Browsing by Author "Hägele, Lorena"

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Open Access
CRISPRi enables fast growth followed by stable aerobic pyruvate formation in Escherichia coli without auxotrophy
(2021) Ziegler, Martin; Hägele, Lorena; Gäbele, Teresa; Takors, Ralf
CRISPR interference (CRISPRi) was applied to enable the aerobic production of pyruvate in Escherichia coli MG1655 under glucose excess conditions by targeting the promoter regions of aceE or pdhR. Knockdown strains were cultivated in aerobic shaking flasks and the influence of inducer concentration and different sgRNA binding sites on the production of pyruvate was measured. Targeting the promoter regions of aceE or pdhR triggered pyruvate production during the exponential phase and reduced expression of aceE. In lab‐scale bioreactor fermentations, an aceE silenced strain successfully produced pyruvate under fully aerobic conditions during the exponential phase, but loss of productivity occurred during a subsequent nitrogen‐limited phase. Targeting the promoter region of pdhR enabled pyruvate production during the growth phase of cultivations, and a continued low‐level accumulation during the nitrogen‐limited production phase. Combinatorial targeting of the promoter regions of both aceE and pdhR in E. coli MG1655 pdCas9 psgRNA_aceE_234_pdhR_329 resulted in the stable aerobic production of pyruvate with non‐growing cells at YP/S = 0.36 ± 0.029 gPyruvate/gGlucose in lab‐scale bioreactors throughout an extended nitrogen‐limited production phase.
Open Access
Getting the right clones in an automated manner : an alternative to sophisticated colony-picking robotics
(2024) Hägele, Lorena; Pfleger, Brian F.; Takors, Ralf
In recent years, the design-build-test-learn (DBTL) cycle has become a key concept in strain engineering. Modern biofoundries enable automated DBTL cycling using robotic devices. However, both highly automated facilities and semi-automated facilities encounter bottlenecks in clone selection and screening. While fully automated biofoundries can take advantage of expensive commercially available colony pickers, semi-automated facilities have to fall back on affordable alternatives. Therefore, our clone selection method is particularly well-suited for academic settings, requiring only the basic infrastructure of a biofoundry. The automated liquid clone selection (ALCS) method represents a straightforward approach for clone selection. Similar to sophisticated colony-picking robots, the ALCS approach aims to achieve high selectivity. Investigating the time analogue of five generations, the model-based set-up reached a selectivity of 98 ± 0.2% for correctly transformed cells. Moreover, the method is robust to variations in cell numbers at the start of ALCS. Beside Escherichia coli , promising chassis organisms, such as Pseudomonas putida and Corynebacterium glutamicum , were successfully applied. In all cases, ALCS enables the immediate use of the selected strains in follow-up applications. In essence, our ALCS approach provides a ‘low-tech’ method to be implemented in biofoundry settings without requiring additional devices.
Open Access
Optimization and kinetic modeling of a fed-batch fermentation for mannosylerythritol lipids (MEL) production with moesziomyces aphidis
(2022) Beck, Alexander; Vogt, Franziska; Hägele, Lorena; Rupp, Steffen; Zibek, Susanne
Mannosylerythritol lipids are glycolipid biosurfactants with many interesting properties. Despite the general interest in those molecules and the need for a robust process, studies on their production in bioreactors are still scarce. In the current study, the fermentative production of MEL in a bioreactor with Moesziomyces aphidis was performed using a defined mineral salt medium. Several kinetic process parameters like substrate consumption rates and product formation rates were evaluated and subsequently enhanced by increasing the biomass concentration through an exponential fed-batch strategy. The fed-batch approaches resulted in two to three fold increased dry biomass concentrations of 10.9-15.5 g/L at the end of the growth phase, compared with 4.2 g/L in the batch process. Consequently, MEL formation rates were increased from 0.1 g/Lh up to around 0.4 g/Lh during the MEL production phase. Thus, a maximum concentration of up to 50.5 g/L MEL was obtained when oil was added in excess, but high concentrations of residual fatty acids were also present in the broth. By adjusting the oil feeding to biomass-specific hydrolysis and MEL production rates, a slightly lower MEL concentration of 34.3 g/L was obtained after 170 h, but at the same time a very pure crude lipid extract with more than 90% MEL and a much lower concentration of remaining fatty acids. With rapeseed oil as substrate, the ideal oil-to-biomass ratio for full substrate conversion was found to be around 10 goil/gbiomass. In addition, off-gas analysis and pH trends could be used to assess biomass growth and MEL production. Finally, kinetic models were developed and compared to the experimental data, allowing for a detailed prediction of the process behavior in future experiments.