This document discusses industrial microbiology and the production of various products through microbial fermentation processes. It describes how microorganisms are used to produce pharmaceuticals, chemicals, enzymes and other industrial products. Specific examples discussed include antibiotics, vitamins, amino acids, alcoholic beverages and biofuels. The document also covers topics like isolating and screening microorganisms, secondary metabolite production, large-scale fermentation processes, and genetic engineering techniques used to modify microbes for industrial applications.

1. © Angel L. Salaman. PhD
ISOLATION AND
SCREENING OF
SECONDARY METABOLITES
Angel L. Salaman, PhD
angelsalaman@yahoo.com

2. 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 high-yielding
microbial variants

3. 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
 Allow genetic manipulation

4. 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

5. PRODUCTION AND SCALE
 Primary metabolite
 Produced during exponential growth
 Example: alcohol
 Secondary metabolite
 Produced during stationary phase

6. 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

7. © Angel L. Salaman. PhD
FIGURE 15.1
Primary
metabolite
Secondary
metabolite
Alcohol
Penicillin
Cells
Sugar
Cells
Sugar
Time Time
Alcohol,sugar,orcellnumber
Penicillin,sugar,orcellnumber

8. 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

9. PRODUCTION AND SCALE
 Fermentor is where the microbiology process takes
place
 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

10. © Angel L. Salaman. PhD
FIGURE 15.2A

11. © Angel L. Salaman. PhD
FIGURE 15.2B
Steam
Sterile
seal
Motor
pH pH controller
Acid–base
reservoir and
pump
Viewing
port
Filter
Exhaust
Impeller
(mixing)
Cooling
jacket
External
cooling
water in
External
cooling
water out
Culture
broth
Steam in
Valve
Harvest
Sparger (high-
pressure air
for aeration)
Sterile air

12. © Angel L. Salaman. PhD
FIGURE 15.2C

13. © Angel L. Salaman. PhD
FIGURE 15.3

14. 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
Microbes are obtained from nature in pure culture
 Assayed for products that inhibit growth of test bacteria

15. © Angel L. Salaman. PhD
FIGURE 15.4A
I. Isolation
Sterile glass spreader
Colonies of
Streptomyces
species
Nonproducing
organisms
Zones of
growth inhibition
Producing
organisms
Spread a soil
dilution on a plate
of selective medium
Incubation
Overlay with an
indicator organism
Incubate

16. ANTIBIOTICS: ISOLATION, YIELD,
AND PURIFICATION
 Cross-streak method
 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

17. © Angel L. Salaman. PhD
FIGURE 15.4B II. Testing Activity Spectrum
Streak antibiotic producer
across one side of plate
Incubate to permit growth
and antibiotic production
Cross-streak with test organisms
Incubate to permit
test organisms to grow
Antibiotic diffuses
into agar
Streptomyces cell mass
Growth of test organism
Inhibition zones where
sensitive test organisms
did not grow

18. INDUSTRIAL PRODUCTION OF
PENICILLINS AND TETRACYCLINES
 Penicillins are β-lactam antibiotics
 Natural and biosynthetic penicillins
 Semisynthetic penicillins
 Broad spectrum of activity
 Penicillin production is typical of a secondary
metabolite
 Production only begins after near-exhaustion of carbon
source
 High levels of glucose repress penicillin production

19. © Angel L. Salaman. PhD
FIGURE 15.6
Glucose
feeding
Nitrogen
feeding
Cells
Lactose
Ammonia
Penicillin
Fermentation time (h)
Biomass(g/liter),carbohydrate,
ammonia,penicillin(g/liter×10)
100
90
80
70
60
50
40
30
20
10
0
20 40 60 80 100 120 140

20. 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

21. VITAMINS AND AMINO ACIDS
 Production of vitamins is second only to antibiotics
in terms of total pharmaceutical sales
 Vitamin B12 produced exclusively by microorganisms
 Deficiency results in pernicious anemia
 Cobalt is present in B12
 Riboflavin can also be produced by microbes

22. 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)

23. 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

24. © Angel L. Salaman. PhD
FIGURE 15.10
Starch oligosaccharides
Time (h)
Percentenzyme
activityremaining
Pullulanase
90°C
100°C
110°C
110°C plus Ca2+
100
10
1
1 2 3 4
Pullulanase is used predominantly in conjunction with other enzymes that break down starch (glucoamylase). It is produced as an extracellular, cell surface-
anchored lipoprotein by Gram-negative bacteria of the genus Klebsiella. Type I pullulanases specifically attack α-1,6 linkages, while type II pullulanases are also
able to hydrolyse α-1,4 linkages. It is also produced by some other bacteria and archaea. Pullulanase is used as a processing aid in grain processing
biotechnology (production of ethanol and sweeteners).

