Preparation of holo- and malonyl-[acyl-carrier-protein] in a manner suitable for analog development

•We demonstrate the lab-scale production of holo-ACP and malonyl-ACP to high titers.•The ACP and ACP synthase polypeptides form a discrete oligomer.•The beta-ketoacyl synthase FabH efficiently utilize our malonyl-ACP preparation.•We describe a fluorescence-based assay to monitor FabH activity.