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. 2017 Jan 25;6(5):793–805. doi: 10.1021/acssynbio.6b00230

Figure 5.

Figure 5

Enhanced poly(3-hydroxybutyrate) synthesis in recombinant Escherichia coli carrying Modules I and II. (a) Three enzymes are necessary for de novo synthesis of poly(3-hydroxybutyrate) (PHB). In Cupriavidus necator, from which the cognate genes were harnessed, PHB accumulation depends on the sequential activity of a 3-ketoacyl-coenzyme A (CoA) thiolase (PhaA), a NADPH-dependent 3-acetoacetyl-CoA reductase (PhaB1), and a PHB synthase (PhaC1). PhaA and PhaB1 catalyze the condensation of two molecules of acetyl-CoA to 3-acetoacetyl-CoA and the reduction of this intermediate to R-(−)-3-hydroxybutyryl-CoA (3-HB-CoA), respectively. PhaC1 polymerizes 3-HB-CoA monomers to PHB by releasing one CoA-SH molecule per monomer added. Note that acetyl-CoA can also be used in the major fermentation pathway of E. coli, that produces acetate. The main metabolic blocks within the biochemical network are identified with different colors in the outline: the Embden–Meyerhof–Parnas (EMP) pathway, red; the pentose phosphate (PP) pathway, purple; and the tricarboxylic acid (TCA) cycle and gluconeogenesis, green. Abbreviations of metabolic intermediates are as shown in the caption to Figure 1; other abbreviations are as follows: 6PG, 6-phosphogluconate; acetyl-CoA, acetyl-coenzyme A; OAA, oxaloacetate; and 2-OX, 2-oxoglutarate. (b) Glucose consumption profile and (c) PHB accumulation by E. coli BW25113 carrying plasmid pAeT41 (i.e., constitutively expressing the phaC1AB1 gene cluster from C. necator) transformed with plasmids carrying the genes or modules indicated (see Table 2 for further details). Cells were grown aerobically in LB medium added with glucose at 10 g L–1 for 24 h. Each parameter is reported as the mean value ± standard deviation from duplicate measurements in at least three independent experiments. CDW, cell dry weight. Significant differences (P < 0.05, as evaluated by means of the Student’s t test) in the pairwise comparison of a given recombinant to the control strain, carrying the empty pSEVA224 vector, are indicated by an asterisk.