<jats:title>ABSTRACT</jats:title> <jats:p> To elevate the production level of heterologous polyketide in <jats:italic>Streptomyces venezuelae</jats:italic> , an additional copy of the positive regulatory gene <jats:italic>pikD</jats:italic> was introduced into the pikromycin (Pik) polyketide synthase (PKS) deletion mutant of <jats:italic>S. venezuelae</jats:italic> ATCC 15439 expressing tylosin PKS genes. The resulting mutant strain showed enhanced production of both tylactone (TL) and desosaminyl tylactone (DesTL) of 2.7- and 17.1-fold, respectively. The notable increase in DesTL production strongly suggested that PikD upregulates the expression of the desosamine ( <jats:italic>des</jats:italic> ) biosynthetic gene cluster. In addition, two hydroxylated forms of DesTL were newly detected from the extract of this mutant. These hydroxylated forms presumably resulted from a PikD-dependent increase in expression of the <jats:italic>pikC</jats:italic> gene that encodes P450 hydroxylase. Gene expression analysis by reverse transcriptase PCR and bioconversion experiments of 10-deoxymethynolide, narbonolide, and TL into the corresponding desosaminyl macrolides indicated that PikD is a positive regulator of the <jats:italic>des</jats:italic> and <jats:italic>pikC</jats:italic> genes, as well as the Pik PKS genes. These results demonstrate the role of PikD as a pathway-specific positive regulator of the entire Pik biosynthetic pathway and its usefulness in the development of a host-vector system for efficient heterologous production of desosaminyl macrolides and novel hydroxylated compounds.