Conversion of acetate and glyoxylate to fumarate by a cell-free synthetic enzymatic biosystem

Abstract

Fumarate is a value-added chemical that is widely used in food, medicine, material, and agriculture industries. With the rising attention to the demand for fumarate and sustainable development, many novel alternative ways that can replace the traditional petrochemical routes emerged. The in vitro cell-free multi-enzyme catalysis is an effective method to produce high value chemicals. In this study, a multi-enzyme catalytic pathway comprising three enzymes for fumarate production from low-cost substrates acetate and glyoxylate was designed. The acetyl-CoA synthase, malate synthase, and fumarase from Escherichia coli were selected and the coenzyme A achieved recyclable. The enzymatic properties and optimization of reaction system were investigated, reaching a fumarate yield of 0.34 mM with a conversion rate of 34% after 20 h of reaction. We proposed and realized the conversion of acetate and glyoxylate to fumarate in vitro using a cell-free multi-enzyme catalytic system, thus providing an alternative approach for the production of fumarate.

Keywords: Acetate; Cell-free; Fumarate; Glyoxylate; Multi-enzyme catalysis.

Fig. 1

(A) Schematic illustration of the in vitro multi-enzyme catalytic pathway that converts acetate and glyoxylate to fumarate. (B) Fumarate production under initial conditions. The reaction mixture contained 0.01 U/mL Acs, 0.01 U/mL GlcB, 0.2 U/mL FumC, 10 mM MgCl₂, 0.2 mM CoA, 1 mM ATP, 1 mM acetate, and 1 mM glyoxylate.

Fig. 2

Optimization of acetyl-CoA synthesis-related enzymes for higher production of fumarate. (A), Effect of Acs concentration from 0 to 0.05 U/mL on fumarate production. (B), Effect of AckA concentration from 0 to 0.10 U/mL on fumarate production. (C), Effect of Pta concentration from 0 to 0.010 U/mL on fumarate production. (D) The comparison of two acetyl-CoA synthesis pathways for fumarate production. The points in the red circle indicate the optimal condition for each parameter. The other components in the reaction mixture were as follows, 0.01 U/mL GlcB, 0.2 U/mL FumC, 10 mM MgCl₂, 0.2 mM CoA, 1 mM ATP, 1 mM acetate, and 1 mM glyoxylate. The reaction time was 16 h.

Fig. 3

Optimization of fumarate synthesis-related enzymes for higher production of fumarate. (A), Effect of GlcB concentration from 0 to 0.020 U/mL on fumarate production. (B), Effect of FumC concentration from 0 to 0.6 U/mL on fumarate production. The points in the red circle indicate the optimal condition for each parameter. The other components in the reaction mixture were as follows, 0.02 U/mL Acs, 10 mM MgCl₂, 0.2 mM CoA, 1 mM ATP, 1 mM acetate, and 1 mM glyoxylate. The reaction time was 16 h.

Fig. 4

Optimization of other components for higher production of fumarate. (A), Effect of CoA concentration from 0 to 0.4 mM on fumarate production. (B) Effect, of ATP concentration from 0 to 5 mM on fumarate production. (C), Effect of Tris-HCl concentration from 50 to 250 mM on fumarate production. (D), Effect of MgCl₂ concentration from 1 to 10 mM on fumarate production. The reaction mixture contained 0.02 U/mL Acs, 0.01 U/mL GlcB, and 0.4 U/mL FumC. Both acetate and glyoxylate were added at the concentration of 1 mM.

Fig. 5

Fumarate production under optimized conditions. (A), Comparison of initial and optimized conditions for fumarate production. (B), Effect of substrate concentration on the conversion rate.