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Abstract Carbon-negative synthesis of biochemical products has the potential to mitigate global CO₂emissions. An attractive route to do this is the reverse β-oxidation (r-BOX) pathway coupled to the Wood-Ljungdahl pathway. Here, we optimize and implement r-BOX for the synthesis of C4-C6 acids and alcohols. With a high-throughput in vitro prototyping workflow, we screen 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity. Implementation of these pathways intoEscherichia coligenerates designer strains for the selective production of butanoic acid (4.9 ± 0.1 gL⁻¹), as well as hexanoic acid (3.06 ± 0.03 gL⁻¹) and 1-hexanol (1.0 ± 0.1 gL⁻¹) at the best performance reported to date in this bacterium. We also generateClostridium autoethanogenumstrains able to produce 1-hexanol from syngas, achieving a titer of 0.26 gL⁻¹in a 1.5 L continuous fermentation. Our strategy enables optimization of r-BOX derived products for biomanufacturing and industrial biotechnology.