While chemical steps involved in bioactive cembranoid biosynthesis have been examined, the corresponding enzymatic mechanisms leading to their formation remain elusive. In the tobacco plant, Nicotiana tabacum, a putative cembratriene-ol synthase (CBTS) initiates the catalytic cascade that lead to the biosynthesis of cembratriene-4,6-diols, which displays antibacterial- and anti-proliferative activities. We report here on structural homology models, functional studies, and mechanistic explorations of this enzyme using a combination of biosynthetic and computational methods. This approach guided us to develop an efficient de novo production of five bioactive non- and monohydroxylated cembranoids. Our homology models in combination with quantum and classical simulations suggested putative principles of the CBTS catalytic cycle, and provided a possible rationale for the formation of premature olefinic side products. Moreover, the functional reconstruction of a N. tabacum-derived class II P450 with a cognate CPR, obtained by transcriptome mining provided for production of bioactive cembratriene-4,6-diols. Our combined findings provide mechanistic insights into cembranoid biosynthesis, and a basis for the sustainable industrial production of highly valuable bioactive cembranoids.

虽然已经研究了涉及生物活性的类鲨生物合成的化学步骤，但导致其形成的相应酶促机制仍然难以捉摸。在烟草植物中，烟草，推定西柏三烯醇合酶（CBTS）启动催化级联导致西柏三烯-4,6-二醇，其显示antibacterial-和抗增殖活性的生物合成。我们在这里报道了使用生物合成和计算方法相结合的这种酶的结构同源性模型，功能研究和机理探索。这种方法引导我们开发了有效的从头开始生产五种具有生物活性的非和单羟基化的类天柏。我们的同源性模型与量子模拟和经典模拟相结合，提出了CBTS催化循环的推定原理，并为形成过早的烯烃副产物提供了可能的原理。此外，通过转录组挖掘获得的具有烟草同源的CPR的烟草来源的IIP450类的功能性重建提供了生物活性的cembratriene-4，6-二醇的生产。我们的综合研究结果提供了对斜方肌生物合成的机械洞察力，并为可持续工业生产高价值的生物活性斜方肌奠定了基础。