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

ABSTRACT: Carbon-negative synthesis of biochemical products has the potential to mitigate global CO₂ emissions. 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 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 d

SUBMITTER: Vogeli B 

PROVIDER: S-EPMC9160091 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature