The γ-butyrolactone motif is found in many natural signaling molecules and other specialized metabolites. A prominent example is the potent aquatic phytotoxin cyanobacterin, which has a highly functionalized γ-butyrolactone core structure. The enzymatic machinery that assembles cyanobacterin and structurally related natural products (herein termed furanolides) has remained elusive for decades. Here, we elucidate the biosynthetic process of furanolide assembly. The cyanobacterin biosynthetic gene cluster was identified by targeted bioinformatic screening and validated by heterologous expression in Escherichia coli. Full functional evaluation of the recombinant key enzymes in vivo and in vitro, individually and in concert, provided in-depth mechanistic insights into a streamlined C–C bond-forming cascade that involves installation of compatible reactivity at seemingly unreactive Cα positions of amino acid precursors. Our work extends the biosynthetic and biocatalytic toolbox for γ-butyrolactone formation, provides a general paradigm for furanolide biosynthesis and sets the stage for their targeted discovery, biosynthetic engineering and enzymatic synthesis.

γ-丁内酯基序存在于许多天然信号分子和其他特殊代谢物中。一个突出的例子是强效水生植物毒素蓝藻素，它具有高度功能化的 γ-丁内酯核心结构。几十年来，组装蓝藻素和结构相关的天然产物（本文称为呋喃内酯）的酶机制一直难以捉摸。在这里，我们阐明了呋喃内酯组装的生物合成过程。通过靶向生物信息学筛选鉴定蓝藻素生物合成基因簇，并通过大肠杆菌中的异源表达进行验证. 重组关键酶在体内和体外的完整功能评估，单独和协同，提供了对流线型 C-C 键形成级联的深入机制洞察，该级联涉及在氨基酸前体看似不反应的 Cα 位置安装相容的反应性. 我们的工作扩展了 γ- 丁内酯形成的生物合成和生物催化工具箱，为呋喃内酯生物合成提供了一个通用范例，并为其靶向发现、生物合成工程和酶促合成奠定了基础。