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Genetic Tools for the Enhancement of Probiotic Properties

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  • Authors: Laura Ortiz-Velez1, Robert Britton2
  • Editors: Robert Allen Britton3, Patrice D. Cani4
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    Affiliations: 1: Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030; 2: Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030; 3: Baylor College of Medicine, Houston, TX 77030; 4: Université catholique de Louvain, Louvain Drug Research Institute, Brussels 1200, Belgium
    • Source: microbiolspec September 2017 vol. 5 no. 5 doi:10.1128/microbiolspec.BAD-0018-2016
  • Received 13 April 2017 Accepted 22 July 2017 Published 22 September 2017
  • Robert Britton, [email protected]
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  • Abstract:

    The genus is a diverse group of microorganisms, many of which are of industrial and medical relevance. Several species have been used as probiotics, organisms that when present in sufficient quantities confer a health benefit to the host. A significant limitation to the mechanistic understanding of how these microbes provide health benefits to their hosts and how they can be used as therapeutic delivery systems has been the lack of genetic strategies to efficiently manipulate their genomes. This article will review the development and employment of traditional genetic tools in lactobacilli and highlight the latest methodologies that are allowing for precision genome engineering of these probiotic organisms. The application of these tools will be key in providing mechanistic insights into probiotics as well as maximizing the value of lactobacilli as either a traditional probiotic or as a platform for the delivery of therapeutic proteins. Finally, we will discuss concepts that we consider relevant for the delivery of engineered therapeutics to the human gut.

  • Citation: Ortiz-Velez L, Britton R. 2017. Genetic Tools for the Enhancement of Probiotic Properties. Microbiol Spectrum 5(5):BAD-0018-2016. doi:10.1128/microbiolspec.BAD-0018-2016.

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/content/journal/microbiolspec/10.1128/microbiolspec.BAD-0018-2016
2017-09-22
2021-04-17

Abstract:

The genus is a diverse group of microorganisms, many of which are of industrial and medical relevance. Several species have been used as probiotics, organisms that when present in sufficient quantities confer a health benefit to the host. A significant limitation to the mechanistic understanding of how these microbes provide health benefits to their hosts and how they can be used as therapeutic delivery systems has been the lack of genetic strategies to efficiently manipulate their genomes. This article will review the development and employment of traditional genetic tools in lactobacilli and highlight the latest methodologies that are allowing for precision genome engineering of these probiotic organisms. The application of these tools will be key in providing mechanistic insights into probiotics as well as maximizing the value of lactobacilli as either a traditional probiotic or as a platform for the delivery of therapeutic proteins. Finally, we will discuss concepts that we consider relevant for the delivery of engineered therapeutics to the human gut.

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Genetic Tools for the Enhancement of Probiotic Properties
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Citation: Ortiz-Velez L, Britton R. 2017. Genetic tools for the enhancement of probiotic properties. microbiolspec 5(5): doi:10.1128/microbiolspec.BAD-0018-2016
/content/microbiolspec/10.1128/microbiolspec.BAD-0018-2016.fig1
FIGURE 1
Schematic representation of dsDNA recombineering in . A piece of dsDNA harboring the -- cassette ( and sites, red; Cat/CM marker, yellow) and regions with homology to the genomic insertion site (H1, green; H2, blue) is transformed into expressing an exonuclease (Lp0642), a possible host nuclease inhibitor (Lp0640), and an ssDNA binding protein (Lp0641). Once the dsDNA fragments are integrated into the genome, which renders the cells resistant to CM (CM), bacteria are transformed with a plasmid that induces the expression of the recombinase Cre to recombine the sites. This recombination deletes the CM marker contained inside the sites, rendering the cells CM-sensitive and leaving a modified site (72).
microbiolspec/5/5/BAD-0018-2016-fig1_thmb.gif
microbiolspec/5/5/BAD-0018-2016-fig1.gif
Image of FIGURE 1

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FIGURE 1

Schematic representation of dsDNA recombineering in . A piece of dsDNA harboring the -- cassette ( and sites, red; Cat/CM marker, yellow) and regions with homology to the genomic insertion site (H1, green; H2, blue) is transformed into expressing an exonuclease (Lp0642), a possible host nuclease inhibitor (Lp0640), and an ssDNA binding protein (Lp0641). Once the dsDNA fragments are integrated into the genome, which renders the cells resistant to CM (CM), bacteria are transformed with a plasmid that induces the expression of the recombinase Cre to recombine the sites. This recombination deletes the CM marker contained inside the sites, rendering the cells CM-sensitive and leaving a modified site (72).

