Biotechnology Annual Review 8, 227-265(2002)
Antibiotics
V. Běhal
Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague,
Czech Republic.
Abstract. The chapter informes about different types of antibiotics, their structure, biosynthesis and their
regulation. Industrial cultivation and isolation of antibiotics is described in the chapter. Search for
microorganisms producing antibiotics and preparation of high-producing strains is described. Resistance
against antibiotics in producing microorganisms and pathogens is discussed.
Keywords: antibiotics, secondary metabolites, overproduction, biological activity, regulation of
production, resistance.
Introduction
Antibiotics as representatives of biologically active substances from microorganisms
Antibiotics are defined as microbial products that inhibit growth of other
microorganisms. After the antibiotic effect of penicillin had been observed by Fleming,
a number of other antibiotics were discovered. The main producers are soil
microorganisms as actinomycetes [1], moulds anf fungi (Fig. 1). New antibiotics being
searched for the microorganisms were found to produce a broad spectrum of compounds
having various effects on living organisms. Some of them have occupied a weighty
position as medicines and agricultural drugs and for animal health. One microorganism
can produce several compounds with different biological activity (staurosporine) [2]
and, on the contrary, one compound can be produced by several microorganisms.
Besides to traditional antibiotics, compounds with different biological activities are
synthesized by various microorganisms: coccidiostatics used in poultry farming,
antiparasitic compounds with a broad spectrum of the activity against nematodes and
arthropods, substances with the antitumor activity, immunosuppressors, thrombolytics
(staphylokinase), herbicides, pesticides, compounds affecting blood pressure, etc [3].
For medicine are important enzyme inhibitors synthesized by microorganisms [4].
They are used as inhibitors of enzymes produced by resistant strains that decompose the
antibiotic during application of antibiotics. These enzyme inhibitors can be also used for
inhibition of undesirable enzyme activities in human metabolism that cause some
illnesses. Many enzyme inhibitors are protease inhibitors, variously active against
pepsin, papain, trypsin, chymotrypsin, catepsin, elastase, renin, etc. Inhibitors of
glucosidases, cyclic AMP phosphodiesterase, different carbohydrases, esterases,
kinases, phosphatases, etc. have been isolated from microorganisms. The enzyme
inhibitors that participate in the biosynthesis of cholesterol and fat are also used in
medicine.
Several thousands of compounds having different biological activities have so far
been listed and new compounds are still isolated from microorganisms. There is a
widespread acceptance that microorganisms are an unlimited source of new substances
with many potential therapeutic applications. A great number of those compounds,
however, are toxic and thus cannot be used for human and veterinary therapy.
Role of antibiotics in producing microorganisms
Antibiotics are the typical secondary metabolites produced by microorganisms.
Secondary metabolites are meant products of microorganisms (also plants) which are
not essential for basic metabolic processes such as reproduction and growth [5]. On the
other hand, in the case of many secondary compounds, pieces of evidence of their role
in the metabolism of the producer have been brought. These compounds often function
as the so-called signal molecules, used to control the producer’s metabolism. One of the
functions attributed to antibiotics is a suppression of the competing microorganisms in
the environment. Thus the antibiotic-producing microorganisms have an advantage in
competing for nutrients with the other microorganisms but antibiotic activity is only one
from many other biological activities of secondary microbial products. However, the
function of antibiotics in the environment can be observed only with difficulty.
Use of antibiotics in human, veterinary and plant medicine
Antibiotics are very often used in medicine for suppression of pathogenic
bacteria, fungi and viral diseases. Their use marked a revolution in medicine, saved
millions of lives and helped reduce some, rather frequent diseases such as tuberculosis.
An efficient, antiprotozoan antibiotic, however, has not yet been discovered.
Antibacterial antibiotics are sometimes used in the case of viral diseases to protect the
weakened macroorganism against a subsequent bacterial infection. As mentioned in the
introductory part, some antibiotics are also used as cancerostatics or for curing some
other illnesses.
In a similar way as in human medicine, antibiotics are also employed in veterinary
medicine. Besides, antibiotics are added to various feeding mixtures used in poultry and
animal farming to keep the animals in good health. If the antibiotics are used, higher
farming yields are often reached. However, the administration of antibiotics should be
stopped a certain time before the animal is slaughtered and the meat consumed since the
residues of antibiotics should not enter the human diet. To avoid the production of
strains resistant to the antibiotics used in human medicine, special antibiotics allowed
to be employed in veterinary medicine and animal production have been singled out and
are no more used in human medicine (chlortetracycline, bacitracin, tylosin, etc.).
Side effects of antibiotics.
In addition to their positive effects, antibiotics can also have negative effects.
Besides various allergies linked with the use of antibiotics, the human organism can
sometimes suffer a damage when treated with them. Sometimes toxic compounds can be
formed when antibiotics are transformed in the organism. Tetracyclines, that form
complexes with calcium, can, for example, inflict damage on the formation of tooth
enamel in children, on the condition they are frequently used during the period of teeth
growth. A number of newly discovered antibiotics cannot be used for therapy because of
their excessive toxicity. Fortunately, the first antibiotic to be used on a massive scale,
penicillin, has relatively moderate side effects on the human organism.
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Biosynthesis
In spite of variety of their structures, antibiotics are synthesized from simple
building units amino acids, acetate, propionate, sugars, nucleotides which are used in
living organisms for the biosynthesis of cellular structures. According to their structure
and type of biosynthesis, antibiotics are classified to form several groups.
Peptide and peptide-derived antibiotics
Peptides
Microorganisms produce a number of peptides that have the biological activity.
In contrast to biologically active peptides of higher organisms, where they function as
hormones, the function of microbial peptides in microorganisms is not known. They are
included in the group of secondary metabolites. They differ from the biologically active
peptides of higher organisms in having often D-amino acids in their molecules. Besides
their antibiotic activity, another interesting feature of the peptide antibiotics is the fact
that they are not synthesized on ribosomes, as other peptides, but on enzyme complexes
called peptide synthetases [6,7].
Chemical structure. Amino acids linked by the peptide bond form the basal structure of
any peptide antibiotic. The peptide chain is often cyclic or branched. In addition to Lamino acids, other compounds can also be present in the molecule, such as D-amino
acids, organic acids, pyrimidines and sugar molecules. Numbers of derivatives are
known to exist in the case of some peptide antibiotics, that differ in both amino acid
substitutions and substituents bound to the amino acids.
The linear molecule of gramicidin A and the cyclic molecule of gramicidin S belong
to the structurally simplest peptide antibiotics. Bacitracins are an example of cyclic
peptides having a side chain (Fig. 2). In the molecule of bleomycins, the sugars Lglucose and 3-O-carbamoyl-D-mannose are found. Peptide antibiotics containing an
atom of iron or phosphorus in the molecule have also been isolated. If two molecules of
cysteine are present in the peptide antibiotic, they are linked by a sulfide bridge.
Another cyclic polypeptide (heptapeptide) is iturin, an antifungal antibiotic, produced by
Bacillus subtilis, effective against plant pathogens [8].
A special type of compounds are enniatines. Their molecule consists of three residues
of branched amino acids, L-valine, L-leucine and L-isoleucine, and three residues of D2-hydroxyisovaleric acid (D-Hyiv) [9]. The amino acids and D-Hyiv are linked by
alternating amide and ester bonds. The amide bonds are finally N-methylated.
Molecular conformation is important for the biological activity of peptide antibiotics.
This is true mainly for the peptides capable of formation of chelates with metals. The
studies showed three-dimensional molecular structures with many hydrogen bonds [10].
In the case of valinomycin (L-Val-D-Hyiv-D-Val-L-Lac)3, that is able to selectively
transport K+ and Rb+ ions across natural and artificial membranes, the molecule is
symmetrical in three dimensions, if it forms a complex with the metal. If it is not in the
form of the complex, it has only a pseudocentral symmetry.
3
Biosynthesis. The biosynthesis of peptide antibiotics takes place on a multienzyme
complex [11]. The individual amino acids are activated using ATP to form aminoacyl
adenylates. The aminoacyl groups are transferred to the enzyme thiol groups where they
are bound as thioesters. The structural arrangement of the thiol groups in the synthetases
determines the order of amino acids in the peptide. The formation of peptide bond is
mediated by 4-phosphopantetheine, that is an integral part of the multifunctional
multienzyme.
The way how the order of the amino acids in the molecule is regulated is not known.
It is probably determined by the tertiary configuration of the enzyme. This specificity,
however, is not very high since the microorganisms mostly produce a mixture of
peptides differing only in one or several amino acids in the chain.
Enzymes. Gramicidin S synthetase is an enzyme consists of two complementary
enzymes having molecular weights of 100 kD and 280 kD.
Bacitracin synthetase. The enzyme consists of three subunits [12] (Fig. 3) having
molecular weights of 200, 210 and 360 kD [13]. Each subunit contains
phosphopantetheine. The enzyme A activates the first five amino acids of bacitracin, the
enzyme B activates L-Lys and L-Orn, and the enzyme C activates the other five amino
acids. D-amino acids are produced by racemization of their L-forms directly on the
enzyme complex. Initiation and elongation start on the subunit A up to the pentapeptide,
independently of the presence of the subunits B and C. The pentapeptide is transferred
to the subunit B where two other amino acids are added. The heptapeptide is
subsequently transferred to the subunit C where the biosynthesis of bacitracin is
finished. The cyclization is achieved by binding the asparagine carboxy group to the εamino group of lysine, whereas, to the α-amino group of the same lysine, the isoleucine
carboxyl group is bound [7,14].
Mechanism of action. The antibiotic activity of bacitracin results in an efficient
inhibition of proteosynthesis and cell wall synthesis but other effects such as an
interference with cytoplasmic membrane components and cation-dependent antifungal
effects have been observed as well. In the case of gramicidin S, hemolytic effects,
inhibition of protein phosphatases and interaction with nucleotides have been observed,
in addition to the antibacterial activity. Even though antibiotics normally have several
mechanisms of their action, the primary one is thought to be the effect observed at the
lowest concentration of all. The peptide antibiotics are efficient mainly against Grampositive bacteria.
ß-Lactams.
The main representatives of ß-lactams are penicillins and cephalosporins [15,16].
Penicillins have a thiazoline ß-lactam ring in the molecule and differ, one from another,
by side chains linked via the amino group. Main types of the penicillin molecule are
shown in Fig. 4.
Cephalosporins [17] have a basic structure similar to that of penicillins and the
derivatives are also formed by a variation of the side chain (Fig. 5).
The thiazolidine ß-lactam ring is synthesized using three amino acids: L-α-amino
adipic acid, L-cystein and L-valine by α-aminoadipyl-cysteine-valine synthetases [18].
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By condensation of these three amino acids, a tripeptide is formed. It is transformed to
the molecule of penicillin or cephalosporin through subsequent transformations. The
principial works about enzymes of β-lactams biosynthetic pathways were done by
Abraham and his collegues [19].
Clavulanic acid also belongs to ß-lactams (6). This acid has a bicyclic ring structure
resembling that of penicillin, except that oxygen replaces sulfur in the five-membered
ring. Clavulanic acid is an irreversible inhibitor of many ß-lactamases. The discovery of
clavulanic acid was a starting point for the development of penicillin analogues, able to
inactivate these enzymes.
Biological activity. Penicillins are especially active against Gram-positive bacteria but
some semisynthetic penicillins, such as ampicillin, that is lipophilic as compared to, for
example, benzyl penicillin, are also effective against Gram-negative bacteria. This effect
is explained by their easier entering the cells of Gram-negative bacteria that have a high
lipid content in the cell wall. ß-lactam antibiotics interfere with the synthesis of bacterial
cell wall and thus inhibit bacterial growth. Such a mechanism of action does little harm
to the macroorganism to which ß-lactams are applied.
Glycopeptides.
At present several hundreds compounds belonging to glycopeptides are known,
including semisynthetic derivatives. The best known of all is vancomycin (Fig. 7) [20]
that is effective against Gram-positive bacteria. This antibiotic is widely used in
medicine, especially against ß-lactam resistant microorganisms. Vancomycin is not
absorbed from the gastrointestinal tract and is used to treat enterocolitis caused mainly
by Clostridium difficile.
Vancomycin is produced by several tens of microorganisms, of which Amycolotopsis
orientalis is used for commercial production. Glycopeptides are composed of either
seven modified or unusual aromatic amino acids or a mix of aromatic and aliphatic
amino acids. By the substitution of amino acids in the amino acids core, derivatives of
amino glycosides are formed. In vancomycin the aminosugar vancosamine is bound to
the amino acids core. The removal of aminosugar reduces the activity of vancomycin
two- to fivefold. Glycopeptides lose the majority of their antibiotic activity after
splitting of sugar [21].
Polyketide-derived antibiotics
A large group of antibiotics includes compounds that are synthesized by
polymerization of acetate units and subsequent cyclization of the polyketo chain, that
has been formed before or is just being formed, to provide six carbon atoms containing
aromatic rings or macrocyclic lactone ring. The terminal group need not be an acetate
but also pyruvate, butyrate, ethyl malonate, paraminobenzoic acid, etc. In the early
phase, the formation of polyketo chain is similar to that taking place during the
biosynthesis of fatty acids, and is catalyzed by the enzyme polyketosynthase [22]. A
principal role is played by the Acyl Carrier Protein (ACP) [23]. The ACP prosthetic
group in many microorganisms is 4´-phosphopantothenic acid. Its terminal groups and
acyls produced by polymerization are bound via the -SH group. The acyls are transferred
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to the other -SH group, that is a part of the cysteine molecule. Polyketosynthase has not
yet been isolated and its properties have been deduced from the analyses of DNA
sequences of cloned genes. Polyketosynthases include two distinct groups located either
in domains on multifunctional proteins or present on individual, monofunctional
proteins.
6-Methyl salicylic acid
6-Methyl salicylic acid (6MS) represents the simplest polyketide, that is formed
by condensation and subsequent aromatisation of one acetylCoA molecule and three
malonylCoA molecules. This compound was isolated from Penicillium patulum. By
other metabolic steps 6MS is transformed to produce a toxin called patulin. The
synthesis of 6MS takes place on an enzymatic complex called 6MS synthetase [24,25].
Tetracyclines
The chemical structure of typical tetracyclines is shown in Fig. 8 and their
biosynthesis in Figs. 9 and 10 [26]. Chlortetracycline and tetracycline are produced by
the actinomycete Streptomyces aureofaciens, whereas oxytetracycline and tetracycline
by the actinomycete Streptomyces rimosus. As seen in Fig. 9, the tetracycline molecule
is synthesized from one molecule of malonic acid semiamide and eight molecules of
malonate. In the early steps, the synthesis is similar to the biosynthesis of fatty acids, but
the keto groups are not reduced and aromatic rings are formed to yield 6-methyl
pretetramide. This compound is the first known intermediate of the tetracycline
biosynthesis that is further transformed to yield one of the tetracycline molecules. As to
the enzymes transforming the intermediates of chlortetracycline and tetracycline
biosynthesis, the last three have been described [27,28]: S-adenosylmethionine:4dedimethylamino-4-aminotetracycline N-methyltransferase methylating the amino group
in
position
4,
anhydrotetracycline
oxygenase
and
NADP:tetracycline
5a(11a)dehydrogenase (tetracycline dehydrogenase). For more extensive coverage of
research, articles by Běhal [29] and Běhal and Hunter [30] can by consulted.
Tetracyclines act as inhibitors of proteosynthesis. They are considered to be widespectrum antibiotics, that are efficient against both Gram-positive and Gram-negative
bacteria. However, having significant side effects on the human macroorganism, they
are preferably used only in the case other, less toxic antibiotics are not effective.
