Figure 15.2b

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Transcript Figure 15.2b

LECTURE PRESENTATIONS
For BROCK BIOLOGY OF MICROORGANISMS, THIRTEENTH EDITION
Michael T. Madigan, John M. Martinko, David A. Stahl, David P. Clark
Chapter 15
Commercial Products
and Biotechnology
Lectures by
John Zamora
Middle Tennessee State University
© 2012 Pearson Education, Inc.
I. Putting Microorganisms to Work
• 15.1 Industrial Products and the Microorganisms
That Make Them
• 15.2 Production and Scale
© 2012 Pearson Education, Inc.
15.1 Industrial Products and the
Microorganisms That Make Them
• Industrial microbiology
– Uses microorganisms, typically grown on a large
scale, to produce products or carry out chemical
transformation
– Originated with alcoholic fermentation processes
• Later on, processes such as production of
pharmaceuticals, food additives, enzymes, and
chemicals were developed
– Major organisms used are fungi and Streptomyces
– Classic methods are used to select for highyielding microbial variants
© 2012 Pearson Education, Inc.
15.1 Industrial Products and the
Microorganisms That Make Them
• Properties of a useful industrial microbe include
– Produces spores or can be easily inoculated
– Grows rapidly on a large scale in inexpensive
medium
– Produces desired product quickly
– Should not be pathogenic
– Amenable to genetic manipulation
© 2012 Pearson Education, Inc.
15.1 Industrial Products and the
Microorganisms That Make Them
• Microbial products of industrial interest include
–
–
–
–
–
Microbial cells
Enzymes
Antibiotics, steroids, alkaloids
Food additives
Commodity chemicals
• Inexpensive chemicals produced in bulk
• Include ethanol, citric acid, and many others
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15.2 Production and Scale
• Primary metabolite
– Produced during exponential growth
– Example: alcohol
• Secondary metabolite
– Produced during stationary phase
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15.2 Production and Scale
• Secondary metabolites
–
–
–
–
–
Not essential for growth
Formation depends on growth conditions
Produced as a group of related compounds
Often significantly overproduced
Often produced by spore-forming microbes
during sporulation
© 2012 Pearson Education, Inc.
Primary
metabolite
Cells
Alcohol
Sugar
Penicillin, sugar, or cell number
Alcohol, sugar, or cell number
Figure 15.1
Secondary
metabolite
Sugar
Cells
Penicillin
Time
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Time
15.2 Production and Scale
• Secondary metabolites are often large
organic molecules that require a large
number of specific enzymatic steps for
production
– Synthesis of tetracycline requires at least 72
separate enzymatic steps
– Starting materials arise from major
biosynthetic pathways
© 2012 Pearson Education, Inc.
15.2 Production and Scale
• Fermentor is where the microbiology process
takes place (Figure 15.2a and b)
• Any large-scale reaction is referred to as a
fermentation
– Most are aerobic processes
• Fermentors vary in size from 5 to 500,000 liters
– Aerobic and anaerobic fermentors
• Large-scale fermentors are almost always
stainless steel
– Impellers and spargers supply oxygen
(Figure 15.2c)
© 2012 Pearson Education, Inc.
Figure 15.2a
© 2012 Pearson Education, Inc.
Figure 15.2b
Motor
pH
Steam
Sterile
seal
pH controller
Acid–base
reservoir and
pump
Viewing
port
Filter
Exhaust
Impeller
(mixing)
External
cooling
water out
Cooling
jacket
Culture
broth
External
cooling
water in
Sparger (highpressure air
for aeration)
Steam in
Sterile air
Valve
Harvest
© 2012 Pearson Education, Inc.
Figure 15.2c
© 2012 Pearson Education, Inc.
15.2 Production and Scale
• Industrial Fermentors
– Closely monitored during production run
– Growth and product formation must be measured
– Environmental factors must be controlled and
altered as needed
• Including temperature, pH, cell mass, nutrients,
and product concentration
– Data on the process must be obtained in real time
© 2012 Pearson Education, Inc.
