E. coli

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14 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

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Biochemical Engineering

CEN 551

Instructor: Dr. Christine Kelly


Utilizing Genetically Engineering
Organisms (Chapter 14)

Schedule


Posters due on Tuesday, April 13, 3
weeks from today. Format for editing
(black and white, not on poster board).


Poster Presentations: Saturday
afternoon, April 17, 3 weeks from this
Saturday.


Oral presentations: April 15, 20, 22, 27.

April 15: Mittal, Sameer, Xu, Anitescu

April 20: Meka, Chapeaux, Chang, Price

April 22: Pasenello, Prantil, Lu, Menon

April 27 Sayut, Reis


If your project is similar to your presentation
during the semester


do not repeat your
presentation.

Presentations should be about 20 minutes
long.

Outline


Host
-
Vector Systems


Genetic Instability


Plasmid Design


Predicting Host
-
Vector
Interactions


Regulatory Constraints


Metabolic Engineering


Protein engineering

Products from Genetically
Engineering Organisms


Proteins


Most current industrial efforts.


Human therapeutics
-

over 200 in clinical trials.


Food processing.


Industrial catalysts.


$32 billion by 2006.


Nonproteins


Metabolic engineering, altering existing pathways
with an enhanced capacity to produce a metabolite.

Constraints based on product type


Pharmaceutical


Objective is safety and efficacy.


Purity, authenticity, posttranslational
possessing.


Cost result of research and clinical trials


not manufacturing. Cost of manufacturing
not as an important issue.


Animal feed supplements or pharmaceuticals


Purity is requirement.


Cost important also.


Industrial


Low manufacturing cost critical.


Can tolerate lower levels of purity.


Food Processing


Safety important.


Purity requirements less stringent than
pharmaceuticals.


Volume is large.


Cost important for penetrating the market.

Host Organisms


E. coli


Gram positive bacteria


Lower eukaryotic cells


Mammalian cells


Insect
-
baculovirus system


Transgenic animals


Plants and plant cell culture

E. coli


If no post translational modifications
necessary,
E. coli

selected.


Physiology and genetics well understood.


Wide range of vectors, hosts, promoters
available.


High growth rates, high cell density
achievable.


Will grow on simple and inexpensive
media.


Glucose feeding regime important to
regulate the production of inhibitory by
-
products like acetate.


Does not secrete proteins.


Inclusion bodies: misfolded recombinant
protein, must be lysed and resolubilized.


Disulfide bridges.


Methionine first amino acid, in mammals
methionine removed in post translational
processing.

Gram
-
positive Bacteria


Bacillus subtilis

most studied gram
positive organisms.


Contains no outer membrane.


Very effective excreter of proteins.


Produces many proteases.


Limited range of vectors and
promoters.


Plasmids less stable.

Lower Eukaryotic Cells
(Yeast and Fungi)

Saccharomyces cerevisiae



extensively used in food and industrial
fermentations.


High cell densities, high growth rate (25% of
E. coli
).


Simple glycosylation, but hyperglycosylates.


GRAS (generally accepted as safe) list.


Secretion bottlenecks can occur.

Pichia pastoris

and
Hansenula polymorpha


Methanol as sole carbon and energy source.


Very strong promoter.


Simple glycosylation, less likely to
hyperglycosylate.


Very high cell density.

Aspergillus


Good protein secretion.


Filamentous growth


bioreactor production
more problematic.

Mammalian Cells


Authenticity


posttranslational processing.


Readily excreted.


Slow growing.


Expensive media.


CHO most common.


Must have transformed cell line.


Vectors derived from primate viruses


concern
about reversion.


Quality changes upon scale
-
up.

Insect Cell


Baculovirus
System


Small scales.


Good for characterization studies.


Very strong promoter.


Secreted and glycosylated proteins
produced at lower levels than
intracellular proteins.


Naturally continuous


not transformed.


Not pathogenic.

Transgenic Animals


Animals engineering to express the
protien in specific fluids (milk or
urine).


High concentrations of complex
proteins.


Sheep, goats, pigs most common.


Costly.

Plants


Inexpensive.


Fewer safety concerns.


Scale up simple


more acres.


Edible delivery.


Low expression levels.


Glycosylation incomplete.


Long lead times.


Corn common.


Also plant cell cultures


Taxol.

Genetic Instability


Maximum target
-
protein
production vs. well growing
culture.


Production of lots of
recombinant protein is always
detrimental to the cell.

Cells lose the capacity to make the
target protein


they often grow
more quickly that the original
strain.


Segregational loss.


Structural instability.


Host cell regulatory mutations.


Growth rate ratio.


Segregational loss


Cells divide


daughter cell receives no
plasmids.


Plasmids can be “low copy number” or
“high copy number”.


High copy number randomly distributed
between daughter cells.


Low probability of daughter receiving no
plasmids.


Affected by many process variables.

Structural Instability


Retain the plasmid, but alter to reduce the
harmful effects of the plasmid.


