Biotechnology 2 lecture notes
INTRODUCTION
Biotechnology can be divided into three generations:
1. Traditional/classical biotechnology: production of beer, cheese, bread and soy sauce
2. Industrial biotechnology: antibiotics, amino acids, citrate, acetone and butanol (we know
that microorganisms are involved in the processes here).
3. Modern biotechnology: gene technology, which can be divided into pharmaceutical (human
proteins), (industrial) enzymes, fine chemical, food, detergent and plants
Butanol/acetone fermentation timeline:
1861, Louis Pasteur He found that organisms produced butanol during fermentation
1905, Schardinger He used the production of butanol for the production of rubber.
He also described that acetone was a by-product of
fermentation.
A few years later, Weizmann He showed that Clostridium acetobutylicum (forms spores)
produced large amounts of acetone.
Great War (WW 1) The acetone was used for the production of cordite (an
explosive).
Around 1930 The production of acetone/butanol was at a large scale.
Around 1960 The production stopped because crude oil was now used, which
was very cheap
Ethene production:
Petrochemical Biotechnological
Uses crude oil Uses biomass
Crude oil is cracked using oil biorefinery Glucose -> 2 ethanol -> 2 ethene
Shortage of crude oil Food vs fuel debate
The feasibility of biotechnological processes is based on the market, the politics and the competition
(besides the process itself).
Competition is based on availability of the substrate (price), the relation between price and volume,
the purity of the product/concentration in water and the production of carbon dioxide.
Modern biotechnology is based on consumer acceptance/marketing. Especially medical applications,
industrial applications and food ‘free from GMO’ are points for debates. This must be regulated:
approval takes time and time is money.
Biotechnology can be classified into colours:
1. Red biotechnology (medical/healthcare)
2. Green biotechnology (agricultural/agro-food)
3. Blue biotechnology (marine)
, 4. White biotechnology (biobased technology: industrial, replacing petrochemical production
lines, use renewable resources, constant and slow growth).
Models are made for sustainability: profit (economy), people (socially responsible) and of course the
environment/planet. The driving forces behind biobased technology (which is circular) are the
sustainability, the supply of raw materials, the supply of energy and the consumer behaviour.
Barriers of white technology:
Technical: biomass availability, storage/transport
Economical: price regime of agricultural policy
Political/legal: intellectual property, regulation on products (e.g. packaging material), novel
products (uncertain regulation)
Social: competition with food.
DESIGN PRODUCTION ORGANISMS (USING THE EXAMPLE OF ENZYME PRODUCTION)
A design is based on a sequence of events: product -> host -> construct -> cultivation -> downstream
processing -> formulation. A host must be chosen using the criteria of production, safety, knowledge
and post-translational processes.
Fungi can be used for the production at low pH. Yeasts are used for the production of alcohols. The
post-translational modifications are important for medical purposes (pharmacology). These
modifications are used for the production of human proteins.
Production: the easiest way of production is choosing a host which is closely related to the donor.
However, this is not always possible (slow growing organism, high demands for expensive media
etc.). Tests for possible hosts can be performed and the production level can be artificially increased.
A safe host has no virulence factors and no toxins. Organisms have a food grade and a GRAS status.
This status is generally recognised as safe, the product itself must be safe for food production. The
GRAS status is about the product (for example soy sauce), not about the organism itself. The safety is
less important in medical applications (time legislation, because there are already very struct
procedures), but it is very important in food applications.
For the choosing of a host, experience of the host in past large scale processes is a pre. For large scale
systems, cultivation conditions and fermentation behaviour of the organism must be taken into
account. Many companies have preference for certain organisms and they speak about ‘platform
preference’. Platform preference is about the organism used for production by companies (called
PLUGBUG system in the past).
Certain post-translational processes can occur:
Folding SS bridges by extra disulfide oxidoreductases
Introduction extra chaperone proteins for the folding
Secretion* also helps for good folding. Secretion is also useful
because the product can be taken out of the cell without breaking this
cell.
Peptide chain An important example for this is human insulin, where parts of the peptide
adjustment chain are cut off.
Sugar residues This is really important for cell recognition (pharmaceutical). For this, closely
related hosts are needed to avoid side effects.
*Secretion: add host sequences and a secretion vector (often already with a secretion sequence in it).
