Stabilizing displayed proteins on vegetative Bacillus subtilis cells

Abstract

Microbes engineered to display heterologous proteins could be useful biotechnological tools for protein engineering, lignocellulose degradation, biocatalysis, bioremediation and biosensing. Bacillus subtilis is a promising host to display proteins, as this model Gram-positive bacterium is genetically tractable and already used industrially to produce enzymes. To gain insight into the factors that affect displayed protein stability and copy-number, we systematically compared the ability of different protease-deficient B. subtilis strains (WB800, BRB07, BRB08 and BRB14) to display a Cel8A-LysM reporter protein in which the Clostridium thermocellum Cel8A endoglucanase is fused to LysM cell wall binding modules. Whole-cell cellulase measurements and fractionation experiments demonstrate that genetically eliminating extracytoplasmic bacterial proteases improves Cel8A-LysM displays levels. However, upon entering stationary phase, for all protease-deficient strains, the amount of displayed reporter dramatically decreases, presumably as a result of cellular autolysis. This problem can be partially overcome by adding chemical protease inhibitors, which significantly increase protein display levels. We conclude that strain BRB08 is well-suited for stably displaying our reporter protein, as genetic removal of its extracellular and cell wall-associated proteases leads to the highest levels of surface accumulated Cel8A-LysM without causing secretion stress or impairing growth. A two-step procedure is presented that enables the construction of enzyme coated vegetative B. subtilis cells that retain stable cell-associated enzyme activity for nearly 3 days. The results of this work could aid the development of whole cell display systems that have useful biotechnological applications.

Surface display system for probiotics and its application in aquaculture

“…2008; Huang et al . 2018). Moreover, the C‐terminal end of molecular chaperon prsA of B. subtilis fused with Clostridium thermocellum cellulase (celA) can make more than 85% of total cellulase activity be in surface‐displayed format (Kim et al .…”

Genetically Engineered Proteins to Improve Biomass Conversion: New Advances and Challenges for Tailoring Biocatalysts

“…Even though this section focused on eukaryotic models, it is worth noting that several approaches were also taken in prokaryotic hosts to pursue a more efficient protein secretion and display, given that purification of such enzymes is often troublesome due to the typical formation of inclusion bodies in these systems [126]. Works by Yildirim et al, in which a recombinant endoglucanase was efficiently engineered for periplasmic and extracellular targeting by a native secretion signal of E. coli [138], and Huang et al, who showed the advantages of using protease-deficient strains of B. subtilis for cell-surface display of heterologous endoglucanase [139], gave insights for overcoming current limitations of prokaryotic systems.…”