microbial production of chemotherapeutic agents

MICROBIAL PRODUCTION OF
CHEMOTHERAPEUTIC AGENTS

 Antibiotic
Antibiotic as originally defined was a chemical
substance produced by various species of
microorganisms that was capable of inhibiting the
growth or kill the microorganism.
 Antimicrobial Agents
Antimicrobial agents is a chemical substance which
inhibit or kill the microorganism.

Introduction
 Antibiotics are a special category of chemotherapeutic
agents that are administered to fight infections, usually
microbial, of humans and animals.
 the classical definition of antibiotics associates it with
secondary metabolite, the term antibiotic today
includes many similar but chemically synthesized
chemotherapeutic compounds also.
 Over 8000 antibiotics are known and several hundreds
discovered yearly.
 Nearly 3000 antibiotically active substances have been
detected in lichens, algae, higher animals and plants.

 Each year about 300 new antibiotically active
substances are detected of which 30-35% are
secondary components from fermentation with
known antibiotics.
 only 123 are currently being produced by
fermentation. In addition, some 50 antibiotics are
produced as semisynthetic antibiotics.
 Three antibiotics, viz., phosphonomycin,
pyrrolnitrin and chloramphenicol are produced
completely synthetically.

Examples of organisms capable of
producing antibiotics

Ideal Characteristics of
antibiotic
 Wide spectrum: it must be active against a
wide range of pathogens
 Prevent the development of resistant forms:
pathogens should not easily gain resistance
to the antibiotic in question
 Selective nature: it must act only against the
target and not the host organism
 Not disturb the normal gut flora when orally
administered

STREPTOMYCIN
 Streptomycin is an aminoglycoside antibiotic
produced by selected strains of Streptomyces
griseus.
 The antibiotic works by inhibiting the
synthesis of DNA and proteins.

CHEMISTRY
 It is basic in nature, with solubility in water at the rate
of 20 g/L.
 It is stable to pH changes.
 It can withstand boiling temperature.
 Being a base, streptomycin is usually produced as
salt, normally of HCl and sulfate.
 One unit of streptomycin is equal to 1 μg of free base.
 Streptomycin is composed of 3 subunits: (i)
aminocyclitol (= streptidine), (ii) L-streptose, and (iii)
Nmethyl- L-glucosamine.

Some streptomycins and their R
groups

USES
 In the treatment of tuberculosis, urinary tract
infection, systemic infection by Gram positive
bacteria
 for bacteria that have gained resistance to
penicillin.
 Non-medical uses include preparation of
selective media
 in cloning experiments
 as laboratory standard for quantitative
analysis of streptomycin.

LIMITATIONS
 The antibiotic exerts a neurotoxic reaction upon
prolonged use.
 It can lead to hearing loss and loss of balance (that
is, it is ototoxic).
 Streptomycin may sometimes damage kidney also.
 The drug may lead to development of
streptomycin-resistant forms. It is therefore
advisable to use the drug along with p-
aminosalicylic acid or isoniazid.
 Dihydrostreptomycin has lesser side effects than
streptomycin.

MODE OF ACTION
 It affect protein synthesis, the target of this
antibiotic is the 30S subunit of the 70S ribosome
of the prokaryotes.
 It strongly inhibits initiation and elongation of
peptide chains.
 It also causes misreading of mRNA thereby
leading to insertion of wrong amino acids
 Finally, under the influence of streptomycin,
some molecules of nucleic acids (e.g., rRNA,
tRNA, and denatured DNA) also act as mRNA
although they ordinarily do not have this
property.

BIOSYNTHESIS OF STREPTOMYCIN
 It follows three pathway.
 Glucose act as sole source of carbon.
 It involves 28 enzymes.
 The final intermediate of the pathway,
streptomycin phosphate, is biologically inactive but
becomes active following removal of the
phosphate group.
 Many organisms synthesize
mannosidostreptomycin before the actual
formation of streptomycin.

 mannosidostreptomycin is degraded by the
organism’s own enzyme
mannosidostreptomycinase to yield streptomycin.
 In fermentation, the concentration of
mannosidostreptomycin can reach up to 40%.
 Biosynthesis of streptomycin is regulated by an
inducer called Factor A.
 The antibiotic is synthesized in the idiophase and
this occurs only after factor A has reached a critical
concentration.

