Production of polyhydroxyalkanoates from intact triacylglycerols by genetically engineered Pseudomonas

• Published in: Applied microbiology and biotechnology 2001-09, Vol.56 (5/6), p.664-669
• Main Authors: Solaiman, D.K.Y, Ashby, R.D, Foglia, T.A
• Format: Article
• Language: English
• Subjects: Bacteria; Biological and medical sciences; Biotechnology; Chromatography; Coconut oil; Culture Media; Fermentation; fluorescence; Fundamental and applied biological sciences. Psychology; Gas chromatography; gel chromatography; genes; genetic engineering; Genetic Engineering - methods; Genetically modified organisms; lard; Lipase - genetics; Mass spectrometry; Methods. Procedures. Technologies; Microbial engineering. Fermentation and microbial culture technology; Microbiology; mixed culture; molecular weight; plasmids; Polyesters - metabolism; Polyhydroxyalkanoates; polyhydroxyalkanoic acid; Polymers; Pseudomonas; Pseudomonas - genetics; Pseudomonas - growth & development; Pseudomonas - metabolism; Pseudomonas oleovorans; Pseudomonas putida; Pseudomonas putida - genetics; Pseudomonas putida - growth & development; Pseudomonas putida - metabolism; Recombination, Genetic; triacylglycerol; Triacylglycerols; Triglycerides - metabolism
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s002530100692

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 82-170x10(3) g/mol and 464-693x10(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.