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Item type:Publication, Access status: with files , A Consecutive Genome Engineering Method Reveals a New Phenotype and Regulation of Glucose and Glycerol Utilization in Clostridium Pasteurianum(Wiley, 2025-01-03); ; ; ; Clostridium pasteurianum is a microorganism for production of 1,3-propanediol (1,3-PDO) and butanol, but suffers from lacking genetic tools for metabolic engineering to improve product titers. Furthermore, previous studies of C. pasteurianum have mainly focused on single genomic modification. The aim of this work is the development and application of a method for modification of multiple gene targets in the genome of C. pasteurianum. To this end, a new approach for consecutive genome engineering is presented for the first time using a method based on endogenous CRISPR-Cas machineries. A total of three genome modifications were consecutively introduced in the same mutant and the effect of combined changes on the genome was observed by 39% decreased specific glycerol consumption rate and 29% increased 1,3-PDO yield in mixed substrate fermentations at laboratory scale in comparison to the wildtype strain. Additionally, examination of the phenotype of the generated mutant strain led to discovery of 2,3-butanediol (2,3-BDO) production of up to 0.48 g L−1, and this metabolite was not reported to be produced by C. pasteurianum before. The developed procedure expands the genetic toolkit for C. pasteurianum and provides researchers an additional method which contributes to improved genetic accessibility of this strain.Publicationtype: Journal Article30 14 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Design of cell expansion processes for adherent-growing cells with mDoE-workflowAdherent cells, mammalian or human, are ubiquitous for production of viral vaccines, in gene therapy and in immuno-oncology. The development of a cell-expansion process with adherent cells is challenging as scale-up requires the expansion of the cell culture surface. Microcarrier (MC)-based cultures are still predominate. However, the development of MC processes from scratch possesses particular challenges due to their complexity. A novel approach for the reduction of development times and costs of cell propagation processes is the combination of mathematical process models with statistical optimization methods, called model-assisted Design of Experiments (mDoE). In this study, an mDoE workflow was evaluated successfully for the design of a MC-based expansion process of adherent L929 cells at a very early stage of development with limited prior knowledge. At the start, the analytical methods and the screening of appropriate MCs were evaluated. Then, cause-effect relationships (e.g., cell growth related to medium conditions) were worked out, and a mathematical process model was set-up and adapted to experimental data for modeling purposes. The model was subsequently used in mDoE to identify optimized process conditions, which were proven experimentally. An eight-fold increase in cell yield was achieved basically by reducing the initial MC concentration.Publicationtype: Journal ArticleScopus© Citations 2 54 113 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Novel synthetic co-culture of Acetobacterium woodii and Clostridium drakei using CO₂ and in situ generated H₂ for the production of caproic acid via lactic acidAcetobacterium woodii is known to produce mainly acetate from CO₂ and H₂, but the production of higher value chemicals is desired for the bioeconomy. Using chain-elongating bacteria, synthetic co-cultures have the potential to produce longer-chained products such as caproic acid. In this study, we present first results for a successful autotrophic co-cultivation of A. woodii mutants and a Clostridium drakei wild-type strain in a stirred-tank bioreactor for the production of caproic acid from CO₂ and H₂ via the intermediate lactic acid. For autotrophic lactate production, a recombinant A. woodii strain with a deleted Lct-dehydrogenase complex, which is encoded by the lctBCD genes, and an inserted D-lactate dehydrogenase (LdhD) originating from Leuconostoc mesenteroides, was used. Hydrogen for the process was supplied using an All-in-One electrode for in situ water electrolysis. Lactate concentrations as high as 0.5 g L–1 were achieved with the AiO-electrode, whereas 8.1 g L–1 lactate were produced with direct H₂ sparging in a stirred-tank bioreactor. Hydrogen limitation was identified in the AiO process. However, with cathode surface area enlargement or numbering-up of the electrode and on-demand hydrogen generation, this process has great potential for a true carbon-negative production of value chemicals from CO₂.Publicationtype: Journal ArticleScopus© Citations 8 44 178 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Scaling-down biopharmaceutical production processes via a single multi-compartment bioreactor (SMCB)(Wiley-VCH, 2023-01); ; ; ; Biopharmaceutical production processes often use mammalian cells in bioreactors larger than 10,000 L, where gradients of shear stress, substrate, dissolved oxygen and carbon dioxide, and pH are likely to occur. As former tissue cells, producer cell lines such as Chinese hamster ovary (CHO) cells sensitively respond to these mixing heterogeneities, resulting in related scenarios being mimicked in scale-down reactors. However, commonly applied multi-compartment approaches comprising multiple reactors impose a biasing shear stress caused by pumping. The latter can be prevented using the single multi-compartment bioreactor (SMCB) presented here. The exchange area provided by a disc mounted between the upper and lower compartments in a stirred bioreactor was found to be an