Hybrid Computational-Experimental Framework for Microbial Chemical Synthesis via Enzyme Channeling; Final rept

摘要

The immediate scientific objectives, which have changed significantly from our original proposal due to overlap with a pre-existing ONR YIP Award, include: (1) To engineer metabolic enzymes into functional multi- protein assemblies. We have explored the use of eukaryotic signaling scaffolds for in vivo enzyme assembly. Note: the original proposal focused on using TGase-mediated enzymatic cross-linking to accomplish enzyme assembly. The efficacy of these channels will be demonstrated for efficient metabolic conversion of renewable resources (e.g., glycerol) to 1,2-propanediol. (2) Enable combinatorial channel engineering via intracellular metabolite sensors. We have engineered a protein conformational switch based on the green fluorescent protein note: that the original proposal sought to develop RNA aptamer-based switches that we expect will dynamically respond to a broad concentration range of specific metabolites including R-1,2-PD. (3) Computational design of optimal metabolic systems. We have shown through simulation that our synthetic channels locally improve the catalytic efficiency of the 1,2-propanediol enzyme assembly compared to the unchanneled case (Conrado et al., 2007 Metab Eng). To design optimal precursor flux to the 1,2- PD channel, we will develop new network design tools that can be used to computationally develop metabolic architectures that take full advantage of engineered assemblies.