《化学合成中生物技术的革新》（英文版） 6 Production and diversification of antibiotics

147 Production and diversification of antibiotics 6.1 An introduction to antibiotics 148 6. 2 General strategies for the production of antibiotics 153 6.3 a brief history of penicillin production 6.4 Antibacterial mode of action of B-lactam antibiotics 6.5 Biosynthesis of penicillins and cephalosporins 6.6 Semi-synthetic penicillins 6.7 Cephalosporin diversification 6.8 Alternative strategies for product diversification 181 Summary and objectives Appendix 6.1 Examples of biotransformation of antibiotics(data abstracted from Sebek, O. K"Antibiotics"in Biotechnology Volume 6a, edited by Kieslich, K. 1984 Verlag Chemie, weihe

1 47 Production and diversification of antibiotics 6.1 An introduction to antibiotics 6.2 General strategies for the production of antibiotics 6.3 A brief history of peNcillin production 6.4 Antibacterial mode of action of fl-lactam antibiotics 6.5 Biosynthesis of penicillins and cephalosporins 6.6 Semi-synthetic penicillins 6.7 Cephalosporin diversification 6.8 Alternative strategies for product diversification Summary and objectives Appendix 6.1 Examples of biotransformation of antibiotics (data abstracted from Sebek, 0. K. "Antibiotics" in Biotechnology - Volume 6a, edited by Kieslich, K. 1984 Verlag Chemie, Weinheim). 148 153 156 164 165 168 179 181 186 187

hapter 6 Production and diversification of antibiotics 6.1 An introduction to antibiotics The discovery and production of antibiotics has been of tremendous importance to human and animal health care. Prior to their discovery about half a century ago, many bacterial infections caused debilitating diseases and fatalities were high. The discovery of antibiotics was a major step in the treatment of infectious diseases, especially those caused by bacteria. today about 50,000 tonnes of antibiotics are produced annually About a third of this consists of penicillins, whilst tetracyclines make up about a quarter of the market The first of the antibiotics that found practical use as a therapeutic was penicillin. The success of penicillin initiated a vast screening process all over the world, which resulted in the isolation of a large number of antibiotic substances from various natural sources. Many of these compounds were produced by micro-organisms and prove to be lethal for other micro-organisms. Many of these compounds were also very toxic to humans and could not be used therapeutically. Nevertheless a large number of classes of useful compounds were produced. The chemical structures of members of some of the most important classes are shown in Figure 6.1. ∏ Examine Figure 6.1 and see if you can identify the p-lactam structure in the first four structures shown B-lactam ring The structure you are looking for is a four membered ring containing three carbon atoms and a nitrogen atom in the ring. The structure you should have identified is You will see that this structure contains a tertiary amide You should examine the other structures shown, but we would not expect you to remember the details of these structures. You should, however be aware of the gene quinolones, tetracyclines, sulphonamides treptomycin

148 Chapter 6 Production and diversification of antibiotics 6.1 An introduction to antibiotics The discovery and production of antibiotics has been of tremendous importance to human and animal health care. Prior to their dimvery about half a century ago, many bacterial infections caused debilitating diseases and fatalities were high. The discovery of antibiotics was a major step in the treatment of infectious diseases, especially those caused by bacteria. Today about 50,OOO tonnes of antibiotics are produced annually. About a third of this consists of peNcillins, whilst tetracyclines make up about a quarter of the market. The first of the antibiotics that found practical use as a therapeutic was penicillin. The success of penicillin initiated a vast screening process all over the world, which resulted in the isolation of a large number of antibiotic substances from various ~Wal sources. Many of these compounds were produced by micro-organisms and prove to be lethal for other micro-organisms. Many of these compounds were also very toxic to humans and could not be used thempeutically. Nevertheless a large number of classes of useful compounds were produced. The chemical structures of members of some of the most impo+nt classes are shown in Figure 6.1. Examine Figure 6.1 and see if you can identify the f3-lactam structure in the first rI four structures shown. The structure you are loolung for is a four membered ring containing three carbon atoms and a nitrogen atom in the ring. The structure you should have identified is ptactamring You will see that this structure contains a tertiary amide. You should examine the other structures shown, but we would not expect you to remember the details of these structures. You should, however, be aware of the general forms of quinolones; tetracyclines; majordasses glycopeptides; of mlibiilics sulphonamides; aminoglycosides; macrolides; streptomycin

Production and diversification of antibiotics 14 lactam antibiotics (a monobactam (a ca non-B-lactam antibiotics )人 (a betraying Figure 6. 1 The chemical structure of some members of the important classes of antibiotics

Production and diversification of antibiotics 149 Figure 6.1 The chemical structure of some members of the important classes of antibiotics

150 Chapter 6 HoN HO HO chloramphenicol H2N H (a glycopeptide <>- 6N bactrim (a sulphonamide) (an aminoglycoside)Ho- NH erythromycin A (a macrolide Figure 6.1 Continued ∏ From these structures, would you expect each of these groups of antibiotics to act in the same way in target organisms

