Synthetic scaffolds that permit spatial and temporal organization of enzymes in living cells are a promising post-translational strategy for controlling the flow of information in both metabolic and signaling pathways. Here, we describe the use of plasmid DNA as a stable, robust and configurable scaffold for arranging biosynthetic enzymes in the cytoplasm of Escherichia coli. This involved conversion of individual enzymes into custom DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences. When expressed in cells that carried a rationally designed DNA scaffold comprising corresponding zinc finger binding sites, the titers of diverse metabolic products, including resveratrol, 1,2-propanediol and mevalonate were increased as a function of the scaffold architecture. These results highlight the utility of DNA scaffolds for assembling biosynthetic enzymes into functional metabolic structures. Beyond metabolism, we anticipate that DNA scaffolds may be useful in sequestering different types of enzymes for specifying the output of biological signaling pathways or for coordinating other assembly-line processes such as protein folding, degradation and post-translational modifications.

S. aureus, we examined the effects of mutations that prevent specific modifications of cell wall (dltA) and cell membrane (mprF) polyanions. In comparison to the parent strain, isogenic dltA ؊ bacteria are ϳ30 -100؋ more sensitive to PLA 2 , whereas mprF ؊ bacteria are <3-fold more sensitive. Differences in PLA 2 sensitivity of intact bacteria reflect differences in cell wall, not cell membrane, properties since protoplasts from all three strains are equally sensitive to PLA 2 . A diminished positive charge in PLA 2 reduces PLA 2 binding and antibacterial activity. In contrast, diminished cell wall negative charge by substitution of (lipo)teichoic acids with D-alanine reduces antibacterial activity of bound PLA 2 , but not initial PLA 2 binding. Therefore, the potent antistaphylococcal activity of Group IIA PLA 2 depends on cationic properties of the enzyme that promote binding to the cell wall, and polyanionic properties of cell wall (lipo)teichoic acids that promote attack of membrane phospholipids by bound PLA 2 .