Development of genetic modification tools to enable bioengineering of clostridial species for improved healthcare, chemical commodity and biofuel manufacture.
Submitting InstitutionUniversity of Nottingham
Unit of AssessmentBiological Sciences
Summary Impact TypeTechnological
Research Subject Area(s)
Biological Sciences: Genetics
Technology: Medical Biotechnology
Medical and Health Sciences: Medical Microbiology
Summary of the impact
Bacteria of the Clostridium genus are of pathogenic, medical and
industrial importance. Development by University of Nottingham School of
Life Science researchers of three patented methods for genetic
manipulation of clostridial species has led to licensing agreements for
commercial exploitation of the methodology to enhance strains for chemical
commodity and biofuel production and for targeted cancer therapy. These
methods are providing significant world-wide impact by facilitating
commercial R&D investment and technology developments in fields
ranging from healthcare, through chemicals manufacture, to the
Historically, the bacterial genus Clostridium is most often
associated with debilitating and life-threatening diseases such as
botulism, tetanus, gangrene, antibiotic associated diarrhoea and food
poisoning. However, the vast majority of clostridia are entirely benign,
and far from being the scourge of humankind, may well be its saviour.
Clostridia occupy many specialised biological niches and have evolved a
plethora of bio-catalytic abilities that can be exploited for humankind's
benefit. Many species are being pursued as cell factories for production
of biofuels and chemicals through alternative processes to traditional
petro-chemical routes. Yet others are being explored as tumour delivery
vehicles for anti-cancer drugs.
Tools for manipulating clostridial genomes (for gene knock-out and
knock-in) are essential for generating the functional knowledge needed to
combat clostridial pathogens, and for genetic enhancement of
strains used in chemical production and cancer therapy. Without those
tools, in the 20 years prior to breakthrough research at Nottingham, just
five mutants had ever been made in the biobutanol organism C.acetobutylicum,
only one in the pathogen C.difficile and none in the pathogens C.botulinum
or C.sporogenes. The majority of these mutants were made by single
cross-over plasmid insertion, and consequently were genetically unstable
due to plasmid excision.
The problem of achieving stable integration was solved in 2006 by
Professor Nigel Minton's Clostridia Research Group, under a combination of
BBSRC, MRC and industrial funding, by developing an intron retargeting
system, the ClosTron1,2. The engineered plasmids (eg.
pMTL007C-E2) encode a mobile group II intron that can be directed to
insert into virtually any target DNA substrate by simply making a handful
of nucleotide changes to the intron encoding region. Through the use of a
Retrotransposition-Activated Marker (RAM) based on the ermB gene,
successful insertion is selected by acquisition of erythromycin or
lincomycin resistance. The necessary intron changes are designed using a
`free to access' online re-targeting algorithm at a purpose built website
(www.clostron.com). Sequences can
be ordered through this website with DNA2.0 (www.dna20.com)
for both synthesis of the retargeted region and its cloning into
the ClosTron vector. The paper describing the ClosTron1 is the
most cited paper in the Journal of Microbiology Methods since its
publication in 2007. ClosTron has revolutionised clostridial molecular
biology3 and it is now the most widely used gene tool within
the clostridial community. A patent protecting the system was filed in
However, the ClosTron tool has a significant drawback: it is an
insertional mutagen and can therefore cause detrimental genetic effects.
To overcome this limitation, in 2009-2010, new recombination-based methods
were devised. Of most significance is Allele-Coupled Exchange (ACE) which
allows the insertion of DNA fragments of any size or complexity into
clostridial genomes4. Following integration of the ACE plasmid
by single-crossover recombination, the system is designed so that the
second recombination event leads to a plasmid borne allele becoming
`coupled' to a genome located allele, generating a new selectable allele
for isolation of double-crossover cells. Use of highly asymmetric homology
arms dictates the order of recombination events. Moreover, ACE generates
mutants of a heterologous pyrE gene5 or another marker, CodA7
subsequently developed at Nottingham, that can be used as negative
selection markers in classical allelic exchange strategies. ACE and CodA
patents were filed prior to publication, in 200910 and 201011.
