초록
<P><B>Abstract</B></P> <P>Microbial fumarate production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here, we report a modular engineering approach that systematically removed metabolic pathway bottlenecks and led to significant titer improvements in a multi-gene fumarate metabolic pathway. On the basis of central pathway architecture, yeast fumarate biosynthesis was re-cast into three modules: reduction module, oxidation module, and byproduct module. We targeted reduction module and oxidation module to the cytoplasm and the mitochondria, respectively. Combinatorially tuning pathway efficiency by constructing protein fusions RoMDH-P160A and KGD2-SUCLG2 and optimizing metabolic balance by controlling genes <I>RoPYC</I>, <I>RoMDH-P160A</I>, <I>KGD2-SUCLG2</I> and <I>SDH1</I> expression strengths led to significantly improved fumarate production (20.46g/L). In byproduct module, synthetizing DNA-guided scaffolds and designing sRNA switchs enabled further production improvement up to 33.13g/L. These results suggest that modular pathway engineering can systematically optimize biosynthesis pathways to enable an efficient production of fumarate.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Modular pathway engineering was used to produce fumarate. </LI> <LI> Fusion proteins were constructed to improve the production of fumarate. </LI> <LI> Gene expression strengths cassettes were optimized to increase fumarate. </LI> <LI> DNA-guided scaffolds and RNA switchs were used to enhance fumarate production. </LI> <LI> The final strain TGFA091-16 could produce 33.13g/L fumarate. </LI> </UL> </P>