초록
<P>Fumarate is a well-known biomass building block compound. However, the poor catalytic efficiency of fumarase is one of the major factors preventing its widespread production. To address this issue, we selected residues <SUP>159</SUP>HPND<SUP>162</SUP> of fumarase from <I>Rhizopus oryzae</I> as targets for site-directed mutagenesis based on molecular docking analysis. Twelve mutants were generated and characterized in detail. Kinetic studies showed that the <I>K</I><SUB><I>m</I></SUB> values of the P160A, P160T, P160H, N161E, and D162W mutants were decreased, whereas <I>K</I><SUB><I>m</I></SUB> values of H159Y, H159V, H159S, N161R, N161F, D162K, and D162M mutants were increased. In addition, all mutants displayed decreased catalytic efficiency except for the P160A mutant, whose <I>k</I><SUB><I>cat</I></SUB>/<I>K</I><SUB><I>m</I></SUB> was increased by 33.2%. Moreover, by overexpressing the P160A mutant, the engineered strain T.G-PMS-P160A was able to produce 5.2 g/L fumarate. To further enhance fumarate production, the acid tolerance of T.G-PMS-P160A was improved by deleting <I>ade12</I>, a component of the purine nucleotide cycle, and the resulting strain T.G(△ade12)-PMS-P160A produced 9.2 g/L fumarate. The strategy generated in this study opens up new avenues for pathway optimization and efficient production of natural products.</P>