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Establishment of markerless gene deletion tools in thermophilic Bacillus smithii and construction of multiple mutant strains.

Bosma EF, van de Weijer AH, van der Vlist L, de Vos WM, van der Oost J, van Kranenburg R - Microb. Cell Fact. (2015)

Bottom Line: Thermophilic microorganisms have several operational advantages as a production host over mesophilic organisms, such as low cooling costs, reduced contamination risks and a process temperature matching that of commercial hydrolytic enzymes, enabling simultaneous saccharification and fermentation at higher efficiencies and with less enzymes.The described markerless gene deletion system paves the way for more extensive metabolic engineering of B. smithii.This enables the development of this species into a platform organism and provides tools for studying its metabolism, which appears to be different from its close relatives such as B. coagulans and other bacilli.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB, Wageningen, The Netherlands. elleke.bosma@wur.nl.

ABSTRACT

Background: Microbial conversion of biomass to fuels or chemicals is an attractive alternative for fossil-based fuels and chemicals. Thermophilic microorganisms have several operational advantages as a production host over mesophilic organisms, such as low cooling costs, reduced contamination risks and a process temperature matching that of commercial hydrolytic enzymes, enabling simultaneous saccharification and fermentation at higher efficiencies and with less enzymes. However, genetic tools for biotechnologically relevant thermophiles are still in their infancy. In this study we developed a markerless gene deletion method for the thermophile Bacillus smithii and we report the first metabolic engineering of this species as a potential platform organism.

Results: Clean deletions of the ldhL gene were made in two B. smithii strains (DSM 4216(T) and compost isolate ET 138) by homologous recombination. Whereas both wild-type strains produced mainly L-lactate, deletion of the ldhL gene blocked L-lactate production and caused impaired anaerobic growth and acid production. To facilitate the mutagenesis process, we established a counter-selection system for efficient plasmid removal based on lacZ-mediated X-gal toxicity. This counter-selection system was applied to construct a sporulation-deficient B. smithii ΔldhL ΔsigF mutant strain. Next, we demonstrated that the system can be used repetitively by creating B. smithii triple mutant strain ET 138 ΔldhL ΔsigF ΔpdhA, from which also the gene encoding the α-subunit of the E1 component of the pyruvate dehydrogenase complex is deleted. This triple mutant strain produced no acetate and is auxotrophic for acetate, indicating that pyruvate dehydrogenase is the major route from pyruvate to acetyl-CoA.

Conclusions: In this study, we developed a markerless gene deletion method including a counter-selection system for thermophilic B. smithii, constituting the first report of metabolic engineering in this species. The described markerless gene deletion system paves the way for more extensive metabolic engineering of B. smithii. This enables the development of this species into a platform organism and provides tools for studying its metabolism, which appears to be different from its close relatives such as B. coagulans and other bacilli.

No MeSH data available.


Related in: MedlinePlus

Schaeffer-Fulton staining on ET 138 wild-type and mutants. Cultures used were grown aerobically overnight in LB2 medium at 55°C and subsequently kept at room temperature for 24 h, after which the staining was performed. Pink-stained cells indicate intact cells, whereas spores are green–blue. a strain ET 138 wild-type, in which sporulation is observed. b strain ET 138 ΔldhL, in which sporulation is observed. c Strain ET 138 ΔldhL ΔsigF, where no sporulation is observed due to removal of the sigF gene.
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Fig3: Schaeffer-Fulton staining on ET 138 wild-type and mutants. Cultures used were grown aerobically overnight in LB2 medium at 55°C and subsequently kept at room temperature for 24 h, after which the staining was performed. Pink-stained cells indicate intact cells, whereas spores are green–blue. a strain ET 138 wild-type, in which sporulation is observed. b strain ET 138 ΔldhL, in which sporulation is observed. c Strain ET 138 ΔldhL ΔsigF, where no sporulation is observed due to removal of the sigF gene.

Mentions: To confirm that strain ET 138 ΔldhL ΔsigF was unable to form spores, a Schaeffer-Fulton staining was performed (Figure 3). In the wild-type and the ldhL-mutant (Figure 3a, b) many spores were observed as indicated by the presence of green spheres, whereas no spores were observed in the ldhL-sigF-double mutant (Figure 3c). Pasteurisation of cultures of the wild-type and the ΔldhL-strain resulted in colony counts of >5 × 105 and 3 × 105 per mL of cells, respectively. As expected, no colonies were observed after Pasteurisation of a culture of the ΔldhL ΔsigF double mutant, while colony counts for the control treatment were >5 × 105 per mL of cells for all three strains. Both assays confirm that the removal of the sigF gene results in a sporulation-deficient phenotype.Figure 3


Establishment of markerless gene deletion tools in thermophilic Bacillus smithii and construction of multiple mutant strains.

