Synthetic scaffold based on a cohesin–dockerin interaction for improved production of 2,3-butanediol in Saccharomyces cerevisiae.

Substrate channeling is a process of transferring an intermediate from one enzyme to the next enzyme without diffusion into the bulk phase, thereby leading to an enhanced reaction rate. Here, we newly designed substrate channeling modules in Saccharomyces cerevisiae based on a high affinity interaction between dockerin and cohesin domains, which is a key process in the formation of cellulosome structure. Synthetic scaffolds containing two, three, or seven cohesin domains were constructed, and the assembly of dockerin-tagged proteins onto the scaffolds was confirmed by pull-down assay and bimolecular fluorescent complementation (BiFC) assay in vivo. This system was applied to produce 2,3-butanediol in S. cerevisiae by using dockerin-tagged AlsS, AlsD, and Bdh1 enzymes, resulting in a gradual increase in 2,3-butanediol production depending on the number of cohesin domains in the scaffold.