Abstract：
The step catalyzed by terpene synthases is a well-recognized and significant bottleneck in engineered terpenoid bioproduction. Consequently, substantial efforts have been devoted towards increasing metabolic flux catalyzed by terpene synthases, employing strategies such as gene overexpression and protein engineering. Notably, numerous studies have demonstrated remarkable titer improvements by applying translational fusion, typically by fusing the terpene synthase with a prenyl diphosphate synthase that catalyzes the preceding step in the pathway. The main appeal of the translational fusion approach lies in its simplicity and orthogonality to other metabolic engineering tools. However, there is currently limited understanding of the underlying mechanism of flux enhancement, owing to the unpredictable and often protein-specific effects of translational fusion. In this chapter, we discuss practical considerations when engineering translationally fused terpene synthases, drawing insights from our experience and existing literature. We also provide detailed experimental workflows and protocols based on our previous work in budding yeast (Saccharomyces cerevisiae). Our intention is to encourage further research into the translational fusion of terpene synthases, anticipating that this will contribute mechanistic insights not only into the activity, behavior, and regulation of terpene synthases, but also of other enzymes.
摘要：
萜烯合酶催化的步骤是工程萜类生物生产中公认的重要瓶颈。因此,大量的努力已经致力于增加由萜烯合酶催化的代谢通量，采用基因过表达和蛋白质工程等策略。值得注意的是,通过应用翻译融合，许多研究已经证明了显著的滴度改善，通常通过将萜烯合酶与催化该途径的前一步的异戊烯二磷酸合酶融合。平移融合方法的主要吸引力在于其简单性和与其他代谢工程工具的正交性。然而,目前对通量增强的潜在机制的理解有限，由于翻译融合的不可预测且通常是蛋白质特异性的作用。在这一章中,我们讨论了工程翻译融合萜烯合酶时的实际考虑因素，从我们的经验和现有文献中汲取见解。我们还根据我们以前在发芽酵母（酿酒酵母）中的工作提供详细的实验工作流程和方案。我们的目的是鼓励进一步研究萜烯合酶的翻译融合，预计这不仅会对活动产生机械论的见解，行为，和调节萜烯合酶，还有其他酶。
