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

Elleke F Bosma,Laurens Van Der Vlist,Antonius H P Van De Weijer,Richard Van Kranenburg,John Van Der Oost,Willem M De Vos

doi:10.1186/s12934-015-0286-5

Elleke F Bosma, Laurens Van Der Vlist + Show 4 more

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https://doi.org/10.1186/s12934-015-0286-5

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Abstract

BackgroundMicrobial 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.ResultsClean deletions of the ldhL gene were made in two B. smithii strains (DSM 4216T 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.ConclusionsIn 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0286-5) contains supplementary material, which is available to authorized users.

Highlights

Microbial conversion of biomass to fuels or chemicals is an attractive alternative for fossil-based fuels and chemicals
Despite the aforementioned advantages of thermophiles, mesophilic model organisms such as Escherichia coli and Saccharomyces cerevisiae are still preferred production organisms, as these are well-studied and genetic tools are available to enable their use as versatile platform organisms [7, 8]
Most engineering efforts in thermophiles have so far been directed at ethanol production, but recently examples for chemical production have been shown such as Thermoanaerobacterium aotearoense for lactate production [11], Bacillus licheniformis for 2,3-butanediol production [12], and Bacillus coagulans for d-lactate production [13, 14]

Summary

Introduction

Microbial conversion of biomass to fuels or chemicals is an attractive alternative for fossil-based fuels and chemicals. 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. The development of genetic tools for thermophilic organisms is crucial to fully understand their metabolic versatility and to establish a thermophilic production platform for green chemical and fuel production. Markerless gene deletions should be made such that no antibiotic resistance genes or other scars are introduced into the target genome This is especially important when working with thermophilic organisms as the number of available markers is limited, requiring re-use of the marker [9, 10]

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