Pho regulon promoter-mediated transcription of the key pathway gene aroGFbr improves the performance of an L-phenylalanine-producing Escherichia coli strain.
DAHP synthase (EC 4.1.2.15) is one of the key enzymes involved in aromatic amino acid biosynthesis in Escherichia coli. An approximately twofold decrease in DAHP synthase activity level was detected in the late growth phase of the L-phenylalanine (Phe)-producing E. coli strain, in which this enzyme encoded by aroG4 is resistant to feedback inhibition. An additional copy of aroG4 that is controlled by promoters of E. coli phoA or pstS genes was integrated into the chromosome of the Phe producer. The choice of promoter was based on the detected activation of the Pho regulon that occurs in response to the depletion of soluble inorganic orthophosphate (P(i)) in the medium, provided that the optical density of the Phe-producing culture did not exceed 70% of its maximum value. Pho-mediated aroG4 transcription increased both the accumulation of Phe and the level of DAHP synthase activity in the late stage of batch cultivation on glucose in P(i)-limited conditions. Disruption of rpoS led to the improved performance of a P(phoA)-aroG4 strain. The pstS promoter that is recognized by the σ(70)/σ(S)-associated core RNA polymerase resulted in the stable maintenance of DAHP synthase activity during long-drawn fed-batch cultivation of the RpoS(+) strain carrying the P(pstS)-aroG4.
Doroshenko
Vera G
VG
Ajinomoto-Genetika Research Institute, 1st Dorozhny pr-d, 1-1, Moscow, 117545, Russian Federation. Vera_Doroshenko@agri.ru
Tsyrenzhapova
Irina S
IS
Krylov
Alexander A
AA
Kiseleva
Evgeniya M
EM
Ermishev
Vladimir Yu
VY
Kazakova
Svetlana M
SM
Biryukova
Irina V
IV
Mashko
Sergey V
SV
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000235:genetics / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D055786:Gene Knockout Techniques; D033902:Periplasmic Binding Proteins / Q000235:genetics; D010649:Phenylalanine / Q000096:biosynthesis*; D028044:Phosphate-Binding Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D018085:Regulon; D012808:Sigma Factor / Q000235:genetics; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D033902:Periplasmic Binding Proteins; D028044:Phosphate-Binding Proteins; C090133:PstS protein, E coli; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria; D010649:Phenylalanine; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli