Учебная работа. Semisynthetic and synthetic antibiotics

Introduction

Timeliness.The mass production of antibiotics began during World War II with streptomycin and penicillin. Now most antibiotics are produced by staged fermentations in which strains of microorganisms producing high yields are grown under optimum conditions in nutrient media in fermentation tanks holding several thousand gallons. The mold is strained out of the fermentation broth, and then the antibiotic is removed from the broth by filtration, precipitation, and other separation methods. In some cases new antibiotics are laboratory synthesized, while many antibiotics are produced by chemically modifying natural substances; many such derivatives are more effective than the natural substances against infecting organisms or are better absorbed by the body, e.g., some semisynthetic penicillins are effective against bacteria resistant to the parent substance.

Despite the wide variety of known antibiotics, less than 1% of antimicrobial agents have medical or commercial value. For example, whereas penicillin has a high therapeutic indexas it does not generally affect human cells, this is not so for many antibiotics[1].Other antibiotics simply lack advantage over those already in use, or have no other practical applications.

Academic novelty. Useful antibiotics are often discovered using a screening process. Most antibiotics identified in such a screen are already known and must therefore be disregarded. The remainder must be tested for their selective toxicities and therapeutic activities, and the best candidates can be examined and possibly modified.A more modern version of this approach is a rational design program. This involves screening directed towards finding new natural products that inhibit a specific target, such as an enzyme only found in the target pathogen, rather than tests to show general inhibition of a culture. Streptomyces violatus showed the highest antimicrobial activity in static cultures after 7 days incubation at 30°C. The antibacterial substance was more active against Bacillus subtilis and Staphyllococcus aureus than Escherichia coli or Sarcina lutea. Growth of S. violatus and production of antibiotic in a starch-nitrate medium were monitored over a period of 14 days. The organism produced a blue pigment associated with the antibiotic appearance in the cultures. Optimization of antibiotic production in batch cultures has been carried out. Substitution of starch by glycerol at a concentration of 12.5 g/l showed 1.32-fold increase of antibiotic production. Cultures containing sodium nitrate (2.5g/l) showed the highest antibiotic production followed by peptone, alanine, monosodium glutamate or phenylalanine. The presence of ferrous sulphate and manganese chloride improved the production of the antibiotic. An inoculum size of 4×106 spores/ml and initial pH 7.0 at 30°C were optimum for a maximum antibiotic production of 268µg/ml in the culture filtrates of S. Violatus.

The purpose of my research is improvement of antibiotic production using Streptomyces vielatus by optimization of the cultural conditions.

Basic part

1. Analytical review

1.1 Antibiotics as representatives of biologically active substances from microorganisms

Antibiotics are defined as microbial products that inhibit growth of other microorganisms. After the antibiotic effect of penicillin had been observed by Fleming, a number of other antibiotics were discovered. The main producers are soil microorganisms as actinomycetes moulds anf fungi. New antibiotics being searched for the microorganisms were found to produce a broad spectrum of compounds having various effects on living organisms. Some of them have occupied a weighty position as medicines and agricultural drugs and for animal health. One microorganism can produce several compounds with different biological activity (staurosporine) and, on the contrary, one compound can be produced by several microorganisms. Besides to traditional antibiotics, compounds with different biological activities are synthesized by various microorganisms: coccidiostatics used in poultry farming, antiparasitic compounds with a broad spectrum of the activity against nematodes and arthropods, substances with the antitumor activity, immunosuppressors, thrombolytics (staphylokinase), herbicides, pesticides, compounds affecting blood pressure, etc [2].

For medicine are important enzyme inhibitors synthesized by microorganisms. They are used as inhibitors of enzymes produced by resistant strains that decompose the antibiotic during application of antibiotics. These enzyme inhibitors can be also used for inhibition of undesirable enzyme activities in human metabolism that cause some illnesses. Many enzyme inhibitors are protease inhibitors, variously active against pepsin, papain, trypsin, chymotrypsin, catepsin, elastase, renin, etc. Inhibitors of glucosidases, cyclic AMP phosphodiesterase, different carbohydrases, esterases, kinases, phosphatases, etc. have been isolated from microorganisms. The enzyme inhibitors that participate in the biosynthesis of cholesterol and fat are also used in medicine.

