How we produce antibiotics from different living organisms

1. Antibiotic production
Dr. Muhammad Tahir
Associate Professor
Department of Biotechnology
CUI-Vehari

2. Antibiotic production
• Antibiotic Production
• The first antibiotic was discovered in 1896 by Ernest Duchesne
and "rediscovered" by Alexander Fleming in 1928 from the
filamentous fungus Penicilium notatum.
• Antimicrobial agents are; antibacterials, antivirals, antifungal
and antiparasitic

3. • less than 1% of antimicrobial agents have any medical or commercial value
• Penicillin
• Why are human cells not affected by Penicillin? There is a constant search for new
antibiotics the most-prescribed drugs big business can take 15 years to land in the
market
Identify the useful antibiotics
• Screening
• Isolates of a large number of microorganisms are cultured
• Tested for production of diffusible products which inhibit the growth of test organisms
• Tested for their selective toxicities and therapeutic activities
• Examined and possibly modified
• A more modern version - Rational Design Program
• Finding new natural products
• Inhibit specific targets (e.g. a particular step of a metabolic pathway)

4. Industrial production techniques
• Process of fermentation
• Secondary metabolites
• The population size must be controlled very carefully to ensure
that maximum yield is obtained before the cells die
• Extracted and purified to a crystalline product

5. Strains used for production
• Microorganisms used in fermentation are rarely identical to the
wild type
• Genetically modified
• To yield the maximum amounts of antibiotics;
• Selection and further reproduction of the higher yielding strains
over many generations can raise yields by 20-fold or more.
• Gene amplification
• Via vectors such as plasmids

10. How penicillin amount can be increased
• When penicillin was first made at the end of the second world
war using the fungus Penicilium notatum, the process made 1
mg dm-3. (1 mg/Liter)
• Today, using a different species (P. chrysogenum) and a better
extraction procedures the yield is 50 g dm-3. (50 g/Liter)

11. Media for penicillin production from
Penicillium chrysogenum
• Penicillin is produced by the fungus Penicillium chrysogenum which
requires lactose, other sugars, and a source of nitrogen (in this case a
yeast extract) in the medium to grow well.
• Like all antibiotics, penicillin is a secondary metabolite, so is only produced
in the stationary phase.
What sort of fermenter does it require?
• It requires a batch fermenter, and a fed-batch process is normally used to
prolong the stationary period and so increase production.
• In contrast with a continuous process, a batch process does not deliver its
product continuously, instead delivering it in discrete amounts. A batch process
is a process in which the product comes out in groups and not continuously.
• In fed-batch operations, intermittent or continuous feeding of nutrients is used
to supplement the reactor contents and provide control over the substrate
concentration

13. Three things should be kept in mind
before choosing the raw materials
1. An abundant growth of mycelium
2. Maximum accumulation of penicillin
3. Ease of extraction and purification of the antibiotics
Raw materials: 1. Carbon source 2. Nitrogen source 3. Mineral source 4.
Corn – steep liquor 5. Precursor metabolites
• Extraction and Purification
• Removal of mycelium: A rotary vacuum filter is used to remove mycelium
by filtration
• To extract penicillin, the pH of the filtrate is adjusted to 2-2.5 then antibiotic
is extracted back into an aqueous buffer at pH 7-7.5
• The resulting solution is again acidified and re-extracted with an organic
solvent.

14. Treatment of crude extract:
• Involves the formation of an appropriate penicillin salt followed
by charcoal treatment and lastly crystallization

17. Few primary factors
• Reactor size:
• Optimum rate of production
• Reactor configuration: Mechanical agitation
Mode of operation
• Fed batch or continuous Condition inside the reactor:
• Temperature, pH etc
Economic Requirements:
• Easy to operate aseptically
• Reasonably flexible regarding process requirements
• Lower power consumption
• Stable under fluctuating conditions
• Cheap, robust, simple

18. Downstream processing
• Downstream processing is relatively easy
• Since penicillin is secreted into the medium (to kill other cells)
• So there is no need to break open the fungal cells
• However, the product needs to be very pure, since it being used
as a therapeutic medical drug
• So it is dissolved and then precipitated as a potassium salt to
separate it from other substances in the medium

21. Modification in penicillin
• The resulting penicillin (called penicillin G) can be;
• Chemically and enzymatically modified to make a variety of
penicillins with slightly different properties
• These semi-synthetic penicillins include penicillin V, penicillin O,
ampicillin and amoxycillin.

22. Streptomycin Production
• Streptomycin was isolated by Waksman in 1944, and its
activity against M. tuberculosis ensured its use as a primary
drug in the treatment of tuberculosis.
• Streptomycin also shows activity against other types of bacteria,
for example against various Gram-negative bacteria and some
strains of staphylococci.

