出增强的热稳定性（在60°C下t1/2 = 44.2小时），但最初仅产生15%的Rh1。半理性工程产生了I83A/F285M变体，使产量提高到92%。为了实现经济可行性，I83A/F285M变体与蔗糖合酶（AtSUS1）共表达，使得能够使用具有成本效益的蔗糖进行UDP-葡萄糖再生。这种整合在1 mM PPT时实现了99.9%的产量。分子动力学模拟表明，I83A/F285M扩大的结合口袋入口有助于提高Rh1产量。我们的研究结果为Rh1的高效生物合成提供了策略，并为经济可行的生产铺平了道路。
