3. One can be a good biologist without necessarily knowing much
about microorganisms, but one cannot be a good microbiologist
without a fair basic knowledge of biology!
Stanier, R. Y., Doudoroff, M.
4. This is an era of explosive growth of analysis and manipulation of
microbial genomes as well as of invention of many new, creative
ways in which both microorganisms and their genetic endowment
are utilized.
The umbrella of microbial biotechnology covers many scientific
activities, ranging from
production of recombinant human hormones
microbial insecticides
mineral leaching
bioremediation of toxic wastes etc.
The purpose of this course is to convey the impact, the
extraordinary breadth of applications, and the multidisciplinary
nature of this technology. The common denominator to the
subjects discussed is that in all instances, prokaryotes or fungi
provide the indispensable component.
5. Microorganisms are important for many reasons such as
They produce things that are of value to us
Large material like proteins, carbohydrates, nucleic
acids and even cells.
Small molecules like primary metabolites that are
essential for vegetative growth and secondary
metabolites (non essential).
Why Microorganisms?
6. Microorganisms produce an array of metabolites but in
minute quantity.
Regulatory mechanisms that keep a check on over
production of metabolites.
However,
Industrial biotechnologist seek for such wasteful strain
that will over produce a certain compound which can be
isolated and marketed.
After desired strain has been found a development
program is initiated to improve titers by modification of
cultural conditions by mutations or by recombinant
DNA technology.
Metabolite production
7. The main reason to use a microorganisms over plant and
animal for synthesis of a compound is that microorganisms
can be manipulated to increase the production to even
1000 fold for small metabolites.
8. Primary metabolites
These are small molecules produced by living cells; they
are intermediates or end products of the pathway of
intermediary metabolism such as
Building blocks for essential macromolecules, or are
converted into coenzymes.
TRADITIONAL MICROBIAL
BIOTECHNOLOGY
9. Alcohols (ethanol)
Amino acids (monosodium glutamate, lysine, threonine,
phenyl alanine, tryptophan)
Flavour nucleotides (5’- guanylic acid, 5’- inosinic acid)
Organic acid (acetic,propionic, succinic, fumaric and lactic )
Polyols (glycerol, mannitol, xylitol etc.)
Polysaccharides (xanthan, gellen)
Sugars (fructose, ribose, sorbose)
Vitamins (Ribo flavin B2, cyanocobalamin (B12), biotin)
Examples of primary metabolites used in food and feed
industries are
10. Mutants
Auxotrophic mutants
‘‘A mutant strain of microorganism that will proliferate
only when the medium is supplemented with some specific
substance not required by wild-type organisms ’’
Amino acid production in which regulatory mechanism
is bypassed by auxotrophic mutants by partially starving
them for a requirement.
D
A B C
E
C - objective product
Enzyme 2
Enzyme 1
11. In parent strain enzyme 1 is subjected by cumulative
feed back regulation by end product D and E.
A mutant is obtained that lacks enzyme 3.
D must be supplied in the medium.
If D is supplied in growth limiting concentrations,
commulative feed back mechanism is broken and C is
over produced.
Example: Inosine 5- mono-phospahte (IMP production)
12. Produce mutants that are resistant to toxic analog of a
metabolite i.e. antimetabolite.
Due to feed back mechanism the presence of primary
metabolite inhibits over production of itself.
Analog mimics the metabolite in chemical and structural
properties.
Strains are first grown at different conc, of an analog.
Those isolates that are resistant to an analog can over
produce the metabolite.
Examples, amino acid, vitamins and antibiotic
production.
Resistance to toxic metabolite
13. Outward permeability i.e. how much conc. per litre.
example., sodium glutamate an amino acid
Annual production is 1.2 billion employing different bacteria
like Corneybacterium and Brevebacterium.
From sugur conc. of 100g/ lit. have been achieved.
Glutamic Acid
Over production of glutamic acid is inhibited by feed back
mechanisim.
It is only regulated by change in conformity of cell membrane
by biotin limitation process (biotin auxotrophs) that result in
phospholipid deficient cell membrane.
Efflux is carried out by special system through a carrier that
is dependent on membrane potential.
Fermentation
14. In E.coli Threonine, lysine and methionine produced by
a tight system of 3 enzymes through feed back
mechanism. Naturally this does not lead to
overproduction of any amino acid at commercial level.
Commercially C. glutamicum is used for commercial
production of lysine. Homoserine dehydrogenase is
removed genetically and threonine and methionine are
provided in limited amount in the media.
Assignment diagrammatic representation of L-lysine
production in both E.coli and mutant strain.
Lysine
15. No feed back repression of aspartate kinase occurs in
lysine over producers.
The first and second enzyme in lysine production are
neither repressed or inhibited by lysine conc.
L- lysine decarboxylase is absent in over producers
World market for amino acids
L-glutamate US dollar 915 million
L-lysine US dollar 450 million
L-phenylalanine US dollar 198 million
L-aspartate US dollar 43 million
Difference between lysine over
producers and E.coli
16. Recombinant technology along with mutations and
selection procedures have led to production of amino
acids to these levels g/l
L-Threonine 100
L-Isoleucine 40
L- leucine 34
L-valine 31 etc.
Recombinant DNA technology
17. Riboflavin (vitamin B2)
Over producer are two yeast Eremothecium ashbyii and
Ashbya gossypii (20g/l)
Candidia and recombinant Bacillus subtilis strains have
improved yeild by 30g/l.
Vitamins
18. Produced by Propionibacterium shermani and
Pseudomonas denitrificans
P. shermanii fermentation first step is under anaerobic
conditions without addition of 5,6 benzimidazole
resulting in inhibition of B12 and accumulation of
intermediate cobinamide.
Later under aerobic conditions precursor is added and
B12 is synthesized.
With Pseudomonas denitrificans fermentation entire
process takes place under low oxygen content.
Production level is 150mg/l and world market value of
71 million US dollar.
Vitamin B12
19. Strains of Serratia marcescens after recombination
have produced yield of biotin upto 600mg/l.
Fungi
Mainly used for the production of organic acids e.g. 1 billion
pounds of citric acid (CA) produced per year of 1.4 million US
dollar market value.
Produced by Embden-Meyerhof pathway and the first step of
TCA cycle
Control of production is by inhibition of phosphofructokinase by
citric acid.
Commercially Aspergillus niger is used for CA production in iron
and manganese deficient media.
High level of CA is also associated with high intracellular conc. of
fructose 2-6, bisphosphate, an activator of glycolysis.
Assignment : diagrammatic representation of CA production.
Biotin
20. Other factors effecting CA production
High CA production by Inhibition off isocitrate
dehydrogenase by CA.
Low pH (1.7-2.0), inhibits glucose oxidase that would
normally produce gluconic acid. After 4 -5 days 80%
sugar is converted to CA with titers of 100g/l.
Higher pH values (3.0) leads to production of oxalic
acid and gluconic acid instead of CA.
CA from hydrocarbons using Candida yeast have
reported to yield 150-170% of CA and titre upto 225g/l.
21. Ethyl alcohol is a Primary metabolite produced by
fermentation of sugars.
Saccharomyces cerevicae for fermentation of hexoses.
Kluyveromyces fragilis or Candida for lactose and pentoses.
Under optimum conditions 10-12% of alcohol by volume can
be produced.
After this conc. ethanol intolerance inhibit further
conversion.
With special yeast 20% yield can be achieved by after
several month and years of fermentation.
Fermentation by bacteria include Zymomonas and
Clostridium thermocellum .
Recombinant E.coli has given 43% (v/v) of yield.
Alcohols
22. Microbes have enzyme systems for almost every type of
reaction.
In bioconversion a compound is converted to another
compound that is structurally related to the initial
compound i.e. stereospecific.
Bioconversions yield 90-100% conversion.
Bioconverting-organisms
23. Very important from health and nutrition point of view.
Includes antibiotics, other medicinals, toxins,
biopesticides and other plant and animal growth factors.
Produced by restricted group of microbes and chemicals
mixtures formed as closely related member of a
chemical family.
Secondary metabolites
24. Antibiotics
The best known group of secondary metabolites
In 1996 over 160 antibiotics are known to be produced and world
market value of US 23 billion dollar.
targets include DNA replication (actinomycin, bleomycin and
griseofulvin)
transcription (rifamycin)
translation by 70-S ribosomes (chloramphenicol, tetracycline,
lincomycin, erythromycin and streptomycin)
transcription by 80-S ribosomes (cyclohexamide)
transcription by 70- and 80-S ribosomes (puromycin and fusidic
acid)
cell wall synthesis (cycloserine, bacitracin, penicillin, cephalosporin
and vancomycin)
cell membranes (surfactants including: polymyxin and
amphotericin; channel forming ionophores, such as linear
gramicidin; and mobile carrier ionophores, such as monensin).
25. The search for new antibiotics continues, in order to:
combat
evolving pathogens,
natural resistance
bacteria and fungi,
previously susceptible microbes that have developed
resistance
improve pharmacological properties
combat tumors
viruses and parasites
and discover safer more potent and broad spectrum
compounds.
Antibiotics are used not only for chemotherapy in human
and veterinary medicine, but also for growth promotion in
farm animals and for the protection of plants
26. In nature, secondary metabolites are important to the
organisms that produce them, functioning as:
(1) Sex hormones
(2) Ionophores
(3) competitive weapons against other bacteria, fungi,
amoebae, insects and plants
(4) agents of symbiosis
(5) effectors of differentiation.
Non-antibiotic agents
27. Despite the testing of thousands of synthetic
compounds, only a few promising structures were
found.
As new lead compounds became more and
more difficult to find, microbial broths filled the void
and microbial products increased in importance in the
therapy of non-microbial diseases.
polyethers: monensin, lasalocid and salinomycin dominate
the coccidiostat market
These are also chief growth promoters in use for ruminant
animals.
avermectins, another group of Streptomycete products with a
market of more than US$1 billion per year, have high activity
against helminths and arthropods.
28. Inhibitors such as statins, including lovastatin (also known
as mevinolin) and pravastatin: fungal products that are used
as cholesterol-lowering agents in humans and animals.
Lovastatin in its hydroxy acid form, is a potent competitive
inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A
reductase from liver.
Other wellknown enzyme inhibitors include:
Clavulanic acid, a penicillinase-inhibitor that protects
penicillin from inactivation by resistant pathogens
Acarbose, a natural inhibitor of intestinal glucosidase, which
is produced by an actinomycete of the genus Actinoplanes.
Acarbose decreases hyperglycemia and triglyceride synthesis
in adipose tissue, the liver and the intestinal wall of patients
suffering from diabetes, obesity and type IV hyperlipidemia.
29. In commercial or near-commercial use are biopesticides, including
Biofungicides e.g. kasugamycin, polyoxins,
Bioinsecticides e.g. nikkomycin, spinosyns
Bioherbicides such as bialaphos
Antihelminthics e.g. avermectin
Coccidiostats, ruminant-growth promoters (monensin, lasalocid,
salinomycin)
plant-growth regulators (gibberellins),
immunosuppressants for organ transplants (cyclosporin A, FK-506,
rapamycin)
Anabolic agents in farm animals (zearelanone)
Uterocontractants (ergotalkaloids)
Antitumor agents (doxorubicin, daunorubicin, mitomycin,
bleomycin).
Biopesticides
30. Tropophase and idiophase
In batch culture, most secondary metabolite processes
have a distinct growth phase (trophophase) followed by a
production phase (idiophase). In other fermentations, the two
phases overlap; the timing depends on
1. The nutritional environment presented to the culture
2. The growth rate or both.
A delay in antibiotic production until after trophophase helps
the producing organism because the microbe is sometimes
sensitive to its own antibiotic during growth.
Resistance mechanisms that develop in producing
microorganisms include:
1. enzymatic modification of the antibiotic,
2. alteration of the cellular target of the antibiotic and
3. decreased uptake of the excreted antibiotic.
31. Directed biosynthesis
The manipulation of the culture media in any development
program often involves the testing of hundreds of additives as
possible limiting precursors of the desired product.
Occasionally, a precursor that increases production of the
secondary metabolite is found.
The precursor may also direct the fermentation towards the
formation of one specific desirable product: this is known as
directed biosynthesis.
Examples of directed biosynthesis include the use of
1. Phenylacetic acid in the fermentation of benzylpenicillin
2. Specific amino acids in the production of actinomycins and
tyrocidins.
Stimulatory precursors include:
1. Methionine, as an inducer in cephalosporin C formation
2. Valine, in tylosin production
3. Tryptophan for ergot-alkaloid production.
32. In many fermentations, however, precursors show no
activity because their syntheses are not rate-limiting.
In screening of additives often revealed dramatic
effects, both stimulatory and inhibitory, of non-
precursor molecules on the production of secondary
metabolites.
These effects are usually due to the interaction of
these compounds with the regulatory mechanisms
existing in the fermentation organism.
Antibiotic biosynthesis ends via the decay of
antibiotic synthetases or because of feedback
inhibition and repression of these enzymes.
33. Because the regulatory mechanisms are genetically
determined, mutations have had a major effect on the
production of secondary metabolites. Indeed, it is the
chief factor responsible for the 100–1000-fold increases
obtained in the production of antibiotics from their initial
discovery to the present time.
These tremendous increases in fermentation productivity
and the resulting decreases in costs have come about
mainly by random mutagenesis and screening for higher-
producing microbial strains.
Mutation has also served to:
(1) shift the proportion of metabolites produced in a
fermentation broth to a more favorable distribution;
(2) elucidate the pathways of secondary metabolism; and
(3) yield new compounds.
34. Modern biotechnology is now over 25 years old.
In addition to recombinant DNA technology, modern microbial
biotechnology encompasses
fermentation,
microbial physiology,
high-throughput screening for novel metabolites,
strain improvement,
bioreactor design
Downstream processing,
cell immobilization (enzyme engineering),
cell fusion,
metabolic engineering,
in vitro mutagenesis (protein
engineering)
directed evolution of enzymes (applied molecular evolution).
Modern microbial biotechnology
35. With the revolutionary exploitation of microbial genetic discoveries in the
1970s, 1980s and 1990s.
The major microbial hosts for production of recombinant proteins are
E. coli,
B. subtilis,
S. cerevisiae,
Pichia pastoris,
Hansenula polymorpha
Aspergillus niger.
The use of recombinant microorganisms provided the techniques and
experience necessary for the successful application of higher
organisms, such as
mammalian and insect cell culture,
transgenic animals and plants as hosts for the production of glycosylated
recombinant proteins.
Recombinant microorganisms
36. The progress in biotechnology has been truly remarkable. Within
four years of the discovery of recombinant DNA technology,
genetically engineered bacteria were making human insulin and
human growth hormone.
This led to an explosion of investment activity in new companies,
mainly dedicated to innovation via genetic approaches.
Newer companies entered the scene in various niches such as
biochemical engineering
downstream processing.
Today, biotechnology in the USA is represented by some
1300 companies with revenues of US$19.6 billion, of which sales
represent US$13.4 billion and approximately 153 000 employees
Progress
37. Canada
The number of biotechnology companies reached 282 in
1998,
employing 10 000 workers and with revenues of
approximately US$1.1 billion.
Japan
biotechnology sales were approximately US$10 billion,
mainly by
established pharmaceutical, food and beverage companies.
European
biotechnology moved rapidly in the 1990s, after years of
lagging behind and, in 1998, 1178 biotechnology companies
existed with 45 000 employees, and revenues of US$3.7
billion.
38. The major thrust of recombinant DNA technology has been in the area of rare
mammalian peptides, such as
hormones,
growth factors,
enzymes,
antibodies and
biological response modifiers.
