isolation and screening of secondary metabolites
DESCRIPTION
Isolation of MacromoleculesTRANSCRIPT
© Angel L. Salaman. PhD
ISOLATION AND SCREENING OF SECONDARY METABOLITESAngel L. Salaman, PhD
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
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
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
PRODUCTION AND SCALE
Primary metabolite Produced during exponential growth Example: alcohol
Secondary metabolite Produced during stationary phase
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
© Angel L. Salaman. PhD
FIGURE 15.1
Primarymetabolite
Secondarymetabolite
Alcohol
Penicillin
Cells
Sugar
Cells
Sugar
Time Time
Alc
oh
ol,
sug
ar, o
r ce
ll n
um
ber
Pen
icill
in, s
ug
ar, o
r ce
ll n
um
ber
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
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
© Angel L. Salaman. PhD
FIGURE 15.2B
Steam
Sterile seal
Motor
pH pH controller
Acid–basereservoir andpump
Viewing port
Filter
Exhaust
Impeller(mixing)
Coolingjacket
Externalcoolingwater in
Externalcoolingwater out
Culturebroth
Steam in
Valve
Harvest
Sparger (high-pressure airfor aeration)
Sterile air
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
© Angel L. Salaman. PhD
FIGURE 15.4A
I. Isolation
Sterile glass spreader
Colonies ofStreptomycesspecies
Nonproducingorganisms
Zones ofgrowth inhibition
Producingorganisms
Spread a soildilution on a plateof selective medium
Incubation
Overlay with anindicator organism
Incubate
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
© Angel L. Salaman. PhD
FIGURE 15.4B II. Testing Activity Spectrum
Streak antibiotic produceracross one side of plate
Incubate to permit growthand antibiotic production
Cross-streak with test organisms
Incubate to permit test organisms to grow
Antibiotic diffusesinto agar
Streptomyces cell mass
Growth of test organism
Inhibition zones wheresensitive test organismsdid not grow
INDUSTRIAL PRODUCTION OF PENICILLINS AND TETRACYCLINES
Penicillins are -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
© Angel L. Salaman. PhD
FIGURE 15.6
Glucosefeeding
Nitrogenfeeding
Cells
Lactose
Ammonia
Penicillin
Fermentation time (h)
Bio
mas
s (g
/lite
r), c
arb
oh
ydra
te,
amm
on
ia, p
enic
illin
(g
/lite
r
10) 100
90
80
70
60
50
40
30
20
10
020 40 60 80 100 120 140
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
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
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)
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
© Angel L. Salaman. PhD
FIGURE 15.10
Starch oligosaccharides
Time (h)P
erce
nt
enzy
me
acti
vity
rem
ain
ing
Pullulanase
90°C100°C110°C110°C plus Ca2
100
10
11 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).
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
© Angel L. Salaman. PhD
FIGURE 15.13
Stems removedGrapes crushed
Must
Juice sits in contactwith skins for 16–24 h
Press
Yeast
White wine Red wine
Pomace(discard)
Yeast
Fermentation vat10–15 days
Aging 5 months
Racking
Clarifying agents
Filtration
Bottling
Stems removedGrapes crushed
Must
Fermentation vat 3 weeks(pulp is not removed)
Press
Pomace(discard)
Aging in barrels
Racking
Transfer to clean barrels3 times per year
Clarifying agents
2 years
Settling tank
Filtration
Bottling: Age in bottles6 months or more Sodium metabisulfite : It is used as a disinfectant, antioxidant and
preservative agent
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.
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
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
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).
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
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.
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.
FINDING THE RIGHT CLONE
Special procedures are needed to detect the foreign gene in the cloning host
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.
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.
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.
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
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.
• This can be accomplished by using reverse transcriptase to synthesize cDNA from the mature mRNA encoding the protein of interest.
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.
© Angel L. Salaman. PhD
Production of Insulin: The Beginnings of Commercial Biotechnology
PRACTICAL APPLICATIONS OF GENETIC ENGINEERING
• 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.
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.
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.
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.
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.
GENETICALLY ENGINEERED VACCINES
Many recombinant vaccines have been produced. These include live recombinant, vector, subunit, and DNA vaccines
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.