, Hosts for industrial applications:
E. coli E. coli can incorrectly fold human proteins
The have the same inactive proteins as humans
Some strains produce toxins
K12, a type of E. coli, is not a type of pathogen, it doesn’t produce toxins.
This type can thus safely be used in the lab.
Disadvantages: a prokaryote, which forms a lot of proteins. They
therefore form inclusion bodies*. These inclusion bodies are nowadays
seen as an advantage, because these bodies can easily be purified.
Bacillus Have experience at large scale (for example with food fermentation and
species they are used in the antibiotics)
Produces a lot of exo enzymes. This is both an advantage (good
secretion) and a disadvantage. Disadvantage: the exo enzymes are often
exo proteases, which will break up your protein. Exo proteases can be
knocked out, but the bacillus needs these for growth.
Has a good food grade
No post-translational modifications possible.
Saccharomyce Have a large scale experience (biomass production)
s cerevisiae They don’t respond to phage
They also have a good food grade
Post-translational modifications are possible (they can, however,
hyperglycosylate)
They have a low level of secretion
They are crab tree positive, see explanation below.
Aspergillus Also has large scale experience for the citrate production
niger Don’t respond to phage
Have a good food grade
Good secretion
Can undergo post-translational modifications
They produce exo enzymes (and thus exo proteases).
They produce degradation polymers.
Plant cell High production level
culture Don’t respond to phage
They produce incorrect modifications
It is also difficult to extract the proteins in the cell (due to the cell wall).
Mammal cell Sometimes the only alternative
culture Produces correct modifications
systems It is an expensive, complex media (many need embryo serums). For
complex media, the compounds are not defined, so you don’t know what
compounds need to be filtered out.
There is a risk for contamination, if there is an infection, the culture
needs to be thrown away. The chance of an infection with complex
media is very high.
There is slow growth.
*
Inclusion bodies: bag of clustered proteins that are wrongly fold.
Crab tree positive: grow in the absence and presence of oxygen. In the presence of oxygen, only citric
acid is produced (in theory and in the lab). In reality (large scale), the organisms also produced
ethanol. In Saccharomyces cerevisiae, the enzyme pyruvate dehydrogenase, used for the citric acid
production, is the slowest enzyme in the metabolism. Therefore, a lot of ethanol can be made. Some
INTRODUCTION
Biotechnology can be divided into three generations:
1. Traditional/classical biotechnology: production of beer, cheese, bread and soy sauce
2. Industrial biotechnology: antibiotics, amino acids, citrate, acetone and butanol (we know
that microorganisms are involved in the processes here).
3. Modern biotechnology: gene technology, which can be divided into pharmaceutical (human
proteins), (industrial) enzymes, fine chemical, food, detergent and plants
Butanol/acetone fermentation timeline:
1861, Louis Pasteur He found that organisms produced butanol during fermentation
1905, Schardinger He used the production of butanol for the production of rubber.
He also described that acetone was a by-product of
fermentation.
A few years later, Weizmann He showed that Clostridium acetobutylicum (forms spores)
produced large amounts of acetone.
Great War (WW 1) The acetone was used for the production of cordite (an
explosive).
Around 1930 The production of acetone/butanol was at a large scale.
Around 1960 The production stopped because crude oil was now used, which
was very cheap
Ethene production:
Petrochemical Biotechnological
Uses crude oil Uses biomass
Crude oil is cracked using oil biorefinery Glucose -> 2 ethanol -> 2 ethene
Shortage of crude oil Food vs fuel debate
The feasibility of biotechnological processes is based on the market, the politics and the competition
(besides the process itself).
Competition is based on availability of the substrate (price), the relation between price and volume,
the purity of the product/concentration in water and the production of carbon dioxide.
Modern biotechnology is based on consumer acceptance/marketing. Especially medical applications,
industrial applications and food ‘free from GMO’ are points for debates. This must be regulated:
approval takes time and time is money.
Biotechnology can be classified into colours:
1. Red biotechnology (medical/healthcare)
2. Green biotechnology (agricultural/agro-food)
3. Blue biotechnology (marine)
, 4. White biotechnology (biobased technology: industrial, replacing petrochemical production
lines, use renewable resources, constant and slow growth).
Models are made for sustainability: profit (economy), people (socially responsible) and of course the
environment/planet. The driving forces behind biobased technology (which is circular) are the
sustainability, the supply of raw materials, the supply of energy and the consumer behaviour.
