Cell-free prototyping enables implementation of optimized reverse β-oxidation pathways in heterotrophic and autotrophic bacteria

An attractive route for carbon-negative synthesis of biochemical products is the reverse beta-oxidation pathway coupled to the Wood-Ljungdahl pathway. Here the authors use a high-throughput in vitro prototyping workflow to screen 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity. 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 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 into Escherichia coli generates 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 generate 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 r-BOX derived products for biomanufacturing and industrial biotechnology.

Automated summary

The study demonstrates the use of a high-throughput in vitro prototyping workflow to screen unique pathway combinations and identify enzyme sets for enhanced product selectivity. The combination of reverse beta-oxidation (r-BOX) pathway and Wood-Ljungdahl pathway allows for selective synthesis of C4-C6 acids and alcohols in Escherichia coli and Clostridium autoethanogenum.