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.

E. coli HB101 pRK2013 Sm R , hsdR-M + , proA2, leuB6, thi-1, recA; harboring plasmid pRK2013 Ditta et al., 1980 E. coli DH5α pYTSK01K_0G7 DH5α harboring plasmid pYTSK01K_0G7 This study E. coli DH5α pVLT33-PA_rhlABC DH5α harboring plasmid pVLT33-PA_rhlABC Wittgens et al., 2017 E. coli DH5α pBNT Km DH5α harboring plasmid pBNTmcs(t)Km This study S. cerevisiae VL6-48 pYTSK10K_0G7_rhlAB VL6-48 harboring plasmid pYTSK10K_0G7_rhlAB This study E. coli DH5α pYTSK10K_1G7_rhlAB DH5α harboring plasmid pYTSK10K_1G7_rhlAB This study E. coli DH5α pRhon5Hi-2-eyfp DH5α harboring plasmid pRhon5Hi-2-eyfp Troost et al., 2019 S. cerevisiae VL6-48 pYTSK40K_1G7_rhlAB VL6-48 harboring plasmid pYTSK40K_1G7_rhlAB This study E. coli DH5α pYTSK40K_0G7_rhlAB DH5α harboring plasmid pYTSK40K_0G7_rhlAB This study E. coli S17-1 pYTSK40K_1G7_rhlAB S17-1 harboring plasmid pYTSK40K_1G7_rhlAB This study E. coli DH5αλpir pSK02 DH5α λpir harboring Tn7 delivery vector pSK02 for chromosomal integration Bator et al., 2020b E. coli DH5α pSW-2 DH5α harboring plasmid pSW-2 encoding I-SceI nuclease Martinez-Garcia and de Lorenzo, 2011 E. coli DH5αλpir pTNS-1 DH5α λpir harboring plasmid pTNS-1 Choi et al., 2005 E. coli DH5αλpir p pha DH5α λpir harboring plasmid p pha Mato Aguirre, 2019 E. coli PIR2 pEMG-flag1 PIR2 harboring plasmid pEMG-flag1 Blesken et al., 2020 E. coli PIR2 pEMG-flag2 PIR2 harboring plasmid pEMG-flag2 Blesken et al., 2020 E. coli DH5αλpir pMaW03 DH5α λpir harboring plasmid pMaW03 This study P. putida chassis strains P. putida KT2440 Wild type Bagdasarian et al., 1981 P. putida KT2440 flag PP_4328-PP_4344 and PP_4351-PP_4397 Blesken et al., 2020 P. putida KT2440 phaG PP_1408 This study P. putida KT2440 pha PP_5003-PP_5008 Blesken et al., 2020 P. putida KT2440 phaG pha PP_1408 and PP_5003-PP_5008 (phaC1ZC2DFI) This study P. putida KT2440 flag pha PP_4328-PP_4344, PP_4351-PP_4397 and PP_5003-PP_5008 This study P. putida strains used for biosurfactant production P. putida KT2440 pPS05 KT2440 harboring plasmid pPS05 Tiso et al., 2016 P. putida KT2440 SK4 rhlAB, attTn7 integrated, P 14ffg This study P. putida KT2440 sal mRL E SK40 rhlAB, eYFP, attTn7 integrated, P nagAa /nagR, Gm R , tnsABCD This study P. putida KT2440 flag SK4 PP_4328-PP_4344 and PP_4351-PP_4397, rhlAB, attTn7 integrated, P 14ffg Blesken et al., 2020 P. putida KT2440 phaG SK4 PP_1408, rhlAB, attTn7 integrated, P 14ffg This study P. putida KT2440 pha SK4 PP_5003-PP_5008, rhlAB, attTn7 integrated, P 14ffg Blesken et al., 2020 P. putida KT2440 phaG pha SK4 PP_1408 and PP_5003-PP_5008, rhlAB, attTn7 integrated, P 14ffg This study P. putida KT2440 flag pha SK4 PP_4328-PP_4344 and PP_4351-PP_4397 and PP_5003-PP_5008, rhlAB, attTn7 integrated, P 14ffg

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.