Adv. Appl. Microbiol. 47, 113-156, 2000.
Bioactive Products from Streptomyces
VLADISLAV BĚHAL
Institute of Microbiology
Academy of Sciences of the Czech Republic
Prague, Czech Republic
I. Introduction
II. Chemistry and biosynthesis
A. Peptide and peptide-derivative antibiotics
B. Polyketide derivatives
C. Other groups of bioactive products
III. Genetics and molecular genetics
A. Preparation of high production microorganisms
B. Genetic manipulation of secondary metabolites producers
IV. Obtaining new bioactive secondary metabolites
A. Isolation from natural resources
B. Producers of bioactive compounds
C. Screening
D. Semisynthetic and synthetic bioactive products
E. Hybrid bioactive products and combinatorion biosynthesis
V. Regulation of secondary metabolites production
A.Growth phases of microbial culture
B. Control of fermentation by basal nutrients
C. How signals from the medium are received
D. Regulation by low molecular compounds
E. Autoregulators
F. Regulation by metal ions
VI. Resistance to secondary metabolites
A. Resistance of bioactive secondary metabolites producers
B. Resistance in pathogenic microorganisms
VII. References
I. Introduction
A. ANTIBIOTICS AND OTHER BIOACTIVE PRODUCTS
Medicine of twentieth century, especially its second half, was transformed by the
discovery of antibiotics and other bioactive secondary metabolites produced by
microorganisms. Antibiotics are defined as microbial products that inhibit the growth of
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other microorganisms. After the antibacterial effect of penicillin had been observed by
Fleming, a number of other antibiotics were discovered, mainly those produced by soil
Streptomyces and moulds. Moreover, a broad spectrum of natural products having other
effects on living organisms were found in microorganisms. In addition to standard
antibiotics, the following compounds have also been found: coccidiostatics used in
poultry farming, antiparasitic compounds with a broad spectrum of activity against
nematodes and arthropods, substances with antitumor activity, immunosuppressors,
thrombolytics (staphylokinase), compounds affecting blood pressure, end so forth.
Microbial metabolites also exhibit good herbicide and pesticide activities and are
biodegradable. However, microbial herbicides and pesticides only exceptionally used
(e.g. bialaphos) due to their high price.
Another special group of natural products are the enzyme inhibitors synthesized by
microorganisms (Umezawa et al., 1976). These compounds can inhibit antibiotic
derading enzymes, as well as certain enzyme activities in human metabolism that cause
illness. 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 also isolated from Streptomyces. The enzyme inhibitors
that block synthesis of cholesterol are also important. Other exhibit the
immunosuppressive effects, the most famous of them being cyclosporin A (a cyclic
undecapeptide) produced by filamentous fungi. Some macrolide antibiotics, isolated
from Streptomyces, are also immunosuppressives.
Several thousands biologically active compounds have been deseribed and each year
new compounds are isolated from microorganisms. Microorganisms are a virtually
unlimited source of novel chemical structures with many potential therapeutic
applications.
The therm "secondary metabolite" used for some microbial products Bu´Lock
(1961) and suitability of this therm discused Bennett and Bentley (1989). Secondary
metabolites are meant compounds that the microorganism can synthesize but they are
not essential for basic metabolic processes such as growth and reproduction.
Nevertheless many secondary compounds function as the so-called signal molecules,
used to control the producer’s metabolism. Another function attributed to antibiotics is a
suppression of competing microorganisms in the environment whereby the antibioticproducing microorganisms have an advantage in competing for nutrients with the other
microorganisms.
The production of secondary metabolites in microorganisms isolated from nature is
rather low in most cases.To be usable for the commercial production of secondary
metabolites, high yilding strains need to be selected through multiple mutations of the
strain´s genetic material, optimization of culture conditions and genetic engineering.
II. Chemistry and biosynthesis
In spite of variety of their structures, bioactive secondary metabolites are synthesized
from simple building units used in living organisms for the biosynthesis of cellular
structures. These units include amino acids, acetate, propionate, sugars, nucleotides, etc.
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According to their structure and type of biosynthesis, secondary metabolites are
classified to form several groups.
A. PEPTIDE AND PEPTIDE-DERIVATIVE ANTIBIOTICS
Microorganisms produce a number of peptides as secondary metabolites. These
peptide antibiotics are not synthetized on ribosomes but on enzyme complexes called
peptide synthetases (Lipmann et al., 1971; Laland and Zimmer, 1973). In peptide
antibiotic the peptide chain is often cyclic or branched. In addition to L-amino acids,
other compounds can also be present in the molecule, such as D-amino acids, organic
acids, pyrimidines and sugar molecules. The wellknown bioactive peptides, gramicidins
and bacitracins are produced by different strains of Bacillus licheniformis and Bacillus
brevis but some of them are produced by Streptomyces (Kleinkauf and von Doehren,
1986).
The linear molecule of gramicidin A (Fig. 1) and the cyclic molecule of gramicidin S
(Fig. 2) belong to the structurally simplest class of peptide antibiotics. Bacitracins are an
example of cyclic peptides having a side chain (Fig. 3). In the molecule of bleomycin,
the sugars L-glucose 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. The -CO-O- bond in the antibiotic molecule is present in lactones. Such
antibiotics are represented especially by the group of actinomycins that contain a
phenoxazine dicarboxylic group bearing two peptide chains.
The enniatine molecule consists of three residues of branched amino acids, L-valine,
L-leucine and L-isoleucine, and three residues of D-2-hydroxyisovaleric acid (D-Hyiv)
(Billich and Zocher, 1987). 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.
especially for the peptides capable of formating of chelates with metals. Studies showed
three-dimensional molecular structures with many hydrogen bonds (Iitaka, 1978). In the
case of valinomycin (L-Val-D-Hyiv-D-Val-L-Lac)3, which transports 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.
The biosynthesis of peptide antibiotics takes place on a multienzyme complex.
Kleinkauf and von Doehren,1983; Kleinkauf and von Doehren, 1986) 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 bonds is mediated by 4phosphopantetheine, an integral part of the multifunctional multienzyme. The
intermediate peptides are also bound to the synthetases by the thioester bond.
The way in which 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.
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Our knowledge of the biosynthesis of peptide antibiotics comes mostly from the
study of the gramicidin S and bacitracin synthetases.
Gramicidin S synthetase consists of two complementary enzymes having molecular
weights of 100 kD and 280 kD while bacitracin synthetase consists of three subunits
(Roland et al., 1977) (Fig. 4) having molecular weights of 200, 210 and 360 kD (Ishiara
et al., 1975). Each subunit contains phosphopantetheine. Enzyme A activates the first
five amino acids of bacitracin, 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 enzyme complex. Initiation and elongation start on subunit A
up to the pentapeptide, independently of the presence of the subunits B and C. The
pentapeptide is transferred to subunit B where two other amino acids are added. The
heptapeptide is subsequently transferred to subunit C where the biosynthesis of
bacitracin is finished. The cyclization is achieved by binding the asparagine carboxy
group to the epsilon-amino group of lysine, whereas, the isoleucine carboxyl group is
bound to the alpha-amino group of the same lysine (Laland et al., 1978).
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 action, the
primary one is defined to be the effect observed at the lowest active concentration. The
peptide antibiotics are efficient mainly against Gram-positive bacteria.
The b-lactams are peptide derived secondary metabolites. They are produced by
different microorganisms . Several review sumarise reseach in these area (Martin and
Liras, 1989; Jensen and Demain, 1995). The main producers are fungi (penicillins) but
they are produced also by Strepromyces ( clavulanic acid) and Cephalosporium
(cephalosporins). The main representatives of ß-lactams are penicillins and
cephalosporins. Penicillins have a thiazoline ß-lactam ring in the molecule and differ,
one from another, by side chains linked via the amino group (Fig. 5). Cephalosporins
have a basic structure similar to that of penicillins and the derivatives are also formed by
a variation of the side chain.
The thiazolidine ß-lactam ring is synthesized using three amino acids: L-alpha-amino
adipic acid, L-cystein and L-valine. By condensation of these three amino acids, a
tripeptide is formed. It is transformed to the molecule of penicillin or cephalosporin
through subsequent transformations (Fig. 6).
Clavulanic acid, produced by Streptomyces clavuligerus, also belongs to ßlactamfamily (Reading and Cole, 1977). This acid has a bicyclic ring structure
resembling that of penicillin, except that oxygen replaces sulfur in the five-membered
ring (Fig. 7.). 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.
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
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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.
Another example of amino acid bioactive substances are the glycopeptides including
semisynthetic derivatives (Zmijewski Jr. and Fayreman, 1995). The best known of all is
vancomycin (Fig. 8) (Harris and Harris, 1982), effective against gram-positive bacteria.
This antibiotic is widely used in medicine, especially against ß-lactam resistant strains.
Vancomycin is not absorbed from the gastrointestinal tract and is used to treat
enterocolitis caused mainly by Clostridium difficile.
Vancomycin is produced by many species, 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 acid core, derivatives of amino glycosides are
formed. In vancomycin the aminosugar vancosamine is bound to the amino acid core.
The removal of aminosugar reduces the activity of vancomycin two- to fivefold. The
sugars seem to play an important role in imparting the enhanced pharmacokinetic
properties for vancomycin-type, glycopeptide antibiotics.
B. POLYKETIDE-DERIVATIVES
Polyketides are a large group of secondary metabolites synthesized by decarboxylative
condensation malonyl units often with subsequent cyclization of the polyketo chain .
The starter group may be an acetate but also pyruvate, butyrate, ethyl malonate,
paraaminobenzoic acid, etc. The formation of the initial polyketo chain is similar to that
taking place during the biosynthesis of fatty acids, and is catalyzed by polyketide
synthases. Simple carboxylic acids are activated as thioesters (acyl-SCoA) which are
carboxylated to form malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA and after
decarboxylation polymerized. ( Lynen, and Reichert, 1951; Lynen, 1959; Lynen and
Tada, 1961). A principal role is played by the Acyl Carrier Protein (ACP) (Goldman
and Vagelos, 1962). ACP detected throughout the growth of Streptomyces glaucescens
was purified to homogenity and found to behave like many othes ACPs from bacteria
and plants (Sumers et al. 1995). 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 to the other -SH group, that is a
part of the cysteine molecule. Polyketide synthases have not yet been isolated and their
properties have been deduced from the analyses of DNA sequences of cloned genes.
