Exploring epoxyketone synthases and their biosynthetic potential Marlene Rothe 1 , Callum Bullock 1,2 , Josh W. Cartwright 1 , Lona Alkhalaf 1 , Fabrizio Alberti 1,2 , Gregory L. Challis 1,3,4 1 Department of Chemistry, University of Warwick, UK, 2 School of Life Sciences, University of Warwick, UK, 3 Monash University, Australia, 4 Warwick Integrative Synthetic Biology Centre, University of Warwick, UK Current treatments for different types of cancer include natural product derived epoxyketones which act as proteasome inhibitors. 1 The chemical syntheses of epoxyketone drugs are very inefficient due to loss of half of the material in the unselective formation of the epoxyketone warhead. 2,3 Epoxyketone natural products are found in a variety of natural organism and are biosynthesized by epoxyketone synthases, which enantio-selectively form the epoxyketone from α-dimethyl-β-keto acids (Fig. 1). 3,4 The aim of this work is to enable a chemoenzymatic and more sustainable production of the epoxyketone drug candidates and an interdisciplinary approach was taken to investigate epoxyketone synthases and their biosynthetic potential. In vitro enzyme assays with known epoxyketone synthase EpnF and synthetic epoxyketone precursors showed the production of the desired epoxyketones, indicating that EpnF accepts native and tested non-native substrates. Additionally, bioinformatic experiments revealed the presence of genes encoding novel epoxyketone synthases in known Streptomyces strains. Nanopore sequencing of two Streptomyces strains confirmed the presence of potential epoxyketone biosynthetic gene clusters and metabolite expression and extraction showed epoxyketone production in one of the strains. Further experiments with the novel epoxyketone synthases and EpnF will reveal the full biosynthetic potential of these enzymes for the chemoenzymatic production of potential proteasome inhibitor drugs. Genetic engineering and A-domain swap in epoxyketone biosynthetic gene clusters could enable de novo construction of bacterial pathways for natural product derived epoxyketone proteasome inhibitors.
Figure 1 General reaction from α-dimethyl-β-keto acids to epoxyketone natural products catalysed by epoxyketone synthases. References 1. Lawasut et al., Curr. Hematol. Malig. Rep. , 2012, 7 , 258–266.
2. Elofsson et al., Chem. Biol. , 1999, 6 , 811–822. 3. Zhou et al., J. Med. Chem., 2009, 52 , 3028–3038. 4. Zabala et al., Am. Chem. Soc., 2016, 138 , 4342−4345.
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