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Advanced (long-chain) fuels and chemicals are generated from short-chain metabolic intermediates through pathways that require carbon-chain elongation. The condensation reactions mediating this carbon-carbon bond formation can be catalysed by enzymes [...]

The fermentative metabolism of D-glucuronic acid (glucuronate) in Escherichia coil was investigated with emphasis on the dissimilation of pyruvate via pyruvate formate-lyase (PFL) and pyruvate dehydrogenase (PDH). In silico and in vivo metabolic flux analysis (MFA) revealed that PFL and PDH share the dissimilation of pyruvate in wild-type MG1655. Surprisingly, it was found that PDH supports fermentative growth on glucuronate in the absence of PFL. The PDH-deficient strain (Pdh--) exhibited a slower transition into the exponential phase and a decrease in specific rates of growth and glucuronate utilization. Moreover, a significant redistribution of metabolic fluxes was found in PDH- and PFL-deficient strains. Since no role had been proposed for PDH in the fermentative metabolism of E. coli, the metabolic differences between MG1655 and Pdh--were further investigated. An increase in the oxidative pentose phosphate pathway (ox-PPP) flux was observed in response to PDH deficiency. A comparison of the ox-PPP and PDH pathways led to the hypothesis that the role of PDH is the supply of reducing equivalents. The finding that a PDH deficiency lowers the NADH : [NAD.sup.+] ratio supported the proposed role of PDH. Moreover, the NADH : [NAD.sup.+] ratio in a strain deficient in both PDH and the ox-PPP (Pdh--Zwf--) was even lower than that observed for Pdh--. Strain Pdh--Zwf--also exhibited a slower transition into the exponential phase and a lower growth rate than Pdh--. Finally, a transhydrogenase-deficient strain grew more slowly than wild-type but did not show the slower transition into the exponential phase characteristic of Pdh--mutants. It is proposed that PDH fulfils two metabolic functions. First, by creating the appropriate internal redox state (i.e. appropriate NADH : [NAD.sup.+] ratio), PDH ensures the functioning of the glucuronate utilization pathway. Secondly, the NADH generated by PDH can be converted to NADPH by the action of transhydrogenases, thus serving as biosynthetic reducing power in the synthesis of building blocks and macromolecules. DOI 10.1099/mic.0.036251-0

Escherichia coli was engineered for the efficient conversion of glycerol to D-lactic acid (D-lactate) and understand the pathways mediating the metabolism of glycerol in wild-type strains. The strain exhibited maximum volumetric and specific productivities for D-lactate production and the engineered homolactic route was observed to generate 1 to 2 mol of ATP per mol of D-lactate and is redox balanced and hence represents a viable metabolic pathway.