초록
<P><B>Abstract</B></P> <P>Lignin represents an untapped feedstock for the production of fuels and chemicals, but its intrinsic heterogeneity makes lignin valorization a significant challenge. In nature, many aerobic organisms degrade lignin-derived aromatic molecules through conserved central intermediates including catechol and protocatechuate. Harnessing this microbial approach offers potential for lignin upgrading in modern biorefineries, but significant technical development is needed to achieve this end. Catechol and protocatechuate are subjected to aromatic ring cleavage by dioxygenase enzymes that, depending on the position, <I>ortho</I> or <I>meta</I> relative to adjacent hydroxyl groups, result in different products that are metabolized through parallel pathways for entry into the TCA cycle. These degradation pathways differ in the combination of succinate, acetyl-CoA, and pyruvate produced, the reducing equivalents regenerated, and the amount of carbon emitted as CO<SUB>2</SUB>—factors that will ultimately impact the yield of the targeted product. As shown here, the ring-cleavage pathways can be interchanged with one another, and such substitutions have a predictable and substantial impact on product yield. We demonstrate that replacement of the catechol <I>ortho</I> degradation pathway endogenous to <I>Pseudomonas putida</I> KT2440 with an exogenous <I>meta</I>-cleavage pathway from <I>P. putida</I> mt-2 increases yields of pyruvate produced from aromatic molecules in engineered strains. Even more dramatically, replacing the endogenous protocatechuate <I>ortho</I> pathway with a <I>meta</I>-cleavage pathway from <I>Sphingobium</I> sp. SYK-6 results in a nearly five-fold increase in pyruvate production. We further demonstrate the aerobic conversion of pyruvate to <SMALL>L</SMALL>-lactate with a yield of 41.1±2.6% (wt/wt). Overall, this study illustrates how aromatic degradation pathways can be tuned to optimize the yield of a desired product in biological lignin upgrading.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We engineered <I>Pseudomonas putida</I> strains to degrade lignin monomers using different pathways. </LI> <LI> We compared yields of pyruvate and lactate produced from benzoate or <I>p</I>-coumarate. </LI> <LI> Catechol and PCA <I>meta</I>-cleavage pathways outperformed the <I>ortho</I>-cleavage pathways. </LI> <LI> Conversion of pyruvate to lactate resulted in greater overall product yields. </LI> <LI> Yields from the PCA 4,5 <I>meta</I>-cleavage were 75.1% of the theoretical maximum. </LI> </UL> </P>