title: Production of polyhydroxyalkanoates from intact triacylglycerols by genetically engineered Pseudomonas.
abstract: Pseudomonas putida and P oleovorans have been extensively studied for their production of medium-chain-length (mcl)-polyhydroxyalkanoates (PHA). These bacteria are incapable of metabolizing triacylglycerols (TAGs). We have constructed recombinant P. putida and P. oleovorans that can utilize TAGs as substrates for growth and mcl-PHA synthesis. A recombinant plasmid, pCN51lip-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 recombinant P. putida produced mcl-PHA at a crude yield of 0.9-1.6 g/l with lard or coconut oil (Co) as substrate. While P. oleovorans transformant did not produce mcl-PHA, a mixed-culture fermentation approach with the wild-type and recombinant strains afforded polymer production from Co at a crude yield of 0.5 g/l. Compositional analysis by gas chromatography/mass spectrometry showed that beta-hydroxyoctanoate (31-45 mol %) and beta-hydroxydecanoate (28-35 mol %) were the dominant repeat units of the TAG-based PHA. The number-average and weight-average molecular masses of the PHAs as determined by gel permeation chromatography were 82-170 x 10(3) g/mol and 464-693 x 10(3) g/mol, respectively. The recombinant approach can greatly increase the number of organisms that can be used to produce PHA from fat and oil substrates.
authors.affiliation: US Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, PA 19038, USA. dsolaiman@arserrc.gov
authors.forename: D K
authors.initials: DK
authors.lastname: Solaiman
authors.forename: R D
authors.initials: RD
authors.lastname: Ashby
authors.forename: T A
authors.initials: TA
authors.lastname: Foglia
mesh_terms: D003470:Culture Media; D005818:Genetic Engineering / Q000379:methods*; D008049:Lipase / Q000235:genetics; D011091:Polyesters / Q000378:metabolism*; D011549:Pseudomonas / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D016958:Pseudomonas putida / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D011995:Recombination, Genetic; D014280:Triglycerides / Q000378:metabolism*
chemical_list: D003470:Culture Media; D011091:Polyesters; D014280:Triglycerides; D008049:Lipase