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Poly-[(R)-3-hydroxyalkanoates] (PHAs) are biodegradable polyesters produced by diverse microbial strains and genetically modified organisms. Increasing our understanding of different metabolic pathways within PHA-producing organisms is highly desired so as to bolster PHAs as economically competitive alternatives to petroleum-based plastics. Some pseudomonads, including Pseudomonas putida, Pseudomonas aeruginosa, and Pseudomonas oleovorans, commonly biosynthesize PHA polyesters composed of side chains containing between 6-14 carbons (medium chain length or MCL PHAs) derived from intracellular fatty acid feedstocks. The metabolic link between fatty acids and PHA biosynthesis is the enzyme PhaG, which was reported to exhibit 3-hydroxyacyl-ACP:CoA transferase activity. However, recent studies have suggested PhaG can alternatively function as a 3-hydroxyacyl-ACP thioesterase to produce free 3-hydroxyfatty acids which, coupled with a 3-hydroxyfatty acid:CoA ligase (AlkK), yields the CoA-activated substrates needed for polymerization by the PHA synthase, PhaC. In this study, we hypothesize that PhaG acts preferentially as a 3- hydroxyacyl-ACP thioesterase, effectively increasing the pool of free 3-hydroxyfatty acids available for their downstream CoA activation and polymerization. To test our hypothesis we cloned phaG (PP 1408) from P. putida KT2440 into expression plasmids, and heterologously expressed/purified PhaG fused with either N or C terminal polyhistidine tags. To investigate the enzyme's activity in vitro, an N-acetylcysteamine (SNAC) thioester of rac-3-hydroxydecanoic acid was synthesized. Additionally, we are performing the enantioselective syntheses of (R)-3-hydroxydecanoic acid and its SNAC analog to carry out a thorough kinetic analysis of PhaG.

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