L-Tyrosine production by deregulated strains of Escherichia coli

Abstract

The excretion of the aromatic amino acid L: -tyrosine was achieved by manipulating three gene targets in the wild-type Escherichia coli K12: The feedback-inhibition-resistant (fbr) derivatives of aroG and tyrA were expressed on a low-copy-number vector, and the TyrR-mediated regulation of the aromatic amino acid biosynthesis was eliminated by deleting the tyrR gene. The generation of this L: -tyrosine producer, strain T1, was based only on the deregulation of the aromatic amino acid biosynthesis pathway, but no structural genes in the genome were affected. A second tyrosine over-producing strain, E. coli T2, was generated considering the possible limitation of precursor substrates. To enhance the availability of the two precursor substrates phosphoenolpyruvate and erythrose-4-phosphate, the ppsA and the tktA genes were over-expressed in the strain T1 background, increasing L: -tyrosine production by 80% in 50-ml batch cultures. Fed-batch fermentations revealed that L: -tyrosine production was tightly correlated with cell growth, exhibiting the maximum productivity at the end of the exponential growth phase. The final L: -tyrosine concentrations were 3.8 g/l for E. coli T1 and 9.7 g/l for E. coli T2 with a yield of L: -tyrosine per glucose of 0.037 g/g (T1) and 0.102 g/g (T2), respectively.

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…Finally, the tyrR-knockout strain harboring feedback-resistant (fbr) aroG fbr and tyrA fbr with overexpressed ppsA and tktA produced 621 mg L −1 of tyrosine in a 50 mL batch culture. [134] The usage of feedback resistant DAHP synthase was also effective in yeast for enhanced p-coumaric acid production. [136] The authors overexpressed various feedback resistant DAHP synthases (ARO4 K229L from S. cerevisiae, aroF and two mutant variants of aroG from E. coli) and chorismate mutases (ARO7 G141S from S. cerevisiae, a chorismate mutase from C. guilliermonii, and tyrA from the E. coli).…”

“…Before the mutagenesis, transcriptional regulation was eliminated by deleting tyrR and amplifying aroG/tyrA in E. coli (Figure 10). [134] Feedback resistant tyrA and aroG mutants were also cloned and expressed. By substituting Asp146 to Asn in AroG and Met53 to Ile, Ala354 to Val in TyrA, both enzymes became feedbackresistant to tyrosine.…”

Glycolysis and Its Metabolic Engineering Applications

“…15,61 Deletion of CsrA, additional overexpression of feedbackinhibitionresistant aromatic path way enzymes like AroG fbr (DAHP synthase) and TyrA fbr (chorismate mutase/prephenate dehydrogenase), and/or deletion of transcriptional repressor gene tyrR or trpR, will further enhance the production of aro matic compounds. 60 …”

“…57,59 Overexpression of native PEP synthetase ppsA will recycle pyruvate back to PEP pool and enhance availability of PEP for aromatic pathway. 60,61 In E. coli, carbon storage regulator protein CsrA is global regulator that negatively impacts PEP synthesis by repressing pckA and ppsA while activating pykF, which channels flux away from PEP. 14,62,63 Repression or disruption of csrA will result in increased levels of PEP and increased production of aromatic amino acids.…”

Bench-top fermentative production of plant benzylisoquinoline alkaloids using a bacterial platform

“…The L-tyrosine over-producing strain was constructed by modifying the E. coli BL21(DE3) strain. It has a disrupted tyrR gene, which encodes a repressor of several genes related to the shikimic acid pathway, 7 and was modified by introduction of four overexpressed enzymes: feedback resistant (fbr) 3-deoxyd-arabino-heptulosonate-7-phosphate synthase (fbr-DAHPS: aroG fbr ), fbr-chorismate mutase/prephenate dehydrogenase (fbr-CM/ PDH: tyrA fbr ), phosphoenolpyruvate synthetase (PEPS: ppsA), and transketolase (TKT: tktA) 8,9 ( Fig. 1).…”