Biocatalytic Synthesis of 4-Hydroxyvaleric Acid from Levulinic Acid by Engineered 3-Hydroxybutyrate Dehydrogenase from Alcaligenes faecalis

Enzymatic conversion of levulinic acid (LA) to 4-hydroxyvaleric acid (4HV) was performed by an engineered oxidoreductase. LA, the reactant of this study, is a versatile chemical building block produced from cellulosic biomass, and can be converted into valuable chemicals and fuels. 4HV, the product of this study, can be used as a monomer of bio-polyester and a precursor of bio-fuels. Since there is no enzyme reported in nature that shows substantial catalytic activity toward LA, engineering of the substrate specificity of an enzyme is required to yield the enzymatic reaction. 3-Hydroxybutyrate dehydrogenase (3HBDH) from Alcaligenes faecalis was selected as a target enzyme for the engineering. A rational design approach was applied for the engineering of the substrate specificity of the enzyme. The substrate specificity of the enzyme was engineered via the mutational studies, and a double mutant exhibiting 33-fold improved catalytic activity toward LA was obtained as a best mutant with ~99% enzymatic conversion of LA to 4HV.