Heterologous production of NRPs: From the identification of the biosynthesis genes to the productive heterologous yeast

Non-ribosomal peptides (NRPs) represent a huge group of microbial secondary metabolites with broad and potent biological activities such as antibiotics, antifungal, anti-inflammatory, immunosuppressive, etc. These natural products are assembled by NonRibosomal Peptide Synthetases (NRPSs), multi-enzyme structures divided into different domains. They are encoded by large genes encompassing several kb, usually organized in operons (prokaryotes) or in clusters (eukaryotes). Numerous NRP producer organisms are still unknown and understudied despite the promising activities of the NRPs they synthesize. Moreover, some of these organisms are not easily cultivable or have not immediate applicability to industrial processes. Therefore, to gain suitable production rates of these compounds of interest, one option consists in working with a heterologous production using more efficient strains. Based on the example of a non-ribosomal pentapeptide produced initially by a filamentous fungus, we elaborated a general methodology to develop an eukaryotic microbial platform in a yeast for the heterologous production of NRPs. This is a multiple steps methodology starting with the identification of all the genes involved in the biosynthesis of the NRPs from the native producer, not restricted to nrps genes. Bioinformatics analyses of genomes and DNA-sequences with tools dedicated and tailored to the modular organization of NRPSs enabled to identify NRPSs structures and to predict their biological activities. A second issue is related to the choice of the host organism which, besides being an efficient producer, needs to display a codon fitness with the foreign genes. The final step deals with the major advances of bioengineering approaches enabling the transformation of the chosen strain to embed such complex biosynthesis pathways.