Prenylated tryptophan-containing cyclic dipeptides are found in different fungi and serve as precursors for the biosynthesis of diverse biologically active secondary metabolites. They show distinct and usually higher biological and pharmacological activities than the respective non-prenylated dipeptides. Successful production of such compounds were achieved by a new approach based on the coexpression of ftmPS, a non-ribosomal peptide synthetase from Neosartorya fischeri, with three cyclic dipeptide prenyltransferase genes from different biosynthetic gene clusters in Aspergillus nidulans. The genes are expressed under the control of constitutive gpdA promoter and trpC terminator. Expression of ftmPS alone resulted in the formation of the expected cyclic dipeptide brevianamide F with a yield of up to 36.9 mg l(-1). Introducing the reverse C2-prenyltransferase gene cdpC2PT as well as the reverse C3-prenyltransferase gene cdpNPT into a ftmPS mutant yielded reversely C2- and C3-prenylated derivatives, respectively. Coexpression of ftmPS with the reverse C3-prenyltransferase gene cdpC3PT resulted in the formation of N1-regularly, C2-, and C3-reversely prenylated derivatives. The prenyl transfer reactions catalyzed by CdpC2PT, CdpNPT, and CdpC3PT observed in this study correspond well to those detected with purified proteins. The yields of the detected prenylated products were found to be up to 12.2 mg l(-1). The results presented in this study show the potential of synthetic biology for production of prenylated compounds.