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. 2018 Sep 27:11:264.
doi: 10.1186/s13068-018-1260-3. eCollection 2018.

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

Ching-Ning Huang  1 , Wolfgang Liebl  1 , Armin Ehrenreich  1
Affiliations

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

Ching-Ning Huang et al. Biotechnol Biofuels. .

Abstract

Background: Clostridium 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.

Results: In 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.

Conclusions: The 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.

Keywords: 5-Fluorocytosine; Butyrate kinase; CodB/codA; Phosphotransbutyrylase conjugation; Xylulose kinase.

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Figures

Fig. 1
Fig. 1
Schematic representation of deletion vector construction. a pCN3, a shuttle vector for C. saccharobutylicum NCP262 in which the antibiotic cassette of pKVM4 is replaced by the catP gene from pJIR750. b pCN6, a suicide vector to delete the hsdR1 gene, where the pE194ts replicon is replaced by hsdR1 homologous arms. c pCN8, where the homologous arms of pCN6 are replaced by those hsdR2. d pChN1, a deletion vector for the hsdR2 where the codBA operon genes of pCN8 are replaced by those from C. ljungdahlii. e pChN, a deletion vector cassette produced by removing the hsdR2 homologous arms from pChN1
Fig. 2
Fig. 2
Gene replacement via allelic exchange at the hsdR1, hsdR2, hsdR3, xylB, and ptb–buk loci. PCR confirmation of the different double-crossover deletion mutants using external primers annealing to the chromosome upstream and downstream of each deletion cassette. Strains (a) ΔhsdR1. b ΔhsdR1 ΔhsdR2. c ΔhsdR1 ΔhsdR2 ΔhsdR3. d ΔhsdR1 ΔhsdR2 ΔxylB. e ΔhsdR1 ΔhsdR2 Δptb Δbuk. ΔhsdR1: 2141 bp (a, b, c, d, e), WT of hsdR1: 5553 bp (a), catP gene: 622 bp (a, b, c, d, e). ΔhsdR2: 2064 bp (b, c, d, e), WT of hsdR2: 5259 bp (b) ΔhsdR3: 2078 bp (c), WT of hsdR3: 5010 bp (c). ΔxylB: 2081 bp (d), WT of xylB: 3549 bp (d). Δptb Δ buk: 2042 bp (e), and WT of ptb–buk: 4026 bp (e)
Fig. 3
Fig. 3
Growth of C. saccharobutylicum Ch1 (a) and C. saccharobutylicum Ch1 ΔxylB (b) on different carbon sources. Cells were grown in 30 ml of MES-MM supplemented with 0.001% yeast extract and 40 g/l d-glucose (black circle), 40 g/l l-Arabinose (black square) or 40 g/l d-xylose (white up-pointing triangle)
Fig. 4
Fig. 4
General diagram representing gene replacement via allelic exchange at the target gene. a C. saccharobutylicum NCP262 genomic regions surrounding CLSA_RS14125 (hsdR2). The deletion vector pChN1, containing approximately 1 kbp of upstream and downstream sequences of hsdR2 and the codBA operon from C. ljungdahlii. b Counterselection strategy with the 5-FC/codBA system resulting in a marker-less deletion mutant lacking CLSA_RS14125 (hsdR2) between the two flanking regions
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