초록
<P><B>Abstract</B></P> <P>Adipic acid is an important dicarboxylic acid mainly used for the production of nylon 6–6 fibers and resins. Previous studies focused on the biological production of adipic acid directly from different substrates, resulting in low yields and titers. In this study, a five-step reverse adipate-degradation pathway (RADP) identified in <I>Thermobifida fusca</I> has been reconstructed in <I>Escherichia coli</I> BL21 (DE3). The resulting strain (Mad136) produced 0.3 g L<SUP>−1</SUP> adipic acid with a 11.1% theoretical yield in shaken flasks, and we confirmed that the step catalyzed by 5-Carboxy-2-pentenoyl-CoA reductase (<I>Tfu_1647</I>) as the rate-limiting step of the RADP. Overexpression of <I>Tfu_1647</I> by pTrc99A carried by strain Mad146 produced with a 49.5% theoretical yield in shaken flasks. We further eliminated pathways for major metabolites competing for carbon flux by CRISPR/Cas9 and deleted the succinate-CoA ligase gene to promote accumulation of succinyl-CoA, which is the precursor for adipic acid synthesis. The final engineered strain Mad123146, which could achieve 93.1% of the theoretical yield in the shaken flask, was able to produce 68.0 g L<SUP>−1</SUP> adipic acid by fed-batch fermentation. To the best of our knowledge, these results constitute the highest adipic acid titer reported in <I>E. coli</I>.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A five-step reverse adipate-degradation pathway (RADP) was reconstructed in <I>Escherichia coli</I>. </LI> <LI> 5-Carboxy-2-pentenoyl-CoA reductase was the rate-limiting step of the RADP. </LI> <LI> Strain Mad123146 achieved 93.1% of the theoretical yield in the shaken flask. </LI> <LI> A high production of 68.0 g L<SUP>−1</SUP> adipic acid was produced by fed-batch fermentation. </LI> </UL> </P>