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Metabolic Engineering of Escherichia coli for Efficient Production of 2‑Pyrone-4,6-dicarboxylic Acid from Glucose
journal contribution
posted on 2018-08-10, 00:00 authored by Zi Wei Luo, Won Jun Kim, Sang Yup Lee2-Pyrone-4,6-dicarboxylic acid (PDC)
is a pseudoaromatic dicarboxylic acid and is a promising biobased
building block chemical that can be used to make diverse polyesters
with novel functionalities. In this study, Escherichia coli was metabolically engineered to produce PDC from glucose. First,
an efficient biosynthetic pathway for PDC production from glucose
was suggested by in silico metabolic flux simulation.
This best pathway employs a single-step biosynthetic route to protocatechuic
acid (PCA), a metabolic precursor for PDC biosynthesis. On the basis
of the selected PDC biosynthetic pathway, a shikimate dehydrogenase
(encoded by aroE)-deficient E. coli strain was engineered by introducing heterologous genes of different
microbial origin encoding enzymes responsible for converting 3-dehydroshikimate
(DHS) to PDC, which allowed de novo biosynthesis
of PDC from glucose. Next, production of PDC was further improved
by applying stepwise rational metabolic engineering strategies. These
include elimination of feedback inhibition on 3-deoxy-d-arabino-heptulosonate-7-phosphate
synthase (encoded by aroG) by overexpressing a feedback-resistant
variant, enhancement of the precursor phosphoenolpyruvate supply by
changing the native promoter of the ppsA gene with
the strong trc promoter, and reducing accumulation
of the major byproduct DHS by overexpression of a DHS importer (encoded
by shiA). Furthermore, cofactor (NADP+/NADPH) utilization was manipulated through genetic modifications
of the E. coli soluble pyridine nucleotide transhydrogenase
(encoded by sthA), and the resultant impact on PDC
production was investigated. Fed-batch fermentation of the final engineered E. coli strain allowed production of 16.72 g/L of PDC
from glucose with the yield and productivity of 0.201 g/g and 0.172
g/L/h, respectively, representing the highest PDC production performance
indices reported to date.
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biobased building block chemicalPDC biosynthetic pathwaypseudoaromatic dicarboxylic acidPDC production performance indices3- deoxy-d-arabino-heptulosonate -7-phosphate synthaseorigin encoding enzymespyridine nucleotide transhydrogenase. coliNADPEscherichia coliglucosePDC productionE . coliPCADHSNADPHprecursor phosphoenolpyruvate supplyE . coli strain
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