초록
<P>2-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, <I>Escherichia coli</I> was metabolically engineered to produce PDC from glucose. First, an efficient biosynthetic pathway for PDC production from glucose was suggested by <I>in silico</I> 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 <I>aroE</I>)-deficient <I>E. coli</I> strain was engineered by introducing heterologous genes of different microbial origin encoding enzymes responsible for converting 3-dehydroshikimate (DHS) to PDC, which allowed <I>de novo</I> 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-<SMALL>D</SMALL>-arabino-heptulosonate-7-phosphate synthase (encoded by <I>aroG</I>) by overexpressing a feedback-resistant variant, enhancement of the precursor phosphoenolpyruvate supply by changing the native promoter of the <I>ppsA</I> gene with the strong <I>trc</I> promoter, and reducing accumulation of the major byproduct DHS by overexpression of a DHS importer (encoded by <I>shiA</I>). Furthermore, cofactor (NADP<SUP>+</SUP>/NADPH) utilization was manipulated through genetic modifications of the <I>E. coli</I> soluble pyridine nucleotide transhydrogenase (encoded by <I>sthA</I>), and the resultant impact on PDC production was investigated. Fed-batch fermentation of the final engineered <I>E. coli</I> 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.</P><BR>[FIG OMISSION]</BR>