Integrated engineering of core β-oxidation reversal and termination pathways for the production of fuels and chemicals

The growing demand for the development of technologies capable of producing advanced fuels and chemicals from sustainable feedstocks has resulted in the exploitation and engineering of biological systems enabling the synthesis of longer carbon chain length compounds from 2- and 3-carbon metabolic intermediates. While several pathways have been investigated for this purpose, the recently engineered reversal of the β-oxidation cycle provides an attractive platform that can support the synthesis of short-, medium-, and long-chain products at high yields. However, fully capitalizing on the intrinsic capabilities of this carbon-carbon elongation platform requires the ability to both control and diversify product synthesis. The work presented here highlights our recent efforts in which the utility of this platform is demonstrated for the synthesis of medium-chain length (C₆-C₁₀) products by engineering key pathway components. Through the selection of an appropriate thiolase(s) and manipulation of required termination pathways for product synthesis, both product type and chain length can be refined. Furthermore, integration of ω-oxidation pathways within this framework enables increased product diversity by facilitating a route to ω-functionalized products. The combinatorial engineering of these key pathway components was exploited for the synthesis of carboxylic acids (hexanoic, octanoic, and decanoic acids), alcohols (hexanol, octanol, and decanol), ω-hydroxyacids (6-hydroxyhexanoic, 8-hydroxyoctanoic, and 10-hydroxydecanoic acids), and dicarboxylic acids (adipic, suberic, and sebacic acids). These results demonstrate the versatile nature of a β-oxidation reversal and highlight several key aspects and control points that can be further manipulated to fine-tune the synthesis of various industrially relevant fuels and chemicals.