title: Metabolic flux between unsaturated and saturated fatty acids is controlled by the FabA:FabB ratio in the fully reconstituted fatty acid biosynthetic pathway of Escherichia coli.
abstract: The entire fatty acid biosynthetic pathway of Escherichia coli, starting from the acetyl-CoA carboxylase, has been reconstituted in vitro from 14 purified protein components. Radiotracer analysis verified stoichiometric conversion of acetyl-CoA and NAD(P)H to the free fatty acid product, allowing implementation of a facile spectrophotometric assay for kinetic analysis of this multienzyme system. At steady state, a maximal turnover rate of 0.5 s(-1) was achieved. Under optimal turnover conditions, the predominant products were C16 and C18 saturated as well as monounsaturated fatty acids. The reconstituted system allowed us to quantitatively interrogate the factors that influence metabolic flux toward unsaturated versus saturated fatty acids. In particular, the concentrations of the dehydratase FabA and the β-ketoacyl synthase FabB were found to be crucial for controlling this property. Via changes in these variables, the percentage of unsaturated fatty acid produced could be adjusted between 10 and 50% without significantly affecting the maximal turnover rate of the pathway. Our reconstituted system provides a powerful tool for understanding and engineering rate-limiting and regulatory steps in this complex and practically significant metabolic pathway.
authors.affiliation: Department of Chemistry and ‡Department of Chemical Engineering, Stanford University , Stanford, California 94305, United States.
authors.forename: Xirui
authors.initials: X
authors.lastname: Xiao
authors.forename: Xingye
authors.initials: X
authors.lastname: Yu
authors.forename: Chaitan
authors.initials: C
authors.lastname: Khosla
mesh_terms: D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000378:metabolism*; D000103:Acetyl-CoA Carboxylase / Q000032:analysis; D053898:Biosynthetic Pathways / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D054889:Fatty Acid Synthase, Type II / Q000032:analysis / Q000378:metabolism*; D005227:Fatty Acids / Q000096:biosynthesis*; D005231:Fatty Acids, Unsaturated / Q000096:biosynthesis*; D006836:Hydro-Lyases / Q000378:metabolism*; D007700:Kinetics; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
chemical_list: D029968:Escherichia coli Proteins; D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; C490369:FabB protein, E coli; D006836:Hydro-Lyases; C047118:3-hydroxyacyl-(acyl-carrier-protein) dehydratase; D054889:Fatty Acid Synthase, Type II; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein