Extracellular secretion of a recombinant therapeutic peptide by Bacillus halodurans utilizing a modified flagellin type III secretion system

<p>Abstract</p> <p>Background</p> <p>Through modification of the flagellin type III secretion pathway of <it>Bacillus halodurans </it>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.</p> <p>Results</p> <p>An 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 <it>spo0A, lytC </it>and <it>lytE </it>genes, giving rise to strain <it>B. halodurans </it>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.</p> <p>Conclusions</p> <p>The type III flagellar secretion system of <it>B. halodurans </it>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.</p

Analysis of genes and enzymes involved in the degradation of cellulose and proteins by Butyrivibrio fibrisolvens H17c

Bibliography: pages 147-169.Butyrivibrio fibrisolvens H17c is a gram-negative obligate anaerobic bacterium found in the rumen of most ruminants. The aim of this thesis was to investigate the enzymes produced by B. fibrisolvens H17c involved in the degradation of cellulose, xylan, and protein. A library of chromosomal DNA fragments from B. fibrisolvens H17c was established in the plasmid pEcoR251, an Escherichia coli positive selection vector. The library was screened for genes expressing cellulase, xylanase, and protease activity. Two genes expressing endo-β-1,4-glucanase and cellodextrinase activity were cloned in E. coli as host. The gene expressing endo-β-1,4-glucanase activity (end1) was cloned on a recombinant plasmid pES400. The end1 gene was located on a 6.8 kb DNA fragment and expressed from its own promoter in the E. coli host. It was shown that 64% of the endoglucanase activity was located in the periplasm of the E. coli host. TnphoA mutagenesis indicated the presence of a functional E. coli-like signal peptide. The nucleotide sequence of end1 was determined and the amino acid sequence (547 amino acids) deduced. The catalytic domain of End1 showed very good similarity to the catalytic domain of the Clostridium thermoceiium EGE endoglucanase. End1 also has a non-catalytic domain similar to the binding domains of the CenA and Cex cellulases from Ceilulomonas fimi The gene expressing cellodextrinase activity (ced1) was cloned on a recombinant plasmid pES500. This gene was located on a 3.55 kb fragment and was also expressed from its own promoter in the E. coli host. The Ced1 enzyme was also exported to the periplasm of the E. coli host, but did not contain a functional E. coli-like signal peptide. The nucleotide sequence was determined and the deduced amino acid sequence (547 residues) showed high similarity to the catalytic domain of the C. thermocellum EGD endoglucanase. The proteins of End1 and Ced1 showed no similarity. The End1 and Ced1 enzymes were characterized using a range of different substrates. The End1 enzyme showed optimal activity at pH 5.6 and 45°C. Optimal activity for the Ced1 enzyme was obtained at pH 6.6 and 50°C. The proteolytic activity of B. fibrisolvens H17c was characterized using gelatin-SD5-PAGE. Ten bands of protease activity with apparent molecular weights ranging between 42 000 and 101 000 were detected at different stages during the growth cycle. The effect of protease inhibitors indicated that all ten protease bands were serine proteases. Optimal activity was observed between pH 6.0 to 7.5 and at a temperature of 50°C. The proteolytic activity of B. fibrisolvens H17c varied depending on the type of carbohydrate substrate in the medium, and was positively correlated with the growth rate

Evaluation of the Staphylococcus aureus Class C Nonspecific Acid Phosphatase (SapS) as a Reporter for Gene Expression and Protein Secretion in Gram-Negative and Gram-Positive Bacteria▿

A phosphatase secreted by Staphylococcus aureus strain 154 has previously been characterized and classified as a new member of the bacterial class C family of nonspecific acid phosphatases. As the acid phosphatase activity can be easily detected with a cost-effective plate screen assay, quantitatively measured by a simple enzyme assay, and detected by zymography, its potential use as a reporter system was investigated. The S. aureus acid phosphatase (sapS) gene has been cloned and expressed from its own regulatory sequences in Escherichia coli, Bacillus subtilis, and Bacillus halodurans. Transcriptional and translational fusions of the sapS gene with selected heterologous promoters and signal sequences were constructed and expressed in all three of the host strains. From the range of promoters evaluated, the strongest promoter for heterologous protein production in each of the host strains was identified, i.e., the E. coli lacZ promoter in E. coli, the B. halodurans alkaline protease promoter in B. subtilis, and the B. halodurans σD promoter in B. halodurans. This is the first report on the development of a class C acid phosphatase gene as a reporter gene with the advantage of being able to function in both gram-positive and gram-negative host strains

Impact of Inactivated Extracellular Proteases on the Modified Flagellin Type III Secretion Pathway of Bacillus halodurans▿

The flagellin type III secretion pathway of Bacillus halodurans BhFC01 (Δhag) was modified by the inactivation of fliD. An in-frame flagellin gene fusion polypeptide construct was expressed, and the heterologous peptides were secreted as flagellin fusion monomers. The stability of the secreted monomers was significantly enhanced through gene-targeted inactivation of extracellular proteases