Metabolomic and kinetic investigations on the electricity-aided production of butanol by strains.

In this contribution, we studied the effect of electro-fermentation on the butanol production of strains by a targeted metabolomics approach. Two strains were examined: an electrocompetent wild type strain (R525) and a mutant strain (dhaB mutant) lacking formation of 1,3-propanediol (PDO). The dhaB-negative strain was able to grow on glycerol without formation of PDO, but displayed a high initial intracellular NADH/NAD ratio which was lowered subsequently by upregulation of the butanol production pathway.

Integrated laboratory evolution and rational engineering of GalP/Glk-dependent for higher yield and productivity of L-tryptophan biosynthesis.

L-Tryptophan (Trp) is a high-value aromatic amino acid with diverse applications in food and pharmaceutical industries. Although production of Trp by engineered has been extensively studied, the need of multiple precursors for its synthesis and the complex regulations of the biosynthetic pathways make the achievement of a high product yield still very challenging. Metabolic flux analysis suggests that the use of a phosphoenolpyruvate:sugar phosphotransferase system (PTS) independent glucose uptake system, i.

Model-assisted DoE software: optimization of growth and biocatalysis in Saccharomyces cerevisiae bioprocesses.

Bioprocess development and optimization are still cost- and time-intensive due to the enormous number of experiments involved. In this study, the recently introduced model-assisted Design of Experiments (mDoE) concept (Möller et al. in Bioproc Biosyst Eng 42(5):867, https://doi.

Dynamic parameter estimation and prediction over consecutive scales, based on moving horizon estimation: applied to an industrial cell culture seed train.

Bioprocess modeling has become a useful tool for prediction of the process future with the aim to deduce operating decisions (e.g. transfer or feeds).

Introduction of glycine synthase enables uptake of exogenous formate and strongly impacts the metabolism in Clostridium pasteurianum.

Autotrophic or mixotrophic use of one-carbon (C1) compounds is gaining importance for sustainable bioproduction. In an effort to integrate the reductive glycine pathway (rGP) as a highly promising pathway for the assimilation of CO and formate, genes coding for glycine synthase system from Gottschalkia acidurici were successfully introduced into Clostridium pasteurianum, a non-model host microorganism with industrial interests. The mutant harboring glycine synthase exhibited assimilation of exogenous formate and reduced CO formation.

Structure-based dynamic analysis of the glycine cleavage system suggests key residues for control of a key reaction step.

Molecular shuttles play decisive roles in many multi-enzyme systems such as the glycine cleavage system (GCS) for one-carbon (C1) metabolism. In GCS, a lipoate swinging arm containing an aminomethyl moiety is attached to protein H and serves as a molecular shuttle among different proteins. Protection of the aminomethyl moiety in a cavity of protein H and its release induced by protein T are key processes but barely understood.

Digital Twins and Their Role in Model-Assisted Design of Experiments.

Rising demands for biopharmaceuticals and the need to reduce manufacturing costs increase the pressure to develop productive and efficient bioprocesses. Among others, a major hurdle during process development and optimization studies is the huge experimental effort in conventional design of experiments (DoE) methods. As being an explorative approach, DoE requires extensive expert knowledge about the investigated factors and their boundary values and often leads to multiple rounds of time-consuming and costly experiments.

Activation and competition of lipoylation of H protein and its hydrolysis in a reaction cascade catalyzed by the multifunctional enzyme lipoate-protein ligase A.

Protein lipoylation is essential for the function of many key enzymes but barely studied kinetically. Here, the two-step reaction cascade of H protein lipoylation catalyzed by the multifunctional enzyme lipoate-protein ligase A (LplA) was quantitatively and differentially studied. We discovered new phenomena and unusual kinetics of the cascade: (a) the speed of the first reaction is faster than the second one by two orders of magnitude, leading to high accumulation of the intermediate lipoyl-AMP (Lip-AMP); (b) Lip-AMP is hydrolyzed, but only significantly at the presence of H protein and in competition with the lipoylation; (c) both the lipoylation of H protein and its hydrolysis is enhanced by the apo and lipoylated forms of H protein and a mutant without the lipoylation site.

Co-cultures with integrated in situ product removal for lactate-based propionic acid production.

Propionic acid (PA) is a valuable organic acid for the food and feed industry, but no bioproduction at industrial scale exists so far. As product inhibition is a major burden for bioprocesses producing organic acids, in situ product removal (ISPR) is desirable. Here, we demonstrate a new strategy to produce PA with a co-culture coupled with ISPR using electrodialysis.

Measurement of length distribution of beta-lactoglobulin fibrils by multiwavelength analytical ultracentrifugation.

The whey protein beta-lactoglobulin is the building block of amyloid fibrils which exhibit a great potential in various applications. These include stabilization of gels or emulsions. During biotechnological processing, high shear forces lead to fragmentation of fibrils and therefore to smaller fibril lengths.

Efficient Optimization of Process Strategies with Model-Assisted Design of Experiments.

Conventional design of experiments (DoE) methods require expert knowledge about the investigated factors and their boundary values and mostly lead to multiple rounds of time-consuming and costly experiments. The combination of DoE with mathematical process modeling in model-assisted DoE (mDoE) can be used to increase the mechanistic understanding of the process. Furthermore, it is aimed to optimize the processes with respect to a target (e.

Estimation of Process Model Parameters.

Cell culture technology has become a substantial domain of modern biotechnology, particularly in the pharmaceutical market. Today, products manufactured from cells itself dominate the biopharmaceutical industry. In addition, a limited number of products made of in vitro cultivated cells for regenerative medicine were launched to the market.

Near-Physiological Cell Cycle Synchronization with Countercurrent Centrifugal Elutriation.

The bioreactor conditions and cell diversity in mammalian cell cultures are often regarded as homogeneous. Recently, the influence of various kinds of heterogeneities on production rates receives increasing attention. Besides spatial gradients within the cultivation system, the variation between cell populations and the progress of the cells through the cell cycle can affect the dynamics of the cultivation process.

Quantitative analysis of glycine related metabolic pathways for one-carbon synthetic biology.

Glycine is an essential one-carbon (C1) metabolite nested in a complex network of cellular metabolism. Glycine and its related metabolic pathways have important biochemical and biomedical implications and have thus been studied for a long time. However, quantitative and systems level knowledge about the interactions and regulations of the pathways are severely limited, especially for the purpose of reengineering the relevant pathways for C1-based biotechnological processes using synthetic biology and metabolic engineering approaches.

An Aldolase-Catalyzed New Metabolic Pathway for the Assimilation of Formaldehyde and Methanol To Synthesize 2-Keto-4-hydroxybutyrate and 1,3-Propanediol in .

Formaldehyde (HCHO) is an important intermediate in the metabolism of one-carbon (C1) compounds such as methanol, formate, and methane. The ribulose monophosphate (RuMP) pathway is the most-studied HCHO assimilation route and the 3-hexulose-6-phosphate synthase (Hps) plays an important role for HCHO fixation. In this study, we proposed and selected a pyruvate-dependent aldolase to channel HCHO into 2-keto-4-hydroxybutyrate as an important intermediate for biosynthesis.

Characterization and Engineering of a Clostridium Glycine Riboswitch and Its Use To Control a Novel Metabolic Pathway for 5-Aminolevulinic Acid Production in .

A riboswitch, a regulatory RNA that controls gene expression by specifically binding a ligand, is an attractive genetic element for the control of conditional gene expression and metabolic pathways. In this study, we identified a glycine riboswitch located in the 5'-untranslated regions of a glycine:proton symporter gene in . The glycine riboswitch is shown to contain two tandem aptamers and to function as an activator of expression of genes fused to its expression platform.

Redox governed electro-fermentation improves lipid production by the oleaginous yeast Rhodosporidium toruloides.

Electro-fermentation (EF) is a promising technique to increase the performance of bioprocesses. Here, the effect of EF on the lipid production by the yeast Rhodosporidium toruloides is studied. First, an in silico analysis was performed to unveil possible lipid yield increase and metabolic shifts by EF.

