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1. Characterizing the effect of expression of an acetyl-CoA synthetase insensitive to acetylation on co-utilization of glucose and acetate in batch and continuous cultures of E. coli W

Author

Katharina Novak, Lukas Flöckner, Anna Maria Erian, Philipp Freitag, Christoph Herwig, and Stefan Pflügl

Subjects
E. coli W, Acetyl-CoA-synthetase, Acetate (Acs), Metabolic engineering, Protein acetylation, Biomass composition, Microbiology, QR1-502
Abstract

Abstract Background Due to its high stress tolerance and low acetate secretion, Escherichia coli W is reported to be a good production host for several metabolites and recombinant proteins. However, simultaneous co-utilization of glucose and other substrates such as acetate remains a challenge. The activity of acetyl-CoA-synthetase, one of the key enzymes involved in acetate assimilation is tightly regulated on a transcriptional and post-translational level. The aim of this study was to engineer E. coli W for overexpression of an acetylation insensitive acetyl-CoA-synthetase and to characterize this strain in batch and continuous cultures using glucose, acetate and during co-utilization of both substrates. Results Escherichia coli W engineered to overexpress an acetylation-insensitive acetyl-CoA synthetase showed a 2.7-fold increase in acetate uptake in a batch process containing glucose and high concentrations of acetate compared to a control strain, indicating more efficient co-consumption of glucose and acetate. When acetate was used as the carbon source, batch duration could significantly be decreased in the overexpression strain, possibly due to alleviation of acetate toxicity. Chemostat cultivations with different dilution rates using glucose revealed only minor differences between the overexpression and control strain. Accelerostat cultivations using dilution rates between 0.20 and 0.70 h−1 indicated that E. coli W is naturally capable of efficiently co-utilizing glucose and acetate over a broad range of specific growth rates. Expression of acetyl-CoA synthetase resulted in acetate and glucose accumulation at lower dilution rates compared to the control strain. This observation can possibly be attributed to a higher ratio between acs and pta-ackA in the overexpression strain as revealed by gene expression analysis. This would result in enhanced energy dissipation caused by an imbalance in the Pta-AckA-Acs cycle. Furthermore, yjcH and actP, genes co-transcribed with acetyl-CoA synthetase showed significant down-regulation at elevated dilution rates. Conclusions Escherichia coli W expressing an acetylation-insensitive acetyl-CoA synthetase was shown to be a promising candidate for mixed feed processes using glucose and acetate. Comparison between batch and continuous cultures revealed distinct differences in glucose-acetate co-utilization behavior, requiring additional investigations such as multi-omics analysis and further engineering towards even more efficient co-utilization strains of E. coli W.

Published
2018
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2. Metabolic engineering of Escherichia coli W for isobutanol production on chemically defined medium and cheese whey as alternative raw material

Author

Philipp Freitag, Juliane Baar, Stefan Pflügl, and Katharina Novak

Subjects
Butanols, Pulsed fed-batch, Isobutanol adaptation, Bioengineering, Promotor fine-tuning, Raw material, medicine.disease_cause, Applied Microbiology and Biotechnology, Constitutive promotor, Metabolic engineering, chemistry.chemical_compound, Cheese, Whey, Escherichia coli, medicine, Food science, Expression vector, Bioenergy/Biofuels/Biochemicals - Original Paper, Chemistry, Isobutanol, Escherichia coli Proteins, Substrate (chemistry), Culture Media, Chemically defined medium, Metabolic Engineering, Aeration, Plasmids, Biotechnology
Abstract

The aim of this study was to establish isobutanol production on chemically defined medium in Escherichia coli. By individually expressing each gene of the pathway, we constructed a plasmid library for isobutanol production. Strain screening on chemically defined medium showed successful production in the robust E. coli W strain, and expression vector IB 4 was selected as the most promising construct due to its high isobutanol yields and efficient substrate uptake. The investigation of different aeration strategies in combination with strain improvement and the implementation of a pulsed fed-batch were key for the development of an efficient production process. E. coli W ΔldhA ΔadhE Δpta ΔfrdA enabled aerobic isobutanol production at 38% of the theoretical maximum. Use of cheese whey as raw material resulted in longer process stability, which allowed production of 20 g l−1 isobutanol. Demonstrating isobutanol production on both chemically defined medium and a residual waste stream, this study provides valuable information for further development of industrially relevant isobutanol production processes. Electronic supplementary material The online version of this article (10.1007/s10295-020-02319-y) contains supplementary material, which is available to authorized users.

Published
2020

3. Surgical stabilization of serial rib fractures is advantageous in patients with relevant traumatic brain injury

Author

Philipp Freitag, Cornelius Bechmann, Lars Eden, Rainer Meffert, and Thorsten Walles

Subjects
Male, Injury Severity Score, Rib Fractures, Thoracic Injuries, Brain Injuries, Traumatic, Emergency Medicine, Humans, Orthopedics and Sports Medicine, Surgery, Length of Stay, Critical Care and Intensive Care Medicine, Retrospective Studies
Abstract

Purpose To evaluate the clinical benefit of surgical stabilization of rib fractures (SSRF) in polytrauma patients with serial rib fractures. Methods Retrospective single-center cohort analysis in trauma patients. Serial rib fracture was defined as three consecutive ribs confirmed by chest computer tomography (CT). Study cohort includes 243 patients that were treated conservatively and 34 patients that underwent SSRF. Demographic patient data, trauma mechanism, injury pattern, Injury Severity Score (ISS), Glasgow Coma Scale (GCS) and hospital course were analyzed. Two matched pair analyses stratified for ISS (32 pairs) and GCS (25 pairs) were performed. Results The majority of patients was male (74%) and aged 55 ± 20 years. Serial rib fractures were associated with more than 6 broken ribs in average (6.3 ± 3.7). Other thoracic bone injury included sternum (18%), scapula (16%) and clavicula (13%). Visceral injury consisted of pneumothorax (51%), lung contusion (33%) and diaphragmatic rupture (2%). Average ISS was 22 ± 7.3. Overall hospital stay was 15.9 and ICU stay 7.4 days. In hospital, mortality was 13%. SSRF did not improve hospital course or postoperative complications in the complete study cohort. However, patients with a significantly reduced GCS (7.6 ± 5.3 vs 11.22 ± 4.8; p = 0.006) benefitted from SSRF. Matched pair analysis stratified for GCS showed shorter ICU stays (9 vs 15 days; p = 0.005) including shorter respirator time (143 vs 305 h; p = 0.003). Conclusion Patients with serial rib fractures and simultaneous moderate or severe traumatic brain injury benefit from surgical stabilization of rib fractures.

Published
2021

4. High rate 2,3-butanediol production with Vibrio natriegens

Author

Martin Gibisch, Stefan Pflügl, Anna Maria Erian, and Philipp Freitag

Subjects
Environmental Engineering, Sucrose, biology, Renewable Energy, Sustainability and the Environment, Chemistry, 020209 energy, Acetoin, Substrate (chemistry), Bioengineering, 02 engineering and technology, Chemostat, 010501 environmental sciences, Vibrio natriegens, biology.organism_classification, 01 natural sciences, chemistry.chemical_compound, Yield (chemistry), 0202 electrical engineering, electronic engineering, information engineering, 2,3-Butanediol, Sugar beet, Food science, Waste Management and Disposal, 0105 earth and related environmental sciences
Abstract

The aim of this study was to investigate if the high substrate turnover rates of Vibrio natriegens enable 2,3-butanediol production at high productivities. Introduction of a heterologous pathway led to 2,3-butanediol production at yields comparable to Escherichia coli using glucose, sucrose and sugar beet molasses. Microaerobic fed-batch cultivations maintained at 0–1% dissolved oxygen yielded a combined diol titer of 49.9 g L−1 of 2,3-butanediol and acetoin and a production rate of 3.9 g L−1 h−1. Optimization of the oxygen supply in chemostat cultivations increased the product yield to 70% of the theoretical maximum at volumetric and specific production rates of 2 g L−1 h−1 and 0.66 g g−1 h−1, respectively. In conclusion, this study could for the first time demonstrate V. natriegens as novel host for 2,3-butanediol production with high volumetric and specific productivity, underlining its potential for microbial chemical production.

Published
2020

5. Familie, Rente, Pflege: Die Sozialpolitik der Großen Koalition auf dem Prüfstand

Author

Philipp Freitag

Subjects
Sociology and Political Science, Social Sciences (miscellaneous)
Abstract

In der laufenden Legislaturperiode wurden weitreichende sozialpolitische Masnahmen in den Bereichen Familie (Elterngeld Plus), Rentenversicherung (Rentenpaket) und Pflegeversicherung (Pflegestarkungsgesetz I, Gesetz zur besseren Vereinbarkeit von Familie, Pflege und Beruf) verabschiedet. Die beschlossenen Leistungsausweitungen sind angesichts des demographischen Wandels im Hinblick auf die Sicherung der langfristigen finanziellen Stabilitat der sozialen Sicherungssysteme kritisch zu beurteilen. Zu verteilungspolitisch fragwurdigen Effekten fuhren die abschlagsfreie Rente mit 63 Jahren und das Elterngeld, welches in der reformierten Fassung zudem verstarkt erhebliche Anreizwirkung in Bezug auf die (gleiche) Aufteilung von Erwerbs- und Familienarbeitszeit zwischen den Eltern entfaltet. Reformbedarf besteht im Hinblick auf die angemessene beitrags- und leistungsseitige Berucksichtigung von Erziehungsleistungen im Rahmen der sozialen Sicherungssysteme. Family, Pension, Care: The Social Policy of th...

Published
2015

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