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  2. CE508 – Metabolic Engineering • Instructor • Mattheos Koffas

  3. Course Information • Lectures • M, W, F 11:00-11:50 am 106 Talbert • Office Hours • Monday 9:30-11:00 am 904 Furnas Hall • By appointment or drop-in

  4. Textbook Metabolic Engineering, Principles and Methodologies G.N. Stephanopoulos, A.A. Aristidou, J. Nielsen Academic Press, 1998 ISBN: 0-12-666260-6

  5. Recommended Bibliography • Fundamentals of Biochemistry by Voet & Voet • Genes by Benjamin Lewin • Protein Purification by Robert K. Scopes • Computational Analysis of Biochemical Systems by Eberhard O. Voit

  6. Course Grade • The grade of the course will be based on a final paper delivered by the end of the semester and an oral presentation.

  7. Projects • Project titles will be handed by the end of September. • Groups of two students- arranged by the students themselves- will pick one of the projects to work on. • The main goal is to gather literature information about the project and prepare a report summarizing findings. • A presentation by all groups will be scheduled on the last day of classes.

  8. Course Outline • Molecular Biology and Protein Chemistry • Introduction to Metabolic Engineering • The Basic Principle of Life- from DNA to Proteins • Enzyme and Protein Chemistry • Protein Purification • Transcription and RNA • DNA replication • Plasmids and Cloning Vectors • Molecular Biology tools • Theoretical Section • S-System representation of Enzymes and Metabolic Pathways • Metabolic Flux Analysis • Metabolic Control Analysis • Metabolic Flux Optimization

  9. Course Objectives • To demonstrate some of the experimental and theoretical tools available that help identify and optimize bioengineering processes at the metabolic level.

  10. The essence of Metabolic Engineering • What is Metabolic Engineering: it is the directed improvement of product formation or cellular properties through the modification of specific biochemical reaction(s) or the introduction of new one(s) with the use of recombinant DNA technology. • Other terms used: molecular breeding; pathway engineering and cellular engineering. • A two step process: • Modification of metabolic pathways • Assessment of physiological state of transformed organisms

  11. The essence of Metabolic Engineering • An essential characteristic of the preceding definition is the specificity of the particular biochemical reactions targeted for modification or to be introduced: • Once biochemical reaction targets have been identified, established molecular biology techniques are applied in order to amplify, inhibit or delete the corresponding enzymes.

  12. Flux Quantification Analysis of Flux Control ANALYSIS Metabolic Networks MODIFICATION recombinant DNA technology Cell improvement METABOLIC ENGINEERING

  13. The Cell as a factory • We treat the cell as a chemical factory, with an input and an output. D S A E B C P P1

  14. Metabolic Engineering as a Directed Evolution strategy • In biology, evolution is the sequence of events involved in the development of a species or taxonomic group of organisms. • Metabolic Engineering does exactly the same, only in a more controlled and faster way: develops new living organisms by altering the metabolism of existing ones. In that respect, Metabolic Engineering can be viewed as a method for in vitro evolution. • As in every engineering field, there is an analytical and a synthetic component.

  15. Analysis and Synthesis • Historically, the synthetic component of metabolic engineering appeared first, through the application of molecular biology tools. The main enabling technology is the recombinant DNA technology that refers to DNA that has been artificially manipulated to combine genes from two different sources. That way, well-defined genetic backgrounds are constructed. • However, the analytical component of metabolic engineering, that was emphasized later, offers a more significant engineering component: • How does one identify the targets for genetic engineering? Is there a rational process to identify the most promising targets for metabolic manipulation?

  16. Genome Sequence Analysis and Synthesis

  17. Analysis and Synthesis (cont.) • The identification of targets for genetic modification offers a directionality in cell improvement. • On the synthetic side, another novel aspect is the focus on integrated metabolic pathways instead of individual reactions. Notion of metabolic network.

  18. Metabolic Pathway- Metabolic Flux • We define a metabolic pathway to be any sequence of feasible and observable biochemical reactions steps connecting a specified set of input and output metabolites. • The pathway flux is then defined as the rate at which input metabolites are processed to form output metabolites.

  19. Metabolic Pathway- Metabolic Flux (cont.) • The concept of flux is not new to engineers. Material and energy fluxes, balances and their control are part of the core of the chemical engineering curriculum. • The combination of analytical methods to quantify fluxes and their control with molecular biological techniques to implement suggested genetic modifications is the essence of metabolic engineering.

  20. Metabolic Nodes • At a metabolic branch point, or metabolic node, a metabolite I can be used by two different pathways. • Nearly any network architecture can be constructed by connecting various unbranched pathways at particular branch points, often building a complex interweaving of branches.

  21. Metabolic Flux • The flux is a fundamental determinant of cell physiology. • For the linear pathway of the figure, the flux J1 is equal to the rates of the individual reactions at steady state. • During a transient, the individual reaction rates are not equal and the pathway flux is variable and ill-defined.