Polyketide synthases include two distinct groups located either in domains on
multifunctional proteins or present on individual, monofunctional proteins (McDaniel
et al., 1993, Shen and Hutchinson, 1993). The structure and function of polyketide
synthase in antibiotics overwie Robinson (1991) and Bentley and Bennett (1999).
6-Methyl salicylic acid (6MS) represents one of the simplest polyketides formed by
condensation and subsequent aromatisation of one acetylCoA molecule and three
malonylCoA molecules. This compound was isolated from Penicillium patulum
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(Bu´Lock and Ryan, 1958). By other metabolic steps 6MS is transformed to produce a
toxin called patulin (Sekiguchi, 1983; Sekiguchi et al., 1983). The synthesis of 6MS
takes place on an enzymatic complex called 6MS synthetase (Fig. 9) (Dimroth et al.,
1970,1976).
The chemical structure of sometypical tetracyclines is shown in Fig. 10 and their
biosynthesis in Figs. 11 and 12 (McCormick, 1965). Chlortetracycline (CTC) and
tetracycline (TC) are produced by the actinomycete Streptomyces aureofaciens, whereas
oxytetracycline (OTC) and tetracycline by the actinomycete Streptomyces rimosus. For a
more extensive coverage of research, articles by Běhal et al. (1983), Běhal (1987) and
Běhal and Hunter (1995) should be 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 where other, less toxic antibiotics are not effective.
Anthracyclines are synthesized in a similar way as tetracyclines, however, they often
have one or several sugar residues in the molecule. Most often deoxy-sugars,
synthesized from glucose, are present in the anthracycline molecule. Daunorubicin and
doxorubicin (adriamycin) (Fig. 13) are excellent antitumor agents, which are widely
used in the treatment of a number of solid tumors and leukemias in human.
Unfortunately, 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, the drugs were linked to high
molecular compounds such as dextran (Levi-Schaff et al., 1982; Tanaka, 1994), DNA
(Campeneere, 1979), and others. Anthracyclines are produced by many Streptomyces
(Grein, 1987) and genetics of their production is well elaborated (Hutchinson, 1995).
Macrolides are usually classified to include: proper macrolides having 12-, 14- or 16membered 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. Both macrolides and polyenes are biosynthesized 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. Macrolides usually possess an
antibacterial activity whereas polyens are mostly fungicides.
Erythromycins produced by Saccharopolyspora erythrea (Fig. 14), together
with oleandomycin and picromycin, belong to the best known 14-membered lactone ring
macrolides (Harris et al., 1965). Macrolides with a 16-membered ring are represented by
tylosin (Fig. 15) (Omura et al., 1975), 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, pyruvate 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
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groups of the polyketide chain, from which macrolides are formed, are normally
transformed more frequently.
Nystatin is the best known polyene secondary metabolite (Fig. 16). Candicidine is
another well known secondary metabolite belonging to the polyene group. Its molecule
includes p-aminoacetophenone as the terminal group. 4-amino benzoic acid (PABA)
was identified as a precursor of the aromatic part of candicidine molecule (Liu et al.,
1972, Martin, 1977).
The sugars found in macrolide and polyene molecules are not usuallyencountered in
microbial cells. They include both basic and neutral sugar molecules and L-forms are
often found. So far, 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 (Corcoran and Chick, 1966), 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 (Martin and Gil, 1979). 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 secondary metabolites.
Avermectins consist of a 16-membered, macrocyclic lactone to which the disaccharide
oleandrose is bound (Fig. 17) (Burg, R.W., 1979; Miller, T.W., 1979). 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 active against a broad spectrum
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 and 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 (MacNeil, 1995; Ikeda
and Omura, 1995).
Polyethers form a large group of structural related natural products mainly
produced by Streptomyces (Birch and Robinson, 1995). They are potent coccidiostats
(monensin, salinomycin) and are used in the agricultural arena.(Westley, 1977).
Polyethers are compouns possesing the ability to form lipid-soluble complexes that
provide a vehicle for a wide variety of cations to traverse lipid barrieres. This ionbearing property led to their being named ionophores (Moore and Pressman, 1994).
Backbones of polyethers are synthetized from acetate, propionate and butyrate
(monensin A) units. Isobutyrate and n-butyrate are efficiently incorporated into
polyether antibiotics (Pospíšil et al., 1983). Incorporation of isobutyrate was explained
by formal conversion of isobutyryl-CoA into n-butyryl-CoA or methylmalonyl-CoA by
isobutyryl-CoA mutase and methylmalonyl-CoA mutase, respectively.
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C. OTHER GROUPS OF BIOACTIVE PRODUCTS
Chloramphenicol (Fig. 18) is produced by Streptomyces venezuelae (Vining and
Westlake, 1984). At present, however, 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.
Streptomycin (Fig. 19) is a well-known aminoglycoside antibioticoriginaly
discovered by Selnon Waksman. 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 steps of streptomycin
biosynthesis were disclosed mainly by Walker (Walker and Walker, 1971), who also
studied the relevant enzymes (Walker, 1975).
The importance of streptomycin consists mainly in its ability to suppress
Mycobacterium tuberculosis, resulting in effective suppression of tuberculosis,
especially in developed countries.
Bialaphos is formed from two L-alanine residues and the amino acid
phosphinothricine. The latter compound is synthesized by streptomycetes from
acetylCoA and phosphoenolpyruvate, and subsequently methylated using methionine as
the methyl donor (Bayer et al., 1972; Ogawa et al., 1973). The producing
microorganisms are Streptomyces hygroscopicus and Streptomyces viridochromogenes.
Bialaphos, as well as phosphinothricine, inhibits the activity of glutamine synthetase.
III. Genetics and molecular genetics
A. PREPARATION OF HIGH PRODUCTION MICROORGANISMS
The structural genes encoding the enzymes that synthesize secondary metabolites are
mostly located on chromosomes They are often organized in gene clusters (Binnie et al.,
1989; Malpartida and Hopwood, 1984; Lotvin et al., 1992; Martin, 1992). Resistance of
the producer to its own products are located either at the beginning or at the end of the
cluster, often in both positions. In addition to the resistance and structural genes,
regulatory genes are important in secondary metabolites production, however, they
function is poorly understand.
Microorganisms that are isolated from nature (wild type strains) produce small
amounts of secondary metabolites. Sometimes during selection and subsequent
cultivation in the laboratory, a changes occur, making the cultivated strain non-identical
with the original strain. In such cases it should be remarked that the term wild type
strain only refers to the fact that the strain did not undergo an “artificial” genetic change.
In order for the commercial production of secondary metabolites could be profitable,
higher levels of the secondary metabolites synthesis are reached via genetic changes of
producers. Mutants are isolated by exposure of spores to UV irradiation, X-rays, γ-rays,
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α-particles or chemical mutagens (nitrogen mustards, N-methyl-N´-nitro-N-nitroso
guanidine). Combined mutagenesis using various mutagens is often used. The surviving
spores give rise to individual colonies of isolates, whose capability of secondary
metabolites production is then tested. Mutants that exhibit poor growth and sporulation
ability are not suitable candidates for further improvement, even if their secondary
metabolites production may exceed that of the original strain. Today’s high production
strains, that synthesize as high as 10 000-fold levels of secondary metabolites, compared
to the original strains, are the result of many year of costly strain improvement.
Unfortunately, these high production strains can revert to lose their overproduction
though spontaneous mutagenesis.
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 a secondary metabolite intermediate whose transformation stopped due to the
absence of the corresponding enzyme. By crossing these mutants, some biosynthetic
pathways used to synthesize secondary metabolites were elucidated, e.g. tetracyclines
(McCormick et all.1960).
Loss of the capability of secondary metabolite 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 (Hotta et al, 1977; Okanishi,
1979; Akagava et al., 1979; Boronin et al., 1974; Ikeda et al., 1982).
B. GENETIC MANIPULATION OF SECONDARY METABOLITES PRODUCERS
Structural genes for a number of secondary metabolites have been cloned into host
microorganisms. Similarly, genes for secondary metabolites 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 secondary metabolites or of their high levels. The secondary
metabolites biosynthesis is a very complex process that requires not only the structural
genes for ESM but also the genes for regulation of their biosynthesis. Moreover, the
overproduction of a secondary metabolite has to be coordinated with the primary
metabolism of the producing microorganism.
The cloning of structural genes and genes for resistance to the own secondary
metabolite enables us to work out genetic maps of the producers. On the basis of those
maps, hybrid clusters combined of two and more clusters of different secondary
metabolites can be created. Consequently, semisynthetic secondary metabolites 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 (Hopwood and Sherman, 1990). As a result, a great similarity of
polyketide synthases from various streptomycetes was evidenced (Malpartida et al.,
1987; Butler et al., 1990).
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IV. Obtaining new bioactive secondary metabolites
A. ISOLATION FROM NATURAL RESOURCES
In spite of the fact that several thousands of compounds isolated from
microorganisms having some biological activity are known, new substances are still
saught by pharmaceutical companies. The probability of finding a new compound that
would be usable as a new antibiotic or another biologically active compound is low, so a
great number of microorganisms have to be screened. A rough estimation says that
about 100 000 microorganisms are screened for the presence of biologically active
compounds per year. Modern screens are highly automated. The selection methods
used, the targets, and the methods of detection of the biological activity are normally
not published.
Preparation of a new biologically active compound and its introduction into clinical
practice requires the cooperation of scientists from various scientific disciplines and
years of clinical trials. This effort can be divided into three parts:(Yarbrough et al.,
1993):
1. microbiology
-collection of source samples (soil)
-isolation of diverse microbes
-fermentation to enhance diversity
-reproduce fermentation
-enhance the production for isolation
-taxonomy of the organism
2. molecular biology/pharmacology
-target selection
-screen design/implementation
-high through-put screening
-identification of active compounds
-efficacy studies
-mechanism of action
3. chemistry
-active compound identification
-characterisation/dereplication
-isolation/purification
-structure elucidation.
B. PRODUCERS OF BIOACTIVE COMPOUNDS
About 70 % of the known bioactive substances are produced by Streptomyces and the
rest mainly by moulds and non-filamentous bacteria. With an increasing spectrum of
efficiency of microbial metabolites, new, non-traditional sources of such compounds
have been tappede. These include the microorganisms living under extreme conditions
(high and low temperatures, etc.), sea living microorganisms, and multicelular plants
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and animals. Another important source of new compounds are the mutants of producers
of known active substances, e.g. blocked mutants.
