title: Expression of an acetyl-CoA synthase and a CoA-transferase in Escherichia coli to produce modified taxanes in vivo.
abstract: Previous in vitro studies revealed that the 10-deacetylbaccatin III 10beta-O-acetyltransferase (DBAT) from Taxus can catalyze the transfer of acetyl, propionyl or n-butyryl from CoA to the C10-hydroxyl of 10-deacetylbaccatin III. Accordingly, Escherichia coli JM109 were transformed to recombinantly express dbat, and this enzyme function was coupled to that of acetyl-CoA synthase (acs, EC 6.2.1.1) expressed from and regulated by genes encoded on the bacterial chromosome. Incubation of the bacteria with 10-deacetylbaccatin III and increasing concentrations of acetic acid revealed an in vivo conversion ( approximately 10%) of substrate to natural product baccatin III (C10-acetylated), which was remarkably similar to the relative conversion without acid supplementation. Incubation of the modified E. coli with 5 mM propionic acid, revealed a fivefold increase in the conversion ( approximately 13%) of 10-deacetylbaccatin III to 10-deacetyl-10-propionylbaccatin III, compared to approximately 2% conversion in the absence of exogenous propionate. To produce the butyrylbaccatin III analog in vivo, bacteria were engineered to co-express the dbat and atoAD (EC 2.8.3.8) genes; the latter encodes an acetoacetate: acetyl-CoA CoA-transferase that activates butyrate to butyryl CoA. The bacteria were incubated with 10-deacetylbaccatin III and 25-100 mM butyrate, and a maximum of approximately 2.6% conversion to 10-butyrylbaccatin III was observed compared to approximately 0.6% conversion when no exogenous butyrate was supplied.
authors.affiliation: Departments of Chemistry, Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA.
authors.forename: Catherine
authors.initials: C
authors.lastname: Loncaric
authors.forename: Amanda F
authors.initials: AF
authors.lastname: Ward
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Walker
mesh_terms: D000470:Alkaloids / Q000737:chemistry / Q000378:metabolism; D002087:Butyrates / Q000737:chemistry / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism*; D015256:Coenzyme A-Transferases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015394:Molecular Structure; D013379:Substrate Specificity; D043823:Taxoids / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000470:Alkaloids; D002087:Butyrates; D029968:Escherichia coli Proteins; D043823:Taxoids; C073950:baccatin III; C042213:10-deacetylbaccatine III; D015256:Coenzyme A-Transferases; D003066:Coenzyme A Ligases