Bielefeld University, Faculty of Biology, Center for Biotechnology (CeBiTec), Universitätsstrasse 27, 33615, Bielefeld, Germany.
Bielefeld University, Faculty of Biology, Center for Biotechnology (CeBiTec), Universitätsstrasse 27, 33615, Bielefeld, Germany.
Metab Eng. 2022 Sep;73:82-90. doi: 10.1016/j.ymben.2022.06.002. Epub 2022 Jun 16.
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 with a maximal productivity of 78 mg L day 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.
二萜类化合物是一大类结构多样的天然化合物，主要作为特殊的植物代谢物存在。由于其具有多样化的生物功能，它们是各种工业相关应用（如生物制药、营养保健品和香料）的重要来源。然而，利用其天然宿主进行商业生产受到丰度低、可培养性有限和提取困难的限制，而精确的立体化学结构对化学合成构成了特殊挑战。由于其天然 2-C-甲基-D-赤藓醇 4-磷酸（MEP）途径向光合活性色素的碳通量较高，绿藻作为高效、可持续的异源底盘，具有巨大的潜力用于可持续合成植物源性二萜类化合物。在本研究中，创新性的合成生物学和高效的代谢工程策略被系统地结合起来，重新定向 MEP 途径的代谢通量，以在 C. reinhardtii 中高效异源合成二萜类化合物。工程化 1-脱氧-D-木酮糖 5-磷酸合酶（DXS）作为 MEP 途径的主要限速酶，并过表达二萜合酶融合蛋白，增加了高价值二萜类化合物的产量。采用完全自养的高密度细胞培养方法，在 2.5L 光生物反应器中实现了高达 656mg/L 的高价值二萜类化合物——西罗莫司的高效、可持续生产，最大生产率为 78mg/L·天，这与经过高度工程化的酵母达到的西罗莫司产量相当。因此，这项工作代表着在建立强大的光合绿色细胞工厂方面取得了突破，可用于工业生物技术的竞争应用。