25. III. ALCOHOLIC BEVERAGES AND
BIOFUELS
 Wine
 Brewing and Distilling
 Biofuels

26. 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, glass-lined steel,
or stone
 White wine is made from white grapes or red
grapes that have had their skin removed
 Red wine is aged for months or years
 White wine is often sold without aging

27. © Angel L. Salaman. PhD
FIGURE 15.12B

28. © Angel L. Salaman. PhD
FIGURE 15.12C

29. © Angel L. Salaman. PhD
FIGURE 15.12D

30. © Angel L. Salaman. PhD
FIGURE 15.13
Stems removed
Grapes crushed
Must
Juice sits in contact
with skins for 16–24 h
Press
Yeast
White wine Red wine
Pomace
(discard)
Yeast
Fermentation vat
10–15 days
Aging 5 months
Racking
Clarifying
agents
Filtration
Bottling
Stems removed
Grapes crushed
Must
Fermentation vat 3 weeks
(pulp is not removed)
Press
Pomace
(discard)
Aging in barrels
Racking
Transfer to clean barrels
3 times per year
Clarifying
agents
2 years
Settling tank
Filtration
Bottling: Age in bottles
6 months or more Sodium metabisulfite : It is used as a disinfectant, antioxidant and
preservative agent

31. BREWING AND DISTILLING
 Brewing is the term used to describe the manufacture
of alcoholic beverages from malted grains. Yeast is
used to produce beer
 Two main types of brewery yeast strains
 Top fermenting — Ale is a type of beer brewed from
malted barley using a warm-fermentation with a strain of
brewers' yeast.The yeast will ferment the beer quickly,
giving it a sweet, full bodied and fruity taste.
 Bottom fermenting — Lager (German: storage) is a type
of beer that is fermented and conditioned at low
temperatures.

32. © Angel L. Salaman. PhD
FIGURE 15.14

33. BREWING AND DISTILLING
 Distilled alcoholic beverages are made by heating
previously fermented liquid to a temperature that
volatilizes most of the alcohol
 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

34. BIOFUELS
 Ethanol Biofuels
 Ethanol is a major industrial commodity chemical
 Over 60 billion liters of alcohol are produced yearly from
the fermentation of feed stocks
 Gasohol and E-85
 Petroleum Biofuels
 Production of butanol
 Synthesis of petroleum from green algae

35. © Angel L. Salaman. PhD
FIGURE 15.17

36. BOTOX® (ALLERGAN) MYOBLOC®
(SOLSTICE NEUROSCIENCES, INC),
DYSPORT® (BIOPHARM LIMITED), OR
XEOMIN® (MERZ PHARMA GMBH & CO.)
 BOTOX COSMETIC® (onabotulinumtoxinA for injection) is a
sterile, vacuum-dried form of purified botulinum neurotoxin type
A complex, (AB5-type exotoxin) produced by the bacterium
Bordetella pertussis, which causes whooping cough produced from
a culture of the Hall strain of Clostridium botulinum grown in a
medium containing N-Z amine, glucose and yeast extract.
 Pertussis toxin (PT) is a proteBOTOX® Cosmetic is a
prescription medicine that is injected into muscles and used to
improve the look of moderate to severe frown lines between the
eyebrows (glabellar lines) in people 18 to 65 years of age for a
short period of time (temporary).

37. EXPRESSING MAMMALIAN
GENES IN BACTERIA

38. PRODUCTS FROM GENETICALLY
ENGINEERED MICROORGANISMS
 Expressing Mammalian Genes in Bacteria
 Ex. Production of Genetically Engineered
Somatotropin
 Other Mammalian Proteins and Products
 Genetically Engineered Vaccines
 Mining Genomes
 Engineered Metabolic Pathways

39.  Successful genetic engineering depends not only
on being able to carry out molecular cloning but
also on knowledge of replication, transcription,
translation, and the regulatory aspects that
control all of these processes.

41. HOSTS FOR CLONING VECTORS
 The choice of a cloning host depends on the final
application. In many cases, the host can be a
prokaryote, but in others it is essential that the
host be a eukaryote.
 Any host must be able to take up DNA, and there
are a variety of techniques by which this can be
accomplished, both natural and artificial.

43. Nucleic acid gun for transfection of certain
eukaryotic cells.

44. FINDING THE RIGHT CLONE
 Special procedures are needed to detect the
foreign gene in the cloning host

45.  If the gene is expressed, the presence of the
foreign protein itself, as detected either by its
activity or by reaction with specific antibodies, is
evidence that the gene is present. However, if the
gene is not expressed, its presence can be
detected with a nucleic acid probe.

47. SHUTTLE VECTORS
 allow cloned DNA to be moved between unrelated
organisms. A shuttle vector is a cloning vector
that can stably replicate in two different
organisms.

48. SPECIALIZED VECTORS
 Many cloned genes are not expressed efficiently
in a new host. Expression vectors have been
developed for both prokaryotic and eukaryotic
hosts
 These vectors contain genes that will increase
the level of transcription of the cloned gene and
make its transcription subject to specific
regulation. Signals to improve the efficiency of
translation may also be present in the expression
vector.