B. SCREENING
The enterprise of screening microbial metabolites 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 antibiotic efficiency consisted of the
application of test extract to wells made in agar medium layer in Petri dishes to which
the sensitive (target) microorganism was inoculated. Most often Staphylococcus aureus,
Sarcina lutea, Klebsiella pneumoniae, Salmonella gallinarium, Pseudomonas spp.,
Bacillus subtilis, and Candida albicans were used. In case a compound with an
antibiotic activity towards the testing microorganism was put into the well, it diffused
through the agar medium and a halo was formed around the well, as a result of the
suppressed growth of the microorganism. This classic plate assay has been modified and
improved in many ways.
The tests of other biological activities require different and frequently sophisticated
methods. This is true especially when enzyme inhibitors are a case in point. Thus,
Ogawara et al. (1986) chose a tyrosine protein kinase associated with the malignant
transformation of the cell caused by retroviruses as the target in a biochemical screen,
they 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 (Take et al., 1989).
D. SEMISYNTHETIC AND SYNTHETIC BIOACTIVE PRODUCTS
Natural products can be modified in various ways. The unspecificity of the enzyme
systems facilitates the synthesis of certain secondary metabolites though the addition of
selectedprecursors to the growth medium. Thus, the reaction equilibrium can be shifted
to promote the production of the derivative required, e.g. the prepareation of penicillins
with different side chains. The individual derivatives of penicillin and cephalosporin
have slightly different antimicrobial spectra and are active against microorganisms
resistant to other derivatives. The structuure of polypeptide antibiotics can also be
modified by the addition of amino acids to the growth
Replacement of a part of the metabolite molecule can be accomplished chemically or
enzymatically. In this way, semisynthetic penicillins, cephalosporins, tetracyclines and
other antibiotics can be prepared. The production of semisynthetic penicillins and
cephalosporins is facilitated by the fact that 6-amino penicillanic and 7-amino
cephalosporanic acids are easily prepared.
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The side chain is removed by the action of an enzyme or by a chemical hydrolysis
(Fig. 20) then another acyl is bound chemically or enzymatically to the amino group in
position 6 (penicillins) or 7 (cephalosporins).
Semisynthetic tetracyclines, pyrolinomethyltetracycline, metamycin and doxycycline,
exhibit a greater solubility and somewhat different antimicrobial spectrum, as compared
to the original tetracyclines.
New derivatives of aminoglycosides also have been obtained by chemical and
enzymatic modifications.
As the majority of bioactive products 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.
E. HYBRID BIOACTIVE PRODUCTS
Genetic engineering methods have recently advanced so much that now we can
suitably combine structural genes of two or even more bioactive secondary metabolites
producers. If these genes are expressed, a hybrid bioactive products is synthesized, ore
that cannot be found in nature (Hutchinson, 1987, 1988; Tomich, 1988; Hopwood,
1993). Hopwood et al. (1985, 1986,) used this method with the genes of actinorhodin
synthesis and obtained related hybrid macrolides, mederhodin A and B,
dihydromederhodin A and dihydrogranatirhodin. A new anthracyclines were produced
when a DNA segment was cloned from Streptomyces purpurascenc ATCC 25489 close
to a region that hybridized to a probe containing part of the actinorhodin polyketide
synthase Streptomyces galilaeus ATCC 31615 (Niemi et al., 1994).
V. Regulation of secondary metabolites production
A. GROWTH PHASES OF Stepromyces
In the cultures of Streptomyces capable of secondary metabolite production several
growth phases representing different physiological statescan be distinguished:
1. Preparatory phase (lag phase) - the biomass increase is low, the culture is adapting
to the new environment.
2. Growth phase (the term logarithmic phase is not suitable for most Streptomyces
since their growth curves are not exponential functions) - intensive growth is taking
place, accompanied by a low secondary metabolite synthesis. This phase is roughly
equivalent to "trophophase".
3. Transition phase - characterized by a decreased growth rate; the secondary
metabolite production is started. The enzymes of secondary metabolism are
synthesized (Běhal, 1986a; Běhal, 1986b) and proteosynthesis slowed down.
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4. Production phase - characterized by a significant reduction of the growth rate
(sometimes growth is even completely ceased), a negligible change in the biomass
concentration, and an intensive synthesis of the secondary metabolite. This phase is
some times called "idiophase".
Producers of secondary metabolites mostly belong filamentous bacteria or fungi,
which means that in their culture cells of various age and at different stages of
development are present. The microorganisms grow in pellets, inside which the
cultivation conditions differ from those on the pellet surface (nutrient concentrations,
oxygen concentration, etc.). An increase in dry weight does always correlation with an
increase growth since, in streptomycetes, often a thickening of the cell wall or
glycocalyx formation occur that increase the dry weight value without rising the number
of cell (Voříšek et al., 1983). Since individual cells of a fermentation can be at different
stages of development, (i.e. in different physiological states). The physiological state of
the whole culture represents an average of physiological states of the individual cells.
B. CONTROL OF FERMENTATION BY BASAL NUTRIENTS
In order to reach a high yieldof secondary metabolite, sufficient biomass is required.
Moreover to danger of contamination is diminished and the economic parameters of the
fermentation device are optimal if the growth is rapid. For this purpose, readily
utilizable sources of carbon, nitrogen and phosphorus sources (e.g. molasses, corn
starch, etc) are used. However, production of the secondary metabolite does not usually
take place until one or more nutrients become limited.Thefore, the culture medium
should be designed in such a way that after the biomass increased sufficiently, at least
one of the nutrient sources will become depleted. Carbon source, nitrogen source and
phosphate limitation have been described as important triggers in different systems.
Most secondary metabolites 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 fermenter. Thus a prolonged
cultivation can be accomplished that enables us to increase the yield of the secondary
metabolite. The inflow of nutrients makes possible keep their optimal levels. An
example of how a production cultivation of Streptomyces aureofaciens can look like is
shown in Fig. 21 (Běhal, 1987). In cultivations whose course is well known, the nutrient
inflow is programmed in advance
The inhibition of penicillin synthesis by glucose was observed shortly after its
discovery in media containing glucose and lactose (Demain, 1974). 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 (Demain, 1983). 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 secondary metabolites synthesis by
readily utilizable sugars probably consists in a repression of enzymes of secondary
metabolism (Revilla, G. et al., 1986; Erban, et al., 1983).
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Readily utilizable nitrogen sources can also negatively influence the production of
secondary metabolites.Ammonium ions often decrease secondary metabolite synthesis
and, therefore, their concentration in production media is limited while, soy flour,
peanut flour and other substances are preffered nitrogen sources. These latter nitrogen
sources are more similar to those used by the microorganisms producing secondary
metabolites in nature. Readily utilizable nitrogen sources repress enzymes of secondary
metabolism in Cephalosporium acremonium (Shen et al., 1984) during the biosynthesis
of cephalosporin and in Streptomyces clavuligerus producing cephamycin (Demain and
Brana, 1986). Similarly, the inhibition of biosyntheses of leucomycin (Omura et al.,
1980a), tylosin (Omura et al., 1980b), and erythromycin (Flores and Sánches, 1985) are
explained by the repression of enzymes of secondary metabolism. Ammonium salts also
inhibit the activity of anhydrotetracycline oxygenase isolated from S. aureofaciens
(Běhal et al., 1983).
The overproduction of most secondary metabolites can be achieved only if phosphate
is limited. Inorganic phosphate has to be carefully added in doses to the medium so as to
accomplish an optimal ratio between biomass production and secondary metabolite
biosynthesis. When bound to organic compounds normally added to medium (soy flour,
etc.), phosphate does not affect secondary metabolite production. In general secondary
metabolite biosynthesis is started when the concentration of phosphate decreased below
a certain level. At this point, the producer culture undergoes a shift from the
physiological state characteristic for the growth phase to that of the overproduction
phase.
Inorganic phosphate also causes a repression of the synthesis of enzymes of secondary
metabolism (Běhal et al., 1979b; Madry and Pape; 1981, Martin et al., 1981). After
phosphate was depleted from the medium, a significant decrease of the rate of
proteosynthesis was observed during tetracycline (Běhal,1982). If phosphate was kept
above the threshold concentration, the significant decrease of the rate of protein
synthesis did not occur and ESM 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 enzymes of secondary metabolism levels in the culture and an
acceleration of proteosynthesis.
C. HOW SIGNALS FROM THE MEDIUM ARE RECEIVED
Reception of signals from the environment, that result in the initiation of the
secondary metabolite synthesis does not significantly differ from the transduction 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 ( Doull and Vining, 1995). With some structural
varietion, these proteins are characterized by common mechanistic features and
conserved amino acid sequences.
The two-component system consists of a cytoplasmic membrane-linked, sensortransmitter 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-end is
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found outside the cytoplasm. A specific effector is capable of binding directly to this Nend. 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 histidine-protein kinases, capable of autophosphorylation
at their C-ends on receiving a proper signal. The phosphorylated protein becomes a
donor in reactions transferring phosphorus. The acceptor is the cytoplasmic, responseregulator protein. Two-component signal proteins thus transfer the information
concerning the conditions that can affect the cell action.
D. REGULATION BY LOW MOLECULAR COMPOUNDS
The expression of structural genes is also regulated by some low molecular
compounds. The mechanism of their action is not understood. For example tryptophan
exhibited a stimulatory effect on the production of mucidin in the basidiomycete
Oudemansiella mucida (Nerud et al., 1984) and actinomycin in Streptomyces parvulus
(Troast et al., 1980). Methionine was found to promote the synthesis of cephalosporin C
(Nuesch et al., 1973). Neither tryptophan nor methionine were used as building units for
these metabolites.
Benzyl thiocyanate is one of the low molecular compounds that affect the
chlortetracycline biosynthesis. It increases the production of both chlortetracycline and
tetracycline in S. aureofaciens, although, it does not influence the production of
oxytetracycline in S. rimosus. The effect on the metabolism of S. aureofaciens is
multiple (Novotná et al., 1995), including a number of enzymes, including the enzymes
of secondary metabolism (Běhal et al., 1982). Benzyl thiocyanate is able to raise the
level of secondary metabolite 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 enzyme level and chlortetracycline production are
increased 10 to 20-fold, as compared to high production strains where the increase is
only twofold. .