15.2 Production and Scale
• Scale-up
– The transfer of a process from a small laboratory
scale to large-scale commercial equipment
– Major task of the biochemical engineer
• Requires knowledge of the biology of producing
organism and the physics of fermentor design and
operation
– Many challenges in scale-up arise from aeration
and mixing
• Flask  laboratory fermentor  pilot plant 
commercial fermentor (Figure 15.3)
© 2012 Pearson Education, Inc.
Figure 15.3
© 2012 Pearson Education, Inc.
II. Drugs, Other Chemicals, and Enzymes
• 15.3 Antibiotics: Isolation, Yield, and Purification
• 15.4 Industrial Production of Penicillins and
Tetracyclines
• 15.5 Vitamins and Amino Acids
• 15.6 Enzymes as Industrial Products
© 2012 Pearson Education, Inc.
15.3 Antibiotics: Isolation, Yield, and
Purification
• Antibiotics
– Compounds that kill or inhibit the growth of other
microbes
– Typically secondary metabolites
– Most antibiotics in clinical use are produced by
filamentous fungi or actinomycetes
– Still discovered by laboratory screening
(Figure 15.4a)
• Microbes are obtained from nature in pure culture
• Assayed for products that inhibit growth of test
Animation: Isolation and Screening
bacteria
of Antibiotic Producers
© 2012 Pearson Education, Inc.
Figure 15.4a
I. Isolation
Spread a soil
dilution on a plate
of selective medium
Sterile glass spreader
Incubation
Colonies of
Streptomyces
species
Nonproducing
organisms
Zones of
growth inhibition
Producing
organisms
© 2012 Pearson Education, Inc.
Overlay with an
indicator organism
Incubate
15.3 Antibiotics: Isolation, Yield, and
Purification
• Cross-streak method (Figure 15.4b)
– Used to test new microbial isolates for
antibiotic production
– Most isolates produce known antibiotics
– Most antibiotics fail toxicity and therapeutic
tests in animals
– Time and cost of developing a new antibiotic
is approximately 15 years and $1 billion
• Involves clinical trials and U.S. FDA approval
• Antibiotic purification and extraction often
involves elaborate methods
© 2012 Pearson Education, Inc.
Figure 15.4b
II. Testing Activity Spectrum
Streak antibiotic producer
across one side of plate
Incubate to permit growth
and antibiotic production
Antibiotic diffuses
into agar
Streptomyces cell mass
Cross-streak with test organisms
Incubate to permit
test organisms to grow
Growth of test organism
Inhibition zones where
sensitive test organisms
did not grow
© 2012 Pearson Education, Inc.
15.4 Industrial Production of Penicillins
and Tetracyclines
• Penicillins are -lactam antibiotics
– Natural and biosynthetic penicillins (Figure 15.5)
– Semisynthetic penicillins
• Broad spectrum of activity
• Penicillin production is typical of a secondary
metabolite
– Production only begins after near-exhaustion of
carbon source (Figure 15.6)
– High levels of glucose repress penicillin
production
© 2012 Pearson Education, Inc.
Figure 15.5
Add
precursor I
Biosynthetic
penicillin I
Add
precursor II
Biosynthetic
penicillin II
Penicillin
fermentation
Add
precursor
III
Chemical or
enzymatic
treatment
of penicillin G
Biosynthetic
penicillin III
Natural penicillins
(for example,
penicillin G)
6-Aminopenicillanic acid
Add side
chains
chemically
Semisynthetic penicillins
(for example, ampicillin,
amoxycillin, methicillin)
© 2012 Pearson Education, Inc.
Figure 15.6
Biomass (g/liter), carbohydrate,
ammonia, penicillin (g/liter  10)
Glucose
feeding
100
Nitrogen
feeding
90
80
Penicillin
70
60
50
40
30
Cells
20
Lactose
10
Ammonia
0
20 40 60 80 100 120 140
Fermentation time (h)
© 2012 Pearson Education, Inc.
15.4 Industrial Production of Penicillins
and Tetracyclines
• Biosynthesis of tetracycline has a large
number of enzymatic steps
– More than 72 intermediates
– More than 300 genes involved!
– Complex biosynthetic regulation (Figure 15.7)
© 2012 Pearson Education, Inc.
