Metabolic Flux Analysis

This review illustrates the need to use nuclear magnetic resonance (NMR) spectroscopy for the quantitative analysis of small molecules in their crude forms and in mixtures.

We provide the basic concepts of quantitative NMR (qNMR), a brief description of important acquisition and processing parameters responsible for obtaining high-quality, reproducible NMR spectra in order to maximize accuracy, and the latest referencing techniques used for quantitative analysis.

We also describe methods that are used for quantitative analysis including calibration-curve and standard-addition methods. Further, we briefly address validation of qNMR spectroscopy and its major applications in various scientific disciplines

Produksi asam asetat dengan agen hayati Lactobacillus spp. banyak digunakan dalam industri berbasis hayati. Untuk meningkatkan proses produksi menjadi skala industri, diperlukan perancangan scale-up pada bioreaktor. Pada perancangan scale-up pada bioreaktor dilakukan beberapa pendekatan, yang meliputi black box stoichiometry model, analisis termodinamika proses reaksi biokimia, analisis fluks metabolik reaksi biokimia (MFA), serta model kinetika mekanistik. Hasil analisis termodinamika reaksi biokimia produksi asam asetat didapatkan untuk katabolisme ΔH° = 679.4 kJ/mol, ΔS° = -2.146 kJ/mol. K, dan ΔG° = 39.8 kJ/mol, serta untuk anabolisme ΔH° = 8.61 kJ/mol, ΔS° = 8.61 kJ/mol, dan ΔG° = 8.61 kJ/mol. Nilai laju reaksi fluks produksi asam asetat dan biomassa didapatkan masing-masing sebesar 0.01843 mmol/g DW.jam dan 0.00153 mmol/g DW.jam. Model pertumbuhan Lactobacillus spp. yang digunakan dalam bioreaktor ini merupakan model pertumbuhan Monod. Tren laju pertumbuhan biomassa menunjukkan peningkatan konsentrasi biomassa yang sebanding dengan penurunan jumlah substrat sesuai laju penggunaan substrat. Laju pembentukan produk juga mengalami peningkatan seiring bertambahnya waktu, namun peningkatannya jauh lebih besar daripada peningkatan konsentrasi biomassa. Hal ini dapat terjadi karena laju pembentukan produk tidak berkorelasi secara linier dengan laju pertumbuhan biomassa, melainkan korelasinya adalah secara eksponensial. Jenis bioreaktor yang digunakan adalah Aerated Stirred Tank Reactor (ASTR), dengan nilai 𝑃𝑔=0,1285 W dan 𝑘𝐴,𝐺𝑎=1.079,139/h untuk pengoperasian bioreaktor.

The meiotic developmental pathway in yeast enables both differentiation of vegetative cells into haploid spores that ensure long-term survival, and recombination of the parental DNA to create genetic diversity. Despite the importance of proper metabolic regulation for the supply of building blocks and energy, little is known about the reprogramming of central metabolic pathways in meiotically differentiating cells during passage through successive developmental stages. Metabolic regulation during meiotic differentiation in budding yeast was analysed by integrating information on genome-wide transcriptional activity, 26 enzymatic activities in the central metabolism, the dynamics of 67 metabolites, and a metabolic flux analysis at mid-stage meiosis. Analyses of mutants arresting sporulation at defined stages demonstrated that metabolic reprogramming is tightly controlled by the progression through the developmental pathway. The correlation between transcript levels and enzymatic acti...

The analysis of primary and secondary nitrogen metabolism in plants by nuclear magnetic resonance (NMR) spectroscopy is comprehensively reviewed. NMR is a versatile analytical tool, and the combined use of 1H, 2H, 13C, 14N and 15N NMR allows detailed investigation of the acquisition, assimilation and metabolism of nitrogen. The analysis of tissue extracts can be complemented by the in vivo NMR analysis of functioning tissues and cell suspensions, and by the application of solid state NMR techniques. Moreover stable isotope labelling with 2H-, 13C- and 15N-labelled precursors provides direct insight into specific pathways, with the option of both time-course and steady state analysis increasing the potential value of the approach. The scope of the NMR method, and its contribution to studies of plant nitrogen metabolism, are illustrated with a wide range of examples. These include studies of the GS/GOGAT pathway of ammonium assimilation, investigations of the metabolism of glutamate, glycine and other amino acids, and applications to tropane alkaloid metabolism. The continuing development of the NMR technique, together with potential applications in the emerging fields of metabolomics and metabolic flux analysis, leads to the conclusion that NMR will play an increasingly valuable role in the analysis of plant nitrogen metabolism.

Rab25, by altering trafficking of critical cellular resources, influences cell metabolism and survival during stress conditions. Overall, perturbations in the vesicular trafficking machinery change cellular bioenergetics that can be directly measured in real time as Oxygen Consumption Rate, OCR (mitochondrial respiration) and Extracellular Acidification Rate, ECAR (glycolysis) by an extracellular flux analyzer (XF96, Seahorse Biosciences, MA). Additionally, overall turnover of glucose, lactate, as well as glutamine and glutamate can be measured biochemically using the YSI2900 Biochemistry Analyzer (YSI Incorporated, Life Sciences, OH). A combination of these two methods allows a precise and quantitative approach to interrogate the role of Rab25 as well as other Rab GTPases in central carbon energy metabolism.

Advances in metabolic engineering are enabling the creation of a large number of cell factories. However, high-throughput platforms do not yet exist for rapidly analyzing the metabolic network of the engineered cells. To fill the gap, we developed an integrated solution for fluxome profiling of large sets of biological systems and conditions. This platform combines a robotic system for (13)C-labelling experiments and sampling of labelled material with NMR-based isotopic fingerprinting and automated data interpretation. As a proof-of-concept, this workflow was applied to discriminate between Escherichia coli mutants with gradual expression of the glucose-6-phosphate dehydrogenase. Metabolic variants were clearly discriminated while pathways that support metabolic flexibility towards modulation of a single enzyme were elucidating. By directly connecting the data flow between cell cultivation and flux quantification, considerable advances in throughput, robustness, release of resources...

The precise quantification of intracellular metabolic flow rates is of fundamental importance in bio(techno)logy and medical research. The gold standard in the field is metabolic flux analysis (MFA) with 13C-labeling experiments. 13C-MFA workflows orchestrate several, mainly human-in-the-loop, software applications, integrating them with plenty of heterogeneous information. In practice, this had posed a major practical barrier for evaluating, interpreting and understanding isotopic data from carbon labeling experiments. Graphical modeling, interactive model exploration and visual data analysis are the key to overcome this limitation. We have developed a first-of-its-kind graphical tool suite providing scientists with an integrated software framework for all aspects of 13C-MFA. Almost 30 modules (plug-ins) have been implemented for the Omix visualization software. Several advanced graphical workflows and ergonomic user interfaces support major domain-specific modeling and proofreadin...

The current energy demands continuously lead us to the quest of developing alternative energy sources to compete with the depleting fossil fuel reserves. In the recent years algae have garnered attention as third generation biofuel. However, low biomass concentration and metabolite accumulation results in low biofuel yield, thereby limiting its application. The present study reviews three possible approaches for enhancing biofuel production from algae: biochemical engineering, genetic engineering and application of omics/metabolic flux. Biochemical engineering depends on changing different physiological conditions such as increasing cultivation temperature or applying nitrogen stress to route the carbon flux towards metabolite accumulation. Genetic engineering exploits the knowledge of the major molecules for metabolite accumulation to create recombinant microalgal strains, which overexpresses specific enzymes or blocks certain pathways to improve metabolism of targeted biomolecules. Omics and flux analysis helps to mathematically analyze these manipulations and interpret different possible responses in silico, based on which experiments can be conducted in-vivo. Currently biochemical engineering is the most recognized technique for metabolite improvement in algae. However, these approaches have inhibitions which can be reduced by genetic engineering, metabolic flux or omics strategies. It was analyzed that integration of these approaches may help us to overcome the current limitations of algal biofuel production. The present paper discusses the merits, success rates and constraints of each of these approaches. The article explicitly discusses how combining these engineering techniques may help us to enhance the starch/carbohydrate and the lipid content in algae for establishing it as a promising energy feedstock for the future.

We describe here a novel methodology for rapid diagnosis of metabolic changes, which is based on probabilistic equations that relate GC-MS-derived mass distributions in proteinogenic amino acids to in vivo enzyme activities. This metabolic flux ratio analysis by GC-MS provides a comprehensive perspective on central metabolism by quantifying 14 ratios of fluxes through converging pathways and reactions from [1-13C] and [U-13C]glucose experiments. Reliability and accuracy of this method were experimentally verified by successfully capturing expected flux responses of Escherichia coli to environmental modifications and seven knockout mutations in all major pathways of central metabolism. Furthermore, several mutants exhibited additional, unexpected flux responses that provide new insights into the behavior of the metabolic network in its entirety. Most prominently, the low in vivo activity of the Entner-Doudoroff pathway in wild-type E. coli increased up to a contribution of 30% to glu...

A review of the use of elementary flux modes (EFMs) and their applications in metabolic engineering covered with yield space analysis (YSA) is presented. EFMs are an invaluable tool in mathematical modeling of biochemical processes. They are described from their inception in 1994, followed by various improvements of their computation in later years. YSA constitutes another precious tool for metabolic network modeling, and is presented in details along with EFMs in this article. The application of these techniques is discussed for several case studies of metabolic network modeling provided in respective original articles. The article is concluded by some case studies in which the application of EFMs and YSA turned out to be most useful, such as the analysis of intracellular polyhydroxyalkanoate (PHA) formation and consumption in Cupriavidus necator, including the constraint-based description of the steady-state flux cone of the strain's metabolic network, the profound analysis of...