Dissecting and exploiting fungal polyketide-nonribosomal peptide synthetase

Project Outline

The biosynthesis of polyketides in fungi is catalysed by iterative polyketide synthases (iPKSs). These remarkable, multi-domain enzymes are capable of generating a plethora of structurally complex polyketide scaffolds, often possessing potent biological activities.1 Fungal iPKSs closely resemble the mammalian fatty acid synthase (mFAS) in both domain architecture and catalysis.2,3 An iterative cycle of decarboxylative condensations between malonyl extender units bound to the acyl carrier protein (ACP) domain and the growing acyl chain is catalysed by the ketosynthase (KS) domain; with extender units continually loaded onto the ACP domain by an acyl transferase (AT) domain. Intermediates and extender units are covalently tethered via a thioester bond to a 4’-phosphopantetheine (Ppant) prosthetic ‘arm’ that is post-translationally appended to the ACP domain.4

The thiol-template mechanism employed by iPKSs allows effective collaboration with non-ribosomal peptide synthetases (NRPSs), giving rise to both PKS-NRPS and NRPS-PKS hybrids. This permits incorporation of (non-)proteinogeneic amino acids into the polyketide scaffold, greatly increasing the chemical complexity of the final product, but requiring the ACP domain to bridge communication between PKS and NRPS enzymology. These hybrid systems offer huge combinatorial potential for bioengineering approaches. However, the limited engineering work that has been attempted on these systems resulted in erroneous products and dramatically reduced yields.5 This is likely due to incompatible ACP-C domain combinations, the interface between which is not yet understood.

This project aims to understand the molecular basis for ACP-C domain communication and substrate tolerance, providing a knowledgebase that will allow informed biosynthetic engineering and construction of novel hybrids. This will be investigated using a highly interdisciplinary approach, utilising protein biochemistry, structural biology, chemical synthesis, protein mass spectrometry and yeast-based pathway engineering.

This project will be jointly supervised by Dr. Matthew Jenner (Protein Biochemistry, Mass Spectrometry, Structural Biology, Molecular Biology) and Dr. Mark Greenhalgh (Chemical Synthesis).

References

[1] J. Nat. Prod. 2020, 83, 770 [2] Org. Biomol. Chem. 2007, 5, 2010 [3] J. Org. Chem. 2012, 77, 9933 [4] Nat. Prod. Rep., 2018, 35, 1046 [5] J. Am. Chem. Soc. 2014, 136, 17882.

Techniques

• Bioinformatics.
• Molecular cloning.
• Recombinant protein overproduction and purification.
• Organic synthesis.
• Intact/structural protein mass spectrometry techniques.
• Protein biochemistry.
• X-ray crystallography of proteins.