A roadmap for gene system development in Clostridium

Minton, Nigel P. and Ehsaan, Muhammad and Humphreys, Christopher M. and Little, Gareth T. and Baker, Jonathan and Henstra, Anne M. and Liew, Fungmin and Kelly, Michelle and Sheng, Lili and Schwarz, Katrin and Zhang, Ying (2016) A roadmap for gene system development in Clostridium. Anaerobe, 41 . pp. 104-112. ISSN 1095-8274

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Abstract

Clostridium species are both heroes and villains. Some cause serious human and animal diseases, those present in the microbiota contribute to health and wellbeing, while others represent useful industrial chassis for the production of chemicals and fuels. To understand, counter or exploit, there is a fundamental requirement for effective systems that may be used for directed or random genome modifications. We have formulated a simple roadmap whereby the necessary gene systems maybe developed and deployed. At its heart is the use of 'pseudo-suicide' vectors and the creation of a pyrE mutant (a uracil auxotroph), initially aided by ClosTron technology, but ultimately made using a special form of allelic exchange termed ACE (Allele-Coupled Exchange). All mutants, regardless of the mutagen employed, are made in this host. This is because through the use of ACE vectors, mutants can be rapidly complemented concomitant with correction of the pyrE allele and restoration of uracil prototrophy. This avoids the phenotypic effects frequently observed with high copy number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. Once available, the pyrE host may be used to stably insert all manner of application specific modules. Examples include, a sigma factor to allow deployment of a mariner transposon, hydrolases involved in biomass deconstruction and therapeutic genes in cancer delivery vehicles. To date, provided DNA transfer is obtained, we have not encountered any clostridial species where this technology cannot be applied. These include, Clostridium difficile, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium botulinum, Clostridium perfringens, Clostridium sporogenes, Clostridium pasteurianum, Clostridium ljungdahlii, Clostridium autoethanogenum and even Geobacillus thermoglucosidasius.

Item Type: Article
Keywords: Restriction modification; gene transfer; ClosTron; allelic exchange; counterselection marker; pyrE; knock-out; knock-in; Fluoroorotic acid
Schools/Departments: University of Nottingham UK Campus > Faculty of Medicine and Health Sciences > School of Medicine > Nottingham Digestive Diseases Centre
University of Nottingham UK Campus > Faculty of Medicine and Health Sciences > School of Life Sciences
Identification Number: https://doi.org/10.1016/j.anaerobe.2016.05.011
Depositing User: Eprints, Support
Date Deposited: 23 Aug 2016 16:23
Last Modified: 16 Oct 2016 02:56
URI: http://eprints.nottingham.ac.uk/id/eprint/35934

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