Several thousands of compounds having different biological activities have so far been listed and new compounds are still isolated from microorganisms. There is a widespread acceptance that microorganisms are an unlimited source of new substances with many potential therapeutic applications. A great number of those compounds, however, are toxic and thus cannot be used for human and veterinary therapy.

Role of antibiotics in producing microorganisms.Antibiotics are the typical secondary metabolites produced by microorganisms. Secondary metabolites are meant products of microorganisms (also plants) which are not essential for basic metabolic processes such as reproduction and growth [3]. On the other хэнд, in the case of many secondary compounds, pieces of evidence of their role in the metabolism of the producer have been brought. These compounds often function as the so-called signal molecules, used to control the producer’s metabolism. One of the functions attributed to antibiotics is a suppression of the competing microorganisms in the environment. Thus the antibiotic-producing microorganisms have an advantage in competing for nutrients with the other microorganisms but antibiotic activity is only one from many other biological activities of secondary microbial products. However, the function of antibiotics in the environment can be observed only with difficulty.

1.1.1 Use of antibiotics in human, veterinary and plant medicine

Antibiotics are very often used in medicine for suppression of pathogenic bacteria, fungi and viral diseases. Their use marked a revolution in medicine, saved millions of lives and helped reduce some, rather frequent diseases such as tuberculosis. An efficient, antiprotozoan antibiotic, however, has not yet been discovered. Antibacterial antibiotics are sometimes used in the case of viral diseases to protect the weakened macroorganism against a subsequent bacterial infection. As mentioned in the introductory part, some antibiotics are also used as cancerostatics or for curing some other illnesses.

In a similar way as in human medicine, antibiotics are also employed in veterinary medicine. Besides, antibiotics are added to various feeding mixtures used in poultry and animal farming to keep the animals in good health. If the antibiotics are used, higher farming yields are often reached. However, the administration of antibiotics should be stopped a certain time before the animal is slaughtered and the meat consumed since the residues of antibiotics should not enter the human diet. To avoid the production of strains resistant to the antibiotics used in human medicine, special antibiotics allowed to be employed in veterinary medicine and animal production have been singled out and are no more used in human medicine (chlortetracycline, bacitracin, tylosin, etc.).

Side effects of antibiotics.In addition to their positive effects, antibiotics can also have negative effects. Besides various allergies linked with the use of antibiotics, the human organism can sometimes suffer a damage when treated with them. Sometimes toxic compounds can be formed when antibiotics are transformed in the organism. Tetracyclines, that form complexes with calcium, can, for example, inflict damage on the formation of tooth enamel in children, on the condition they are frequently used during the period of teeth growth. A number of newly discovered antibiotics cannot be used for therapy because of their excessive toxicity. Fortunately, the first antibiotic to be used on a massive scale, penicillin, has relatively moderate side effects on the human organism.

Biosynthesis.In spite of variety of their structures, antibiotics are synthesized from simple building units amino acids, acetate, propionate, sugars, nucleotides which are used in living organisms for the biosynthesis of cellular structures. According to their structure and type of biosynthesis, antibiotics are classified to form several groups.

1.1.2 Peptide and peptide-derived antibiotics

Peptides.Microorganisms produce a number of peptides that have the biological activity. In contrast to biologically active peptides of higher organisms, where they function as hormones, the function of microbial peptides in microorganisms is not known. They are included in the group of secondary metabolites. They differ from the biologically active peptides of higher organisms in having often D-amino acids in their molecules. Besides their antibiotic activity, another interesting feature of the peptide antibiotics is the fact that they are not synthesized on ribosomes, as other peptides, but on enzyme complexes called peptide synthetases [3-4].

Chemical structure. Amino acids linked by the peptide bond form the basal structure of any peptide antibiotic. The peptide chain is often cyclic or branched. In addition to L-amino acids, other compounds can also be present in the molecule, such as D-amino acids, organic acids, pyrimidines and sugar molecules. Numbers of derivatives are known to exist in the case of some peptide antibiotics, that differ in both amino acid substitutions and substituents bound to the amino acids.

The linear molecule of gramicidin A and the cyclic molecule of gramicidin S belong to the structurally simplest peptide antibiotics. Bacitracins are an example of cyclic peptides having a side chain (Fig. 2). In the molecule of bleomycins, the sugars 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. Another cyclic polypeptide (heptapeptide) is iturin, an antifungal antibiotic, produced by Bacillus subtilis, effective against plant pathogens [4].

A special type of compounds are enniatines. Their molecule consists of three residues of branched amino acids, L-valine, L-leucine and L-isoleucine, and three residues of D-2-hydroxyisovaleric acid (D-Hyiv). The amino acids and D-Hyiv are linked by alternating amide and ester bonds. The amide bonds are finally N-methylated.

Biosynthesis. The biosynthesis of peptide antibiotics takes place on a multienzyme complex [5]. The individual amino acids are activated using ATP to form aminoacyl adenylates. The aminoacyl groups are transferred to the enzyme thiol groups where they are bound as thioesters. The structural arrangement of the thiol groups in the synthetases determines the order of amino acids in the peptide. The formation of peptide bond is mediated by 4-phosphopantetheine, that is an integral part of the multifunctional multienzyme.

The way how the order of the amino acids in the molecule is regulated is not known. It is probably determined by the tertiary configuration of the enzyme. This specificity, however, is not very high since the microorganisms mostly produce a mixture of peptides differing only in one or several amino acids in the chain.

Enzymes. Gramicidin S synthetase is an enzyme consists of two complementary enzymes having molecular weights of 100 kD and 280 kD.

Bacitracin synthetase. The enzyme consists of three subunits having molecular weights of 200, 210 and 360 kD [5]. Each subunit contains phosphopantetheine. The enzyme A activates the first five amino acids of bacitracin, the enzyme B activates L-Lys and L-Orn, and the enzyme C activates the other five amino acids. D-amino acids are produced by racemization of their L-forms directly on the enzyme complex. Initiation and elongation start on the subunit A up to the pentapeptide, independently of the presence of the subunits B and C. The pentapeptide is transferred to the subunit B where two other amino acids are added. The heptapeptide is subsequently transferred to the subunit C where the biosynthesis of bacitracin is finished. The cyclization is achieved by binding the asparagine carboxy group to the е-amino group of lysine, whereas, to the б-amino group of the same lysine, the isoleucine carboxyl group is bound.

Mechanism of action. The antibiotic activity of bacitracin results in an efficient inhibition of proteosynthesis and cell wall synthesis but other effects such as an interference with cytoplasmic membrane components and cation-dependent antifungal effects have been observed as well. In the case of gramicidin S, hemolytic effects, inhibition of protein phosphatases and interaction with nucleotides have been observed, in addition to the antibacterial activity. Even though antibiotics normally have several mechanisms of their action, the primary one is thought to be the effect observed at the lowest concentration of all. The peptide antibiotics are efficient mainly against Gram-positive bacteria

Я-Lactams.The main representatives of Я-lactams are penicillins and cephalosporins. Penicillins have a thiazoline Я-lactam ring in the molecule and differ, one from another, by side chains linked via the amino group.

Cephalosporins [6] have a basic structure similar to that of penicillins and the derivatives are also formed by a variation of the side chain.

The thiazolidine Я-lactam ring is synthesized using three amino acids: L-б-amino adipic acid, L-cystein and L-valine by б-aminoadipyl-cysteine-valine synthetases . By condensation of these three amino acids, a tripeptide is formed. It is transformed to the molecule of penicillin or cephalosporin through subsequent transformations. The principial works about enzymes of в-lactams biosynthetic pathways were done by Abraham and his collegues.

Clavulanic acid also belongs to Я-lactams . This acid has a bicyclic ring structure resembling that of penicillin, except that oxygen replaces sulfur in the five-membered ring. Clavulanic acid is an irreversible inhibitor of many Я-lactamases. The discovery of clavulanic acid was a starting

Biological activity. Penicillins are especially active against Gram-positive bacteria but some semisynthetic penicillins, such as ampicillin, that is lipophilic as compared to, for example, benzyl penicillin, are also effective against Gram-negative bacteria. This effect is explained by their easier entering the cells of Gram-negative bacteria that have a high lipid content in the cell wall. Я-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 Я-lactams are applied.

Glycopeptides.

At present several hundreds compounds belonging to glycopeptides are known, including semisynthetic derivatives. The best known of all is vancomycin [20] that is effective against Gram-positive bacteria. This antibiotic is widely used in medicine, especially against Я-lactam resistant microorganisms. Vancomycin is not absorbed from the gastrointestinal tract and is used to treat enterocolitis caused mainly by Clostridium difficile.

Vancomycin is produced by several tens of microorganisms, of which Amycolotopsis orientalis is used for commercial production. Glycopeptides are composed of either seven modified or unusual aromatic amino acids or a mix of aromatic and aliphatic amino acids.

Polyketide-derived antibiotics.A large group of antibiotics includes compounds that are synthesized by polymerization of acetate units and subsequent cyclization of the polyketo chain, that has been formed before or is just being formed, to provide six carbon atoms containing aromatic rings or macrocyclic lactone ring. The terminal group need not be an acetate but also pyruvate, butyrate, ethyl malonate, paraminobenzoic acid, etc. In the early phase, the formation of polyketo chain is similar to that taking place during the biosynthesis of fatty acids, and is catalyzed by the enzyme polyketosynthase. A principal role is played by the Acyl Carrier Protein (ACP) [7]. The ACP prosthetic group in many microorganisms is 4ґ-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. Polyketosynthase has not yet been isolated and its properties have been deduced from the analyses of DNA sequences of cloned genes. Polyketosynthases include two distinct groups located either in domains on multifunctional proteins or present on individual, monofunctional proteins.

6-Methyl salicylic acid.6-Methyl salicylic acid (6MS) represents the simplest polyketide, that is formed by condensation and subsequent aromatisation of one acetylCoA molecule and three malonylCoA molecules. This compound was isolated from Penicillium patulum. By other metabolic steps 6MS is transformed to produce a toxin called patulin. The synthesis of 6MS takes place on an enzymatic complex called 6MS synthetase [7-8].

Tetracyclines. Chlortetracycline and tetracycline are produced by the actinomycete Streptomyces aureofaciens, whereas oxytetracycline and tetracycline by the actinomycete Streptomyces rimosus. The tetracycline molecule is synthesized from one molecule of malonic acid semiamide and eight molecules of malonate. In the early steps, the synthesis is similar to the biosynthesis of fatty acids, but the keto groups are not reduced and aromatic rings are formed to yield 6-methyl pretetramide. This compound is the first known intermediate of the tetracycline biosynthesis that is further transformed to yield one of the tetracycline molecules. As to the enzymes transforming the intermediates of chlortetracycline and tetracycline biosynthesis, the last three have been described: S-adenosylmethionine:4-dedimethylamino-4-aminotetracycline N-methyltransferase methylating the amino group in position 4, anhydrotetracycline oxygenase and NADP:tetracycline 5a(11a)dehydrogenase (tetracycline dehydrogenase). For more extensive coverage of research, articles by Bмhal and Bмhal and Hunter [8] can by consulted.

Tetracyclines act as inhibitors of proteosynthesis. They are considered to be wide-spectrum antibiotics, that are efficient against both Gram-positive and Gram-negative bacteria. However, having significant side effects on the human macroorganism, they are preferably used only in the case other, less toxic antibiotics are not effective.

Anthracyclines.Anthracyclines are synthesized in a similar way as other polyketides. They often have one or several sugar residues in the molecule, most often deoxy-sugars, synthesized from glucose, are present in the anthracycline molecule. As to their biological activity, daunorubicin and doxorubicin (adriamycin) are rather important. They are excellent antitumor agents, which are widely used in the treatment of a number of solid tumors and leukemias in human. However, these drugs have dose limiting toxicities such as cardiac damage and bone marrow inhibition. In recent years, a variety of drug delivery systems for anthracyclines have been reported. In most cases, the drugs were linked to high molecular compounds such as dextran , DNA and others.

Macrolides and polyenes.Macrolides are usually classified to include: proper macrolides having 12-, 14- or 16-membered macrocyclic lactone ring to which at least one sugar is bound, and polyenes having 26- to 38-atom lactone ring containing 2 to 7 unsaturated bonds. Besides the sugars bound to the lactone ring, an additional aromatic part is normally present in the polyene molecule. As to the biosynthesis, however, both macrolides and polyenes are synthesized in the same way using identical building units.

Macrolides represent a broad group of compounds and new substances have been incessantly added to the list, including hybrid compounds. A number of derivatives of the basic structure can be produced by one microorganism, on the other хэнд, however, the compounds can also be found in different microorganisms. Macrolides usually possess an antibacterial activity whereas polyenes are mostly fungicides.

