Restriction-deficient mutants and marker-less genomic modification for metabolic engineering of the solvent producer Clostridium saccharobutylicum

Abstract

BackgroundClostridium saccharobutylicum NCP 262 is a solventogenic bacterium that has been used for the industrial production of acetone, butanol, and ethanol. The lack of a genetic manipulation system for C. saccharobutylicum currently limits (i) the use of metabolic pathway engineering to improve the yield, titer, and productivity of n-butanol production by this microorganism, and (ii) functional genomics studies to better understand its physiology.ResultsIn this study, a marker-less deletion system was developed for C. saccharobutylicum using the codBA operon genes from Clostridium ljungdahlii as a counterselection marker. The codB gene encodes a cytosine permease, while codA encodes a cytosine deaminase that converts 5-fluorocytosine to 5-fluorouracil, which is toxic to the cell. To introduce a marker-less genomic modification, we constructed a suicide vector containing: the catP gene for thiamphenicol resistance; the codBA operon genes for counterselection; fused DNA segments both upstream and downstream of the chromosomal deletion target. This vector was introduced into C. saccharobutylicum by tri-parental conjugation. Single crossover integrants are selected on plates supplemented with thiamphenicol and colistin, and, subsequently, double-crossover mutants whose targeted chromosomal sequence has been deleted were identified by counterselection on plates containing 5-fluorocytosine. Using this marker-less deletion system, we constructed the restriction-deficient mutant C. saccharobutylicum ΔhsdR1ΔhsdR2ΔhsdR3, which we named C. saccharobutylicum Ch2. This triple mutant exhibits high transformation efficiency with unmethylated DNA. To demonstrate its applicability to metabolic engineering, the method was first used to delete the xylB gene to study its role in xylose and arabinose metabolism. Furthermore, we also deleted the ptb and buk genes to create a butyrate metabolism-negative mutant of C. saccharobutylicum that produces n-butanol at high yield.ConclusionsThe plasmid vectors and the method introduced here, together with the restriction-deficient strains described in this work, for the first time, allow for efficient marker-less genomic modification of C. saccharobutylicum and, therefore, represent valuable tools for the genetic and metabolic engineering of this industrially important solvent-producing organism.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1260-3) contains supplementary material, which is available to authorized users.

Screening and application of inducible promoters in Ruminiclostridium papyrosolvens

“…The lactose-inducible promoter Plac from C. perfringens has been used in Clostridium difficile, Clostridium beijerinckii (Hong et al 2018) and Clostridium ljungdahlii (Huang et al 2018). The arabinose-inducible promoter Para from C. acetobutylicum has also been applied in the ClosTron system for gene mutation of R. cellulolyticum.…”

A review on green liquid fuels for the transportation sector: a prospect of microbial solutions to climate change

“…Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 are the most studied solventogenic Clostridium species. Clostridium saccharobutylicum and Clostridium saccharoperbutylacetonium have been receiving attention recently for the production of large amounts of butanol during ABE fermentation (Dürre, 2005;Dong et al, 2018;Huang et al, 2018;Foulquier et al, 2019). Clostridium species such as Clostridium ljungdahlii and Clostridium butyricum have the capacity to utilize syngas and hemicellulose, respectively, for acetone and butanol production (Montoya et al, 2001).…”

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

“…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 electroporation for in frame deletions use a replicative plasmid (typically containing a pIMP13 origin of replication from Bacillus subtilis that is functional in Clostridia ) due to the low frequency of transformation of solventogenic Clostridia [13, 14].…”

“…Creating a method for the rapid deletion, insertion, or modification of genes would require the use of a small suicide vector (to improve the transformation efficiency by electroporation), a replacement cassette consisting of two sequences homologous to the selected regions around the target DNA sequence and a counter selection marker such as upp , codA, or pyrE . One way to increase the transformation efficiency of solventogenic Clostridia is to remove the restriction modification system naturally present in the bacterium [5, 10, 14, 19]. Restrictionless, markerless mutants of solventogenic Clostridia have already been constructed for two species, C. acetobutylicum [10] and C. saccharobutylicum [5].…”

“…One way to increase the transformation efficiency of solventogenic Clostridia is to remove the restriction modification system naturally present in the bacterium [5, 10, 14, 19]. Restrictionless, markerless mutants of solventogenic Clostridia have already been constructed for two species, C. acetobutylicum [10] and C. saccharobutylicum [5]. Although a transformation efficiency of 10 4 /μg DNA has previously been reported when using electroporation for a restrictionless mutant of C. acetobutylicum [10], the transformation efficiency of a restrictionless mutant of C. saccharobutylicum has not been measured [5].…”