Figure 15.7
Inoculum
(spores on
agar slant or in
sterile soil)
Agar plates
Medium
2% Meat extract; 0.05%
asparagine; 1% glucose;
0.5% K2HPO4; 1.3% agar
Growth in
optimal medium
Spores as
inoculum
Shake flask
2% Corn steep liquor;
3% sucrose; 0.5% CaCO3
24 h
Prefermentor
Medium mimics
production
medium
Same as for shake culture
19–24 h
pH 5.2–6.2
Fermentor
60–65 h
pH 5.8–6.0
1% Sucrose; 1% corn steep
liquor; 0.2% (NH4)2HPO4;
0.1% CaCO3;
0.025% MgSO4
0.005% ZnSO4
0.00033% and each of
CuSO4, MnCl2
Production
medium, no
glucose, low
phosphate
Antibiotic
purification from
broth after cell
removal
Chlortetracycline
© 2012 Pearson Education, Inc.
15.5 Vitamins and Amino Acids
• Production of vitamins is second only to
antibiotics in terms of total pharmaceutical
sales
– Vitamin B12 produced exclusively by
microorganisms (Figure 15.8a)
• Deficiency results in pernicious anemia
• Cobalt is present in B12
– Riboflavin can also be produced by microbes
(Figure 15.8b)
© 2012 Pearson Education, Inc.
Figure 15.8a
B12
© 2012 Pearson Education, Inc.
Figure 15.8b
Flavin ring
Riboflavin
© 2012 Pearson Education, Inc.
15.5 Vitamins and Amino Acids
• Amino acids
– Used as feed additives in the food industry
– Used as nutritional supplements in
nutraceutical industry
– Used as starting materials in the chemical
industry
– Examples include
• Glutamic acid (MSG)
• Aspartic acid and phenylalanine (aspartame
[NutraSweet])
• Lysine (food additives; Figure 15.9)
© 2012 Pearson Education, Inc.
Figure 15.9
Methionine
Threonine
Isoleucine
ATP
Aspartate
Aspartyl-P
Aspartokinase
Feedback
inhibition
Aspartate
semialdehyde
Diaminopimelate
Lysine
AEC:
Lysine:
© 2012 Pearson Education, Inc.
15.6 Enzymes as Industrial Products
• Exoenzymes
– Enzymes that are excreted into the medium
instead of being held within the cell; they are
extracellular
– Can digest insoluble polymers such as cellulose,
protein, and starch
• Enzymes are useful as industrial catalysts
– Produce only one stereoisomer
– High substrate specificity
© 2012 Pearson Education, Inc.
15.6 Enzymes as Industrial Products
• Enzymes are produced from fungi and bacteria
– Bacterial proteases are used in laundry
detergents (can also contain amylases, lipases,
and reductases)
• Isolated from alkaliphilic bacteria
• Amylases and glucoamylases are also
commercially important
– Produce high-fructose syrup
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15.6 Enzymes as Industrial Products
• Extremozymes
– Enzymes that function at some environmental
extreme (e.g., pH or temperature; Figure 15.10)
– Produced by extremophiles
© 2012 Pearson Education, Inc.
Figure 15.10
Percent enzyme
activity remaining
100
10
Starch
Pullulanase
oligosaccharides
90°C
100°C
110°C
110°C plus Ca2
1
1
2
Time (h)
© 2012 Pearson Education, Inc.
3
4
15.6 Enzymes as Industrial Products
• Immobilized enzymes are attached to a solid
surface
– Used in the starch processing industry
• Three ways to immobilize an enzyme
(Figure 15.11)
– Bonding of enzyme to a carrier
– Cross-linking of enzyme molecules
– Enzyme inclusion
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Figure 15.11
Carrier-bound
enzyme
Enzyme inclusion in
fibrous polymers
© 2012 Pearson Education, Inc.
Cross-linked
enzyme
Enzyme inclusion
in microcapsules
III. Alcoholic Beverages and Biofuels
• 15.7 Wine
• 15.8 Brewing and Distilling
• 15.9 Biofuels
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15.10 Wine
• Most wine is made from grapes
• Wine fermentation occurs in fermentors
ranging in size from 200 to 200,000 liters
– Fermentors are made of oak, cement, glasslined steel, or stone (Figure 15.12b, c, and d)
• White wine is made from white grapes or red
grapes that have had their skin removed
(Figure 15.13)
• Red wine is aged for months or years
• White wine is often sold without aging
© 2012 Pearson Education, Inc.
