1. Pharmaceutical Production of Antibiotics

2. Pharmaceutical Products and the Microorganisms That Make Them
Uses microorganisms, typically grown on a large scale, to produce products or carry out chemical transformation
Originated with fermentation processes
Major organisms used are fungi
Classic methods are used to select for high-yielding microbial variants

3. Properties of the Microorganisms
Properties of a useful industrial microbe include
Produces spores or can be easily inoculated
Grows rapidly on a large scale in inexpensive medium
Produces desired product quickly
Should not be pathogenic
Amenable to genetic manipulation

4. Industrial Products Produced by Microorganisms
Microbial products of industrial interest include
Microbial cells
Enzymes
Antibiotics, steroids, alkaloids
Food additives
Commodity chemicals
Inexpensive chemicals produced in bulk
Include ethanol, citric acid, and many others

5. Production and Scale Primary metabolite Secondary metabolite
Produced during exponential growth
Example: alcohol
Secondary metabolite
Produced during stationary phase

6. Production and Scale Secondary metabolites Not essential for growth
Formation depends on growth conditions
Produced as a group of related compounds
Often significantly overproduced
Often produced by spore-forming microbes during sporulation

7. Primary metabolite Secondary metabolite Alcohol Penicillin Cells
Alcohol, sugar, or cell number
Penicillin, sugar, or cell number
Sugar
Cells
Sugar
Figure 15.1 Contrast between production of primary and secondary metabolites.
Penicillin
Time
Time

8. Production and Scale
Secondary metabolites are often large organic molecules that require a large number of specific enzymatic steps for production
Synthesis of tetracycline requires at least 72 separate enzymatic steps
Starting materials arise from major biosynthetic pathways

9. Production and Scale
Fermentor is where the microbiology process takes place
Any large-scale reaction is referred to as a fermentation
Most are aerobic processes
Fermentors vary in size from 5 to 500,000 liters
Aerobic and anaerobic fermentors
Large-scale fermentors are almost always stainless steel
Impellers and spargers supply oxygen

10. Fermenters are large vessels that monitor and react to changes allowing ideal conditions for bacterial growth to be maintained by;
Supplying oxygen for respiration
Stirring the suspension to maintain even temperature, oxygen and nutrient distribution
Water cooling system to remove excess heat produced during respiration
Monitoring pH etc to react to any changes

11. Figure 15.2 Fermentors. Motor Steam pH pH controller
Acid–base reservoir and pump
Sterile seal
Viewing port
Filter
Exhaust
Impeller (mixing)
External cooling water out
Cooling jacket
External cooling water in
Culture broth
Figure 15.2 Fermentors.
Sparger (high- pressure air for aeration)
Steam in
Sterile air
Valve
Harvest

12. Large scale microbe production
Large scale production of microorganisms has many problems
The bacteria need to be
Kept at the right temperature
Have nutrients supplied ie food, oxygen
Have waste products removed ie waste and carbon dioxide

13. Antibiotics: Isolation, Yield, and Purification
Compounds that kill or inhibit the growth of other microbes
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

14. Antibiotics: Isolation, Yield, and Purification

15. The classical method for searching for antibiotics
By random search in the soil
The soil is a vast repository of microorganisms and it is to the soil that search is turned when antibiotics are being sought

16. The primary screening
Several methods have been employed in primary screening
The crowded plate
The cross-streak method

17. The crowded plate
A heavy aqueous suspension (1:10; 1:100) of soil is plated on agar.
Organisms showing clear zones around themselves are isolated for further study.
This method has the disadvantage that slow- growing antibiotic-producing organisms such as actinomycetes are usually over grown and are therefore hardly isolated.

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

19. The cross-streak method
This method is used for testing individual isolates, especially actinomycetes which may be obtained from soil without any previous knowledge of their antibiotic- producing potential.
The purified isolate is streaked across the upper third of plate containing a medium which supports its growth as well as that of the test organisms.
A variety of media may be used for streaking the antibiotic producer.
It is allowed to grow for up to seven days, in which time any antibiotic produced would have diffused a considerable distance from the streak.
Test organisms are streaked at right angles to the original isolates and the extent of the inhibition of the various test organisms observed.

