A novel strategy for L-arginine production in engineered Escherichia coli

Abstract

Background: L-arginine is an important amino acid with applications in diverse industrial and pharmaceutical fields. N-acetylglutamate, synthesized from L-glutamate and acetyl-CoA, is a precursor of the L-arginine biosynthetic branch in microorganisms. The enzyme that produces N-acetylglutamate, N-acetylglutamate synthase, is allosterically inhibited by L-arginine. L-glutamate, as a central metabolite, provides carbon backbone for diverse biological compounds besides L-arginine. When glucose is the sole carbon source, the theoretical maximum carbon yield towards L-arginine is 96.7%, but the experimental highest yield was 51%. The gap of L-arginine yield indicates the regulation complexity of carbon flux and energy during the L-arginine biosynthesis. Besides endogenous biosynthesis, N-acetylglutamate, the key precursor of L-arginine, can be obtained by chemical acylation of L-glutamate with a high yield of 98%. To achieve high-yield production of L-arginine, we demonstrated a novel approach by directly feeding precursor N-acetylglutamate to engineered Escherichia coli.

Results: We reported a new approach for the high yield of L-arginine production in E. coli. Gene argA encoding N-acetylglutamate synthase was deleted to disable endogenous biosynthesis of N-acetylglutamate. The feasibility of external N-acetylglutamate towards L-arginine was verified via growth assay in argA^- strain. To improve L-arginine production, astA encoding arginine N-succinyltransferase, speF encoding ornithine decarboxylase, speB encoding agmatinase, and argR encoding an arginine responsive repressor protein were disrupted. Based on overexpression of argDGI, argCBH operons, encoding enzymes of the L-arginine biosynthetic pathway, ~ 4 g/L L-arginine was produced in shake flask fermentation, resulting in a yield of 0.99 mol L-arginine/mol N-acetylglutamate. This strain was further engineered for the co-production of L-arginine and pyruvate by removing genes adhE, ldhA, poxB, pflB, and aceE, encoding enzymes involved in the conversion and degradation of pyruvate. The resulting strain was shown to produce 4 g/L L-arginine and 11.3 g/L pyruvate in shake flask fermentation.

Conclusions: Here, we developed a novel approach to avoid the strict regulation of L-arginine on ArgA and overcome the metabolism complexity in the L-arginine biosynthesis pathway. We achieve a high yield of L-arginine production from N-acetylglutamate in E. coli. Co-production pyruvate and L-arginine was used as an example to increase the utilization of input carbon sources.

Keywords: Escherichia coli; L-Arginine; Metabolic engineering; N-acetylglutamate.

Fig. 1

Major metabolic pathways associated with l-arginine biosynthesis in E. coli and metabolic engineering approaches applied to overproduce l-arginine. Dotted gray arrows with indicate the deletion of the relevant genes. Black thick arrows indicate increased fluxes by directly overexpressing the corresponding genes. Black dashes indicate the negative feedback inhibition mechanisms. The role of ArgR repressor regulation on l-arginine production is shown in the gray box. Abbreviations: G6P glucose 6-phosphate, F6P fructose 6-phosphate, DHAP dihydroxyacetone phosphate, G3P glyceraldehyde 3-phosphate, n-acetyl-glu-P n-acetylglutamate-phosphate, n-acetyl-sa n-acetylglutamate-semialdehyde, CPS carbamoyl phosphate

Fig. 2

Effects of gene argA deletion on cell growth. a Growth curves of strain N1 (E. coli ΔargA) in M9 minimal medium supplemented with 5 g/L glucose (without yeast extract), -Arg means the absence of l-arginine. + Arg means the presence of l-arginine. b Growth curves of strain N1 and BW25113 in M9 minimal medium added with different concentrations of n-acetylglutamate (0 g/L, 1 g/L, 5 g/L, 10 g/L, 20 g/L). Error bars are the standard deviation for three independent experiments

Fig. 3

Effects of removing the l-arginine degradation pathway and transcriptional repressor ArgR on l-arginine production from glucose and l-ornithine. a Plasmid p-AGR-1 was constructed for l-arginine overproduction. b l-arginine production and acetate formation of strains BW25113 and N5-N8. c Cell growth of strains BW25113 and N5-N8. N5: BW25113 with empty plasmid pZE; N6: BW25113 ΔargA ΔspeF ΔspeB, overexpressionof argIGH; N7: BW25113 ΔargA ΔspeF ΔspeB ΔastA, overexpression of argIGH; N8: BW25113 ΔargA ΔspeF ΔspeB ΔastA ΔargR, overexpression of argIGH. Error bars are the standard deviation for three independent experiments

Fig. 4

Effects of enhancing the carbamoyl phosphate supply on l-arginine production from glucose and l-ornithine. a Plasmids p-AGR-1 and p-AGR-2 were constructed for l-arginine overproduction. b l-arginine production and acetate formation of strains N8-N10. c Cell growth and l-arginine yield from l-ornithine of strains N8-N10. N8: BW25113 ΔargA ΔspeF ΔspeB ΔastA ΔargR, overexpression of argIGH; N9: BW25113 ΔargA ΔspeF ΔspeB ΔastA ΔargR, overexpression of argIGH, carAB; and N10: BW25113 ΔargA ΔspeF ΔspeB ΔastA ΔargR, with empty plasmid pZE. Error bars are the standard deviation for three independent experiments

Fig. 5

Production of l-arginine in strain N11 from glucose and n-acetylglutamate. a Plasmids p-AGR-3 and p-AGR-4 were constructed for l-arginine overproduction. b l-arginine production and acetate formation of strain N11. c Cell growth of strain N11. N11: BW25113 ΔargA ΔspeF ΔspeB ΔastA ΔargR, overexpression of argCBH and argDGI. Error bars are the standard deviation for three independent experiments

Fig. 6

Comparison of l-arginine, pyruvate, acetate titers, and the cell growth in strains N11 and N12. N12: BW25113 ΔargA ΔspeF ΔspeB ΔastA ΔargR, ΔldhA ΔadhE ΔaceE ΔpoxB ΔpflB, overexpression of argCBH and argDGI. Error bars are the standard deviation for three independent experiments