Bosma EF, van de Weijer AH, van der Vlist L, de Vos WM, van der Oost J, van Kranenburg R - Microb. Cell Fact. (2015)

Schaeffer-Fulton staining on ET 138 wild-type and mutants. Cultures used were grown aerobically overnight in LB2 medium at 55°C and subsequently kept at room temperature for 24 h, after which the staining was performed. Pink-stained cells indicate intact cells, whereas spores are green–blue. a strain ET 138 wild-type, in which sporulation is observed. b strain ET 138 ΔldhL, in which sporulation is observed. c Strain ET 138 ΔldhL ΔsigF, where no sporulation is observed due to removal of the sigF gene.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494709&req=5

Fig3: Schaeffer-Fulton staining on ET 138 wild-type and mutants. Cultures used were grown aerobically overnight in LB2 medium at 55°C and subsequently kept at room temperature for 24 h, after which the staining was performed. Pink-stained cells indicate intact cells, whereas spores are green–blue. a strain ET 138 wild-type, in which sporulation is observed. b strain ET 138 ΔldhL, in which sporulation is observed. c Strain ET 138 ΔldhL ΔsigF, where no sporulation is observed due to removal of the sigF gene.
Mentions: To confirm that strain ET 138 ΔldhL ΔsigF was unable to form spores, a Schaeffer-Fulton staining was performed (Figure 3). In the wild-type and the ldhL-mutant (Figure 3a, b) many spores were observed as indicated by the presence of green spheres, whereas no spores were observed in the ldhL-sigF-double mutant (Figure 3c). Pasteurisation of cultures of the wild-type and the ΔldhL-strain resulted in colony counts of >5 × 105 and 3 × 105 per mL of cells, respectively. As expected, no colonies were observed after Pasteurisation of a culture of the ΔldhL ΔsigF double mutant, while colony counts for the control treatment were >5 × 105 per mL of cells for all three strains. Both assays confirm that the removal of the sigF gene results in a sporulation-deficient phenotype.Figure 3

Bottom Line: Thermophilic microorganisms have several operational advantages as a production host over mesophilic organisms, such as low cooling costs, reduced contamination risks and a process temperature matching that of commercial hydrolytic enzymes, enabling simultaneous saccharification and fermentation at higher efficiencies and with less enzymes.The described markerless gene deletion system paves the way for more extensive metabolic engineering of B. smithii.This enables the development of this species into a platform organism and provides tools for studying its metabolism, which appears to be different from its close relatives such as B. coagulans and other bacilli.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB, Wageningen, The Netherlands. elleke.bosma@wur.nl.

ABSTRACT

Background: Microbial conversion of biomass to fuels or chemicals is an attractive alternative for fossil-based fuels and chemicals. Thermophilic microorganisms have several operational advantages as a production host over mesophilic organisms, such as low cooling costs, reduced contamination risks and a process temperature matching that of commercial hydrolytic enzymes, enabling simultaneous saccharification and fermentation at higher efficiencies and with less enzymes. However, genetic tools for biotechnologically relevant thermophiles are still in their infancy. In this study we developed a markerless gene deletion method for the thermophile Bacillus smithii and we report the first metabolic engineering of this species as a potential platform organism.

Results: Clean deletions of the ldhL gene were made in two B. smithii strains (DSM 4216(T) and compost isolate ET 138) by homologous recombination. Whereas both wild-type strains produced mainly L-lactate, deletion of the ldhL gene blocked L-lactate production and caused impaired anaerobic growth and acid production. To facilitate the mutagenesis process, we established a counter-selection system for efficient plasmid removal based on lacZ-mediated X-gal toxicity. This counter-selection system was applied to construct a sporulation-deficient B. smithii ΔldhL ΔsigF mutant strain. Next, we demonstrated that the system can be used repetitively by creating B. smithii triple mutant strain ET 138 ΔldhL ΔsigF ΔpdhA, from which also the gene encoding the α-subunit of the E1 component of the pyruvate dehydrogenase complex is deleted. This triple mutant strain produced no acetate and is auxotrophic for acetate, indicating that pyruvate dehydrogenase is the major route from pyruvate to acetyl-CoA.

Conclusions: In this study, we developed a markerless gene deletion method including a counter-selection system for thermophilic B. smithii, constituting the first report of metabolic engineering in this species. The described markerless gene deletion system paves the way for more extensive metabolic engineering of B. smithii. This enables the development of this species into a platform organism and provides tools for studying its metabolism, which appears to be different from its close relatives such as B. coagulans and other bacilli.

No MeSH data available.


Related in: MedlinePlus