Pho regulon promoter-mediated transcription of the key pathway gene aroG Fbr improves the performance of an l-phenylalanine-producing Escherichia coli strain

Abstract

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.

Construction of a novel phenol synthetic pathway in Escherichia coli through 4-hydroxybenzoate decarboxylation

“…The precursor for phenol production of the TPL-pathway is tyrosine. Production of tyrosine is strictly controlled by feedback inhibition of this aromatic amino acid to DAHP synthase, chorismate mutase, and prephenate dehydrogenase (Ikeda 2006;Merino et al 2008;Yakandawala et al 2008), which makes it difficult to realize a high production level (Doroshenko et al 2010;Na et al 2013). In contrast, 4-hydroxybenzonate is an intermediate of the ubiquinol biosynthetic pathway in E. coli.…”

Metabolic Engineering of a Novel Muconic Acid Biosynthesis Pathway via 4-Hydroxybenzoic Acid in Escherichia coli

“…The genes, aroF, aroH, and aroG, which code for the enzymes for this step, are feedback inhibited by the aromatic amino acids Tyr, Trp, and Phe, respectively (1,22,23). Deregulation of the feedback inhibition is essential to divert glucose to the aromatic amino acid pathway, and mutations were reported in the literature for the aroF and aroG genes that reduce or eliminate the feedback inhibition (12,24). In this study, the single amino acid mutant aroF gene, renamed as aroF FBR , was overexpressed.…”

Engineering Escherichia coli to overproduce aromatic amino acids and derived compounds

“…Introduction of plasmid-encoded copies of aroF fbr and aroG fbr combined with additional plasmid-cloned gene tktA , or their chromosomal integrations in gene clusters, have resulted in increased carbon flow from the CCM to the SHK pathway for the production of L-PHE [11],[55],[56], L-TYR [5],[57],[58] and L-TRP [59]-[61]. Positive results were also obtained with the insertion of an aroG fbr gene into the chromosome of an L-PHE producing strain while being controlled by a promoter that is active during late cultivation stages, in order to counteract the fall of DAHPS activity in stationary phase [62]. …”