Dissection and Engineering of Modular Polyketide Synthase Extender Unit Specificity Motifs.

Polyketide synthases (PKSs) are privileged enzymatic platforms for generating natural products. Yet, efforts to redesign their acyltransferase (AT) domains for incorporation of non-native extender units remain constrained by an incomplete understanding of the sequence elements that govern specificity. EryAT6 is the methylmalonyl-CoA-specific terminal AT domain of the erythromycin PKS (DEBS). Two conserved motifs within EryAT6, the large- and small-subunit motifs (LSM and SSM), suggest that short sequence segments encode key extender unit selectivity, but their modularity has not been systematically explored. Here, we delineate the sequence-function relationships underlying extender unit selection by combining mutagenesis, motif swapping, and functional reconstitution in Ery6TE. Substitution of ten nonconserved residues across the LSM and SSM revealed some positions that strongly influence the incorporation of larger extender units. Triple-residue combinations exhibited cooperativity, with the T739A/Y744R/S746G mutant increasing the portion of butylmalonyl-CoA-derived product by 42-fold. Motif exchanges showed that both LSM and SSM can reprogram AT selectivity in AT-swapped Ery6TE chimeras, enabling the formation of butyl-substituted pyrones even when the parent domain swap was inactive. Together, these results identify compact determinants of extender unit specificity and establish motif-level engineering as a strategy to access diverse polyketides.