Escherichia coli is one of the favourite hosts for recombinant protein production and is recognized as an excellent model for biofilm studies. High cell density cultures (HCDC) of this bacterium enable attractive volumetric production yields and cells growing in biofilms share some of the challenges of conventional high cell density planktonic cultures. This work assesses the production potential of E. coli JM109(DE3) biofilm cells expressing a model protein, the enhanced green fluorescent protein (eGFP), from a recombinant plasmid. A control strain harbouring the same plasmid backbone but lacking the eGFP gene was used to assess the impact of heterologous protein production on biofilm formation. Results show that specific eGFP production from biofilm cells was about 30 fold higher than in planktonic state. Moreover, eGFP-expressing cells had enhanced biofilm formation compared to control cells. Volumetric production values were 2 fold higher than those previously reported with the same protein and are within the range of what can be obtained by conventional HCDC in the production of soluble proteins. Although the cellular density that was obtained was lower than in conventional HCDC (0.5 fold), this novel system reached good production values which are likely to be improved after optimization of culture conditions.
1. Introduction
The Gram-negative bacterium Escherichia coli is a preferred host for the production of recombinant proteins [1,2] due to its fast growth at high cell densities, simple nutrient requirements, well-known genetics and the availability of a large number of cloning vectors and mutant host strains [3]. This bacterium has the ability to accumulate many recombinant proteins to at least 20% of the total cell protein [4] and, in some cases, to translocate them from the cytoplasm to the periplasm [5]. E. coli cultivation in high cell density cultures (HCDC) presents many advantages such as reduced culture volume, enhanced downstream processing and lower production costs [6]. Despite these advantages, there are still many challenges that have to be addressed in HCDC and these include insufficient oxygen transfer, specific culture medium requirements, reduced mixing efficiency in the reactor, accumulation of carbon dioxide which decreases growth rates and increased acetate production [5,6]. Recombinant protein production by biofilm cells shares some of the challenges of conventional HCDC, namely in diffusion of nutrients and oxygen through the biofilm and also in the accumulation of toxic waste products [7,8].
Recombinant protein expression in E. coli biofilms was pioneered by Huang et al. [9- 11] who have studied the production of -galactosidase in E. coli DH5α carrying a plasmid containing the tac promoter. Later, O’Connell et al. [12] have described the first system for high level heterologous protein production in E. coli biofilm cells using a pUC-based vector for the expression of enhanced green fluorescent protein (eGFP). Despite the enormous potential of this expressing system, heterologous protein production from E. coli biofilm cells remains largely unexplored.
5. Conclusions
Using E. coli cells in HCDC is a very attractive way of producing heterologous proteins by improving volumetric productivities and reducing capital investment and operation costs in production facilities. Producing heterologous proteins in biofilm cells shares some of the challenges of HCDC, but some studies have shown that biofilm cells have a superior specific producing capacity when compared to their planktonic counterparts. This work shows that E. coli biofilm cells can produce a model heterologous protein at much higher levels than planktonic cells and that the cellular densities and volumetric productivities are already within the range of what can be obtained by HCDC, even without optimization of culture conditions. Future work will address the optimization of these conditions in order to harness the power of these productive biofilms.