Anthracyclines
Anthracyclines are synthesized in a similar way as other polyketides ([31]. They
often have one or several sugar residues in the molecule, most often deoxy-sugars,
synthesized from glucose, are present in the anthracycline molecule. As to their
biological activity, daunorubicin and doxorubicin (adriamycin) (Fig. 11) are rather
important. They are excellent antitumor agents, which are widely used in the treatment
of a number of solid tumors and leukemias in human. However, these drugs have dose
limiting toxicities such as cardiac damage and bone marrow inhibition. In recent years, a
variety of drug delivery systems for anthracyclines have been reported. In most cases,
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the drugs were linked to high molecular compounds such as dextran [32,33], DNA [34]
and others.
Macrolides and polyenes
Macrolides are usually classified to include: proper macrolides having 12
-, 14- or 16-membered macrocyclic lactone ring to which at least one sugar is bound,
and polyenes having 26- to 38-atom lactone ring containing 2 to 7 unsaturated bonds.
Besides the sugars bound to the lactone ring, an additional aromatic part is normally
present in the polyene molecule. As to the biosynthesis, however, both macrolides and
polyenes are synthesized in the same way using identical building units.
Macrolides represent a broad group of compounds and new substances have been
incessantly added to the list, including hybrid compounds [35]. A number of derivatives
of the basic structure can be produced by one microorganism, on the other hand,
however, the compounds can also be found in different microorganisms. Macrolides
usually possess an antibacterial activity whereas polyenes are mostly fungicides.
Erythromycins produced by Saccharopolyspora erythrea (Fig. 12), together with
oleandomycin and picromycin, belong to the best known 14-membered lactone ring
macrolides. A novel erythromycin was prepared by the recombinant Saccharopolyspora
erythrea strain [36,37]. Macrolides with a 16-membered ring are represented by tylosin
(Fig. 13) [38], that is produced by Streptomyces fradiae, as well as by leucomycin,
spiramycin, etc.
The synthesis of lactone ring is similar to that observed in the case of other
polyketides. In contrast to aromatics, propionate and butyrate units are more often used
in the biosynthesis, instead of acetate ones. The greatest difference, however, consists in
the fact that, instead of aromatic rings, a lactone ring is formed. Keto- and methyl
groups of the polyketide chain, from which macrolides are formed, are normally
transformed more frequently.
Nystatin is the best known polyene antibiotic (Fig. 14) [39]. Candicidine is another
well known antibiotic belonging to the polyene group. Its molecule includes paminoacetophenone as the terminal group. 4-amino benzoic acid (PABA) was identified
as a precursor of the aromatic part of candicidine molecule [40,41].
The sugars found in macrolide and polyene molecules are not encountered in the
structures of microbial cells. They include both basic and neutral sugar molecules.
Often, L-forms are found. Sofar, at least 15 different sugars have been described to
occur in macrolides and polyenes. All of them are 6-deoxy sugars; some of them are Nmethylated, others have the methyl on either the oxygen or carbon atom. As it has
been repeatedly proven [42,43], glucose is primarily incorporated into macrolide sugar
residues. Also in Streptomyces griseus, glucose, mannose and galactose were
incorporated to a greater extent into the mycosamine candicidine, as compared to its
aglycon [44]. The transformation of glucose to a corresponding sugar takes place in the
form of the nucleoside diphosphate derivatives, which is similar to the situation found in
the case of other antibiotics.
Avermectins
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The molecule of avermectins [45] consists of a 16-membered, macrocyclic
lactone to which the disaccharide oleandrose is bound (Fig. 15). Avermectins are
produced by Streptomyces avermitillis. The macrocyclic ring of avermectins is
synthesized, as other polyketides, by producing a chain from acetate, propionate and
butyrate building units. Oleandrose (2,6-dideoxy-3-O-methylated hexose) is synthesized
from glucose.
Avermectins are potent antiparasitic compounds with a broad spectrum against
nematode and anthropod parasites. They lack antifungal and antibacterial activities.
They bind to a specific, high-affinity site present in nematodes but not in vertebrates. Its
dosage for animal and human is extremely low. Ivermectin (22,23-dihydroavermectin
B1) is a semisynthetic compound which is used to control internal and external parasites
in animals. It is the most potent anthelmintic compound of all. Avermectins are also
employed in human medicine and plant protection. Detailed reviews on the uses and
biosynthesis of avermectins can be found in recent monographs [46,47].
Chloramphenicol
Chloramphenicol (Fig. 16) is produced by Streptomyces venezuelae [48].
However, at present the antibiotic is commercially produced using a fully synthetic
process. In contrast to polyketides, the aromatic ring of chloramphenicol molecule is
synthesized from glucose via chorismic acid and p-amino benzoic acid in the microbe.
Aminoglycosides
Streptomycin (Fig. 17) is a well-known representative of aminoglycoside
antibiotics. It is synthesized by many streptomycetes to produce a number of derivatives.
The molecule of streptomycin consists of three components: streptidine, L-streptose and
N-methyl-L-glucosamine. None of these components has been found in the primary
metabolism of microorganisms. The biosynthesis of streptomycin was disclosed mainly
by Walker [49], who also studied the enzymes participating in the biosynthesis of
streptomycin [50].
The importance of streptomycin consists mainly in its efficiency to suppress
Mycobacterium tuberculosis. A massive use of streptomycin resulted in effective
suppression of tuberculosis, especially in developed countries. Recently, however, the
disease caused by M. tuberculosis has been found to increase again due to the
occurrence of strains resistant to streptomycin.
Antiviral compounds
Recently also compouds active against viruses have been discovered. Sattabacins
and sattazolins, isolated from Bacillus sp. [51] and fattivirin A1, isolated from
Streptomyces microflavus [52] are active against Herpes simplex viruses. Inhibitors of
HIV are intensively looked out in microorganisms. Inhibitors of HIV-1 protease were
detected in fungus Chrysosporium merdarium P-5626 [53]. A compound which has an
inhibitory effect on HIV-1 replication in chronically infected cells as well as actualy
infected cells was isolated (after screeninng 10,000 microorganism products) from the
culture supernatant of Streptomyces sp. Mer-2487 [54]. A hydroxyl benzaldehyde
8
compound, active against influenza virus in vitro, was isolated from Aspergillus terreus
[55]. Rhodopseudomonas capsulata produces a virucide substance which inactivated
polio virus, Sindbis virus, some fish viruses, without causing any damage to the host
cells [56].
Genetics
High production strains
The genes coding the enzymes that synthesize antibiotics are mostly located on
chromosomes. These genes are called structural genes and the enzymes taking part in
the antibiotic synthesis are called the enzymes of secondary metabolism. The structural
genes are organized to form one cluster. This situation has been observed in all cases
described so far [57,58,59]. The expression of structural genes is controlled in a similar
way as in the case of other genes. Next to a cluster of the structural genes, the genes
coding for the resistance of the producer to its own antibiotic are located. Those genes
are situated either at the beginning or at the end of the cluster, often in both positions. In
the case the resistance genes are present in the two positions, different types of
resistance are included as a rule. In addition to the structural genes, regulation genes also
determine the antibiotic production. They are often located on plasmids. The genetic
control of antibiotic biosynthesis is poorly known. The type of control where the
antibiotic synthesis is inhibited by the own product can serve as an example. As a result,
the product’s cellular concentration is maintained at a physiologically tolerable level
and, consequently, the producing microorganism is prevented from being self damaged
by high concentrations of the product, that are toxic.
Microorganisms that are isolated in nature produce small amounts of antibiotics. They
are called “wild type strains”. The term “wild strain”, however, does not reflect the fact
that, during selection and subsequent cultivation in the laboratory, a change could occur
making it non-identical with the original strain. The term wild type strain thus only
refers to the fact that the strain did not undergo an “artificial” genetic change in the
laboratory and was duly conserved and maintained.
