MICROBIAL BIOTECHNOLOGY-
MICROBIAL PRODUCTS FOR
COMMERCIAL USE
SEMINAR BY
PRAKRITI B
MSc MB
MSRCASC
CONTENTS
1. VACCINES
2. MICROBIAL INSECTICIDES
3. SECONDARY METABOLITES IN BACTERIA & FUNGI
VACCINES
VACCINES
• Vaccine (L. vacca = cow) is a preparation/suspension or extract of
dead/attenuated (weakened) disease causing microbe which on
inoculation into a healthy person provides temporary/permanent
active/passive immunity by inducing antibodies formation.
• Thus antibody provoking agents are called vaccines.
• Pasteur coined the term vaccine
• Vaccine is used to boost the body’s immune system and prevent the
serious life threatening diseases.
• Vaccine can be prepared against bacteria or even eukaryotic
protozoans, fungi, however most successful vaccines have been
developed against viruses.
• Historically, these vaccines have eliminated fatal infections such as
smallpox, almost eliminated polio, and saved many individuals from
typhus, tetanus, hepatitis A and B, measles, rotavirus diseases, etc.
• However, still successful vaccines are yet to be developed for many
deadly diseases that cause chronic infections such as AIDS, hepatitis
C, tuberculosis, malaria, herpes, etc…
• Vaccination is immunization against infectious disease through the
administration of vaccines for the production of
active (protective) immunity in humans or other animals.
IDEAL CHARACTERISTICS OF A VACCINE:
[Link]: Vaccine must be safe and must not itself causes illness or death
[Link]: vaccine must protect against illness resulting from exposure
to live pathogen
[Link] protection: protection against illness must last for years
[Link] neutralizing antibodies
[Link] protective T cells
[Link] cost
[Link] stability
[Link] of administration
[Link] side effect or Very few side effects
TYPES OF VACCINES:
WHOLE-ORGANISM SUBUNIT NUCLEIC ACID
VACCINE VACCINE VACCINES
•Inactivated (Killed) •Polysaccharide •DNA plasmid
Vaccine - Typhoid Vaccine – vaccines - SARS
•Live-attenuated Streptococcus coronavirus
vaccines - TB pneumoniae •mRNA vaccines –
•Chimeric vaccine – (Conjugated Vaccines) Zika Virus (in
(Dengue + Zika) Virus •Toxoid Vaccines - monkeys & mice)
Diphtheria
•Recombinant Vaccines
RECOMBINANT VACCINE:
• A recombinant vaccine is a vaccine produced through recombinant
DNA technology. This involves inserting the DNA encoding an antigen
(such as a bacterial surface protein) that stimulates an immune response
into bacterial or mammalian cells, expressing the antigen in these cells
and then purifying it from them.
• TYPES:
1. Subunit recombinant vaccines:
These are the components of the pathogenic organisms. Subunit vaccines
include proteins, peptides and DNA.
2. Attenuated recombinant vaccines:
These are the genetically modified pathogenic organisms (bacteria or
viruses) that are made non-pathogenic and used as vaccines.
3. Vector recombinant vaccines:
These are the genetically modified viral vectors that can be used as
vaccines against certain pathogens.
Some of the developments made in the production of recombinant
vaccines against certain diseases are briefly described in the
following slides:
[Link]
Subunit Recombinant Vaccines:
• As already stated, subunit recombinant vaccines are the components
(proteins, peptides, DNAs) of the pathogenic organisms.
• The advantages of these vaccines include their purity in preparation,
stability and safe use.
• The disadvantages are — high cost factor and possible alteration in native
conformation. Scientists carefully evaluate the pros and cons of subunit
vaccines for each disease, and proceed on the considered merits.
HEPATITIS B:
• Hepatitis B is a widespread disease in man. It primarily affects
liver causing chronic hepatitis, cirrhosis and liver cancer.
• Hepatitis B virus is a 42 nm particle, called Dane particle.
• It consists of a core containing a viral genome (DNA) surrounded
by a phospholipid envelope carrying surface antigens.
(42nm)
HBsAg gene
Attenuated Recombinant Vaccine
• It is now possible to genetically engineer the organisms (bacteria or
viruses) and use them as live vaccines, and such vaccines are referred to
as attenuated recombinant vaccines.
• The genetic manipulations for the production of these vaccines are
broadly of two types:
1. Deletion or modification of virulence genes of pathogenic organisms.
2. Genetic manipulation of non-pathogenic organisms to carry and express
antigen determinants from pathogenic organisms.
CHOLERA:
• Cholera is an intestinal disease characterized by diarrhea, dehydration,
abdominal pain and fever.
• It is caused by the bacterium, Vibrio cholera
• On entering the small intestine, V. cholera colonizes and starts producing
large amounts of a toxic protein, a hexameric enterotoxin.
• The currently used cholera vaccine is composed of phenol-killed
V. cholera.
• The immuno-protection, lasting for 3-6 months is just moderate.
• Attempts are being made to develop better vaccines.
Restriction
digestion
ligation
Vector Recombinant Vaccines:
• Some of vectors can be genetically modified and employed as
vaccines against pathogens.
