It describes the application of microbes in medical biotechnology

1. Chapter 6
Application of microbes in medical
biotechnology

2. 6.1. Antibiotics
 Antibiotic: A substance produced by a
microbe that, in small amounts, kill or
inhibits another microbe
 Antibiotics
 Typically secondary metabolites
 Most antibiotics in clinical use are
produced by filamentous fungi or
actinomycetes
 Still discovered by laboratory screening
Microbes are obtained from nature in
pure culture
Assayed for products that inhibit growth
of test bacteria

3. Examples of organisms capable of
producing antibiotics

4. IdealCharacteristicsof 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

5. I. Isolation
Sterile glass spreader
Colonies of
Streptomyces
species
Nonproducing
organisms
Zones of
growth inhibition
Producing
organisms
Spread a soil
dilution on a plate
of selective medium
Incubation
Overlay with an
indicator organism
Incubate

6. Antibiotics: Isolation, Yield, and
Purification
 Cross-streak method
◦ Used to test new microbial isolates for
antibiotic production
◦ Most isolates produce known antibiotics
◦ Most antibiotics fail toxicity and
therapeutic tests in animals
◦ Time and cost of developing a new
antibiotic is approximately 15 years and $1
billion
 Involves clinical trials and U.S. FDA approval
 Antibiotic purification and extraction
often involves elaborate methods

7. II. Testing Activity Spectrum
Streak antibiotic producer
across one side of plate
Incubate to permit growth
and antibiotic production
Cross-streak with test organisms
Incubate to permit
test organisms to grow
Antibiotic diffuses
into agar
Streptomyces cell mass
Growth of test organism
Inhibition zones where
sensitive test organisms
did not grow
Isolation and screening of
antibiotic producers.

8. Industrial Production of Penicillins
and Tetracyclines
 Penicillins are -lactam antibiotics
◦ Natural and biosynthetic penicillins
◦ Semisynthetic penicillins
 Broad spectrum of activity
 Penicillin production is typical of a
secondary metabolite
◦ Production only begins after near-exhaustion
of carbon source
◦ High levels of glucose repress penicillin
production

9. Add
precursor I
Add
precursor II Add
precursor
III
Chemical or
enzymatic
treatment
of penicillin G
Add side
chains
chemically
6-Aminopenicillanic acid
(for example,
penicillin G)
(for example, ampicillin,
amoxycillin, methicillin)
Penicillin
fermentation
Biosynthetic
penicillin I
Biosynthetic
penicillin II
Biosynthetic
penicillin III
Semisynthetic penicillins
Natural penicillins
Industrial production of penicillins.

10. Glucose
feeding
Nitrogen
feeding
Cells
Lactose
Ammonia
Penicillin
Fermentation time (h)
Biomass
(g/liter),
carbohydrate,
ammonia,
penicillin
(g/liter

10)
100
90
80
70
60
50
40
30
20
10
0
20 40 60 80 100 120 140
Kinetics of
the penicillin
fermentation
with
Penicillium
chrysogenu
m.

11. Industrial Production of Penicillins and
Tetracyclines
 Biosynthesis of tetracycline has a large
number of enzymatic steps
◦ More than 72 intermediates
◦ More than 300 genes involved!
◦ Complex biosynthetic regulation

12. Medium
Chlortetracycline
Inoculum
(spores on
agar slant or in
sterile soil)
Agar plates
Shake flask
Prefermentor
Fermentor
Antibiotic
purification from
broth after cell
removal
2% Meat extract; 0.05%
asparagine; 1% glucose;
0.5% K2HPO4; 1.3% agar
2% Corn steep liquor;
3% sucrose; 0.5% CaCO3
Same as for shake culture
1% Sucrose; 1% corn steep
liquor; 0.2% (NH4)2HPO4;
0.1% CaCO3;
0.025% MgSO4
0.005% ZnSO4
0.00033% and each of
CuSO4, MnCl2
Spores as
inoculum
24 h
19–24 h
pH 5.2–6.2
60–65 h
pH 5.8–6.0
Growth in
optimal medium
Medium mimics
production
medium
Production
medium, no
glucose, low
phosphate
Production scheme for
chlortetracycline using
Streptomyces aureofaciens.