In order that the commercial production of antibiotics could be profitable, higher
levels of the antibiotic synthesis are reached via genetic changes. Most often the spores
are exposed to UV irradiation, X-rays, gamma-rays, α-particles or chemical mutagens
(nitrogen mustards, N-methyl-N´-nitro-N-nitroso guanidine). A combined mutagenesis
using various mutagens is currently used. Having germinated, the surviving spores give
rise to individual colonies of isolates, whose capability of antibiotic production is then
tested. Those of the mutants that exhibit poor growth and sporulation ability are not
suitable candidates for further improvement, even though their antibiotic production
may exceed that of the original strain. Today’s high production strains, that synthesize
as high as 10 000-fold levels of antibiotics, compared to the original strains, must be
seen as a result of many year’s, costly efforts. Unfortunately, these high production
strains can revert to lose their overproduction. The high production levels have thus to
be maintained, at least by a permanent selection of the high production strains during
9
storage, when spontaneous mutagenesis can take place, without involvement of any
mutagenic agent.
Multiplication of the structural genes is not an important factor increasing the
antibiotic production. Mutations resulting in an increased antibiotic synthesis mostly
affect the regulatory genes. Hopwood and co-workers [60] transferred the genes for the
production of actinorhodine to a low production, wild type strain using a plasmid. Even
though the number of copies of the structural gene increased only twofold, the
production of the antibiotic rose 30-40 times. The increase of the antibiotic production
has to be accompanied by an increase of resistance to the own product.
When high production strains are prepared by mutagenesis, a type of mutant that loses
some of the structural genes can also be obtained. Such a mutant can exhibit a higher
level of an antibiotic intermediate whose transformation stopped due to the absence of
the corresponding enzyme. By crossing these mutants, some biosynthetic pathways used
to synthesize antibiotics were elucidated, e.g. tetracyclines [61].
Loss of the capability of antibiotic production in the strains where extrachromosomal
DNA was removed (e.g. by using acriflavine or ethidium bromide) suggests that the
regulatory genes are located on plasmids [62,63,64,47].
Strains that lost the ability to produce other secondary metabolites and nondesirable
derivatives of the antibiotic in question are also selected during the process of
improvement of high production strains. In this way the metabolic pathways that
unnecessarily consume the building units used for the antibiotic synthesis can be
eliminated and, also purification of the antibiotic product from other compounds and
impurities can thus be facilitated.
Genetic manipulation of antibiotic producers
The structural genes for a number of antibiotics have been cloned into host
microorganisms. Similarly, genes for antibiotic resistance and other regulatory genes
have also been cloned. Streptomyces lividans was found to be a suitable acceptor of
foreign genetic material, in which a low degree of restriction of this genetic material
exists. This microorganism can host various plasmids and phage vectors. However, at
the same time, this microorganism was found not to be usable for the synthesis of
various antibiotics or of high antibiotic levels. The antibiotic biosynthesis is a very
complex process that requires not only the structural genes for enzymes of secondary
metabolism but also the genes for regulation of their biosynthesis. Moreover, the
overproduction of an antibiotic has to be coordinated with the primary metabolism of
the producing microorganism.
The cloning of structural genes and genes for resistance to the own antibiotic enables
us to work out genetic maps of the antibiotic producers. On the basis of those maps,
hybrid clusters combined of two and more clusters of different antibiotics can be
created. Consequently, semisynthetic antibiotics can be produced that may possess new
biological activities or an antibiotic activity against resistant strains.
Polyketide synthase genes of microorganisms producing various polyketides have
also been hybridized [22]. As a result, a great similarity of polyketide synthases from
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various streptomycetes was evidenced and new polyketide antibiotics were synthetized
[58].
Search for new antibiotics
Isolation from nature
At present several thousands of compounds having some biological activity
have been obtained from microorganisms isolated from nature. As the probability of
finding a new compound that would be usable as a new antibiotic is as little as one in
ten thousand, a great number of microorganisms have to be checked. A rough estimation
says that about 100 000 microorganisms is screened for the presence of biologically
active compounds per year. Well equipped laboratories study about 30 different
biological activities. The requirements for new antibiotics result from the occurrence of
resistant strains of pathogenic microorganisms, that are no more sensitive to known
antibiotics used in the clinical practice. It is mostly big pharmaceutical companies that
look for new antibiotics. Their search for new compounds is highly automated. The
selection methods used and the methods of detection of the biological activity are
normally not published.
Preparation of a new antibiotic and its introduction into the clinical practice requires
cooperation of scientists from various scientific disciplines. They can be divided into
three groups [65]: microbiology (colection of source samples, isolation of diverse
microbes, fermentation to enhance the production, taxonomy), pharmacology (target
selection, screen design, high-troughput screening, identification of active compounds,
efficacy studies, mechanism of action), chemistry (active compounds identification,
characterization/replication, isolation/purification, structure elucidation).
Producers of antibiotics and other biologically active compounds
The majority of the known antibiotics are produced by actinomycetes, fungi and
by moulds. With an increasing spectrum of efficiency of microbial metabolites, new,
non-traditional sources of such compounds have been looked for. Tropical soils have an
enormous biodiversity and they are a rich source of new antibiotics [124]. Various
species of microorganisms have been checked including the microorganisms living
under extreme conditions (high and low temperatures, etc.), sea living microorganisms
[66,67], and higher marine organisms. Marine microorganisms live in a quite different
environment from their terrestrial counterparts and would thus be expected to have a
different metabolic pathways and to produce compounds, which posses unique
structures and activities.
The enterprise of screening microbial metabolite for new leads, first exploited by
antibiotic researchers and today expanded to virtually all fields of therapeutic interest,
has proven successful and will continue as an important avenue to new drug discovery.
The original method for determination of the antibiotic efficiency consisted in the
application of an extract from the microorganism studied to wells made in a 0.5 cm,
agar medium layer in Petri dishes or plates to which the testing microorganism was
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subsequently inoculated. Most often Staphylococcus aureus, Sarcina lutea, Klebsiella
pneumoniae, Salmonella gallinarium, Pseudomonas spp., Bacillus subtilis, and Candida
albicans were used.
The tests of other biological than antibiotic activities require sophisticated methods.
This is true especially when enzyme inhibitors are looked for. Thus, Ogawara et al. [68]
chose a tyrosine protein kinase associated with the malignant transformation of the cell
caused by retroviruses as the target in a biochemical screen and found genistein, an
isoflavone from Pseudomonas, exhibiting a specific inhibitory activity. Production of
target enzymes using recombinant DNA methodology has dramatically expanded the
number of potential targets that can be feasibly screened. A screen for the inhibitors of
HIV reverse transcriptase is an example. The enzyme was produced in Escherichia coli,
purified by affinity chromatography, and used to test natural products for the activity
[69].
Semisynthetic and synthetic antibiotics
After the structures of the antibiotics discovered had been determined and
microbial strains resistant to them detected, possible variations of the molecules of
known antibiotics were studied. Several methods have been used to accomplish such
variations.
Biosynthetic antibiotics.
The unspecificity of the enzyme systems able to synthesize antibiotics was used,
together with the addition of precursors to the growth medium. Thus, the reaction
equilibrium was shifted to promote the production of the derivative required. In this
way, penicillins with different side chains were prepared. Addition of amino acids to
the growth medium can affect the amino acid composition of polypeptide antibiotics.
The individual derivatives of penicillin and cephalosporin have slightly different
antimicrobial spectra and are capable of suppression of microorganisms resistant to
other derivatives.
Semisynthetic antibiotics.
Replacement of a part of the antibiotic molecule can be accomplished chemically
or enzymatically. In this way, semisynthetic penicillins, cephalosporins, tetracyclines,
etc. were prepared. The production of semisynthetic penicillins and cephalosporins was
facilitated by the fact that 6-amino penicillanic and 7-amino cephalosporanic acids (Fig.
18) could be easily prepared. The side chain is removed by the action of an enzyme or
by a chemical hydrolysis and to the amino group in position 6 (penicillins) or 7
(cephalosporins), that was made free in the previous step, another acyl is bound
chemically or enzymatically. In such a way, various penicillins and cephalosporins have
been prepared to be effective against microorganisms resistant to original compounds.
