CE508 – LECTURE ONE
INTRODUCTION TO METABOLIC
ENGINEERING
Chapter 1 of textbook
CE508 – Metabolic Engineering
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Instructor
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Mattheos Koffas
Course Information
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Lectures
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M, W, F 11:00-11:50 am
106 Talbert
Office Hours
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Monday 9:30-11:00 am
904 Furnas Hall
By appointment or drop-in
Textbook
Metabolic Engineering,
Principles and
Methodologies
G.N. Stephanopoulos, A.A.
Aristidou, J. Nielsen
Academic Press, 1998
ISBN: 0-12-666260-6
Recommended Bibliography
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Fundamentals of Biochemistry by Voet & Voet
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Genes by Benjamin Lewin
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Protein Purification by Robert K. Scopes
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Computational Analysis of Biochemical
Systems by Eberhard O. Voit
Course Grade
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The grade of the course will be based
on a final paper delivered by the end of
the semester and an oral presentation.
Projects
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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.
Course Outline
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Molecular Biology and Protein Chemistry
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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
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S-System representation of Enzymes and Metabolic Pathways
Metabolic Flux Analysis
Metabolic Control Analysis
Metabolic Flux Optimization
Course Objectives
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To demonstrate some of the
experimental and theoretical tools
available that help identify and optimize
bioengineering processes at the
metabolic level.
The essence of Metabolic
Engineering
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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
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of metabolic pathways
Assessment of physiological state of transformed organisms
The essence of Metabolic
Engineering
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An essential characteristic of the
preceding definition is the specificity of
the particular biochemical reactions
targeted for modification or to be
introduced:
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Once biochemical reaction targets have been
identified, established molecular biology
techniques are applied in order to amplify,
inhibit or delete the corresponding enzymes.
METABOLIC ENGINEERING
Flux Quantification
ANALYSIS
MODIFICATION
recombinant
DNA technology
Analysis of Flux
Control
Metabolic
Networks
Cell improvement
The Cell as a factory
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We treat the cell as a chemical factory, with an input
and an output.
D
S
A
B
E
C
P1
P
Metabolic Engineering as a
Directed Evolution strategy
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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.
Analysis and Synthesis
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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, welldefined genetic backgrounds are constructed.
However, the analytical component of metabolic
engineering, that was emphasized later, offers a more
significant engineering component:
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How does one identify the targets for genetic engineering? Is
there a rational process to identify the most promising targets
for metabolic manipulation?
Analysis and Synthesis
Genome Sequence
Analysis and Synthesis (cont.)
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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.
Metabolic Pathway- Metabolic
Flux
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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.
Metabolic Pathway- Metabolic
Flux (cont.)
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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.
Metabolic Nodes
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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.
Metabolic Flux
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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.
Metabolic Flux
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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.
Lumping Metabolic Fluxes
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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.
Metabolic Flux Analysis
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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:
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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.
Step one
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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.
Step two
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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.
Step three
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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.
Implementation
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After the key parameters of flux control
have been determined, one needs to
implement those changes, usually by
applying genetic modifications.
Genetic engineering
Metabolic Engineering is
an interdisciplinary field
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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.
The new Paradigm ShiftGenomics 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.
Importance of Metabolic
Engineering
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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.
Contributions of Metabolic
Engineering
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Petroleum-derived thermoplastics.
Polysaccharides
Enzymes/Proteins
Antibiotics
Vitamins
Amino Acids
Pigments
Several other high-value chemicals.
Metabolic Engineering versus
Bioengineering
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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.
Metabolic Engineering and
Chemical Engineering
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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.
Brief History of Biotechnology
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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.
Biotechnology today
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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