Self-Assembled Amyloid-Like Oligomeric-Cohesin Scaffoldin for Augmented Protein Display on the Saccharomyces cerevisiae Cell Surface

Abstract

In this study, a molecular self-assembly strategy to develop a novel protein scaffold for amplifying the extent and variety of proteins displayed on the surface of Saccharomyces cerevisiae is presented. The cellulosomal scaffolding protein cohesin and its upstream hydrophilic domain (HD) were genetically fused with the yeast Ure2p N-terminal fibrillogenic domain consisting of residues 1 to 80 (Ure2p 1-80 ). The resulting Ure2p 1-80 -HD-cohesin fusion protein was successfully expressed in Escherichia coli to produce self-assembled supramolecular nanofibrils that serve as a novel protein scaffold displaying multiple copies of functional cohesin domains. The amyloid-like property of the nanofibrils was confirmed via thioflavin T staining and atomic force microscopy. These cohesin nanofibrils attached themselves, via a green fluorescent protein (GFP)-dockerin fusion protein, to the cell surface of S. cerevisiae engineered to display a GFP-nanobody. The excess cohesin units on the nanofibrils provide ample sites for binding to dockerin fusion proteins, as exemplified using an mCherry-dockerin fusion protein as well as the Clostridium cellulolyticum CelA endoglucanase. More than a 24-fold increase in mCherry fluorescence and an 8-fold increase in CelA activity were noted when the cohesin nanofibril scaffold-mediated yeast display was used, compared to using yeast display with GFPcohesin that contains only a single copy of cohesin. Self-assembled supramolecular cohesin nanofibrils created by fusion with the yeast Ure2p fibrillogenic domain provide a versatile protein scaffold that expands the utility of yeast cell surface display. Yeast surface display provides a promising platform for protein engineering (9). The ability to anchor and display functional proteins on the yeast cell surface is also being exploited for developing novel whole-cell biocatalysts (11). Although whole-cell biocatalysis using yeast surface display is generally considered an established technology, challenges remain regarding the efficient display of protein complexes and the further increase in the amount of protein that can be displayed on the yeast surface without destabilizing the cell. To this end, unique properties of protein building blocks found in natural higher-order protein complexes may be exploited to create novel synthetic protein chimeras that self-assemble into nano-scaffolds on the yeast surface to display increased levels and varieties of proteins. One such protein building block is the cohesin (Coh) domain, which is found with multiple copies in cellulosomes.The cellulosome is an extracellular multienzyme complex found on the cell surface of anaerobic bacteria such as Clostridium cellulolyticum, facilitating highly efficient hydrolysis of amorphous and crystalline cellulose. It is composed of numerous cellulolytic enzymes and other functional units assembled, via highaffinity interactions between the cohesin and dockerin domains (22), on a protein scaffold (scaffoldin) that contains multiple copies of cohesin. Cellulases and r...

Tunable nano‐oleosomes derived from engineered Yarrowia lipolytica

“…E. coli BL21(DE3)TrxB was used for the production of GFP-dockerin (GD), mCherry-dockerin (MD), and the Clostridium cellulolyticum CelA endoglucanase, from pETGD, pETMD, and pJFAc (Fierobe et al, 2001;Han et al, 2012), respectively. E. coli BL21(DE3)TrxB was used for the production of GFP-dockerin (GD), mCherry-dockerin (MD), and the Clostridium cellulolyticum CelA endoglucanase, from pETGD, pETMD, and pJFAc (Fierobe et al, 2001;Han et al, 2012), respectively.…”

“…The plasmid pINA1269 and Y. lipolytica host strain Polg (Madzak et al, 2000) were used for expression of mCherry and the oleosin-fusion proteins. Growth conditions for E. coli and S. cerevisiae were described in Han et al (2012). For brevity, the GFPnanobody displaying S. cerevisiae is termed ''Yn yeast'' hereinafter.…”

“…Strains and Vectors E. coli strain DH5a was used for plasmid manipulation and propagation. E. coli BL21(DE3)TrxB was used for the production of GFP-dockerin (GD), mCherry-dockerin (MD), and the Clostridium cellulolyticum CelA endoglucanase, from pETGD, pETMD, and pJFAc (Fierobe et al, 2001;Han et al, 2012), respectively. The plasmid pINA1269 and Y. lipolytica host strain Polg (Madzak et al, 2000) were used for expression of mCherry and the oleosin-fusion proteins.…”

“…To evaluate the feasibility of this method, dockerin-tagged GFP (GD) and mCherry (MD) were mixed and incubated with the OHC-oleosomes. Further evidence of efficient co-display came from experiments that utilized the Yn yeast (Han et al, 2012). As shown in Figure 5a, GFP and mCherry fluorescence colocalized to the same oleosomes.…”

“…Excess fluorescent proteins were removed after repeated washing and centrifugation. We have shown recently that GFPnanobody can be efficiently displayed on yeast surface using the a-agglutinin system (Han et al, 2012). Larger oleosomes are more visible in the fluorescence micrograph; however, they do not reflect the average size of the oleosomes.…”