|Ashby, Richard - Rick|
Submitted to: American Chemical Society National Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 5/20/2003
Publication Date: 8/1/2003
Citation: Solaiman, D., Ashby, R.D., Foglia, T.A. 2003. Molecular engineering in the production of poly(hydroxyalkanoates) [abstract]. American Chemical Society National Meeting. Paper No. AGFD 159.
Technical Abstract: Poly(hydroxyalkanoates) (PHAs), the biodegradable polymers synthesized by many microorganisms, have been the subject of intense studies because of their potential applications as ecologically advantageous substitutes for the petroleum-based thermoplastics and elastomers. There is a continued need to lower the production cost and to enhance the material properties of PHAs to render them more attractive to commercial adoption. Genetic construction of transgenic plants to bulk-produce PHAs could result in economical production of the polymers. An in-depth understanding of the biosynthesis and degradation pathways of PHA could greatly facilitate the construction of PHA-producing transgenic plants. We have constructed and studied chimeric genes from Pseudomonas resinovorans PHA synthase genes (phaC1 and phaC2) to investigate the structure-function relationship of PHA synthase enzymes responsible for the polymerization of PHAs. Two chimeric genes (pha7 and pha8) afforded PHA synthesis when expressed in E. coli LS1298 (fadB). The repeat-unit compositions of the resultant PHAs were similar to those of the polymers obtained with the native phaC1 and phaC2 genes. We found, however, two deletion mutants of pha7 and pha8 that were capable of synthesizing PHA with different repeat-unit compositions from those obtained from the native or parental chimeric genes. We have also constructed PHA depolymerase (PhaZ)-knockout strains of P. resinovorans to examine the role of PHA degradation activity in the production and properties of PHAs. To this end, we constructed two transposon-insertional phaZ mutants (i.e., FOAC001 and FOAC002) of P. resinovorans NRRL B-2649. We found that the PHA contents of B-2649 and FOAC001 decreased drastically when cells were grown for 5 days in a high-N medium or subjected to a nitrogen-source shift-up (NSU) condition. No such change was observed with FOAC002 in which the PHA polymerase activity was inactivated. We further observed that the molecular weights of the PHAs in 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 results demonstrated that metabolic engineering of the PHA degradation pathway could provide means to affect the accumulation and properties of the polymers.