Gao Hai-Yun, Zhao Huan, Hu Tian-Yuan, Jiang Zhou-Qian, Xia Meng, Zhang Yi-Feng, Lu Yun, Liu Yuan, Yin Yan, Chen Xiao-Chao, Luo Yun-Feng, Zhou Jia-Wei, Wang Jia-Dian, Gao Jie, Gao Wei, Huang Lu-Qi
School of Traditional Chinese Medicine, Capital Medical University, Beijing, China.
School of Pharmacy, College of Medicine, Hangzhou Normal University, Hangzhou, China.
Front Bioeng Biotechnol. 2022 Feb 7;10:805429. doi: 10.3389/fbioe.2022.805429. eCollection 2022.
Friedelin, the most rearranged pentacyclic triterpene, also exhibits remarkable pharmacological and anti-insect activities. In particular, celastrol with friedelin as the skeleton, which is derived from the medicinal plant , is a promising drug due to its anticancer and antiobesity activities. Although a previous study achieved friedelin production using engineered , strains capable of producing high-level friedelin have not been stably engineered. In this study, a combined strategy was employed with integration of endogenous pathway genes into the genome and knockout of inhibiting genes by CRISPR/Cas9 technology, which successfully engineered multiple strains. After introducing an efficient OSC1, all strains with genetic integration (, , , , , or ) showed a 3.0∼6.8-fold increase in friedelin production compared with strain BY4741. Through further double knockout of inhibiting genes, only strains GD1 and GD3 produced higher yields. Moreover, strains GQ1 and GQ3 with quadruple mutants (; ; ; ) displayed similar increases. Finally, the dominant strain GQ1 with OSC1 was cultured in an optimized medium in shake flasks, and the final yield of friedelin reached 63.91 ± 2.45 mg/L, which was approximately 65-fold higher than that of the wild-type strain BY4741 and 229% higher than that in ordinary SD-His-Ura medium. It was the highest titer for friedelin production to date. Our work provides a good example for triterpenoid production in microbial cell factories and lays a solid foundation for the mining, pathway analysis, and efficient production of valuable triterpenoids with friedelin as the skeleton.
木栓酮是重排程度最高的五环三萜，也具有显著的药理活性和抗昆虫活性。特别是以木栓酮为骨架的雷公藤红素，它来源于药用植物，因其抗癌和抗肥胖活性而成为一种有前景的药物。尽管先前的一项研究利用工程菌株实现了木栓酮的生产，但能够高产木栓酮的菌株尚未得到稳定改造。在本研究中，采用了一种联合策略，即将内源性途径基因整合到基因组中，并通过CRISPR/Cas9技术敲除抑制基因，成功改造了多个菌株。引入高效的OSC1后，所有基因整合菌株（，，，，，或）的木栓酮产量与BY4741菌株相比提高了3.0至6.8倍。通过进一步双敲除抑制基因，只有GD1和GD3菌株产量更高。此外，具有四重突变体（；；；）的GQ1和GQ3菌株也有类似的产量增加。最后，将带有OSC1的优势菌株GQ1在摇瓶中的优化培养基中培养，木栓酮的最终产量达到63.91±2.45mg/L，比野生型菌株BY4741高出约65倍，比普通SD-His-Ura培养基高出229%。这是迄今为止木栓酮生产的最高滴度。我们的工作为微生物细胞工厂中三萜类化合物的生产提供了一个很好的例子，并为以木栓酮为骨架的有价值三萜类化合物的挖掘、途径分析和高效生产奠定了坚实的基础。
