Project Outline

Natural products represent a major source of powerful and innovative therapeutic agents for the treatment of human, animal and plant diseases.¹ Many of these products, including broad-spectrum and last resort antibiotics such as erythromycin, vancomycin and taxol are of polyketide, nonribosomal peptide and terpene origin. The structural complexity of these molecules still provides stimulating and challenging targets for synthetic organic chemists. Moreover, synthetic biology is currently seen as a highly attractive avenue for complex natural product preparation as well as structural diversification.² This latter is highly desirable in view of developing new bioactive molecules of increased efficiency and/or novel activities.³

Polyketide and nonribosomal peptide products are biosynthesized in microorganisms and plants by multifunctional enzymes, the polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) respectively. These utilize simple and abundant building blocks (e.g. acyl and amino acyl) and iterative condensation reactions to generate highly chemically functionalised polyketide/peptide chains, which can be further enzymatically tailored to yield the final natural products.^{4, 5} In the Tosin group at Warwick Chemistry we have recently found that small synthetic molecules that mimic the building blocks utilized in polyketide and nonribosomal peptide assembly can react with ‘stalled’ enzyme-bound biosynthetic intermediates and lead to the formation of ‘unnatural’ products: ^6-8 these are structurally complex molecules that are still made by a PKS enzymatic assembly but that are structurally different from the original products. Unnatural products may be as medicinally/commercially valuable as existing natural products, if not superior.^{3, 6}

The overall aim of this PhD project is to generate novel unnatural products from simple synthetic precursors (‘synthons’) and genetically engineered bacteria. The following specific research objectives will be pursued:

1. the preparation of a library of novel chemical synthons bearing functionalities that cannot be made biosynthetically (organic synthesis, under the supervision of Dr Manuela Tosin);
2. the genetic engineering of Streptomyces bacteria to allow/facilitate the chemical synthons cellular uptake and their incorporation into selected polyketide/ nonribosomal peptide/terpene pathways (molecular biology, under the supervision of Dr Christophe Corre);

the generation of unnatural products in vitro and in vivo, by providing enzymes/ engineered bacterial cells with the chemical synthons, followed by product isolation and characterization (microbiology and analytical chemistry, under the supervision of Dr Manuela Tosin). Preliminary bioassays of the novel products will be undertaken, either in the host lab(s) or through collaborations.

References

1. D. J. Newman et al., J. Nat. Prod. 2012, 75, 311-335.
2. M. R. Seyedsayamdost et al., ACS Synth. Biol. 2014, 3, 745-747.
3. R. J. M. Goss et al., Nat. Prod. Rep. 2012, 29, 870-889.
4. K. J. Weissman, Methods Enzymol. 2009, 459, 3-16.
5. E. A. Felnagle et al., Mol. Pharmaceutics 2008, 5, 191- 211.
6. E. Riva et al., Angew. Chem. Int. Ed. 2014, 53, 11944-9 (VIP article).
7. C. H. Y. Ho et al., Chem. Commun. 2017, 53, 7088-7091.
8. D. J. Leng et al., Chem. Commun. 2021, 5, 2293-2296.

Techniques

• Organic chemistry: synthesis, purification and characterisation of organic compounds;
• Microbiology: growing and handling of E. coli and Actinobacteria strains;
• Molecular biology: gene cloning and genetic disruptions in Actinobacteria using a PCR targeting methodology;
• Protein chemistry and enzymology: protein expression in heterologous hosts, enzyme activity: reconstitution and characterisation;
• Analytical chemistry: identification, isolation and characterisation of natural products and biosynthetic intermediates by LC-MS, HPLC, HR-MS, GC, NMR.