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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 as phaCAB) encodes the essential proteins for the production of P(3HB) in the native producer, Ralstonia eutropha. The three genes of the phaCAB operon are phaC, which encodes the polyhydroxyalkanoate (PHA) synthase, phaA, which encodes a 3-ketothiolase, and phaB, which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase. In this study, the effect of gene order of the phaCAB operon (phaABC, phaACB, phaBAC, phaBCA, phaCAB, and phaCBA) on an expression plasmid in genetically engineered E. 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 the pha genes relative to the promoter. The most balanced production result was achieved in the strain harboring the phaBCA 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. P olyhydroxyalkanoates (PHAs) are polyesters synthesized by more than 200 species of bacteria and are accumulated as intracellular carbon and energy storage materials (13,16,23,24). PHAs have attracted much attention as environmentally friendly plastic materials because these plastics are derived from renewable biomass resources and are completely mineralized to CO 2 and H 2 O by microorganisms in the environment after use (29).A homopolymer of (R)-3-hydroxybutyrate [P(3HB)] is the most common type of PHA that bacteria accumulate in nature, and its production has been extensively studied. Ralstonia eutropha is the most widely studied native producer of P(3HB), and in this bacterium the polymer is synthesized from acetyl coenzyme A (acetyl-CoA) by the reaction of three enzymes: 3-ketothiolase (PhaA), NADPH-dependent acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). The weight-average molecular weight (M w ) of P(3HB) produced by native PHA-producing bacteria is usually in the range of 0.2 ϫ 10 6 to 2 ϫ 10 6 (24). However, this P(3HB) is a brittle and rigid material with low flexibility because of its high crystallinity. On the other hand, recombinant Escherichia coli harboring the R. eutropha pha operon (phaC-phaAphaB) is capable of synthesizing ultrahigh-molecular-weight P(3HB) [UHMW-P(3HB)] with an M w exceeding 3 ϫ 10 6 from glucose (1,2,5,8,9,10). The higher molecular weight gives UHMW-P(3HB) higher mechanical strength, and unlike lowermolecular-weight P(3HB) polymers, UHMW-P(...