Abstract
BackgroundEscherichia coli is commonly used in academia and industry for expressing recombinant proteins because of its well-characterized molecular genetics and the availability of numerous expression vectors and strains. One important issue during recombinant protein production is the so-called ‘metabolic burden’: the material and energy normally reserved for microbial metabolism which is sapped from the bacterium to produce the recombinant protein. This material and energy drain harms biomass formation and modifies respiration. To the best of our knowledge, no research has investigated so far whether a single amino acid exchange in a recombinant protein affects the metabolic burden phenomenon. Thus, in this study, 15 E. coli BL21(DE3) clones expressing either the fusion tags, a recombinant wild type lipase, or 13 different lipase variants are investigated to quantitatively analyze the respective effects of single amino acid exchanges at different positions on respiration, biomass and protein production of each clone. Therefore, two small-scale online monitoring systems, namely a Respiration Activity MOnitoring System (RAMOS) and a microtiter plate based cultivation system (BioLector) are applied.ResultsUpon expression of all enzyme variants, strong variations were found in the Oxygen Transfer Rate (OTR), biomass and protein (lipase) production of the respective E. coli clones. Two distinct patterns of respiration behavior were observed and, so, the clones could be classified into two groups (Type A and B). Potential factors to explain these patterns were evaluated (e.g. plasmid copy number, inclusion body formation). However, no decisive factor could yet be identified. Five distinct cultivation phases could be determined from OTR curves which give real-time information about carbon source consumption, biomass and protein production. In general, it was found that the quantity of product increased with the duration of active respiration.ConclusionsThis work demonstrates that single amino acid exchanges in a recombinant protein influence the metabolic burden during protein production. The small-scale online monitoring devices RAMOS and BioLector enable the real-time detection of even smallest differences in respiration behavior, biomass and protein production in the E. coli clones investigated. Hence, this study underscores the importance of parallel online monitoring systems to unveil the relevance of single amino acid exchanges for the recombinant protein production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0191-y) contains supplementary material, which is available to authorized users.
Highlights
Escherichia coli is commonly used in academia and industry for expressing recombinant proteins because of its well-characterized molecular genetics and the availability of numerous expression vectors and strains
This study aims to investigate the influence of single amino acid exchanges at different positions of a recombinant enzyme on metabolic activity and expression of the host E. coli BL21(DE3) using two small-scale online monitoring devices
A quantitative evaluation of the Oxygen Transfer Rate (OTR) as a function of time allowed the classification of the clones into two types of respiration behavior named Type A and Type B
Summary
Escherichia coli is commonly used in academia and industry for expressing recombinant proteins because of its well-characterized molecular genetics and the availability of numerous expression vectors and strains. One important issue during recombinant protein production is the so-called ‘metabolic burden’: the material and energy normally reserved for microbial metabolism which is sapped from the bacterium to produce the recombinant protein This material and energy drain harms biomass formation and modifies respiration. Among many available microbial systems, Escherichia coli is the most commonly used prokaryotic expression system for the production of recombinant proteins. This is due to its well-known genetics, its ability to grow rapidly to high cell densities on inexpensive mineral media, as well as the large number of available cloning vectors and optimized host strains [1,2]. Mineral autoinduction media have a defined chemical composition which allows a better understanding of metabolic processes during induction and protein expression
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