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

Abstract

ABSTRACTUltrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] [UHMW-P(3HB)] synthesized by genetically engineeredEscherichia coliis 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 thephaCABoperon 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 thephaCABoperon (phaABC,phaACB,phaBAC,phaBCA,phaCAB, andphaCBA) on an expression plasmid in genetically engineeredE. coliwas 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 thephagenes relative to the promoter. The most balanced production result was achieved in the strain harboring thephaBCAexpression 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.