Engineering Pseudomonas aeruginosa for (R)-3-hydroxydecanoic acid production.

(R)-3-hydroxyalkanoic acids (R-3HAs) play a crucial role as essential chemicals serving as precursors or intermediates in the synthesis of a wide range of valuable compounds, such as pharmaceuticals, antibiotics, and food additives. Despite their significance, achieving industrial-scale production of R-3HAs, particularly medium-chain-length (mcl) R-3HAs, has been challenging due to the absence of suitable strains with efficient biosynthesis pathways. This study focuses on achieving the production of mcl R-3HA monomers by leveraging the "substrate pool" of R-3-(R-3-hydroxyalkanoyloxy) alkanoic acids (HAAs) which is synthesized by HAAs synthase RhlA. The process involved truncating the rhamnolipids synthesis pathway in Pseudomonas aeruginosa PAO1 by knocking out downstream genes rhlB and rhlC, leading to the accumulation and collection of intermediate HAAs from the culture supernatant. To enhance the production of HAAs further, a series of key genes in the β-oxidation pathway were knocked out, resulting in a titer of approximately 18 g/L. Subsequently, hydrolysis of HAAs was conducted under alkaline conditions, where the dimers could be rapidly and efficiently converted into monomers. The hydrolysis process was completed in 2.5 h at 80 °C using a 0.5 M NaOH solution. The primary hydrolysis product identified through GC-MS analysis was (R)-3-hydroxydecanoic acid (R-3HD) with a purity of 95%.