Construction of hybrid peptide synthetases by module and domain fusions

Nonribosomal peptide synthetases are modular enzymes that assemble peptides of diverse structures and important biological activities. Their modular o rganization provides a great potential for the rational design of novel com pounds by recombination of the biosynthetic genes, Here we describe the ext ension of a dimodular system to trimodular ones based on whole-module fusio n. The recombinant hybrid enzymes were purified to monitor product assembly in vitro. We started from the first two modules of tyrocidine synthetase, which catalyze the formation of the dipeptide DPhe-Pro, to construct such h ybrid systems. Fusion of the second, proline-specific module with the ninth and tenth modules of the tyrocidine synthetases, specific for ornithine an d leucine, respectively, resulted in dimodular hybrid enzymes exhibiting th e combined substrate specificities. The thioesterase domain was fused to th e terminal module. Upon incubation of these dimodular enzymes with the firs t tyrocidine module, TycA, incorporating DPhe, the predicted tripeptides DP he-Pro-Orn and oPhe-Pro-Leu were obtained at rates of 0.15 min(-1) and 2.1 min(-1). The internal thioesterase domain was necessary and sufficient to r elease the products from the hybrid enzymes and thereby facilitate a cataly tic turnover. Our approach of whole-module fusion is based on an improved d efinition of the fusion sites and overcomes the recently discovered editing function of the intrinsic condensation domains. The stepwise construction of hybrid peptide synthetases from catalytic subunits reinforces the inhere nt potential for the synthesis of novel, designed peptides.