Enzyme-constrained metabolic model and metabolic engineering of for the development of sustainable production processes.

Constraint-based genome-scale models (GEMs) of microorganisms provide a powerful tool for predicting and analyzing microbial phenotypes as well as for understanding how these are affected by genetic and environmental perturbations. Recently, MATLAB and Python-based tools have been developed to incorporate enzymatic constraints into GEMs. These constraints enhance phenotype predictions by accounting for the enzyme cost of catalyzed model´s reactions, thereby reducing the space of possible metabolic flux distributions.

Towards closed carbon loop fermentations: Cofeeding of Yarrowia lipolytica with glucose and formic acid.

A novel fermentation process was developed in which renewable electricity is indirectly used as an energy source in fermentation, synergistically decreasing both the consumption of sugar as a first generation carbon source and emission of the greenhouse gas CO . As an illustration, a glucose-based process is co-fed with formic acid, which can be generated by capturing CO from fermentation offgas followed by electrochemical reduction with renewable electricity. This "closed carbon loop" concept is demonstrated by a case study in which cofeeding formic acid is shown to significantly increase the yield of biomass on glucose of the industrially relevant yeast species Yarrowia lipolytica.

A 9-pool metabolic structured kinetic model describing days to seconds dynamics of growth and product formation by Penicillium chrysogenum.

A powerful approach for the optimization of industrial bioprocesses is to perform detailed simulations integrating large-scale computational fluid dynamics (CFD) and cellular reaction dynamics (CRD). However, complex metabolic kinetic models containing a large number of equations pose formidable challenges in CFD-CRD coupling and computation time afterward. This necessitates to formulate a relatively simple but yet representative model structure.

Proliferating bloodstream-form Trypanosoma brucei use a negligible part of consumed glucose for anabolic processes.

Our quantitative knowledge of carbon fluxes in the long slender bloodstream form (BSF) Trypanosoma brucei is mainly based on non-proliferating parasites, isolated from laboratory animals and kept in buffers. In this paper we present a carbon balance for exponentially growing bloodstream form trypanosomes. The cells grew with a doubling time of 5.

Identification of informative metabolic responses using a minibioreactor: a small step change in the glucose supply rate creates a large metabolic response in Saccharomyces cerevisiae.

In this study, a previously developed mini-bioreactor, the Biocurve, was used to identify an informative stimulus-response experiment. The identified stimulus-response experiment was a modest 50% shift-up in glucose uptake rate (qGLC) that unexpectedly resulted in a disproportionate transient metabolic response. The 50% shift-up in qGLC in the Biocurve resulted in a near tripling of the online measured oxygen uptake (qO2) and carbon dioxide production (qCO2) rates, suggesting a considerable mobilization of glycogen and trehalose.

Cytosolic NADPH balancing in Penicillium chrysogenum cultivated on mixtures of glucose and ethanol.

The in vivo flux through the oxidative branch of the pentose phosphate pathway (oxPPP) in Penicillium chrysogenum was determined during growth in glucose/ethanol carbon-limited chemostat cultures, at the same growth rate. Non-stationary (13)C flux analysis was used to measure the oxPPP flux. A nearly constant oxPPP flux was found for all glucose/ethanol ratios studied.

Metabolite and reaction inference based on enzyme specificities.

Motivation: Many enzymes are not absolutely specific, or even promiscuous: they can catalyze transformations of more compounds than the traditional ones as listed in, e.g. KEGG.

Fast dynamic response of the fermentative metabolism of Escherichia coli to aerobic and anaerobic glucose pulses.

The response of Escherichia coli cells to transient exposure (step increase) in substrate concentration and anaerobiosis leading to mixed-acid fermentation metabolism was studied in a two-compartment bioreactor system consisting of a stirred tank reactor (STR) connected to a mini-plug-flow reactor (PFR: BioScope, 3.5 mL volume). Such a system can mimic the situation often encountered in large-scale, fed-batch bioreactors.

Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry.

