Citrobacter freundii as a test platform for recombinant cellulose degradation systems.
Cellulosic biomass represents a huge reservoir of renewable carbon, but converting it into useful products is challenging. Attempts to transfer cellulose degradation capability to industrially useful micro-organisms have met with limited success, possibly due to poorly understood synergy between multiple cellulases. This is best studied by co-expression of many combinations of cellulases and associated proteins. Here, we describe the development of a test platform based on Citrobacter freundii, a cellobiose-assimilating organism closely related to Escherichia coli. Standard E. coli cloning vectors worked well in Cit. freundii. Expression of cellulases CenA and Cex of Cellulomonas fimi in Cit. freundii gave recombinant strains which were able to grow at the expense of cellulosic filter paper or microcrystalline cellulose (Avicel) in a mineral medium supplemented with a small amount of yeast extract. Periodic physical agitation of the cultures was highly beneficial for growth at the expense of filter paper. This provides a test platform for the expression of combinations of genes encoding biomass-degrading enzymes to develop effective genetic cassettes for degradation of different biomass streams.


CONCLUSIONS
Biofuels have been shown to be the best sustainable and alternative source of fuel to replace fossil fuels. Of the different types of feedstocks used for producing biofuels, lignocellulosic biomass is the most abundant. Converting this biomass to useful products has met with little success. Different approaches are being used and microbial platforms are the most promising and sustainable method. This study shows that Citrobacter freundii is a better test platform than Escherichia coli for testing various combinations of cellulases for the development of microbial systems for biomass conversion.
School of Biological Sciences, University of Edinburgh, Edinburgh, UK.|Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan.
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SS
Lakhundi
School of Biological Sciences, University of Edinburgh, Edinburgh, UK.|School of Basic & Biomedical Sciences, University of Health and Allied Sciences, Ho, Ghana.
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http://orcid.org/0000-0001-5816-5172
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Duedu
School of Biological Sciences, University of Edinburgh, Edinburgh, UK.
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Cain
School of Biological Sciences, University of Edinburgh, Edinburgh, UK.
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R
Nagy
School of Biological Sciences, University of Edinburgh, Edinburgh, UK.
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J
Krakowiak
School of Biological Sciences, University of Edinburgh, Edinburgh, UK.
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CE
French
D056804:Biofuels; D018533:Biomass; D050260:Carbohydrate Metabolism; D002475:Cellobiose / Q000378:metabolism*; D044602:Cellulases / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D016971:Citrobacter freundii / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D008031:Lignin / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D056804:Biofuels; D011994:Recombinant Proteins; C036909:lignocellulose; D002475:Cellobiose; D002482:Cellulose; D008031:Lignin; D044602:Cellulases; C109691:microcrystalline cellulose
biodegradation; biofuels; gene expression; plasmids; recombinant protein