VACCINIA VIRUS:
• Vaccinia viruses is basically the vaccine that was originally used by
Jenner for the eradication of smallpox. The molecular biology of this
virus has been clearly worked out. Vaccinia virus contains a double-
stranded DNA (187 kb) that encodes about 200 different proteins. The
genome of this virus can accommodate stretches of foreign DNA
which can be expressed along with the viral genes.
Advantages:
• Authenticated antigens that closely resemble natural
antigens can be produced.
• The virus can replicate in the host cells. This enables
the amplification of the antigens for their action on B-
lymphocytes and T-lymphocytes.
• There is a possibility of vaccinating several diseases
with one recombinant vaccinia virus.
Disadvantages:
• 1. The most important limitation is the yet unknown risks of using
these vaccines in humans.
• 2. There may be serious complications of using recombinant viral
vaccines in immunosuppressed individuals such as AIDS patients.
MICROBIAL
INSECTICIDE
S
MICROBIAL INSECTICIDES:
• Single celled organisms like Bacteria, fungi, actinomycetes, viruses and
protozoa have been mass produced and formulated in a manner similar
to insecticides.
• These are also called as Bio-insecticides.
• Microbial insecticides can be :
1. Microbially produced toxic substance
2. The organism itself
Bacterial Insecticides:
• Several bacterial pathogens of different insects are being used as
insecticides.
• These are Bacillus, Clostridium, Pseudomonas, Enterobacter, Proteus,
Serratia etc.
• Out of these, Bacillus thuringiensis has been used extensively.
• The bacterium has been shown to be successful against a wide range
of more than 150 insects.
BACILLUS THURIENGIENSIS:
• It is a gram positive, spore forming, aerobic bacteria.
• Produces a toxin or crystal protein (BT toxin or CRY protein) that kills
certain insects.
• The commercial Bt products are produced as dust, wettable powder and
emulsifiable concentrates.
• The BT toxin or the CRY protein is produced when the bacteria sporulates.
Mode of action:
• To kill an insect pest, [Link] must be ingested by the target
organism, otherwise there will be no effect on the pest.
• After ingestion, the CRY protein is broken down in the midgut by proteases
into smaller toxic peptic molecules (under high pH) which binds to the
receptors and results in cell disruption.
[Link]
Target organisms:
Cry toxins have specific action against insect species of the orders:
1. Lepidoptera family (moths, butterflies)
2. Diptera family (flies, mosquitoes)
3. Coleoptera family (beetles)
4. Hymenoptera family ( wasps, bees, ants, sawflies)
5. Nematodes
Advantages:
• Reduces the use of chemical pesticides or insecticides.
• It will not kill beneficial insects such as butterflies and other pollinators.
• It is non toxic to humans, pets and other wildlife.
Fungal Insecticides:
• More than 750 species are known to infect insects.
• Mostly causing disease to insects.
• Some examples include: Metarhizium anisopliae, Beaveria bassiana,
Verticellum lecanii, Nomuraea rileyi, etc…
Nomuraea rileyi:
• It is a cosmopolitan occurrence and pathogenic to a number of
economically important lepidopterous pests.
• It is formulated as wettable powder.
• It could be multiplied on polished rice grains and crushed sorghum.
Beaveria bassiana:
• Deuteromycotina fungus, which is commonly found in soil throughout
the world.
• Mass produced on locally available grains and other solid substrate.
• Formulated as wettable powder, water dispersible granules, and oil based
emulsifiable suspension.
• Useful against Coffee berry borer, Diamond back moth, Thrips,
Grasshoppers, white flies, aphid, codling moth, etc..
Metarhizium anisopliae:
• It is a very widely distributed soil inhabiting fungi.
• Its spores can be formulated as dust and sprayable formulation.
• It is used to control termites, mosquitoes, leaf hoppers, beetles, etc.
Verticellum lecanii:
• It is known as “white – halo” fungus because of the white mycelial
growth on the edges of infected scale insects.
• It can be multiplied on medium based on locally available grains and
tubers.
• It is formulated as wettable powder.
• It is effective against coffee green bug, and certain other homopterans.
MODE OF ACTION:
Actinomycete insecticides:
• Actinomycetes are a group of unicellular filamentous bacteria that
form a branching network of filaments and produce spores.
Avermectin:
• From Streptomyces avermectin.
• Major homologues: A1a, A2a, B1a, B2a.
• Minor homologues: A1b, A2b, B1b, B2b.
• Effective against caterpillars, grasshoppers, etc..
Milbemectin:
• S. hygroscopium var aureolacromosus.
• Has 2 main groups: A3, A4
• Effective against bugs, thrips, etc..
Spinosad:
• Derived from S. spinosa
• Effective against caterpillars and thrips.
Viral Insecticides:
• Insect specific viruses can be used as effective natural controls of
several caterpillar pests.
• Examples: Baculovirus, Granulosis virus, cytoplasm polyhedrosis virus,
etc..
Baculovirus:
• Rod shaped DNA virus.
• Includes NPV and GV.