Semisynthetic tetracyclines, pyrolinomethyltetracycline, metamycin and doxycycline,
exhibit a greater solubility and somewhat different antimicrobial spectrum, as compared
to the original tetracyclines [70]. New derivatives of aminoglycosides have been
obtained by chemical and enzymatic modifications.
12
Chemical synthesis of antibiotics
As the majority of antibiotics have rather complex structures, their chemical
synthesis is mostly more expensive than the production by fermentation. An exception
to the rule seems to be chloramphenicol, that is normally prepared using a chemical
synthesis.
Hybrid antibiotics
Using of genetic engineering we can combine structural genes of different
antibiotic producers to obtaining new products which are not present in nature
[71,72,73]. If these genes are expressed, a hybrid antibiotic is synthesized, that cannot
be found in nature. Hopwood et al. [74,60] used this method with the genes of
actinorhodin synthesis and obtained related hybrid macrolides, mederhodin A and B,
dihydromederhodin A and dihydrogranatirhodin. Niemi et al. [123] prepared new
anthracyclines by combination of DNA Streptomyces purpurascens ATCC 15489 and
Streptomyces galilaeus ATCC 31615. A next hybrid anthracycline antibiotic was
produced by expression of dnrK encoding carminomycin 4-O-methyltransferase in an
epelomycin-producing Streptomyces violaceus [75].
Resistance to antibiotics
The antibiotic resistance is usually looked at from two angles: first, how the microbial
strains arise, that obtain the resistance during the treatment of the macroorganism with
the antibiotic, second, the resistance of microorganisms producing antibiotics that build
up their resistance against the product of their own which, synthesized at high
concentrations, would damage the producer. The ways of how these two types of
resistance are achieved are often similar, even though the aims are different. Whereas a
resistant microorganism is most often capable of transforming the antibiotic or even
degrading it completely, the resistance of producing microorganisms has to ensure that
the antibiotic will not be destroyed.
Resistance of antibiotic producers
The basic metabolic processes of microorganisms producing antibiotics are not
inhibited, if the antibiotics are synthesized at low concentrations, observed in strains
isolated from nature. By strain improvement, mutants have been able to reach 100 to
1000-fold antibiotic yields, as related to a volume unit of the fermentation medium.
Genome changes of the improved strains include a number of deletions and
amplifications in the chromosomal DNA. Changes in extrachromosomal DNA were also
detected.
Low production strains, whose resistance to the own product is low (i.e. higher
concentrations of the product inhibit their growth), regulate the antibiotic production,
e.g. by inhibiting the enzyme activities that participate in the synthesis of the antibiotic.
In high production strains, such a control is lost and the strains have to find a way how
to survive in the presence of a high concentration of the antibiotic without decomposing
it.
13
As mentioned above, the genes for resistance to the own product are often located at
the beginning of the cluster of structural genes. As a result, they are expressed
simultaneously with the structural genes. However, the genes of newly gained
resistances are mostly located on plasmids.
Antibiotics hit active centres of enzymes which makes the enzymes inactive.
However, if the enzyme active centre is modified, the antibiotic cannot bind to it and the
resistance comes into existence. It is not known whether a decreased ability to bind the
antibiotic results from a posttranslational modification of the active centre or if resistant
molecules of the enzyme are synthesized de novo.
Many antibiotics inhibit protein synthesis, the target site being at the ribosome level.
Often, the functions of Tu and G elongation factors are also impaired, together with the
synthesis of guanosin penta- and tetraphosphates that is significantly reduced. The
antibiotic producers (mostly actinomycetes), as well as the bacteria against which the
antibiotic is used, protect themselves by posttranscriptional modification of rRNA.
Adenine is methylated to obtain N6-dimethyladenine rRNA in 23S. Such modified
ribosomes do not bind the antibiotic. In other cases, adenine is methylated to yield 2-Omethyladenosine [76,77]. However, methylation modified ribosomes can be sensitive to
the effect of other antibiotics. The genes coding for methylases, that catalyze
methylation of adenine in some streptomycetes, were cloned into Streptomyces lividans
and the ribosomes of the mutants prepared were resistant towards the corresponding
antibiotics.
The most important mechanism of resistance observed in the antibiotic producers
seems to be the transport of the antibiotic from the cell to the environment. In the case
of high production rates, probably no protection of the active centres could be
sufficiently effective. In addition, the antibiotic produced would gradually fill up the
interior of the cell. In Streptomyces rimosus, an oxytetracycline producer, genes for the
enzymes increasing the antibiotic transport rate precede the structural genes on the
chromosome. Genes for the resistance consisting in the protection of ribosomes via the
synthesis of an unidentified protein are located at the end of the structural gene cluster
[78].
Antibiotic producers also have to solve the problem of a reverse flow of the antibiotic
into the cell. Some antibiotics bind to the cell wall, others are complexed in the medium
(tetracyclines in the presence of Ca2+ ions). Cytoplasmic membranes of resistant strains
are often less sensitive to the effect of antibiotics. This kind of resistance is thought to
be connected with the content of phospholipids in the cell. In Bacillus colistinus, a
colistin producer, the content of phospholipids in the cell-free extract increased with the
sensitivity to the antibiotic.
Antibiotic producers can use several types of resistance at one time. Tetracyclines,
that strongly inhibit proteosynthesis, interfere with the binding of the ternary complex of
amino acyl-tRNA-EFTu-GTP to ribosomes. The genes for resistance were cloned into
Streptomyces griseus, sensitive to tetracyclines, using pOA15 as the vector plasmid.
After mapping the plasmids in resistant strains using restriction nucleases, two types of
plasmids capable of transfer of different types of resistance were found. One type
consisted in an increased ability of tetracycline transport to the medium, the other in an
increased resistance of ribosomes to the effect of tetracyclines. These ribosomes bore a
14
compound(s), bound to their surface, that could be removed by washing with 1 M
NH4Cl solution. The ribosomes lost their resistance after the washing, which was
demonstrated with both the ribosomes of Streptomyces griseus and those of the original
strain of Streptomyces rimosus. The two types of resistance were both constitutive and
inducible [78]. The proteosynthesis inhibiting concentrations of chlortetracycline in
Streptomyces aureofaciens are higher in the production phase, as compared to the
growth phase [79]. Thus, the resistance can be increased even during the fermentation
process.
Another way how the antibiotic producers can avoid the effect of their products is by
situating the distal enzymes of the antibiotic biosynthetic pathway (synthases) outside
the cell, most often in the periplasm. In Streptomyces aureofaciens, a higher proportion
of the outside terminal tetracycline synthase was found in production strains under high
production conditions in periplasm, as compared to low production conditions [80].
Resistance in pathogenic microorganisms
Shortly after antibiotics were introduced into clinical practice on a massive scale,
strains of hitherto-sensitive microorganisms started to appear, that required the use of
much higher antibiotic concentrations or, even, were completely resistant to these
antibiotics. The resistant strains originated from clones that survived the antibiotic
treatment, especially if the treatment was terminated before all pathogenic
microorganisms were killed or the antibiotic was applied at sublethal doses. There are
several ways how microorganisms can gain resistance [81].
In most resistant microorganisms, the main mechanisms of resistance are
detoxification or inactivation of the antibiotic, change of the target site, blocking of the
transport of the antibiotic out of the cell.
Penicillins and cephalosporins are degraded using three ways:
a) by the enzyme penicillin amidase that cleaves the amidic bond by which the side
chain is bound to the β-lactam ring,
b) by the enzyme acetyl esterase that hydrolyzes the acetyl group at C-3 on the
dihydrazine ring of cephalosporins,
c) by the enzyme β-lactamase that catalyzes hydrolysis of the β-lactam ring of
penicillins and cephalosporins.
Penicillin amidases are rarely used by microorganisms to build up resistance to βlactam antibiotics. They are often employed for the synthesis of semisynthetic
antibiotics. Acetyl esterase is also not important from the point of view of antibiotic
resistance. In most cases, β-lactam antibiotics are inactivated by β-lactamase that
destroys one of the important sites for their antibiotic activity; the damage is
irreversible.