Erythromycins produced by Saccharopolyspora erythrea, together with oleandomycin and picromycin, belong to the best known 14-membered lactone ring macrolides. A novel erythromycin was prepared by the recombinant Saccharopolyspora erythrea strain [9]. Macrolides with a 16-membered ring are represented by tylosin, that is produced by Streptomyces fradiae, as well as by leucomycin, spiramycin, etc.

The synthesis of lactone ring is similar to that observed in the case of other polyketides. In contrast to aromatics, propionate and butyrate units are more often used in the biosynthesis, instead of acetate ones. The greatest difference, however, consists in the fact that, instead of aromatic rings, a lactone ring is formed. Keto- and methyl groups of the polyketide chain, from which macrolides are formed, are normally transformed more frequently.Nystatin is the best known polyene antibiotic. Candicidine is another well known antibiotic 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.

The sugars found in macrolide and polyene molecules are not encountered in the structures of microbial cells. They include both basic and neutral sugar molecules. Often, L-forms are found. Sofar, at least 15 different sugars have been described to occur in macrolides and polyenes. All of them are 6-deoxy sugars; some of them are N-methylated, others have the methyl on either the oxygen or carbon atom. As it has been repeatedly proven , 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 . The transformation of glucose to a corresponding sugar takes place in the form of the nucleoside diphosphate derivatives, which is similar to the situation found in the case of other antibiotics.

Avermectins.The molecule of avermectins [10] consists of a 16-membered, macrocyclic lactone to which the disaccharide oleandrose is bound (Fig. 15). Avermectins are produced by Streptomyces avermitillis. The macrocyclic ring of avermectins is synthesized, as other polyketides, by producing a chain from acetate, propionate and butyrate building units. Oleandrose (2,6-dideoxy-3-O-methylated hexose) is synthesized from glucose.

Avermectins are potent antiparasitic compounds with a broad spectrum against nematode and anthropod parasites. They lack antifungal and antibacterial activities. They bind to a specific, high-affinity Site present in nematodes but not in vertebrates. Its dosage for animal and human is extremely low. Ivermectin (22,23-dihydroavermectin B1) is a semisynthetic compound which is used to control internal and external parasites in animals. It is the most potent anthelmintic compound of all. Avermectins are also employed in human medicine and plant protection. Detailed reviews on the uses and biosynthesis of avermectins can be found in recent monographs.

Chloramphenicol.Chloramphenicol is produced by Streptomyces venezuelae [11]. However, at present the antibiotic is commercially produced using a fully synthetic process. In contrast to polyketides, the aromatic ring of chloramphenicol molecule is synthesized from glucose via chorismic acid and p-amino benzoic acid in the microbe.

Aminoglycosides.Streptomycin (Fig.4) is a well-known representative of aminoglycoside antibiotics. It is synthesized by many streptomycetes to produce a number of derivatives. The molecule of streptomycin consists of three components: streptidine, L-streptose and N-methyl-L-glucosamine. None of these components has been found in the primary metabolism of microorganisms. The biosynthesis of streptomycin was disclosed mainly by Walker [12], who also studied the enzymes participating in the biosynthesis of streptomycin .

The importance of streptomycin consists mainly in its efficiency to suppress Mycobacterium tuberculosis. A massive use of streptomycin resulted in effective suppression of tuberculosis, especially in developed countries. Recently, however, the disease caused by M. tuberculosis has been found to increase again due to the occurrence of strains resistant to streptomycin.

Antiviral compounds.Recently also compouds active against viruses have been discovered. Sattabacins and sattazolins, isolated from Bacillus sp. and fattivirin A1, isolated from Streptomyces microflavus are active against Herpes simplex viruses. Inhibitors of HIV are intensively looked out in microorganisms. Inhibitors of HIV-1 protease were detected in fungus Chrysosporium merdarium P-5626. A compound which has an inhibitory effect on HIV-1 replication in chronically infected cells as well as actualy infected cells was isolated (after screeninng 10,000 microorganism products) from the culture supernatant of Streptomyces sp. Mer-2487. A hydroxyl benzaldehyde compound, active against influenza Virus in vitro, was isolated from Aspergillus terreus. Rhodopseudomonas capsulata produces a virucide substance which inactivated polio Virus, Sindbis virus, some fish viruses, without causing any damage to the host cells.

