Cargando…

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

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Bosma, Elleke F, van de Weijer, Antonius H P, van der Vlist, Laurens, de Vos, Willem M, van der Oost, John, van Kranenburg, Richard
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4494709/
https://www.ncbi.nlm.nih.gov/pubmed/26148486
http://dx.doi.org/10.1186/s12934-015-0286-5
_version_ 1782380142236008448
author Bosma, Elleke F
van de Weijer, Antonius H P
van der Vlist, Laurens
de Vos, Willem M
van der Oost, John
van Kranenburg, Richard
author_facet Bosma, Elleke F
van de Weijer, Antonius H P
van der Vlist, Laurens
de Vos, Willem M
van der Oost, John
van Kranenburg, Richard
author_sort Bosma, Elleke F
collection PubMed
description 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-015-0286-5) contains supplementary material, which is available to authorized users.
format Online
Article
Text
id pubmed-4494709
institution National Center for Biotechnology Information
language English
publishDate 2015
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-44947092015-07-08 Establishment of markerless gene deletion tools in thermophilic Bacillus smithii and construction of multiple mutant strains Bosma, Elleke F van de Weijer, Antonius H P van der Vlist, Laurens de Vos, Willem M van der Oost, John van Kranenburg, Richard Microb Cell Fact Research 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-015-0286-5) contains supplementary material, which is available to authorized users. BioMed Central 2015-07-07 /pmc/articles/PMC4494709/ /pubmed/26148486 http://dx.doi.org/10.1186/s12934-015-0286-5 Text en © Bosma et al. 2015 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Bosma, Elleke F
van de Weijer, Antonius H P
van der Vlist, Laurens
de Vos, Willem M
van der Oost, John
van Kranenburg, Richard
Establishment of markerless gene deletion tools in thermophilic Bacillus smithii and construction of multiple mutant strains
title Establishment of markerless gene deletion tools in thermophilic Bacillus smithii and construction of multiple mutant strains
title_full Establishment of markerless gene deletion tools in thermophilic Bacillus smithii and construction of multiple mutant strains
title_fullStr Establishment of markerless gene deletion tools in thermophilic Bacillus smithii and construction of multiple mutant strains
title_full_unstemmed Establishment of markerless gene deletion tools in thermophilic Bacillus smithii and construction of multiple mutant strains
title_short Establishment of markerless gene deletion tools in thermophilic Bacillus smithii and construction of multiple mutant strains
title_sort establishment of markerless gene deletion tools in thermophilic bacillus smithii and construction of multiple mutant strains
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4494709/
https://www.ncbi.nlm.nih.gov/pubmed/26148486
http://dx.doi.org/10.1186/s12934-015-0286-5
work_keys_str_mv AT bosmaellekef establishmentofmarkerlessgenedeletiontoolsinthermophilicbacillussmithiiandconstructionofmultiplemutantstrains
AT vandeweijerantoniushp establishmentofmarkerlessgenedeletiontoolsinthermophilicbacillussmithiiandconstructionofmultiplemutantstrains
AT vandervlistlaurens establishmentofmarkerlessgenedeletiontoolsinthermophilicbacillussmithiiandconstructionofmultiplemutantstrains
AT devoswillemm establishmentofmarkerlessgenedeletiontoolsinthermophilicbacillussmithiiandconstructionofmultiplemutantstrains
AT vanderoostjohn establishmentofmarkerlessgenedeletiontoolsinthermophilicbacillussmithiiandconstructionofmultiplemutantstrains
AT vankranenburgrichard establishmentofmarkerlessgenedeletiontoolsinthermophilicbacillussmithiiandconstructionofmultiplemutantstrains