isolation and screening of secondary metabolites

© Angel L. Salaman. PhD

ISOLATION AND SCREENING OF SECONDARY METABOLITESAngel L. Salaman, PhD

[email protected]

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


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


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


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


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


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


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


FIGURE 15.2A


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


FIGURE 15.2C


FIGURE 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

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


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


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


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


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).

III. ALCOHOLIC BEVERAGES AND BIOFUELS

Wine Brewing and Distilling Biofuels

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


FIGURE 15.12B


FIGURE 15.12C


FIGURE 15.12D


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.


FIGURE 15.14

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


FIGURE 15.17

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).

EXPRESSING MAMMALIAN GENES IN BACTERIA

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.

Nucleic acid gun for transfection of certain eukaryotic cells.

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.

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


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

• Lists some genetically engineered vaccines.

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

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.

isolation and screening of secondary metabolites

Science

white blood cells

genetically engineered vaccines

scale secondary metabolites

recombinant dna technology

recombinant vaccines

rheumatoid arthritis

large scale

alcoholic beverages