The redefined role of PhaG and CoA ligases in medium-chain-length 3-hydroxy acid and polyhydroxyalkanoate production in Pseudomonas putida

Under nutrient starvation conditions, many microorganisms such as Pseudomonas putida store excess carbon in medium-chain-length polyhydroxyalkanoate ( mcl -PHA) granules. Biosynthesis of PHAs from the fatty acid biosynthetic pathway requires PhaG, which has long been thought to encode a 3-hydroxyacyl-ACP:CoA transferase, and PhaC, the PHA polymerase. Although this pathway has been extensively studied, the exact role of PhaG remains inconclusive. In this work we present in vitro biochemical and in vivo genetic evidence demonstrating that PhaG functions as a 3-hydroxyacyl-ACP thiolase, producing 3-hydroxyacids rather than 3-hydroxyacyl-CoA. We identified two CoA ligases, fadD1 and alkK , essential for conversion of mcl -3-hydroxyacids to 3-hydroxyacyl-CoA, and thus PHA production. Taken together, this redefines the PHA biosynthetic pathway to include PhaG-dependent hydrolysis of hydroxyacyl-ACP, ligation of the 3-hydroxyacid to coenzyme-A, and polymerization by PhaC. Using these insights, we engineered P. putida to produce 3.7 g/L extracellular 3-hydroxyacids, which can be used for chemical synthesis of performance-advantaged polymers.