Insights into the biosynthesis of icumazole, unveiling a distinctive family of crotonyl-CoA carboxylase/reductase

Icumazoles are potent antifungal polyketides with intriguing struc-tural features. Here, we present the polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) hybrid biosynthetic gene clus-ter 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 unpre-cedently utilizes both nicotinamide adenine dinucleotide phosphate (NADPH) and NADH as cofactors with similar efficiency, diverging from the NADPH-specific characteristic of canonical CCRs. Further-more, a sequence similarity network-based and phylogenetic bio-informatic survey reveals that the IcuL-like CCRs are evolutionarily separated from canonical CCRs.