Functional assembly of a multi-enzyme methanol oxidation cascade on a surface-displayed trifunctional scaffold for enhanced NADH production.

We report a simple and low-cost strategy that allows the sequential and site-specific assembly of a dehydrogenase-based multi-enzyme cascade for methanol oxidation on the yeast surface using the high-affinity interactions between three orthogonal cohesin-dockerin pairs. The multi-enzyme cascade showed 5 times higher NADH production rate than the non-complexed enzyme mixture, a result of efficient substrate channeling.