Rational design of GDP-d-mannose mannosyl hydrolase for microbial l-fucose production

Backgroundl-Fucose is a rare sugar that has beneficial biological activities, and its industrial production is mainly achieved with brown algae through acidic/enzymatic fucoidan hydrolysis and a cumbersome purification process. Fucoidan is synthesized through the condensation of a key substance, guanosine 5 '-diphosphate (GDP)-l-fucose. Therefore, a more direct approach for biomanufacturing l-fucose could be the enzymatic degradation of GDP-l-fucose. However, no native enzyme is known to efficiently catalyze this reaction. Therefore, it would be a feasible solution to engineering an enzyme with similar function to hydrolyze GDP-l-fucose.ResultsHerein, we constructed a de novo l-fucose synthetic route in Bacillus subtilis by introducing heterologous GDP-l-fucose synthesis pathway and engineering GDP-mannose mannosyl hydrolase (WcaH). WcaH displays a high binding affinity but low catalytic activity for GDP-l-fucose, therefore, a substrate simulation-based structural analysis of the catalytic center was employed for the rational design and mutagenesis of selected positions on WcaH to enhance its GDP-l-fucose-splitting efficiency. Enzyme mutants were evaluated in vivo by inserting them into an artificial metabolic pathway that enabled B. subtilis to yield l-fucose. WcaH(R36Y/N38R) was found to produce 1.6 g/L l-fucose during shake-flask growth, which was 67.3% higher than that achieved by wild-type WcaH. The accumulated l-fucose concentration in a 5 L bioreactor reached 6.4 g/L.ConclusionsIn this study, we established a novel microbial engineering platform for the fermentation production of l-fucose. Additionally, we found an efficient GDP-mannose mannosyl hydrolase mutant for L-fucose biosynthesis that directly hydrolyzes GDP-l-fucose. The engineered strain system established in this study is expected to provide new solutions for l-fucose or its high value-added derivatives production.