title: Linker Flexibility Facilitates Module Exchange in Fungal Hybrid PKS-NRPS Engineering.
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.
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Kongens Lyngby, Denmark.
authors.forename: Maria Lund
authors.initials: ML
authors.lastname: Nielsen
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Kongens Lyngby, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Isbrandt
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Kongens Lyngby, Denmark.
authors.forename: Lene Maj
authors.initials: LM
authors.lastname: Petersen
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Kongens Lyngby, Denmark.
authors.forename: Uffe Hasbro
authors.initials: UH
authors.lastname: Mortensen
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Kongens Lyngby, Denmark.
authors.forename: Mikael Rørdam
authors.initials: MR
authors.lastname: Andersen
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Kongens Lyngby, Denmark.
authors.forename: Jakob Blæsbjerg
authors.initials: JB
authors.lastname: Hoof
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Kongens Lyngby, Denmark.
authors.forename: Thomas Ostenfeld
authors.initials: TO
authors.lastname: Larsen
mesh_terms: D001233:Aspergillus nidulans / Q000201:enzymology / Q000235:genetics*; D001688:Biological Products; D003572:Cytochalasins / Q000096:biosynthesis; D015971:Gene Expression Regulation, Enzymologic; D005813:Genes, Synthetic / Q000235:genetics; D005818:Genetic Engineering; D020082:Magnaporthe / Q000201:enzymology / Q000235:genetics; D010453:Peptide Synthases / Q000096:biosynthesis / Q000235:genetics*; D048630:Polyketide Synthases / Q000096:biosynthesis / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D001688:Biological Products; D003572:Cytochalasins; C007215:cytochalasin E; D048630:Polyketide Synthases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase