Polyketides, known for their diverse structures and potent pharmaceutical activities, underpin a wide array of clinical applications, including antibiotics, anticancer agents, and immunosuppressants. Their structural diversity arises from polyketide synthases (PKSs), modular enzymatic assembly lines that offer significant potential for genetic engineering to produce novel derivatives with improved therapeutic properties [1]. However, engineered PKSs often exhibit low activity, resulting in poor yields of the desired derivatives. In our previous work, we deactivated the reduction domain in module 4 of the monensin PKS, which enabled the detection of oxidized derivatives but revealed a biosynthetic bottleneck [2]. High intermediate accumulation suggested a substrate transfer inefficiency between modules 4 and 5, potentially linked to the substrate specificity of the ketosynthase (KS) domain in module 5.
To address this, we inspected the KS domain sequences of monensin PKS in comparison to those of other polyether-forming PKSs, identifying fingerprint motifs likely influencing substrate specificity. Guided by multiple sequence alignments, we performed site-directed mutagenesis on the KS5 domain and introduced point mutations into two engineered mutants: the KR40-null mutant (with the ketoreductase domain in module 4 deactivated) and the DH40-null mutant (with the dehydratase domain in module 4 deactivated). The same mutations were introduced in the A495-WT mutant as a control [3]. Targeted LC-MS-based metabolomics was employed to evaluate the impact on product spectrum.
Our findings highlight the critical role of KS domains in PKS assembly line efficiency. Strategic alterations at key active-site residues can improve overall productivity, modify substrate preference, or influence stereospecificity, paving the way for more efficient production of diverse polyketide derivatives.
References:
[1] Cremosnik, G. S., Liu, J., & Waldmann, H. (2020). Guided by evolution: from biology oriented synthesis to pseudo natural products. Natural Product Reports, 37(11), 1497-1510.
[2] Grote, M., Kushnir, S., Pryk, N., Möller, D., Erver, J., Ismail-Ali, A., & Schulz, F. (2019). Identification of crucial bottlenecks in engineered polyketide biosynthesis. Organic & biomolecular chemistry, 17(26), 6374-6385.
[3] Kushnir, S., Sundermann, U., Yahiaoui, S., Brockmeyer, A., Janning, P., & Schulz, F. (2012). Minimally invasive mutagenesis gives rise to a biosynthetic polyketide library. Angewandte Chemie International Edition, 51(42), 10664-10669.