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

PLoS One. 2016 Aug 23;11(8):e0161199. doi: 10.1371/journal.pone.0161199. eCollection 2016.

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.

MeSH terms

  • Aspergillus nidulans / enzymology
  • Aspergillus nidulans / genetics*
  • Biological Products
  • Cytochalasins / biosynthesis
  • Gene Expression Regulation, Enzymologic
  • Genes, Synthetic / genetics
  • Genetic Engineering*
  • Magnaporthe / enzymology
  • Magnaporthe / genetics
  • Peptide Synthases / biosynthesis
  • Peptide Synthases / genetics*
  • Polyketide Synthases / biosynthesis
  • Polyketide Synthases / genetics*
  • Substrate Specificity

Substances

  • Biological Products
  • Cytochalasins
  • cytochalasin E
  • Polyketide Synthases
  • Peptide Synthases
  • non-ribosomal peptide synthase

Grants and funding

This work was supported by the Novo Nordisk Foundation (http://www.novonordiskfonden.dk/en). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.