Metabolic Engineering Notes

Scope of Metabolic Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Classical Metabolic Engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Penicillin as an Example. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
General Strategy of Classical Metabolic Engineering. . . . . . . . . . . . . . . . . . . . . . . . . 4
Lysine Product: Unlocking Cellular Control of Metabolism . . . . . . . . . . . . . . . . . . 5
Screening of Mutants with the Desired Phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Selection of Mutants with the Target Trait. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
High Throughput Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Recombinant DNA based Metabolic Engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
General Strategy of Metabolic Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Engineering Lysine Producing Microorganism . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Scope of Metabolic Engineering

Many intermediates of biochemical pathways are involved in more
than one reaction forming a vast interconnected network. These Scope of metabolic engineering
different reactions channel the resources to meet various needs in the • Modify host cells, host multicellular organisms,
or product
cells. Cells must regulate the distribution of the resources in response • Improved production, in selectivity or in
to their changing needs. In general, the microorganisms have evolved quantity, of chemicals already produced by the
to be not wasteful of their resources. Metabolic engineering is to host organism
• Extended substrate range for growth and
overcome those regulations, to make cells to produce products that product formation
they may not need for their own well being, or produce those products • Addition of new catabolic activities for
degradation of toxic chemicals
in such a large quantity that greatly exceeds their own needs. In a
• Production of chemicals new to the host
way by metabolically engineering organisms we make them become organism
wasteful in using their resources. Such manipulation of an organism’s • Modification of cell properties
metabolic pathway has traditionally been achieved through mutating
cells, but is now done by using recombinant DNA technology.

Almost all sorts of organisms can be subjected to metabolic

engineering including bacteria, molds or multi-cellular organisms.
Metabolic engineering is frequently used to improve the efficiency of
product formation. One may change the selectivity of a pathway to

Metabolic Engineering Notes  

favor one product. An organism may produce two or three products,
but only one of them is desirable. The undesirable products can be
eliminated to enrich the product. Both the productivity (or production
rate) and the yield can be greatly improved through metabolic
engineering. Having a single product also improves purification.
Metabolic engineering can also be used to change the substrate used.
An organism cultured a substrate that is too expensive for the product
produced can be metabolically engineered to make them grow on an
inexpensive substrate. This has been achieved for the use of cellulosic
biomass to produce ethanol. Ethanol fermenting bacterium can only
use six-carbon sugar. However, the cellulosic agricultural cellulose
biomass consists of a large percentage of 5-carbon sugar in addition
to glucose. The micro-orgranism was engineered to use the 5-carbon
sugar so that it can convert both 6- and 5-carbon sugar to ethanol.
This can change the economic outlook of the process of biomass
conversion to ethanol or biofuel formation. Bacteria and plants have
also been metabolically engineered to degrade or absorb heavy
metal and store it inside the cell so the heavy metal can thus be
removed from the surrounding environment.. In other examples cells
are engineered to degrade toxic organic material. Some cells can
degrade PCP (Penta-chloro phenol) into a non-toxic compound, such
as Carbon Dioxide and HCL or Hydrochloric Acid. They are often
also susceptible to high levels of PCP. By metabolic engineering they
may be made to tolerate higher levels of PCP. The novel application
of metabolic engineering is to make cells to make new chemicals that
are foreign even to themselves. This can be done by introducing new
enzymes to divert the metabolic intermediates to a new pathway,
to create a link by introducing an entirely new set of enzymes to
create a new pathway between two otherwise uncoupled pathways.
By combinatorial techniques one can shuffle one take segments of
protein from a different enzyme and recombine them to form new
enzymes and potentially from an organism to create new pathways.

Metabolic engineering is also used to modify the cells themselves.