Figure 15.12b
© 2012 Pearson Education, Inc.
Figure 15.12c
© 2012 Pearson Education, Inc.
Figure 15.12d
© 2012 Pearson Education, Inc.
Figure 15.13
Stems removed
Grapes crushed
Stems removed
Grapes crushed
Must
Must
Yeast
Juice sits in contact
with skins for 16–24 h
Fermentation vat 3 weeks
(pulp is not removed)
Press
Press
Pomace
(discard)
Yeast
Fermentation vat
10–15 days
Pomace
(discard)
Aging in barrels
Racking
Aging 5 months
Racking
Transfer to clean barrels
3 times per year
2 years
Clarifying
agents
Settling tank
Clarifying
agents
Filtration
Filtration
Bottling
Bottling: Age in bottles
6 months or more
White wine
© 2012 Pearson Education, Inc.
Red wine
15.8 Brewing and Distilling
• Brewing is the term used to describe the
manufacture of alcoholic beverages from malted
grains (Figure 15.14)
• Yeast is used to produce beer
• Two main types of brewery yeast strains
– Top fermenting — ales
– Bottom fermenting — lagers
© 2012 Pearson Education, Inc.
Figure 15.14
© 2012 Pearson Education, Inc.
15.8 Brewing and Distilling
• Distilled alcoholic beverages are made by
heating previously fermented liquid to a
temperature that volatilizes most of the
alcohol (Figure 15.16)
– Whiskey, rum, brandy, vodka, gin
• >50,000,000,000 liters of ethanol are
produced yearly for industrial purposes
– Used as an industrial solvent and gasoline
supplement
© 2012 Pearson Education, Inc.
Figure 15.16
© 2012 Pearson Education, Inc.
15.9 Biofuels
• Ethanol Biofuels
– Ethanol is a major industrial commodity chemical
– Over 60 billion liters of alcohol are produced
yearly from the fermentation of feedstocks
(Figure 15.17a and b)
– Gasohol and E-85
• Petroleum Biofuels
– Production of butanol
– Synthesis of petroleum from green algae
(Figure 15.17c)
© 2012 Pearson Education, Inc.
Figure 15.17
© 2012 Pearson Education, Inc.
IV. Products from Genetically Engineered
Microorganisms
• 15.10 Expressing Mammalian Genes in Bacteria
• 15.11 Production of Genetically Engineered
Somatotropin
• 15.12 Other Mammalian Proteins and Products
• 15.13 Genetically Engineered Vaccines
• 15.14 Mining Genomes
• 15.15 Engineering Metabolic Pathways
© 2012 Pearson Education, Inc.
15.10 Expressing Mammalian Genes
in Bacteria
• Biotechnology
– Use of living organisms for industrial or
commercial applications
• Genetically modified organism (GMO)
– An organism whose genome has been altered
• Genetic engineering allows expression of
eukaryotic genes in prokaryotes (e.g., insulin)
• This is achieved by
– Cloning the gene via mRNA (Figure 15.18)
– Finding the gene via the protein (Figure 15.19)
© 2012 Pearson Education, Inc.
Figure 15.18
Poly(A) tail
mRNA
Addition of primer
Oligo
dT primer
Reverse transcription
to form
single-stranded cDNA
cDNA
Hairpin loop
Removal of RNA
with alkali
DNA polymerase I
to form doublestranded cDNA
Nuclease
Single-strand-specific
nuclease
Double-stranded cDNA
Clone
© 2012 Pearson Education, Inc.
Figure 15.19
Protein
Possible mRNA codons
DNA oligonucleotides (possible probes)
and so on
Preferred DNA sequence (based on the
organism’s codon bias)
© 2012 Pearson Education, Inc.
