Development of host microorganisms for heterologous expression of polyketide synthases (PKS) that possess the intrinsic capacity to overproduce polyketides with a broad spectrum of precursors supports the current demand for new tools to create novel chemical structures by combinatorial engineering of modular and other classes of PKS. Streptomyces fradiae is an ideal host for development of generic polyketide-overproducing strains because it contains three of the most common precursors-malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA-used by modular PKS, and is a host that is amenable to genetic manipulation. We have expanded the utility of an overproducing S. fradiae strain for engineered biosynthesis of polyketides by engineering a biosynthetic pathway for methoxymalonyl-ACP, a fourth precursor used by many 16-membered macrolide PKS. This was achieved by introducing a set of five genes, fkbG-K from Streptomyces hygroscopicus, putatively encoding the methoxymalonyl-ACP biosynthetic pathway, into the S. fradiae chromosome. Heterologous expression of the midecamycin PKS genes in this strain resulted in 1 g/l production of a midecamycin analog. These results confirm the ability to engineer unusual precursor pathways to support high levels of polyketide production, and validate the use of S. fradiae for overproduction of 16-membered macrolides derived from heterologous PKS that require a broad range of precursors.