Development of an electrotransformation protocol for genetic manipulation of Clostridium pasteurianum

Biotechnology for Biofuels

Michael E Pyne¹  Murray Moo-Young¹  Duane A Chung²  C Perry Chou¹ 

[1] Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, ON, N2L 3G1, Canada

[2] Centurion Biofuels, Corp., Rm. 5113 Michael G. DeGroote Centre for Learning and Discovery, 1280 Main Street West, Hamilton, ON, L8S 4K1, Canada

关键词: Transformation;    Restriction;    Methylation;    Glycerol;    Genetic engineering;    Electroporation;    Clostridium pasteurianum;    Biobutanol;    Butanol;    Biofuels;   

Others  :  798104
DOI  :  10.1186/1754-6834-6-50

 received in 2013-01-11, accepted in 2013-04-04,  发布年份 2013

【 摘 要 】

Background

Reducing the production cost of, and increasing revenues from, industrial biofuels will greatly facilitate their proliferation and co-integration with fossil fuels. The cost of feedstock is the largest cost in most fermentation bioprocesses and therefore represents an important target for cost reduction. Meanwhile, the biorefinery concept advocates revenue growth through complete utilization of by-products generated during biofuel production. Taken together, the production of biofuels from low-cost crude glycerol, available in oversupply as a by-product of bioethanol production, in the form of thin stillage, and biodiesel production, embodies a remarkable opportunity to advance affordable biofuel development. However, few bacterial species possess the natural capacity to convert glycerol as a sole source of carbon and energy into value-added bioproducts. Of particular interest is the anaerobe Clostridium pasteurianum, the only microorganism known to convert glycerol alone directly into butanol, which currently holds immense promise as a high-energy biofuel and bulk chemical. Unfortunately, genetic and metabolic engineering of C. pasteurianum has been fundamentally impeded due to lack of an efficient method for deoxyribonucleic acid (DNA) transfer.

Results

This work reports the development of an electrotransformation protocol permitting high-level DNA transfer to C. pasteurianum ATCC 6013 together with accompanying selection markers and vector components. The CpaAI restriction-modification system was found to be a major barrier to DNA delivery into C. pasteurianum which we overcame by in vivo methylation of the recognition site (5’-CGCG-3’) using the M.FnuDII methyltransferase. With proper selection of the replication origin and antibiotic-resistance marker, we initially electroporated methylated DNA into C. pasteurianum at a low efficiency of 2.4 × 10¹ transformants μg^-1 DNA by utilizing conditions common to other clostridial electroporations. Systematic investigation of various parameters involved in the cell growth, washing and pulse delivery, and outgrowth phases of the electrotransformation procedure significantly elevated the electrotransformation efficiency, up to 7.5 × 10⁴ transformants μg^-1 DNA, an increase of approximately three order of magnitude. Key factors affecting the electrotransformation efficiency include cell-wall-weakening using glycine, ethanol-mediated membrane solubilization, field strength of the electric pulse, and sucrose osmoprotection.

Conclusions

C. pasteurianum ATCC 6013 can be electrotransformed at a high efficiency using appropriately methylated plasmid DNA. The electrotransformation method and tools reported here should promote extensive genetic manipulation and metabolic engineering of this biotechnologically important bacterium.

【 授权许可】

   
2013 Pyne et al.; licensee BioMed Central Ltd.

【 图 表 】

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