INTRODUCTION TO METABOLIC ENGINEERING Chapter 1 of textbook

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INTRODUCTION TO METABOLIC ENGINEERINGChapter 1
of textbook

• CE508 LECTURE ONE

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CE508 Metabolic Engineering

• Instructor
• Mattheos Koffas

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Course Information

• Lectures
• M, W, F 1100-1150 am
• 106 Talbert
• Office Hours
• Monday 930-1100 am
• 904 Furnas Hall
• By appointment or drop-in

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Textbook

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

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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

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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.

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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.

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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

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Course Objectives

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

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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

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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.

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METABOLIC ENGINEERING
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The Cell as a factory

• We treat the cell as a chemical factory, with an
  input and an output.

D
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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.

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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?

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Analysis and Synthesis
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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.

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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.

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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.

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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.

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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.

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Metabolic Flux

• For the branched pathway splitting at
  intermediate I, we have two additional fluxes for
  each of the branching pathways, related by
  J1J2J3 at steady state.

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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.

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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.

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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.

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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.

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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.

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Implementation

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

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Genetic engineering
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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.

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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, 34999.
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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.

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Contributions of Metabolic Engineering

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

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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.

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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.

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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.

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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

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