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.

Figure 1. The biosynthetic pathway of fatty acids and derivatives in E. coli

(A) The overall reaction scheme involving the acetyl-CoA carboxylase (ACC), the fatty acid synthase (FAS) and the thioesterase (TE). (B) Biotinylation of the biotin carboxyl carrier protein (AccB) is catalyzed by BirA. The resulting holo-AccB participates in ACC-catalyzed carboxylation of acetyl-CoA. (C) Production of saturated fatty acids from acetyl-CoA and malonyl-CoA, catalyzed by the 9-component FAS (FabA, FabB, FabD, FabF, FabG, FabH, FabI, FabZ, ACP) and the TE (TesA). (D) Unsaturated fatty acids are synthesized via the isomerization of a common intermediate, trans-2-decenoyl-ACP, in a reaction catalyzed by FabA, a dual-function isomerase and dehydratase. Subsequent elongation of the cis-3-decenoyl-ACP product traps the cis-olefin, leading to eventual release of monounsaturated fatty acids.

Figure 2. Comparison of radioactive and spectrophotometric assays in measuring activities of reconstituted fatty acid synthases

(A) Reference FAS, consisting of 1 μM FabABDFGHIZ, 10 μM holo-ACP, and 10 μM TesA. (B) Optimized FAS, consisting of 1 μM FabABDFGH, 10 μM FabIZ, 30 μM holo-ACP, and 30 μM TesA. Reaction conditions were otherwise identical: 100 μM acetyl-CoA, 500 μM malonyl-CoA, and 1.3 mM NADPH. Under optimal conditions, the initial rate of fatty acid production was missed when a radioactive assay was used. See also Table 1 for a comparison of the measured rate constants.

Figure 3. Reconstitution of acetyl-CoA carboxylase

(A) SDS-PAGE analysis of the acetyl-CoA carboxylase subunits. AccA, holo-AccB, AccC and AccD. AccB migrates around 20 kDa in a Tris-glycine SDS-PAGE system, because of its long Pro- and Ala-rich segment between residues 34 and 101. (B) Effect of acetyl-CoA concentration on the initial reaction velocity of the complete reconstituted system shown in Figure 1. Reaction condition: 30 μM AccABCD, 1 μM FabABDFGH, 10 μM FabIZ, 30 μM holo-ACP, 30 μM TesA, 15 mM bicarbonate, 1 mM ATP, 5 mM MgCl₂, and 1.3 mM NADPH. (C) Dependence of overall turnover rate on acetyl-CoA carboxylase concentration in the complete reconstituted system. Reactions contain varying concentrations of the carboxylase complex, 1 μM FabABDFGH, 10 μM FabIZ, 30 μM holo-ACP, 30 μM TesA (TE), 600 μM acetyl-CoA, 15 mM sodium bicarbonate, 1 mM ATP, 5 mM MgCl₂, and 1.3 mM NADPH.

Figure 4. Effect of ACC activity on the initial reaction velocity of the complete reconstituted system shown in Figure 1

(A) Titration of AccAD complex. (B) Titration of AccB subunit. (C) Titration of AccC subunit. Reactions contain varying concentrations of ACC subunits or complex in question, 1 μM FabABDFGH, 10 μM FabIZ, 30 μM holo-ACP, 30 μM TesA (TE), 600 μM acetyl-CoA, 15 mM sodium bicarbonate, 1 mM ATP, 5 mM MgCl₂, and 1.3 mM NADPH.

Figure 5. Kinetic properties of overall reconstituted pathway shown in Figure 1

(A) Kinetic linearity of overall reconstituted pathway. Reaction conditions: 2–10 μM AccAD, AccC, 6–30 μM holo-AccB, 0.2–1 μM FabABDFGH, 2–10 μM FabIZ, 6–30 μM holo-ACP, 6–30 μM TesA, 600 μM acetyl-CoA, 15 mM bicarbonate, 1 mM ATP, 5 mM MgCl₂, and 1.3 mM NADPH. (B) Palmitic acid product formation in 5-fold dilution system. (C) Initial reaction rates of optimized reconstituted systems with an individual enzyme concentration doubled. Experiments were performed in two-fold dilution system.

Figure 6. GC-MS and kinetic analysis of factors responsible for unsaturated fatty acid production

(A) Individual GC-MS traces correspond to the following experiments: (1) optimized system (2); optimized system without FabA; (3) optimized system without FabZ but with 10 μM FabA; (4) optimized system with 10 μM each of FabA and FabZ; (5) optimized system with 10 μM FabB; and (6) optimized system with 1 μM FabI. Labeled peaks correspond to BSTFA derivatized fatty acids produced by TesA-catalyzed hydrolysis. All other unlabeled peaks are impurities most likely derived from the plastics involved in the product extraction and storage process. (B) Relative rates of fatty acid biosynthesis under the different conditions described in part A. The graph also includes data for the synthase lacking FabZ (bar 7), which has a low but measurable turnover rate.

Figure 7. Quantitative analysis of the effect of FabB:FabA ratio on unsaturated fatty acid production

The bars correspond to the relative abundance of unsaturated fatty acid in the product mixture (left y-axis), and the black squares represent the initial turnover rate of the overall pathway (right y-axis). All data are means ± s.d. (n=3). For experimental details, see text.

Figure 8. GC-FID chromatographic analysis of product profiles under varying FabB:FabA ratios

The relevant assay conditions are: (1) 1 μM FabB, 10 μM FabA; (2) 1 μM FabB, 1 μM FabA; (3) 3 μM FabB, 1 μM FabA; (4) 5 μM FabB, 1 μM FabA; (5) 7 μM FabB, 1 μM FabA; (6) 10 μM FabB, 1 μM FabA; and (7) 10 μM FabB, 10 μM FabA. Chromatographic peaks were identified through comparison of retention time with authentic standards.