Establishment of Escherichia coli Strain for Production of 5-Aminolevulinic Acid through Metabolic Engineering

Abstract

Escherichia coli W3110 strain was metabolically engineered to produce5-aminolevulinic acid (ALA), C5 non-standard amino acid is widely used in agriculturaland medical industries. First, Rhodobacter sphaeroideshemA gene catalyzing thecondensation of succinyl-CoA and glycine to make ALA was codon-optimized andcloned into high copy number plasmid pKE112. Second, plasmid pKE112hemA wasintroduced into the lacI-deleted WL3110 strain; the WL3110 (pKE112hemA) strainproduced 0.249 ± 0.027 g/L of ALA. Third, in silico knock-out simulation was carriedout to identify additional gene knock-out targets to further improve ALA production.The gcvTHP genes (glycine cleavage system) were predicted as knockout targets.The JW01 strain (WL3110 ΔgcvTHP) harboring pKE112hemA produced 1.17 ± 0.035g/L of ALA, which was 4.7 times higher than that obtained with the base strain.Finally, in order to increase the succinyl-CoA pool, the glyoxylate shunt flux wasenhanced by the deletion of the iclR and sdhAB genes, while the TCA cycle fluxwas reinforced by the deletion of ptsG gene. The JW03 strain (JW01 ΔiclR ΔsdhABΔptsG) harboring pKE112hemA was able to produce 1.72 ± 0.001 g/L of ALA. Fed-batchculture of the JW03 (pKE112hemA) strain resulted in the production of 5.77 g/Lof ALA in 41 h. (Development of systems metabolic engineering platform technologiesfor biorefineries funded by the Ministryof Education, Science and Technology)