초록
<P><B>Abstract</B></P> <P>4-Hydroxybenzoate (4HBA) is a valuable platform intermediate for the production of commodity and fine chemicals, including protocatechuate, <I>cis,cis</I>-muconic acid, adipic acid, terephthalic acid, phenol, vanillin, and 4-hydroxybenzyl alcohol glycoside (gastrodin). Here we describe rational engineering of the shikimate and related pathways in <I>Corynebacterium glutamicum</I> ATCC13032 for over-producing 4HBA. As an approach to increase the carbon flux to 4HBA, we first introduced a mutated chorismate-pyruvate lyase (CPL<SUP>pr</SUP>) and feedback-resistant 3-deoxy-<SMALL>D</SMALL>-arabinoheptulosonate-7-phosphate synthases encoded by <I>ubiC<SUP>pr</SUP> </I> and <I>aroF<SUP>fbr</SUP>/aroG<SUP>fbr</SUP> </I>, respectively, from <I>Escherichia coli</I> along with blockage of carbon flux to the biosynthetic pathways for aromatic amino acids and the catabolic pathway for 4HBA by deletion of the genes <I>trpE</I> (encoding anthranilate synthase I), <I>csm</I> (chorismate mutase), and <I>pobA</I> (4HBA hydroxylase). In particular, CPL<SUP>pr</SUP> less sensitive to product inhibition was incorporated into the microorganism to enhance the conversion of chorismate to 4HBA. The subsequent steps involved expression of <I>aroE</I> (shikimate kinase) and <I>aroCKB</I> in the shikimate pathway and deletion of <I>qsuABD</I> coding for enzymes involved in the quinate/shikimate degradation pathway. Finally, to reduce accumulation of pathway intermediates, shikimate and 3-dehydroshikimate, shikimate-resistant AroK from <I>Methanocaldococcus jannaschii</I> was introduced. The resulting strain was shown to produce 19.0 g/L (137.6 mM) of 4HBA with a molar yield of 9.65% after 65 h in a fed-batch fermentation. The engineered strain can also be effectively applied for the production of other products derived from the shikimate pathway.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Rational engineering of the shikimate and related pathways for 4-hydroxybenzoate production. </LI> <LI> Pathway extending into 4-hydroxybenzoate by introduction of a feedback-resistant chorismate-pyruvate lyase. </LI> <LI> Optimization of shikimate pathway established. </LI> <LI> Modulation of the quinate/shikimate degradation pathway established. </LI> <LI> Strategy to reduce accumulation of pathway intermediates generated. </LI> </UL> </P>