  22. Metabolic Flux • For the branched pathway splitting at intermediate I, we have two additional fluxes for each of the branching pathways, related by J1=J2+J3 at steady state.

  23. Lumping Metabolic Fluxes • Some cells in nature contain more than one different enzymes that can lead from the same input substrate to the same output product. • If the fluxes through these enzymatic reactions cannot be determined independently, their inclusion provides no additional information. In this case, it is better if these reactions are lumped together.

  24. Metabolic Flux Analysis • The determination of metabolic fluxes in vivo has been termed Metabolic Flux Analysis (MFA). • There are three steps in the process of systematic investigation of metabolic fluxes and their control: • Development of means to observe metabolic pathways and measure their fluxes. • Introduction of well-defined perturbations to the bioreaction network and pathway flux determination at the new state. • Analysis of flux perturbation results. Perturbation results will determine the biochemical reaction(s) within the metabolic network that critically determine the metabolic flux.

  25. Step one • The development of means to obtain flux measurements still tends to be problem specific. Radio or isotopomer labeling tend to be two popular methods for elucidating metabolic fluxes.

  26. Step two • Introduction of perturbations refers to the targeted change of enzymatic activities involved in a metabolic pathway. • The application of such perturbations tends to be problem specific. Several experimental methods have been proposed to that end. • Such perturbations provide means to determine, among other things, the flexibility of metabolic nodes.

  27. Step three • Fluxes at the new state need to be determined. • Analysis of the data obtained will provide a clear view of the way fluxes are controlled intracellularly. • The understanding of metabolic flux control provides the basis for rational modification of metabolic pathways.

  28. Implementation • After the key parameters of flux control have been determined, one needs to implement those changes, usually by applying genetic modifications.

  29. Genetic engineering

  30. Metabolic Engineering is an interdisciplinary field • Biochemistry has provided the basic metabolic maps and all the information on enzyme properties. • Genetics and molecular biology provide the tools for applying modifications. • Cell physiology has provided a more integrated view of cellular metabolic function.

  31. The new Paradigm Shift- Genomics and postgenomic era The new paradigm, now emerging, is that all the ‘genes’ will be known (in the sense of being resident in databases available electronically), and that the starting point of a biological investigation will be theoretical. An individual scientist will begin with a theoretical conjecture, only then turning to experiment to follow or test that hypothesis. Walter Gilbert. 1991. Towards a paradigm shift in biology. Nature, 349:99.

  32. Importance of Metabolic Engineering • The rapid increase of global population and living standards, combined with a limited ability of the traditional chemical industry to reduce its manufacturing costs and negative environmental impact make biotechnological manufacturing technologies the only alternative and the choice of the future. • Within this context, Metabolic Engineering provides the biotech industry with tools for rational strain design and optimization. This brings about significant shifts in manufacturing costs and the yields of desired products.

  33. Contributions of Metabolic Engineering • Petroleum-derived thermoplastics. • Polysaccharides • Enzymes/Proteins • Antibiotics • Vitamins • Amino Acids • Pigments • Several other high-value chemicals.

  34. Metabolic Engineering versus Bioengineering • Bioengineering (or biochemical engineering) targets optimization of processes that utilize living organisms or enzymes (biocatalysts) for production purposes. • Metabolic engineering focuses on optimizing the biocatalyst itself. • In this sense, Metabolic Engineering is equivalent to catalysis in the chemical processing industry.

  35. Metabolic Engineering and Chemical Engineering • Just as many chemical processes became a reality only after suitable catalysts were developed, the enormous potential of biotechnology will be realized when process biocatalysts become more readily available, to a significant extend through metabolic engineering. • Chemical engineering, is the most suitable engineering discipline to apply engineering approaches to the study of biological systems and to eventually bring biocatalysts to large scale production.

  36. Brief History of Biotechnology • Man has been manipulating living things to solve problems and improve his way of life for millennia. • Early agriculture concentrated on producing food. Plants and animals were selectively bred and microorganisms were used to make food items such as beverages, cheese and bread. • The late eighteenth century and the beginning of the nineteenth century saw the advent of vaccinations. • At the end of the nineteenth century microorganisms were discovered, Mendel's work on genetics was accomplished, and institutes for investigating fermentation and other microbial processes were established by Koch, Pasteur, and Lister. • Biotechnology at the beginning of the twentieth century began to bring industry and agriculture together. During World War I, fermentation processes were developed that produced acetone from starch and paint solvents The advent of World War II brought the manufacture of penicillin. The biotechnological focus moved to pharmaceuticals. The "cold war" years were dominated by work with microorganisms in preparation for biological warfare as well as antibiotics and fermentation processes.

  37. Biotechnology today • Biotechnology is currently being used in many areas including agriculture, bioremediation, food processing, and energy production. Production of insulin and other medicines is accomplished through cloning of vectors that now carry the chosen gene. Immunoassays are used by farmers to aid in detection of unsafe levels of pesticides, herbicides and toxins on crops and in animal products. In agriculture, genetic engineering is being used to produce plants that are resistant to insects, weeds and plant diseases