50. REPORTER GENES
 are incorporated into vectors because they encode
proteins that are readily detected. These genes
can be used to signal the presence or absence of a
particular genetic element or its location. They
can also be fused to other genes or to the
promoter of other genes so that expression can be
studied

52. EXPRESSION OF MAMMALIAN
GENES IN BACTERIA
 It is possible to achieve very high levels of
expression of mammalian genes in prokaryotes.
However, the expressed gene must be free of
introns.

53. • This can be accomplished
by using reverse
transcriptase to synthesize
cDNA from the mature
mRNA encoding the protein
of interest.

54.  One can also use the amino acid sequence of a
protein to design and synthesize an
oligonucleotide probe that encodes it. This
process is in effect reverse translation.

56. • Fusion proteins are often used to stabilize or
solubilize the cloned protein.

57. © Angel L. Salaman. PhD
Production of Insulin: The Beginnings of
Commercial Biotechnology
PRACTICAL APPLICATIONS
OF GENETIC ENGINEERING

58. • The first human protein made
commercially using engineered bacteria
was human insulin, but many other
hormones and human proteins are now
being produced. In addition, many
recombinant vaccines have been
produced.

60. OTHER MAMMALIAN PROTEINS
AND PRODUCTS
 Many human proteins that were formerly
extremely expensive to produce because they
were found in human tissues only in small
amounts can now be made in large amounts from
the cloned gene in a suitable expression system.

63. ANAKINRA® (AMGEN LTD)
 Anakinra (Kineret) is an interleukin-1 (IL-1) receptor antagonist.
Anakinra blocks the biologic activity of naturally occurring IL-1,
including inflammation and cartilage degradation associated with
rheumatoid arthritis, by competitively inhibiting the binding of
IL-1 to the Interleukin-1 type receptor, which is expressed in
many tissues and organs. IL-1 is produced in response to
inflammatory stimuli and mediates various physiologic responses,
including inflammatory and immunologic reactions. IL-1
additionally stimulates bone reabsorption and induces tissue
damage like cartilage degradation as a result of loss of
proteoglycans.
 In patients with rheumatoid arthritis the natural IL-1 receptor
antagonist is not found in effective concentrations in synovium
and synovial fluid to counteract the elevated IL-1 concentrations
in these patients.
 It is produced by recombinant DNA technology using an E coli
bacterial expression system.

64. ENBREL® (AMGEN LTD)
 ENBREL treats moderate to severe rheumatoid arthritis,
adult chronic moderate to severe plaque psoriasis in
patients who are candidates for systemic therapy or
phototherapy, psoriatic arthritis, ankylosing spondylitis,
and moderately to severely active polyarticular juvenile
idiopathic arthritis.
 Enbrel (etanercept) is a dimeric fusion protein consisting of
the extracellular ligand-binding portion of the human 75
kilodalton (p75) tumor necrosis factor receptor (TNFR)
linked to the Fc portion of human IgG1. The Fc component
of etanercept contains the CH2 domain, the CH3 domain
and hinge region, but not the CH1 domain of IgG1.
Etanercept is produced by recombinant DNA technology in
a Chinese hamster ovary (CHO) mammalian cell
expression system. It consists of 934 amino acids and has
an apparent molecular weight of approximately 150
kilodaltons.

65. NEUPOGEN™ (AMGEN LTD)
 Neupogen (filgrastim) is a form of a protein that stimulates
the growth of white blood cells in your body. White blood
cells help your body fight against infection.
 Filgrastim is a granulocyte colony-stimulating factor (G-
CSF) analog used to stimulate the proliferation and
differentiation of granulocytes. It is produced by
recombinant DNA technology. The gene for human
granulocyte colony-stimulating factor is inserted into the
genetic material of Escherichia coli. The G-CSF then
produced by E. coli is different from G-CSF naturally made
in humans.
 Neupogen is used to treat neutropenia, a lack of certain
white blood cells caused by cancer, bone marrow
transplant, receiving chemotherapy, or by other conditions.

66. GENETICALLY ENGINEERED
VACCINES
 Many recombinant vaccines have been produced.
These include live recombinant, vector, subunit,
and DNA vaccines

67. • Lists some genetically engineered vaccines.

68. •Production of recombinant
vaccinia virus and its use as a
recombinant vaccine.

69. GENETIC ENGINEERING IN
ANIMAL AND HUMAN GENETICS
 Genetic engineering can be used to develop
transgenic organisms capable of producing
proteins of pharmaceutical value
 The techniques of genetic engineering are also
applied to identifying individuals using DNA
fingerprinting
 One of the great hopes of genetic engineering is
gene therapy, in which functional copies of a
gene can be supplied to an individual to treat
human genetic disease.