Sesquiterpene scaffolds are the core backbones of many medicinally and industrially important natural products. A plethora of sesquiterpene synthases, widely present in bacteria, fungi, and plants, catalyze the formation of these intricate structures often with multiple stereocenters starting from linear farnesyl diphosphate substrates. Recent advances in next-generation sequencing and metabolomics technologies have greatly facilitated gene discovery for sesquiterpene synthases. However, a major bottleneck limits biochemical characterization of recombinant sesquiterpene synthases: the absolute structural elucidation of the derived sesquiterpene products. Here, we report the identification and biochemical characterization of LphTPS-A, a sesquiterpene synthase from the red macroalga Laurencia pacifica. Using the combination of transcriptomics, sesquiterpene synthase expression in yeast, and microgram-scale nuclear magnetic resonance-coupled crystalline sponge X-ray diffraction analysis, we resolved the absolute stereochemistry of prespatane, the major sesquiterpene product of LphTPS-A, and thereby functionally define LphTPS-A as the first bourbonane-producing sesquiterpene synthase and the first biochemically characterized sesquiterpene synthase from red algae. Our study showcases a workflow integrating multiomics approaches, synthetic biology, and the crystalline sponge method, which is generally applicable for uncovering new terpene chemistry and biochemistry from source-limited living organisms.

中文翻译：

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通过微克级NMR耦合的晶体海绵X射线衍射分析定义的红色藻类波旁烷倍半萜烯合酶

倍半萜骨架是许多医学和工业上重要的天然产物的核心骨架。广泛存在于细菌，真菌和植物中的大量倍半萜烯合酶催化这些复杂结构的形成，这些结构通常具有从线性法呢基二磷酸底物开始的多个立体中心。下一代测序和代谢组学技术的最新进展极大地促进了倍半萜烯合酶的基因发现。然而，一个主要的瓶颈限制了重组倍半萜烯合酶的生化特性：衍生倍半萜烯产物的绝对结构解析。在这里，我们报告的鉴定和生化特征LphTPS-A，从红色大藻Laurencia太平洋的倍半萜烯合酶。结合转录组学，倍半萜烯合酶在酵母中的表达以及微克级核磁共振耦合的结晶海绵X射线衍射分析，我们解析了LphTPS-A的主要倍半萜烯产品前庚烷的绝对立体化学，从而在功能上进行了定义LphTPS-A是第一个产生波旁烷的倍半萜烯合酶，也是第一个从红藻中生化鉴定的倍半萜烯合酶。我们的研究展示了一个集成了多组学方法，合成生物学和结晶海绵方法的工作流程，该方法通常适用于从来源有限的生物中发现新的萜烯化学和生物化学。