Source: microbiolspec September 2017 vol. 5 no. 5 doi:10.1128/microbiolspec.BAD-0018-2016
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/content/microbiolspec/10.1128/microbiolspec.BAD-0018-2016.fig2
FIGURE 2
Schematic representation of ssDNA recombineering in . An oligomer harboring the mutation (red) and regions with homology to the genomic insertion site (blue) is transformed into cells expressing RecT. The oligomer is incorporated into the lagging strand being synthesized at the DNA replication fork, generating a mutant with a mixed genotype (GT). Screening of the mutant is done by MAMA-PCR, using two oligomers that amplify from a WT sequence (black) and a third oligomer (red-blue) that only amplifies when the mutation is incorporated. Thus, two amplicons are generated in the case of the mutant (lane 1), whereas only one amplicon is generated in the case of the WT (lanes 2 to 5). After the mixed genotype is identified, single colony purification is done to isolate cells containing only the mutation (pure genotype).
microbiolspec/5/5/BAD-0018-2016-fig2_thmb.gif
microbiolspec/5/5/BAD-0018-2016-fig2.gif
Image of FIGURE 2

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FIGURE 2

Schematic representation of ssDNA recombineering in . An oligomer harboring the mutation (red) and regions with homology to the genomic insertion site (blue) is transformed into cells expressing RecT. The oligomer is incorporated into the lagging strand being synthesized at the DNA replication fork, generating a mutant with a mixed genotype (GT). Screening of the mutant is done by MAMA-PCR, using two oligomers that amplify from a WT sequence (black) and a third oligomer (red-blue) that only amplifies when the mutation is incorporated. Thus, two amplicons are generated in the case of the mutant (lane 1), whereas only one amplicon is generated in the case of the WT (lanes 2 to 5). After the mixed genotype is identified, single colony purification is done to isolate cells containing only the mutation (pure genotype).

Source: microbiolspec September 2017 vol. 5 no. 5 doi:10.1128/microbiolspec.BAD-0018-2016
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FIGURE 3
Overview of recombineering/CRISPR-Cas9 genome engineering in . An oligomer harboring the mutation (red) and regions with homology to the genomic insertion site (blue) is transformed into expressing RecT, Cas9, and its tracrRNA. The oligomer targets the lagging strains and is incorporated into one of the DNA strands at the replication fork. When cells divide, a mixed population of WT and mutant cells is generated. A plasmid expressing guide RNA (pgRNA) that targets the WT sequence is introduced into the cell. Cas9 is directed by the gRNA to cleave DNA of cells that did not incorporate the mutations (WT), whereas cells that incorporate the mutation are void of cleavage (mutant).
microbiolspec/5/5/BAD-0018-2016-fig3_thmb.gif
microbiolspec/5/5/BAD-0018-2016-fig3.gif
Image of FIGURE 3

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FIGURE 3

Overview of recombineering/CRISPR-Cas9 genome engineering in . An oligomer harboring the mutation (red) and regions with homology to the genomic insertion site (blue) is transformed into expressing RecT, Cas9, and its tracrRNA. The oligomer targets the lagging strains and is incorporated into one of the DNA strands at the replication fork. When cells divide, a mixed population of WT and mutant cells is generated. A plasmid expressing guide RNA (pgRNA) that targets the WT sequence is introduced into the cell. Cas9 is directed by the gRNA to cleave DNA of cells that did not incorporate the mutations (WT), whereas cells that incorporate the mutation are void of cleavage (mutant).

Source: microbiolspec September 2017 vol. 5 no. 5 doi:10.1128/microbiolspec.BAD-0018-2016
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