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Microbial systems are being increasingly developed as production hosts for a wide variety of chemical compounds. Broader adoption of microbial synthesis is hampered by a limited number of high-yielding natural pathways for molecules with the desired physical properties, as well as the difficulty in functionally assembling complex biosynthetic pathways in heterologous hosts. Here, we address both of these challenges by reporting the adaptation of the butanol biosynthetic pathway for the synthesis of odd-chain molecules and the development of a complementary modular toolkit to facilitate pathway construction, characterization, and optimization in engineered Escherichia coli. The modular feature of our pathway enables multientry and multiexit biosynthesis of various odd-chain compounds at high efficiency. By varying combinations of the pathway and toolkit enzymes, we demonstrate controlled production of propionate, trans-2-pentenoate, valerate, and pentanol, compounds with applications that include biofuels, antibiotics, biopolymers, and aroma chemicals. Importantly, and in contrast to a previously used method to identify limitations in heterologous amorphadiene production, our bypass strategy was effective even without the presence of freely membrane-diffusible substrates. This approach should prove useful for optimization of other pathways that use CoA-derivatized intermediates, including fatty acid β-oxidation and the mevalonate pathway for isoprenoid synthesis.

Controlled biosynthesis of odd-chain fuels and chemicals via engineered modular metabolic pathways