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.
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Cultivations of mammalian cells are routinely conducted in shake flasks. In contrast to instrumented bioreactors, reliable options for non-invasive, time-resolved monitoring of the culture status in shake flasks are lacking. The Respiration Activity Monitoring Respiration Activity Monitoring System system was used to determine the oxygen transfer rate (OTR) in shake flasks. It was proven that the OTR could be regarded as equal to the oxygen uptake rate as the change of the dissolved oxygen concentration in the liquid phase over time was negligibly small. Thus, monitoring the oxygen transfer rate (OTR) was used to increase the information content from shake flask experiments. The OTR of a Chinese hamster ovary cell line was monitored by applying electrochemical sensors. Glass flasks stoppered with cotton plugs and polycarbonate flasks stoppered with vent-caps were compared in terms of mass transfer characteristics and culture behavior. Similar mass transfer resistances were determined for both sterile closures. The OTR was found to be well reproducible within one experiment (standard deviation <10%). It correlated with changes in cell viability and depletion of carbon sources, thus, giving more profound insights into the cultivation process. Culture behavior in glass and polycarbonate flasks was identical. Monitoring of the OTR was applied to a second culture medium. Media differed in the maximum OTR reached during cultivation and in the time when all carbon sources were depleted. By applying non-invasive, parallelized, time-resolved monitoring of the OTR, the information content and amount of data from shake flask experiments was significantly increased compared to manual sampling and offline analysis. The potential of the technology for early-stage process development was demonstrated.
Microbial induced calcite precipitation (MICP) based on ureolysis has a high potential for many applications, e.g. restoration of construction materials. The gram-positive bacterium Sporosarcina pasteurii is the most commonly used microorganism for MICP due to its high ureolytic activity. However, Sporosarcina pasteurii is so far cultivated almost exclusively in complex media, which only results in moderate biomass concentrations at the best. Cultivation of Sporosarcina pasteurii must be strongly improved in order to make technological application of MICP economically feasible. The growth of Sporosarcina pasteurii DSM 33 was boosted by detecting auxotrophic deficiencies (L-methionine, L-cysteine, thiamine, nicotinic acid), nutritional requirements (phosphate, trace elements) and useful carbon sources (glucose, maltose, lactose, fructose, sucrose, acetate, L-proline, L-alanine). These were determined by microplate cultivations with online monitoring of biomass in a chemically defined medium and systematically omitting or substituting medium components. Persisting growth limitations were also detected, allowing further improvement of the chemically defined medium by the addition of glutamate group amino acids. Common complex media based on peptone and yeast extract were supplemented based on these findings. Optical density at the end of each cultivation of the improved peptone and yeast extract media roughly increased fivefold respectively. A maximum OD600 of 26.6 ± 0.7 (CDW: 17.1 ± 0.5 g/L) was reached with the improved yeast extract medium. Finally, culture performance and media improvement was analysed by measuring the oxygen transfer rate as well as the backscatter during shake flask cultivation.
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