Substrate Channeling for the 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. In the first part, 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. In the second part, we investigated the effect of substrate channeling on the flux partitioning at a metabolic branch point, focusing on pyruvate metabolism. As a platform strain for the channeling of pyruvate flux, PYK1-Coh-Myc strain was constructed in which PYK1 gene encoding pyruvate kinase is tagged with cohesin domain. By tethering the pyruvate-forming enzyme Pyk1 to heterologous pyruvate-converting enzymes, lactate dehydrogenase and α-acetolactate synthase, using high-affinity cohesin-dockerin interaction, pyruvate flux was successfully redirected toward desired pathways producing lactic acid and 2,3-butanediol, respectively, with a concomitant decrease in ethanol production even without genetic attenuation of the ethanol-producing pathway.