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Title: EFFECT OF INACTIVATION OF POLY(HYDROXYALKANOATE) DEPOLYMERASE GENE ON THE PROPERTIES OF POLY(HYDROXYALKANOATES) IN PSEUDOMONAS RESINOVORANS

Author
item Solaiman, Daniel
item Ashby, Richard - Rick
item Foglia, Thomas

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/12/2003
Publication Date: 11/1/2003
Citation: Solaiman, D., Ashby, R.D., Foglia, T.A. 2003. Effect of inactivation of poly(hydroxyalkanoate) deploymerase gene on the properties of poly(hydroxyalkanoates) in pseudomonas resinovorans. Applied Microbiology and Biotechnology. 62:536-543.

Interpretive Summary: Poly(hydroxyalkanoates) (PHAs) are a kind of polymer produced by and stored inside bacteria. Depending on their chemical make-up, these biopolymers can have the properties of hard plastics, stretchy elastomers or sticky adhesives. PHAs are potentially useful as ecologically beneficial substitutes for the petroleum-based polymers used in many consumer products. The major factors preventing their widespread commercial applications are the cost of production and the performance properties of these materials. Controlling the activities of the genes that govern the synthesis and breakdown of PHA provides a possible route to improve the yields and thus the production cost of the polymers. We report here the construction and characterization of two modified strains (designated as FOAC001 and FOAC002) of a bacterium, called Pseudomonas resinovorans, that has a defect in the gene responsible for the breakdown of PHA. This bacterium is of interest because of its natural ability to use agricultural fats and oils to produce PHA. Our study further showed that FOAC002 is able to maintain a constant level of the polymer under a nutrient-rich growth condition in which the normal strain loses virtually all of its PHA. Furthermore, unlike the normal strain, the modified strains produce smaller size polymers than that synthesized by the normal strain. Although still unsuitable for use in bio-lubricant formulation, further work may lead to production of even smaller size polymers to satisfy the need of this particular application. The study has thus generated important information from which biotechnology for the production of a potentially useful type of PHA can be developed in cells that will not in turn degrade the product. In the long run, the consumers will benefit from the availability of ecologically sound bio-based products derived from PHA.

Technical Abstract: The phaZ gene of Pseudomonas resinovorans codes for a PHA depolymerase enzyme. Two phaZ mutants of Pseudomonas resinovorans NRRL B-2649, called FOAC001 and FOAC002, were constructed by an in vitro transposition procedure followed by chromosomal integration via the homologous recombination mechanism. A detailed mapping of the transposon insertion sites and an analysis of the resultant sequences showed that putative fusion polypeptides PhaZFOAC001 (239 amino-acid residues) and PhaZFOAC002 (85 amino-acid residues) could result from the mutant phaZ genes of FOAC001 and FOAC002, respectively. In vivo PHA degradation data indicated that PhaZFOAC001 might still retain a partial PHA depolymerization activity, while the PhaZFOAC002 is completely devoid of this function. The cell yields and PHA contents of B-2649, FOAC001 and FOAC002 were similar when the cells were grown either under a limiting nitrogen-source (low-N) condition for up to 5 days or in excess N-source (high-N) for 3 days. A dramatic decrease in PHA contents was observed in the PhaZ-active B-2649 and FOAC001 cells on a prolonged cell growth (5 days) in high-N medium or on a nitrogen-source shift-up condition. The repeat-unit compositions of the PHA's from FOAC001 and FOAC002 exhibited a slightly lower beta hydroxyoctanoate and higher beta hydroxytetradecenoate contents than that of the wild-type B-2649 when grown under a high-N condition. While the molecular masses of the PHA's from FOAC001 and FOAC002 did not vary under any conditions used in this study, those of the wild-type B-2649 were markedly increased in cells either grown for 5 days under a high-N condition or subjected to a nitrogen-source shift-up condition. These phaZ mutants thus provide a valuable system to study the influence of the PHA depolymerase on the accumulation and properties of mcl-PHA.