GENERAL PRODUCTION METHOD
a. MICROBIAL STRAIN
 Streptomyces griseus is used for production of
streptomycin
 Walksman’s discovered the species and till now it
has been using
 The productivity of Streptomyces griseus has
increased by over 100 fold.
 Classical mutation programs are used for the
improvement of the strain

b. CULTURE MEDIUM
 Glucose is the carbon source of choice, because it is precursor
for streptomycin.
 The preferred nitrogen source is soybean flour meal.
 Minerals are automatically inclusive because of the complex
nature of the medium.

PRODUCTION
 The inoculum is built up in a stepwise manner at 27°C.
 The process starts with the plate culturing of
lyophilized spore cultures in soy flour agar medium.
 Incubation is done at 27°C for 2-3 weeks.
 The spores are then transferred to shaker flask.
 After growth for some time the whole is again
transferred to propagator for biomass build up.
 The medium is sterilized as usual.
 The fermenter is inoculated at the rate of 5-10%
vol/vol.The process is aerobic.

 Inadequate supply of air (O2) leads to accumulation of
lactate and pyruvate, which is undesirable.
 The pH is maintained at around 7 and fermentation carried
out at 27°C.
 The fermentation is triphasic.Trophophase lasts for 24 hrs.
 The pH increases preferential utilization of soybean meal.
 Growth and concomitant accumulation of Factor A also
rapid.
 Idiophase lasts for 2-7 days during which streptomycin is
rapidly synthesized. Glucose utilization is very rapid.
 The third phase marks the cessation of antibiotic synthesis.
 Cells begin to lyse, and pH rises due to NH3 liberation.
 Harvesting is done before the third phase commences.
 The yield is about 1200 μg/ml.

RECOVERY
 The broth (beer) is filtered in rotary vacuum filter to
remove mycelia.
 Water is added to the liquor in the ratio 1:1 and passed
through adsorption column.
 Through the same column, EDTA solution is passed to
remove metal ions.
 The adsorbed, pure streptomycin is eluted from the
column with 2.5 N H2SO4.
 Further processing entails decolorizing with carbon,
antigen removal by filtration, concentration, and
drying.
 The final product is either sulfate- or hydrochloride salt
of streptomycin.
 The purity will be of the order of 98%.

Introduction
 Tetracyclines are a group of broad-spectrum antibiotics
 They can be prepared microbiologically as well as
chemically
 Tetracycline has low toxicity and good oral absorption.
 It is bacteriostatic and requires high dosage.
 Examples ofTetracycline and organism

Uses
 This antibiotic is used in the treatment of
shigellosis, salmonellosis, typhoid fever,
brucellosis, etc.
 It is also used in feed to eliminate parasites (and
thus help weight gain in animals).
 The antibiotic also finds use in the preservation of
fish (the ice in which the fish is kept is treated with
tetracycline).

PRODUCTION OF TETRACYCLINE
 Tetracycline can be produced chemically as well as
microbiologically.
 The microbial production of all tetracyclines is
similar.
 Chlortetracycline production, however, is
comparatively simpler than the production of
other tetracyclines.
 In particular, production of tetracycline is very
sensitive to chloride content in the medium: it
leads to the production of chlortetracycline rather
than the tetracycline

FERMENTATION
 For trade fermentations, UV mutants of
Streptomyces aureofaciens are used.
 The organism comes in the form of lyophilized
spores.
 The inoculum preparation requires several stages.
 Starting from the spores dried on sand or lyophil
vials, one or more shake flask stages may be used
and then one or two inoculum tank stages.
 The sporulation medium, inoculum build-up
medium and the production medium have different
compositions

 During inoculum build-up, the organism remains in the
shake-flask for 24 hours at 28°C.
 The final propagator uses medium of the same
composition.
 About 5% inoculum is added and propagation carried
out for 19-24 hours at pH 5.2-6.2.
 The main fermenter receives 2-10% of inoculum from
the final propagator.
 The fermenter has a nominal capacity of 5000 to 15000
gallons.
 Fermentation is carried out in sterilized medium (121°C
for 12 hour).
 The main fermentation runs for 60-65 hours at 28°C.

 The pH is around 5.8-6.
 It is a submerged fermentation requiring 0.5-2
vol/vol/min of aeration.
 Agitation is carried out with mechanical agitators.
 Lard is used as antifoam.
 Glucose is generally not used in the main
fermentation as this exerts catabolite repression.
 The yield is around 15000 units per ml.

microbial production of chemotherapeutic agents

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