Engineered Modular Polyketide Synthases Elucidate How Enoylreductases Collaborate with Downstream Acyl Carrier Proteins and Ketosynthases to Set Stereocenters

platforms. ER-inserted modules were observed to generate both unsaturated and saturated intermediates. Increased production of the saturated intermediate was realized when the downstream KS was also swapped for a KS from an ER-containing module. To access products with desired methyl group orientations, it was necessary to consider how the methyl groups interact with the KS of the module that installed them, as well as with the KS of the downstream module. Additionally, we interconverted d- and l-type ERs by swapping a 14-residue loop and propose the molecular basis for how ERs set stereocenters. This research provides new strategies for controlling the orientations of alkyl substituents in complex polyketides and enables access to stereochemical libraries of polyketide drug leads.