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Rational design of improved pharmabiotics.

Sleator RD, Hill C - J. Biomed. Biotechnol. (2009)

Bottom Line: Herein we review the most recent advances in probiotic research and applications with particular emphasis on the novel concept of patho-biotechnology: the application of pathogen-derived (ex vivo and in vivo) stress survival strategies for the design of more technologically robust and effective probiotic cultures with improved biotechnological and clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Alimentary Pharmabiotic Centre, University College Cork, Ireland. roy.sleator@cit.ie

ABSTRACT
Herein we review the most recent advances in probiotic research and applications with particular emphasis on the novel concept of patho-biotechnology: the application of pathogen-derived (ex vivo and in vivo) stress survival strategies for the design of more technologically robust and effective probiotic cultures with improved biotechnological and clinical applications.

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Related in: MedlinePlus

Growth of a proline hyperproducing strain of E. coli expressing a mutated version of the listerial proB gene relative to a wild-type E. coli in M9 minimal medium of elevated osmolarity. Growth (as determined by turbidity using a Spectra max 340 spectrophotometer, Molecular Devices), was measured both in the presence (closed symbols) and absence (open symbols) of 4% NaCl. (□, ▪) CSH26C control strain, (Δ, ▲) CSH26proBmut. Each point represents the mean value of three independent experiments.
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Related In: Results  -  Collection


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fig5: Growth of a proline hyperproducing strain of E. coli expressing a mutated version of the listerial proB gene relative to a wild-type E. coli in M9 minimal medium of elevated osmolarity. Growth (as determined by turbidity using a Spectra max 340 spectrophotometer, Molecular Devices), was measured both in the presence (closed symbols) and absence (open symbols) of 4% NaCl. (□, ▪) CSH26C control strain, (Δ, ▲) CSH26proBmut. Each point represents the mean value of three independent experiments.

Mentions: We employed this technique to engineer proline hyperproducing strains of E. coli with a significantly increased ability to tolerate elevated osmolarities (Figure 5). Bacterial proline synthesis from glutamate occurs via three enzymatic reactions, catalyzed by γ-glutamyl kinase (GK) (proB product), γ-glutamyl phosphate reductase (GPR) (proA product), and 1Δ-pyrroline-5-carboxylate reductase (P5C) (proC product). For both prokaryotic and eukaryotic systems proline synthesis from glutamate is regulated by feedback inhibition of the first enzyme in the pathway (GK). Thus, it is possible to isolate proline hyperproducing strains by screening for isolates exhibiting reduced proline-mediated feedback inhibition of GK activity (as a consequence of single-base-pair substitutions in the proB gene). This was achieved by passaging the listerial proBA operon through E. coli XL1-Red thus creating a bank of randomly mutated proBA operons. The resulting gene bank was then transformed into E. coli CSH26 (a proline auxotroph) and successful transformants were screened for proline hyperproduction. Three independent proline overproducing mutants were obtained (each carrying point mutations at a different location within the proB gene). These strains, heterologously expressing the mutated listerial proBA operon, were shown to be considerably more osmotolerant than strains expressing the wild-type listerial proBA [23]. Thus, while complementation with wild-type listerial proBA offers a significant degree of osmoprotection, the bioengineered proBA operon is far more effective, proving that the directed evolution approach provides a new dimension to the patho-biotechnology concept. It is of course entirely likely that this directed evolution approach may well dispense with the need for pathogens altogether as a source of stress survival systems in favour of selectively enhancing the probiotic's own gene complement.


Rational design of improved pharmabiotics.

Sleator RD, Hill C - J. Biomed. Biotechnol. (2009)

Growth of a proline hyperproducing strain of E. coli expressing a mutated version of the listerial proB gene relative to a wild-type E. coli in M9 minimal medium of elevated osmolarity. Growth (as determined by turbidity using a Spectra max 340 spectrophotometer, Molecular Devices), was measured both in the presence (closed symbols) and absence (open symbols) of 4% NaCl. (□, ▪) CSH26C control strain, (Δ, ▲) CSH26proBmut. Each point represents the mean value of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2742647&req=5

fig5: Growth of a proline hyperproducing strain of E. coli expressing a mutated version of the listerial proB gene relative to a wild-type E. coli in M9 minimal medium of elevated osmolarity. Growth (as determined by turbidity using a Spectra max 340 spectrophotometer, Molecular Devices), was measured both in the presence (closed symbols) and absence (open symbols) of 4% NaCl. (□, ▪) CSH26C control strain, (Δ, ▲) CSH26proBmut. Each point represents the mean value of three independent experiments.
Mentions: We employed this technique to engineer proline hyperproducing strains of E. coli with a significantly increased ability to tolerate elevated osmolarities (Figure 5). Bacterial proline synthesis from glutamate occurs via three enzymatic reactions, catalyzed by γ-glutamyl kinase (GK) (proB product), γ-glutamyl phosphate reductase (GPR) (proA product), and 1Δ-pyrroline-5-carboxylate reductase (P5C) (proC product). For both prokaryotic and eukaryotic systems proline synthesis from glutamate is regulated by feedback inhibition of the first enzyme in the pathway (GK). Thus, it is possible to isolate proline hyperproducing strains by screening for isolates exhibiting reduced proline-mediated feedback inhibition of GK activity (as a consequence of single-base-pair substitutions in the proB gene). This was achieved by passaging the listerial proBA operon through E. coli XL1-Red thus creating a bank of randomly mutated proBA operons. The resulting gene bank was then transformed into E. coli CSH26 (a proline auxotroph) and successful transformants were screened for proline hyperproduction. Three independent proline overproducing mutants were obtained (each carrying point mutations at a different location within the proB gene). These strains, heterologously expressing the mutated listerial proBA operon, were shown to be considerably more osmotolerant than strains expressing the wild-type listerial proBA [23]. Thus, while complementation with wild-type listerial proBA offers a significant degree of osmoprotection, the bioengineered proBA operon is far more effective, proving that the directed evolution approach provides a new dimension to the patho-biotechnology concept. It is of course entirely likely that this directed evolution approach may well dispense with the need for pathogens altogether as a source of stress survival systems in favour of selectively enhancing the probiotic's own gene complement.

Bottom Line: Herein we review the most recent advances in probiotic research and applications with particular emphasis on the novel concept of patho-biotechnology: the application of pathogen-derived (ex vivo and in vivo) stress survival strategies for the design of more technologically robust and effective probiotic cultures with improved biotechnological and clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Alimentary Pharmabiotic Centre, University College Cork, Ireland. roy.sleator@cit.ie

ABSTRACT
Herein we review the most recent advances in probiotic research and applications with particular emphasis on the novel concept of patho-biotechnology: the application of pathogen-derived (ex vivo and in vivo) stress survival strategies for the design of more technologically robust and effective probiotic cultures with improved biotechnological and clinical applications.

Show MeSH
Related in: MedlinePlus