A notable example is the genetically modified organisms (GMO)
especially in plants. In the last decade many crops (corn, soybean)
have been genetically modified so that they harbor a foreign protein
that can cause insects to die after ingesting the leaves or other parts
of the plant. These metabolically engineered plants reduce the need
of insecticide. GMO is also controversial as their potential long-
term impact on the environment is not known. Another example
of metabolic engineering to change cell properties is the reduction
of the tendency of cells to commit suicide (apoptosis). Many

  Metabolic Engineering Notes

higher organisms, especially in the mammalian cells, under adverse
105
cultivation conditions can trigger a process called atoptosis that is
essentially committing suicide. In rDNA protein production such 104 $18/kg
(15,000
process is not desirable because we would like cells to stay alive as ton)
$150/kg
long as possible. One can metabolically engineer cells to suppress 103
(3000 ton)
Penicillin
the apoptotic mechanism to render cells having a lesser tendency to Titer 102
(mg/l)
commit suicide and prolong culture time and increase the productivity.
$11,000/kg
101 (2.3 ton)
Classical Metabolic Engineering
100

Penicillin as an Example 1940 1950 1960

Year
1970 1980

4
Traditional metabolic engineering has changed the picture of Engineering invotion enabled the large scale production and reduced the cost of products

penicillin production Its increased availability at a lower cost greatly

improved human health care in the middle of the 20th century. When  Repeated mutations were necessary to create strains
it was first produced in the 1930’s, the antibiotic titer in the culture of the mold Penicillium chrysogenum which produce
high titers of penicillin; that became the foundation
was very, very low. Now the titer in the production scale bioreactor of a commercial process and changed human health
is about 5 times of what was initially discovered more than 70 years care.
ago. We can also see the quality produced is tremendously higher
 Radiation and chemical agents were employed
also about 3000 times. The price also has reduced drastically. In by investigators to induce mutations in the
effect, initially penicillin was such a precious compound when microorganism.
it was first discovered. Now penicillin is a commodity chemical.
No industrialized country can produce penicillin economically
nowadays. It is produced in China, India and other developing
countries. The improvements in the productivity can be attributed
to engineering work that allowed sufficient supply of oxygen and
removed metabolic heat in large scale processes. Equally important
has been the contribution from the microbiologists on increasing the
productivity of the microorganism by a few thousand fold. When the
productivity and cost of materials are plotted against time, one sees
that in the early stages of development the rate of process enhancement
is very high. After a period of slower increase, the product becomes
a commodity chemical. This time the slope is lower Basically, good
engineering makes processes so good that it eventually one has to
phase out the process as it becomes too cheap a product. Just as in
the case of Penicillin, no Western world can produce Penicillin at an
economic level any more. So, through metabolic engineering one
aims is to discover new compounds and focus on products which
are at an early stage of development that has a higher profit margin.

An example of classical metabolic engineering is the increase

of Penicillin productivity from the initial isolates to industrial
producers. The classical metabolic engineering largely relies on
random mutagenesis in which the microorganisms were exposed

Metabolic Engineering Notes 

to the radiation by UV light or other chemical agents to increase
the mutation frequency. A small number of those mutations
affect genes related to antibiotic biosynthesis resulting in either
higher or lower antibiotic production. By employing screening or
other methods the ones which have higher productivity than the
original parental cells were isolated and verified. The process to
achieve the 100-fold increase in productivity was long and tedious.
General methodology of metabolic engineering
1. Identify the target phenotype or trait
General Strategy of Classical Metabolic
Engineering 2. Increase the frequency of occurrence of
gene(s) that may confer the phenotype
A general procedure of classical metabolic engineering is described – Increase the mutation frequency in producing
in slide 8. Ideally the desired phenotype or the trait should be clearly cells by Mutagen treatment (UV, X-ray,
chemical mutagen) (Classical method)
defined. Preferably the biochemical pathway or other cell properties – Introduce additional gene(s) (that may
leading to the desired trait is also known. This is not always necessary. If already exist or absent in the host cell)
there is a good method to differentiate organisms harboring the desired known to give cells the desired properties
(Genetic Engineering)
trait from others, An example is, again, the Penicillin story. One didn’t – Introduce genetic element to inactivate or
know how Penicillin biosynthetic pathway, many high producers activate the gene by random insertion of
extra sequence
were isolated by simply treating the microorganism with mutagens
and then looked for ones that produced more antibiotics. However, 3. Identify the mutants (clones) that have the
with the modern approach of using recombinant DNA to modify the desired trait.
microorganisms, one often needs to know the pathway in order to Two general means
design the new genetic make-up to be introduced to the microorganism. • Screening
• Selection

