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

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.

Complete genome sequence of thermophilic Bacillus smithii type strain DSM 4216T

“…This is in accordance with the absence of 2,3-butanediol production [3, 4]. The anabolic acetolactate synthase small and large subunit genes ilvBH (also called α-acetohydroxyacid synthase) are present.…”

Development and implementation of rapid metabolic engineering tools for chemical and fuel production in Geobacillus thermoglucosidasius NCIMB 11955

“…Comparisons to other mutagenesis methods are not straightforward, as published protocols generally do not provide sufficient details. Estimates of two previously published methods for thermophilic bacilli [15, 52] suggest that mutant generation takes anywhere from 7 to 10 days. CRISPR/Cas9 genome editing has yet to be reported in Geobacillus , but two recent exemplifications in B. subtilis [53, 54] indicate that mutant generation is accomplished in 5–7 days.…”

Contributory roles of two l-lactate dehydrogenases for l-lactic acid production in thermotolerant Bacillus coagulans

“…In this study, ldh L1 knockout resulted in the complete loss of LDH function affecting intracellular NADH distribution and eliminating the primary route for NADH oxidation. The redox imbalance that resulted from the elimination of the main NAD + -regeneration pathway affected cellular metabolism and thus impaired growth rates2021.…”