Epothilone (Epo) D, an antitumor agent currently in clinical trials, is a hybrid natural product produced by the combined action of nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS). In the epothilone biosynthetic pathway, EpoB, a 165 kDa NRPS is inserted into an otherwise entirely PKS assembly line, forming two hybrid NRPS-PKS interfaces. In light of the terminal linker effect previously identified in PKS, the N- and C-terminal sequences of EpoB were examined for their roles in propagating the incipient natural product. Eight amino acid residues at EpoB C terminus, in which six are positively charged, were found to be a key component of the C-terminal linker effect. A minimal sequence of 56 residues at EpoB N terminus was required for elongating the acetyl group from the acyl carrier protein (ACP) of EpoA to form methylthiazolyl-S-EpoB.