Icumazoles are potent antifungal polyketides with intriguing structural features. Here, we present the polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) hybrid biosynthetic gene cluster of icumazoles. Surprisingly, an unusual nonterminal thioesterase domain divides the PKS/NRPS assembly line. The succeeding PKS modules potentially form a rare precursor 4-methyl-2-hexenoyl-ACP, thus deviating from the previously proposed polyoxypeptin pathway. The 4-methyl-2-hexenoyl-ACP is further reductively carboxylated to 2-methylbutylmalonyl-ACP, essential for icumazole biosynthesis by IcuL. We characterize IcuL and its homologs TgaD and Leu10 in vitro, suggesting a stricter substrate specificity of this new family of crotonyl carboxylases/reductases (CCRs) than found in canonical ones. Intriguingly, we also find that TgaD unprecedently utilizes both nicotinamide adenine dinucleotide phosphate (NADPH) and NADH as cofactors with similar efficiency, diverging from the NADPH-specific characteristic of canonical CCRs. Furthermore, a sequence similarity network-based and phylogenetic bioinformatic survey reveals that the IcuL-like CCRs are evolutionarily separated from canonical CCRs.

Icumazole 是一种有效的抗真菌聚酮化合物，具有有趣的结构特征。在这里，我们介绍了 icumazole 的聚酮化合物合酶 (PKS)/非核糖体肽合成酶 (NRPS) 杂交生物合成基因簇。令人惊讶的是，一个不寻常的非末端硫酯酶结构域将 PKS/NRPS 装配线分开。随后的 PKS 模块可能形成罕见的前体 4-甲基-2-己烯酰-ACP，从而偏离先前提出的聚氧肽素途径。4-甲基-2-己烯酰基-ACP 进一步还原羧化为 2-甲基丁基丙二酰基-ACP，这是 IcuL 进行艾克唑生物合成所必需的。我们在体外表征 IcuL 及其同系物 TgaD 和 Leu10, 表明这个新的巴豆酰羧化酶/还原酶 (CCR) 家族的底物特异性比在经典家族中发现的更严格。有趣的是，我们还发现 TgaD 前所未有地利用烟酰胺腺嘌呤二核苷酸磷酸 (NADPH) 和 NADH 作为辅因子，效率相似，这与典型 CCR 的 NADPH 特异性特征不同。此外，基于序列相似性网络和系统发育生物信息学调查表明，IcuL 样 CCR 在进化上与规范 CCR 分开。