Prediction of recombinant protein production by <i>Escherichia coli</i> derived online from indicators of metabolic burden

Mühlmann, Martina; Forsten, Eva; Noack, Saskia; Büchs, Jochen

doi:10.1002/btpr.2704

Cited by 22 publications

(20 citation statements)

References 51 publications

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“…This behavior was considered in the theoretical model. The scattered light in the main culture does not reach the same level as in the preculture, since induced cells suffer from a metabolic burden …”

Section: Resultsmentioning

confidence: 99%

“…By integration of OTRs over time, the total oxygen transferred (OT) was calculated. OT is a good quantitative indicator for the total consumed substrate and therefore for the produced biomass (assuming a constant yield coefficient) . This approach is advantageous since the matrix at the bottom of the wells of the fed‐batch MTP prevents the optical measurement of scattered light.…”

Section: Methodsmentioning

confidence: 99%

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Precultures Grown under Fed‐Batch Conditions Increase the Reliability and Reproducibility of High‐Throughput Screening Results

Keil

Landenberger

Dittrich

et al. 2019

Biotechnology Journal

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One essential task in bioprocess development is strain selection. A common screening procedure consists of three steps: first, the picking of colonies; second, the execution of a batch preculture and main culture, e.g., in microtiter plates (MTPs); and third, the evaluation of product formation. Especially during the picking step, unintended variations occur due to undefined amounts and varying viability of transferred cells. The aim of this study is to demonstrate that the application of polymer‐based controlled‐release fed‐batch MTPs during preculture eliminates these variations. The concept of equalizing growth through fed‐batch conditions during preculture is theoretically discussed and then tested in a model system, namely, a cellulase‐producing Escherichia coli clone bank containing 32 strains. Preculture is conducted once in the batch mode and once in the fed‐batch mode. By applying the fed‐batch mode, equalized growth is observed in the subsequent main culture. Furthermore, the standard deviation of cellulase activity is reduced compared to that observed in the conventional approach. Compared with the strains in the batch preculture process, the first‐ranked strain in the fed‐batch preculture process is the superior cellulase producer. These findings recommend the application of the fed‐batch MTPs during preculture in high‐throughput screening processes to achieve accurate and reliable results.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Precultures Grown under Fed‐Batch Conditions Increase the Reliability and Reproducibility of High‐Throughput Screening Results

Keil

Landenberger

Dittrich

et al. 2019

Biotechnology Journal

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“…This increase continues after induction, indicating no strong metabolic burden. Typically, a strong metabolic burden is reflected by visible changes in the OTR after induction (Rahmen et al, 2015a,b;Ihling et al, 2018;Mühlmann et al, 2018). The maximum oxygen transfer capacity (OTRmax) under the chosen cultivation conditions was determined to be 52 mmol/L/h according to Meier et al (2016).…”

Section: Early Induction Leads To Higher Productivitymentioning

confidence: 99%

Integration of Genetic and Process Engineering for Optimized Rhamnolipid Production Using Pseudomonas putida

Tiso

Ihling

Kubicki

et al. 2020

Front. Bioeng. Biotechnol.

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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

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“…From the decrease of the oxygen partial pressure, the OTR is calculated. Due to the very low changes in dissolved oxygen during batch cultivation, the OTR can be assumed to be equal to the oxygen uptake rate . For parallel offline sampling, conventional Erlenmeyer flasks, equipped with a cotton plug, were cultivated at the same conditions as the RAMOS‐flasks.…”

Section: Methodsmentioning

confidence: 99%

Scale‐up of a Type I secretion system in E. coli using a defined mineral medium

Ihling

Uhde²,

Scholz

et al. 2019

Biotechnology Progress

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Secretion of heterologous proteins into the culture supernatant in laboratory strains of Escherichia coli is possible by utilizing a Type I secretion system (T1SS). One prominent example for a T1SS is based on the hemolysin A toxin. With this system, heterologous protein secretion has already been achieved. However, no cultivations in a defined mineral medium and in stirred tank bioreactors have been described in literature up to now, hampering the broad applicability of the system. In this study, a mineral medium was developed for cultivation under defined conditions. With this medium, the full potential and advantage of a secretion system in E. coli (low secretion of host proteins, no contamination with proteins from complex media compounds) can now be exploited. Additionally, quantification of the protein amount in the supernatant was demonstrated by application of the Bradford assay. In this work, host cell behavior was described in small scale by online monitoring of the oxygen transfer rate. Scalability was demonstrated by stirred tank fermentation yielding 540 mg/L HlyA1 in the supernatant. This work enhances the applicability of a protein secretion system in E. coli and paves the way for an industrial application.

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Prediction of recombinant protein production by Escherichia coli derived online from indicators of metabolic burden

Cited by 22 publications

References 51 publications

Precultures Grown under Fed‐Batch Conditions Increase the Reliability and Reproducibility of High‐Throughput Screening Results

Precultures Grown under Fed‐Batch Conditions Increase the Reliability and Reproducibility of High‐Throughput Screening Results

Integration of Genetic and Process Engineering for Optimized Rhamnolipid Production Using Pseudomonas putida

Scale‐up of a Type I secretion system in E. coli using a defined mineral medium

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