Another element of metabolic engineering is to increase the

frequency of occurrence of the mutants. Like the example shown
with the Penicillin, one can use chemical or radiation to increase the
mutant occurrence frequency. Modern metabolic engineering uses
recombinant DNA techniques to introduce additional copy of the target
genes or add new genes to the microorganism. Sometimes recombinant
DNA techniques are used to “knock down” or “knock out” an existing
gene in the microorganism. For example, to channel carbon in an
organism producing both acetic acid and ethanol one may knock out
a gene involved in acetic acid synthesis to produce only ethanol. After
mutagenesis or genetic engineering, the result population is a mixture
of cells in which maybe only a few will have the desired property or
has the correct genetic make-up. One will have to sort out which ones
to choose for further verification and characterization. In general, the
procedure of finding them can be classified into 2 types of methods:
screening and selection. Screening is to allow most if not all in the
population to grow and one aims to find the desired ones by some
phenotypic trait. Selection uses selection pressure to kill off most
of the unwanted ones to enrich the desired ones. One then confirms
presence of the desired trait with the smaller number of isolates.

  Metabolic Engineering Notes

Lysine Product: Unlocking Cellular Control
of Metabolism Identifying the target- example: Lysine
Biosynthesis Pathway
Slide number 9 shows an example of how to identify the target. Lysine Aspartate inhibition

is an essential amino acid for most animals. Cattle obtain Lysine from Aspertate Kinase
repression

Aspartyl Phosphate
components of the feed, such as soybean meal. Sometimes a lysine Aspertate Semialdehyde Dehydrogenase
Aspartate Semialdehyde
supplement is used especially when the cost of other protein sources
is high. Lysine is produced through a fermentation process using a
Homoserine Dehydrogenase
Homoserine

bacterium called Corymebacterum. The microorganism typically Homoserine Phosphate

uses glucose and ammonium as carbon and nitrogen sources. They can Methionine Threonine

incorporate an amino group to oxaloacetic acid in TCA cyclte to form

Lysine
aspartic acid. Aspartic acid can then be converted through aspartate Isoleucine

family pathway to lysine. However, in the process of producing The parental microorganism produces lysine, methionine and isoleucine. If lysine is the desired
product, one can block the branch leading to homoserine. However, the resulting mutant will require 9
lysine, methionine, threonine, and isoleucine are also formed. This methionine and isoleucine for growth. They are thus methionine and isoleucine auxotrophs.

metabolic pathway is subjected to feedback regulation, Feedback

inhibition that regulates the enzyme activities and feedback repression
that affect the Synthesis of the enzyme. Both feedback repression and
feedback inhibition on Aspartate pathway reduces the production of
lysine. Threonine and lysine together inhibit Aspertate Kinase, the
first enzyme in the pathway while threonine and methionine repress
their own biosynthetic enzyme (also in the first step after the bunching
point). Isoleucine inhibits the first enzyme in its own biosynthetic
branch. These regulations modulate the biosynthetic rate to prevent
excessive formation of the amino acids. If one amino acid is no
longer used for the protein synthesis it accumulates intracellularly.
When that happens, the feedback inhibition and repression reduce
the over-accumulation and waste of resources. For the production
of anubi acuds as industrial chemicals, these regulations are needed
to overcome to allow cells to produce in great excess. In the case
of lysine production stopping the flux toward the branch from
Aspartate Semialdehyde to methionine and threonine isoleucine will
Classical Industrial Producer Improvement
increase the yield of lysine. However, this will render the cell to
• Mutagenesis and Screening/Selection
need exogenous supply of methionine, threonine, and isoleucine in – Looking for phenotype, not knowing the enzyme(s)
order to grow. In principle, only methionine and threonine need to or other mechanisms that has been changed
• Possible cause of increased production:
be supplied as threonine can lend to isoleucine. Additionally, the – Deregulation of the production pathway
feedback regulation of lysine should be removed to allow for lysine – Increased supply of Precursor (raw material)
to accumulate and be excreted without causing feedback regulation. –Enhanced transport of product out
–Increased resistance or tolerance to product concentration
–Amplification of genes involved in the biosynthesis
In addition to channeling fluxes at a branching point in a pathway
and to relax feedback regulations It may be necessary to increase
the supply of the precursor in the case of lysine, aspartic acid.
Furthermore, the overproduction of lysine should not merely lead
to accumulation at a high level intracellularly. A high intracellular
level may change the pH or cause other physiological damaging

Metabolic Engineering Notes 

effects. The transport of the product outside of the cell is thus
important. Alternatively one may alter cellular machinery to
make them more tolerant to the higher product concentration
inside the cell. There are also the cases that the entire machinery
of making the product, the entire pathway, may be amplified.
After introducing mutations or other alterations in a
pathway, selection or screening is used to find the mutants.