E. AUTOREGULATORS
Streptomycetes low-molecular, diffusible compounds have been discovered that
regulate the metabolism of producing strain (Horinuchi and Beppu, 1990; Horinuchi
and Beppu, 1992). The most famous of them is factor A, γ-butyrolactone (Fig. 22), that
was discovered in Streptomyces griseus (Khokhlov et al., 1969; Khokhlov, 1982). A
non-producing strain started the synthesis of streptomycin after factor A was added to
the culture simultaneously, the coltura formed aerial mycelium. Factor A is synthesized
by many streptomycetes but the regulatory effect was observed only in Streptomyces
griseus, Streptomyces bikiniensis and Streptomyces actuosus (Ohkishi et al., 1988). The
addition of factor A to blocked mutants of Streptomyces griseus JA 5142, caused
resumption of the synthesis of anthracyclines and leukaemomycin (anthracycline type
antibiotic) (Graefe et al., 1983). The resistance to streptomycin linked with an enzymatic
phosphorylation of the antibiotic is also induced by factor A (Hara and Beppu, 1982).
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Analogues of factor A have also been found, all of them being γ-butyrolactones.
Virginiae butanolides were detected in Streptomyces virginiae (Yanagimoto et al.,
1979). Factor I was isolated from Streptomyces sp. FR1-5 (Sato et al., 1989) and its
effective concentration was 0.6 ng/ml culture. Most of the factor A analogues, however,
were not biologically active.
Factor B was isolated from the yeast Saccharomyces cerevisiae. This substance was
capable of eliciting the production of rifamycin in a blocked mutant of Nocardia sp.
(Fig. 23) (Kawaguchi et al., 1984). Factor B was effective at a concentration of 10-8 M,
with one molecule eliciting a synthesis of about 1500 molecules of the rifamycin. 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. Activity was observed with those that contained a C2 -C12 acyl
moiety; octylester was the most effective of them (Kawaguchi et al., 1988). 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 (Szabo et al., 1967).
Factor C is a protein having a molecular weight of about 34 500 D, and is rich in
hydrophobic amino acids.
The effect of autoregulators is easily observable if they elicit morphological changes
such as the formation of aerial mycelium. Carbazomycinal and
6-methoxcarbazomycinal, isolated from Streptoverticillium species, inhibit of the aerial
mycelium formation at a concentration of 0.5 to 1 microgram per ml. Autoregulators
affecting sporulation were found in Streptomyces venezuelae (Scribner et al., 1973),
Streptomyces avermitilis (Novák et al., 1992), and Streptomyces viridochromogenes
NRRL B-1551 (Hirsch and Ensign, 1978). From the same strain of Streptomyces
viridochromogenes, germicidin was isolated by Petersen and coworkers (1993). The
compound had an inhibitory effect on the germination of arthrospores of Streptomyces
viridochromogenes at a concentration as low as 40 picogram per ml. Germicidin (6-(2butyl)-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 and also from the supernatant of a submerged culture.
Mutants of Streptomyces cinnamonensis resistant to high concentrations of butyrate
and isobutyrate produce an anti-isobutyrate (AIB) factor that is excreted into the culture
medium (Pospíšil, 1991). On plates, AIB factor efficiently counteracted toxic
concentrations of isobutyrate, acetate, propionate, butyrate, 2-methylbutyrate, valerate,
and isovalerate in Streptomyces cinnamonensis and other Streptomyces species.
F. REGULATION BY PHOSPHORYLATED NUCLEOTIDES
Global control mechanisms for secondary metabolites biosynthesis have been
investigated. 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 secondary metabolite synthesis is linked with a decrease of
the intracellular ATP level. Such a relationship was observed in Streptomyces
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aureofaciens and Streptomyces fradiae during the production of tetracycline (Janglová
et al., 1969; Čurdová et al., 1976) and tylosin (Madry et al., 1979; Vu-Truong et al.,
1980), 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 accompanies active primary
metabolism. A slow down of growth and primary metabolism is accompanied by a
decrease of the ATP level.
The role of cAMP in the metabolism of secondary metabolites producers was also
studied, especially in connection with glucose regulation. Hitherto, no indication has
been obtained suggesting a specific role of cAMP in the regulation of secondary
metabolites production (Cortéz et al., 1986; Chatterjee and Vining, 1981).
G. REGULATION BY METAL IONS
Metal ions act as a part of enzyme active centers. The optimal concentrations of metal
ions for cultivation of the secondary metabolites producing strains have usually been
determined empirically. In complex media it is generally not necessary to add specific
metal ions, however in defined media their presence is essential.
VI. Resistance to bioactive products
Resistance against bioactive products has been studied mainly in antibiotic
producers. Antibiotic resistance is usually looked at from two angles: first, the
emergence of drug rezsstant strain and second, "self resistance" of antibiotics producing
strains. The ways in which these two types of resistance are achieved is often similar.
A. RESISTANCE OF SECONDARY METABOLITES PRODUCERS
Basic metabolic processes of wild type, secondary metabolite producing
microorganisms are not inhibited if the secondary metabolires are synthesized at low
concentrations. After strain improvement, strains with 100 to 1000-fold increases
insecondary metabolite yields have been isolated. Genome changes of the improved
strains include a number of deletions and amplifications in the chromosomal DNA, as
well as changes in extrachromosomal DNA.
Low production strains, whose resistance to the own product is low (i.e. higher
concentrations of the product inhibit their growth), regulate the secondary metabolite
production by inhibiting the enzyme activities that participate in the synthesis of the
secondary metabolite. In high production strains, such controls are lost and the strains
have to find a way how to survive in the presence of a high concentration of the
antibiotic (Vining, 1979).
The genes for self resistance are often located at the beginning of the cluster of
structural genes. As a result, they are expressed simultaneously with the structural
genes. The genes of newly gained resistances, however, are mostly located on plasmids.
Some antibioticsfunction by hitting active centres of enzymes. However, if active
centre is modified, the antibiotic cannot bind to it and then resistance comes into
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existence. It is not known whether a decreased ability to bind the secondary metabolites
results from a posttranslational modification of the active centre or if resistant
molecules of the enzyme are synthesized de novo. Clear evidence in support of the latter
situation has sofar been brought.
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
reduced synthesis of guanosine penta- and tetraphosphates (Weiser et al., 1981). The
antibiotic producers (mostly Streptomyces), 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 the 23S subunit. Such
modified ribosomes do not bind the antibiotic. In other cases, adenine is methylated to
yield 2-O-methyladenosine (Cundliffe and Thompson, 1979; Mikulík et al.,1983;
Thompson et al., 1982). 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 secondary metabolites
producers seems to be export from the cell to the environment. 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(Ohnuki et al., 1985).
Producers bioactive secondary metabolites also have to solve the problem of a reverse
flow of products into the cell. Some secondary metabolites are 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
secondary metabolites. This kind of resistance is thought to be connected with the
content of phospholipids in the cell.
Secondary metabolite producers can use several types of resistance simultaneously.
Tetracyclines, that strongly inhibit protein synthesis, interfere with the binding of the
ternary complex of amino acyl-tRNA-EFTu-GTP to ribosomes (Gavrilova et al., 1976).
The genes for resistance were cloned into Streptomyces griseus, sensitive to
tetracyclines, using pOA15 as the vector plasmid (Ohnuki et al., 1985). 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
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. The inhibiting concentrations of chlortetracycline in Streptomyces
aureofaciens are higher in the production phase as compared to the growth phase (Běhal
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et al., 1979a). Thus, the resistance can be increased even during the fermentation
process.
Another way secondnary metabolite producers can avoid the effect of their products is
to situate the distal enzymes of secondary metabolite biosynthetic pathway (synthases)
outside the cell, most often in the periplasm. In Streptomyces aureofaciens, a higher
proportion of the terminal enzyme of tetracycline synthase was found under high
production conditions in periplasm, as compared to low production conditions (Erban et
al.,1985).
B. RESISTANCE IN PATHOGENIC MICROORGANISMS
Shortly after antibiotics were introduced into clinical practice on a massive scale,
strains of hitherto-sensitive microorganisms started to appear. These resistant strains
required the use of much higher antibiotic concentrations or, 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 in which microorganisms can gain resistance (Ogawara,
1981). These include:
1. Creation of an alternative metabolic pathway producing a compound whose
biosynthesis is blocked by the bioactive metabolite; 2. Production of a metabolite that
can antagonize the inhibitory effects of the bioactive metabolite; 3. Increase of the
amount of the enzyme inhibited by the secondary metabolite; 4. Decrease of the cell’s
metabolic requirement for the reaction inhibited by the secondary metabolite; 5.
Detoxification or inactivation of the bioactive metabolite; 6. Change of the target site; 7.
Blocking of the transport of the bioactive metabolite into the cell.
In most resistant microorganisms, the mechanisms of resistance mentioned in the
items 5, 6 and 7 are encountered.
Penicillins and cephalosporins are degraded using three ways: by the enzyme
penicillin amidase that cleaves the amidic bond by which the side chain is bound to the
β-lactam ring; by the enzyme acetyl esterase that hydrolyzes the acetyl group at C-3 on
the dihydrazine ring of cephalosporins and 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, however these enzymes 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-lactams are inactivated by β-lactamase that
destroys one of the important sites for their antibiotic activity; the damage is
irreversible.
Β-lactamases, however, are not only synthesized by microorganisms that came into
contact with penicillins. Constitutive synthesis of these enzymes have been found in
three quarters of all streptomyces strains, (Ogawara et al., 1978). One can suppose that
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20
the genes for the synthesis of β-lactamases were transferred horizontally. Recent studies
indicate frequent and promiscuous gene transfer even between distantly related bacterial
species. A possibility of direct transfer from a streptomycete to a pseudomonad, for
example, may seem 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 (Davis, 1992).
Another way of inactivating a bioactive metabolite molecule is N-acetylation of the
amino group or O-phosphorylation of the hydroxyl. Bialaphos was found to be
inactivated by acetylation. These substance itself is not toxic but, in the cell,
phosphinothricine is liberated that inhibits glutamine synthetases, key enzymes of the
inorganic nitrogen assimilation pathway.
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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.
Gramicidin A
Gramicidin S
Bacitracin
Bacitracin synthetase
Penicillins
Cephalosporins
Clavulanic acid
Vancomycin
6-methyl salicylic acid synthetase
Tetracyclines
Tetracycline biosynthesis
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Fig. 12.
Fig. 13.
Fig. 14.
Fig. 15.
Fig. 16.
Fig. 17.
Fig. 18.
Fig.19.
Fig. 20.
Fig. 21.
Fig. 22.
Fig. 23.
