Linker Flexibility Facilitates Module Exchange in Fungal Hybrid PKS-NRPS Engineering

• Published in: PloS one 2016-08, Vol.11 (8), p.e0161199-e0161199
• Main Authors: Nielsen, Maria Lund, Isbrandt, Thomas, Petersen, Lene Maj, Mortensen, Uffe Hasbro, Andersen, Mikael Rørdam, Hoof, Jakob Blæsbjerg, Larsen, Thomas Ostenfeld
• Format: Article
• Language: English
• Subjects: Aspergillus; Aspergillus nidulans; Aspergillus nidulans - enzymology; Aspergillus nidulans - genetics; Biological activity; Biological Products; Biology and Life Sciences; Biosynthesis; Chain transfer; Complexity; Cytochalasins - biosynthesis; Enzymes; Flexibility; Fungi; Gene Expression Regulation, Enzymologic; Genes; Genes, Synthetic - genetics; Genetic Engineering; Hybrids; Intermediates; Magnaporthe - enzymology; Magnaporthe - genetics; Magnaporthe oryzae; Natural products; Optimization; Oryza; Peptide Synthases - biosynthesis; Peptide Synthases - genetics; Peptide synthesis; Peptides; Physical Sciences; Polyketide Synthases - biosynthesis; Polyketide Synthases - genetics; Polyketides; Proteins; Research and Analysis Methods; Studies; Substrate Specificity; Synthetic biology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0161199

Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) each give rise to a vast array of complex bioactive molecules with further complexity added by the existence of natural PKS-NRPS fusions. Rational genetic engineering for the production of natural product derivatives is desirable for the purpose of incorporating new functionalities into pre-existing molecules, or for optimization of known bioactivities. We sought to expand the range of natural product diversity by combining modules of PKS-NRPS hybrids from different hosts, hereby producing novel synthetic natural products. We succeeded in the construction of a functional cross-species chimeric PKS-NRPS expressed in Aspergillus nidulans. Module swapping of the two PKS-NRPS natural hybrids CcsA from Aspergillus clavatus involved in the biosynthesis of cytochalasin E and related Syn2 from rice plant pathogen Magnaporthe oryzae lead to production of novel hybrid products, demonstrating that the rational re-design of these fungal natural product enzymes is feasible. We also report the structure of four novel pseudo pre-cytochalasin intermediates, niduclavin and niduporthin along with the chimeric compounds niduchimaeralin A and B, all indicating that PKS-NRPS activity alone is insufficient for proper assembly of the cytochalasin core structure. Future success in the field of biocombinatorial synthesis of hybrid polyketide-nonribosomal peptides relies on the understanding of the fundamental mechanisms of inter-modular polyketide chain transfer. Therefore, we expressed several PKS-NRPS linker-modified variants. Intriguingly, the linker anatomy is less complex than expected, as these variants displayed great tolerance with regards to content and length, showing a hitherto unreported flexibility in PKS-NRPS hybrids, with great potential for synthetic biology-driven biocombinatorial chemistry.