15.10 Expressing Mammalian Genes
in Bacteria
• Protein synthesis in a foreign host is subject
to other problems
– Degradation by intracellular proteases
– Toxicity to prokaryotic host
– Formation of inclusion bodies
• Fusion of a target protein with a carrier
protein facilitates protein purification (Figure
15.20)
© 2012 Pearson Education, Inc.
Figure 15.20
Ptac
Encodes
Shine–Dalgarno
lacI
malE
Encodes protease
cleavage site
Polylinker
lacZ
pBR322
origin
M13 origin
© 2012 Pearson Education, Inc.
Ampicillin resistance
15.11 Production of Genetically
Engineered Somatotropin
• Insulin was the first human protein made
commercially by genetic engineering
• Somatotropin, a growth hormone, is another
widely produced hormone (Figure 15.21)
– Cloned as cDNA from the mRNA
– Recombinant bovine somatotropin (rBST) is
commonly used in the dairy industry;
stimulates milk production in cows
© 2012 Pearson Education, Inc.
Figure 15.21
Bacterial promoter
and RBS
BST mRNA
from cow
Bovine somatotropin mRNA
Expression vector
Convert BST
mRNA to cDNA
using reverse
transcriptase
Inject rBST
into cow
to increase
milk yield
Bovine somatotropin cDNA
rBST
Transform
into cells of
Escherichia coli
© 2012 Pearson Education, Inc.
Commercial
production
15.12 Other Mammalian Proteins
and Products
• Many mammalian proteins are produced by
genetic engineering
– These include hormones and proteins for
blood clotting and other blood processes
© 2012 Pearson Education, Inc.
15.13 Genetically Engineered Vaccines
• Recombinant vaccines
– Vector vaccine
– Subunit vaccine
– DNA vaccine
• Polyvalent vaccine
– A single vaccine that immunizes against two
different diseases
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15.13 Genetically Engineered Vaccines
• Vector vaccine
– Vaccine made by inserting genes from a
pathogenic virus into a relatively harmless carrier
virus (e.g., vaccinia virus; Figure 15.22)
Animation: Production of Recombinant Vaccina Virus
© 2012 Pearson Education, Inc.
Figure 15.22
Cloning plasmid
Foreign DNA
Foreign DNA
inserted into
tdk gene
Part of vaccinia
tdk gene
tdk
Insert into
host cell
Wild-type vaccinia
virus DNA
Host cell
with defective
tdk gene
Foreign DNA
Recombinant vaccinia
virus DNA
© 2012 Pearson Education, Inc.
Recombination
Select with
5-bromo-dU
15.13 Genetically Engineered Vaccines
• Subunit vaccines
– Contain only a specific protein or proteins from a
pathogenic organism (e.g., coat protein of a virus)
– Preparation of a viral subunit vaccine
• Fragmentation of viral DNA by restriction enzymes
• Cloning of viral coat protein genes into a suitable
vector
• Provision of proper conditions for expression
(promoter, reading frame, and ribosome-binding
site)
• Reinsertion and expression of the viral genes in a
microbe
© 2012 Pearson Education, Inc.
15.13 Genetically Engineered Vaccines
• DNA vaccine (genetic vaccine)
– Vaccine that uses the DNA of a pathogen to
elicit an immune response
– Defined fragments of genomic DNA or specific
genes encoding immunogenic proteins are
used
• They are cloned into a plasmid or viral vector
and delivered by injection
© 2012 Pearson Education, Inc.
15.14 Mining Genomes
• Gene mining
– The process of isolating potentially useful novel
genes from the environment without culturing the
organism
– To do so, DNA (or RNA) is directly isolated from
the environment and cloned into appropriate
expression vectors, and the library is screened for
activities of interest (Figure 15.23)
© 2012 Pearson Education, Inc.
Figure 15.23
Collect DNA
samples from
different
environments
Construct
gene library
Transform host
cells and plate
on selective
media
Screen library
for reactive
colonies
Large DNA
inserts in BAC
Plates of differential media
Analyze and sequence
positive clones
© 2012 Pearson Education, Inc.
Vector
15.15 Engineering Metabolic Pathways
• Transgenic organism
– An organism that contains a gene from
another organism
© 2012 Pearson Education, Inc.
