The oxidized kaurene (Ox-Kau) compounds are the core structures of many important diterpenoids with biological activities and economical values. However, easy access to diverse Ox-Kau products is still limited by low natural abundance, and large-scale manufacture remain challenging due to lack of proper heterologous production. To achieve an abundant source alternative to natural extracts, we here report a highly effective Escherichia coli-based platform for the de novo production of multiple Ox-Kau molecules from simple carbon source. Pathway optimization in prokaryotic cells through modification of transmembrane CYP450 oxidases, cytochrome b₅ co-expression and AlphaFold-based protein engineering improved a 50-fold yield of steviol (1.07 g L⁻¹), a key intermediate in the kaurenoid biosynthesis. Combinatorial biosynthetic strategy further led to a series of oxidized derivatives (20–600 mg L⁻¹) with rich oxygenated functional groups on C3, C7, C16 and C19 previously hard to be introduced. Our engineered strains not only laid a foundation for realizing the industrial fermentation of gram-scale ent-kaurene diterpenoids, but also provided a reliable platform for characterization and utilization of kaurene-modifying oxidases, which may generate naturally rare or unnatural ent-kaurenoids with potential bioactivity.

氧化贝壳杉（Ox-Kau）化合物是许多重要的二萜类化合物的核心结构，具有生物活性和经济价值。然而，由于缺乏适当的异源生产，难以获得多种 Ox-Kau 产品仍然受到自然丰度低的限制，并且大规模生产仍然具有挑战性。为了实现天然提取物的丰富来源替代品，我们在此报告了一种高效的基于大肠杆菌的平台，用于从简单的碳源从头生产多种 Ox-Kau 分子。通过修饰跨膜 CYP450 氧化酶、细胞色素b ₅共表达和基于 AlphaFold 的蛋白质工程优化原核细胞中的途径，将甜菊醇的产量提高了 50 倍（1.07 g L ^-1)，是类贝壳杉类生物合成的关键中间体。组合生物合成策略进一步导致了一系列氧化衍生物（20-600 mg L ^-1），在以前很难引入的C3、C7、C16和C19上具有丰富的氧化官能团。我们的工程菌不仅为实现克级对角-贝壳杉烯二萜的工业化发酵奠定了基础，而且为贝壳杉烯修饰氧化酶的表征和利用提供了可靠的平台，可能产生天然稀有或非天然的对角-贝壳杉类化合物具有潜力。生物活性。