Wang Rong-Feng, Liu Zheng, Wang Xin-Meng, Gao Wei, Wang Jia-Dian, Hu Ya-Ting, Huang Lu-Qi
School of Traditional Chinese Medicine, Capital Medical University Beijing 100069, China.
National Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences Beijing 100700, China.
Zhongguo Zhong Yao Za Zhi. 2024 Dec;49(24):6667-6675. doi: 10.19540/j.cnki.cjcmm.20240918.101.
Tripterifordin and neotripterifordin are important ent-kaurane diterpenoids in the Chinese medicinal herb Tripterygium wilfordii, possessing significant anti-HIV(human immunodeficiency virus) activity. On the basis of elucidating the natural biosynthetic pathways of these compounds, heterologous production with microbial cell factories can help to alleviate the reliance on plant resources and provide abundant raw materials for sustainable production. TwKO is the first CYP450 enzyme involved in the biosynthesis of tripterifordin and neotripterifordin. This study aimed to enhance the catalytic activity of TwKO by site-directed mutagenesis to benefit the production of tripterifordin and neotripterifordin in yeast. The AlphaFold DB established based on the AlphaFold 2 was employed to obtain the protein model of TwKO. According to multiple sequence alignments and principles of natural evolution, the key residues influencing the binding of TwKO to the substrate were identified. Subsequently, functional characterization of the mutants were conducted in Saccharomyces cerevisiae. A total of 71 mutants were obtained, among which 11 and 11 mutants had the abilities of enhancing the production of 16α-hydroxy-ent-kaurenol and 16α-hydroxy-ent-kaurenoic acid, respectively. In addition, 10 mutants could increase the proportion of the oxidation product of 16α-hydroxy-ent-kaurenol. In particular, R304 was identified as a key residue affecting the catalytic specificity of TwKO, the mutation of which led to the specific prodiction of 16α-hydroxy-ent-kaurenol. This study was the first to reveal the key residue affecting the catalytic activity of TwKO and obtained the mutants with increased TwKO activity, lay a foundation for the biosynthesis of tripterifordin and neotripterifordin.
雷公藤红素和新雷公藤红素是中药雷公藤中重要的对映-贝壳杉烷二萜类化合物，具有显著的抗HIV（人类免疫缺陷病毒）活性。在阐明这些化合物的天然生物合成途径的基础上，利用微生物细胞工厂进行异源生产有助于减轻对植物资源的依赖，并为可持续生产提供丰富的原材料。TwKO是参与雷公藤红素和新雷公藤红素生物合成的首个CYP450酶。本研究旨在通过定点诱变提高TwKO的催化活性，以利于酵母中雷公藤红素和新雷公藤红素的生产。基于AlphaFold 2建立的AlphaFold DB被用于获得TwKO的蛋白质模型。根据多序列比对和自然进化原理，确定了影响TwKO与底物结合的关键残基。随后，在酿酒酵母中对突变体进行功能表征。共获得71个突变体，其中分别有11个和11个突变体具有提高16α-羟基对映-贝壳杉烯醇和16α-羟基对映-贝壳杉烯酸产量的能力。此外，有10个突变体可以增加16α-羟基对映-贝壳杉烯醇氧化产物的比例。特别地，R304被确定为影响TwKO催化特异性的关键残基，其突变导致16α-羟基对映-贝壳杉烯醇的特异性产生。本研究首次揭示了影响TwKO催化活性的关键残基，并获得了TwKO活性增强的突变体，为雷公藤红素和新雷公藤红素的生物合成奠定了基础。
