Production of Ready-To-Use Functionalized Sup35 Nanofibrils Secreted by Komagataella pastoris

Amyloid nanofibrils are excellent scaffolds for designable materials that can be endowed with biotechnologically relevant functions. However, most of all excellent ideas and concepts that have been reported in the literature might never see real-world implementation in biotechnological applications. One bottleneck is the large-scale production of these materials. In this paper, we present an attempt to create a generic and scalable platform for producing ready-to-use functionalized nanofibrils directly from a eukaryotic organism. As a model material, we assembled Sup35(1-61) amyloid nanofibrils from Saccharomyces cerevisiae decorated with the Z-domain dimer, which has a high affinity toward antibody molecules. To this end, Komagataella pastoris was engineered by inserting gene copies of Sup35(1-61) and the protein chimera Sup35(1-61)-ZZ into the genome. This strain has the capability to constantly secrete amyloidogenic proteins into the extracellular medium, where the mature functionalized fibrils form, with a production yield of 35 mg/L culture. Another striking feature of this strategy is that the separation of the fibril material from the cells requires only centrifugation and resuspension in saline water. The fast production rates, minimal hands-on time, and high stability of the assembled material are some highlights that make the direct assembly of functionalized fibrils in the extracellular medium an alternative to production methods that are not suitable for large-scale production of designed amyloids.