Metabolic engineering of Escherichia coli BL21 (DE3) for de novo production of l-DOPA from d-glucose

Production of l-tyrosine is gaining grounds as the market size of 3,4-dihydroxyphenyl-l-alanine (l-DOPA) is expected to increase due to increasing cases of Parkinson's disease a neurodegenerative disease. Attempts to overproduce l-tyrosine for conversion to l-DOPA has stemmed on the overexpressing of critical pathway enzymes, an introduction of feedback-resistant enzymes, and deregulation of transcriptional regulators. An E. coli BL21 (DE3) was engineered by deleting tyrR, ptsG, crr, pheA and pykF while directing carbon flow through the overexpressing of galP and glk. TktA and PpsA were also overexpressed to enhance the accumulation of E4P and PEP. Directed evolution was then applied on HpaB to optimize its activity. Three mutants, G883R, G883A, L1231M, were identified to have improved activity as compared to the wild-type hpaB showing a 3.03-, 2.9- and 2.56-fold increase in l-DOPA production respectively. The use of strain LP-8 resulted in the production of 691.24 mg/L and 25.53 g/L of l-DOPA in shake flask and 5 L bioreactor, respectively. Deletion of key enzymes to channel flux towards the shikimate pathway coupled with the overexpression of pathway enzymes enhanced the availability of l-tyrosine for L-DOPA production. Enhancing the activity of HpaB increased l-DOPA production from glucose and glycerol. This work demonstrates that increasing the availability of l-tyrosine and enhancing enzyme activity ensures maximum l-DOPA productivity.