Coumarins are a vast family of natural products with diverse biological activities. Cinnamyl-CoA ortho-hydroxylases (CCHs) catalyze the gateway and rate-limiting step in coumarin biosynthesis. However, engineering CCHs is challenging due to the large size of the substrates and the vague structure-activity relationship. Herein, directed evolution and structure-guided engineering were performed to engineer a CCH (AtF6′H from Arabidopsis thaliana) using a fluorescence-based screening method, yielding the transplantable surface mutations and the substrate-specific pocket mutations with improved activity. Structural analysis and molecular dynamics simulations elucidated the conformational changes that led to increased catalytic efficiency. Applying appropriate variants with the optimized upstream biosynthetic pathways improved the titers of three simple coumarins by 5 to 22-fold. Further introducing glycosylation modules resulted in the production of four coumarin glucosides, among which the titer of aesculin was increased by 15.7-fold and reached 3 g/L in scale-up fermentation. This work unleashed the potential of CCHs and established an Escherichia coli platform for coumarins production.

中文翻译：

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通过蛋白质和代谢工程建立香豆素生产平台


香豆素是一大类天然产物，具有不同的生物活性。肉桂酰辅酶 A 邻羟化酶 （CCH） 催化香豆素生物合成中的网关和限速步骤。然而，由于衬底尺寸大且构效关系模糊，工程 CCH 具有挑战性。在此，进行了定向进化和结构引导工程，使用基于荧光的筛选方法设计了 CCH （来自拟南芥的 AtF6'H），产生了活性更高的可移植表面突变和底物特异性口袋突变。结构分析和分子动力学模拟阐明了导致催化效率提高的构象变化。将适当的变体与优化的上游生物合成途径相结合，将三种简单香豆素的滴度提高了 5 至 22 倍。进一步引入糖基化模块，生产了 4 种香豆素葡萄糖苷，其中七叶素滴度提高了 15.7 倍，在放大发酵中达到 3 g/L。这项工作释放了 CCH 的潜力，并建立了用于香豆素生产的大肠杆菌平台。