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
Parameters of an industrial fermentation of S. aureofaciens
1 - chlortetracycline production (g/l); 2 - ATC-oxygenase (pkat/ mg
proteins x 2); 3 - NH3-nitrogen (g/l x 0.1); sucrose (g/l x 10);
5 - pH; ammonium supplement were added at points A and B
Factor A
Factor B
Adv. Appl. Microbiol. 47, 113-156, 2000.
Bioactive Products from Streptomyces
VLADISLAV BĚHAL
Institute of Microbiology
Academy of Sciences of the Czech Republic
Prague, Czech Republic
I. Introduction
II. Chemistry and biosynthesis
A. Peptide and peptide-derivative antibiotics
B. Polyketide derivatives
C. Other groups of bioactive products
III. Genetics and molecular genetics
B. Preparation of high production microorganisms
B. Genetic manipulation of secondary metabolites producers
IV. Obtaining new bioactive secondary metabolites
A. Isolation from natural resources
B. Producers of bioactive compounds
C. Screening
D. Semisynthetic and synthetic bioactive products
E. Hybrid bioactive products and combinatorion biosynthesis
V. Regulation of secondary metabolites production
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A.Growth phases of microbial culture
B. Control of fermentation by basal nutrients
C. How signals from the medium are received
D. Regulation by low molecular compounds
E. Autoregulators
F. Regulation by metal ions
VI. Resistance to secondary metabolites
A. Resistance of bioactive secondary metabolites producers
B. Resistance in pathogenic microorganisms
VII. References
I. Introduction
C. ANTIBIOTICS AND OTHER BIOACTIVE PRODUCTS
Medicine of twentieth century, especially its second half, was transformed by the
discovery of antibiotics and other bioactive secondary metabolites produced by
microorganisms. Antibiotics are defined as microbial products that inhibit the growth of
other microorganisms. After the antibacterial effect of penicillin had been observed by
Fleming, a number of other antibiotics were discovered, mainly those produced by soil
Streptomyces and moulds. Moreover, a broad spectrum of natural products having other
effects on living organisms were found in microorganisms. In addition to standard
antibiotics, the following compounds have also been found: coccidiostatics used in
poultry farming, antiparasitic compounds with a broad spectrum of activity against
nematodes and arthropods, substances with antitumor activity, immunosuppressors,
thrombolytics (staphylokinase), compounds affecting blood pressure, end so forth.
Microbial metabolites also exhibit good herbicide and pesticide activities and are
biodegradable. However, microbial herbicides and pesticides only exceptionally used
(e.g. bialaphos) due to their high price.
Another special group of natural products are the enzyme inhibitors synthesized by
microorganisms (Umezawa et al., 1976). These compounds can inhibit antibiotic
derading enzymes, as well as certain enzyme activities in human metabolism that cause
illness. 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 also isolated from Streptomyces. The enzyme inhibitors
that block synthesis of cholesterol are also important. Other exhibit the
immunosuppressive effects, the most famous of them being cyclosporin A (a cyclic
undecapeptide) produced by filamentous fungi. Some macrolide antibiotics, isolated
from Streptomyces, are also immunosuppressives.
Several thousands biologically active compounds have been deseribed and each year
new compounds are isolated from microorganisms. Microorganisms are a virtually
unlimited source of novel chemical structures with many potential therapeutic
applications.
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The therm "secondary metabolite" used for some microbial products Bu´Lock
(1961) and suitability of this therm discused Bennett and Bentley (1989). Secondary
metabolites are meant compounds that the microorganism can synthesize but they are
not essential for basic metabolic processes such as growth and reproduction.
Nevertheless many secondary compounds function as the so-called signal molecules,
used to control the producer’s metabolism. Another function attributed to antibiotics is a
suppression of competing microorganisms in the environment whereby the antibioticproducing microorganisms have an advantage in competing for nutrients with the other
microorganisms.
The production of secondary metabolites in microorganisms isolated from nature is
rather low in most cases.To be usable for the commercial production of secondary
metabolites, high yilding strains need to be selected through multiple mutations of the
strain´s genetic material, optimization of culture conditions and genetic engineering.
II. Chemistry and biosynthesis
In spite of variety of their structures, bioactive secondary metabolites are synthesized
from simple building units used in living organisms for the biosynthesis of cellular
structures. These units include amino acids, acetate, propionate, sugars, nucleotides, etc.
According to their structure and type of biosynthesis, secondary metabolites are
classified to form several groups.
A. PEPTIDE AND PEPTIDE-DERIVATIVE ANTIBIOTICS
Microorganisms produce a number of peptides as secondary metabolites. These
peptide antibiotics are not synthetized on ribosomes but on enzyme complexes called
peptide synthetases (Lipmann et al., 1971; Laland and Zimmer, 1973). In peptide
antibiotic the peptide chain is often cyclic or branched. In addition to L-amino acids,
other compounds can also be present in the molecule, such as D-amino acids, organic
acids, pyrimidines and sugar molecules. The wellknown bioactive peptides, gramicidins
and bacitracins are produced by different strains of Bacillus licheniformis and Bacillus
brevis but some of them are produced by Streptomyces (Kleinkauf and von Doehren,
1986).
The linear molecule of gramicidin A (Fig. 1) and the cyclic molecule of gramicidin S
(Fig. 2) belong to the structurally simplest class of peptide antibiotics. Bacitracins are an
example of cyclic peptides having a side chain (Fig. 3). In the molecule of bleomycin,
the sugars L-glucose 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. The -CO-O- bond in the antibiotic molecule is present in lactones. Such
antibiotics are represented especially by the group of actinomycins that contain a
phenoxazine dicarboxylic group bearing two peptide chains.
The enniatine molecule consists of three residues of branched amino acids, L-valine,
L-leucine and L-isoleucine, and three residues of D-2-hydroxyisovaleric acid (D-Hyiv)
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(Billich and Zocher, 1987). 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.
especially for the peptides capable of formating of chelates with metals. Studies showed
three-dimensional molecular structures with many hydrogen bonds (Iitaka, 1978). In the
case of valinomycin (L-Val-D-Hyiv-D-Val-L-Lac)3, which transports 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.
The biosynthesis of peptide antibiotics takes place on a multienzyme complex.
Kleinkauf and von Doehren,1983; Kleinkauf and von Doehren, 1986) 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 bonds is mediated by 4phosphopantetheine, an integral part of the multifunctional multienzyme. The
intermediate peptides are also bound to the synthetases by the thioester bond.
The way in which 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.
Our knowledge of the biosynthesis of peptide antibiotics comes mostly from the
study of the gramicidin S and bacitracin synthetases.
Gramicidin S synthetase consists of two complementary enzymes having molecular
weights of 100 kD and 280 kD while bacitracin synthetase consists of three subunits
(Roland et al., 1977) (Fig. 4) having molecular weights of 200, 210 and 360 kD (Ishiara
et al., 1975). Each subunit contains phosphopantetheine. Enzyme A activates the first
five amino acids of bacitracin, 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 enzyme complex. Initiation and elongation start on subunit A
up to the pentapeptide, independently of the presence of the subunits B and C. The
pentapeptide is transferred to subunit B where two other amino acids are added. The
heptapeptide is subsequently transferred to subunit C where the biosynthesis of
bacitracin is finished. The cyclization is achieved by binding the asparagine carboxy
group to the epsilon-amino group of lysine, whereas, the isoleucine carboxyl group is
bound to the alpha-amino group of the same lysine (Laland et al., 1978).
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 action, the
primary one is defined to be the effect observed at the lowest active concentration. The
peptide antibiotics are efficient mainly against Gram-positive bacteria.
The b-lactams are peptide derived secondary metabolites. They are produced by
different microorganisms . Several review sumarise reseach in these area (Martin and
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Liras, 1989; Jensen and Demain, 1995). The main producers are fungi (penicillins) but
they are produced also by Strepromyces ( clavulanic acid) and Cephalosporium
(cephalosporins). The main representatives of ß-lactams are penicillins and
cephalosporins. Penicillins have a thiazoline ß-lactam ring in the molecule and differ,
one from another, by side chains linked via the amino group (Fig. 5). Cephalosporins
have a basic structure similar to that of penicillins and the derivatives are also formed by
a variation of the side chain.
The thiazolidine ß-lactam ring is synthesized using three amino acids: L-alpha-amino
adipic acid, L-cystein and L-valine. By condensation of these three amino acids, a
tripeptide is formed. It is transformed to the molecule of penicillin or cephalosporin
through subsequent transformations (Fig. 6).
Clavulanic acid, produced by Streptomyces clavuligerus, also belongs to ßlactamfamily (Reading and Cole, 1977). This acid has a bicyclic ring structure
resembling that of penicillin, except that oxygen replaces sulfur in the five-membered
ring (Fig. 7.). 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.
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.
Another example of amino acid bioactive substances are the glycopeptides including
semisynthetic derivatives (Zmijewski Jr. and Fayreman, 1995). The best known of all is
vancomycin (Fig. 8) (Harris and Harris, 1982), effective against gram-positive bacteria.
This antibiotic is widely used in medicine, especially against ß-lactam resistant strains.
Vancomycin is not absorbed from the gastrointestinal tract and is used to treat
enterocolitis caused mainly by Clostridium difficile.
Vancomycin is produced by many species, 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 acid core, derivatives of amino glycosides are
formed. In vancomycin the aminosugar vancosamine is bound to the amino acid core.
The removal of aminosugar reduces the activity of vancomycin two- to fivefold. The
sugars seem to play an important role in imparting the enhanced pharmacokinetic
properties for vancomycin-type, glycopeptide antibiotics.
B. POLYKETIDE-DERIVATIVES
Polyketides are a large group of secondary metabolites synthesized by decarboxylative
condensation malonyl units often with subsequent cyclization of the polyketo chain .
The starter group may be an acetate but also pyruvate, butyrate, ethyl malonate,
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paraaminobenzoic acid, etc. The formation of the initial polyketo chain is similar to that
taking place during the biosynthesis of fatty acids, and is catalyzed by polyketide
synthases. Simple carboxylic acids are activated as thioesters (acyl-SCoA) which are
carboxylated to form malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA and after
decarboxylation polymerized. ( Lynen, and Reichert, 1951; Lynen, 1959; Lynen and
Tada, 1961). A principal role is played by the Acyl Carrier Protein (ACP) (Goldman
and Vagelos, 1962). ACP detected throughout the growth of Streptomyces glaucescens
was purified to homogenity and found to behave like many othes ACPs from bacteria
and plants (Sumers et al. 1995). 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 to the other -SH group, that is a
part of the cysteine molecule. Polyketide synthases have not yet been isolated and their
properties have been deduced from the analyses of DNA sequences of cloned genes.
