|Ashby, Richard - Rick|
Submitted to: UJNR Food & Agricultural Panel Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 7/10/2010
Publication Date: 8/22/2010
Citation: Solaiman, D., Ashby, R.D. 2010. Recent Progress on the Construction and Testing of a Fusion Poly(hydroxyalkanoate) Synthase Gene. UJNR Food & Agricultural Panel Proceedings. p. 16. (Full paper published on a memory stick.)
Technical Abstract: Poly(hydroxyalkanoates) (PHAs) are biodegradable polyesters produced by some bacteria. Two genes in Allochromatium vinosum, phaE and phaC, respectively code for the two subunits of the enzyme complex, PHA synthase, which catalyzes the polymerization of precursors into PHA. We hypothesized that by constructing a translationally read-through phaEC gene that encodes a fused enzyme complex, the enzyme activity and consequently PHA productivity may be increased. Accordingly, phaEC was constructed first in E. coli using a combination of a PCR method and the conventional “cut-and-paste” approach employing restriction endonucleases and DNA ligase. The fused gene was subcloned into an expression vector, pBS29-P2, and expressed in Pseudomonas chlororaphis. PHA polymers produced using glucose or glucose plus oleic acid as substrates were analyzed by GC/MS to determine their repeat-unit compositions. The cloned phaEC gene did not affect the compositions of PHAs from glucose-grown P. chlororaphis. It did, however, cause a marked shift of repeat-unit distribution to the longer-chain fatty acids when glucose and oleic acid were simultaneously used as growth substrates.