School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, People's Republic of China.
Centre Laboratory, Guangdong Pharmaceutical University, Guangzhou, 510006, People's Republic of China.
Planta. 2018 Mar;247(3):613-623. doi: 10.1007/s00425-017-2797-2. Epub 2017 Nov 14.
The THSG biosynthetic pathway in F. multiflora was characterized, and enzymatic activities responsible for the resveratrol synthesis, hydroxylation, and glycosylation reactions involved in THSG biosynthesis were confirmed in vitro. The biosynthetic origin of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucopyranoside (THSG) and the enzymes involved in THSG biosynthesis in Fallopia multiflora were studied using stable isotope labeling and biocatalytic methods. UPLC-MS-based analyses were used to unravel the isotopologue composition of the biosynthetic intermediates and products, as well as to detect the products of the enzyme assay experiments. In this study, C-labeled L-phenylalanine (L-PHE), sodium pyruvate (SP), and sodium bicarbonate (SB) were used as putative precursors in the feeding experiment. Labeling of polydatin (PD) and THSG using [C]L-PHE and [C]L-PHE confirmed that the p-coumaric moiety of PD and THSG was derived from PHE. The results of the feeding experiments with [C] SB and [2, 3-C] SP suggested that PD and THSG were derivatives of resveratrol that were synthesized by glycosylation and hydroxylation. We developed methods using total crude protein extracts (soluble and microsomal) for comprehensive and simultaneous analysis of resveratrol synthase, glycosyltransferase, and hydroxylase activities in various tissue types of wild F. multiflora and callus cultures. The activity of each tested enzyme was confirmed in one or more tissue types or cell cultures in vitro. The results of the enzyme activity experiments and the distributions of PD and THSG were used to determine the main site and pathway of THSG biosynthesis in F. multiflora.
本研究采用稳定同位素标记和生物催化方法，研究了何首乌 THSG 的生物合成途径，并对涉及 THSG 生物合成的酶促反应进行了体外鉴定。利用超高效液相色谱-串联质谱（UPLC-MS）分析，揭示了生物合成中间体和产物的同位素组成，并检测了酶促反应实验的产物。在这项研究中，使用 C 标记的 L-苯丙氨酸（L-PHE）、丙酮酸钠（SP）和碳酸氢钠（SB）作为饲喂实验中的可能前体。用 [C]L-PHE 和 [C]L-PHE 对虎杖苷（PD）和 THSG 进行标记，证实 PD 和 THSG 的对羟基桂皮酸部分来源于 PHE。用 [C]SB 和 [2,3-C]SP 进行的饲喂实验结果表明，PD 和 THSG 是由虎杖苷经糖基化和羟化合成的白藜芦醇衍生物。我们开发了使用总粗蛋白提取物（可溶性和微粒体）的方法，用于全面和同时分析野生何首乌和愈伤组织培养物中各种组织类型的白藜芦醇合酶、糖基转移酶和羟化酶的活性。在体外的一种或多种组织类型或细胞培养物中，对每种测试酶的活性进行了确认。酶活性实验和 PD 及 THSG 的分布结果用于确定 THSG 在何首乌中的主要生物合成部位和途径。