A new sensitive and accurate analytical method has been developed for quantification of intracellular nucleotides in complex biological samples from cultured cells of different microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. This method is based on ion pair reversed phase liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry (IP-LC-ESI-ID-MS/MS. A good separation and low detection limits were observed for these compounds using dibutylamine as volatile ion pair reagent in the mobile phase of the LC.

Dynamic 13C-tracer study of storage carbohydrate pools in aerobic glucose-limited Saccharomyces cerevisiae confirms a rapid steady-state turnover and fast mobilization during a modest stepup in the glucose uptake rate.

In this research, two dynamic (13)C-labeling experiments confirmed turnover and rapid mobilization of stored glycogen and trehalose in an aerobic glucose-limited chemostat (D=0.05 h(-1)) culture of Saccharomyces cerevisiae. In one experiment, the continuous feed to an aerobic glucose-limited chemostat culture of S.

Energetic and metabolic transient response of Saccharomyces cerevisiae to benzoic acid.

Saccharomyces cerevisiae is known to be able to adapt to the presence of the commonly used food preservative benzoic acid with a large energy expenditure. Some mechanisms for the adaptation process have been suggested, but its quantitative energetic and metabolic aspects have rarely been discussed. This study discusses use of the stimulus response approach to quantitatively study the energetic and metabolic aspects of the transient adaptation of S.

Model reduction and a priori kinetic parameter identifiability analysis using metabolome time series for metabolic reaction networks with linlog kinetics.

In this work, we present a time-scale analysis based model reduction and parameter identifiability analysis method for metabolic reaction networks. The method uses the information obtained from short term chemostat perturbation experiments. We approximate the time constant of each metabolite pool by their turn-over time and classify the pools accordingly into two groups: fast and slow pools.

Isotopic non-stationary 13C gluconate tracer method for accurate determination of the pentose phosphate pathway split-ratio in Penicillium chrysogenum.

Current (13)C labeling experiments for metabolic flux analysis (MFA) are mostly limited by either the requirement of isotopic steady state or the extremely high computational effort due to the size and complexity of large metabolic networks. The presented novel approach circumvents these limitations by applying the isotopic non-stationary approach to a local metabolic network. The procedure is demonstrated in a study of the pentose phosphate pathway (PPP) split-ratio of Penicillium chrysogenum in a penicillin-G producing chemostat-culture grown aerobically at a dilution rate of 0.

Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export.

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate.

Quantitative analysis of metabolites in complex biological samples using ion-pair reversed-phase liquid chromatography-isotope dilution tandem mass spectrometry.

A rapid, sensitive and selective ion-pair reversed-phase liquid chromatography-electrospray ionization isotope dilution tandem mass spectrometry (IP-LC-ESI-ID-MS/MS) was developed for quantitative analysis of free intracellular metabolites in cell cultures. As an application a group of compounds involved in penicillin biosynthesis pathway of Penicillium chrysogenum cells, such as penicillin G (PenG), 6-aminopenicillanic acid (6-APA), benzylpenicilloic acid (PIO), ortho-hydroxyphenyl acetic acid (o-OH-PAA), phenylacetic acid (PAA), 6-oxopipeidine-2-carboxylic acid (OPC), 8-hydroxypenicillic acid (8-HPA), L-alpha-(delta-aminoadipyl)-L-alpha-cystenyl-D-alpha-valine (ACV) and isopenicillin N (IPN) were chosen. (13)C-labeled analogs of the metabolites were added to the sample solutions as internal standards (I.

Quantitative physiological study of the fast dynamics in the intracellular pH of Saccharomyces cerevisiae in response to glucose and ethanol pulses.

Considering the effects of pH on many aspects of cell metabolism, such as its role in signaling processes and enzyme kinetics, it is indispensable to include the measurement of the dynamics of the intracellular pH, when studying the fast dynamic response of cells to perturbations. It has been shown previously that the intracellular pH rapidly drops following an increase in external glucose concentration [Kresnowati, M.T.

Dynamic in vivo metabolome response of Saccharomyces cerevisiae to a stepwise perturbation of the ATP requirement for benzoate export.

