Non-ribosomal peptide synthetases are complex multimodular biosynthetic machines that assemble various important and medically relevant peptide antibiotics. An interesting subgroup comprises the cyclodepsipeptide synthetases from fungi synthesizing cyclohexa- and cyclo-octadepsipeptides with antibacterial, anthelmintic, insecticidal, and anticancer properties; some are marketed drugs. We exploit the modularity of these highly homologous synthetases by fusing the hydroxy-acid-activating module of PF1022 synthetase with the amino-acid-activating modules of enniatin and beauvericin synthetase, thus yielding novel hybrid synthetases. The artificial synthetases expressed in Escherichia coli and the fungus Aspergillus niger yielded new cyclodepsipeptides, thus paving the way for the exploration of these derivatives for their bioactivity.

The heterologous expression of genes or gene clusters in microbial hosts, followed by metabolic engineering of biosynthetic pathways, is key to access industrially and pharmaceutically relevant compounds in an economically affordable and sustainable manner. Therefore, platforms need to be developed, which provide tools for the controlled synthesis of bioactive compounds. The Gram-positive bacterium Bacillus subtilis is a promising candidate for such applications, as it is generally regarded as a safe production host, its physiology is well investigated and a variety of tools is available for its genetic manipulation. Furthermore, this industrially relevant bacterium provides a high secretory potential not only for enzymes but also for primary and secondary metabolites. In this study, we present the first heterologous expression of an eukaryotic non-ribosomal peptide synthetase gene (esyn) coding for the biosynthesis of the small molecule enniatin in B. subtilis. Enniatin is a pharmaceutically used cyclodepsipeptide for treatment of topical bacterial and fungal infections. We generated various enniatin-producing B. subtilis strains, allowing for either single chromosomal or plasmid-based multi-copy expression of the esyn cluster under the control of an acetoin-inducible promoter system. Optimization of cultivation conditions, combined with modifications of the genetic background and multi-copy plasmid-based esyn expression, resulted in a secretory production of enniatin B. This work presents B. subtilis as a suitable host for the expression of heterologous eukaryotic non-ribosomal peptide synthetases (NRPS) clusters.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-014-6199-0) contains supplementary material, which is available to authorized users.

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The cover picture shows the non‐ribosomally synthesized cyclodepsipeptides PF1022, beauvericin and enniatin with high pharmacological importance because of their various bioactivities. Today, PF1022 and derivatives are under trials for use for treatment of neglected diseases like filariasis, which is of considerable interest due to the more recent development of resistances against the anthelmintic avermectin. Based on the modular organization of NRPS we considered each module to be a piece of a jigsaw puzzle that can be replaced by the corresponding piece of a homologous NRPS enzyme: the first module of the PF1022 was exchanged for the respective module of the enniatin and beauvericin synthetases to create new‐to‐nature cyclodepsipeptides with promising bioactivities. We were able to express the different artificial NRPS enzymes of fungal origin in both, Escherichia coli and the filamentous fungus Aspergillus niger. An anthelmintic bioactivity assay revealed promising bioactivity against microfilariae of Dirofilaria immitis. More information can be found in the communication by R. D. Süssmuth et al. on page 283 in Issue 4, 2016 (DOI: 10.1002/cbic.201500649).