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@article{Ehsaan:2016:10.1186/s13068-015-0410-0,
author = {Ehsaan, M and Kuit, W and Zhang, Y and Cartman, ST and Heap, JT and Winzer, K and Minton, NP},
doi = {10.1186/s13068-015-0410-0},
journal = {Biotechnology for Biofuels},
pages = {4--4},
title = {Mutant generation by allelic exchange and genome resequencing of the biobutanol organism Clostridium acetobutylicum ATCC 824},
url = {http://dx.doi.org/10.1186/s13068-015-0410-0},
volume = {9},
year = {2016}
}

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TY  - JOUR
AB  - BACKGROUND: Clostridium acetobutylicum represents a paradigm chassis for the industrial production of the biofuel biobutanol and a focus for metabolic engineering. We have previously developed procedures for the creation of in-frame, marker-less deletion mutants in the pathogen Clostridium difficile based on the use of pyrE and codA genes as counter selection markers. In the current study we sought to test their suitability for use in C. acetobutylicum. RESULTS: Both systems readily allowed the isolation of in-frame deletions of the C. acetobutylicum ATCC 824 spo0A and the cac824I genes, leading to a sporulation minus phenotype and improved transformation, respectively. The pyrE-based system was additionally used to inactivate a putative glycogen synthase (CA_C2239, glgA) and the pSOL1 amylase gene (CA_P0168, amyP), leading to lack of production of granulose and amylase, respectively. Their isolation provided the opportunity to make use of one of the key pyrE system advantages, the ability to rapidly complement mutations at appropriate gene dosages in the genome. In both cases, their phenotypes were restored in terms of production of granulose (glgA) and amylase (amyP). Genome re-sequencing of the ATCC 824 COSMIC consortium laboratory strain used revealed the presence of 177 SNVs and 49 Indels, including a 4916-bp deletion in the pSOL1 megaplasmid. A total of 175 SNVs and 48 Indels were subsequently shown to be present in an 824 strain re-acquired (Nov 2011) from the ATCC and are, therefore, most likely errors in the published genome sequence, NC_003030 (chromosome) and NC_001988 (pSOL1). CONCLUSIONS: The codA or pyrE counter selection markers appear equally effective in isolating deletion mutants, but there is considerable merit in using a pyrE mutant as the host as, through the use of ACE (Allele-Coupled Exchange) vectors, mutants created (by whatever means) can be rapidly complemented concomitant with restoration of the pyrE allele. This avoids the phenotypic eff
AU  - Ehsaan,M
AU  - Kuit,W
AU  - Zhang,Y
AU  - Cartman,ST
AU  - Heap,JT
AU  - Winzer,K
AU  - Minton,NP
DO  - 10.1186/s13068-015-0410-0
EP  - 4
PY  - 2016///
SN  - 1754-6834
SP  - 4
TI  - Mutant generation by allelic exchange and genome resequencing of the biobutanol organism Clostridium acetobutylicum ATCC 824
T2  - Biotechnology for Biofuels
UR  - http://dx.doi.org/10.1186/s13068-015-0410-0
UR  - http://www.ncbi.nlm.nih.gov/pubmed/26732067
UR  - http://hdl.handle.net/10044/1/32825
VL  - 9
ER  -

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