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Bacillus subtilis
Besides the Gram-negative bacterium Escherichia coli, Bacillus subtilis, the representative of the low GC group of the Gram-positive bacteria, is the best-studied microorganism. B. subtilis does not produce endotoxins and has been classified by the American FDA as GRAS (generally regarded as safe) organisms; these organisms can be used in the food industry.
B. subtilis and its close relatives (e.g., B. licheniformis and B. amyloliquefaciens) have been used for decades for the industrial production of technical enzymes including proteases, lipases and starch-degrading enzymes. These technical enzymes are secreted into the growth medium and can be enriched and purified rather easily. By the classical method of induced mutagenesis followed by screening, super producers have been isolated which are able to secrete up to 20g of the desired protein into the culture medium.
The genome of B. subtilis was published in 1997, and the function of most of its genes is known. Most of its physiology has been unravelled including the major catabolic and anabolic pathways. Genetic manipulation of its genome is an easy task. Plasmids able to replicate stably are known, foreign genes can be inserted into the bacterial genome at many ectopic sites. In addition, chromosomal genes can be deleted or replaced by foreign genes. Under starving conditions, B. subtilis cells form endospores allowing the storage of industrially important strains as spores at room temperature. B. subtilis spores are extremely resistant against many physical and chemical stress factors such as UV irradiation, desiccation, heat, cold and oxidative stress. Furthermore, spores constitute excellent bioparticles for surface display of passenger proteins.
For industrial application involving biotechnological processes, B. subtilis has several advantages over E. coli.
Enzyme production: The intracellular production of any enzyme is possible. We have developed expression vectors allowing overproduction of recombinant enzymes representing up to 45% of the total cellular protein. The problem of forming inclusion bodies is less prominent in B. subtilis since it has been shown that the major classes of promiscuous chaperones keep many overproduced proteins soluble.
Secretion: Secretion of recombinant proteins into the culture medium is rather tedious and rarely used since these proteins have to cross two membranes. Since B. subtilis cells are surrounded only by one membrane, proteins destined for secretion have to be equipped with an appropriate signal peptide to allow secretion into the medium. Here, three different mechanisms have been described and can be used depending on the recombinant protein.
Surface display on B. subtilis cells: Anchoring of proteins on the outside of E. coli cells is possible, but these proteins have to cross two membranes. In the case of B. subtilis cells, proteins have to cross just one membrane as already mentioned, and we have established a method allowing the covalent anchoring of up to 240,000 protein molecules on the cell wall.
Surface display on spores: Besides storage of important strains as spores at room temperature, any foreign protein can be anchored on the spore surface. Since synthesis and anchoring occurs within the cytoplasm, molecular chaperones are present to assist protein folding. It should also be possible to engineer B. subtilis cells in such a way that they are able to catalyze disulfide bonds within the cytoplasm.
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