essential design parameter. Mimicking the mixing power input at a large scale on a small scale allowed the installation of similar mixing times in the SMCB. The particularities of the disc geometry may also be considered, finally leading to a converged decision tree. The work flow identifies a sharply contoured operational field comprising disc designs and power input to install the same mixing times on a large scale in the SMCB without the additional shear stress caused by pumping. The design principle holds true for both nongassed and gassed systems.Publicationtype: Journal ArticleScopus© Citations 11 124 289 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Generation and evaluation of input values for computational analysis of transport processes within tissue cultures(Wiley-VCH, 2022-11-04); ; ; ; Techniques for tissue culture have seen significant advances during the last decades and novel 3D cell culture systems have become available. To control their high complexity, experimental techniques and their Digital Twins (modelling and computational tools) are combined to link different variables to process conditions and critical process parameters. This allows a rapid evaluation of the expected product quality. However, the use of mathematical simulation and Digital Twins is critically dependent on the precise description of the problem and correct input parameters. Errors here can lead to dramatically wrong conclusions. The intention of this review is to provide an overview of the state-of-the-art and remaining challenges with respect to generating input values for computational analysis of mass and momentum transport processes within tissue cultures. It gives an overview on relevant aspects of transport processes in tissue cultures as well as modelling and computational tools to tackle these problems. Further focus is on techniques used for the determination of cell-specific parameters and characterization of culture systems, including sensors for on-line determination of relevant parameters. In conclusion, tissue culture techniques are well-established, and modelling tools are technically mature. New sensor technologies are on the way, especially for organ chips. The greatest remaining challenge seems to be the proper addressing and handling of input parameters required for mathematical models. Following Good Modelling Practice approaches when setting up and validating computational models is, therefore, essential to get to better estimations of the interesting complex processes inside organotypic tissue cultures in the future.Publicationtype: Review ArticleScopus© Citations 1 56 126 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Enhanced sequestration of carbon dioxide into calcium carbonate using pressure and a carbonic anhydrase from alkaliphilic Coleofasciculus chthonoplastesCO2 in the atmosphere is a major contributor to global warming but at the same time it has the potential to be a carbon source for advanced biomanufacturing. To utilize CO2, carbonic anhydrase has been identified as a key enzyme. Furthermore, attempts have been made to accelerate the sequestration via pressure. This study aims to combine both approaches to achieve high sequestration rates. The carbonic anhydrase of the alkaliphilic cyanobacterium Coleofasciculus chthonoplastes (cahB1) and bovine carbonic anhydrase (BCA) are introduced into a high-pressure reactor to catalyze the hydration of CO2 at up to 20 bar. The reactor is filled with a CaCl2 solution. Due to the presence of Ca2+, the hydrated CO2 precipitates as CaCO3. The impact of the carbonic anhydrase is clearly visible at all pressures tested. At ambient pressure a CO2 sequestration rate of 243.68 kgCaCO3/m3 h for cahB1 was achieved compared to 150.41 kgCaCO3/m3 h without enzymes. At 20 bar the rates were 2682.88 and 2267.88 kgCaCO3/m3 h, respectively. The study shows the benefit of a combined CO2 sequestration process. To examinate the influence of the enzymes on the product formation, the precipitated CaCO3 was analyzed regarding the crystalline phase and morphology. An interchange of the crystalline phase from vaterite to calcite was observed and discussed.Publicationtype: Journal ArticleScopus© Citations 6 106 425 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Advances in automated real-time flow cytometry for monitoring of bioreactor processesFlow cytometry and its technological possibilities have greatly advanced in the past decade as analysis tool for single cell properties and population distributions of different cell types in bioreactors. Along the way, some solutions for automated real-time flow cytometry (ART-FCM) were developed for monitoring of bioreactor processes without operator interference over extended periods with variable sampling frequency. However, there is still great potential for ART-FCM to evolve and possibly become a standard application in bioprocess monitoring and process control. This review first addresses different components of an ART-FCM, including the sampling device, the sample-processing unit, the unit for sample delivery to the flow cytometer and the settings for measurement of pre-processed samples. Also, available algorithms are presented for automated data analysis of multi-parameter fluorescence datasets derived from ART-FCM experiments. Furthermore, challenges are discussed for integration of fluorescence-activated cell sorting into an ART-FCM setup for isolation and separation of interesting subpopulations that can be further characterized by for instance omics-methods. As the application of ART-FCM is especially of interest for bioreactor process monitoring, including investigation of population heterogeneity and automated process control, a summary of already existing