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Metabolomics and Application of Flux in Metabolic Engineering

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  2. 2. • Metabolism is the set of life-sustaining chemical transformations within the cells of living organisms. These enzyme-catalyzed reactions allow organisms to grow and reproduce, maintain their structures, and respond to their environments. The word metabolism can also refer to all chemical reactions that occur in living organisms, including digestion and the transport of substances into and between different cells, in which case the set of reactions within the cells is called intermediary metabolism or intermediate metabolism. • Metabolism is usually divided into two categories. Catabolism, that breaks down organic matter and harvests energy by way of cellular respiration, and anabolism that uses energy to construct components of cells such as proteins and nucleic acid • Metabolic profiling (metabolomics) is the measurement in biological systems of low-molecular-weight metabolites and their intermediates that reflects the dynamic response to genetic modification and physiological, pathophysiological, and/or developmental stimuli. • Metabolome refers to the complete set of small-molecule metabolites (such as metabolic intermediates, hormones and other signaling molecules, and secondary metabolites) to be found within a biological sample, such as a single organism. • Metabolites are the intermediates and products of metabolism. Within the context of metabolomics, a metabolite is usually defined as any molecule less than 1 kDa in size. A primary metabolite is directly involved in the normal growth, development, and reproduction. A secondary metabolite is not directly involved in those processes, but usually has important ecological function. Examples include antibiotics and pigments. By contrast, in human-based metabolomics, it is more common to describe metabolites as being either endogenous (produced by the host organism) or exogenous. Metabolites of foreign substances such as drugs are termed Xeno-metabolites. • Each type of cell and tissue has a unique metabolic ‘fingerprint’ that can elucidate organ or tissue-specific information, while the study of bio-fluids can give more generalized though less specialized information. Commonly used bio-fluids are urine and plasma. TERMINOLOGY USED IN METABOLOMICS
  3. 3. In their efforts to understand the human body and cure disease, scientists thought knowing all the 20,ooo to 25,000 genes in the genome would lead to answers. But the genome encodes—as a rough approximation—more than a million proteins, each with a special function. Even more frustrating, changes to those proteins (from bonding with lipids, carbohydrates, and so on) lead to more than 10 million functionally distinct modified proteins. Compared to unraveling the effects and associations with illness of these numerous entities, metabolomics has a decisive edge: there are only, at current best guess, about 3,000 to 6,000 metabolites of interest. METABOLOMICS
  4. 4. “A gene makes transcripts, transcripts make proteins, and proteins (or enzymes) make metabolites.” Many steps separate a gene from its ultimate expression, perhaps as a disease, but because metabolites are “downstream of genetic variation, transcriptional changes, and post-translational modifications of proteins,” they capture what is actually happening in the body: “They are the most proximal reporters of any disease status or phenotype.” They also capture the environment. “If you eat some noxious metabolite in your Big Mac,” your blood will reflect that. Genes won’t.
  5. 5. METABOLIC NETWORK Cellular metabolism is represented by a large number of metabolic reactions that are involved in the conversion of the carbon source into building blocks needed for macromolecular biosynthesis. Furthermore, there are specific reactions that ensure the constant supply of Gibbs free energy via ATP and redox equivalents (generally in the form of the cofactor NADPH) needed for biosynthesis of macromolecules. This large number of metabolic reactions forms a so-called metabolic network inside the cells, and as a result of reconstruction of the complete metabolic networks in different bacteria and in the yeast Saccharomyces cerevisiae, more insight into the function of complete metabolic networks has been obtained. These reconstructed metabolic networks can be used for detailed studies of metabolic functions and the effect of gene deletions.