To exemplify the ease, rapidity and iterative capability of ACE to insert
and extend large operon sequences, the entire lambda genome (48.5 kb) was
inserted into the C. acetobutylicum genome through the sequential
delivery of three overlapping fragments of 28, 18 and 6.5 kb4.
ACE has most recently been used within the BBSRC Sustainable BioEnergy
Centre (BSBEC) to introduce genes encoding components (scaffold &
hydrolases) capable of degrading cellulose into the genome of the
biobutanol organism, C. acetobutylicum6.
Significantly, ACE has now been extended to [text removed for
publication] Geobacillus, allowing [text removed for publication]
strain to be recreated in just 30 days by 3 simple genetic manipulations.
These technologies, therefore, translate beyond Clostridium
References to the research
Publications (UoN authors in bold, key author(s) underlined)
1. Heap JT, Pennington OJ, Cartman ST, Carter
GP, Minton NP (2007). The ClosTron: A universal gene
knock-out system for the genus Clostridium. J Microbiol
Methods. 70: 452-64. doi: 10.1016/j.mimet.2007.05.021
2. Heap JT, Kuehne SA, Ehsaan M, Cartman ST,
Cooksley CM, Scott JC, Minton NP. (2010) The
ClosTron: Mutagenesis in Clostridium refined and streamlined. J
Microbiol Methods. 78: 79-85. doi:
3. Kuehne SA, Cartman ST, Heap JT, Kelly M,
Cockayne A and Minton NP (2010) The role of toxin A and
toxin B in Clostridium difficile infection. Nature 467(7316):
711-713. doi:10.1038/nature09397. Included in REF2.
4. Heap JT, Ehsaan M, Cooksley CM, Ng Y-K,
Cartman ST, Winzer K, Minton NP (2012)
Integration of DNA into bacterial chromosomes from plasmids without a
counter-selection marker. Nucleic Acids Research 40(8) e59;
doi:10.1093/nar/gkr1321. Included in REF2.
5. Ng YK, Ehsaan M, Philip S, Collery MM, Janoir C, Collignon A,
Cartman ST, Minton NP. (2013) Expanding the
repertoire of gene tools for precise manipulation of the Clostridium
difficile genome: allelic exchange using pyrE alleles. PLoS
One 8(2):e56051. doi: 10.1371/journal.pone.0056051
6. Kovács K, Willson BJ, Schwarz K, Heap JT, Jackson A, Bolam DN,
Winzer K, Minton NP (2013) Secretion and assembly of
functional mini-cellulosomes from synthetic chromosomal operons in Clostridium
acetobutylicum ATCC 824. Biotechnol Biofuels 6(1):117-130.
7. CartmanST, Kelly ML, Heeg D, Heap JT,
Minton NP. (2012) Precise manipulation of the Clostridium
difficile chromosome reveals a lack of association between the tcdC
genotype and toxin production. Appl Environ Microbiol 78(13):4683-4690.
8. Bradshaw M, Marshall KM, Heap JT, Tepp WH, Minton NP,
Johnson EA (2010) Construction of a nontoxigenic Clostridium botulinum
strain for food challenge studies. Appl Environ Microbiol 76(2)
9. Heap JT and Minton NP (2007) DNA Molecules and
Methods; WO2007148091; Morvus Technology Ltd, published 27 Dec 2007.
10. Heap JT, Minton NP (2009) Methods;
WO/2009/101400; University of Nottingham; Published 20 Aug 2009
11. Cartman ST, Minton NP (2010) Method of double
crossover homologous recombination in Clostridia; WO/2010/084349;
University of Nottingham; published 29 July 2010.
