2-(Furan-2-yl)-2-oxoacetic acid (2-FOA) is a critical building block for the C7 side chain of cefuroxime, a second-generation antibiotic. Traditional chemical synthesis of 2-FOA involves harsh conditions and toxic reagents, creating significant environmental challenges. Here, we developed an alternative biocatalytic approach to produce 2-FOA, beginning with the condensation of furfural and formaldehyde to form 2-furyl hydroxymethyl ketone (2-FHMK), followed by enzymatic oxidation to 2-FOA. To address the rate-limiting oxidation step, we engineered a galactose oxidase from Fusarium graminearum (FgGOase) through error-prone PCR and site-saturation mutagenesis. The optimal variant M13 exhibited 46-fold and 102-fol... More
2-(Furan-2-yl)-2-oxoacetic acid (2-FOA) is a critical building block for the C7 side chain of cefuroxime, a second-generation antibiotic. Traditional chemical synthesis of 2-FOA involves harsh conditions and toxic reagents, creating significant environmental challenges. Here, we developed an alternative biocatalytic approach to produce 2-FOA, beginning with the condensation of furfural and formaldehyde to form 2-furyl hydroxymethyl ketone (2-FHMK), followed by enzymatic oxidation to 2-FOA. To address the rate-limiting oxidation step, we engineered a galactose oxidase from Fusarium graminearum (FgGOase) through error-prone PCR and site-saturation mutagenesis. The optimal variant M13 exhibited 46-fold and 102-fold enhancements in oxidation efficiency toward 2-FHMK and the intermediate aldehyde, respectively, compared to wild-type. Molecular dynamics simulations revealed that the introduced mutations improved substrate binding through increased hydrophobic interactions, along with enhanced hydration. The engineered variant M13 was then integrated into a complete biocatalytic pathway, achieving 2-FOA production at a concentration of 14.6 g/L with 86.7% conversion. Our study addressed the inherent limitations of the FgGOase while demonstrating its potential as a model for developing efficient "through-oxidation" biocatalysts, offering a sustainable alternative biocatalytic approach to 2-FOA synthesis. It emphasised the potential of enzyme engineering in diversifying sustainable chemical processes.
