Research paperDNA-guided assembly of a five-component enzyme cascade for enhanced conversion of cellulose to gluconic acid and H2O2

•Direct conversion of cellulose to gluconic acid and H2O2 using a DNA-guided five-enzyme cascade.•Enhanced H2O2 production by over 11-fold using the assembled BlgA and GOX cascade.•Enhanced H2O2 production using the assembled five-enzyme cascade.

Enzymatic fuel cells have received considerable attention because of their potential for direct conversion of abundant raw materials such as cellulose to electricity. The use of multi-enzyme cascades is particularly attractive as they offer the possibility of achieving a series of complex reactions at higher efficiencies. Here we reported the use of a DNA-guided approach to assemble a five-component enzyme cascade for direct conversion of cellulose to gluconic acid and H2O2. Site-specific co-localization of β-glucosidase and glucose oxidase resulted in over 11-fold improvement in H2O2 production from cellobiose, highlighting the benefit of substrate channeling. Although a more modest 1.5-fold improvement in H2O2 production was observed using a five-enzyme cascade, due to H2O2 inhibition on enzyme activity, these results demonstrated the possibility to enhance the production of gluconic acid and H2O2 directly from cellulose by DNA-guided enzyme assembly.