Expanding Biosynthetic Pathways Based on Thermodynamic Preferences

Whole-cell biocatalysts have the ability to catalyze reactions at ambient temperatures and in aqueous solutions. Inside a cell, various cofactors that contain stored energy drive reactions forward. Metabolic flux toward a desired final product can drastically increased by coupling these cofactors even if the reaction is thermodynamically unfavorable at ambient temperatures. Another advantage of whole-cell biocatalysts is the ability to achieve multi-part syntheses, whereby multiple intermediates are generated in parallel in the same host and are combined into a final product. In addition, a multitude of linear biosynthetic pathways have been developed and optimized. These existing pathways as well as new pathways can be introduced in parallel into a single host to produce chemical compounds which microbes naturally produce in trace amounts or not at all. Since enzymes often have promiscuous activity, by changing the flux toward different precursors, the final product of a multi-part biosynthetic pathway can be altered in a combinatorial fashion. Here we explored the potential of multi-part synthesis in Escherichia coli by introducing parallel biosynthetic pathways. Systematic comparison of the components in each step of the biosynthetic pathway and careful matching of genes, pathway, and chemical toxicity to the chosen host enabled specific and efficient production of chemical compounds.