Metabolic engineering for efficient supply of acetyl-CoA from different carbon sources in Escherichia coli

Abstract

Background: Acetyl-CoA is an important metabolic intermediate and serves as an acetylation precursor for the biosynthesis of various value-added acetyl-chemicals. Acetyl-CoA can be produced from glucose, acetate, or fatty acids via metabolic pathways in Escherichia coli. Although glucose is an efficient carbon source for acetyl-CoA production, the pathway from acetate to acetyl-CoA is the shortest and fatty acids can produce acetyl-CoA through fatty acid oxidation along with abundant NADH and FADH₂. In this study, metabolically engineered E. coli strains for efficiently supplying acetyl-CoA from glucose, acetate, and fatty acid were constructed and applied in one-step biosynthesis of N-acetylglutamate (NAG) from glutamate and acetyl-CoA.

Results: A metabolically engineered E. coli strain for NAG production was constructed by overexpressing N-acetylglutamate synthase from Kitasatospora setae in E. coli BW25113 with argB and argA knockout. The strain was further engineered to utilize glucose, acetate, and fatty acid to produce acetyl-CoA. When glucose was used as a carbon source, the combined mutants of ∆ptsG::glk, ∆galR::zglf, ∆poxB::acs, ∆ldhA, and ∆pta were more efficient for supplying acetyl-CoA. The acetyl-CoA synthetase (ACS) pathway and acetate kinase-phosphate acetyltransferase (ACK-PTA) pathway from acetate to acetyl-CoA were investigated, and the ACK-PTA pathway showed to be more efficient for supplying acetyl-CoA. When fatty acid was used as a carbon source, acetyl-CoA supply was improved by deletion of fadR and constitutive expression of fadD under the strong promoter CPA1. Comparison of acetyl-CoA supply from glucose, acetate and palmitic acid revealed that a higher conversion rate of glutamate (98.2%) and productivity (an average of 6.25 mmol/L/h) were obtained when using glucose as a carbon source. The results also demonstrated the great potential of acetate and fatty acid to supply acetyl-CoA, as the molar conversion rate of glutamate was more than 80%.

Conclusions: Metabolically engineered E. coli strains were developed for NAG production. The metabolic pathways of acetyl-CoA from glucose, acetate, or fatty acid were optimized for efficient acetyl-CoA supply to enhance NAG production. The metabolic strategies for efficient acetyl-CoA supply used in this study can be exploited for other chemicals that use acetyl-CoA as a precursor or when acetylation is involved.

Keywords: Acetate; Acetyl-CoA; Fatty acid; Glucose; N-Acetylglutamate.

Fig. 1

Overview of acetyl-CoA metabolism in Escherichia coli and the biosynthesis pathway of NAG

Fig. 2

Effects of N-acetylglutamate synthase (NAGS) from different species on NAG production. a Effects of NAGS reported in references on NAG production. b Effects of NAGS not previously reported on NAG production. Ec = E. coli, Pa = Pseudomonas aeruginosa, Xc = Xanthomonas campestris, Cg = Corynebacterium glutamicum, Sc = Streptomyces coelicolor, Mt = Mycobacterium tuberculosis, Tt = Thermus thermophilus, Mr = Meiothermus ruber, Ks = Kitasatospora setae, Dd = Deinococcus deserti

Fig. 3

Production of NAG by engineered strain from glutamate and glucose. Engineered host strains transformed with plasmid pNAG19 were induced and suspended in a reaction mixture containing 50 mM sodium glutamate and 50 mM glucose. The bioconversion reactions were performed at 37 °C and 200 rpm for 8 h

Fig. 4

Production of NAG by engineered strain from glutamate and acetate. The bioconversion medium used here contained 50 mM sodium glutamate and 100 mM sodium acetate. The bioconversion reactions were performed at 37 °C and 200 rpm. H₂SO₄ was added at different reaction times to adjust the pH value

Fig. 5

Production of NAG by engineered strain from glutamate and fatty acid. Concentrations of 50 mM sodium glutamate and 15 mM palmitic acid were used for NAG production. The bioconversion reactions were performed at 37 °C and 200 rpm

Fig. 6

Comparison of glucose, acetate, and fatty acid as source of acetyl-CoA. The reaction mixture containing 50 mM sodium glutamate and 1 × M9 salts buffer were used. a Glucose (80 mM) was supplemented at rates of 20, 20, 20, 10, and 10 mM at 0, 1, 2, 4, and 6 h of bioconversion. b Acetate (160 mM) was supplemented at rates of 30, 30, 30, 30, 20, and 20 mM at 0, 2, 4, 6, 8, and 10 h of bioconversion. Additionally, 2 mM glucose was added at 0 h of bioconversion to supply ATP for the acetylation of acetate. c Palmitic acid (20 mM) was supplemented at 5 mM at each addition at 0, 3, 6, and 9 h of bioconversion

Fig. 7

Bioconversion for NAG by engineered strain 0419. a Scale-up bioconversion of NAG production in a 1-L fermenter. The black square indicates the concentration of NAG. The hollow circle represents the concentration of glutamate. The black circle represents the concentration of glucose consumed. b Substrate inhibition. The concentrations of glutamate used were: 1: 50 mM; 2: 100 mM; 3: 200 mM; 4: 500 mM; 5: 1 M. The glucose concentration was 50 mM. The reaction was carried out for 2 h. c Product inhibition. The concentration of NAG at 0 h represented the initially added concentration. In detail, the concentrations of NAG added initially were: 1: 0 mM; 2: 20 mM; 3: 40 mM; 4: 70 mM; 5: 100 mM. The bioconversion reactions were performed at 37 °C and 200 rpm for 2 h