Predicted Incorporation of Non‐native Substrates by a Polyketide Synthase Yields Bioactive Natural Product Derivatives

Abstract

The polyether ionophore monensin is biosynthesized by a polyketide synthase that delivers a mixture of monensins A and B by the incorporation of ethyl- or methyl-malonyl-CoA at its fifth module. Here we present the first computational model of the fifth acyltransferase domain (AT5_mon) of this polyketide synthase, thus affording an investigation of the basis of the relaxed specificity in AT5_mon, insights into the activation for the nucleophilic attack on the substrate, and prediction of the incorporation of synthetic malonic acid building blocks by this enzyme. Our predictions are supported by experimental studies, including the isolation of a predicted derivative of the monensin precursor premonensin. The incorporation of non-native building blocks was found to alter the ratio of premonensins A and B. The bioactivity of the natural product derivatives was investigated and revealed binding to prenyl-binding protein. We thus show the potential of engineered biosynthetic polyketides as a source of ligands for biological macromolecules

Flexible enzymatic activation of artificial polyketide extender units by Streptomyces cinnamonensis into the monensin biosynthetic pathway

“…a, compound 1a), as previously described (Bravo‐Rodriguez et al . ). These were two MatB*‐expressing variants and the original premonensin‐producer S. cinnamonensis A495 as control.…”

“…), premonensin (Bravo‐Rodriguez et al . ; Ismail‐Ali et al . ) and kirromycin (ethyl: kirromycin) (Musiol‐Kroll et al .…”

Recent advances in the biosynthesis of unusual polyketide synthase substrates

“…This played a pivotal role in establishing if there were any “non-native” substrates that could be recognized by the AT domain of module 5 (AT5mon), thus enabling informed decisions to be made with regards to precursor-directed biosynthesis of the monensins. 60 Various malonyl-SNAC analogues, including propargyl-SNAC and propyl-, butryl- and allylmalonyl-SNAC, were fed to a premonensin producing strain, S. cinnamonensis A495, resulting in the selective incorporation of these precursors by AT5mon adding to the series of premonensin derivatives produced ( 54 — 57 ) by the strain (Figure 8A). 60 …”

“…60 Various malonyl-SNAC analogues, including propargyl-SNAC and propyl-, butryl- and allylmalonyl-SNAC, were fed to a premonensin producing strain, S. cinnamonensis A495, resulting in the selective incorporation of these precursors by AT5mon adding to the series of premonensin derivatives produced ( 54 — 57 ) by the strain (Figure 8A). 60 …”

Harnessing natural product assembly lines: structure, promiscuity, and engineering

“…Similarly to specialized biosynthetic pathways, these strategies also offer the potential to generate polyketide analogues in situ that are modified with “click” handles. For example, a variety of extender units have been provided as the SNAC thioester and installed into several polyketide natural products by feeding these cell permeable substrates into producing organisms and utilizing inherent extender unit promiscuity of PKSs via precursor directed biosynthesis [12,117,148]. Alternatively, structure-guided mutagenesis, driven by the recently described MatB crystal structure [55], has afforded malonyl-CoA synthetase mutants with broad specificity and provide a facile route to non-natural and non-native extender units, including those modified with such chemical handles for ‘click chemistry’ ( Figure 5d ) [71,73].…”