Diterpenoids display a large and structurally diverse class of natural compounds mainly found as specialized plant metabolites. Due to their diverse biological functions they represent an essential source for various industrially relevant applications as biopharmaceuticals, nutraceuticals, and fragrances. However, commercial production utilizing their native hosts is inhibited by low abundances, limited cultivability, and challenging extraction, while the precise stereochemistry displays a particular challenge for chemical synthesis. Due to a high carbon flux through their native 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway towards photosynthetically active pigments, green microalgae hold great potential as efficient and sustainable heterologous chassis for sustainable biosynthesis of plant-derived diterpenoids. In this study, innovative synthetic biology and efficient metabolic engineering strategies were systematically combined to re-direct the metabolic flux through the MEP pathway for efficient heterologous diterpenoid synthesis in C. reinhardtii. Engineering of the 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) as the main rate-limiting enzyme of the MEP pathway and overexpression of diterpene synthase fusion proteins increased the production of high-value diterpenoids. Applying fully photoautotrophic high cell density cultivations demonstrate potent and sustainable production of the high-value diterpenoid sclareol up to 656 mg L⁻¹ with a maximal productivity of 78 mg L⁻¹ day⁻¹ in a 2.5 L scale photobioreactor, which is comparable to sclareol titers reached by highly engineered yeast. Consequently, this work represents a breakthrough in establishing a powerful phototrophic green cell factory for the competetive use in industrial biotechnology.

二萜类化合物显示出一大类结构多样的天然化合物，主要以特殊植物代谢物的形式存在。由于其多样化的生物学功能，它们代表了各种工业相关应用的重要来源，如生物制药、营养品和香料。然而，利用其天然宿主的商业生产受到低丰度、有限的可培养性和具有挑战性的提取的抑制，而精确的立体化学对化学合成显示出特殊的挑战。由于通过其天然 2-C-甲基-D-赤藓糖醇 4-磷酸 (MEP) 途径向光合活性色素产生的高碳通量，绿色微藻作为植物源二萜类化合物可持续生物合成的高效和可持续异源基础具有巨大潜力。在这项研究中， 莱茵衣藻。将 1-脱氧-D-木酮糖 5-磷酸合酶 (DXS) 作为 MEP 途径的主要限速酶进行工程改造，二萜合酶融合蛋白的过表达增加了高价值二萜类化合物的产量。应用完全光自养的高细胞密度培养证明了在 2.5 L 规模的光生物反应器中有效且可持续地生产高达 656 mg L ^-1的高价值二萜类紫苏醇， 最大生产力为 78 mg L ^-1  day ^-1  ，这与高度工程化酵母达到的香紫苏醇滴度。因此，这项工作代表了建立强大的光养绿色细胞工厂以在工业生物技术中竞争使用的突破。