Abstract

Abstract Malonyl‐coenzyme A (CoA) is a key precursor for the biosynthesis of multiple value‐added compounds by microbial cell factories, including polyketides, carboxylic acids, biofuels, and polyhydroxyalkanoates. Owing to its role as a metabolic hub, malonyl‐CoA availability is limited by competition in several essential metabolic pathways. To address this limitation, we modified a genome‐reduced Pseudomonas putida strain to increase acetyl‐CoA carboxylation while limiting malonyl‐CoA utilization. Genes involved in sugar catabolism and its regulation, the tricarboxylic acid (TCA) cycle, and fatty acid biosynthesis were knocked‐out in specific combinations towards increasing the malonyl‐CoA pool. An enzyme‐coupled biosensor, based on the rppA gene, was employed to monitor malonyl‐CoA levels in vivo. RppA is a type III polyketide synthase that converts malonyl‐CoA into flaviolin, a red‐colored polyketide. We isolated strains displaying enhanced malonyl‐CoA availability via a colorimetric screening method based on the RppA‐dependent red pigmentation; direct flaviolin quantification identified four engineered strains had a significant increase in malonyl‐CoA levels. We further modified these strains by adding a non‐canonical pathway that uses malonyl‐CoA as precursor for poly(3‐hydroxybutyrate) biosynthesis. These manipulations led to increased polymer accumulation in the fully engineered strains, validating our general strategy to boost the output of malonyl‐CoA–dependent pathways in P . putida .