School of Chemistry Chemical Engineering and Life Sciences, Wuhan University of Technology, No. 122, Lo Lion Road, Wuhan, 430070, Hubei, China.
Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
Microb Cell Fact. 2022 Sep 19;21(1):195. doi: 10.1186/s12934-022-01921-2.
Karst-adapted plant, Lysionotus pauciflours accumulates special secondary metabolites with a wide range of pharmacological effects for surviving in drought and high salty areas, while researchers focused more on their environmental adaptations and evolutions. Nevadensin (5,7-dihydroxy-6,8,4'-trimethoxyflavone), the main active component in L. pauciflours, has unique bioactivity of such as anti-inflammatory, anti-tubercular, and anti-tumor or cancer. Complex decoration of nevadensin, such as hydroxylation and glycosylation of the flavone skeleton determines its diversity and biological activities. The lack of omics data limits the exploration of accumulation mode and biosynthetic pathway. Herein, we integrated transcriptomics, metabolomics, and microbial recombinant protein system to reveal hydroxylation and glycosylation involving nevadensin biosynthesis in L. pauciflours.
Up to 275 flavonoids were found to exist in L. pauciflorus by UPLC-MS/MS based on widely targeted metabolome analysis. The special flavone nevadensin (5,7-dihydroxy-6,8,4'-trimethoxyflavone) is enriched in different tissues, as are its related glycosides. The flavonoid biosynthesis pathway was drawn based on differential transcripts analysis, including 9 PAL, 5 C4H, 8 4CL, 6 CHS, 3 CHI, 1 FNSII, and over 20 OMTs. Total 310 LpCYP450s were classified into 9 clans, 36 families, and 35 subfamilies, with 56% being A-type CYP450s by phylogenetic evolutionary analysis. According to the phylogenetic tree with AtUGTs, 187 LpUGTs clustered into 14 evolutionary groups (A-N), with 74% being E, A, D, G, and K groups. Two LpCYP82D members and LpUGT95 were functionally identified in Saccharomyces cerevisiae and Escherichia coli, respectively. CYP82D-8 and CYP82D-1 specially hydroxylate the 6- or 8-position of A ring in vivo and in vitro, dislike the function of F6H or F8H discovered in basil which functioned depending on A-ring substituted methoxy. These results refreshed the starting mode that apigenin can be firstly hydroxylated on A ring in nevadensin biosynthesis. Furthermore, LpUGT95 clustered into the 7-OGT family was verified to catalyze 7-O glucosylation of nevadensin accompanied with weak nevadensin 5-O glucosylation function, firstly revealed glycosylation modification of flavones with completely substituted A-ring.
Metabolomic and full-length transcriptomic association analysis unveiled the accumulation mode and biosynthetic pathway of the secondary metabolites in the karst-adapted plant L. pauciflorus. Moreover, functional identification of two LpCYP82D members and one LpUGT in microbe reconstructed the pathway of nevadensin biosynthesis.
适应喀斯特地貌的植物，虎舌红 Lysionotus pauciflours 积累了具有广泛药理作用的特殊次生代谢产物，以在干旱和高盐地区生存，而研究人员则更关注其环境适应和进化。虎舌红中的主要活性成分虎舌红苷（5,7-二羟基-6,8,4'-三甲氧基黄酮）具有独特的抗炎、抗结核和抗肿瘤或癌症的生物活性。黄酮骨架的羟化和糖基化等复杂修饰决定了其多样性和生物活性。缺乏组学数据限制了对其积累模式和生物合成途径的探索。在此，我们整合了转录组学、代谢组学和微生物重组蛋白系统，以揭示 L. pauciflours 中虎舌红苷生物合成涉及的羟化和糖基化。
通过基于广泛靶向代谢组学分析的 UPLC-MS/MS，发现 275 种黄酮类化合物存在于虎舌红中。特殊的黄酮类化合物虎舌红苷（5,7-二羟基-6,8,4'-三甲氧基黄酮）在不同组织中含量丰富，其相关糖苷也同样如此。基于差异转录物分析绘制了黄酮类生物合成途径，包括 9 个 PAL、5 个 C4H、8 个 4CL、6 个 CHS、3 个 CHI、1 个 FNSII 和 20 多个 OMTs。总共 310 个 LpCYP450s 被分类为 9 个家族、36 个科和 35 个亚科，通过系统进化分析，其中 56%为 A 型 CYP450s。根据与 AtUGTs 的系统进化树，187 个 LpUGTs 聚类为 14 个进化群（A-N），其中 74%为 E、A、D、G 和 K 组。两个 LpCYP82D 成员和 LpUGT95 分别在酿酒酵母和大肠杆菌中被功能鉴定。CYP82D-8 和 CYP82D-1 可分别在体内和体外特异性地羟化 A 环的 6-或 8-位，不喜欢发现于罗勒中的 F6H 或 F8H 的功能，因为罗勒中的 F6H 或 F8H 的功能取决于 A 环取代的甲氧基。这些结果刷新了虎舌红苷生物合成中首先在 A 环上羟化芹黄素的起始模式。此外，LpUGT95 聚类到 7-OGT 家族被证实可催化虎舌红苷的 7-O-葡萄糖基化，同时伴有较弱的虎舌红苷 5-O-葡萄糖基化功能，首次揭示了完全取代 A 环的黄酮类化合物的糖基化修饰。
代谢组学和全长转录组关联分析揭示了适应喀斯特地貌的植物 L. pauciflorus 中次生代谢物的积累模式和生物合成途径。此外，两个 LpCYP82D 成员和一个 LpUGT 在微生物中的功能鉴定重建了虎舌红苷生物合成途径。
