Redirecting metabolic fluxes through cofactor engineering: Role of CoA-esters pool during L(-)-carnitine production by Escherichia coli.
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.
Bernal
Vicente
V
Departamento de Bioquímica y Biología Molecular B e Inmunología, Facultad de Química, Universidad de Murcia, Apdo. Correos 4021, 30100 Murcia, Spain.
Masdemont
Beatriz
B
Arense
Paula
P
Cánovas
Manuel
M
Iborra
José Luis
JL
D000105:Acetyl Coenzyme A / Q000378:metabolism; D002331:Carnitine / Q000096:biosynthesis* / Q000378:metabolism; D002952:Citric Acid Cycle / Q000235:genetics; D015256:Coenzyme A-Transferases / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D009876:Operon / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*
D000105:Acetyl Coenzyme A; D015256:Coenzyme A-Transferases; D002331:Carnitine