1.1.3 Genetics

High production strains.The genes coding the enzymes that synthesize antibiotics are mostly located on chromosomes. These genes are called structural genes and the enzymes taking part in the antibiotic synthesis are called the enzymes of secondary metabolism. The structural genes are organized to form one cluster. This situation has been observed in all cases described so far. The expression of structural genes is controlled in a similar way as in the case of other genes. Next to a cluster of the structural genes, the genes coding for the resistance of the producer to its own antibiotic are located. Those genes are situated either at the beginning or at the end of the cluster, often in both positions. In the case the resistance genes are present in the two positions, different types of resistance are included as a rule. In addition to the structural genes, regulation genes also determine the antibiotic production. They are often located on plasmids. The genetic control of antibiotic biosynthesis is poorly known. The type of control where the antibiotic synthesis is inhibited by the own product can serve as an example. As a result, the products cellular concentration is maintained at a physiologically tolerable level and, consequently, the producing microorganism is prevented from being self damaged by high concentrations of the product, that are toxic.

Multiplication of the structural genes is not an important factor increasing the antibiotic production. Mutations resulting in an increased antibiotic synthesis mostly affect the regulatory genes. Hopwood and co-workers [12-13] transferred the genes for the production of actinorhodine to a low production, wild type strain using a plasmid. Even though the number of copies of the structural gene increased only twofold, the production of the antibiotic rose 30-40 times. The increase of the antibiotic production has to be accompanied by an increase of resistance to the own product.

When high production strains are prepared by mutagenesis, a type of mutant that loses some of the structural genes can also be obtained. Such a mutant can exhibit a higher level of an antibiotic intermediate whose transformation stopped due to the absence of the corresponding enzyme. By crossing these mutants, some biosynthetic pathways used to synthesize antibiotics were elucidated, e.g. tetracyclines .Genetic manipulation of antibiotic producers.The structural genes for a number of antibiotics have been cloned into host microorganisms. Similarly, genes for antibiotic resistance and other regulatory genes have also been cloned. Streptomyces lividans was found to be a suitable acceptor of foreign genetic material, in which a low degree of restriction of this genetic material exists. This microorganism can host various plasmids and phage vectors. However, at the same time, this microorganism was found not to be usable for the synthesis of various antibiotics or of high antibiotic levels. The antibiotic biosynthesis is a very complex process that requires not only the structural genes for enzymes of secondary metabolism but also the genes for regulation of their biosynthesis. Moreover, the overproduction of an antibiotic has to be coordinated with the primary metabolism of the producing microorganism.

Polyketide synthase genes of microorganisms producing various polyketides have also been hybridized. As a result, a great similarity of polyketide synthases from various streptomycetes was evidenced and new polyketide antibiotics were synthetized.

1.1.4 Search for new antibiotics

Isolation from nature.At present several thousands of compounds having some biological activity have been obtained from microorganisms isolated from nature. As the probability of finding a new compound that would be usable as a new antibiotic is as little as one in ten thousand, a great number of microorganisms have to be checked. A rough estimation says that about 100 000 microorganisms is screened for the presence of biologically active compounds per year. Well equipped laboratories study about 30 different biological activities. The requirements for new antibiotics result from the occurrence of resistant strains of pathogenic microorganisms, that are no more sensitive to known antibiotics used in the clinical practice. It is mostly big pharmaceutical companies that look for new antibiotics. Their search for new compounds is highly automated. The selection methods used and the methods of detection of the biological activity are normally not published.

Preparation of a new antibiotic and its introduction into the clinical practice requires cooperation of scientists from various scientific disciplines. They can be divided into three groups : microbiology (colection of source samples, isolation of diverse microbes, fermentation to enhance the production, taxonomy), pharmacology (target selection, screen design, high-troughput screening, identification of active compounds, efficacy studies, mechanism of action), chemistry (active compounds identification, characterization/replication, isolation/purification, structure elucidation). Producers of antibiotics and other biologically active compounds.The majority of the known antibiotics are produced by actinomycetes, fungi and by moulds. With an increasing spectrum of efficiency of microbial metabolites, new, non-traditional sources of such compounds have been looked for. Tropical soils have an enormous biodiversity and they are a rich source of new antibiotics [14].

The tests of other biological than antibiotic activities require sophisticated methods. This is true especially when enzyme inhibitors are looked for. Thus, Ogawara chose a tyrosine protein kinase associated with the malignant transformation of the cell caused by retroviruses as the target in a biochemical screen and found genistein, an isoflavone from Pseudomonas, exhibiting a specific inhibitory activity. Production of target enzymes using recombinant DNA methodology has dramatically expanded the number of potential targets that can be feasibly screened. A screen for the inhibitors of HIV reverse transcriptase is an example. The enzyme was produced in Escherichia coli, purified by affinity chromatography, and used to test natural products for the activity.

