DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency

Abstract

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.

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

“…We hypothesize that engineered supramolecular enzyme assemblies can be similarly created as a kinetic trap to enable fast and efficient methanol utilization. Unlike other reports based on the use of a scaffold (24,28), which tend to produce large and disordered multienzyme complexes, we take advantage of the decameric nature of Mdh3 from B. methanolicus to construct a scaffoldless supramolecular enzyme complex composed of Mdh3 and a previously validated Hps-Phi fusion from Mycobacterium gastri to enhance methanol conversion to F6P. Our ability to enhance F6P synthesis is ideally suited for the recently proposed methanol condensation cycle (29), which combines the enzymes from the RuMP pathway and the nonoxidative glycolysis pathway for the in vitro synthesis of platform chemicals from methanol.…”

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…[54] Approaches using DNA or RNA scaffolds were also proven to be effective for microbial production of biomolecules. [140] Improvement of resveratrol production in yeast was achieved by the scaffold engineering. [141] In the same manner with Figure 7c, nine scaffolds with varying numbers of Src homology 3 domain (SH3) and PSD95/DlgA/Zo-1 (PDZ) domains were tested for assembling 4-coumarate: 4-coumarate-CoA ligase 1 (4CL1) and STS to convert p-coumaric acid into resveratrol.…”

Improved Production of l -Threonine in Escherichia coli by Use of a DNA Scaffold System

“…4 and 5) can be explained by the enforced proximity and substrate channeling effect facilitated by our DNA scaffold system through the following specific effects: (i) increasing the local concentration of intermediates around the enzymes on the DNA scaffold, (ii) preventing the loss of intermediates to diffusion or by competing reactions, and (iii) circumventing feedback inhibition on other pathways due to the rapid conversion of feedback inhibitors (14,18,27). Therefore, the production of L-threonine through multiple enzymatic reactions could be achieved as a single-step reaction by assembly lines on a DNA scaffold.…”

“…The number of enzymes bound to the DNA scaffold and order of enzymes can be controlled in a designable manner by simply changing the sequence of binding sites on the DNA scaffold, and the relative position of enzymes to each other and the distance between the binding sites for each enzyme might be also controllable to some extent. Through the artificial enzymatic cascade on a DNA scaffold, the metabolic efficiency of a desired pathway should be significantly improved because of the increased local concentrations of substrates around enzymes and reduced loss of intermediates to competing pathways (14,27).…”

“…Recently, Dueber et al (18) expressed scaffolds built from the interaction domains of metazoan signaling proteins to assemble metabolic enzymes and reported significant increases in the production of mevalonate and, separately, glucaric acid. Delebecque et al (26) created RNA aptamerbased scaffolds to control the spatial organization of two metabolic enzymes involved in biological hydrogen production, and Conrado et al (27) reported a DNA-based scaffold system to promote improved catalytic efficiency: application of the DNA scaffold to produce resveratrol, 1,2-propandiol, and mevalonate significantly enhanced the titer of these metabolites. These reports demonstrate the potential of scaffold systems as a powerful tool for metabolic engineering.…”