Polyketide synthases include two distinct groups located either in domains on
multifunctional proteins or present on individual, monofunctional proteins (McDaniel
et al., 1993, Shen and Hutchinson, 1993). The structure and function of polyketide
synthase in antibiotics overwie Robinson (1991) and Bentley and Bennett (1999).
6-Methyl salicylic acid (6MS) represents one of the simplest polyketides formed by
condensation and subsequent aromatisation of one acetylCoA molecule and three
malonylCoA molecules. This compound was isolated from Penicillium patulum
(Bu´Lock and Ryan, 1958). By other metabolic steps 6MS is transformed to produce a
toxin called patulin (Sekiguchi, 1983; Sekiguchi et al., 1983). The synthesis of 6MS
takes place on an enzymatic complex called 6MS synthetase (Fig. 9) (Dimroth et al.,
1970,1976).
The chemical structure of sometypical tetracyclines is shown in Fig. 10 and their
biosynthesis in Figs. 11 and 12 (McCormick, 1965). Chlortetracycline (CTC) and
tetracycline (TC) are produced by the actinomycete Streptomyces aureofaciens, whereas
oxytetracycline (OTC) and tetracycline by the actinomycete Streptomyces rimosus. For a
more extensive coverage of research, articles by Běhal et al. (1983), Běhal (1987) and
Běhal and Hunter (1995) should be 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 where other, less toxic antibiotics are not effective.
Anthracyclines are synthesized in a similar way as tetracyclines, however, they often
have one or several sugar residues in the molecule. Most often deoxy-sugars,
synthesized from glucose, are present in the anthracycline molecule. Daunorubicin and
doxorubicin (adriamycin) (Fig. 13) are excellent antitumor agents, which are widely
used in the treatment of a number of solid tumors and leukemias in human.
Unfortunately, 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, the drugs were linked to high
molecular compounds such as dextran (Levi-Schaff et al., 1982; Tanaka, 1994), DNA
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(Campeneere, 1979), and others. Anthracyclines are produced by many Streptomyces
(Grein, 1987) and genetics of their production is well elaborated (Hutchinson, 1995).
Macrolides are usually classified to include: proper macrolides having 12-, 14- or 16membered 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. Both macrolides and polyenes are biosynthesized 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. Macrolides usually possess an
antibacterial activity whereas polyens are mostly fungicides.
Erythromycins produced by Saccharopolyspora erythrea (Fig. 14), together
with oleandomycin and picromycin, belong to the best known 14-membered lactone ring
macrolides (Harris et al., 1965). Macrolides with a 16-membered ring are represented by
tylosin (Fig. 15) (Omura et al., 1975), 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, pyruvate 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 secondary metabolite (Fig. 16). Candicidine is
another well known secondary metabolite belonging to the polyene group. Its molecule
includes p-aminoacetophenone as the terminal group. 4-amino benzoic acid (PABA)
was identified as a precursor of the aromatic part of candicidine molecule (Liu et al.,
1972, Martin, 1977).
The sugars found in macrolide and polyene molecules are not usuallyencountered in
microbial cells. They include both basic and neutral sugar molecules and L-forms are
often found. So far, 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 (Corcoran and Chick, 1966), 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 (Martin and Gil, 1979). 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 secondary metabolites.
Avermectins consist of a 16-membered, macrocyclic lactone to which the disaccharide
oleandrose is bound (Fig. 17) (Burg, R.W., 1979; Miller, T.W., 1979). 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.
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Avermectins are potent antiparasitic compounds active against a broad spectrum
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 and 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 (MacNeil, 1995; Ikeda
and Omura, 1995).
Polyethers form a large group of structural related natural products mainly
produced by Streptomyces (Birch and Robinson, 1995). They are potent coccidiostats
(monensin, salinomycin) and are used in the agricultural arena.(Westley, 1977).
Polyethers are compouns possesing the ability to form lipid-soluble complexes that
provide a vehicle for a wide variety of cations to traverse lipid barrieres. This ionbearing property led to their being named ionophores (Moore and Pressman, 1994).
Backbones of polyethers are synthetized from acetate, propionate and butyrate
(monensin A) units. Isobutyrate and n-butyrate are efficiently incorporated into
polyether antibiotics (Pospíšil et al., 1983). Incorporation of isobutyrate was explained
by formal conversion of isobutyryl-CoA into n-butyryl-CoA or methylmalonyl-CoA by
isobutyryl-CoA mutase and methylmalonyl-CoA mutase, respectively.
C. OTHER GROUPS OF BIOACTIVE PRODUCTS
Chloramphenicol (Fig. 18) is produced by Streptomyces venezuelae (Vining and
Westlake, 1984). At present, however, 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.
Streptomycin (Fig. 19) is a well-known aminoglycoside antibioticoriginaly
discovered by Selnon Waksman. 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 steps of streptomycin
biosynthesis were disclosed mainly by Walker (Walker and Walker, 1971), who also
studied the relevant enzymes (Walker, 1975).
The importance of streptomycin consists mainly in its ability to suppress
Mycobacterium tuberculosis, resulting in effective suppression of tuberculosis,
especially in developed countries.
Bialaphos is formed from two L-alanine residues and the amino acid
phosphinothricine. The latter compound is synthesized by streptomycetes from
acetylCoA and phosphoenolpyruvate, and subsequently methylated using methionine as
the methyl donor (Bayer et al., 1972; Ogawa et al., 1973). The producing
microorganisms are Streptomyces hygroscopicus and Streptomyces viridochromogenes.
Bialaphos, as well as phosphinothricine, inhibits the activity of glutamine synthetase.
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III. Genetics and molecular genetics
A. PREPARATION OF HIGH PRODUCTION MICROORGANISMS
The structural genes encoding the enzymes that synthesize secondary metabolites are
mostly located on chromosomes They are often organized in gene clusters (Binnie et al.,
1989; Malpartida and Hopwood, 1984; Lotvin et al., 1992; Martin, 1992). Resistance of
the producer to its own products are located either at the beginning or at the end of the
cluster, often in both positions. In addition to the resistance and structural genes,
regulatory genes are important in secondary metabolites production, however, they
function is poorly understand.
Microorganisms that are isolated from nature (wild type strains) produce small
amounts of secondary metabolites. Sometimes during selection and subsequent
cultivation in the laboratory, a changes occur, making the cultivated strain non-identical
with the original strain. In such cases it should be remarked that the term wild type
strain only refers to the fact that the strain did not undergo an “artificial” genetic change.
In order for the commercial production of secondary metabolites could be profitable,
higher levels of the secondary metabolites synthesis are reached via genetic changes of
producers. Mutants are isolated by exposure of spores to UV irradiation, X-rays, γ-rays,
α-particles or chemical mutagens (nitrogen mustards, N-methyl-N´-nitro-N-nitroso
guanidine). Combined mutagenesis using various mutagens is often used. The surviving
spores give rise to individual colonies of isolates, whose capability of secondary
metabolites production is then tested. Mutants that exhibit poor growth and sporulation
ability are not suitable candidates for further improvement, even if their secondary
metabolites production may exceed that of the original strain. Today’s high production
strains, that synthesize as high as 10 000-fold levels of secondary metabolites, compared
to the original strains, are the result of many year of costly strain improvement.
Unfortunately, these high production strains can revert to lose their overproduction
though spontaneous mutagenesis.
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 a secondary metabolite intermediate whose transformation stopped due to the
absence of the corresponding enzyme. By crossing these mutants, some biosynthetic
pathways used to synthesize secondary metabolites were elucidated, e.g. tetracyclines
(McCormick et all.1960).
Loss of the capability of secondary metabolite 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 (Hotta et al, 1977; Okanishi,
1979; Akagava et al., 1979; Boronin et al., 1974; Ikeda et al., 1982).
B. GENETIC MANIPULATION OF SECONDARY METABOLITES PRODUCERS
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Structural genes for a number of secondary metabolites have been cloned into host
microorganisms. Similarly, genes for secondary metabolites 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 secondary metabolites or of their high levels. The secondary
metabolites biosynthesis is a very complex process that requires not only the structural
genes for ESM but also the genes for regulation of their biosynthesis. Moreover, the
overproduction of a secondary metabolite has to be coordinated with the primary
metabolism of the producing microorganism.
The cloning of structural genes and genes for resistance to the own secondary
metabolite enables us to work out genetic maps of the producers. On the basis of those
maps, hybrid clusters combined of two and more clusters of different secondary
metabolites can be created. Consequently, semisynthetic secondary metabolites 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 (Hopwood and Sherman, 1990). As a result, a great similarity of
polyketide synthases from various streptomycetes was evidenced (Malpartida et al.,
1987; Butler et al., 1990).
IV. Obtaining new bioactive secondary metabolites
A. ISOLATION FROM NATURAL RESOURCES
In spite of the fact that several thousands of compounds isolated from
microorganisms having some biological activity are known, new substances are still
saught by pharmaceutical companies. The probability of finding a new compound that
would be usable as a new antibiotic or another biologically active compound is low, so a
great number of microorganisms have to be screened. A rough estimation says that
about 100 000 microorganisms are screened for the presence of biologically active
compounds per year. Modern screens are highly automated. The selection methods
used, the targets, and the methods of detection of the biological activity are normally
not published.
Preparation of a new biologically active compound and its introduction into clinical
practice requires the cooperation of scientists from various scientific disciplines and
years of clinical trials. This effort can be divided into three parts:(Yarbrough et al.,
1993):
1. microbiology
-collection of source samples (soil)
-isolation of diverse microbes
-fermentation to enhance diversity
-reproduce fermentation
-enhance the production for isolation
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-taxonomy of the organism
2. molecular biology/pharmacology
-target selection
-screen design/implementation
-high through-put screening
-identification of active compounds
-efficacy studies
-mechanism of action
3. chemistry
-active compound identification
-characterisation/dereplication
-isolation/purification
-structure elucidation.
B. PRODUCERS OF BIOACTIVE COMPOUNDS
About 70 % of the known bioactive substances are produced by Streptomyces and the
rest mainly by moulds and non-filamentous bacteria. With an increasing spectrum of
efficiency of microbial metabolites, new, non-traditional sources of such compounds
have been tappede. These include the microorganisms living under extreme conditions
(high and low temperatures, etc.), sea living microorganisms, and multicelular plants
and animals. Another important source of new compounds are the mutants of producers
of known active substances, e.g. blocked mutants.
D. SCREENING
The enterprise of screening microbial metabolites 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 antibiotic efficiency consisted of the
application of test extract to wells made in agar medium layer in Petri dishes to which
the sensitive (target) microorganism was inoculated. Most often Staphylococcus aureus,
Sarcina lutea, Klebsiella pneumoniae, Salmonella gallinarium, Pseudomonas spp.,
Bacillus subtilis, and Candida albicans were used. In case a compound with an
antibiotic activity towards the testing microorganism was put into the well, it diffused
through the agar medium and a halo was formed around the well, as a result of the
suppressed growth of the microorganism. This classic plate assay has been modified and
improved in many ways.
The tests of other biological activities require different and frequently sophisticated
methods. This is true especially when enzyme inhibitors are a case in point. Thus,
Ogawara et al. (1986) chose a tyrosine protein kinase associated with the malignant
transformation of the cell caused by retroviruses as the target in a biochemical screen,
they 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
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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 (Take et al., 1989).
D. SEMISYNTHETIC AND SYNTHETIC BIOACTIVE PRODUCTS
Natural products can be modified in various ways. The unspecificity of the enzyme
systems facilitates the synthesis of certain secondary metabolites though the addition of
selectedprecursors to the growth medium. Thus, the reaction equilibrium can be shifted
to promote the production of the derivative required, e.g. the prepareation of penicillins
with different side chains. The individual derivatives of penicillin and cephalosporin
have slightly different antimicrobial spectra and are active against microorganisms
resistant to other derivatives. The structuure of polypeptide antibiotics can also be
modified by the addition of amino acids to the growth
Replacement of a part of the metabolite molecule can be accomplished chemically or
enzymatically. In this way, semisynthetic penicillins, cephalosporins, tetracyclines and
other antibiotics can be prepared. The production of semisynthetic penicillins and
cephalosporins is facilitated by the fact that 6-amino penicillanic and 7-amino
cephalosporanic acids are easily prepared.
The side chain is removed by the action of an enzyme or by a chemical hydrolysis
(Fig. 20) then another acyl is bound chemically or enzymatically to the amino group in
position 6 (penicillins) or 7 (cephalosporins).
Semisynthetic tetracyclines, pyrolinomethyltetracycline, metamycin and doxycycline,
exhibit a greater solubility and somewhat different antimicrobial spectrum, as compared
to the original tetracyclines.
New derivatives of aminoglycosides also have been obtained by chemical and
enzymatic modifications.
As the majority of bioactive products 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.
E. HYBRID BIOACTIVE PRODUCTS
Genetic engineering methods have recently advanced so much that now we can
suitably combine structural genes of two or even more bioactive secondary metabolites
producers. If these genes are expressed, a hybrid bioactive products is synthesized, ore
that cannot be found in nature (Hutchinson, 1987, 1988; Tomich, 1988; Hopwood,
1993). Hopwood et al. (1985, 1986,) used this method with the genes of actinorhodin
synthesis and obtained related hybrid macrolides, mederhodin A and B,
dihydromederhodin A and dihydrogranatirhodin. A new anthracyclines were produced
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when a DNA segment was cloned from Streptomyces purpurascenc ATCC 25489 close
to a region that hybridized to a probe containing part of the actinorhodin polyketide
synthase Streptomyces galilaeus ATCC 31615 (Niemi et al., 1994).
V. Regulation of secondary metabolites production
A. GROWTH PHASES OF Stepromyces
In the cultures of Streptomyces capable of secondary metabolite production several
growth phases representing different physiological statescan be distinguished:
5. Preparatory phase (lag phase) - the biomass increase is low, the culture is adapting
to the new environment.
6. Growth phase (the term logarithmic phase is not suitable for most Streptomyces
since their growth curves are not exponential functions) - intensive growth is taking
place, accompanied by a low secondary metabolite synthesis. This phase is roughly
equivalent to "trophophase".
7. Transition phase - characterized by a decreased growth rate; the secondary
metabolite production is started. The enzymes of secondary metabolism are
synthesized (Běhal, 1986a; Běhal, 1986b) and proteosynthesis slowed down.
8. Production phase - characterized by a significant reduction of the growth rate
(sometimes growth is even completely ceased), a negligible change in the biomass
concentration, and an intensive synthesis of the secondary metabolite. This phase is
some times called "idiophase".
Producers of secondary metabolites mostly belong filamentous bacteria or fungi,
which means that in their culture cells of various age and at different stages of
development are present. The microorganisms grow in pellets, inside which the
cultivation conditions differ from those on the pellet surface (nutrient concentrations,
oxygen concentration, etc.). An increase in dry weight does always correlation with an
increase growth since, in streptomycetes, often a thickening of the cell wall or
glycocalyx formation occur that increase the dry weight value without rising the number
of cell (Voříšek et al., 1983). Since individual cells of a fermentation can be at different
stages of development, (i.e. in different physiological states). The physiological state of
the whole culture represents an average of physiological states of the individual cells.
B. CONTROL OF FERMENTATION BY BASAL NUTRIENTS
In order to reach a high yieldof secondary metabolite, sufficient biomass is required.
Moreover to danger of contamination is diminished and the economic parameters of the
fermentation device are optimal if the growth is rapid. For this purpose, readily
utilizable sources of carbon, nitrogen and phosphorus sources (e.g. molasses, corn
starch, etc) are used. However, production of the secondary metabolite does not usually
take place until one or more nutrients become limited.Thefore, the culture medium
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should be designed in such a way that after the biomass increased sufficiently, at least
one of the nutrient sources will become depleted. Carbon source, nitrogen source and
phosphate limitation have been described as important triggers in different systems.
Most secondary metabolites 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 fermenter. Thus a prolonged
cultivation can be accomplished that enables us to increase the yield of the secondary
metabolite. The inflow of nutrients makes possible keep their optimal levels. An
example of how a production cultivation of Streptomyces aureofaciens can look like is
shown in Fig. 21 (Běhal, 1987). In cultivations whose course is well known, the nutrient
inflow is programmed in advance
The inhibition of penicillin synthesis by glucose was observed shortly after its
discovery in media containing glucose and lactose (Demain, 1974). 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 (Demain, 1983). 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 secondary metabolites synthesis by
readily utilizable sugars probably consists in a repression of enzymes of secondary
metabolism (Revilla, G. et al., 1986; Erban, et al., 1983).
Readily utilizable nitrogen sources can also negatively influence the production of
secondary metabolites.Ammonium ions often decrease secondary metabolite synthesis
and, therefore, their concentration in production media is limited while, soy flour,
peanut flour and other substances are preffered nitrogen sources. These latter nitrogen
sources are more similar to those used by the microorganisms producing secondary
metabolites in nature. Readily utilizable nitrogen sources repress enzymes of secondary
metabolism in Cephalosporium acremonium (Shen et al., 1984) during the biosynthesis
of cephalosporin and in Streptomyces clavuligerus producing cephamycin (Demain and
Brana, 1986). Similarly, the inhibition of biosyntheses of leucomycin (Omura et al.,
1980a), tylosin (Omura et al., 1980b), and erythromycin (Flores and Sánches, 1985) are
explained by the repression of enzymes of secondary metabolism. Ammonium salts also
inhibit the activity of anhydrotetracycline oxygenase isolated from S. aureofaciens
(Běhal et al., 1983).
The overproduction of most secondary metabolites can be achieved only if phosphate
is limited. Inorganic phosphate has to be carefully added in doses to the medium so as to
accomplish an optimal ratio between biomass production and secondary metabolite
biosynthesis. When bound to organic compounds normally added to medium (soy flour,
etc.), phosphate does not affect secondary metabolite production. In general secondary
metabolite biosynthesis is started when the concentration of phosphate decreased below
a certain level. At this point, the producer culture undergoes a shift from the
physiological state characteristic for the growth phase to that of the overproduction
phase.
Inorganic phosphate also causes a repression of the synthesis of enzymes of secondary
metabolism (Běhal et al., 1979b; Madry and Pape; 1981, Martin et al., 1981). After
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phosphate was depleted from the medium, a significant decrease of the rate of
proteosynthesis was observed during tetracycline (Běhal,1982). If phosphate was kept
above the threshold concentration, the significant decrease of the rate of protein
synthesis did not occur and ESM 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 enzymes of secondary metabolism levels in the culture and an
acceleration of proteosynthesis.
C. HOW SIGNALS FROM THE MEDIUM ARE RECEIVED
Reception of signals from the environment, that result in the initiation of the
secondary metabolite synthesis does not significantly differ from the transduction 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 ( Doull and Vining, 1995). With some structural
varietion, these proteins are characterized by common mechanistic features and
conserved amino acid sequences.
The two-component system consists of a cytoplasmic membrane-linked, sensortransmitter 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-end is
found outside the cytoplasm. A specific effector is capable of binding directly to this Nend. 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 histidine-protein kinases, capable of autophosphorylation
at their C-ends on receiving a proper signal. The phosphorylated protein becomes a
donor in reactions transferring phosphorus. The acceptor is the cytoplasmic, responseregulator protein. Two-component signal proteins thus transfer the information
concerning the conditions that can affect the cell action.
D. REGULATION BY LOW MOLECULAR COMPOUNDS
The expression of structural genes is also regulated by some low molecular
compounds. The mechanism of their action is not understood. For example tryptophan
exhibited a stimulatory effect on the production of mucidin in the basidiomycete
Oudemansiella mucida (Nerud et al., 1984) and actinomycin in Streptomyces parvulus
(Troast et al., 1980). Methionine was found to promote the synthesis of cephalosporin C
(Nuesch et al., 1973). Neither tryptophan nor methionine were used as building units for
these metabolites.
Benzyl thiocyanate is one of the low molecular compounds that affect the
chlortetracycline biosynthesis. It increases the production of both chlortetracycline and
tetracycline in S. aureofaciens, although, it does not influence the production of
oxytetracycline in S. rimosus. The effect on the metabolism of S. aureofaciens is
multiple (Novotná et al., 1995), including a number of enzymes, including the enzymes
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of secondary metabolism (Běhal et al., 1982). Benzyl thiocyanate is able to raise the
level of secondary metabolite 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 enzyme level and chlortetracycline production are
increased 10 to 20-fold, as compared to high production strains where the increase is
only twofold. .
