Chap 18 Antibiotics
I. Introduction

Used as therapeutics to fight microbial infections.

Penicillin was the first major antibiotic from a microbial source to be commercialized
(during WW II).

Also available for agricultural uses (e.g.
feed additives, growth stimulants).

In 2005, worldwide sales of oral
antibiotics totaled $25.0 billion, including
US sales of $8.5 billion. The area also
continues to grow at a significant rate,
with adult and pediatric prescriptions
growing 8.5% and 10.8%, respectively, in
Nature Biotechnology, 2006
2005. The majority of which falls into 5
groups:
Annual sales of therapeutic antibiotics (1999)
Antibiotic
US sales (billion)
Cephalosporin
3.6
Penicillin
1.2
Fluoroquinolones
0.9
Tetracycline
0.5
Macrolides
0.4
Reference: Strohl WR. 1999. Secondary metabolites; antibiotics. In: Flickinger MC, Drew SW
(Eds) Encyclopedia of bioprocess technology: fermentation, biocatalyst and
bioseparations, Vol 5. Wiley, New York, pp 2348-2365

Penicillin is the largest volume category for therapeutic antibiotics (>65,000 tons). The
volume for veterinary use is close to that for human therapeutics, but the value is less
than 10%.
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
Sources of antibiotics:



From 1950s to 1980s: chemists, microbiologists, and chemical engineers play important
roles in the industry. But in the 90s, enzymologists, protein chemists, molecular
biologists and biochemical engineers are deeply involved.

Biotechnology contributes dramatic changes to the ways antibiotics are manufactured
through fermentation yield improvements, recovery processes and final product
purification.
II. Characteristics of antibacterial drugs1
Cell wall synthesis

-lactam drugs:

Penicillin
1

Natural penicillin (Penicillin G and V):

Cloxacillin and floxacillin:

Methicillin, dicloxicillin:
Chap 21 in “Microbiology, A Human Perspective” (Nester et al., 2001)
2

Ampicillin: Broad-spectrum, extended activity against Gram- bacteria such as
Haemophilus influenzae (嗜血桿菌) and E. coli.

Cephalosporins (e.g. cephalexin, cefaclor…): some can be taken orally, some must be
injected because of instability in stomach acid. The later generations are more
effective against Gram- bacteria and less susceptible to -lactamase.

Vancomycin:
used to treat systemic infections and
antibiotic-associated colitis (結腸炎), poorly absorbed from the intestinal tract
administered intravenously.
Protein synthesis

Aminoglycosides




Toxicity (e.g. hearing loss, kidney damage) limits their use.

Resistance can arise from

Neomycin is commonly used in non-prescription antibiotic ointments (軟膏).
Tetracycline

Against Gram+ and Gram- bacteria

Binds to 30S ribosomal subunit

Resistance is generally due to decreased accumulation, either thru decreased
uptake or increased efflux.

Macrolide

Against Gram+ bacteria that are the most common causes of atypical
pneumonia

3


Used to treat patients who are allergic to -lactam drugs.
Chloramphenicol


Against a wide range of bacteria, but generally used only as a last resort for life
threatening infections to avoid rare but lethal side effects.
Nucleic acid synthesis

Fluoroquinolones (ciprofloxacin, ofloxacin)

Against a wide variety of Gram+ and Gram- bacteria.
.

Primarily used for infections caused by Mycobacterium tuberculosis2 and as a
prophylaxis (預防) for patients who have been exposed to bacteria causing
meningitis (腦膜炎).
Folate synthesis (folate is a key ingredient in the biosynthesis of nucleic acids)

Sulfonamides: against Gram+ and Gram- bacteria. Structurally similar to
Cell membrane integrity
 Polymyxin B:
III.Strain Improvement

Productivity and yield can be increased by introduction of mutations using either UV
radiation or a chemical mutagen such as nitrosoguanidine (NTG).

Mutated strains can be selected by different selective approaches.

Today’s screening programs rely on the use of miniaturization, automation and high
throughput screening to process the large numbers necessary for the recognition of
superior strains.
2
結核分枝桿菌
4
Selection environment for strain improvement
Resistance
Possible effect
Toxic metals e.g. Cu, Cd, Hg
Increase in thiols, glutathione (intracellular
reducing agent)
so the cells are more resistant to
heavy metals.
Deoxyglucose
Reduced glucose regulated metabolism
Carbon dioxide
Tolerate high levels CO2 in fermentation
High salt
Tolerate high-salt raw materials
Polypropylene glycol
Tolerate high levels of antifoam

Genetic engineering techniques can now be used to express selected enzymes in the
cells. Using kinetic studies and the measurement of metabolic pools, presumptive ratelimiting enzyme steps can be identified in the biosynthesis of antibiotics. These genes
can be cloned to remove the metabolic bottleneck to increase the yield.

It is important to have a master cell bank to avoid lot-to-lot variation. Because
repeated transfer of high yielding strains can produce subpopulations with lower
productivity, master cell bank can ensure a common lineage to start cellular
propagation.
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IV.

Fermentation
Fed-batch is often used for the final production stage.
Parameters:

The raw materials for fermentation include corn steep (浸泡) liquor, casein (酪蛋白)
hydrolysate, yeast extract, etc. (Table 18.5). Suitable carbohydrates are sucrose,
glucose, or enzyme hydrolyzed corn syrups. Nitrogen can be supplemented from corn
steep liquor.

Oil (e.g. soy oil, peanut oil, etc. depending on local availability) addition helps control
excessive foaming and air hold-up. Alternatively, antifoams can be used to supplement
or replace oil feeding. Control of foaming is important in obtaining the maximum
volumetric output from a fermenter. The final harvest volume should be  80-85% of
the total fermenter capacity.

pH can be controlled by the addition of acid or base. It can also be controlled by the
metabolism. Excess feeding of sugar in some conditions produce acetic acid, which
lowers the pH.

DO is often controlled by the addition of air because pure O2 is costly and safety is a
concern. Several points need to be noted:

DO should be higher than 20% saturation (usually air saturation) at 1.5-2
atmosphere pressure (to increase solubility).

Air flow rate should be high enough to sweep out as much CO2 as possible,
build-up of which could be detrimental.
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Strains and Recovery

Penicillin G and V are obtained from the fermentation of
 Depending on the nature of mycelium the solids can be removed by filtration or
centrifugation.
Appendix
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