title: Tools for metabolic engineering in Escherichia coli: inactivation of panD by a point mutation.
abstract: l-Aspartate-alpha-decarboxylase (PanD) catalyzes the decarboxylation of aspartate to produce beta-alanine, a precursor of Coenzyme A (CoA). The pyruvoyl-dependent enzyme from Escherichia coli is activated by self-cleavage at serine 25 to generate a 102-residue alpha subunit with the pyruvoyl group at its N terminus and a 24-residue beta subunit with a hydroxy at its C terminus. A mutant form of the panD gene from E. coli in which serine 25 was replaced with an alanine (S25A) was constructed. Assays conducted in vitro and in vivo confirmed that the mutant version was completely inactive and was incapable of undergoing self-cleavage to generate the active form of the enzyme. The S25A panD mutant was used to replace the chromosomal copy of panD in BAP1, a strain of E. coli modified for polyketide production. Comparison of this strain with panD2 mutant strains derived from E. coli SJ16 showed an equivalent dependence on exogenous beta-alanine for growth in liquid medium. Unlike the undefined and leaky panD2 mutation, the panD S25A mutation is defined and tight. The panD S25A E. coli strain enables analysis of intracellular acyl-CoA pools in both defined and complex media and is a useful tool in metabolic engineering studies that require the manipulation of acyl-CoA pools for the heterologous production of polyketides.
authors.affiliation: Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, CA 94545, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Kennedy
authors.forename: James T
authors.initials: JT
authors.lastname: Kealey
mesh_terms: D000214:Acyl Coenzyme A / Q000096:biosynthesis* / Q000378:metabolism; D019943:Amino Acid Substitution; D004917:Erythromycin / Q000031:analogs & derivatives* / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005968:Glutamate Decarboxylase / Q000235:genetics* / Q000378:metabolism; D018942:Macrolides / Q000378:metabolism; D017354:Point Mutation; D015202:Protein Engineering; D015091:beta-Alanine / Q000378:metabolism
chemical_list: D000214:Acyl Coenzyme A; D029968:Escherichia coli Proteins; D018942:Macrolides; D015091:beta-Alanine; C034277:6-deoxyerythronolide B; D004917:Erythromycin; C019104:aspartate-alpha-decarboxylase; D005968:Glutamate Decarboxylase