Screening of Mutants with the Desired

Phenotype
Slide 11 shows how penicillin-producing organism is selected.
After the producing microorganism is subjected to mutagenesis
they are plated on agar plates with indicator organisms which are
sensitive to penicillin the survived penicillin producing mold
form colonies and produce penicillin which prevent the growth
of the indicator organism, creating a clear zone in which no
indicator cells grow. A mutant producing more penicillin also
gives a larger growth inhibition zone, and can be identified and
isolated. Subsequent cultivation of the isolates usual in liquid
medium confirms that what isolated is indeed a high producer.
11

This example shows screening by growth inhibition.

The method can be applied to select microorganisms which secrete
The same principle is used for growth enabling. In
specific nutrient(s) rather than an antibiotic. One can screen for that case the producer produces a nutrient which is
growth promoting activity rather than growth inhibiting activity. In required for indicator organism to grow.
this case, the indicator organism will be one that requires the nutrients This results in a mixed growth of producing organism
secreted by the producer in order to grow. However, in this case both as well as indicator organism. This is not desired as the
the producing organism and the indicator organism will grow, this producing organism is “contamined”.

forming a mixed culture. It will make it harder to isolate the high One possible solution is to separate the producing
producing organism. One way to overcome the problem is to use organism and the indicator organism with a membrane
that is permeable to nutrient, but not penetrated by the
a membrane separating the producing cells and indicator organism.
organisms.

Another screening commonly used is for isolating high antibody

producing mammalian cells. Antibodies are increasingly used in the
treatment of cancer and other diseases. The first step in generating
a recombinant cell producing antibody is to introduce the IgG gene
into the cells. The most frequently used cells used are either Chinese
Hamster ovary (CHO Cells) and myeloma cells (NSO, SPO). Typically
the IgG genes (a heavy chain and a light chain) are put together with
an antibiotic-resistance gene on a plasmid. The cells which acquire
the plasmid and survive the antibiotic selection are very likely to have
the IgG gene. Therefore, after introducing the plasmid into the cell,
cells are exposed to the antibiotic to kill any cell that has not acquired 13

the plasmid and enrich the cells which carry IgG genes. However,

  Metabolic Engineering Notes

even among those cells which have the IgG gene the productivity can
vary over a very wide range, as there are many other factors affecting
IgG antibody production. Therefore, a screening process is usually
done to isolate cells which produce more. A common practice is
to dilute the cells to a low level that in a possoin distribution, the
probability of having more than one cell present in the same well of
a 96 well plate will be very low. Therefore, any well that has cells
growing afteran incubation period will have originated from a single
cell. Each resulting cell population from a well is thus a “clone”. The
supernatant from each well that has cells growing are then taken for
IgG measurement to determine which well produces more antibodies.

Selection of Mutants with the Target Trait

Selection can largely be classified as is negative selection and positive
selection. In a positive selection only the target cell will grow under
the selection condition. While in a negative selection, the target
cells will not grow under the selection condition. Obviously positive
selection is a better method. The example shown in slide 14 is to
isolate yeast which grows in permissive temperature of 23°C but not
in the non-permissive temperature of 36°C. Normally, all the cells
can grow in 36°C. After treating the cells with mutagen, the cells
are plated on agar plates at a low concentration to allow for growth
as a single colony. One then creates something called the Replica-
plate. Using a stamp, by stamping on the agar plate to pick up some
cells from each colony one can transfer the cells to another plate
Chapter 9 of textbook

