Abstract
BackgroundThrough modification of the flagellin type III secretion pathway of Bacillus halodurans heterologous peptides could be secreted into the medium as flagellin fusion monomers. The stability of the secreted monomers was significantly enhanced through gene-targeted inactivation of host cell extracellular proteases. In evaluating the biotechnological potential of this extracellular secretion system an anti-viral therapeutic peptide, Enfuvirtide, was chosen. Currently, Enfuvirtide is synthesised utilizing 106 chemical steps. We used Enfuvirtide as a model system in an effort to develop a more cost-effective biological process for therapeutic peptide production.ResultsAn attempt was made to increase the levels of the fusion peptide by two strategies, namely strain improvement through gene-targeted knock-outs, as well as vector and cassette optimization. Both approaches proved to be successful. Through chromosomal inactivation of the spo0A, lytC and lytE genes, giving rise to strain B. halodurans BhFDL05S, the secretion of recombinant peptide fusions was increased 10-fold. Cassette optimization, incorporating an expression vector pNW33N and the N- and C-terminal regions of the flagellin monomer as an in-frame peptide fusion, resulted in a further 3.5-fold increase in the secretion of recombinant peptide fusions.ConclusionsThe type III flagellar secretion system of B. halodurans has been shown to successfully secrete a therapeutic peptide as a heterologous flagellin fusion. Improvements to both the strain and expression cassette led to increased levels of recombinant peptide, showing promise for a biotechnological application.
Highlights
Through modification of the flagellin type III secretion pathway of Bacillus halodurans heterologous peptides could be secreted into the medium as flagellin fusion monomers
Construction of B. halodurans BhFD05 mutant strains through gene-targeted inactivation Modifications were made to the genome of B. halodurans BhFD05 to generate a number of strains with different genotypes with a view to evaluating their effect on the flagellin type III secretion pathway
By analogy to E. coli and S. typhimurium, genes involved in the hook-basal body (HBB) complex are grouped into class II, and genes whose expression is dependent on class II genes are considered to belong to class III
Summary
Through modification of the flagellin type III secretion pathway of Bacillus halodurans heterologous peptides could be secreted into the medium as flagellin fusion monomers. In a related approach, harnessing the Salmonella type III flagellin pathway, Végh et al [5] used a Salmonella strain defective in flagellin gene expression, but still apparently possessing a functional fliD gene to show heterologous secretion of polypeptide-FliC fusions. They demonstrated that a 22-residue long segment within the disordered N-terminal region of Salmonella flagellin contains the recognition signal for the flagellar export machinery. They found that the nature of the attached polypeptide had a significant effect on secretion efficiency
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