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Ultrahigh-molecular-weight poly[(R )-3-hydroxybutyrate] [UHMW-P(3HB)] synthesized by genetically engineeredEscherichia coli is an environmentally friendly bioplastic material which can be processed into strong films or fibers. An operon of three genes (organized asphaCAB ) encodes the essential proteins for the production of P(3HB) in the native producer,Ralstonia eutropha . The three genes of thephaCAB operon arephaC , which encodes the polyhydroxyalkanoate (PHA) synthase,phaA , which encodes a 3-ketothiolase, andphaB , which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase. In this study, the effect of gene order of thephaCAB operon (phaABC ,phaACB ,phaBAC ,phaBCA ,phaCAB , andphaCBA ) on an expression plasmid in genetically engineeredE. coli was examined in order to determine the best organization to produce UHMW-P(3HB). The results showed that P(3HB) molecular weights and accumulation levels were both dependent on the order of thepha genes relative to the promoter. The most balanced production result was achieved in the strain harboring thephaBCA expression plasmid. In addition, analysis of expression levels and activity for P(3HB) biosynthesis enzymes and of P(3HB) molecular weight revealed that the concentration of active PHA synthase had a negative correlation with P(3HB) molecular weight and a positive correlation with cellular P(3HB) content. This result suggests that the level of P(3HB) synthase activity is a limiting factor for producing UHMW-P(3HB) and has a significant impact on P(3HB) production.

applied and environmental microbiology

Rearrangement of Gene Order in the <i>phaCAB</i> Operon Leads to Effective Production of Ultrahigh-Molecular-Weight Poly[( <i>R</i> )-3-Hydroxybutyrate] in Genetically Engineered Escherichia coli

Rearrangement of Gene Order in the phaCAB Operon Leads to Effective Production of Ultrahigh-Molecular-Weight Poly[( R )-3-Hydroxybutyrate] in Genetically Engineered Escherichia coli