|Ashby, Richard - Rick|
Submitted to: Annual Meeting and Expo of the American Oil Chemists' Society
Publication Type: Abstract Only
Publication Acceptance Date: 3/1/2004
Publication Date: 5/1/2004
Citation: Solaiman, D., Ashby, R.D., Foglia, T.A. 2004. Modulation of poly(hydroxyalkanoates) production and properties by pha depolymerase-knockout mutants of pseudomonas resinovorans [abstract]. Annual Meeting of the American Oil Chemists' Society. p. 26-27.
Technical Abstract: Poly(hydroxyalkanoates) (PHAs) are biodegradable polymers produced by many microorganisms. Pseudomonas resinovorans synthesizes a medium-chain-length (mcl-)PHA that contains as its monomers the 3-hydroxyalka(e)noates with 6- to 14-carbon chains. Mcl-PHAs have the properties of elastomers and adhesives, depending on the compositional distribution of their 3-hydroxyester monomers. To render PHAs more amenable to commercial adoption, there is a need to lower their cost and to enhance their materials properties. Metabolic engineering of the PHA biosynthesis and degradation pathways provides a means to achieve these goals. In this study, the P. resinovorans gene (phaZ; 858 bps) coding for the degradation enzyme of mcl-PHA was inactivated with the aim of improving the yields and modifying the physicochemical properties of the polymer. Accordingly, we constructed two transposon-insertional phaZ mutants (i.e., FOAC001 and FOAC002) of P. resinovorans NRRL B-2649. We then mapped the transposon insertion-sites by sequence determination to nucleotide numbers 664 and 163 of the phaZ gene in FOAC001 and FOAC002, respectively. We further predicted two putative fusion polypeptides [i.e., PhaZ-FOAC001 (239 amino acid residues) and PhaZ-FOAC002 (85 a.a. residues)] by analyzing the sequence data. In vivo studies indicated that PhaZ-FOAC001 is partially active and PhaZ-FOAC002 is inactive. When grown under a limiting nitrogen-source (low-N) condition for up to 5-days or in excess N-source (high-N) for 3 days, P. resinovorans B-2649, FOAC001 and FOAC002 gave similar cell and PHA yields. The PHA contents of B-2649 and FOAC001, however, decreased drastically when cells were grown for 5 days in a high-N medium or subjected to a nitrogen-source shift-up (NSU) condition. Repeat-unit compositional analyses showed that the PHAs of FOAC001 and FOAC002 had slightly lower beta hydroxyoctanoate and higher beta hydroxytetradecenoate contents than those of the wild-type B-2649 but only when grown under a high-N condition. We also found that the molecular masses for the PHAs of FOAC001 and FOAC002 did not vary under all growth conditions used in this study. B-2649, however, produced higher molecular weight PHAs when grown for 5 days under high-N conditions or subjected to an NSU condition. These phaZ mutants provide valuable systems to study how metabolic engineering of the PHA degradation pathway could affect the accumulation and properties of mcl-PHA.