Li Shuyu, Chen Sa, Fu Chunjin, Zhang Jingjing, Xing Jiale, Chai Xin, Zhang Qian, Zhou Jie, Lyu Haining, Xu Chengchao, Liao Jingjing
State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, Artemisinin Research Center, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China.
Key Laboratory of Agricultural Biotechnology of Liaoning Province, College of Biosciences and Biotechnology, Shenyang Agricultural University, Shenyang 110161, China.
Plant Cell Physiol. 2025 Feb 28;66(2):249-259. doi: 10.1093/pcp/pcae147.
Polyphyllins are the active ingredients of the medicinal plant Paris polyphylla. The biosynthesis of different types of polyphyllins all requires the catalysis of glycosyltransferases. Even though significant efforts have been made to identify PpUGTs capable of catalyzing the initial glycosylation reaction, the specific glycosyltransferases responsible for the synthesis of trillin have not been reported in P. polyphylla. Here, we identified a new trillin synthase, named PpUGT91BP1, which was highly expressed in the rhizome. Importantly, PpUGT91BP1 could specifically glycosylate diosgenin but not pennogenin. To improve its catalytic efficiency, we introduced random mutations through error-prone PCR and conducted an activity-based screening. Three mutants with significantly enhanced trillin synthase activity were identified. Finally, we successfully reconstituted trillin biosynthesis in Nicotiana benthamiana, achieving a yield of 3.69 mg/g of plant dry weight using the mutant PpUGT91BP1. Taken together, our results deepen the understanding of the PpUGT91 family's role in polyphyllin biosynthesis in P. polyphylla, facilitating rational selection of better P. polyphylla cultivars and guiding future studies in the metabolic engineering of polyphllins.
重楼皂苷是药用植物云南重楼的活性成分。不同类型重楼皂苷的生物合成均需要糖基转移酶的催化。尽管已做出大量努力来鉴定能够催化初始糖基化反应的PpUGTs，但云南重楼中负责合成重楼皂苷III的具体糖基转移酶尚未见报道。在此，我们鉴定出一种新的重楼皂苷III合酶，命名为PpUGT91BP1，其在根茎中高表达。重要的是，PpUGT91BP1能够特异性地将薯蓣皂苷元糖基化，而不能将原纤细皂苷元糖基化。为了提高其催化效率，我们通过易错PCR引入随机突变并进行基于活性的筛选。鉴定出三个重楼皂苷III合酶活性显著增强的突变体。最后，我们在本氏烟草中成功重建了重楼皂苷III的生物合成，使用突变体PpUGT91BP1时产量达到3.69 mg/g植物干重。综上所述，我们的结果加深了对PpUGT91家族在云南重楼重楼皂苷生物合成中作用的理解，有助于合理选择更好的云南重楼品种，并指导未来重楼皂苷代谢工程的研究。