1.1.5 Semisynthetic and synthetic antibiotics

After the structures of the antibiotics discovered had been determined and microbial strains resistant to them detected, possible variations of the molecules of known antibiotics were studied. Several methods have been used to accomplish such variations.

Biosynthetic antibiotics.

The unspecificity of the enzyme systems able to synthesize antibiotics was used, together with the addition of precursors to the growth medium. Thus, the reaction equilibrium was shifted to promote the production of the derivative required. In this way, penicillins with different side chains were prepared. Addition of amino acids to the growth medium can affect the amino acid composition of polypeptide antibiotics. The individual derivatives of penicillin and cephalosporin have slightly different antimicrobial spectra and are capable of suppression of microorganisms resistant to other derivatives.

Semisynthetic antibiotics.

Replacement of a part of the antibiotic molecule can be accomplished chemically or enzymatically. In this way, semisynthetic penicillins, cephalosporins, tetracyclines, etc. were prepared. The production of semisynthetic penicillins and cephalosporins was facilitated by the fact that 6-amino penicillanic and 7-amino cephalosporanic acids (Fig. 18) could be easily prepared. The side chain is removed by the action of an enzyme or by a chemical hydrolysis and to the amino group in position 6 (penicillins) or 7 (cephalosporins), that was мейд free in the previous step, another acyl is bound chemically or enzymatically. In such a way, various penicillins and cephalosporins have been prepared to be effective against microorganisms resistant to original compounds.

Semisynthetic tetracyclines, pyrolinomethyltetracycline, metamycin and doxycycline, exhibit a greater solubility and somewhat different antimicrobial spectrum, as compared to the original tetracyclines [15]. New derivatives of aminoglycosides have been obtained by chemical and enzymatic modifications.

Chemical synthesis of antibiotics.As the majority of antibiotics have rather complex structures, their chemical synthesis is mostly more expensive than the production by fermentation. An exception to the rule seems to be chloramphenicol, that is normally prepared using a chemical synthesis.

Hybrid antibiotics.Using of genetic engineering we can combine structural genes of different antibiotic producers to obtaining new products which are not present in nature . If these genes are expressed, a hybrid antibiotic is synthesized, that cannot be found in nature. Hopwood et al. [16] used this method with the genes of actinorhodin synthesis and obtained related hybrid macrolides, mederhodin A and B, dihydromederhodin A and dihydrogranatirhodin. Niemi et al. [16] prepared new anthracyclines by combination of DNA Streptomyces purpurascens.

1.2 Resistance to antibiotics

The antibiotic resistance is usually looked at from two angles: first, how the microbial strains arise, that obtain the resistance during the treatment of the macroorganism with the antibiotic, second, the resistance of microorganisms producing antibiotics that build up their resistance against the product of their own which, synthesized at high concentrations, would damage the producer. The ways of how these two types of resistance are achieved are often similar, even though the aims are different. Whereas a resistant microorganism is most often capable of transforming the antibiotic or even degrading it completely, the resistance of producing microorganisms has to ensure that the antibiotic will not be destroyed.

Resistance of antibiotic producers. The basic metabolic processes of microorganisms producing antibiotics are not inhibited, if the antibiotics are synthesized at low concentrations, observed in strains isolated from nature. By strain improvement, mutants have been able to reach 100 to 1000-fold antibiotic yields, as related to a volume unit of the fermentation medium. Genome changes of the improved strains include a number of deletions and amplifications in the chromosomal DNA. Changes in extrachromosomal DNA were also detected.

Low production strains, whose resistance to the own product is low (i.e. higher concentrations of the product inhibit their growth), regulate the antibiotic production, e.g. by inhibiting the enzyme activities that participate in the synthesis of the antibiotic. In high production strains, such a control is lost and the strains have to find a way how to survive in the presence of a high concentration of the antibiotic without decomposing it.As mentioned above, the genes for resistance to the own product are often located at the beginning of the cluster of structural genes. As a result, they are expressed simultaneously with the structural genes. However, the genes of newly gained resistances are mostly located on plasmids. Many antibiotics inhibit protein synthesis, the target Site being at the ribosome level. Often, the functions of Tu and G elongation factors are also impaired, together with the synthesis of guanosin penta- and tetraphosphates that is significantly reduced. The antibiotic producers (mostly actinomycetes), as well as the bacteria against which the antibiotic is used, protect themselves by posttranscriptional modification of rRNA. Adenine is methylated to obtain N6-dimethyladenine rRNA in 23S. Such modified ribosomes do not bind the antibiotic. In other cases, adenine is methylated to yield 2-O-methyladenosine.

