Zhang Shuai, Wang Yingying, Cui Zhirong, Li Qianqian, Kong Lingyi, Luo Jun
Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009, People's Republic of China.
Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009, People's Republic of China.
Plant Physiol Biochem. 2023 Apr;197:107643. doi: 10.1016/j.plaphy.2023.107643. Epub 2023 Mar 16.
Rhamnosyltransferase (RT) and rhamnose synthase (Rhs) are the key enzymes that are responsible for the biosynthesis of rhamnosides and UDP-l-rhamnose (UDP-Rha) in plants, respectively. How to discover such enzymes efficiently for use is still a problem to be solved. Here, we identified HmF3RT, HmRhs1, and HmRhs2 from Hypericum monogynum, which is abundant in flavonol rhamnosides, with the help of a full-length and high throughput transcriptome sequencing platform. HmF3RT could regiospecifically transfer the rhamnose moiety of UDP-Rha onto the 3-OH position of flavonols and has weakly catalytic for UDP-xylose (UDP-Xyl) and UDP-glucose (UDP-Glc). HmF3RT showed well quercetin substrate affinity and high catalytic efficiency with K of 5.14 μM and k/K of 2.21 × 10 S M, respectively. Docking, dynamic simulation, and mutagenesis studies revealed that V129, D372, and N373 are critical residues for the activity and sugar donor recognition of HmF3RT, mutant V129A, and V129T greatly enhance the conversion rate of catalytic flavonol glucosides. HmRhs1 and HmRhs2 convert UDP-Glc to UDP-Rha, which could be further used by HmF3RT. The HmF3RT and HmRhs1 co-expressed strain RTS1 could produce quercetin 3-O-rhamnoside (quercitrin), kaempferol 3-O-rhamnoside (afzelin), and myricetin 3-O-rhamnoside (myricitrin) at yields of 85.1, 110.7, and 77.6 mg L, respectively. It would provide a valuable reference for establishing a better and more efficient biocatalyst for preparing bioactive flavonol rhamnosides by identifying HmF3RT and HmRhs.
鼠李糖基转移酶（RT）和鼠李糖合酶（Rhs）分别是植物中负责鼠李糖苷和UDP-L-鼠李糖（UDP-Rha）生物合成的关键酶。如何高效发现此类酶以供使用仍是一个有待解决的问题。在此，我们借助全长和高通量转录组测序平台，从富含黄酮醇鼠李糖苷的金丝桃中鉴定出了HmF3RT、HmRhs1和HmRhs2。HmF3RT能够区域特异性地将UDP-Rha的鼠李糖部分转移到黄酮醇的3-OH位置，并且对UDP-木糖（UDP-Xyl）和UDP-葡萄糖（UDP-Glc）具有较弱的催化作用。HmF3RT对槲皮素底物表现出良好的亲和力和较高的催化效率，其K值为5.14 μM，k/K值分别为2.21×10⁵ s⁻¹M⁻¹。对接、动力学模拟和诱变研究表明，V129、D372和N373是HmF3RT活性和糖供体识别的关键残基，突变体V129A和V129T极大地提高了催化黄酮醇糖苷的转化率。HmRhs1和HmRhs2将UDP-Glc转化为UDP-Rha，后者可被HmF3RT进一步利用。共表达HmF3RT和HmRhs1的菌株RTS1能够分别以85.1、110.7和77.6 mg L⁻¹的产量生产槲皮素3-O-鼠李糖苷（槲皮苷）、山奈酚3-O-鼠李糖苷（阿福豆苷）和杨梅素3-O-鼠李糖苷（杨梅苷）。通过鉴定HmF3RT和HmRhs，这将为建立更好、更高效的生物催化剂以制备具有生物活性的黄酮醇鼠李糖苷提供有价值的参考。