setups for these purposes is given. Additionally, the general future potential of ART-FCM is addressed.Publicationtype: Review ArticleScopus© Citations 12 17 305 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Phenotype analysis of cultivation processes via unsupervised machine learning: demonstration for Clostridium pasteurianum(Wiley-Blackwell, 2022-02); ; ; ; A novel approach of phenotype analysis of fermentation-based bioprocesses based on unsupervised learning (clustering) is presented. As a prior identification of phenotypes and conditional interrelations is desired to control fermentation performance, an automated learning method to output reference phenotypes (defined as vector of biomass-specific rates) was developed and the necessary computing process and parameters were assessed. For its demonstration, time series data of 90 Clostridium pasteurianum cultivations were used which feature a broad spectrum of solventogenic and acidogenic phenotypes, while 14 clusters of phenotypic manifestations were identified. The analysis of reference phenotypes showed distinct differences, where potential conditionalities were exemplary isolated. Further, cluster-based balancing of carbon and ATP or the use of reference phenotypes as indicator for bioprocess monitoring were demonstrated to highlight the perks of this approach. Overall, such analysis depends strongly on the quality of the data and experimental validations will be required before conclusions. However, the automated, streamlined and abstracted approach diminishes the need of individual evaluation of all noisy dataset and showed promising results, which could be transferred to strains with comparably wide-ranging phenotypic manifestations or as indicators for repeated bioprocesses with clearly defined target.Publicationtype: Journal Article93 159 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Improvement of glycine biosynthesis from one-carbon compounds and ammonia catalyzed by the glycine cleavage system in vitroGlycine cleavage system (GCS) plays a central role in one-carbon (C1) metabolism and receives increasing interest as a core part of the recently proposed reductive glycine pathway (rGlyP) for assimilation of CO2 and formate. Despite decades of research, GCS has not yet been well understood and kinetic data are barely available. This is to a large degree because of the complexity of GCS, which is composed of four proteins (H, T, P, and L) and catalyzes reactions involving different substrates and cofactors. In vitro kinetics of reconstructed microbial multi-enzyme glycine cleavage/synthase system is desired to better implement rGlyP in microorganisms like Escherichia coli for the use of C1 resources. Here, we examined in vitro several factors that may affect the rate of glycine synthesis via the reverse GCS reaction. We found that the ratio of GCS component proteins has a direct influence on the rate of glycine synthesis, namely higher ratios of P protein and especially H protein to T and L proteins are favorable, and the carboxylation reaction catalyzed by P protein is a key step determining the glycine synthesis rate, whereas increasing the ratio of L protein to other GCS proteins does not have significant effect and the ratio of T protein to other GCS proteins should be kept low. The effect of substrate concentrations on glycine synthesis is quite complex, showing interdependence with the ratios of GCS component proteins. Furthermore, adding the reducing agent dithiothreitol to the reaction mixture not only results in great tolerance to high concentration of formaldehyde, but also increases the rate of glycine synthesis, probably due to its functions in activating P protein and taking up the role of L protein in the non-enzymatic reduction of Hox to Hred. Moreover, the presence of some monovalent and divalent metal ions can have either positive or negative effect on the rate of glycine synthesis, depending on their type and their concentration.Publicationtype: Journal ArticleScopus© Citations 7 111 322 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Application of an Escherichia coli triple reporter strain for at-line monitoring of single-cell physiology during L-phenylalanine productionBiotechnological production processes are sustainable approaches for the production of biobased components such as amino acids for food and feed industry. Scale-up from ideal lab-scale bioreactors to large-scale processes is often accompanied by loss in productivity. This may be related to population heterogeneities of cells originating from isogenic cultures that arise due to dynamic non-ideal conditions in the bioreactor. To better understand this phenomenon, deeper insights into single-cell physiologies in bioprocesses are mandatory before scale-up. Here, a triple reporter strain (3RP) was developed by chromosomally integrating the fluorescent proteins mEmerald, CyOFP1, and mTagBFP2 into the L-phenylalanine producing Escherichia coli strain FUS4 (pF81kan) to allow monitoring of growth, oxygen availability, and general stress response of the single cells. Functionality of the 3RP was confirmed in well-mixed lab-scale fed-batch processes with glycerol as carbon source in comparison to the strain without fluorescent proteins, leading to no difference in process performance. Fluorescence levels could successfully reflect the course of related process state variables, revealed population heterogeneities during the transition between different process phases and potentially subpopulations that exhibit superior process performance. Furthermore, indications were found for noise in gene expression as regulation strategy against environmental perturbation.Publicationtype: Journal ArticleScopus© Citations 3 25 143