Grant Funding from 2006 to date:
BBSRC: BB/D522289/1, 2006-2009, £199,304; BB/D522797/1,
2006-2009, £187,191; BB/F003390/1, 2007-2010, £364,436;
BB/E021271/1, 2007-2011, £643,519; BB/G017395/1, 2009-2013, £74,410;
BB/G530341/1, 2009-2013, £26,518; BB/G016224/1, 2009-2014, £2,127,704;
BB/I004475/1, 2010-2013, £452,694; KTP with Green Biologics,
2011-2013, £47,514; BB/L004356/1, TSB SynBio, 2013-2014, £109,539;
BB/L000105/1, ERANET, 2013-2016, £147,227; BB/K020358/1, RICEFUEL,
2013-2016, £1,393,966; BB/J020427/1, India, 2012-2016, £13,692;
BB/L01081X/1, China, 2013-2017, £26,518; BB/K00283X/1, GASCHEM,
MRC: Programme grant on C. difficile virulence (G0601176),
EU FP7: HEALTH-F3-2008-223585; 2008-2011; £632,229; People
Programme 2009 — 2013, £867,641; Marie Curie £100,473.
CLOSTNET: €5 million with Minton as coordinator.
£2.5 million from pharma, biotech, biofuel and chemical commodity
companies; 2009 onwards.
Details of the impact
Impact 1: Technology Transfer, Out-Licensing and Commercial Exploitation
Since publication of the patents, Material Transfer Agreements have been
established with over 250 research and industrial institutions worldwideA
to allow distribution of ClosTron, ACE and CodA reagents. The technologies
have been licensed through the university to 5 companies worldwideA,
with other companies accessing the technologies by collaborative research
with Minton and the Clostridial Research Group. This has enabled further
commercial investment and exploitation by the companies, bringing
financial benefits to them and the university. In addition, patent search
has identified 15 patents published by other research and development
groups that cite use of ClosTron to achieve their inventive step(s)B.
These include genetic manipulations for production of biofuels (including
LanzaTech, Total S.A., Qteros, IFP Energies and TetraVitae) and vaccines
(Wyeth/Pfizer, Altermune and London School of Hygiene and Tropical
Medicine). All these businesses and other organisations are beneficiaries
of the university's research.
Impact 2: Reducing clostridial infection and food contamination
Knowledge of C.difficile and C.botulinum virulence
mechanisms is being enhanced through the generation by Minton's group, and
others, of virulence mutants and their phenotypic characterisation. For
instance, strains of C.difficile expressing recombinant
immunogenic but non-toxigenic toxoids A and B, generated using ClosTron by
Wyeth (subsequently acquired by Pfizer)C have led to
development of a C.difficile vaccine to prevent hospital acquired
infections (currently in Phase 1; http://www.clinicaltrials.gov/ct2/show/NCT01706367).
Wyeth/Pfizer have benefitted from the university's research by being able
to develop the vaccine. Further tangible benefits will arise worldwide for
patients suffering from C.difficile associated infections
(approximately 350,000 in the USD and 15,000 in the UK annuallyE).
The technology will also help to reduce associated deaths (14,000 in the
USD and 2,000 in the UKF annually), and reduce the
estimated annual healthcare burden (~$5 billion USD annually). Similarly,
generation of a non-toxigenic strain of C.botulinum for use by the
food industry as a food processing challenge test strain8
overcomes strict regulations on containment and use of highly toxigenic
strains of C.botulinum, benefitting manufacturers by reducing
costs of food testing, contamination and wastage, as well as reducing
costs and improving food safety for consumers.