13. Mode of action of antibiotics

14. 6.2 Production of vitamins
 Production of vitamins is second only
to antibiotics in terms of total
pharmaceutical sales
◦ Vitamin B12 produced exclusively by
microorganisms
 Deficiency results in pernicious anemia
 Cobalt is present in B12
◦ Riboflavin can also be produced by
microbes

15. A) Vitamin B12 Production

16. Why is Vitamin B12 so Necessary?
 No innate production by human
metabolism, has to be consumed from
outside source
 Major role in DNA synthesis and regulation
 Deficiency leads to:
- pernicious anemia
- fatigue
- depression
- poor memory,
etc.

17. Microbial synthesis possibilities
 Possible Microorganisms to be
utilized:
1. Propionibacterium freudenreichii
2. Propionibacterium shermanii
3. Streptomyces griseus
4. Pseudomonas denitrificans
 Organisms (1),(2) & (3) are anaerobic
while (4) follows aerobic pathway

18. Process Requisites
 Bacteria: Pseudomonas denitrificans
 Substrate: Glucose, Corn steep liquor, Beet
molasses, Maltose syrup
 Temperature: 30oC
 pH: 6.5-7.5
 Duration of fermentation: 2-3 days in pre
culture and 7 days in actual production
fermenter
 Aeration
 Reactor type: Batch(shake flask) on
laboratory scale & Fedbatch on commercial
scale

19. B) Microbial production Riboflavin or
Vitamin B2 Glucose
50% by biotransformation
using Bacillus pumulis
D-ribose
20% production by Chemical synthesis
Riboflavin
1/3rd production by
direct fermentation
Acetone butanol fermentation
Clostridium acetobutylicum
C. butylicum riboflavin as
by product
Ashbya gossypii
Candida famata
Bacillus subtillis (genetically modified)

21. C) Niacin
 NAD and / or NADP are essential for many
oxidative/reductive biochemical reactions
 the niacin synthesized by Streptococcus thermophilus
and Lactobacillus delbrueckii subsp bulgaricus may
originate from nicotinamide fraction arising during
formation of NAD and / or NADP.
 Nicotinic acid is derived by a few bacteria from the
metabolism or breakdown of tryptophan, a pathway which
is dependent on the availability of certain vitamins, e.g,
thiamine, riboflavin and vitamin B6 to activate the
required enzymes.
 As S. thermophilus and lactobacillus delbrueckii subsp
bulgaricus utilizes these vitamins and tryptophan does not
accumulate during yoghurst production, it is possible that
these organism use the vitamins for the synthesis of
niacin.

22. Niacin (Vitamin 3)

23. Pathway for
niacin
production

24. D) Ascorbic acid or Vitamin C
Precursor for its chemical synthesis can be obtained
by biological methods
 pharmaceutical industry (50%),
 food (25%), and
 beverages (15%).
The food and beverage industry predominantly exploits
the antioxidant capacity of L-ascorbic acid to
extend durability, prevent discoloration, and to
protect flavor and nutrient contents of their
products.

25. D-glucose (200g)
Submerged bioreactor fermentation
D-sorbitol
sorbitol dehydrogenase
L-Sorbose
chemical oxidation
2 keto L gulonic acid
Enol form of
2 keto L gulonic acid
acid treatment
L-ASCORBIC ACID (100g)
Acetobacter xylinum,
A,suboxydans
Glucuronic acid
Gluconolactone
L-Gluconolactone
L-ASCORBIC ACID
L-Gluconolactone
dehydrogenase
Reichstein Grussner synthesis
Erwinia sp.
Acetobacter sp.
Gluconobacter sp.
2,5-diketogluconic acid
2-keto L-gluconic acid
L-ASCORBIC ACID
Corynebacterium sp.
2,5-diketogluconic acid
reductase
2,5-diketogluconic acid
Reductase of
Corynebacterium into
Erwinia herbicola
Cloning of gene
Bacillus
megaterium