14
Permissive condition Non-permissive condition
which will have identical spatial distribution of the colony on the
original plate. By placing the replica plates in the two temperatures
cell growth at two different temperatures can be compared.
Lysine Biosynthesis Pathway
Aspartate
Because of its importance in animal nutrition lysine has been an
inhibition
Aspertate Kinase
repression

important biotechnology product over half a century. The early Aspartyl Phosphate
Aspertate Semialdehyde Dehydrogenase

production strains of corynbacterium glutamicum were product Aspartate Semialdehyde

of classical strain improvement through random mutagenesis and Homoserine

Homoserine Dehydrogenase

clever selection. With rDNA technology new production strains Homoserine Phosphate

were generated. Lysine can suppress its own synthesis by inhibiting Methionine Threonine

aspartate kinase. Classical strategy to overcome the inhibition is to Lysine

use analogues. These are chemicals that are structurally very similar Isoleucine
Lysine fermentation in C. glutamicum, lysine plus threonine cause concerted
Mutants resistant to lysine
to lysine but they can not be metabolized or incorporated proteins. feedback inhibition (-----) of aspartokinase. A mutant is obtained that lacks
homoserine dehydrogenase. Threonine plus methionine must now be supplied in analogue, 2-amninoethyl-L-
the medium. If Threonine is added at a growth-limiting concentration, concerted cysteine and Į-chlorocaprolactam
If the culture for the microorganism contains a high concentration of feedback inhibition is broken (-- -- --) and lysine is overproduced. C. glutamicum
lacks the usual feedback inhibition by lysine of dihydrodipicolinate synthetase. overproduce lysine 15

such analogue, aspartate kinase is inhibited and thus shuts down the
biosynthesis of lysine and other amino acids in the aspartate family
pathway. As a result cells do not survive. The selection of the feedback
deregulated mutants entails increasing the mutation frequency, and

Metabolic Engineering Notes 

then growing the organism in high concentrations of lysine analogue.
The majority of the cells will die because of growth inhibition by the
Screening and Selection—Classic methods
analogue. However a few survivors eventually emerge. Many of those • Screening
survivors have the enzyme apartate kinase and have been mutated so – Growth inhibition zone
• For products that inhibit growth of
that they are no longer feedback inhibited by lysine. The method of
susceptible organisms
using analogues is an efficient way of isolating deregulated mutants. – Growth enabling zone
• For products that allow an organism
High Throughput Screening to grown on medium that
is otherwise non-growth
In the past ten years photo-sensing techniques, image analysis, sustaining
and other micro-analytical techniques have found wide spread • Selection
– Positive selection—clones that survive may
application in Life science research. Along with the development carry the desired genotype
of robotic systems the practice of cell screening has changed • Growth on chemical analogs that
drastically. Microbial and biochemical screening was not a good inhibit or repress the production of the
essential product but cannot be used
method for discovery because of the labor involved and the need for growth
of huge numbers of samples to be processed. Now screening has • Growth of antibiotic producer on high
concentration of antibiotics that itself
employed robots or other types of liquid handling systems and
produces (antibiotic is usually also
image analysis systems. Such screening methods are often referred harmful to its producer, high producer
to as high throughput systems. Many such high throughput systems should be more resistant to a high
concentration, so a more resistant
employ reporter genes such as green fluorescent protein (GFP) producer may also produce more.
isolated from Jelly Fish. With GFP as a reporter the cells which – Negative selection—the clones that do not
express GFP also appear green under a fluorescent microscope. grow on some condition may have the
desired trait
GFP can be linked to a promoter. The appearance of GFP is thus • Example: select auxotrophs of A
indicative of the promoter being turned on. GFP can also be fused (which require nutrient A to
grow)
to a target protein. Thus, cells producing the target protein can be
• Take advantage of penicillin G’s
identified or even sorted out by single cell sorting flow cytometer. killing only growing gram (+)
bacteria, not resting bacteria
• Use replica plates (use a stamp to
The robotic system now also widely employed – 96 or 384 well plate. transfer colonies)
Each of them is about 5 x 10 cm contains 96 or 384 wells. Each well can be – In A- plates, all the ones that
used for a sample. For example in isolating cells with high productivity do not need A will divide and
be killed by penicillin. On A+
antibodies, 96 or 384 well plates are commonly used. After cells plates, all will grow. Compare
grow, the robotic system can transfer a small drop of liquid from each the two sets of plates, pick the
ones that grow in A+, not in A-.
well on the culture plate to another plate. The robotic liquid handling
system then performs measurement of the antibody condensation.

