High-Throughput Engineering of Nonribosomal Extension Modules

Abstract

Nonribosomal peptides constitute an important class of natural products that display a wide range of bioactivities. They are biosynthesized by large assembly lines called nonribosomal peptide synthetases (NRPSs). Engineering NRPS modules represents an attractive strategy for generating customized synthetases for the production of peptide variants with improved properties. Here, we explored the yeast display of NRPS elongation and termination modules as a high-throughput screening platform for assaying adenylation domain activity and altering substrate specificity. Depending on the module, display of A–T bidomains or C–A–T tridomains, which also include an upstream condensation domain, proved to be most effective. Reprograming a tyrocidine synthetase elongation module to accept 4-propargyloxy-phenylalanine, a noncanonical amino acid that is not activated by the native protein, illustrates the utility of this approach for altering NRPS specificity at internal sites.

Investigation of the Odilorhabdin Biosynthetic Gene Cluster Using NRPS Engineering

“…In addition to efficient changes of the A domain specificity e.g. through yeast display-based evolutionary optimization [10] or by exchanges of the completete A domain or A subdomain in certain NRPS, [11][12][13][14] recent advances in NRPS engineering have enabled efficient reassembly of different NRPS at several positions and domains within the assembly line. A first concept resembles the 'eXchange Units' (XU), which utilize a specific position within the CÀ A domain linker to assemble different NRPS as well as AÀ TÀ C tri-domains into a given NRPS.…”