Microbial Biotechnology: Scope,

Techniques, Examples

By
Dr. Anum Gul
Microbial Biotechnology

 According to the UN Convention on Biological Diversity, microbial

biotechnology can be defined as any technological application that uses
microbiological systems, microbial organisms, or derivatives thereof, to make
or modify products or processes for specific use.

 The microorganisms used in industrial processes are natural, laboratory-

selected mutant or genetically engineered strains.
Microbial Biotechnology

 Economically valuable products such as alcohols, solvents, organic acids,

amino acids, enzymes, fermented dairy products, food additives, vitamins,
antibiotics, recombinant proteins and hormones, are produced by
microorganisms that are used in chemical, food, pharmaceutical, agricultural,
and other industries.
Microbial Biotechnology

 The umbrella of microbial biotechnology covers many scientific activities,

ranging from production of recombinant human hormones to that of
microbial insecticides, from mineral leaching to bioremediation of toxic
wastes.
History of Microbial Biotechnology
History of Microbial Biotechnology
History of Microbial Biotechnology
Microbial Biotechnology for Pharmaceutical
Needs
Human Therapeutics

Production of Heterologous Proteins

 One of the most dramatic and immediate impacts of genetic engineering was the
production in bacteria of large amounts of proteins encoded by human genes.

 In 1982, insulin, expressed from human insulin genes on plasmids inserted into
Escherichia coli, was the first genetically engineered therapeutic agent to be
approved for clinical use in humans.

 Bacterially produced insulin, used widely in the treatment of diabetes, is

indistinguishable in its structure and clinical effects from natural insulin.
Human Insulin Production
Human Growth Hormone (hGH)

 Human growth hormone (hGH), a protein made naturally by the pituitary gland,
was the second such product.

 Inadequate secretion of hGH in children results in dwarfism.

 Before the advent of recombinant DNA technology, hGH was prepared from
pituitaries removed from human cadavers.

 The supply of such preparations was limited and costly.

Human Growth Hormone (hGH)

 Some patients treated with injections of pituitary hGH developed a disease

caused by a contaminating slow virus, Jakob–Creutzfeldt syndrome, which
leads to dementia and death.

 hGH can be produced in genetically engineered E. coli in large amounts, at

relatively little cost, and free from such contaminants.
Human Tissue Plasminogen Activator (tPA)

 Human tissue plasminogen activator (tPA), a proteolytic enzyme (a “serine” protease)

with an affinity for fibrin clots.

 At the surface of fibrin clots, tPA cleaves a single peptide bond in plasminogen to form
another serine protease, plasmin, which then degrades the clots.

 This clot-degrading property of tPA makes it a life-saving drug in the treatment of

patients with acute myocardial infarction (damage to heart muscle due to arterial
blockage).
Human Tissue Plasminogen Activator (tPA)
Vaccines

A vaccine is a biological preparation that improves human immunity to a

particular disease.

Moreover, a vaccine basically contains an agent that resembles a disease-causing

microorganisms and is often made from weakened or killed forms of the
microorganism or its toxins.
Vaccines

 Among the biologicals, one of the leading and most extensively studied products is
vaccine.

 These are given to generate immune responses against a number of diseases.

 Despite the fact that the safety and effectiveness of vaccines are highly questioned, its
high benefit-to-risk ratio strongly supports its use in providing long-lasting, humoral
and cellular immunity.
Vaccines

 The memory T-cells derived from normal T-cells play a significant role in the
development of long-lasting immunity.

 The T-cells represent an extremely advanced wing of the adaptive immune system,
which is able to differentiate between pathogens and is efficient at progressing or
adapting during the lifetime of an individual such that immunity is enhanced with
each consecutive contact with a pathogen.
Historical Background

 The story behind vaccine development started with the research work done by
Edward Jenner in the late 1700s.
Historical Background

Jenner took pus from the hand of a milkmaid with cowpox,

inoculated an 8-year-old boy with it, and 6 weeks later
inoculated the boy’s arm with smallpox, afterwards observing
that the boy did not catch smallpox.
Type of Vaccines

Vaccines typically consist of either

 Attenuated (live) Vaccine

 Inactivated (killed) Vaccine

Live Attenuated Vaccines

 An attenuated vaccine is a vaccine created by reducing the virulence of a pathogen, but still keeping it viable
(or "live").

 Attenuation takes an infectious agent and alters it so that it becomes harmless or less virulent.

 Examples include measles, mumps and rubella vaccine (MMR vaccine), varicella (chickenpox) vaccine, oral
polio vaccine (OPV).
Inactivated/Killed Vaccine

 Killed vaccines contain killed bacteria or inactivated viruses.

 Inactivated vaccines cannot cause an infection, but they still can stimulate a protective immune response.

 Moreover, viruses can be inactivated with chemicals such as formaldehyde.

 One example of an inactivated vaccine is the inactivated polio vaccine (IPV), which is the shot form of the
polio vaccine, inactivated influenza vaccine.
Biotechnological Approach in Vaccine Production

 With the introduction of recombinant DNA technology, utilizing both

microorganisms and mammalian cells have been exploited enormously for the
production of vaccines in recent years.

 A wide range of host cells, such as bacteria, yeast, and even plants have been
examined as potential sources for engineered vaccines in substantial quantities.

 Tissue culture techniques are also being investigated in genetically modified host
cells to produce large quantities of antigens.
Secondary Metabolites as a Source of Drugs

 Microorganisms produce a huge number of small molecular weight

compounds that are broadly described as secondary metabolites.

 Traditional approach to the discovery of new, naturally occurring

bioactive molecules utilizes “screens.”
Flavonoids

 The natural polyphenolic compounds with low molecular weight represent a

diverse group of secondary metabolites, known as flavonoids, with distinct and
pronounced biological activities.

 Their importance has now been explored comprehensively to ascertain their role as
antimicrobials (antiviral, antibacterial, antifungal, antiprotozoal, etc.), antioxidants
(responsible for beneficial effects on cardiovascular disease), anti-inflammatory,
anti-allergic, anti-cancer, anti-obesity, and others.
Production

 The isolation, extraction, and purification of secondary metabolites is a

difficult task.

 The seasonal and regional variations along with low concentrations of the pure
compound certainly create challenges for researchers.

 Although, in some cases, chemical synthesis usually provides a reasonable

alternative, but the intense reaction environments and disastrous chemicals
required to synthesize flavonoids makes it less acceptable.
Biotechnological Approach

 To meet the increased demand for flavonoids and to effectively utilize their
therapeutic role, microbial production through genetically modified organisms is
becoming progressively imperative from a recombinant manufacturing point of
view.

 E. coli, S. cerevisiae, Streptomyces venezuelae etc. have been extensively studied in

the production of some important flavonoids such as naringenin, kaempferol,
chrysin, genistein, flavanone, dihydroflavonol, flavone, flavonol, quercetin etc.
Antibiotics

Historical background and new challenges

 Microbial production of secondary metabolites and especially

those with therapeutic importance such as “antibiotics” have been
given significant attention worldwide in view of their role in the
management of infectious disease.

 The first therapeutic compound “penicillin,” from the fungal

species, Penicillium notatum, followed by Penicillium
chrysogenum, was discovered by Alexander Fleming in 1928.

 It has been reported to be highly inhibitory effect against

Staphylococcus aureus, a Gram-positive bacteria.
Antibiotics

 In 1942 Selman Waksman suggested a proper definition of the term antibiotics as “a

chemical substance obtained from certain microorganisms, capable of inhibiting the
growth or destroying other microorganism in small concentrations.”

 By the end of 1940, Fleming was able to test the effect of penicillin (benzylpenicillin
or penicillin G) on some other Gram-positive organisms responsible for causing
scarlet fever, pneumonia, meningitis, and diphtheria and one Gram-negative
organism causing gonorrhea, but no prominent place or identification was given to
penicillin.
Antibiotics

 In 1941, Howard Florey and Ernst Boris Chain

completely revolutionized and changed the
concept of infectious disease therapy with the
successful commercial production of penicillin,
this period was called the “Golden Age of
Antimicrobial Therapy.”
Antibiotics

 After penicillin, subsequent research in the field of secondary metabolites,

especially from fungi and actinomycetes (filamentous bacteria), have led to the
discovery of a series of antibiotics for human and veterinary use, such as
cephalosporins, tetracyclines, aminoglycosides, chloramphenicol, macrolides,
and glycopeptides.
Biotechnological Approach

 Soon after the discovery and commercial production of penicillin, scientists

started working on the genetic aspects of antibiotic formation, primarily
because of the increased demand for penicillin to treat the victims of World
War II.

 The initial studies were based on the development of high-yielding mutant

strains using both physical and chemical treatment of the wild strains.
Biotechnological Approach

 With the discovery of the complex mechanisms responsible for the regulation of
antibiotics in various microbial strains, the classic mutation processes were further
linked with genetic engineering techniques.

 This has not only shortened the strains improvement and development time but has
been able to reduce the unnecessary involvement of human resources during the
mutant screening program.

 With the further advancements of new biotechnological approaches and tools,

genetic engineering alone is quite sufficient to modify the strain.
Agriculture

 Methods dependent on microbial biotechnology greatly increase the diversity of

genes that can be incorporated into crop plants and dramatically shorten the time
required for the production of new varieties of plants.

 It is now possible to transfer foreign genes into plant cells.

 Transgenic plants that are viable and fertile can be regenerated from these
transformed cells, and the genes that have been introduced into these transgenic
plants are as stable as other genes in the plant nuclei and show a normal pattern of
inheritance.
Ability to Grow in Harsh Environments

 Tolerance towards environmental stresses are likely to be polygenic traits and as a

consequence may be difficult to transfer from one kind of organism to another.

 Trehalose, a disaccharide of glucose, acts as a compatible solute that stabilizes and

protects proteins and biological membranes in bacteria, fungi, and invertebrates from
damage during desiccation.

 Except for highly desiccation-tolerant “resurrection plants,” most plants do not

accumulate detectable amounts of trehalose.
Ability to Grow in Harsh Environments

 E. coli genes otsA and otsB for trehalose biosynthesis were introduced into indica
rice.

 The constructs were introduced into rice using Agrobacterium-mediated gene transfer.

 Compared with non-transgenic rice, several independent transgenic lines showed

sustained plant growth under drought, salt, or low temperature stress conditions.

 The transgenic rice contained three- to nine fold greater levels of trehalose than the
non-transgenic rice.
Resistance to Insect Pests

 Certain strains of the bacterium Bacillus thuringiensis produce protein endotoxins that
permeabilize the epithelial cells in the gut of the larvae of lepidopteran insects, moths,
and butterflies.

 Genes encoding particular B. thuringiensis endotoxins have been transferred into and
expressed in tobacco, cotton, and tomato.

 In field tests, the transgenic tomato and tobacco plants were only slightly damaged by
caterpillar larvae under conditions that led to total defoliation of control plants.
Resistance to Insect Pests

 A different approach to achieve the same end was to transfer a B. thuringiensis

endotoxin gene into bacteria such as Clavibacter xyli subsp. cynodontis, which
colonizes the interior of plants.

 This organism is generally found inside Bermuda grass plants but can reach
population sizes in excess of 108/gram of stem tissue if purposefully inoculated into
other monocotyledonous species, such as corn.

 Recombinant C. xyli strains expressing the endotoxin show promise in controlling

leaf-and stem-feeding lepidopteran larvae.
Control of Pathogenic Bacteria, Fungi, and Parasitic Nematodes

 The cell walls of many plant pests, such as insects and fungi, contain chitin (poly-
N-acetylglucosamine) as a major structural component.

 Many bacteria (e.g., species of Serratia, Streptomyces, and Vibrio) produce chitin
degrading enzymes (chitinases).

 The control of some fungal diseases by such bacteria has been correlated with the
production of chitinases.

 Genes encoding chitinases from several different soil bacteria have been cloned into
Pseudomonas fluorescens, an efficient colonizer of plant roots.
Preparation of Fermented Foods

 The metabolic end products produced by the microorganism's flavor fermented foods.

 For example, mold-ripened cheese owe their distinctive flavors to the mixture of aldehydes,
ketones, and short-chain fatty acids produced by the fungi.

 Lactic acid bacteria are widely used to produce fermented foods.

 These organisms are also of particular importance in the food fermentation industry
because they produce peptides and proteins (bacteriocins) that inhibit the growth of
undesirable organisms that cause food spoilage and the multiplication of food borne
pathogens.
Nisin

 Nisin inhibits the growth of a wide range of Gram-positive bacteria, including

Listeria, Clostridium, Bacillus, and Enterococci.

 Nisin is designated as a Generally Regarded as Safe (GRAS) food preservative in the

United States and in many other countries around the world.

 It is used in many food products, including pasteurized cheese spreads with fruits,
vegetables, or meats; liquid egg products; dressings and sauces; milk; some beers;
canned foods; and frozen dessert.
Single-Cell Protein

 The term single-cell protein, or SCP, describes the protein-rich cell mass derived from
microorganisms grown on a large scale for either animal or human consumption.

 SCP has a high content of protein containing all the essential amino acids.

 Microorganisms are an excellent source of SCP because of their rapid growth rate,
their ability to use very inexpensive raw materials as carbon sources, and the uniquely
high efficiency, expressed as grams of protein produced per kilogram of raw material,
with which they transform these carbon sources to protein.
Environmental Applications of Microorganisms

 First and foremost, the essential role of microorganisms in the treatment of

wastewater is critical to the wellbeing of life on Earth.

 Bioremediation, biomining, are other large-scale processes in which important

positive environmental outcomes are achieved by directly exploiting the
combined metabolic capabilities of naturally occurring communities of
microorganisms.

 In such applications, the functioning of a particular microbial community can be

influenced through the manipulation of conditions (e.g., nutrients, oxygen
tension, temperature, agitation).
Microbial Whole-Cell Bioreporters

 Bioreporters are intact, living microbial cells that have been genetically engineered
to produce a measurable signal in response to a specific chemical or physical agent
in their environment.

 Bioreporters contain two essential genetic elements, a promoter gene and a reporter
gene.

 The promoter gene is turned on (transcribed) when the target agent is present in the
cell’s environment.
Microbial Whole-Cell Bioreporters

 The promoter gene in a normal bacterial cell is linked to other genes that are then
likewise transcribed and then translated into proteins that help the cell in either
combating or adapting to the agent to which it has been exposed.

 In the case of a bioreporter, these genes, or portions thereof, have been removed and
replaced with a reporter gene.

 Consequently, turning on the promoter gene now causes the reporter gene to be
turned on.
Microbial Whole-Cell Bioreporters

 Activation of the reporter gene leads to production of reporter proteins that

ultimately generate some type of a detectable signal.

 Therefore, the presence of a signal indicates that the bioreporter has sensed a
particular target agent in its environment.
Microbial Whole-Cell Bioreporters

 The great majority of these bioreporters are genetically engineered bacteria within
which a promoter–operator (the sensing element) responds to the stress condition
(toxic organic or inorganic compound, DNA damage, etc.) and changes the level of
expression of a reporter gene that codes for a protein (the signal).

 The protein may be detected either directly (e.g., green fluorescent protein) or
through its catalytic activity (e.g., formation of a fluorescent or chemiluminescent
product).