CE444 – Chemical Process Control

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Dec 12, 2012 (4 years and 8 months ago)

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INTRODUCTION TO METABOLIC
ENGINEERING


Chapter 1 of textbook

CE508


LECTURE ONE

CE508


Metabolic Engineering


Instructor


Mattheos Koffas


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

Textbook

Metabolic Engineering,
Principles and
Methodologies

G.N. Stephanopoulos, A.A.
Aristidou, J. Nielsen

Academic Press, 1998


ISBN: 0
-
12
-
666260
-
6

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

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.

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.

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


Course Objectives


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


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


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.

METABOLIC ENGINEERING

Metabolic

Networks

MODIFICATION

recombinant

DNA technology

ANALYSIS

Flux Quantification


Analysis of Flux

Control

Cell improvement

The Cell as a factory


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

S

A

B

C

P
1

D

E

P

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.

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?

Analysis and Synthesis

Genome Sequence

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.



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.



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 Pathway
-

Metabolic
Flux (cont.)

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.

Metabolic Flux


The flux is a fundamental
determinant of cell
physiology.


For the linear pathway of the
figure, the flux J
1

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


For the branched
pathway splitting at
intermediate I, we have
two additional fluxes for
each of the branching
pathways, related by
J
1
=J
2
+J
3

at steady state.

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.


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.

Metabolic Flux Analysis

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.

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.

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.

Implementation


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


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 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.

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.

Contributions of Metabolic
Engineering


Petroleum
-
derived thermoplastics.


Polysaccharides


Enzymes/Proteins


Antibiotics


Vitamins


Amino Acids


Pigments


Several other high
-
value chemicals.

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.

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.

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.


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