15.15 Engineering Metabolic Pathways
• The production of small metabolites by genetic
engineering typically involves multiple genes that
must be coordinately expressed
• Pathway engineering
– The process of assembling a new or improved
biochemical pathway using genes from one or
more organisms (e.g., indigo; Figure 15.24)
© 2012 Pearson Education, Inc.
Figure 15.24
Tryptophan
Tryptophanase activity
(already in E. coli)
Indole
Naphthalene
oxygenase activity
(from Pseudomonas)
Dihydroxy-indole
Spontaneous
dehydration
Indoxyl
Spontaneous
oxidation by O2
Indigo
© 2012 Pearson Education, Inc.
V. Transgenic Eukaryotes
• 15.16 Genetic Engineering of Animals
• 15.17 Gene Therapy in Humans
• 15.18 Transgenic Plants in Agriculture
© 2012 Pearson Education, Inc.
15.16 Genetic Engineering of Animals
• Genetic engineering can be used to develop
transgenic animals
• Transgenic animals are useful for
– Producing human proteins that require specific
posttranslational modifications
– Medical research
– Improving livestock and other food animals for
human consumption (Figure 15.25)
© 2012 Pearson Education, Inc.
Figure 15.25
© 2012 Pearson Education, Inc.
15.17 Gene Therapy in Humans
• Gene therapy holds promise for tackling many
human genetic diseases
• Gene therapy: introduces a functional copy of a
gene to treat a disease caused by a dysfunctional
version of the gene
• The use of recombinant DNA technology and
conventional genetic studies allows for the
localization of particular genetic defects to
specific regions of the genome
© 2012 Pearson Education, Inc.
15.18 Transgenic Plants in Agriculture
• Plants can be genetically modified through
several approaches, including
– Electroporation
– Particle gun methods
– Use of plasmids from bacterium Agrobacterium
tumefaciens
• Many successes in plant genetic engineering;
several transgenic plants are in agricultural
production
© 2012 Pearson Education, Inc.
15.18 Transgenic Plants in Agriculture
• The plant pathogen Agrobacterium tumefaciens
can be used to introduce DNA into plants
(Figure 15.26)
• A. tumefaciens contains the Ti plasmid, which is
responsible for virulence
• The Ti plasmid contains genes that mobilize DNA
for transfer to the plant
– The segment of the Ti plasmid that is transferred
to the plant is called the T-DNA
© 2012 Pearson Education, Inc.
Figure 15.26
Mobilized
region
Foreign DNA
Kanamycin
resistance
Spectinomycin
resistance
Chromosomes
D-Ti
Transfer to
E. coli cells
Transfer by
conjugation
Origin
A. tumefaciens
Origin E. coli
Cloning vector
Transfer to
plant cells
“Disarmed”
Ti plasmid
E. coli
Nucleus
A. tumefaciens
Plant cell
© 2012 Pearson Education, Inc.
Grow transgenic
plants from
plant cells
15.18 Transgenic Plants in Agriculture
• Tobacco was the first genetically modified (GM)
plant to be grown commercially
– 2005 estimate: >1 billion acres of agricultural land
are used to grow GM crops
• Several areas are targeted for genetic
improvements in plants including herbicide,
insect, and microbial disease resistance as well
as improved product quality
© 2012 Pearson Education, Inc.
15.18 Transgenic Plants in Agriculture
• Plants are engineered to have herbicide
resistance to protect them from herbicides
applied to kill weeds (e.g., glyphosate;
Figure 15.27)
© 2012 Pearson Education, Inc.
Figure 15.27
© 2012 Pearson Education, Inc.
15.18 Transgenic Plants in Agriculture
• One of the most widely used approaches for
genetically engineering insect resistance in
plants involves the introduction of genes
encoding the toxic protein of Bacillus
thuringiensis (Bt toxin; Figure 15.28)
© 2012 Pearson Education, Inc.
Figure 15.28
© 2012 Pearson Education, Inc.
15.18 Transgenic Plants in Agriculture
• Improving product quality is another target
area of genetic engineering of plants
– For example, spoilage delay
• Transgenic plants can also be employed to
produce human proteins for medical use
– Examples: interferon, antibodies, vaccines
© 2012 Pearson Education, Inc.