Creating a large designer cellulosome in yeast to boost ethanol production.

Cellulosic biomass represents a promising feedstock for biofuel and biochemical production. However, its recalcitrant structure strongly hinders enzymatic degradation. Cellulosomes are large multienzyme complexes, highly efficient at degrading cellulose. A cellulase in a cellulosome has a dockerin domain that binds to a cohesin module on the CipA (cellulosome integrating protein A). In a native cellulosome all cohesins are identical, so that the cellulase types and their positions in a CipA cannot be controlled. Here, we constructed the largest designer CipA known to date. Using innovative techniques, we synthesized a designer CipA gene that encodes nine distinct cohesins and two cellulose-binding modules, which we named DCipA2B9C. Then, we fused nine distinct fungal cellulases separately with nine distinct dockerins for their precise positioning on DCipA2B9C to achieve enzyme proximity-effect. We constructed three yeast hosts to compare their performances. First, an enzyme host (EH) secretes nine dockerin-fused cellulases, including endoglucanases (EgIII-a, EgIII-m, and EgIII-c), exoglucanases (CBHII-j and EXG2-r), β-glucosidases (BGS-f and BGS-l), and cellulase boosters, including a LPMO-t and CDH-b. Second, the scaffoldin host (SH) expresses DCipA2B9C. Third, the cellulosome-9 host expresses DCipA2B9C and nine dockerin-fused cellulases. Native-PAGE and ELISA confirmed specific interactions between dockerins and cohesins. Additionally, native-PAGE, SDS-PAGE, and LC-MS verified the successful assembly of the multienzyme complex. Our performance evaluation showed that coculturing of EH and SH outperformed the cellulosome-9 host. It degraded microcrystalline cellulose efficiently to produce 14.29 g/L bioethanol, which surpassed all previously constructed yeast cellulosomes by fourfold or more. In summary, our study provides an effective approach to biomass degradation.