Clostridium Acetobutylicum

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All vectors carrying the ColE1 Gram-negative replicon region and the replication defective pIM13 Gram-positive replicon and catP selective marker catP. The modular integration cassettes, inserted between the SbfI and AscI sites, generally comprise a long (1200 bp) right hand homology arm (RHA) and a shorter (300 bp) left homology arm (LHA).

[1] pyrE-based Vectors

Correction Vector: pMTL-ME6
The 300 bp and 1200 bp regions are in effect a continuous 1500 bp region of homology to the host chromosome from the pyrE locus.

Complementation Vector: pMTL-ME6C
The RHA is composed of the 3′-end of the pyrE gene, while the LHA comprises the 1200 bp region of the chromosome from immediately downstream of pyrE gene encoding CAC0028. The two HAs are separated by a region of DNA comprising lacZ containing multiple cloning site (MCS) region and in which a region of DNA encompassing the transcriptional terminator of the Clostridium pasteurianum ferredoxin gene has been inserted 3’ to the pyrE allele, and preceding CAC0028.

Constitutive Expression Vector: pMTL-ME6X
Compared to pMTL-ME6C, additionally contains a strong promoter (Pfdx) from the C. sporogenes ferredoxin (fdx) gene immediately before the lacZ′ gene.


Orthoganol Expression Vector: pMTL-ME6C-tedR
Equivalent to pMTL-ME6 in which the Pfdx promoter has been replaced with the promoter of the tcdB gene of Clostridoides difficile. This promoter is specific to the C. difficile TcdR sigma factor and is, therefore, not effectively recognised by E.coli sigma factors.

Inducible Expression Vector: pMTL-HZ13-HZ-tcdR
Equivalent to pMTL-ME6, but in this case the Pfdx promoter has been replaced with an lactose-inducible, expression cassette derived from C. perfringens comprising the bgaR gene and PbgaL promoter of the bgaL gene [1].

[2] Other Complementation Vectors

Equivalent regions of the pheA, argH and purD loci were PCR amplified using primers that incorporated the necessary restriction endonuclease recognition sites, and the DNA fragment generated following their cleavage with either SbfI and NotI (LHA) or NdeI and AscI (RHA) cloned in place of the pyrE regions of pMTL-ME6C [2]. Available vectors are:-

  • pMTL-HZ1C (pheA)
  • pMTL-HZ2C (argH)
  • pMTL-BO1C (purD)


[1] Woods C, Humphreys CM, Tomi-Andrino C, Henstra AM, Köpke M, Simpson SD, Winzer K, Minton NP. Required Gene Set for Autotrophic Growth of Clostridium autoethanogenum. Appl Environ Microbiol. 2022 Mar 14:e0247921.

[2] Omorotionmwan BB, Wang H, Baker JP, Gizynski K, Yoo M, Akaluka C, Zhang Y, Minton NP. Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum. Front Bioeng Biotechnol. 2023;11:1218099.