E. AUTOREGULATORS
Streptomycetes low-molecular, diffusible compounds have been discovered that
regulate the metabolism of producing strain (Horinuchi and Beppu, 1990; Horinuchi
and Beppu, 1992). The most famous of them is factor A, γ-butyrolactone (Fig. 22), that
was discovered in Streptomyces griseus (Khokhlov et al., 1969; Khokhlov, 1982). A
non-producing strain started the synthesis of streptomycin after factor A was added to
the culture simultaneously, the coltura formed aerial mycelium. Factor A is synthesized
by many streptomycetes but the regulatory effect was observed only in Streptomyces
griseus, Streptomyces bikiniensis and Streptomyces actuosus (Ohkishi et al., 1988). The
addition of factor A to blocked mutants of Streptomyces griseus JA 5142, caused
resumption of the synthesis of anthracyclines and leukaemomycin (anthracycline type
antibiotic) (Graefe et al., 1983). The resistance to streptomycin linked with an enzymatic
phosphorylation of the antibiotic is also induced by factor A (Hara and Beppu, 1982).
Analogues of factor A have also been found, all of them being γ-butyrolactones.
Virginiae butanolides were detected in Streptomyces virginiae (Yanagimoto et al.,
1979). Factor I was isolated from Streptomyces sp. FR1-5 (Sato et al., 1989) and its
effective concentration was 0.6 ng/ml culture. Most of the factor A analogues, however,
were not biologically active.
Factor B was isolated from the yeast Saccharomyces cerevisiae. This substance was
capable of eliciting the production of rifamycin in a blocked mutant of Nocardia sp.
(Fig. 23) (Kawaguchi et al., 1984). Factor B was effective at a concentration of 10-8 M,
with one molecule eliciting a synthesis of about 1500 molecules of the rifamycin. 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. Activity was observed with those that contained a C2 -C12 acyl
moiety; octylester was the most effective of them (Kawaguchi et al., 1988). 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 (Szabo et al., 1967).
Factor C is a protein having a molecular weight of about 34 500 D, and is rich in
hydrophobic amino acids.
The effect of autoregulators is easily observable if they elicit morphological changes
such as the formation of aerial mycelium. Carbazomycinal and
6-methoxcarbazomycinal, isolated from Streptoverticillium species, inhibit of the aerial
mycelium formation at a concentration of 0.5 to 1 microgram per ml. Autoregulators
affecting sporulation were found in Streptomyces venezuelae (Scribner et al., 1973),
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45
Streptomyces avermitilis (Novák et al., 1992), and Streptomyces viridochromogenes
NRRL B-1551 (Hirsch and Ensign, 1978). From the same strain of Streptomyces
viridochromogenes, germicidin was isolated by Petersen and coworkers (1993). The
compound had an inhibitory effect on the germination of arthrospores of Streptomyces
viridochromogenes at a concentration as low as 40 picogram per ml. Germicidin (6-(2butyl)-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 and also from the supernatant of a submerged culture.
Mutants of Streptomyces cinnamonensis resistant to high concentrations of butyrate
and isobutyrate produce an anti-isobutyrate (AIB) factor that is excreted into the culture
medium (Pospíšil, 1991). On plates, AIB factor efficiently counteracted toxic
concentrations of isobutyrate, acetate, propionate, butyrate, 2-methylbutyrate, valerate,
and isovalerate in Streptomyces cinnamonensis and other Streptomyces species.
F. REGULATION BY PHOSPHORYLATED NUCLEOTIDES
Global control mechanisms for secondary metabolites biosynthesis have been
investigated. 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 secondary metabolite 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 (Janglová
et al., 1969; Čurdová et al., 1976) and tylosin (Madry et al., 1979; Vu-Truong et al.,
1980), 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 accompanies active primary
metabolism. A slow down of growth and primary metabolism is accompanied by a
decrease of the ATP level.
The role of cAMP in the metabolism of secondary metabolites producers was also
studied, especially in connection with glucose regulation. Hitherto, no indication has
been obtained suggesting a specific role of cAMP in the regulation of secondary
metabolites production (Cortéz et al., 1986; Chatterjee and Vining, 1981).
G. REGULATION BY METAL IONS
Metal ions act as a part of enzyme active centers. The optimal concentrations of metal
ions for cultivation of the secondary metabolites producing strains have usually been
determined empirically. In complex media it is generally not necessary to add specific
metal ions, however in defined media their presence is essential.
VI. Resistance to bioactive products
Resistance against bioactive products has been studied mainly in antibiotic
producers. Antibiotic resistance is usually looked at from two angles: first, the
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emergence of drug rezsstant strain and second, "self resistance" of antibiotics producing
strains. The ways in which these two types of resistance are achieved is often similar.
A. RESISTANCE OF SECONDARY METABOLITES PRODUCERS
Basic metabolic processes of wild type, secondary metabolite producing
microorganisms are not inhibited if the secondary metabolires are synthesized at low
concentrations. After strain improvement, strains with 100 to 1000-fold increases
insecondary metabolite yields have been isolated. Genome changes of the improved
strains include a number of deletions and amplifications in the chromosomal DNA, as
well as changes in extrachromosomal DNA.
Low production strains, whose resistance to the own product is low (i.e. higher
concentrations of the product inhibit their growth), regulate the secondary metabolite
production by inhibiting the enzyme activities that participate in the synthesis of the
secondary metabolite. In high production strains, such controls are lost and the strains
have to find a way how to survive in the presence of a high concentration of the
antibiotic (Vining, 1979).
The genes for self resistance are often located at the beginning of the cluster of
structural genes. As a result, they are expressed simultaneously with the structural
genes. The genes of newly gained resistances, however, are mostly located on plasmids.
Some antibioticsfunction by hitting active centres of enzymes. However, if active
centre is modified, the antibiotic cannot bind to it and then resistance comes into
existence. It is not known whether a decreased ability to bind the secondary metabolites
results from a posttranslational modification of the active centre or if resistant
molecules of the enzyme are synthesized de novo. Clear evidence in support of the latter
situation has sofar been brought.
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
reduced synthesis of guanosine penta- and tetraphosphates (Weiser et al., 1981). The
antibiotic producers (mostly Streptomyces), 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 the 23S subunit. Such
modified ribosomes do not bind the antibiotic. In other cases, adenine is methylated to
yield 2-O-methyladenosine (Cundliffe and Thompson, 1979; Mikulík et al.,1983;
Thompson et al., 1982). 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 secondary metabolites
producers seems to be export from the cell to the environment. 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(Ohnuki et al., 1985).
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47
Producers bioactive secondary metabolites also have to solve the problem of a reverse
flow of products into the cell. Some secondary metabolites are 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
secondary metabolites. This kind of resistance is thought to be connected with the
content of phospholipids in the cell.
Secondary metabolite producers can use several types of resistance simultaneously.
Tetracyclines, that strongly inhibit protein synthesis, interfere with the binding of the
ternary complex of amino acyl-tRNA-EFTu-GTP to ribosomes (Gavrilova et al., 1976).
The genes for resistance were cloned into Streptomyces griseus, sensitive to
tetracyclines, using pOA15 as the vector plasmid (Ohnuki et al., 1985). 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
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. The inhibiting concentrations of chlortetracycline in Streptomyces
aureofaciens are higher in the production phase as compared to the growth phase (Běhal
et al., 1979a). Thus, the resistance can be increased even during the fermentation
process.
Another way secondnary metabolite producers can avoid the effect of their products is
to situate the distal enzymes of secondary metabolite biosynthetic pathway (synthases)
outside the cell, most often in the periplasm. In Streptomyces aureofaciens, a higher
proportion of the terminal enzyme of tetracycline synthase was found under high
production conditions in periplasm, as compared to low production conditions (Erban et
al.,1985).
B. RESISTANCE IN PATHOGENIC MICROORGANISMS
Shortly after antibiotics were introduced into clinical practice on a massive scale,
strains of hitherto-sensitive microorganisms started to appear. These resistant strains
required the use of much higher antibiotic concentrations or, 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 in which microorganisms can gain resistance (Ogawara,
1981). These include:
1. Creation of an alternative metabolic pathway producing a compound whose
biosynthesis is blocked by the bioactive metabolite; 2. Production of a metabolite that
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can antagonize the inhibitory effects of the bioactive metabolite; 3. Increase of the
amount of the enzyme inhibited by the secondary metabolite; 4. Decrease of the cell’s
metabolic requirement for the reaction inhibited by the secondary metabolite; 5.
Detoxification or inactivation of the bioactive metabolite; 6. Change of the target site; 7.
Blocking of the transport of the bioactive metabolite into the cell.
In most resistant microorganisms, the mechanisms of resistance mentioned in the
items 5, 6 and 7 are encountered.
Penicillins and cephalosporins are degraded using three ways: by the enzyme
penicillin amidase that cleaves the amidic bond by which the side chain is bound to the
β-lactam ring; by the enzyme acetyl esterase that hydrolyzes the acetyl group at C-3 on
the dihydrazine ring of cephalosporins and 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, however these enzymes 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-lactams are inactivated by β-lactamase that
destroys one of the important sites for their antibiotic activity; the damage is
irreversible.
Β-lactamases, however, are not only synthesized by microorganisms that came into
contact with penicillins. Constitutive synthesis of these enzymes have been found in
three quarters of all streptomyces strains, (Ogawara et al., 1978). One can suppose that
the genes for the synthesis of β-lactamases were transferred horizontally. Recent studies
indicate frequent and promiscuous gene transfer even between distantly related bacterial
species. A possibility of direct transfer from a streptomycete to a pseudomonad, for
example, may seem 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 (Davis, 1992).
Another way of inactivating a bioactive metabolite molecule is N-acetylation of the
amino group or O-phosphorylation of the hydroxyl. Bialaphos was found to be
inactivated by acetylation. These substance itself is not toxic but, in the cell,
phosphinothricine is liberated that inhibits glutamine synthetases, key enzymes of the
inorganic nitrogen assimilation pathway.
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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.
Fig. 20.
Fig. 21.
Gramicidin A
Gramicidin S
Bacitracin
Bacitracin synthetase
Penicillins
Cephalosporins
Clavulanic acid
Vancomycin
6-methyl salicylic acid synthetase
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
Parameters of an industrial fermentation of S. aureofaciens
1 - chlortetracycline production (g/l); 2 - ATC-oxygenase (pkat/ mg
proteins x 2); 3 - NH3-nitrogen (g/l x 0.1); sucrose (g/l x 10);
5 - pH; ammonium supplement were added at points A and B
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58
Fig. 22.
Fig. 23.
Factor A
Factor B
58