The most important mechanism of resistance observed in the antibiotic producers seems to be the transport of the antibiotic from the cell to the environment. In the case of high production rates, probably no protection of the active centres could be sufficiently effective. In addition, the antibiotic produced would gradually fill up the interior of the cell. In Streptomyces rimosus, an oxytetracycline producer, genes for the enzymes increasing the antibiotic transport rate precede the structural genes on the chromosome. Genes for the resistance consisting in the protection of ribosomes via the synthesis of an unidentified protein are located at the end of the structural gene cluster [17].

Antibiotic producers also have to solve the problem of a reverse flow of the antibiotic into the cell. Some antibiotics bind to the cell wall, others are complexed in the medium (tetracyclines in the presence of Ca2+ ions). Cytoplasmic membranes of resistant strains are often less sensitive to the effect of antibiotics. This kind of resistance is thought to be connected with the content of phospholipids in the cell. In Bacillus colistinus, a colistin producer, the content of phospholipids in the cell-free extract increased with the sensitivity to the antibiotic.Another way how the antibiotic producers can avoid the effect of their products is by situating the distal enzymes of the antibiotic biosynthetic pathway (synthases) outside the cell, most often in the periplasm. In Streptomyces aureofaciens, a higher proportion of the outside terminal tetracycline synthase was found in production strains under high production conditions in periplasm, as compared to low production conditions [18].

Resistance in pathogenic microorganisms.Shortly after antibiotics were introduced into clinical practice on a massive scale, strains of hitherto-sensitive microorganisms started to appear, that required the use of much higher antibiotic concentrations or, even, were completely resistant to these antibiotics. The resistant strains originated from clones that survived the antibiotic treatment, especially if the treatment was terminated before all pathogenic microorganisms were killed or the antibiotic was applied at sublethal doses. There are several ways how microorganisms can gain resistance .In most resistant microorganisms, the main mechanisms of resistance are detoxification or inactivation of the antibiotic, change of the target Site, blocking of the transport of the antibiotic out of the cell.

Penicillins and cephalosporins are degraded using three ways:

a) by the enzyme penicillin amidase that cleaves the amidic bond by which the side chain is bound to the в-lactam ring,

b) by the enzyme acetyl esterase that hydrolyzes the acetyl group at C-3 on the dihydrazine ring of cephalosporins,

c) by the enzyme в-lactamase that catalyzes hydrolysis of the в-lactam ring of penicillins and cephalosporins.Penicillin amidases are rarely used by microorganisms to build up resistance to в-lactam antibiotics. They are often employed for the synthesis of semisynthetic antibiotics. Acetyl esterase is also not important from the

1.2.1 Regulation of antibiotic production

Overproduction of secondary metabolites.Microorganisms produce in natural environment only small amount of antibiotics. They have to control the antibiotic synthesis since secondary metabolites at high concentrations are mostly toxic even for their producers. Using high-yielding strains and optimization of fermentation condition we can reach many times higher production. In that case we speak about «overproduction» . Production of antibiotics in factories are at present several thausands higher as production of original strains isolated from nature but this high production is reached only when high-yilding strain is used and special conditons of cultivation are kept. The main factors influence production of antibiotics are discused in next chapters.

Growth phases of microbial culture.A culture of a microorganism capable of antibiotic production, where the overproduction of the antibiotic is taking place, includes several growth phases representing a number of physiological states.

Preparatory phase (lag phase) — the culture is adapting to the new environment, the growth is slow and, evidently, regulatory proteins are being synthesized that, on the basis of the information from the environment, activate the expression of the respective genes during cultivation.

Growth phase — the culture grows intensively, usualy a low amount of antibiotic is synthesized.

Transition phase — growth rate and proteosynthesis slowed down; the antibiotic production is started. The enzymes of secondary metabolism are intensively synthesized .

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