Abstract

Metabolic models and in silico experiments are good approaches with a growing use for predicting cell behavior. Escherichia coli does not accumulate naturally polyhydroxyalkanoates (PHA), however PHA with different monomer composition have been produced by recombinant strains. In this work, an Escherichia coli metabolic model was applied to elementary mode analyses of PHA production evaluating the impact of changes in co-factor specificity of glucose 6-phosphate dehydrogenase (G6PDH) and glyceraldehyde 3-phosphate dehydrogenase. It was evaluated the replacement of the natural NADP-dependent G6PDH by an NAD-dependent or an enzyme presenting dual co-factor specificity. The replacement of NAD-dependent G3PDH by the counterpart non-phosphorilating NADP-dependent was evaluate as well as the co-expression of both kinds of G3PDH. The maximum theoretical 3HA yield from glucose estimated by the model for 3-hydroxyubutyrate (3HB), 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD) and 3-hydroxydodecanoate (3HDd) were, respectively, 0.92, 0.57, 0.41, 0.32 and 0.27 mol/mol in the wild type (NADP-dependent G6PDH/NAD-dependent G3PDH). Smaller values of 3HA yield from glucose were predicted by the expression of a NAD-dependent G6PDH or the enzyme presenting the dual specificity. The expression of a non-phosphorilating NADP-dependent would lead to an improvement only of the 3HHx yield from glucose, reaching 0.67 mol/mol, but not in the yield of other monomers. The expression of both G3PDH enzymes resulted in a 3HB yield from glucose of 1.00 mol/mol based on equal activity of each enzyme. Improvements of the 3HO, 3HD or 3HDd yield from glucose were also detected by the expression of both G3PDH, however a peculiar cyclic conversion of glyceraldehyde 3-phosphate to 3-phosphoglycerate (by NADP-dependent G3PDH) and 3-phosphoglycerate to glyceraldehyde 3-phosphate (by phosphoglycerate mutase and NAD-dependent G3PDH). These cyclic reactions mimic the PntAB transhydrogenase action in a similar way as the cyclic Entner-Doudoroff pathway in Burkholderia or Pseudomonas. It was not predicted yield improvements by the co-expression of G6PDH presenting dual specificity, NAD-dependent G3PHD and non-phosphorilating NADP-dependent G3PDH.