150 Chapter 6 Figure 6.1 _.______ Continued. n in the same way in target organism? From these structures, would you expect each of these pups of antibiotics to act

Production and diversification of antibiotics 151 modes of action You should have concluded that because these structures are very diverse, it is unlike that they will act in the same way. This is, in fact, true; the mode of action differs from one class of antibiotics to another We have listed some modes of action of antibiotics in Table 6.1 AntIbiotic Mode of actlon β Lactams Inhibition of synthesis of, or damage to, cell wall Penicillins Cephalosporins Monobactams Vancomycin Inhibitionof synthesis or metabolism of nucleic acids Polymyxins Inhibition of synthesis or damage to cytoplasmic membrane Polyene antifungals Sulfonamides Modification of energy metabolism Isoniazid Inhibition of protein biosynthesis Chloramphenicol Fusidic acid Table 6.1 Modes of action of antibiotics

Production and diversification of antibiotics 151 modes of adon You should have concluded that because these structures are very diverse, it is unlikely that they will act in the same way. This is, in fact, true; the mode of action differs from one class of antibiotics to another. We have listed some modes of action of antibiotics in Table 6.1. r\ntlbiotlc Mode of action &Lactams Penicillins Cephalosporins Monobactams Carbapenems dancomycin 3acitradn 3ycloserin zosfornycin 2uinolones Sifampin Uitrofurantoins Uitrolmidazoles Polymyxins Polyene antifungals Sulfonamides Trimethopim Dapsone Isoniazid Aminoglycosides Tetracyclines Chloramphenicol Erythromycin Clindamydn Spectinomycin Mupirocin Fusldk add Inhibition of synthesis of, or damage to, cell wall Inhibition of synthesis or metabolism of nucleic acids Inhibition of synthesis or damage to cytoplasmic membrane Modifition of energy metabolism Inhibition of protein biosynthesis Table 6.1 Modes of action of antibiotics

152 Chapter 6 We have this far established that the antibiotics are a diverse group of compounds that are produced industrially in large amounts which are of great value in health care. We need to establish one further point. explain why we do need to continue to find ways of producing new antibiotics? There are many reasons why we need to produce a large variety of antibiotics. Different disease causing micro-organisms have different structures and different metabolisms. Thus you should have anticipated that a particular antibiotic may be effective against a particular type of micro-organism but not against others. For example, traditional enicillins were more effective against Gram-positive bacteria than against Gram-negative bacteria. Furthermore, because of the large numbers of cells involved their rapid rates of growth and the ability to transfer genetic material between often quite unrelated organisms, new varieties of disease-causing micro-organisms arise quite frequently. Amongst the changes that are detected amongst disease-causing resistance to micro-organisms is the development of resistance to antibiotics. This resistance may, for example, depend upon the production of enzymes that destroy the antibiotic or on changes to structural components of cells which result in the antibiotic not being taken up by cells, or on its failure to interact with the target component. The widespread use of antibiotics will itself act as a selection mechanism leading to the proliferation of antibiotic resistant strains. Thus, when penicillin was first introduced, most disease-causing Gram-positive bacteria were sensitive to this antibiotic. Now many such organisms are resistant to this antibiotic. In most cases, this resistance is based pon the production of an enzyme which hydrolyses either the p-lactam ring (B-lactamase)or the secondary amine linking the lactam ring to another moiety (penicillinase). In many instances the genes coding for the resistance factor are encoded in plasmids and are, therefore, readily transmitted from organism to organism. Many strains of bacteria now carry multiple antibiotic resistances It is for these reasons that a search for new antibiotics must continue In this chapter, we will examine strategies for producing antibiotics. We have had to be selective and have chosen to confine discussion largely to the p-lactams, with particular emphasis on the diversification of the primary antibiotics using biotransformation. We ave adopted this strategy in order to produce a manageable study, while enabling to explain the main principles involved chapter We will begin by giving a brief overview of the strategies that may be employed to desirable antibiotics. Then we will give a brief review of the history of the production of penicillin. We will then examine the mode of action of p-lactam antibiotics ind briefly describe the biosynthetic pathways of B-lactam antibiotic production. Subsequently we will examine, in greater depth, the biotransformation of penicillins. A consideration of cephalsporin production will follow and will be compared with the production and diversification of penicillins. In the final part of this chapter we wil fly describe the new p

152 Chapter 6 We have this far established that the antibiotics are a diverse group of compounds that are produced industrially in large amounts which are of great value in health care. We need to establish one further point. Write down two reasons why society needs so many different antibiotics and rI explain why we do need to continue to find ways of producing new antibiotics? There are many reasons why we need to produce a large variety of antibiotics. Different disease causing microorganisms have different structures and different metabolisms. Thus you should have anticipated that a particular antibiotic may be effective against a particular type of micro-organism but not against others. For example, traditional penicillins were more effective against Gram-positive bacteria than against Gram-negative bacteria. