title: Preparation of holo- and malonyl-[acyl-carrier-protein] in a manner suitable for analog development.
abstract: The fatty acid biosynthetic pathway generates highly reduced carbon based molecules. For this reason fatty acid synthesis is a target of pathway engineering to produce novel specialty or commodity chemicals using renewable techniques to supplant molecules currently derived from petroleum. Malonyl-[acyl carrier protein] (malonyl-ACP) is a key metabolite in the fatty acid pathway and donates two carbon units to the growing fatty acid chain during each step of biosynthesis. Attempts to test engineered fatty acid biosynthesis enzymes in vitro will require malonyl-ACP or malonyl-ACP analogs. Malonyl-ACP is challenging to prepare due to the instability of the carboxylate leaving group and the multiple steps of post-translational modification required to activate ACP. Here we report the expression and purification of holo- and malonyl-ACP from Escherichia coli with high yields (>15 mg per L of expression). The malonyl-ACP is efficiently recognized by the E. coli keto-acyl synthase enzyme, FabH. A FabH assay using malonyl-ACP and a coumarin-based fluorescent reagent is described that provides a high throughput alternative to reported radioactive assays.
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, United States.
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Marcella
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, United States.
authors.forename: Fuyuan
authors.initials: F
authors.lastname: Jing
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, United States. Electronic address: abarb@iastate.edu.
authors.forename: Adam W
authors.initials: AW
authors.lastname: Barb
mesh_terms: D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D000213:Acyl Carrier Protein / Q000737:chemistry* / Q000378:metabolism*; D051080:Acyl-Carrier Protein S-Malonyltransferase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D054889:Fatty Acid Synthase, Type II / Q000235:genetics / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D015202:Protein Engineering
chemical_list: D000213:Acyl Carrier Protein; D029968:Escherichia coli Proteins; D000123:Acetyltransferases; C557158:fabH protein, E coli; D051080:Acyl-Carrier Protein S-Malonyltransferase; C494968:fabD protein, E coli; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D054889:Fatty Acid Synthase, Type II
keywords: Biorenewable; FabD; FabH; Fatty acid biosynthesis