Display of Recombinant Proteins on Bacillus subtilis Spores, Using a Coat-Associated Enzyme as the Carrier

Abstract

The display of proteins such as feed enzymes at the surface of bacterial spore systems has a great potential use for animal feed. Feed enzymes increase the digestibility of nutrients, leading to greater efficiency in the manufacturing of animal products and minimizing the environmental impact of increased animal production. To deliver their full potential in the gut, feed enzymes must survive the harsh conditions of the feed preparation and the gastrointestinal tract. The well-documented resistance of spores to harsh environments, together with the ability to use proteins that compose the spore as carriers for the display of passenger proteins, suggests that spores could be used as innovative tools to improve the formulation of bioactive molecules. Although some successful examples have been reported, in which abundant structural proteins of the Bacillus subtilis spore outer-coat layer were used as carriers for the display of recombinant proteins, only one convincing example resulted in the display of functional enzymes. In addition, no examples are available about the use of an inner-coat protein for the display of an active passenger enzyme. In our study, we show that the inner-coat oxalate decarboxylase (OxdD) can expose an endogenous phytase, a commonly used feed enzyme for monogastric animals, in an active form at the spore surface. Importantly, despite the higher abundance of CotG outer-coat protein, an OxdD-Phy fusion was more represented at the spore surface. The potential of OxdD as a carrier protein is further documented through the spore display of a bioactive heterologous passenger, the tetrameric ␤-glucuronidase enzyme from Escherichia coli.Under extreme nutrient deprivation, Bacillus subtilis has the ability to enter a complex differentiation process that culminates with the formation of an extremely resistant spore.

Generation of Multifunctional Magnetic Nanoparticles with Amplified Catalytic Activities by Genetic Expression of Enzyme Arrays on Bacterial Magnetosomes

“…For example, Potot et al [43] displayed GusA monomers on spores of Bacillus subtilis by fusing it to the coat-associated oxalate decarboxylase OxdD, resulting in about 40% of the GusA spores exhibiting catalytic activity. Sheppard et al observed a high activity for GusA when immobilized to living diatom silica by genetic fusion to silaffin protein, resulting in about 0.1% (w/w) enzyme loading of the silica.…”

Progress in Bacillus subtilis Spore Surface Display Technology towards Environment, Vaccine Development, and Biocatalysis

“…The most commonly used outer coat proteins are CotB [Duc le et al, 2007;Hinc et al, 2010b], CotG [Hinc et al, 2010b;Kwon et al, 2007], and CotC [Yuan et al, 2013]. In addition, an inner coat protein, OxdD, was also used by Potot et al [2010] for endogenous phytase exposure. The progressive way for spore surface display is presented in Figure 1.…”

Microbial Community Structure and Functionality in Ruminants Fed the Probiotic Bacillus amyloliquefaciens H57

“…However, it is possible that extracellular compounds produced during fermentation and carried over with the inoculum are responsible for the benefits of H57. The spore coat of B. subtilis can act as a carrier of various enzymes, antigens and even vaccines (Huang et al, 2010;Lee et al, 2010;Potot et al, 2010). The feed enzymes phytase and E. coli β-glucuronidase were successfully displayed on the spore surface of B. subtilis and retained their enzymatic activity by fusing with spore coat proteins CotG and OxdD (Potot et al, 2010).…”

“…The spore coat of B. subtilis can act as a carrier of various enzymes, antigens and even vaccines (Huang et al, 2010;Lee et al, 2010;Potot et al, 2010). The feed enzymes phytase and E. coli β-glucuronidase were successfully displayed on the spore surface of B. subtilis and retained their enzymatic activity by fusing with spore coat proteins CotG and OxdD (Potot et al, 2010). Enzymes attached to the spore coat are stabilised under damaging conditions, such as high temperatures and low pH (Sirec et al, 2012).…”

“…The H57 genome shows it has the potential to grow under anaerobic conditions (Schofield et al, 2016) but it is unclear if it does so, especially as the numbers we found in the rumen were so low. Bacillus subtilis spores can be a carrier for carbohydrate degrading enzymes with the enzymes displayed on the spore coat surface, fusing with spore coat proteins such as CotG and OxdD (Potot et al, 2010). These enzymes retained their enzymatic activity and were stabilised by attachment to the spore coat.…”