  6. 6. • Metabolic Flux is defined as ‘the rate of passage of material, or of a specified substance or molecular fragment, through a given metabolic pathway’ • Flux, or metabolic flux is the rate of turnover of molecules through a metabolic pathway. Flux is regulated by the enzymes involved in a pathway. Within cells, regulation of flux is vital for all metabolic pathways to regulate the metabolic pathway's activity under different conditions. • From the pathway given in the figure, we can measure the uptake rates of glucose, the production rates of carbon dioxide, acetate, ethanol, glycerol, pyruvate, succinate etc. and the rate of synthesis of the key macromolecular pools, i.e. DNA, RNA, protein and lipids and carbohydrates. • Unit of flux is – mmoles/g/h • Why study metabolic flux? METABOLIC FLUX
  7. 7. METABOLIC FLUX ANALYSIS WITH 13-C LABELLING EXPERIMENTS To deeply understand the cells' metabolism is indispensable when aiming at an increased productivity under industrially relevant conditions by targeted genetic manipulation. In 13-C labelling, a defined 13C-labeled substrate is incorporated into the carbon backbone of a wide range of metabolites, the metabolome, either through exchange or by synthesis. The distribution of labeled carbon traversing along metabolic pathways generates a characteristic imprint of labeling patterns whose mass signature is observed by mass spectrometry (MS). Applications: Strain characterization: Generating and improving insights into metabolic pathway activity by comparing flux phenotypes under different environmental conditions and physiological states as well as for a variety of carbon sources. Eg. Strains used in bioremediation Metabolic engineering targets: Identifying pathway bottlenecks in order to maximize metabolite synthesis. This, in turn, can help to derive decisive hypotheses for promising gen targets. Hypothesis validation: Gene function manipulations can be verified on metabolome and fluxome level.
  8. 8. METABOLIC ENGINEERING  Metabolic engineering is the practice of optimizing genetic and regulatory processes within cells to increase the cells' production of a certain substance. These processes are chemical networks that use a series of biochemical reactions and enzymes that allow cells to convert raw materials into molecules necessary for the cell’s survival. Metabolic engineering specifically seeks to mathematically model these networks, calculate a yield of useful products, and pin point parts of the network that constrain the production of these products. Genetic engineering techniques can then be used to modify the network in order to relieve these constraints. Once again this modified network can be modeled to calculate the new product yield.  The ultimate goal of metabolic engineering is to be able to use these organisms to produce valuable substances on an industrial scale in a cost effective manner. Current examples include producing beer, wine, cheese, pharmaceuticals, and other biotechnology products.  The quantitative analysis of the metabolism can be done through concerted analysis of experimental data during fermentation and theoretical calculation using techniques such as Metabolic Flux Analysis (MFA), Metabolic Control Analysis (MCA).  The redesigned optimal metabolic pathways are implemented into certain microorganism to obtain maximum productivity through appropriate recombinant DNA technologies.  Successful metabolic engineering starts with a careful analysis of cellular function; based on the results of this analysis, an improved strain is designed and subsequently constructed by genetic engineering. In recent years some very powerful tools have been developed, both for analysing cellular function and for introducing directed genetic changes.
  9. 9. Metabolic engineering aims at expanding the metabolic capabilities of bacteria to biosynthesize non-natural metabolites in a sustainable, green, and cost-effective fashion. To achieve industry- level biosynthesis of these chemicals, the research theme is divided into three steps: First, artificial metabolic pathways are designed; Second, protein evolution is performed to construct and optimize the designed pathways; Third, metabolic flux is driven to the production of target compounds.
  10. 10. POLYKETIDE SYNTHESIS BY METABOLIC ENGINEERING  Polyketides are a class of secondary metabolites produced by almost all living organisms. These are structurally complex organic compounds that are often highly active biologically. Many pharmaceuticals are derived from or inspired by polyketides. Polyketide antibiotics, antifungals, cytostatics, anticholesteremic, antiparasitics, coccidiostats, animal growth promoters and natural insecticides are in commercial use  Three elements came together during the past year to provide the opportunity to generate new polyketides. • The first was the availability of cloned genes for several metabolic pathways; • The second was a genetically defined host strain able to support the production of polyketides; • The third was the ability to modify specific genes and recombine genes from different pathways using recombinant DNA technology. • These tools culminated in the rational design of new molecules and the biosynthesis of large numbers of new molecules using combinatorial biology. ERYTHROMYCIN AFLATOXIN B1 DOXYCYCLINE (TETRACYCLINE)
  12. 12. REFERENCES Wikipedia