Among those genetically engineered products that have been approved for use
in the USA are
human insulin,
human growth
hormone,
erythropoietin,
antihemophelia factor,
granulocyte-colony stimulating factor,
Granulocyte macrophage-colony
stimulating factor,
Epidermal growth factor and other growth factors, interleukin-2, -, - and -
interferons, and bovine somatotropin.
39. Vaccines
Vaccine production is another important part of the
new technology.
the first subunit vaccine on the market was that of
hepatitis B virus surface antigen produced in yeast.
The great contribution made by recombinant
vaccines is the elimination of the tragic problems
associated with conventional vaccines.
Through reversion of the attenuated pathogen, some
individuals receiving the conventional vaccine not
only failed to be protected, but also came down with
the disease.
40. Combinatorial biosynthesis
Most microbial biosynthetic pathways are encoded by
clustered genes, which facilitates
the transfer of an entire pathway in a single
manipulation.
in fungi, pathway genes are sometimes clustered,
such as the penicillin genes in Penicillium or the
aflatoxin genes in Aspergillus.
For the discovery of new or modified secondary
products, recombinant DNA techniques are being
used to introduce genes for the synthesis of one
product into producers of other antibiotics or into
non-producing strains (combinatorial biosynthesis).
41. Enzyme production
The production of enzyme by fermentation was an established business
before modern microbial biotechnology.
However, recombinant DNA methodology was so perfectly suited to the
improvement of enzyme production technology that it was almost
immediately used by companies involved in manufacturing enzymes.
Industrial enzymes have now reached an annual market of US$1.6 billion.
Important enzymes are proteases,
lipases,
carbohydrases,
recombinant chymosin for cheese manufacture
recombinant lipase for use in detergents.
Recombinant therapeutic enzymes already have a market value of over
US$2 billion, being used for
thromboses,
gastrointestinal
rheumatic disorders,
metabolic diseases and cancer.
plasminogen activator,
human DNAase and
Cerozyme
42. Agriculture
Industrial microbiology through genetic engineering and its associated
disciplines has brought about a revolution in agriculture.
Two bacteria have had a major influence:
Agrobacterium tumefaciens, a bacterium that normally produces crown gall
tumors on dicotyledonous plants and
Bacillus thuringiensis, an insecticidal bacterium.
The tumor-forming genes of A. tumefaciens are present on its tumor-
inducing (Ti) plasmid, along with genes directing the plant to form opines
(nutritional factors required by the bacterium that it cannot produce by
itself).
The Ti vector has been exceedingly valuable for introducing foreign genes
into dicotyledonous plants for production of transgenic plants.
However, the Ti plasmid is not very successful for transferring genes into
monocotyledonous plants, a problem bypassed by, for example, the
development of a particle acceleration gun, which shoots DNA-coated metal
particles into plant cells.
The activity of the insecticidal bacterium, B. thuringiensis, is caused by its
crystal protein produced during sporulation. Crystals and spores have been
applied to plants for many years to protect them against lepidopteran
insects. B. thuringiensis preparations are highly potent, approximately 300
times more active on a molar basis than synthetic pyrethroids and 80 000
times more active than organophosphate insecticides.
43. In the modern biotechnology era, plants resistant to
insects have been produced by expressing forms of the
B. thuringiensis toxin gene in the plant.
Recently developed bioinsecticides include insect
viruses, such as baculoviruses, that are engineered to
produce arthropod toxins.
Transgenic plants, resistant to herbicides, are also
available, as are virus-resistant plants produced by
expressing viral-coat-protein genes in plants.
Interestingly, chemical pesticides against plant viruses
were never available.
44. Conclusion
Although most of the early promises of biotechnology
have been achieved, major challenges remain. We must use
our brains, technology, drive and dedication to solve the
problems of
evolving diseases (e.g. AIDS),
established diseases (cancer and parasitic infection),
Antibiotic resistance development and
environmental pollution, by converting urban, industrial
and agricultural wastes into resources such as liquid fuel.
These efforts will require continued interaction between
different disciplines, major support by governments and
international agencies, as well as an understanding and
supportive public.
