Abstract

Carbon-negative synthesis of biochemical products has the potential to mitigate global CO2 emissions. An attractive route to do this is the reverse {beta}-oxidation (r-BOX) pathway coupled to the Wood-Ljungdahl pathway. Here, we optimized and implemented r-BOX for the synthesis of C4-C6 acids and alcohols. With a high-throughput in vitro prototyping workflow, we screened 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity. Implementation of these pathways into Escherichia coli generated designer strains for the selective production of butanoic acid (4.9 {+/-}0.1 gL-1), 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 generated Clostridium autoethanogenum strains able to produce 1-hexanol from syngas, achieving a titer of 0.26 gL-1 in a 1.5-L continuous fermentation. Our strategy enables optimization of rBOX derived products for biomanufacturing and industrial biotechnology.