Escherichia coli Nissle 1917 (EcN) is a Gram-negative bacterium that is used to treat inflammatory bowel diseases. The probiotic character of EcN is not well-understood, but its ability to produce secondary metabolites plays an important role in its activity. The EcN genome encodes for an aryl polyene (APE) biosynthetic gene cluster (BGC), and APE products have a role in biofilm formation. We show here that this unusual polyketide assembly line synthase produces four APE molecules which are likely cis/trans isomers. Within the APE BGC, two acyl carrier proteins are involved in biosynthesis. Acyl carrier proteins require activation by post-translational modification with a phosphopantetheinyl transferase (PPTase). Through analysis of single, double, and triple mutants of three PPTases, the PPTase-BGC crosstalk relationship in EcN was characterized. Understanding PPTase-BGC crosstalk is important for the engineering of secondary metabolite production hosts and for targeting of PPTases with new antibiotics.