Department of Radiation and Medical Oncology, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, School of Pharmaceutical Sciences, Zhongnan Hospital, Ministry of Education, Wuhan University, Wuhan, 430071, China.
Department of Urology, Renmin Hospital of Wuhan University, No. 238 Jie-Fang Avenue, Wuhan, 430060, China.
Microb Cell Fact. 2024 May 6;23(1):129. doi: 10.1186/s12934-024-02406-0.
Sesterterpenoids are rare species among the terpenoids family. Ophiobolins are sesterterpenes with a 5-8-5 tricyclic skeleton. The oxidized ophiobolins exhibit significant cytotoxic activity and potential medicinal value. There is an urgent need for large amounts of ophiobolins supplication for drug development. The synthetic biology approach has been successfully employed in lots of terpene compound production and inspired us to develop a cell factory for ophiobolin biosynthesis.
We developed a systematic metabolic engineering strategy to construct an ophiobolin biosynthesis chassis based on Saccharomyces cerevisiae. The whole-cell biotransformation methods were further combined with metabolic engineering to enhance the expression of key ophiobolin biosynthetic genes and improve the supply of precursors and cofactors. A high yield of 5.1 g/L of ophiobolin F was reached using ethanol and fatty acids as substrates. To accumulate oxidized ophiobolins, we optimized the sources and expression conditions for P450-CPR and alleviated the toxicity of bioactive compounds to cells through PDR engineering. We unexpectedly obtained a novel ophiobolin intermediate with potent cytotoxicity, 5-hydroxy-21-formyl-ophiobolin F, and the known bioactive compound ophiobolin U. Finally, we achieved the ophiobolin U titer of 128.9 mg/L.
We established efficient cell factories based on S. cerevisiae, enabling de novo biosynthesis of the ophiobolin skeleton ophiobolin F and oxidized ophiobolins derivatives. This work has filled the gap in the heterologous biosynthesis of sesterterpenoids in S. cerevisiae and provided valuable solutions for new drug development based on sesterterpenoids.
倍半萜是萜类化合物家族中的稀有物种。蛇孢菌素是具有 5-8-5 三环骨架的倍半萜烯。氧化蛇孢菌素表现出显著的细胞毒性活性和潜在的药用价值。因此，迫切需要大量的蛇孢菌素来进行药物开发。合成生物学方法已成功应用于许多萜类化合物的生产，并启发我们开发用于蛇孢菌素生物合成的细胞工厂。
我们开发了一种系统的代谢工程策略，基于酿酒酵母构建了蛇孢菌素生物合成底盘。进一步将全细胞生物转化方法与代谢工程相结合，以增强关键蛇孢菌素生物合成基因的表达，并改善前体和辅因子的供应。使用乙醇和脂肪酸作为底物，达到了 5.1 g/L 的蛇孢菌素 F 的高产。为了积累氧化蛇孢菌素，我们优化了 P450-CPR 的来源和表达条件，并通过 PDR 工程缓解了生物活性化合物对细胞的毒性。我们出人意料地获得了一种具有强大细胞毒性的新型蛇孢菌素中间体，5-羟基-21-甲酰基蛇孢菌素 F，以及已知的生物活性化合物蛇孢菌素 U。最终，我们实现了蛇孢菌素 U 的产量为 128.9 mg/L。
我们基于酿酒酵母建立了高效的细胞工厂，实现了蛇孢菌素骨架蛇孢菌素 F 和氧化蛇孢菌素衍生物的从头生物合成。这项工作填补了酿酒酵母中倍半萜类化合物异源生物合成的空白，并为基于倍半萜类化合物的新药开发提供了有价值的解决方案。
