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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

Gel-electrophoresis of PCR products from amplified target genes ldhL (a), sigF (b) and pdhA (c). Order of strains in all three parts of the picture: M: Thermo Scientific 1 kb DNA ladder, 1 DSM 4216T wild-type, 2 DSM 4216T ΔldhL, 3 ET 138 wild-type, 4 ET 138 ΔldhL, 5 ET 138 ΔldhL ΔsigF, 6 ET 138 ΔldhL ΔsigF ΔpdhA. The original gel pictures without cropping are provided in Additional files 1 (for a, b) and 2 (for c). a Amplification of the region 1,000 bp up- and downstream of the ldhL gene using primers BG 3663 and 3669. The wild-type genotype results in a product of 3,036 bp, whereas the complete deletion of the ldhL gene is confirmed by a shift of the product to 2,094 bp. b Amplification of the region 1,000 bp up- and downstream of the sigF gene using primers BG 3990 and 3991. The wild-type genotype results in a product of 3,040 bp, whereas the complete deletion of the sigF gene is confirmed by a shift of the product to 2278 bp. c Amplification of the region 1,000 bp up- and downstream of the pdhA gene using primers BG 4563 and 4564. The wild-type genotype results in a product of 3,390 bp, whereas the complete deletion of the pdhA gene is confirmed by a shift of the product to 2,280 bp.
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Fig2: Gel-electrophoresis of PCR products from amplified target genes ldhL (a), sigF (b) and pdhA (c). Order of strains in all three parts of the picture: M: Thermo Scientific 1 kb DNA ladder, 1 DSM 4216T wild-type, 2 DSM 4216T ΔldhL, 3 ET 138 wild-type, 4 ET 138 ΔldhL, 5 ET 138 ΔldhL ΔsigF, 6 ET 138 ΔldhL ΔsigF ΔpdhA. The original gel pictures without cropping are provided in Additional files 1 (for a, b) and 2 (for c). a Amplification of the region 1,000 bp up- and downstream of the ldhL gene using primers BG 3663 and 3669. The wild-type genotype results in a product of 3,036 bp, whereas the complete deletion of the ldhL gene is confirmed by a shift of the product to 2,094 bp. b Amplification of the region 1,000 bp up- and downstream of the sigF gene using primers BG 3990 and 3991. The wild-type genotype results in a product of 3,040 bp, whereas the complete deletion of the sigF gene is confirmed by a shift of the product to 2278 bp. c Amplification of the region 1,000 bp up- and downstream of the pdhA gene using primers BG 4563 and 4564. The wild-type genotype results in a product of 3,390 bp, whereas the complete deletion of the pdhA gene is confirmed by a shift of the product to 2,280 bp.

Mentions: B. smithii ET 138 can be transformed with E. coli-Bacillus shuttle vector pNW33n with an efficiency of 5 × 103 colonies per µg DNA [15]. To obtain mutants in strain ET 138, we planned to use a protocol similar to that used for Geobacillus thermoglucosidans (recently renamed from G. thermoglucosidasius [16]), which applies pNW33n-derivatives as thermosensitive integration plasmid [17]. To create a markerless l-lactate dehydrogenase (ldhL) knockout strain from which the ldhL gene was entirely deleted, ~1,000 bp regions flanking the ldhL gene and including the start and stop codon were cloned and fused together in plasmid pNW33n. Double homologous recombination of this plasmid with the ET 138 chromosome will fuse the start and stop codons of the gene, thereby removing the entire gene in-frame without leaving any marker (Figure 1). B. smithii ET 138 was transformed with pWUR732 and colonies were transferred once at 55°C on LB2 plates containing chloramphenicol. Subsequent PCR analysis of 7 colonies already showed integration of the plasmid DNA without the temperature increase normally performed with thermosensitive integration systems [17]. A mixture of single crossover integrants via both upstream and downstream regions together with no-integration (either caused by replicating plasmids or randomly integrated plasmids) genotype was observed in one colony, one colony showed a mixture of downstream crossover and wild-type genotype, and five colonies showed no single crossovers but only wild-type genotype. Serial transfer of the colonies containing single crossovers in liquid medium combined with replica plating to identify double recombinants repeatedly resulted in only wild-type double crossover mutants. The mixed genotype persisted after several subculturings on plates containing 7 and 9 µg/mL chloramphenicol in an attempt to obtain pure genotypes. After four transfers, however, also a colony was found that contained a mixture of double crossover knockout genotype together with upstream single crossover and wild-type genotype. After this point, we added glycerol or acetate as carbon sources to allow for a metabolism with minimal impact of the ldhL deletion. After streaking this colony to an LB2 plate containing 10 g/L glycerol, colonies were obtained that had lost the wild-type genotype but contained a mixture of both single crossovers and a double crossover knockout genotypes. A pure double crossover knockout genotype was observed after two transfers on the more defined TVMY medium supplemented with acetate at 65°C, creating strain ET 138 ΔldhL (Figure 2a).Figure 1


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)

