Foulquier et al. Biotechnol Biofuels (2019) 12:31 https://doi.org/10.1186/s13068-019-1364-4 RESEARCH An efcient method for markerless mutant generation by allelic exchange in Clostridium acetobutylicum and Clostridium saccharobutylicum using suicide vectors Celine Foulquier 1† , Ching‑Ning Huang 2† , Ngoc‑Phuong‑Thao Nguyen 1,3 , Axel Thiel 1 , Tom Wilding‑Steel 1 , Julie Soula 1 , Minyeong Yoo 1,4 , Armin Ehrenreich 2 , Isabelle Meynial‑Salles 1 , Wolfgang Liebl 2 and Philippe Soucaille 1,4* 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. saccha- robutylicum 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/inser‑ tion cassette, an antibiotic resistance gene (for the selection of the frst crossing‑over) and upp (from C. acetobutyli- cum) for subsequent use as a counterselectable marker with the aid of 5‑fuorouracil (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 efect. Conclusions: The method described in this study will be useful for functional genomic studies and for the develop‑ ment of industrial strains for the production of biofuels and bulk chemicals. Keywords: Clostridium acetobutylicum, Clostridium saccharobutylicum, upp gene, 5‑FU, Restrictionless, Markerless, Gene deletion, Gene replacement © The Author(s) 2019. This 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. Background In recent years, solventogenic Clostridia have been of interest in the postgenomic era due to the complete sequencing and annotation of their genome [1, 2], sup- plying a wealth of information regarding the metabo- lism of these industrially important strains. Tis global knowledge has prompted new approaches to genetic analysis, functional genomics, and metabolic engineering to develop industrial strains for the production of biofu- els and bulk chemicals. To this end, several reverse genetic tools have been developed for solventogenic Clostridia, including gene inactivation systems based on nonreplicative [3–5] and replicative plasmids [6–10] and the group II intron gene inactivation system [11, 12]. All methods based on elec- troporation for in frame deletions use a replicative plas- mid (typically containing a pIMP13 origin of replication from Bacillus subtilis that is functional in Clostridia) Open Access Biotechnology for Biofuels *Correspondence: soucaille@insa‑toulouse.fr † Celine Foulquier and Ching‑Ning Huang contributed equally to this work 1 LISBP, INSA, University of Toulouse, 135 Avenue de Rangueil, 31077 Toulouse Cedex, France Full list of author information is available at the end of the article