However, β-lactamases are not synthesized only by microorganisms that came
into contact with penicillins, they have been found in three quarters of all streptomyces
strains, their synthesis being constitutive in most of them. One can suppose that the
genes for the synthesis of β-lactamases were transferred from microorganisms that
possess them in nature to newly arisen resistant strains. Recent studies indicate frequent
and promiscuous gene transfer between distantly related bacterial species. A possibility
of direct transfer from a streptomycete to a pseudomonad, for example, may seem
15
unlikely. However, it is not necessary to invoke direct exchanges. It is more reasonable
to imagine that distant exchanges between distantly related organisms result from a
cascade of transfer between related species [82].
Another way of transformation of the antibiotic molecule is N-acetylation of the
amino group or O-phosphorylation of the hydroxyl. Bialaphos [83] was found to be
inactivated by acetylation. This compound itself is not toxic but, in the cell,
phosphinothricine is liberated that inhibits glutamine synthetases, key enzymes of the
inorganic nitrogen assimilation in microorganisms and mainly in plants [84].
Regulation of antibiotic production
Overproduction of secondary metabolites
Microorganisms produce in natural environment only small amount of
antibiotics. They have to control the antibiotic synthesis since secondary metabolites at
high concentrations are mostly toxic even for their producers. Using high-yielding
strains and optimization of fermentation condition we can reach many times higher
production. In that case we speak about "overproduction" . Production of antibiotics in
factories are at present several thausands higher as production of original strains isolated
from nature but this high production is reached only when high-yilding strain is used
and special conditons of cultivation are kept. The main factors influence production of
antibiotics are discused in next chapters.
Growth phases of microbial culture
A culture of a microorganism capable of antibiotic production, where the
overproduction of the antibiotic is taking place, includes several growth phases
representing a number of physiological states.
Preparatory phase (lag phase) - the culture is adapting to the new environment, the
growth is slow and, evidently, regulatory proteins are being synthesized that, on the
basis of the information from the environment, activate the expression of the respective
genes during cultivation.
Growth phase - the culture grows intensively, usualy a low amount of antibiotic is
synthesized.
Transition phase - growth rate and proteosynthesis slowed down; the antibiotic
production is started. The enzymes of secondary metabolism are intensively synthesized
[85,86].
Production phase - growth is practically ceased, dry weight of microorganism is
constant, the antibiotic is intensively synthesized.
Antibiotic producers mostly belong to filamentous microorganisms (Fig 1B), which
means that, in their culture, cells of various age and at different stages of development
are present. The microorganisms grow in pellets (Fig. 1A), inside which the cultivation
conditions differ from those on the pellet surface (nutrient concentrations, oxygen
concentration, etc.). An increase in dry weight does not have to always mean the
biomass growth since, in streptomycetes, often a thickening of the cell wall or
glycocalyx formation occur that increase the dry weight value without rising the cell
number [87]. The individual cells can thus be at different stages of development, i.e. in
16
different physiological states. Therefore, we speak about a physiological state of the
whole culture that represents an average of physiological states of the individual cells.
Regulation by nutrients
In order to reach a high production of an antibiotic, a sufficient biomass yield is
necessary, that is accomplished within a short time, if possible. Thus a danger of
contamination is diminished and the economic parameters of the fermentation device
are kept at its optimum. For this purpose, readily utilizable carbon, nitrogen and
phosphorus sources are used. When they are present in the medium, however, an
overproduction of the antibiotic does not take place. The culture medium should be
designed in such a way that, after the biomass increased sufficiently, at least one of the
basic nutrient sources would become depleted and the culture growth would be
consequently limited. However, this limitation is not well understood.
Regulation by carbon source
The inhibition of antibiotic synthesis by glucose was observed shortly after the
experiments with penicillin producers started. The antibiotic was found to be
synthesized only after glucose was depleted from the medium and lactose started to be
metabolized. Similarly, glycerin was observed to inhibit the biosynthesis of
cephalosporins [88]. Starch, used as the carbon source, did not inhibit the biosynthesis
of penicillin. The concentration of glucose produced from the polysaccharide is that low
that the inhibition does not take place. Using these data, fermentation protocols were
worked out, in which the level of glucose was kept low so as not to inhibit the
antibiotic production. The mechanism of inhibition of the antibiotic synthesis by readily
utilizable sugars probably consists in a repression of enzymes of secondary metabolism
[89]. In addition to lactose, sucrose, starch and fatty acids are also used as carbon
sources in the production phase for the production of antibiotics.
Regulation by nitrogen source
Readily utilizable nitrogen sources present in the culture medium inhibit the
production of antibiotics. Mainly ammonium ions decrease the antibiotic synthesis and,
therefore, their concentration in the production media is limited to be exhausted at the
end of growth phase. Soy flour, peanut flour and other complex substances are used as
nitrogen sources in the production phase of antibiotic fermentations. These nitrogen
sources are not easily utilizable and are similar to those used by the microorganisms
producing antibiotics in nature. Readily utilizable nitrogen sources repress enzymes of
secondary metabolism in Cephalosporium acremonium [90] during the biosynthesis of
cephalosporin and in Streptomyces clavuligerus producing cephamycin [91]. Similarly,
the inhibition of biosyntheses of leucomycin [92], tylosin [93], and erythromycin [94]
are explained by the repression of enzymes of secondary metabolism. Ammonium salts
also inhibit the activity of anhydrotetracycline oxygenase isolated from S. aureofaciens
[95].
17
Regulation by phosphate
Phosphate is used as main regulator of overproduction of antibiotics in factories.
Inorganic phosphate is carefully added in doses to the medium so as to accomplish an
optimal ratio between the biomass production and the capability of antibiotic
biosynthesis. Bound to organic compounds normally added to the medium (soy flour,
etc.), phosphate does not affect the antibiotic production. The antibiotic biosynthesis is
started on the condition the concentration of phosphate in the medium decreased below
a certain level. Consequently, the producer culture undergoes a shift from the
physiological state characteristic for the growth phase to that of the overproduction
phase.
The influence of inorganic phosphate is explained by repression of the synthesis of
enzymes of secondary metabolism [96,97,98]. After the inorganic phosphate was
depleted from the medium, a significant decrease of the rate of proteosynthesis was
observed during the tetracycline biosynthesis and the synthesis of enzymes of secondary
metabolism was commenced [99]. If phosphate was kept above the threshold
concentration, the significant decrease of the rate of protein synthesis did not occur and
enzymes of secondary metabolism were not synthesized. An addition of phosphate to
the medium at the beginning of the production phase, after the phosphorus source was
depleted and the enzymes of secondary metabolism synthesis initiated, resulted in a
decrease of the enzyme levels in the culture and an acceleration of proteosynthesis. The
synthesis of secondary metabolism was resumed after the phosphate was depleted again
from the medium. Production of oxytetracycline by Streptomyces rimosus is controlled,
at least in part, at the level of transcription from promoters overlapped by tandem
repeats similar to those of the DNA-binding sites of the OmpR family [100]. The
phosphate was found to be consumed at a higher rate than expected, with respect to the
actual rate of protein synthesis, and was probably deposited in the cells in the form of
polyphosphates. The capability of absorption of phosphate in the cell seems to be an
important function of soil microorganisms.
Regulation by metal ions.
Metal ions act usually as a part of enzyme active centers. The optimal
concentrations of metal ions for cultivation of the antibiotic producing strains have been
determined empirically. These ions mostly need not be added to complex media. Some
metal ions, present in cultivation media, can substantially decrease production of
antibiotics, for instance iron inhibits production of chlortetracycline. Some
microorganisms are able to grow in iron-depended medium by secreting iron-regulated
chelating compounds as siderophores [101].
Influence of low molecular compounds
The antibiotic production can be regulated by different low molecular
compounds. The mechanism of their action is not understood. Tryptophan exhibited a
stimulatory effect on the production of antibiotics, e.g. mucidin in the basidiomycete
Oudemansiella mucida [102] and actinomycin in Streptomyces parvulus [103].
