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 beta-glucuronidase enzyme from Escherichia coli.

FIG. 1.

(A) Inner- and outer-coat carrier proteins. Morphogenetic proteins SafA (A) and CotE (E) have central roles in the assembly of the inner and outer-coat layers, respectively, and control the assembly of the indicated proteins. The location of SafA and CotE at the cortex-inner coat and inner coat-outer coat interfaces, respectively, is indicated. The following coat carrier proteins, and their locations, are represented: B, CotB; C, CotC; D, OxdD; and G, CotG. D is presumed to be homohexameric; B forms covalently cross-linked dimers, whereas C and G undergo extensive multimerization (n) and cross-linking at the spore surface. The passenger proteins phytase (P) and β-glucuronidase (U) are shown as fusions to D or G. (B) General strategy used to construct the various carrier-passenger fusions. The indicated pDG364 derivatives, carrying the various gene fusions, were linearized and transferred to the nonessential amyE locus through a marker replacement (double-crossover) recombination event. Promoters are represented by arrows, and transcriptional terminators are represented by hairpin structures. L10, linker made of 10 alanine residues; cat, gene encoding chloramphenicol acetyltransferase. The various genes, color-coded to match panel A, are not drawn to scale.

FIG. 2.

Purification of B. subtilis phytase (Phy) and specificity of an anti-Phy antibody. (A) SDS-PAGE analysis (lanes 1 to 3) and immunoblot analysis (lanes 4 to 6) of extracts of E. coli strain SD58 noninduced (lanes 1 and 4), induced with 1 mM IPTG (lanes 2 and 5), and of the His₆-Phy protein (lanes 3 and 6), partially purified by Ni²⁺ affinity chromatography. The asterisk indicates a cross-reactive species or a degradation product of the His₆-phytase. (B) SDS-PAGE (lanes 1 and 2) and immunoblot analysis (lanes 3 and 4) of the cell culture supernatants of the PY79 wild-type strain of B. subtilis (lanes 1 and 3) and of strain AH7666, bearing a Δphy mutation (lanes 2 and 4).

FIG. 3.

Immunofluorescence detection of CotG-Phy and OxdD-Phy protein fusions at the surface of purified spores. FITC, fluorescein isothiocyanate. (A) Phase-contrast microscopy (PC) and fluorescence microscopy (FITC). Scale bars, 2 μm. (B) Fluorescence intensity was quantified on 150 spores. AU, arbitrary units. Purified spores were submitted or not to trypsin proteolysis. noAb, control without primary antibody. Spores were purified from cultures of the following strains: PY79, wild type; SD48, CotG-Phy; and SD50, OxdD-Phy.

FIG. 4.

Abundance of the displayed phytase at the spore surface and specific activity of B. subtilis phytase. (A) Immunofluorescence detection of CotG-Phy and OxdD-Phy protein fusions at the surfaces of purified spores (average signal). (B) Assay of whole-cell extracts prepared from B. subtilis cultures in sporulating medium, 24 h after the onset of sporulation. (C) Assay of purified spores from the same cultures. DW, dry weight. Spores were purified from cultures of the following strains: PY79, wild type; SD48, CotG-Phy; SD50, OxdD-Phy. One unit (U) of phytase was defined as the amount of enzyme required to release 1 μmol of inorganic phosphate from sodium phytate in 1 min.

FIG. 5.

Assay for the activity of GusA at the spore surface, through conversion of the colorless substrate C₁₂FDGlcU (see Materials and Methods) into a yellow fluorescent product. (A) Detection of a functional OxdD-GusA fusion by phase-contrast microscopy (PC) and fluorescence (FITC) microscopy. Bars, 2 μm. (B) The fluorescence intensity was quantified on 150 spores. Purified spores submitted or not to trypsin proteolysis. Spores were purified from cultures of the following strains: PY79, wild type; and SD60, OxdD-GusA. (C) Average fluorescence intensity quantified on 150 spores. AU, arbitrary units.