Production of polyhydroxyalkanoates from intact triacylglycerols by genetically engineered Pseudomonas

Pseudomonas putida and P. oleovorans have been extensively studied for thei r production of medium-chain-length (mcl)-polyhydroxyalkanoates (PHA). Thes e bacteria are incapable of metabolizing triacylglycerols (TAGs). We have c onstructed recombinant P. putida and P. oleovorans that can utilize TAGs as substrates for growth and mcl-PHA synthesis. A recombinant plasmid, pCN51l ip-1, carrying Pseudomonas lipase genes was used to electrotransform these organisms. The transformants expressed TAG-hydrolyzing activity as shown by a rhodamine B fluorescence plate assay. The genetically modified organisms grew in TAG-containing medium to a cell dry weight of 2-4 g/l. The recombi nant P. putida produced mcl-PHA at a crude yield of 0.9-1.6 g/l with lard o r coconut oil (Co) as substrate. While P oleovorans transformant did not pr oduce mcl-PHA, a mixed-culture fermentation approach with the wild-type and recombinant strains afforded polymer production from Co at a crude yield o f 0.5 g/l. Compositional analysis by gas chromatography/mass spectrometry s howed that beta -hydroxyoctanoate (31-45 mol %) and beta -hydroxydecanoate (28-35 mol %) were the dominant repeat units of the TAG-based PHA. The numb er-average and weight-average molecular masses of the PHAs as determined by gel permeation chromatography were 82-170x 10(3) g/mol and 464-693x10(3) g /mol, respectively. The recombinant approach can greatly increase the numbe r of organisms that can be used to produce PHA from fat and oil substrates.