Methionine was found to promote the synthesis of cephalosporin C. Neither tryptophan
nor methionine were used as the building units. When enzymes of secondary
18
metabolism were measured, higher levels were detected in the cells of the producing
strain.
Benzyl thiocyanate increases the production of chlortetracycline and tetracycline in S.
aureofaciens. In contrast, it does not influence the production of oxytetracycline in S.
rimosus. The effect on the metabolism of S. aureofaciens is multiple [104], including a
number of enzymes but the basic influence of benzylthiocyanate at production is the
higher expresion of enzymes of secondary metabolism [89]. This is the reason why
benzyl thiocyanate is able to raise the antibiotic production only if it is added in the lag
phase, growth phase or at the beginning of the production phase. Its effect is more
pronounced in low production strains, where the enzymes of secondary metabolism
level and the antibiotic production are increased 10 to 20-fold, as compared to high
production strains where the increase is only twofold.
In the streptomycete antibiotic producers, low-molecular, diffusible compounds have
been discovered that regulate the metabolism of the producer, where they are present at
very low concentrations, and thus affect both the biochemical and morphological
differentiation. The most famous of them is factor A, gamma-butyrolactone (Fig. 19),
that was discovered in Streptomyces griseus producing streptomycin [105]. A nonproducing strain started the synthesis of streptomycin after factor A was added to the
culture and, in parallel, the formation of aerial mycelium was taking place. Factor A is
synthesized by many streptomycetes but the regulatory effect was observed only in
Streptomyces griseus, Streptomyces bikiniensis and Streptomyces actuosus. As the result
of the addition of factor A to blocked mutants of Streptomyces griseus JA 5142, the
synthesis of anthracyclines and leukaemomycin (anthracycline type antibiotic) was
resumed. [106]. The resistance to streptomycin linked with an enzymatic
phosphorylation of the antibiotic is also induced by factor A [107].
Analogues of factor A have also been found, all of them being gammabutyrolactones. Virginiae butanolides were detected in Streptomyces virginiae [108].
Factor I was isolated from Streptomyces sp. FR1-5 and its effective concentration was
0.6 ng/ml culture. Most of the factor A analogues, however, were not biologically
active.
Factor B (Fig. 19) was isolated from the yeast Saccharomyces cerevisiae. This
substance was capable of eliciting the production of rifamycin in a blocked mutant of
Nocardia sp. This substance was effective at a concentration of 10-8 M, when one
molecule elicited a synthesis of about 1500 molecules of the antibiotic. The structure of
factor B is similar to cAMP but none of the derivatives of known nucleotides exhibited
a comparable effect. Chemically prepared derivatives of factor B have also been tested.
The effect was observed with those that had a C2 -C12 acyl moiety; octylester was the
most effective of them, exhibiting the effect at as low a concentration as 10-10 M [109].
A substitution of guanosine for adenine did not result in a loss of the biological activity
of factor B.
Factor C was isolated from the fermentation medium of Streptomyces griseus. This
compound causes cytodifferentiation of non-differentiating mutants [110]. Factor C is a
protein having a molecular weight of about 34 500 D, whose molecule is rich in
hydrophobic amino acids.
19
The effect of autoregulators is easily observable, if they elicit morphological changes,
such as the formation of aerial mycelium. Carbazomycinal and 6methoxcarbazomycinal, isolated from Streptoverticillium species, were capable of
inhibition of the aerial mycelium formation at a concentration of 0.5 to 1 microgram per
ml. Autoregulators affecting sporulation were found in Streptomyces venezuelae [111],
Streptomyces avermitilis [112]), and Streptomyces viridochromogenes NRRL B1551[113]. From the same strain of Streptomyces viridochromogenes, germicidin was
isolated by Petersen and co-workers [114]. The compound had an inhibitory effect on
the germination of arthrospores of Streptomyces viridochromogenes at a concentration
as low as 40 picograms per ml. Germicidin (6-(2-butyl)-3-ethyl-4-hydroxy-2-pyrone) is
the first known autoregulative inhibitor of spore germination in the genus Streptomyces
and was isolated from the supernatant of germinated spores, but also from the
supernatant of a submerged culture.
Mutants of Streptomyces cinnamonensis resistant to high concentrations of butyrate
and isobutyrate produce an anti-isobutyrate factor, that is excreted into the culture
medium [115]. On plates, anti-isobutyrate factor efficiently counteracted toxic
concentrations of isobutyrate, acetate, propionate, butyrate, 2-methylbutyrate, valerate,
and isovalerate in Streptomyces cinnamonensis and other Streptomyces species.
General control mechanisms have been looked for that operate in the antibiotic
biosynthesis. The energetic state of the cell is thought to be such a general control
mechanism. The intracellular ATP level reflects the content of free energy in the cell. In
some cases, the start of the antibiotic synthesis is linked with a decrease of the
intracellular ATP level. Such a relationship was observed in Streptomyces aureofaciens
and Streptomyces fradiae during the production of tetracycline [116,117] and tylosin
[118], respectively.
Even though the regulatory role of ATP cannot be strictly excluded, the results seem
to support a hypothesis that a higher ATP level is accompanying the active primary
metabolism. A slow down of growth and of the whole primary metabolism would
logically be accompanied by a decrease of the ATP level.
As in the case of ATP, the role of cAMP in the metabolism of antibiotic producers
was also studied, especially in connection with the glucose regulation. Hitherto, no
indication has been obtained suggesting a significant role of cAMP in the regulation of
antibiotic production [119].
Reception of signals from environment
The way of reception of signals from the environment, so that they would be available
to the genetic material of the cell to result in the initiation of the antibiotic synthesis, is
known quite well. It does not significantly differ from the trasduction of signals for
other metabolic processes. Catabolite repression signals or those signalling the
depletion of nitrogen or phosphate or the initiation of sporulation are transducted via
two-component, signal proteins [120]. In spite of some structural varieties, these
proteins are characterized by general mechanistic features and conserved amino acid
sequences. The two-component system consists of a cytoplasmic membrane-linked,
sensor-transmitter protein and a response-regulator protein, located in the cytoplasm.
The sensor-transmitter is composed of a sensor domain located near its N-end; the N-
20
end is found outside the cytoplasm. A specific effector is capable of binding directly to
this N-end. The transmitter domain is located in the cytoplasm to be linked to the sensor
domain via a hydrophobic, amino acid sequence stretching across the membrane. The
sensor-transmitter proteins are normal histidine-protein kinases, capable of
autophosphorylation at its C-end on receiving a proper signal. The phosphorylated
protein becomes a donor in reactions transferring phosphorus. The acceptor is the
cytoplasmic, response-regulator protein.
Two-component signal proteins thus transfer the information concerning the
conditions that can affect the cell action.
Response-regulator characteristic
Response-regulator proteins usually contain 200-500 amino acids. The
phosphorylation of the aspartic acid carboxyl near the N-end of the response regulator
can represent an energy rich bond whose energetic level is believed to decrease through
a conformation change of the protein. The above mentioned type of regulator contains a
DNA binding protein located near its C-end. If the conformation of this protein is
changed, it becomes capable of an interaction with a specific DNA sequence.
Transcription initiation of structural genes
Regulatory proteins, having been bound to specific DNA sequences and having
interacted with RNA polymerase, start the transcription. Regulatory proteins that
activate the transcription of structural genes are probably synthesized already during the
lag phase. Their binding to DNA and a subsequent biosynthesis of the antibiotic depend
mainly on the composition of the growth medium. Provided inorganic phosphate is
present in the medium, the activator becomes phosphorylated and thus incapable of
binding to DNA. In contrast, for example, the activator of the synthesis of glutamine
synthetase, a key enzyme of the assimilation of ammonium salts from the medium and,
consequently, of utmost importance for proteosynthesis, is able to bind to DNA only in
a phosphorylated form. One can hypothesize that a depletion of inorganic phosphate
from the medium does not stop proteosynthesis as a result of a lack of phosphate in the
cell for the biosynthesis of cellular structures, as the phosphate limitation is normally
explained, but rather the presence or absence of phosphate in the medium causes
respective activation or repression of the activators of the enzyme syntheses in primary
or secondary metabolisms. This idea is also supported by the fact that enzymes of
secondary metabolism were synthesized and the antibiotics produced immediately after
the phosphate, that had been added at the beginning of the production phase, was
depleted from the medium and deposited in the cell [89].
