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

Maria Lund Nielsen, Thomas Isbrandt Petersen, Lene Maj Petersen, Uffe Hasbro Mortensen, Mikael Rørdam Andersen, Jakob Blæsbjerg Hoof, Thomas Ostenfeld Larsen

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    Abstract

    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.
    Original languageEnglish
    Article numbere0161199
    JournalP L o S One
    Volume11
    Issue number8
    Number of pages18
    ISSN1932-6203
    DOIs
    Publication statusPublished - 2016

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    This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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