Gel-electrophoresis of PCR products from amplified target genes ldhL (a), sigF (b) and pdhA (c). Order of strains in all three parts of the picture: M: Thermo Scientific 1 kb DNA ladder, 1 DSM 4216T wild-type, 2 DSM 4216T ΔldhL, 3 ET 138 wild-type, 4 ET 138 ΔldhL, 5 ET 138 ΔldhL ΔsigF, 6 ET 138 ΔldhL ΔsigF ΔpdhA. The original gel pictures without cropping are provided in Additional files 1 (for a, b) and 2 (for c). a Amplification of the region 1,000 bp up- and downstream of the ldhL gene using primers BG 3663 and 3669. The wild-type genotype results in a product of 3,036 bp, whereas the complete deletion of the ldhL gene is confirmed by a shift of the product to 2,094 bp. b Amplification of the region 1,000 bp up- and downstream of the sigF gene using primers BG 3990 and 3991. The wild-type genotype results in a product of 3,040 bp, whereas the complete deletion of the sigF gene is confirmed by a shift of the product to 2278 bp. c Amplification of the region 1,000 bp up- and downstream of the pdhA gene using primers BG 4563 and 4564. The wild-type genotype results in a product of 3,390 bp, whereas the complete deletion of the pdhA gene is confirmed by a shift of the product to 2,280 bp.
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Related In: Results  -  Collection

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Fig2: Gel-electrophoresis of PCR products from amplified target genes ldhL (a), sigF (b) and pdhA (c). Order of strains in all three parts of the picture: M: Thermo Scientific 1 kb DNA ladder, 1 DSM 4216T wild-type, 2 DSM 4216T ΔldhL, 3 ET 138 wild-type, 4 ET 138 ΔldhL, 5 ET 138 ΔldhL ΔsigF, 6 ET 138 ΔldhL ΔsigF ΔpdhA. The original gel pictures without cropping are provided in Additional files 1 (for a, b) and 2 (for c). a Amplification of the region 1,000 bp up- and downstream of the ldhL gene using primers BG 3663 and 3669. The wild-type genotype results in a product of 3,036 bp, whereas the complete deletion of the ldhL gene is confirmed by a shift of the product to 2,094 bp. b Amplification of the region 1,000 bp up- and downstream of the sigF gene using primers BG 3990 and 3991. The wild-type genotype results in a product of 3,040 bp, whereas the complete deletion of the sigF gene is confirmed by a shift of the product to 2278 bp. c Amplification of the region 1,000 bp up- and downstream of the pdhA gene using primers BG 4563 and 4564. The wild-type genotype results in a product of 3,390 bp, whereas the complete deletion of the pdhA gene is confirmed by a shift of the product to 2,280 bp.
Mentions: B. smithii ET 138 can be transformed with E. coli-Bacillus shuttle vector pNW33n with an efficiency of 5 × 103 colonies per µg DNA [15]. To obtain mutants in strain ET 138, we planned to use a protocol similar to that used for Geobacillus thermoglucosidans (recently renamed from G. thermoglucosidasius [16]), which applies pNW33n-derivatives as thermosensitive integration plasmid [17]. To create a markerless l-lactate dehydrogenase (ldhL) knockout strain from which the ldhL gene was entirely deleted, ~1,000 bp regions flanking the ldhL gene and including the start and stop codon were cloned and fused together in plasmid pNW33n. Double homologous recombination of this plasmid with the ET 138 chromosome will fuse the start and stop codons of the gene, thereby removing the entire gene in-frame without leaving any marker (Figure 1). B. smithii ET 138 was transformed with pWUR732 and colonies were transferred once at 55°C on LB2 plates containing chloramphenicol. Subsequent PCR analysis of 7 colonies already showed integration of the plasmid DNA without the temperature increase normally performed with thermosensitive integration systems [17]. A mixture of single crossover integrants via both upstream and downstream regions together with no-integration (either caused by replicating plasmids or randomly integrated plasmids) genotype was observed in one colony, one colony showed a mixture of downstream crossover and wild-type genotype, and five colonies showed no single crossovers but only wild-type genotype. Serial transfer of the colonies containing single crossovers in liquid medium combined with replica plating to identify double recombinants repeatedly resulted in only wild-type double crossover mutants. The mixed genotype persisted after several subculturings on plates containing 7 and 9 µg/mL chloramphenicol in an attempt to obtain pure genotypes. After four transfers, however, also a colony was found that contained a mixture of double crossover knockout genotype together with upstream single crossover and wild-type genotype. After this point, we added glycerol or acetate as carbon sources to allow for a metabolism with minimal impact of the ldhL deletion. After streaking this colony to an LB2 plate containing 10 g/L glycerol, colonies were obtained that had lost the wild-type genotype but contained a mixture of both single crossovers and a double crossover knockout genotypes. A pure double crossover knockout genotype was observed after two transfers on the more defined TVMY medium supplemented with acetate at 65°C, creating strain ET 138 ΔldhL (Figure 2a).Figure 1

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