Mutations may arise that result in the
inability to produce the recombinant
protein but retain beneficial plasmid
encoded functions like antibiotic
resistance.


These plasmids are able to grow faster
therefore they take over the culture.

Host Cell Mutations


Alter cellular regulation to
reduce recombinant protein
synthesis.


The mutation confers a growth
advantage to the mutant so that
the mutant will eventually
dominate the culture.

Growth Rate Dominated
Instability


All of the three previous instability
mechanisms become a problem
because of the difference in growth
rate between the altered strain and the
original strain.


Growth rate ratio is a function of
medium (antibiotics, inducer).

Plasmid Design


Origin of replication.

Regulates
reproduction of plasmid and copy number
of plasmid. Different origins for different
host types.


Number of gene copies.

Higher levels of
production with more copies of the gene.
Multiple plasmids or multiple copies on the
same plasmid.
E. coli

typically has 25
-
250
plasmids per cell.


Promoter/Inducer.

Strong promoter means
higher rate of transcription


faster production.
Promoter should be tightly regulated


off = very
little transcription, on = lots of transcription.
Inducer should not be toxic or expensive, easy to
manipulate.


Terminator.

Strong promoters need strong
terminator to prevent read through
(transcription) of the DNA after the gene.


Fusion proteins.

Can fuse small part of host’s
native protein to prevent destruction. Can fuse
handle or tail for affinity chromatography. Can
fuse host’s secretion signal to direct out of the
cell.


Selective pressure.

Antibiotic resistance
or necessary metabolite gene on plasmid to
ensure only the plasmid containing cells
will grow in the bioreactor environment.
Can leak complimenting factor to medium
and cells that lose the plasmid will still
have some complementing factor for
several generations.


Par

and
cer
loci.

Sections of DNA on the
plasmid that promote even distribution of
plasmids to daughter cells.


Predicting Host
-
Vector Interactions
and Genetic Instability


n
+

= cells with plasmid.


n
-

= cells without plasmid.


P = probability of forming a plasmid
free cell.



+
= growth rate of plasmid
containing cells.


R = P

+


Performing a balance around a CSTR on n
+

and n
-
.



Assuming no selective agents present, total
number of cells is approximately constant,
metabolic burden of plasmid is not too
great, and D< 80% of maximum growth
rate, constant delta growth rate and R.


Simplifying for three cases, we can get an
analytical solution for fraction of cells that
do not contain the plasmid.

1.


=

-

-


+

>> R growth
-
rate
instability dominant (eqn. 14.23)

2.



=


R segragational
instability dominant (eqn. 14.24)

3.


< 0 and abs val(

)>>R


effect selective pressure (eqn.
14.25)


Linearize the equations to obtain
parameter R from experimental data,
then use equations to predict plasmid
loss different reactor.


Note. Lots of assumptions!


Can also perform balance on batch
reactor to get an idea of how many
cells will have lost the plasmid by the
end of the batch.

Regulatory Constraints

Regulatory constraints on genetically
engineering organisms are in place to
reduce the chance of release of DNA that
encodes for dangerous substances or
antibiotics into the environment. This
DNA can be taken up by environmental
organisms, and possible these organisms
could then produce the recombinant
compounds.

Containment required depends on

1.
The ability of the host to survive in
the environment

2.
The ability of the vector to cross
species lines or the DNA to be
transformed into another species.

3.
Nature of the recombinant genes.

Metabolic Engineering

Using genetic engineering to…


Make a totally new pathway.


Amplify an existing pathway.


Disable an undesired pathway.


Alter the regulation of a
pathway.

Products from metabolic
engineering


Specialty chemicals (indigo, biotin,
amino acids)


Utilization of alternative substrates
(pentose sugars from
hemicellulose)


Degradation of hazardous wastes.

Why not just use the
natural strain?

Put a pathway under the control of a
regulated promoter


turn on the
pathway when it wouldn’t normally
be turned on. Example: to degrade
hazardous waste to lower
concentrations than would normally
induce the pathway.


Increase the concentration of
enzymes with a strong promoter.


Produce the product in an easier to
grow host.


Combining several pathways.


Patent the organism


cannot
patent ‘unengineered’ organisms.


For protein products (not metabolites), the
protein can be made at the end of batch
culture, not exerting a burden on the cell
during growth.


Metabolites are produced at lower rates,
but produce a high burden.


Nature of the reactor system difficult:
waste degradation not sterile.


Good numerical understanding of pathways
is required. Need to express genes the
‘right’ amount


not just overexpress.


DuPont has commercialized a
process to produce a polymer from
corn with a metabolically engineered
organisms.


Other products include precurser for
vitamin C and xylitol


both
processes that I have worked on.

Protein Engineering


New proteins or altering the amino
acid sequence of existing proteins.


Can require crystal structure of the
protein to examine modifications that
may have benefit.


Driving force for computer modeling
of protein structure from amino acid
sequence.

Site
-
Directed Mutagenesis


Method used to change an amino
acid in a protein sequence.


Rational design of proteins as
opposed to random mutagenesis.