Barriers of white technology:
Technical: biomass availability, storage/transport
Economical: price regime of agricultural policy
Political/legal: intellectual property, regulation on products (e.g. packaging material), novel
products (uncertain regulation)
Social: competition with food.
DESIGN PRODUCTION ORGANISMS (USING THE EXAMPLE OF ENZYME PRODUCTION)
A design is based on a sequence of events: product -> host -> construct -> cultivation -> downstream
processing -> formulation. A host must be chosen using the criteria of production, safety, knowledge
and post-translational processes.
Fungi can be used for the production at low pH. Yeasts are used for the production of alcohols. The
post-translational modifications are important for medical purposes (pharmacology). These
modifications are used for the production of human proteins.
Production: the easiest way of production is choosing a host which is closely related to the donor.
However, this is not always possible (slow growing organism, high demands for expensive media
etc.). Tests for possible hosts can be performed and the production level can be artificially increased.
A safe host has no virulence factors and no toxins. Organisms have a food grade and a GRAS status.
This status is generally recognised as safe, the product itself must be safe for food production. The
GRAS status is about the product (for example soy sauce), not about the organism itself. The safety is
less important in medical applications (time legislation, because there are already very struct
procedures), but it is very important in food applications.
For the choosing of a host, experience of the host in past large scale processes is a pre. For large scale
systems, cultivation conditions and fermentation behaviour of the organism must be taken into
account. Many companies have preference for certain organisms and they speak about ‘platform
preference’. Platform preference is about the organism used for production by companies (called
PLUGBUG system in the past).
Certain post-translational processes can occur:
Folding SS bridges by extra disulfide oxidoreductases
Introduction extra chaperone proteins for the folding
Secretion* also helps for good folding. Secretion is also useful
because the product can be taken out of the cell without breaking this
cell.
Peptide chain An important example for this is human insulin, where parts of the peptide
adjustment chain are cut off.
Sugar residues This is really important for cell recognition (pharmaceutical). For this, closely
related hosts are needed to avoid side effects.
*Secretion: add host sequences and a secretion vector (often already with a secretion sequence in it).
, Hosts for industrial applications:
E. coli E. coli can incorrectly fold human proteins
The have the same inactive proteins as humans
Some strains produce toxins
K12, a type of E. coli, is not a type of pathogen, it doesn’t produce toxins.
This type can thus safely be used in the lab.
Disadvantages: a prokaryote, which forms a lot of proteins. They
therefore form inclusion bodies*. These inclusion bodies are nowadays
seen as an advantage, because these bodies can easily be purified.
Bacillus Have experience at large scale (for example with food fermentation and
species they are used in the antibiotics)
Produces a lot of exo enzymes. This is both an advantage (good
secretion) and a disadvantage. Disadvantage: the exo enzymes are often
exo proteases, which will break up your protein. Exo proteases can be
knocked out, but the bacillus needs these for growth.
Has a good food grade
No post-translational modifications possible.
Saccharomyce Have a large scale experience (biomass production)
s cerevisiae They don’t respond to phage
They also have a good food grade
Post-translational modifications are possible (they can, however,
hyperglycosylate)
They have a low level of secretion
They are crab tree positive, see explanation below.
Aspergillus Also has large scale experience for the citrate production
niger Don’t respond to phage
Have a good food grade
Good secretion
Can undergo post-translational modifications
They produce exo enzymes (and thus exo proteases).
They produce degradation polymers.
Plant cell High production level
culture Don’t respond to phage
They produce incorrect modifications
It is also difficult to extract the proteins in the cell (due to the cell wall).
Mammal cell Sometimes the only alternative
culture Produces correct modifications
systems It is an expensive, complex media (many need embryo serums). For
complex media, the compounds are not defined, so you don’t know what
compounds need to be filtered out.
There is a risk for contamination, if there is an infection, the culture
needs to be thrown away. The chance of an infection with complex
media is very high.
There is slow growth.
*
Inclusion bodies: bag of clustered proteins that are wrongly fold.
Crab tree positive: grow in the absence and presence of oxygen. In the presence of oxygen, only citric
acid is produced (in theory and in the lab). In reality (large scale), the organisms also produced
ethanol. In Saccharomyces cerevisiae, the enzyme pyruvate dehydrogenase, used for the citric acid
production, is the slowest enzyme in the metabolism. Therefore, a lot of ethanol can be made. Some
