High-level over-expression of commercially important proteins in B. subtilis has been difficult to achieve. While there are several different types of B. subtilis plasmids that have been used, such as pUB110, pE194, pMTLBS72, or pSM.beta.1, these plasmids are unstable and don’t segregate well during cell growth, making them relatively difficult to use for gene expression. During large scale fermentation without antibiotic selection, a significant number of cells (50-99.9 percent) lose their plasmids. Even under antibiotic selection, the bacteria may lose their plasmids unless they have this stable segregation system.
UC San Diego investigators have now discovered a sequence that allows a Bacillus subtilis plasmid vector to be stably maintained. It can be used to stably express heterologous proteins in B. subtilis, which was not achievable through plasmid expression before. This new vector contains a plasmid stability determinant functional in B. subtilis that makes it significantly more stable than any other vector available. The plasmid contains a novel type of DNA segregation system that segregates newly replicated plasmids prior to cell division, even without antibiotic selection. The plasmid vector can replicate in both E. coli and B. subtilis.
Claim 1: An isolated recombinant plasmid expression vector comprising a polynucleotide encoding a prokaryote-derived actin like protein (ALP) having at least 90% identity to the polypeptide sequence of SEQ ID NO:1, wherein the ALP confers stability on a mobile genetic element when the ALP is expressed in a prokaryotic cell having a mobile genetic element.
The worldwide market for enzymes produced by strains of Bacillus is estimated to be greater than $1 billion, especially for high-level production of cellulases used to produce biofuels or for other industrial processes.
|United States Of America||Issued Patent||8,636,999||01/28/2014||2008-278|