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An efficient method for markerless mutant generation by allelic exchange in Clostridium acetobutylicum and Clostridium saccharobutylicum using suicide vectors

Authors :
Tom Wilding-Steel
Armin Ehrenreich
Isabelle Meynial-Salles
Minyeong Yoo
Julie Soula
Céline Foulquier
Ching-Ning Huang
Axel Thiel
Wolfgang Liebl
Philippe Soucaille
Ngoc-Phuong-Thao Nguyen
Toulouse Biotechnology Institute (TBI)
Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse)
Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)
Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM)
Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP)
Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse)
Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA)
Tan Tao University (TTU)
University of Nottingham, UK (UON)
613802, KBBE Valor Plus
European Project: 237942,EC:FP7:PEOPLE,FP7-PEOPLE-ITN-2008,CLOSTNET(2009)
Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)
Source :
Biotechnology for Biofuels 1 (12), 1-13. (2019), Biotechnology for Biofuels, Biotechnology for Biofuels, BioMed Central, 2019, 12 (1), pp.1-13. ⟨10.1186/s13068-019-1364-4⟩, Biotechnology for Biofuels, Vol 12, Iss 1, Pp 1-13 (2019), Biotechnology for Biofuels, 2019, 12 (1), pp.1-13. ⟨10.1186/s13068-019-1364-4⟩
Publication Year :
2019

Abstract

Background: Clostridium acetobutylicum and Clostridium saccharobutylicum are Gram-positive, spore-forming, anaerobic bacterium capable of converting various sugars and polysaccharides into solvents (acetone, butanol, and ethanol). The sequencing of their genomes has prompted new approaches to genetic analysis, functional genomics, and metabolic engineering to develop industrial strains for the production of biofuels and bulk chemicals.Results: The method used in this paper to knock-out, knock-in, or edit genes in C. acetobutylicum and C. saccharobutylicum combines an improved electroporation method with the use of (i) restrictionless Δupp (which encodes uracil phosphoribosyl-transferase) strains and (ii) very small suicide vectors containing a markerless deletion/insertion cassette, an antibiotic resistance gene (for the selection of the first crossing-over) and upp (from C. acetobutylicum) for subsequent use as a counterselectable marker with the aid of 5-fluorouracil (5-FU) to promote the second crossing-over. This method was successfully used to both delete genes and edit genes in both C. acetobutylicum and C. saccharobutylicum. Among the edited genes, a mutation in the spo0A gene that abolished solvent formation in C. acetobutylicum was introduced in C. saccharobutylicum and shown to produce the same effect.Conclusions: The method described in this study will be useful for functional genomic studies and for the development of industrial strains for the production of biofuels and bulk chemicals.

Details

Language :
English
ISSN :
17546834
Database :
OpenAIRE
Journal :
Biotechnology for Biofuels 1 (12), 1-13. (2019), Biotechnology for Biofuels, Biotechnology for Biofuels, BioMed Central, 2019, 12 (1), pp.1-13. ⟨10.1186/s13068-019-1364-4⟩, Biotechnology for Biofuels, Vol 12, Iss 1, Pp 1-13 (2019), Biotechnology for Biofuels, 2019, 12 (1), pp.1-13. ⟨10.1186/s13068-019-1364-4⟩
Accession number :
edsair.doi.dedup.....716fb1a8a8176ba7a0888503295ab68d
Full Text :
https://doi.org/10.1186/s13068-019-1364-4⟩