Sporobeads: The Utilization of the Bacillus subtilis Endospore Crust as a Protein Display Platform

Abstract

Upon starvation, the soil bacterium Bacillus subtilis forms an intracellular, metabolically inactive endospore. Its core contains the DNA, encased by three protein layers protecting it against a multitude of environmental threats. The outermost layer, the crust, harbors great potential as a protein-displaying platform: a gene of interest can be translationally fused to a crust protein gene, resulting in endospores displaying the desired protein on their surface. To unlock this potential in a standardized fashion, we designed a suite of 12 vectors (Sporovectors), based on the BioBrick cloning standard. With these vectors, proteins can easily be fused N- or C-terminally to the six crust proteins CotV, CotW, CotX, CotY, CotZ, and CgeA under the control of the strongest crust gene promoter P. All Sporovectors were evaluated with GFP and two different laccases. On the basis of our data, CotY and CotZ represent the best anchor proteins. But there are significant differences in activity and functional stability between the two tested laccases. Our vector suite is a powerful tool to generate and evaluate a vast variety of functionalized endospores. It allows quickly identifying the best anchor and fusion site for the protein of interest. Our findings demonstrate that the crust of B. subtilis endospores is an inexpensive and easy platform for displaying different proteins of interest.

Catalytic Kinetics Considerations and Molecular Tools for the Design of Multienzymatic Cascade Nanoreactors

“…Several vectors for the fusion of proteins to B. subtilis coat proteins have been designed and used for the analysis of the effect of the position in N-or C-terminal involved in the fusion of proteins and as well divers promoters with better expression profiles. [129,130] The transformation of plasmids in B. subtilis occurs by chromosomal integration in a nonessential gene. Additionally, the spores can be used for longterm storage of the transformed strains.…”

Surface display system for probiotics and its application in aquaculture

“…2016), cotX (Wang et al . 2016a), cotY and cotZ (Bartels et al ., 2018) are also used as the surface anchoring protein to mediate spore surface display. Few studies have been made on the inner coat as a carrier protein.…”

Stress-tolerant, recyclable, and autonomously renewable biocatalyst platform enabled by engineered bacterial spores

“…1a, right), analogous to the genetic architectures reported in previous studies. 15-20 We selected a fluorescent reporter protein, mWasabi, as the first target to observe its overall expression level and spore localization in both TIED and endogenous expression systems.…”

“…13,14 So far, a range of enzymes, including α-amylase, lipase, and β-galactosidase, have been installed on the surface of B. subtilis spores. 15-20 However, despite a number of successful proof-of-concept studies, none so far have fully addressed major limitations of this system for practical applications: (1) low enzyme loading density, typically 10 2 –10 5 enzymes per spore, leading to insufficient catalytic activity, and (2) loss of activity over time and in harsh conditions. Furthermore, quantitative and substrate-specific characterizations of catalytic behavior of spore-immobilized enzymes in comparison to their respective free-forms remain elusive.…”