23. Streptomycin Strain
• Streptomyces griseus
• Three phases in the fermentation
1. Mycelial growth
2. Streptomycin production without production of new mycelium
3. Autolysis of mycelium with a rise of pH

24. Phases during fermentation of
streptomycin
• PHASE 1: Rapid growth producing mycelial biomasss. Little
production of Streptomycin is obtained.
• PHASE 2: Low production of mycelium. Streptomycin accumulates
in the medium.
• PHASE 3: Process has completed.
• Finally the mycelium is separated by filtration and antibiotic
recovered.
• Proteolytic activity of the microbe releases NH3 to the medium from
the soybean meal, causing a rise in pH
• The glucose and NH3 released are consumed during this phase.
• The pH remains fairly constant-between 7.6 and 9.0

27. Strain improvement
• There are three major applications of genetic engineering technology to
antibiotic production;
• Strain improvement programs;
• Introduction of genes to produce novel antibiotics; and
• The engineering of microbial strains and enzymes involved in the production
process.
• The growing wealth of knowledge of biosynthetic pathways is allowing a more
rational approach to the genetic manipulation of the producing organisms, and
is also suggesting new approaches to combining biosynthetic pathways from
different organisms either to optimise the production of known antibiotics or to
propose construction of hybrid molecules with potentially improved or novel
characteristics.
• In addition, there continues to be a hope that some of the chemical
manipulations carried out on core molecular structures can be replaced by new
enzymatic approaches to production of these desirable products.

28. Conventional strain improvement
• Strain improvement programmes have been either experimental in nature, i.e. mutation
and selection of organisms for improved production of an antibiotic, or more recently
have been directed by knowledge of the pathways involved in the biosynthetic process.
The challenges in such strain improvement programmes include:
1. Work to enhance production from already engineered strains that are close to the
limits of their biosynthetic capacity
2. Maintenance of these production levels in an industrial processing environment,
where reversion to low levels of production frequently occurs;
3. Adaptation to cheaper sources of raw materials.
• In these programmes, spores of the producing organism are exposed to a variety of
mutagenic agents, either individually or in combination.
Physical agents (UV-irradiation, X-rays, γ –rays)
Chemical agents (Nitrogen mustards, N-methyl-N-nitro-N-nitroso guanidine)
• After treatment, the spores are allowed to germinate and give rise to single colonies.
• These are then tested for antibiotic production and pigment formation.

29. Conventional strain improvement
• In addition, the isolates are tested for growth and sporulation ability.
• Isolates exhibiting poor characteristics are discarded.
• This process requires that a large number of single colony isolates are tested and
suitable screening and analytical methods have to be developed to allow a
healthy judgement to be made about strains selected to go forward for testing on
a larger scale and with the media components used in the production process.
• Also, it is possible to generate desirable characteristics by back crossing different
strains and repeating the selection process.
• Some of the improved strain characteristics that have been selected for by strain
improvement programs include
1. Cultures that grow as pellets rather than filaments
2. Cultures that have lost pigmentation
3. Elimination of side products

30. Genetic engineering
• Manipulation of antibiotic-producing stains with the sequencing of
the genomes of antibiotic producers
• Or at least the sequencing of the gene clusters responsible for
antibiotic biosynthesis.
• With the discovery that many of the biosynthetic genes for a number
of different antibiotic families are clustered on the chromosomes of
the producing organisms, and are regulated together,
• It has become clear that manipulating these genes in a systematic
way might lead to both production improvements and to the
production of novel antibiotics.

31. Genetic engineering
• This is clearly the case with the polyketide gene clusters responsible
for macrolide production.
• The oxidation and dehydration processes that occur stepwise in the
biosynthesis of these molecules can be manipulated by the
appropriate deletion or insertion of genes into the biosynthetic
cassettes.
• In addition, genetic analysis of the higher yielding penicillin and
cephalosporin strains has shown that part of the productivity increase
can be explained by duplication of gene cluster on the same or
different chromosomes of the original low-yielding strains.

32. Genetic engineering
• A challenging area for future exploitation is the creation of hybrid antibiotics by
inserting genes from different organisms.
• A few simple examples have been reported in the anthracycline antibiotic series,
• But the challenge is much greater because of the existing, and difficult to change,
substrate specificity of the biosynthetic enzymes.
• To engineer a strain directly capable of making solvent extractable
cephalosporins, such as cephalosporin G or V, by adding aromatic acetic acids,
such as phenylacetic or phenoxyacetic acid, onto the cephalosporin nucleus
using a combination of the enzymes from the penicillin V and the cephalosporin C
biosynthetic pathway
• Despite considerable effort this has not yet been accomplished.
• Another challenge that still has to be met is the alternative of introducing the
expandase enzyme from Cephalosporium into Penicillium, again leading to direct
production of solvent extractable cephalosporins.