Phenylethanoid glycosides (PhGs) exhibit a multitude of structural variations linked to diverse pharmacological activities. Assembling various PhGs via multienzyme cascades represents a concise strategy over traditional synthetic methods. However, the challenge lies in identifying a comprehensive set of catalytic enzymes. This study explores biosynthetic PhG reconstruction from natural precursors, aiming to replicate and amplify their structural diversity. We discovered 12 catalytic enzymes, including four novel 6'‐OH glycosyltransferases and three new polyphenol oxidases, revealing the intricate network in PhG biosynthesis. Subsequently, the crystal structure of CmGT3 (2.62 Å) was obtained, guiding the identification of conserved residue 144# as a critical determinant for sugar donor specificity. Engineering this residue in PhG glycosyltransferases (FsGT61, CmGT3, and FsGT6) altered their sugar donor recognition. Finally, a one‐pot multienzyme cascade was established, where the combined action of glycosyltransferases and acyltransferases boosted conversion rates by up to 12.6‐fold. This cascade facilitated the reconstruction of 26 PhGs with conversion rates ranging from 5‐100%, and 20 additional PhGs detectable by mass spectrometry. PhGs with extra glycosyl and hydroxyl modules demonstrated notable liver cell protection. This work not only provides catalytic tools for PhGs biosynthesis, but also serves as a proof‐of‐concept for cell‐free enzymatic construction of diverse natural products.

中文翻译：

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利用生物合成网络、蛋白质工程和一锅多酶级联构建苯乙醇苷

苯乙醇苷 (PhG) 表现出多种与不同药理活性相关的结构变异。通过多酶级联组装各种 PhG 是一种优于传统合成方法的简洁策略。然而，挑战在于确定一套全面的催化酶。本研究探索从天然前体中重建生物合成 PhG，旨在复制和放大其结构多样性。我们发现了 12 种催化酶，包括四种新型 6'-OH 糖基转移酶和三种新型多酚氧化酶，揭示了 PhG 生物合成中复杂的网络。随后，获得了 CmGT3 (2.62 Å) 的晶体结构，指导鉴定保守残基 144# 作为糖供体特异性的关键决定因素。在 PhG 糖基转移酶（FsGT61、CmGT3 和 FsGT6）中改造该残基改变了它们的糖供体识别。最后，建立了一锅多酶级联，其中糖基转移酶和酰基转移酶的联合作用将转化率提高了高达 12.6 倍。该级联促进了 26 个 PhG 的重建，转化率范围为 5-100%，并且另外 20 个 PhG 可通过质谱检测。具有额外糖基和羟基模块的 PhG 表现出显着的肝细胞保护作用。这项工作不仅为PhGs生物合成提供了催化工具，而且还为多种天然产物的无细胞酶促构建提供了概念验证。