An Orthogonal and Modular Pathway for the Efficient Synthesis of Functionalized Small Molecules

Anabolic metabolism can produce an array of small molecules, but yields and productivities are often limited by carbon and energy inefficiencies and slow kinetics. Catabolic and fermentative pathways, on the other hand, are carbon and energy efficient but support only a limited product range. To address these limitations, we engineered an orthogonal pathway for the synthesis of functionalized small molecules based on non-decarboxylative Claisen condensation reactions (and subsequent β-reductions) that uses functionalized primers and functionalized extender units and operates in an iterative manner. This carbonâ??carbon elongation mechanism was selected because of its ability to support iterative condensation reactions at high energy (ATP) efficiency, as previously demonstrated in our laboratory (Nature 476, 355-359, 2011). The orthogonality of the newly developed platform enables predictable, tunable, and programmable operation of a pathway that retains the high product diversity, modularity, and combinatorial capabilities of anabolism. Using different Ï?- and Ï?-1-functionalized primers and α-functionalized extender units in combination with various termination pathways, we engineered the synthesis of 18 products from 10 classes in Escherichia coli, including Ï?-phenylalkanoic, α,Ï?-dicarboxylic, Ï?-hydroxy, Ï?-1-oxo, Ï?-1-methyl, 2-methyl, 2-methyl-2-enolic and 2,3-dihydroxy acids, β-hydroxy-Ï?-lactones, and Ï?-1-methyl alcohols (Nature Biotechnology, 2016, 34 (5): 556-561.