Recombinant DNA based Metabolic

Engineering
Since the arrival of recombinant DNA technology the face of
metabolic engineering has changed drastically, random mutagenesis
gave way to targeted gene alteration. In the case that a foreign gene
(or genes) is to be introduced into a host cell or a set of genes are to
be amplified, a DNA segment containing the gene is inserted into
a plasmid, which is circular DNA capable of self replicating in the

  Metabolic Engineering Notes

host cell. The plasmid used usually also harbors a gene encoding for
Metabolic engineering:
resistance to an antibiotic. After a transformation process to create
• Targeted genetic
conditions favoring the uptake of plasmid into the bacterium, a mixed modifications.
• Often applying the same
population usually results, some would have received plasmid, others
strategies, the same set of
not. By growing the cell in a medium containing a lethal concentration targets
of antibiotic, all but those which have received plasmid and express • Better informatic tools,
modeling tools
the antibiotic resistance will survive. The gene of interest is thus • Easier to introduce
introduced into the host cell. In addition to introducing new or extra foreign genes

copies of genes, recombinant DNA technology also allows “knockout”

or deletion of a gene from a chromosome or otherwise renders it
inactive. By manipulating the protein coding DNA sequence, it is 20

also used to alter a protein structure through changes in some amino

acids in the protein. This is often referred to as protein engineering.

General Strategy of Metabolic

Engineering
With the increased capability to modify the biochemical pathways,
new strategies of engineering a pathway emerge. The first line of
modification is generally augmenting the biosynthetic capacity
of the pathway directly involved. This is done by introducing
additional copies of the gene of the rate limiting enzyme(s). In
the case that the pathway involved is a branched pathway, one
can appropriate a larger proportion of the incoming flux to the
desired branch. This can be done by amplifying the gene copy
of the enzyme leading to the target branch by suppressing the
level of enzymes to the other. Normally it is done by the former.

When the flux is restricted by feedback regulation on an enzyme by the

accumulation of the product or an intermediate leading to the product,
one can engineer the regulatory protein or the structural element on
the enzyme that plays the regulatory role. For example, if a repressor
protein is involved, one can modify the binding site of the ligand to
remove its response to feedback regulation or modify the DNA binding
site so that the regulator site on DNA is always at an “on” state. In
the case of feedback inhibition the enzyme often has a binding site
for the ligand (the feedback product). One can engineer the protein
to change its binding characteristics so that the enzyme activity is not
suppressed even at a high level of product. Another approach is to
clone in a gene from another species (called heterologous gene) that
has the same catalytic activity but is not subject to feed back regulation.

There are also cases that one may wish to replace an existing
enzyme with another one (either protein engineered or heterologous)

Metabolic Engineering Notes  

for reasons other than overcoming feedback regulation. The new
characteristics that are more desirable may be for better energetic
efficiency (such as consuming less ATP), better substrate specificity,
(thus less side reactions), better substrate binding characteristics,
(more active even at a low substrate concentration) etc.

Whenever using gene amplification or gene replacement to enhance

the flux of a pathway or a branch in a pathway, it is not unusual that
after one reaction in the pathway is modified, another may become
rate limiting. It is thus not uncommon that multiple steps in a reactor
pathway need to be engineered to increase the flux substantially.

Few biochemical pathways are well isolated from others. Most

derive its precursor from another pathway. Many obtain the raw
materials from multiple pathways. The supply of those precursors
certainly needs to be enhanced. An industrial producer may produce
up to 50,000 times more lysine, than what is needed to make cellular
protein. For those cells the rate of synthesizing oxaloacetic acid,
a precursor to aspartic acid, must also be increased proportionally.

A producer of metabolites may need to impart some precursors directly

used for product synthesis. It also needs to export products out. Few
biological molecules diffuse through the lipid bilayer membrane freely;
transporters are almost invariably used to import substrate and precursors
as well as to export the extracellular product. As the productivity
increases the flux across cellular membrane may become necessary.

Many products when accumulated to a high level can have toxic

effects on the cell. Notable examples are ethanol, organic solvents
or other acids, and many cytotoxic agents such as antibiotics
or antitumor agents. Conferring cells with some resistance
mechanism to its own product is at times necessary to increase
productivity. For example, recombinant mammalian cells producing
therapeutic proteins often produce lactate as a byproduct of
glucose consumption. As cell concentration increases lactate also
accumulates and eventually causes cells to enter decline phase
through induction of an apoptotic process (programmed death).

By expressing antiapoptotic genes in those cells, the death

phase can be delayed and the productivity increased.

10  Metabolic Engineering Notes