Combinatorial biosynthesis of non-ribosomal peptides.

Non-ribosomal peptide synthetases (NRPS) are modular assembly lines catalysing the synthesis of many small peptides in microbes. Genetic replacements of domains or modules in NRPS encoded by gene clusters in Bacillus sp. with corresponding domains or modules from foreign NRPS have led in several cases to the in vivo synthesis of peptides with predicted amino acid substitutions. Fusion points were in variable regions between C- and A- or between T- and C-domains. Successful insertions of whole modules using fusion points in conserved regions internal to functional domains have also been reported. For studying the role of C-, A-, T- and TE (thioesterase)-domains in NRPS, several bi- and trimodular model-NRPS derived from natural NRPS systems were constructed and obtained after expression in E. coli with coexpression of a 4'- phosphopantetheine transferase or in suitable hosts such as the Streptomyces. Such enzymes were shown to catalyse in vitro synthesis of di- and tripeptides, respectively, with and without turnover depending on the presence of Te-domains. The enzymatic analysis revealed the mechanisms of the domains and proved their functional autonomy suggesting the possibility to use any NRPS interdomain region for fusions. Nevertheless, recombinant synthesis of longer and more complex peptides will still be restricted to alteration of existing structures by manipulations of NRPS gene clusters located on chromosomes or artificial chromosomes. Besides targeted replacements of domains and modules, reprogramming of NRPS by altering the substrate specificities of A-domains is a promising tool for the future to get novel peptides.