Redirecting metabolic fluxes through cofactor engineering: Role of CoA-esters pool during L(-)-carnitine production by Escherichia coli |
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Authors: | Bernal Vicente Masdemont Beatriz Arense Paula Cánovas Manuel Iborra José Luis |
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Institution: | Departamento de Bioquímica y Biología Molecular B e Inmunología, Facultad de Química, Universidad de Murcia, Apdo. Correos 4021, 30100 Murcia, Spain. |
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Abstract: | Cofactor engineering, defined as the purposeful modification of the pool of intracellular cofactors, has been demonstrated to be a very suitable strategy for the improvement of L(-)-carnitine production in Escherichia coli strains. The overexpression of CaiB (CoA-transferase) and CaiC (CoA-ligase), both enzymes involved in coenzyme A transfer and substrate activation during the bioprocess, led to an increase in L(-)-carnitine production. Under optimal concentrations of inducer and fumarate (used as electron acceptors) yields reached 10- and 50-fold, respectively, that obtained for the wild type strain. However, low levels of coenzyme A limited the activity of these two enzymes since the addition of pantothenate increased production. Growth on substrates whose assimilation yields acetyl-CoA (such as acetate or pyruvate) further inhibited L(-)-carnitine production. Interestingly, control steps in the metabolism of acetyl-CoA of E. coli were detected. The glyoxylate shunt and anaplerotic pathways limit the bioprocess since strains carrying deletions of isocitrate lyase and isocitrate dehydrogenase phosphatase/kinase yielded 20-25% more L(-)-carnitine than the control. On the other hand, the deletion of phosphotransacetylase strongly inhibited the bioprocess, suggesting that an adequate flux of acetyl-CoA and the connection of the phosphoenolpyruvate-glyoxylate cycle together with the acetate metabolism are crucial for the biotransformation. |
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