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

21. Secondary screening
Organisms showing suitably wide zones of clearing against selected target organisms are cultivated in broth culture in shake flasks using components of the solid medium in which the isolate grew best.
Secondary screening is aimed at eliminating at an early stage any antibiotic which does not appear promising either by virtue of low activity, other undesirable properties or because it has been discovered previously

22. Industrial Production of Penicillins
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

23. Penicillin Fermenters are used to produce large amounts of penicillin
The penicillin mould requires a lot of oxygen
The mould grows rapidly but does not produce penicillin until most of the nutrients are used up
Enough food is provided to let the mould grow then supplies are limited to maximize penicillin production
There is therefore a lag between stating the process and penicillin production

24. Magic Bullet
Penicillin and other beta-lactam antibiotics (named for an unusual, highly reactive lactam ring) are very efficient and have few side effects (apart from allergic reactions in some people.
As an additional advantage, the enzymes attacked by penicillin are found on the outside of the cytoplasmic membrane that surrounds the bacterial cell, so the drugs can attack directly without having to cross this strong barrier

25. Penicillium
The name Penicillium comes from penicillus = brush, and this is based on the  brush-like appearance of the fruiting structures

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

28. Special fermenter Conditions for the production of Penicillin
Most penicillins form filamentous broths that are pseudoplastic in nature. This means they can be difficult to mix due to their high (and not constant) viscosity
Penicillin is an aerobic organism; therefore the rate of oxygen supply is critical to the fermentation
The optimum pH for penicillin growth is Thus the reactor must maintain pH efficiently (this is frequently done by addition of NaOH).
Strain Stability problems do exist and careful strain maintenance is required.
Biomass doubling is about 6h

29. Composition of early media % corn steep liquor (cotton seeds, peanut,
CULTURE MEDIUM
Composition of early media %
corn steep liquor (cotton seeds, peanut,
Linseed or soybean meals) 2-4
lactose, glucose or beet molasses 2-4
CaCO3 or phosphates (buffer)
precursor
Catabolite repression of the enzymes responsible for penicillin biosynthesis occurs in high concentrations of glucose.
Use of precursors to increase penicillin yield.  benzyl penicillin (penicillin G) is desired, phenylacetic acid is added

30. Use of precursors:

32. Production
It requires a batch fermenter, process is normally used to prolong the stationary period and so increase production
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
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

33. Penicillin G
Penicillinase (E.coli)
6 - APA
Side Chain Modification
Amoxycillin
AUGMENTIN
b-lactamase
resistant
Clavulanic acid

34. 6-Aminopenicillanic Acid (6-APA)
6-APA: Raw material for production of new semisynthetic penicillins (amoxycillin and ampicillin)
Fewer side effects
Diminished toxicity
Greater selectivity against pathogens
Broader antimicrobial range including G- -ve
Improved pharmacological properties
Gastric acid stability & oral absorbability
Resistance to beta-lactamases

35. 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

36. Penicillin G
Penicillin G is not stable in the presence of acid (it is therefore said to be acid-labile).
Since our stomach has a lot of hydrochloric acid in it (the pH can be around 2.0), if we were to ingest penicillin G, the compound would be destroyed in our stomach before it could be absorbed into the bloodstream
penicillin G must be taken by intramuscular injection - to get the compound in our bloodstream.
Many of the semi-synthetic penicillins can be taken orally.

37. Use of Biotechnology in Antibiotic improvement

38. Metabolic engineering of bacteria
Increasing biological production of small molecules
Random screening for overproducing strains (genome shuffling)
Rational engineering of pathways

39. Increasing production of antibiotics -traditional methods
Obtain organism that produces a specific compound--Penicillium mold originally made micrograms per liter of culture
Randomly mutagenize the organism and screen for increased production, repeat using top producing organism
Outcome: grams of penicillum per liter of culture (1000-fold increase in production)

40. Advance Method
An alternative to simple random mutagenesis: genome shuffling
Gene shuffling is the process of recombining the starting pool of sequences to generate new gene- sequences that subsequently can be screened for particular desired characteristics.
The shuffling advantage: simultaneous recombination of entire genomes (breeding) with multiple parents

42. Increasing production of a biological compound: rational design
1)Increase production of a naturally produced commercial compound
Modify existing genes
2) Obtain a new organism that can convert an existing compound into a commercial compound
Introduce new genes

43. Method for introducing DNA
Transformation (spontaneous)
Transformation (chemical, electroporation)
Conjugation
Method for replicating DNA
Plasmid replicon
Integration into chromosome (homologous recombination)