| Polyketides are a large class of structurally diverse natural products exhibiting a vast array of biological and pharmacological activities such as antibacterial, antifungal, anticholesterol, antiparasitic, anticancer, and immunosuppressive properties. Polyketide synthases (PKSs) assemble these natural products by condensing an initiating precursor, or starter unit, with a series of additional precursors referred to as extender units. While there are a number of starter units, there are a limited number of known extender units. These include the coenzyme A-linked extender units malonyl-CoA, (2S)-methylmalonyl-CoA, (2S)-ethylmalonyl-CoA, and chloroethylmalonyl-CoA, in addition to the acyl carrier protein (ACP)-linked extender unit (2R)-methoxymalonyl-ACP. One approach in engineering polyketide biosynthetic pathways to produce novel structural derivatives has been to modify the type of extender unit incorporated by the PKS. The incorporation of alternate extender units alters the side chains that extend from the polyketide backbone, potentially changing the interactions of the polyketide with its target. However, this approach has been limited by the relatively few number of extender units available for engineering. In addition, the side chains of the known extender units have little or no hydrogen-bonding potential and limited chemical reactivities for downstream semi-synthetic chemical modifications.;Previously, two additional extender units, (2R)-hydroxymalonyl-ACP and (2S)-aminomalonyl-ACP, were proposed as precursors in the biosynthesis of zwittermicin A, an antibiotic first identified from the biocontrol agent Bacillus cereus UW85. This work describes the in vitro formation of these extender units using heterologously purified enzymes from zwittermicin A biosynthesis. In addition, we characterize two acyltransferases involved in the specific incorporation of each of these extender units. The addition of (2R)-hydroxymalonyl-ACP and (2S)-aminomalonyl-ACP to the repertoire of extender units is promising because their incorporation confers hydrogen-bonding potential and functionalities not available through the use of the other known extender units. Furthermore, these moieties offer chemically reactive handles that can facilitate the generation of additional structural derivatives through downstream semi-synthetic chemistry. This work provides insight into the biosynthesis of an agriculturally important molecule, and it sets the stage for the metabolic engineering of other polyketide biosynthetic pathways to incorporate these unusual extender units in the generation of novel bioactive molecules. |