Technology of antibiotic production
Some antibiotics are commercially produced on a ton scale. The fermentation process
during which microorganisms produce antibiotics is carried out in fermentors having a
volume of several tens of cubic meters. As in any fermentation process, a conserved
strain is used, that is first propagated in the laboratory and then in a plant fermentor. The
21
cells are then used to inoculate production fermentors. The inoculum is most often put
into a 10 to 20-fold volume of the fresh medium.
Isolation of a producing microorganism from one cell
The spores are transferred from an agar slope into a volume of 10 ml of sterile
H2O and, after homogenization, the suspension is diluted to contain 30-50 spores (Fig.
1D) in 1 ml. A volume of 0.25 ml of this suspension is transferred on the surface of a
suitable agar medium on a Petri dish and spread with a sterile glass stick. Colonies, each
of which originates from one cell, grow on the agar. The individual colonies are reinoculated to agar slopes and their antibiotic production is tested.
Conservation of microorganisms
If cultures are conserved for a long time on agar slopes, being repeatedly
transferred from one slope to another, they can degenerate and lose valuable
technological properties. Two types of conservation are recommended for long term
storage of strains: lyophilization (microbial cells or spores are conserved by quick
removal of water by sublimation at a low temperature) or conservation by keeping
cultures at a very low temperature (-70oC) in liquid nitrogen. In both cases cultures keep
their properties for at least 10 years.
Laboratory cultivation
Cultivation in the laboratory, irrespective of the fact whether the microorganism
will finally be used for inoculation of a production fermentor or in laboratory
experiments, is carried out in test tubes or in 200-1000-ml bottles and flasks. The
volume of the culture medium mostly represents about one tenth of the total volume of
the flask. The flasks are sealed with stoppers allowing diffusion of the air into the flasks
to ensure aerobic conditions for growth. At the same time, the stoppers prevent
microorganisms from the environment to penetrate into the flasks (cotton-wool stoppers,
etc.). Producers of antibiotics require a proper aeration, that is important for both the
growth and production of the antibiotic. Therefore, the flask contents is well mixed by
agitation on rotary or reciprocal shakers placed in thermostated rooms or boxes. Strictly
sterile conditions have to be ensured for the cultivation of antibiotic producers since, in
the case of contamination, the producing culture can be suppressed by more rapidly
growing microorganisms.
Cultivation in fermentors
Microbial producers of antibiotics are cultivated in fermentors of various size.
The lower limit of size of laboratory fermentors is about 1 litre. Owing to the use of
complex media, foam is often formed during cultivation and, therefore, the fermentors
are filled with the medium up to one half or two thirds of their maximal capacity. When
the process of antibiotic production is scaled up from the laboratory conditions to those
of true production, basic parameters can be established using several-litre, laboratory
fermentors. However, they should be verified in pilot plant fermentors having a size of
several cubic meters. The basic equipment of both laboratory and pilot plant fermentors
22
is practically the same. They are made of inert materials such as glass and stainless steel,
or their walls are at least lined with an inert material. The fermentors are equipped with
a device keeping the cultivation temperature constant (mainly cooling device is
important in large fermentors) and with an efficient aeration system, since antibiotic
producers require a sufficient oxygen supply for both the growth and synthesis of the
antibiotic. The aeration systems based on intensive stirring are not suitable for
cultivation of antibiotic producers since a majority of them are filamentous
microorganisms that can suffer damage when intensively stirred. The air flowing into
the fermentor has to be sterile. It is sterilized by filtration; most often glass wool or
mineral wool filters are used.
The fermentor is also usually equipped with a device ensuring automatic dosage of
antifoaming agents. Antifoaming agents, which increase interfacial tension, are used for
breaking the foam. Their amount should be minimal because they decrease solubility of
oxygen in the medium. Mixtures of various fatty acids (mainly oleic acid) or some
synthetic oils are used as antifoaming agents. An automatic foam breaker consists of a
probe that ensures the contact with the foam and, at the moment the contact is realized,
an impulse is given by the probe to the injector to add a small amount of the
antifoaming agent to the fermentor.
In order that the pH of the culture could be adjusted during cultivation, fermentors are
equipped with a device ensuring automatic dosage of acids and bases.
Most antibiotics are produced in a fed batch system, i.e. a certain amount of the
culture medium is inoculated with the producing microorganism and, after a time
interval, another dose of nutrients is added to the fermentor. Thus a prolonged
cultivation can be accomplished that enables us to increase the yield of the antibiotic.
The inflow of nutrients makes possible keep their optimal levels. In cultivations whose
course is well known, the nutrient inflow is programmed in advance.
Solid-state fermentation
Solid-state, or substrate, fermentation is characterized by a fermentation process
on a solid support, which has a low moistre content and occurs in a non-septic and
natural state [120]. The use of solid-state technology for the production of antibiotics
has some advantages. Due to the lack of free water, smaller fermentors are required and
the mycelial microorganisms, used predominantely for antibiotic production are well
suited to grow on a solid support. On the contrary, a liquid fermentation process
allows greater control and monitoring of parameters, such pH, heat, nutrient condition
etc [121].
Isolation, separation and purification of antibiotics
Isolation of an antibiotic from the fermentation medium depends on the fact
whether the antibiotic is secreted into the medium or remains in the biomass, inside the
cell or bound to the cell wall. If the antibiotic is bound to the biomass or, in contrast,
present in the broth supernatant, the two phases are separated by filtration or
centrifugation and extracted separately. If the antibiotic is present in both phases the
whole broth is used for extraction. Another isolation step usually includes an extraction
with solvents of different polarities, followed by evaporation of the extracts to dryness.
23
If the antibiotic is extractable by nonpolar solvents, the extraction is preceded by
dehydration, most often using methanol or acetone. By the extraction with nonpolar
solvents, a most part of water soluble compounds present in the medium is eliminated.
The crude isolate obtained is used as a material for further separation processes.
The antibiotic producers often synthesize a number of compounds or derivatives
of the desired compound that have to be separated from the antibiotic produced. The
separation is carried out using standard operations such as an extraction into another
solvent, chromatography techniques and, in the end, precipitation or crystallization. The
purity of the antibiotic compound is checked by thin-layer chromatography, gass
chromatography or HPLC. In the case of new compounds, their structure is determined
using spectroscopical methods such as nuclear magnetic resonance, mass spectroscopy,
and IR, UV or visible-light spectroscopy
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Figures.
Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7.
Fig. 8.
Fig. 9.
Fig. 10.
Fig. 11.
Fig. 12.
Fig. 13.
Fig. 14.
Fig. 15.
Fig. 16.
Fig. 17.
Fig. 18.
Fig.19.
Samples of streptomycetes mycelia were prepared according to standart
procedures used for biological samples preparation for scanning electron
microskopy. The samples were examined in Aquasem electron
microscope (A) or in Philips CM12/STEM electron microscope (B,C and
D) in STM mode. The samples were prepared and analysed in the
Laboratory of Electron Microscopy of the Institute of Microbiology,
Academy of Sicences, CR by Olga Kofroňová and Oldřich Benada.
Bacitracin
Bacitracin synthetase
Penicillins
Cephalosporins
Clavulanic acid
Vancomycin
Tetracyclines
Tetracycline biosynthesis
Tetracycline biosynthesis
Anthracyclines
Erythromycins
Tylosin and Relomycin
Nystatins
Avermectins
A - R5= OCH3; B - R5= OH; 1 - X= -CH=CH-; 2 = X= -CH2-CHOH-;
a - R26= C2H5; b - R26= CH3
Chloramphenicol
Streptomycines
6-aminopenicillanic acid and 7-aminocephalosporanic acid
Factor A, Factor B
33