Mass Production of Highly Active NK Cells for Cancer Immunotherapy in a GMP Conform Perfusion Bioreactor.

NK cells have emerged as promising candidates for cancer immunotherapy, especially due to their ability to fight circulating tumor cells thereby preventing metastases formation. Hence several studies have been performed to generate and expand highly cytotoxic NK cells , e.g.

CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided screening of enzyme variants for a more efficient chorismate pathway in .

Protein engineering plays an increasingly important role in developing new and optimizing existing metabolic pathways for biosynthesis. Conventional screening approach of libraries of gene and enzyme variants is often done using a host strain under conditions not relevant to the cultivation or intracellular conditions of the later production strain. This does not necessarily result in the identification of the best enzyme variant for use in the production strain.

Engineering of Phosphoserine Aminotransferase Increases the Conversion of l-Homoserine to 4-Hydroxy-2-ketobutyrate in a Glycerol-Independent Pathway of 1,3-Propanediol Production from Glucose.

Phosphoserine aminotransferase (SerC) from Escherichia coli (E. coli) MG1655 is engineered to catalyze the deamination of homoserine to 4-hydroxy-2-ketobutyrate, a key reaction in producing 1,3-propanediol (1,3-PDO) from glucose in a novel glycerol-independent metabolic pathway. To this end, a computation-based rational approach is used to change the substrate specificity of SerC from l-phosphoserine to l-homoserine.

Secreted factors from synovial fibroblasts immediately regulate gene expression in articular chondrocytes.

Although articular cartilage degeneration in osteoarthritis represents a major public health problem, there is still no molecular approach to prevent this pathology by blocking specific molecules. We have previously applied genome-wide expression analyses with porcine samples to identify specific markers of either growth plate or articular cartilage. Since the molecular differences were also found in cultured chondrocytes derived from both sites, we utilized primary porcine articular chondrocytes (PPACs) for the present study and analyzed, if and how they respond to synoviocyte-derived molecules.

Enhanced electron transfer of different mediators for strictly opposite shifting of metabolism in Clostridium pasteurianum grown on glycerol in a new electrochemical bioreactor.

Microbial electrosynthesis or electro-fermentation in bioelectrochemical systems (BES) have recently received much attention. Here, we demonstrate with the glycerol metabolism by Clostridium pasteurianum that H from in situ water electrolysis, especially in combination with a redox mediator, provides a simple and flexible way for shifting product selectivity and enhancing product yield in the fermentation process. In particular, we report and quantify for the first time strictly different effects of Neutral Red (NR) and the barely studied redox mediator Brilliant Blue (BB) on the growth and product formation of C.

Direct and highly sensitive measurement of fluorescent molecules in bulk solutions using flow cytometry.

Utilizing flow cytometry to monitor progress of bulk biochemical reactions and concentration of chemical species normally relies on the utilization of cells carrying intrinsic fluorescence or modified beads. We present a method for a simple measurement of the fluorescent marker molecule fluorescein and GFPuv in bulk solutions with high sensitivity using a CytoFLEX flow cytometer and without the need for modified beads. Polystyrene beads were used to trigger measurements based on their high scatter signal, to detect the fluorescence signal from two different fluorophores present in the sample solution.

New bioproduction systems for chemicals and fuels: Needs and new development.

Currently used bioproduction systems for chemicals and fuels are primarily based on sugar-containing substrates. They have inherent limitations regarding substrate sustainability and affordability, product spectrum and yield, and costs for up- and downstream processing. To overcome some of these major burdens new bioproduction strategies and systems are being developed, including biorefinery, electro-biotechnology (use of electricity for biosynthesis) and C1 bioeconomy using synthetic biological systems based on C1 carbon feedstocks such as CO, CO, methane, methanol and formic acid.

Same but different-Scale up and numbering up in electrobiotechnology and photobiotechnology.

Facing energy problems, there is a strong demand for new technologies dealing with the replacement of fossil fuels. The emerging fields of biotechnology, photobiotechnology and electrobiotechnology, offer solutions for the production of fuels, energy, or chemicals using renewable energy sources (light or electrical current e.g.