Impact 3: Novel anti-cancer therapeutic approaches
The anaerobic requirement of C.sporogenes can be used to target
the poorly vascularised centre of a solid tumour mass; early studies
injecting unmodified spores induced partial tumour lysis but showed
systemic toxicity. Development of non-toxigenic C.novyi-NT spores
avoided this toxicity in Phase I trials in 2011 (http://www.clinicaltrials.gov/ct2/show/NCT01118819),
but did not cause complete tumour lysis. Transfecting C.sporogenes
with an E.coli enzyme (via single cross-over plasmid insertion) to
locally activate a prodrug, enhanced tumour lysis (Lui et al. 2002; Gene
Therapy 9: 291-296. doi: 10.1038/sj/gt/3301659). This novel anti-cancer
treatment method, termed `Clostridial-directed Enzyme Prodrug Therapy'
(CDEPT) was patented in 2002 (US20030103952, with Minton as an inventor).
It offers improved, targeted oncolytic efficacy and significantly reduced
side effectsG. However, Regulatory Authority requirements
stipulate that genetic modifications of bacteria that encode therapeutic
enzymes must be stably integrated to prevent gene loss in clinical use.
The knock-in capability of ACE was used for the first time in 20127
to meet this requirement, opening up the pathway to achieve significant
impacts in cancer therapy.
Impact 4: Bioengineering benefits
The capability of these Clostridia genetic manipulation tools is
proving pivotal to gaining a greater understanding of metabolic networks
present in clostridia that are useful in chemical commodity production, as
well as in metabolic engineering approaches to improve product yield.
Industrial exploitation of ACE technology, under licence from the
university, is most visible in development of second generation biofuels.
TMO RenewablesH are engineering C.acetobutylicum and C.thermolyticum
to produce butanol and ethanol from cellulosic and lignocellulosic
feedstock, whereas LanzaTech are developing a microbial fermentation
process for conversion of carbon dioxide to ethanol. TMO Renewables
has a current book value of £10.9 millionH and has recently
signed a $500 million order to build 15 factories across the US to produce
ethanol from household wasteH. Similarly, LanzaTech has
received in excess of $70 million of government and private funding, and
has recently joined a biofuels collaboration with the Indian government
and the Indian Oil CorporationI. Both companies aim to address
the worldwide demand for ethanol (principally as a petroleum blending
additive) which is projected to be nearly 200 billion litres annually by
2022J. First generation production capacity (from maize and
sugar cane fermentation) is currently only 50 billion litres. The impact
of using clostridial genetic engineering tools to expand biofuel and
chemicals production, without impinging on use of agricultural land, not
only benefits the university through technology license fees, and the
companies through access to the technology and revenues generated from it,
but also has significant worldwide environmental benefit.
The beneficiaries of the clostridial bioengineering technology developed
at Nottingham, as a consequence of its myriad of applications, are
extremely diverse and its subsequent benefits far reaching. Its
availability has revolutionised the genetic approaches being undertaken by
academic and industrial researchers alike, aimed at understanding
pathogenic and commercially-important clostridial species through
mutational analysis. It has implications for disease prevention and
treatment, cancer therapy and chemical commodity and biofuels production.
It therefore has direct and consequential impacts in Health, Energy and,
through the generation of sustainable biofuels, the Environment.
Sources to corroborate the impact
A. Numbers of MTAs assigned and licences awarded can be confirmed under
confidentiality by Business Engagement and Innovation Services, University
B. PCT Patent search performed on http://patentscope.wipo.int/search/en/search.jsf
using Full Text search term: EN_ALLTXT:(ClosTron)
D. C.diff infections and deaths in the US 2012: http://www.cdc.gov/hai/eip/pdf/Cdiff-factsheet.pdf
E. Hospital Acquired Infections in the UK 2012:
F. C. difficile deaths, England & Wales 2011: http://www.ons.gov.uk/ons/dcp171778_276892.pdf
G. Wei et al (2008) Cancer Letters 259: 16-27; doi:
J. Global Biofuels predicted markets: http://unctad.org/en/Docs/ditcbcc20091_en.pdf
— in particular Chapter I, pp1-6 for projected ethanol demand and
production capacities and Chapter III, pp29-50 for agricultural benefits
of second generation biofuels.
Corroborative documents and copies of webpages are held on file and are
available on request.