• Pathogenic for lepidoptera, Diptera and Hymenoptera.
• Infection is by ingestion of food.
Protozoan Insecticides:
• These are single celled organisms.
• Some of them parasitise and kill insects.
• Not suited for short time.
• Effective against grasshoppers, mosquitoes.
• They reduce host reproduction or feeding rather than killing the isect.
Vairimorpha necatrix:
• Infects lepidopterans.
• Mortality due to damage of gut followed by septicaemia or by
microsporidia.
• Swelling of fat body is seen.
SECONDARY
METABOLITES IN
BACTERIA AND FUNGI
SECONDARY METABOLITES:
• Many plants, fungi and microbes of certain genera and families
synthesize a number of organic compounds which are not involved in
primary metabolism (photosynthesis, respiration, and protein and lipid
metabolism) and seem to have no direct function in growth and
development of plants.
• Such compounds are called secondary metabolites.
• Secondary metabolites are formed near the stationary phase of growth
and are not involved in growth, reproduction and development.
• The metabolites can be used in industries to develop vaccines,
antibiotics, isolate chemicals for organic synthesis and to obtain amino
acids.
• For e.g., antibiotics, toxins, steroids, alkaloids, pheromones, enzyme
inhibitors, etc..
Characteristics Of Secondary Metabolites
1. They are specifically produced by selected few organisms.
2. They are not essential for the growth and reproduction of organisms
from which they are produced.
3. Environmental factors influence the production of secondary
metabolites.
4. The biosynthetic pathways for most secondary metabolites are not
clearly established.
5. The regulation of the formation of secondary metabolites in more
complex and differs from that of primary metabolites.
Anticancer Compounds From Microbes:
1. Actinomycin D:
• Actinomycin is a polypeptide antibiotic isolated from soil bacteria of
the genus Streptomyces.
• Molecular weight : 1255.42
• It binds to DNA duplexes, thereby interfering with the action of
enzymes engaged in replication and transcription.
• Actinomycin D is one of the old chemotherapy drugs which have
been used in therapy for cancer for many years.
2. Doxorubicin:
• Doxorubicin is an anti-cancer (“antineoplastic” or “cytotoxic”)
chemotherapy drug.
• Doxorubicin is classified as an “anthracycline antibiotic.”
• These are made from natural products produced by species of the soil
fungus Streptomyces.
• It is administered via an intravenous (IV) injection through a central
line or a peripheral venous line, and the drug is given over several
minutes. It can also be given by continuous infusion through a central
catheter line.
• There is no pill form of Doxorubicin.
3. Apratoxin A:
• Source organism: Lyngbya majuscula
• It is a cyclic Depsipeptide.
• Exhibits cytotoxic effects on Human HeLa cervical carcinoma cells by
cell cycle inhibition.
• Coibamide A (on human lung cancer cell), Lyngbyallin B (on human
lymphoma cells) are also isolated from this organism.
4. Notoamides:
• Produced by A. amoenus.
• Showed low to moderate inhibition of two leukemic cell lines.
5. Taxol:
• Produced by the fungus Taxomyces andreanea and later including P.
raistrickii.
• One of the best anticancer compounds used today.
• Taxol is routinely used to treat ovarian, breast and lung tumors as well as
Kaposi’s sarcoma.
6. Stephacidin B:
• It is tryptophan/proline diketopiperazine.
• Stephacidin B is the dimeric form of stephacidin A and is approximate
10-fold more potent.
• It exhibited inhibitory effect on prostate, ovarian, colon, breast and lung
cancer cell lines.
Antidiabetic compounds producdced
by Microbes:
• Diabetes is one of the most common disease that is prevalent all
around the world.
• India has been declared as the diabetes capital of the world and
diabetes has been ranked as the third important cause of global
mortality after cancer and cardiovascular disorders.
• Therefore there is a need for safe and natural products which could
bring some positive effects in controlling this disease.
• So the components produced by microorganisms like bacteria and
fungi are used.
• A large number of probiotics and extracted secondary metabolites
from the Lactobacillus or Bifidobacterium genera, Exiguobacterium
indicum - an Enophytic bacterium, Aspergillus saitoi - a fungus have
proved the antidiabetic properties.
• Endophytic fungi and bacteria are the most common sourced of these
antidiabetic components.
• Acarbose:
o Is a pseudo-oligosaccharide, a bacterial-derived α-glucosidase
inhibitor.
o Naturally produced by Actinoplanes sp. and several other soil bacteria.
o It has shown potential positive effect against type II diabetes.
REFRENCES:
• [Link]
• [Link]
• [Link]
• [Link]
• [Link]
• Complete biosynthetic pathway to the antidiabetic drug acarbose
Takeshi Tsunoda, Arash Samadi, Sachin Burade & Taifo Mahmud
• Anti diabetic Activity of Bacteria and Fungi: A Review - March 2018
• Anticancer and Antifungal Compounds from Aspergillus, Penicillium and Other Filamentous Fungi
Tanja Thorskov Bladt, Jens Christian Frisvad, Peter Boldsen Knudsen, and Thomas Ostenfeld Larsen*
THANK YOU
