초록
<P><B>Abstract</B></P> <P>The L-tryptophan (Trp) biosynthesis pathway is highly regulated at multiple levels. The three types of regulations identified so far, namely repression, attenuation, and feedback inhibition have greatly impacted our understanding and engineering of cellular metabolism. In this study, feed-forward regulation is discovered as a novel regulation of this pathway and explored for engineering <I>Escherichia coli</I> for more efficient Trp biosynthesis. Specifically, indole glycerol phosphate synthase (IGPS) of the multifunctional enzyme TrpC from <I>E. coli</I> is found to be feed-forward inhibited by anthranilate noncompetitively. Surprisingly, IGPS of TrpC from both <I>Saccharomyces cerevisiae</I> and <I>Aspergillus niger</I> was found to be feed-forward activated, for which the glutamine aminotransferase domain is essential. The anthranilate binding site of IGPS from <I>E. coli</I> is identified and mutated, resulting in more tolerant variants for improved Trp biosynthesis. Furthermore, expressing the anthranilate-activated TrpC from <I>A. niger</I> in a previously engineered Trp producing <I>E. coli</I> strain S028 made the strain more robust in growth and more efficient in Trp production in bioreactor. It not only increased the Trp concentration from 19 to 29 g/L within 42 h, but also improved the maximum Trp yield from 0.15 to 0.18 g/g in simple fed-batch fermentations, setting a new level to rationally designed Trp producing strains. The findings are of fundamental interest for understanding and re-designing dynamics and control of metabolic pathways in general and provide a novel target and solution to engineering of <I>E. coli</I> for efficient Trp production particularly.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Discovery of a novel feed-forward regulation in tryptophan biosynthesis. </LI> <LI> IGPS in the multifunctional enzyme TrpC can be inhibited or activated by anthranilate. </LI> <LI> Anthranilate-activated TrpC significantly increases Trp productivity and yield. </LI> <LI> Multiplicity experimentally observed for the first time and overcome by proper design. </LI> </UL> </P>