ABSTRACT
Designer cellulosomes (DCs) are engineered multi-enzyme complexes, comprising carbohydrate-active enzymes attached to a common backbone, the scaffoldin, via high-affinity cohesin-dockerin interactions. The use of DCs in the degradation of renewable biomass polymers is a promising approach for biorefineries. Indeed, DCs have shown significant hydrolytic activities due to the enhanced enzyme-substrate proximity and inter-enzyme synergies, but technical hurdles in DC engineering have hindered further progress towards industrial application. The challenge in DC engineering lies in the large diversity of possible building blocks and architectures, resulting in a multivariate and immense design space. Simultaneously, the precise DC composition affects many relevant parameters such as activity, stability, and manufacturability. Since protein engineers face a lack of high-throughput approaches to explore this vast design space, DC engineering may result in an unsatisfying outcome. This review provides a roadmap to guide researchers through the process of DC engineering. Each step, starting from concept to evaluation, is described and provided with its challenges, along with possible solutions, both for DCs that are assembled in vitro or are displayed on the yeast cell surface. KEY POINTS: ⢠Construction of designer cellulosomes is a multi-step process. ⢠Designer cellulosome research deals with multivariate construction challenges. ⢠Boosting designer cellulosome efficiency requires exploring a vast design space.
