|Ashby, Richard - Rick|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 9/1/2003
Publication Date: 11/1/2003
Citation: Solaiman, D., Ashby, R.D., Foglia, T.A., Rehm, B. 2003. Manipulation of PHA composition by genetic modification [abstract]. 1st International Conference on Biobased Polymers. Paper No. P1-3.
Technical Abstract: Poly(hydroxyalkanoates) (PHA) are produced as short-chain-length (scl; 3- to 5-carbon monomers) or medium-chain-length (mcl; 6- to 14-carbon monomers) molecular species by many organisms. Only a small number of naturally occurring bacteria, mainly Aeromonas species, can produce a limited range of PHA copolymers containing both the short- and medium-chain-length (smcl) monomers. There is a need to improve the microbial production systems for the synthesis of novel smcl-PHAs. In this presentation, we describe the construction of chimeric pha (PHA synthase) genes and the selection of their mutants capable of synthesizing mcl-PHAs having a varied monomer composition. We constructed chimeric genes pha7 and pha8 by exchanging the alpha/beta-hydrolase domain-containing segment of the phaC1 and phaC2 genes of Pseudomonas resinovorans. When expressed in Escherichia coli LS1298 (fadB), the chimeric genes mediated the production of mcl-PHAs containing 73-75% 3-hydroxydecanoate (3-HD) and 25-27% 3-hydroxyoctanoate (3-HO). Two deletion mutants (dpha7 and dpha8) of the chimeric genes were isolated and characterized. E. coli LS1298 expressing these mutants produced mcl-PHAs containing 55-60 mol% 3-HD and 40-45 mol% 3-HO. Cell growth and PHA contents of E. coli harboring phaC1, phaC2, pha7 or dpha7 were similar, suggesting that the PHA compositional variation was unlikely the results of differences in metabolic or enzymatic activity. In vitro enzyme assay study, however, failed to detect the enzymatic activity of the chimeric genes and their mutants. These results nevertheless demonstrated the feasibility of using hybrid PHA synthase genes and/or their mutants to produce PHA polymers with a varied repeat-unit composition.