Resorcylic acid lactones represent a unique class of fungal polyketides and display a wide range of biological activities, such as nanomolar inhibitors of Hsp90 and MAP kinase. The biosynthesis of these compounds is proposed to involve two fungal polyketide synthases (PKS) that function collaboratively to yield a 14-membered macrolactone with a resorcylate core. We report here the reconstitution of Gibberella zeae PKS13, which is the nonreducing PKS associated with zearalenone biosynthesis. Using a small molecule mimic of the natural hexaketide starter unit, we reconstituted the entire repertoire of PKS13 activities in vitro, including starter unit selection, iterative condensation, regioselective C2-C7 cyclization and macrolactone formation. PKS13 synthesized both natural 14-membered and novel 16-membered resorcylic acid lactones, indicating relaxed control in both iterative elongation and macrocyclization. Furthermore, PKS13 exhibited broad starter-unit specificities towards fatty acyl-CoAs ranging in sizes between C6 and C16. PKS13 was also proven to be able to interact with Escherichia coli fatty acid biosynthetic machinery and can be primed with fatty-acyl ACPp at low micromolar concentrations. A number of alkyl pyrones and alkyl resorcylic esters were produced by E. coli BL21(DE3) harboring PKS13 with a highest yield up to 5 mg/L. And we further demonstrated that BL21(DE3) harboring PKS13 can synthesize new polyketides at a yield of 2~3 mg/L, when supplemented with synthetic precursors, which showcases the utility of PKS13 in precursor-directed biosynthesis. PKS13 is therefore a highly versatile polyketide macrolactone synthase that is useful in the engineered biosynthesis of novel polyketides, including resorcylic acid lactones that are not found in nature.