Although much information is available on in vitro role of ATP in regulation, the in vivo kinetics of reactions in which ATP plays a role are only partly known. In order to study such reactions, it is therefore necessary to study the role of ATP in vivo. This study presents an in vivo, targeted perturbation of the ATP flux in aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae, which was accomplished by transiently (20 min) changing the extracellular undissociated benzoic acid concentration via the pH of the culture.

13C-Labeled metabolic flux analysis of a fed-batch culture of elutriated Saccharomyces cerevisiae.

This study addresses the question of whether observable changes in fluxes in the primary carbon metabolism of Saccharomyces cerevisiae occur between the different phases of the cell division cycle. To detect such changes by metabolic flux analysis, a 13C-labeling experiment was performed with a fed-batch culture inoculated with a partially synchronized cell population obtained through centrifugal elutriation. Such a culture exhibits dynamic changes in the fractions of cells in different cell cycle phases over time.

A method for estimation of elasticities in metabolic networks using steady state and dynamic metabolomics data and linlog kinetics.

Background: Dynamic modeling of metabolic reaction networks under in vivo conditions is a crucial step in order to obtain a better understanding of the (dis)functioning of living cells. So far dynamic metabolic models generally have been based on mechanistic rate equations which often contain so many parameters that their identifiability from experimental data forms a serious problem. Recently, approximative rate equations, based on the linear logarithmic (linlog) format have been proposed as a suitable alternative with fewer parameters.

Metabolic flux analysis of a glycerol-overproducing Saccharomyces cerevisiae strain based on GC-MS, LC-MS and NMR-derived C-labelling data.

This study focuses on unravelling the carbon and redox metabolism of a previously developed glycerol-overproducing Saccharomyces cerevisiae strain with deletions in the structural genes encoding triosephosphate isomerase (TPI1), the external mitochondrial NADH dehydrogenases (NDE1 and NDE2) and the respiratory chain-linked glycerol-3-phosphate dehydrogenase (GUT2). Two methods were used for analysis of metabolic fluxes: metabolite balancing and (13)C-labelling-based metabolic flux analysis. The isotopic enrichment of intracellular primary metabolites was measured both directly (liquid chromatography-MS) and indirectly through proteinogenic amino acids (nuclear magnetic resonance and gas chromatography-MS).

Cytosolic NADPH metabolism in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum.

This study addresses the relation between NADPH supply and penicillin synthesis, by comparing the flux through the oxidative branch of the pentose phosphate pathway (PPP; the main source of cytosolic NADPH) in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum. The fluxes through the oxidative part of the PPP were determined using the recently introduced gluconate-tracer method. Significantly higher oxidative PPP fluxes were observed in penicillin-G producing chemostat cultures, indicating that penicillin production puts a major burden on the supply of cytosolic NADPH.

Characterization of an experimental miniature bioreactor for cellular perturbation studies.

A mini bioreactor (3.0 mL volume) has been developed and shown to be a versatile tool for rapidly screening and quantifying the response of organisms on environmental perturbations. The mini bioreactor is essentially a plug flow device transformed into a well-mixed reactor by a recycle flow of the broth.

When transcriptome meets metabolome: fast cellular responses of yeast to sudden relief of glucose limitation.

Within the first 5 min after a sudden relief from glucose limitation, Saccharomyces cerevisiae exhibited fast changes of intracellular metabolite levels and a major transcriptional reprogramming. Integration of transcriptome and metabolome data revealed tight relationships between the changes at these two levels. Transcriptome as well as metabolite changes reflected a major investment in two processes: adaptation from fully respiratory to respiro-fermentative metabolism and preparation for growth acceleration.

Measurement of fast dynamic intracellular pH in Saccharomyces cerevisiae using benzoic acid pulse.

pH affects many processes on cell metabolism, such as enzyme kinetics. To enhance the understanding of the living cells, it is therefore indispensable to have a method to monitor the pH in living cells. To accomplish this, a dynamic intracellular pH measurement method applying low concentration benzoic acid pulse was developed.

13C-labeled gluconate tracing as a direct and accurate method for determining the pentose phosphate pathway split ratio in Penicillium chrysogenum.

In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-13C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum.