Systemic intracellular analysis for balancing complex biosynthesis in a transcriptionally deregulated Escherichia coli l-Methionine producer.

l-Methionine (l-Met) has gained remarkable interest due to its multifaceted and versatile applications in the fields of nutrition, pharmaceuticals and clinical practice. In this study, the fluxes of the challenging l-Met biosynthesis in the producer strain Escherichia coli (E. coli) DM2853 were fine-tuned to enable improved l-Met production.

A Two-Compartment Fermentation System to Quantify Strain-Specific Interactions in Microbial Co-Cultures.

To fulfil the growing interest in investigating microbial interactions in co-cultures, a novel two-compartment bioreactor system was developed, characterised, and implemented. The system allowed for the exchange of amino acids and peptides via a polyethersulfone membrane that retained biomass. Further system characterisation revealed a Bodenstein number of 18, which hints at backmixing.

Polyphosphate Kinases Phosphorylate Thiamine Phosphates.

Polyphosphate kinases (PPKs) catalyze the reversible transfer of the γ-phosphate moiety of ATP (or of another nucleoside triphosphate) to a growing chain of polyphosphate (polyP). In this study, we describe that PPKs of various sources are additionally able to phosphorylate thiamine diphosphate (ThP2) to produce thiamine triphosphate (ThP3) and even thiamine tetraphosphate in vitro using polyP as phosphate donor. Furthermore, all tested PPK2s, but not PPK1s, were able to phosphorylate thiamine monophosphate (ThP1) to ThP2 and ThP3 although at low efficiency.

Monitoring Intracellular Metabolite Dynamics in during Industrially Relevant Famine Stimuli.

Carbon limitation is a common feeding strategy in bioprocesses to enable an efficient microbiological conversion of a substrate to a product. However, industrial settings inherently promote mixing insufficiencies, creating zones of famine conditions. Cells frequently traveling through such regions repeatedly experience substrate shortages and respond individually but often with a deteriorated production performance.

Compartment-specific C metabolic flux analysis reveals boosted NADPH availability coinciding with increased cell-specific productivity for IgG1 producing CHO cells after MTA treatment.

Increasing cell-specific productivities (CSPs) for the production of heterologous proteins in Chinese hamster ovary (CHO) cells is an omnipresent need in the biopharmaceutical industry. The novel additive 5'-deoxy-5'-(methylthio)adenosine (MTA), a chemical degradation product of S-(5'-adenosyl)-ʟ-methionine (SAM) and intermediate of polyamine biosynthesis, boosts the CSP of IgG1-producing CHO cells by 50%. Compartment-specific C flux analysis revealed a fundamental reprogramming of the central metabolism after MTA addition accompanied by cell-cycle arrest and increased cell volumes.

Towards valorization of pectin-rich agro-industrial residues: Engineering of Saccharomyces cerevisiae for co-fermentation of d-galacturonic acid and glycerol.

Pectin-rich plant biomass residues represent underutilized feedstocks for industrial biotechnology. The conversion of the oxidized monomer d-galacturonic acid (d-GalUA) to highly reduced fermentation products such as alcohols is impossible due to the lack of electrons. The reduced compound glycerol has therefore been considered an optimal co-substrate, and a cell factory able to efficiently co-ferment these two carbon sources is in demand.

Compartment-specific metabolome labeling enables the identification of subcellular fluxes that may serve as promising metabolic engineering targets in CHO cells.

C labeling data are used to calculate quantitative intracellular flux patterns reflecting in vivo conditions. Given that approaches for compartment-specific metabolomics exist, the benefits they offer compared to conventional non-compartmented C flux studies remain to be determined. Using compartment-specific labeling information of IgG1-producing Chinese hamster ovary cells, this study investigated differences of flux patterns exploiting and ignoring metabolic labeling data of cytosol and mitochondria.

Micro-aerobic production of isobutanol with engineered .

KT2440 is emerging as a promising microbial host for biotechnological industry due to its broad range of substrate affinity and resilience to physicochemical stresses. Its natural tolerance towards aromatics and solvents qualifies this versatile microbe as promising candidate to produce next generation biofuels such as isobutanol. In this study, we scaled-up the production of isobutanol with from shake flask to fed-batch cultivation in a 30 L bioreactor.

Identifying and Engineering Bottlenecks of Autotrophic Isobutanol Formation in Recombinant by Systemic Analysis.

(, CLJU) is natively endowed producing acetic acid, 2,3-butandiol, and ethanol consuming gas mixtures of CO, CO, and H (syngas). Here, we present the syngas-based isobutanol formation using harboring the recombinant amplification of the "Ehrlich" pathway that converts intracellular KIV to isobutanol. Autotrophic isobutanol production was studied analyzing two different strains in 3-L gassed and stirred bioreactors.

Streamlining the Analysis of Dynamic C-Labeling Patterns for the Metabolic Engineering of as l-Histidine Production Host.

Today's possibilities of genome editing easily create plentitudes of strain mutants that need to be experimentally qualified for configuring the next steps of strain engineering. The application of design-build-test-learn cycles requires the identification of distinct metabolic engineering targets as design inputs for subsequent optimization rounds. Here, we present the pool influx kinetics (PIK) approach that identifies promising metabolic engineering targets by pairwise comparison of up- and downstream C labeling dynamics with respect to a metabolite of interest.

Revisiting the Growth Modulon of Under Glucose Limited Chemostat Conditions.

Increasing the growth rate of the industrial host is a promising target to rise productivities of growth coupled product formation. As a prerequisite, detailed knowledge about the tight regulation network is necessary for identifying promising metabolic engineering goals. Here, we present comprehensive metabolic and transcriptional analysis of ATCC 13032 growing under glucose limited chemostat conditions with μ = 0.

Characterization of Agrobacterium tumefaciens PPKs reveals the formation of oligophosphorylated products up to nucleoside nona-phosphates.

Agrobacterium tumefaciens synthesizes polyphosphate (polyP) in the form of one or two polyP granules per cell during growth. The A. tumefaciens genome codes for two polyphosphate kinase genes, ppk1 and ppk2, of which only ppk1 is essential for polyP granule formation in vivo.

Comparison of l-tyrosine containing dipeptides reveals maximum ATP availability for l-prolyl-l-tyrosine in CHO cells.

Increasing markets for biopharmaceuticals, including monoclonal antibodies, have triggered a permanent need for bioprocess optimization. Biochemical engineering approaches often include the optimization of basal and feed media to improve productivities of Chinese hamster ovary (CHO) cell cultures. Often, l-tyrosine is added as dipeptide to deal with its poor solubility at neutral pH.

Electron availability in CO , CO and H mixtures constrains flux distribution, energy management and product formation in Clostridium ljungdahlii.

Acetogens such as Clostridium ljungdahlii can play a crucial role reducing the human CO footprint by converting industrial emissions containing CO , CO and H into valuable products such as organic acids or alcohols. The quantitative understanding of cellular metabolism is a prerequisite to exploit the bacterial endowments and to fine-tune the cells by applying metabolic engineering tools. Studying the three gas mixtures CO  + H , CO and CO + CO  + H (syngas) by continuously gassed batch cultivation experiments and applying flux balance analysis, we identified CO as the preferred carbon and electron source for growth and producing alcohols.

S-adenosylmethionine and methylthioadenosine boost cellular productivities of antibody forming Chinese hamster ovary cells.

The improvement of cell specific productivities for the formation of therapeutic proteins is an important step towards intensified production processes. Among others, the induction of the desired production phenotype via proper media additives is a feasible solution provided that said compounds adequately trigger metabolic and regulatory programs inside the cells. In this study, S-(5'-adenosyl)- l-methionine (SAM) and 5'-deoxy-5'-(methylthio)adenosine (MTA) were found to stimulate cell specific productivities up to approx.

A universal polyphosphate kinase: PPK2c of Ralstonia eutropha accepts purine and pyrimidine nucleotides including uridine diphosphate.

Polyphosphosphate kinases (PPKs) catalyse the reversible transfer of the γ-phosphate group of a nucleoside-triphosphate to a growing chain of polyphosphate. Most known PPKs are specific for ATP, but some can also use GTP as a phosphate donor. In this study, we describe the properties of a PPK2-type PPK of the β-proteobacterium Ralstonia eutropha.

The Less the Better: How Suppressed Base Addition Boosts Production of Monoclonal Antibodies With Chinese Hamster Ovary Cells.

Biopharmaceutical production processes strive for the optimization of economic efficiency. Among others, the maximization of volumetric productivity is a key criterion. Typical parameters such as partial pressure of CO (pCO) and pH are known to influence the performance although reasons are not yet fully elucidated.

HILIC-Enabled C Metabolomics Strategies: Comparing Quantitative Precision and Spectral Accuracy of QTOF High- and QQQ Low-Resolution Mass Spectrometry.

Dynamic C-tracer-based flux analyses of in vivo reaction networks still require a continuous development of advanced quantification methods applying state-of-the-art mass spectrometry platforms. Utilizing alkaline HILIC chromatography, we adapt strategies for a systematic quantification study in non- and C-labeled multicomponent endogenous extracts by LC-QTOF high resolution (HRMS) and LC-QQQ tandem mass spectrometry (MS/MS). Without prior derivatization, a representative cross-section of 17 central carbon and anabolic key intermediates were analyzed with high selectivity and sensitivity under optimized ESI-MS settings.

From nutritional wealth to autophagy: In vivo metabolic dynamics in the cytosol, mitochondrion and shuttles of IgG producing CHO cells.

To fulfil the optimization needs of current biopharmaceutical processes the knowledge how to improve cell specific productivities is of outmost importance. This requires a detailed understanding of cellular metabolism on a subcellular level inside compartments such as cytosol and mitochondrion. Using IgG1 producing Chinese hamster ovary (CHO) cells, a pioneering protocol for compartment-specific metabolome analysis was applied.

Perfusion cultures require optimum respiratory ATP supply to maximize cell-specific and volumetric productivities.

Perfusion processes are an emerging alternative to common fed-batch processes in the growing biopharmaceutical industry. However, the challenge of maintaining high cell-specific productivities remains. In this study, glucose limitation was applied to two perfusion steady states and compared with a third steady state without any detectable limitation.

Quantitative Profiling of Endogenous Metabolites Using Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry (HILIC-MS/MS).

Dynamic modeling of metabolic reaction networks requires absolute quantification of intracellular and extracellular metabolite concentrations with high precision and accuracy. This chapter presents a robust HILIC-ESI-MS/MS procedure for targeted quantitative profiling of more than 50 polar key metabolites in multicomponent endogenous extracts. Without using ion-pairing-agents or prior derivatization protocols, organic acids, amino acids, sugar phosphates, coenzymes, and nucleotides are measured on a triple quadrupole platform in positive and negative electrospray ionization modes with preoptimized MRM transitions.

Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells.

Biopharmaceuticals are predominantly produced by Chinese hamster ovary (CHO) cells cultivated in fed-batch mode. Hyperosmotic culture conditions (≥ 350 mOsmol kg(∑1) ) resulting from feeding of nutrients may enhance specific product formation rates (qp ). As an improved ATP supply was anticipated to enhance qp this study focused on the identification of suitable miRNA/mRNA targets to increase ATP levels.

Compartment-specific metabolomics for CHO reveals that ATP pools in mitochondria are much lower than in cytosol.

Mammalian cells show a compartmented metabolism. Getting access to subcellular metabolite pools is of high interest to understand the cells' metabolomic state. Therefore a protocol is developed and applied for monitoring compartment-specific metabolite and nucleotide pool sizes in Chinese hamster ovary (CHO) cells.

Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification using tandem mass spectrometry.

Modeling of metabolic networks as part of systems metabolic engineering requires reliable quantitative experimental data of intracellular concentrations. The hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry (HILIC-ESI-MS/MS) method was used for quantitative profiling of more than 50 hydrophilic key metabolites of cellular metabolism. Without prior derivatization, sugar phosphates, organic acids, nucleotides, and amino acids were measured under alkaline and acidic mobile phase conditions with pre-optimized multiple reaction monitoring (MRM) transitions.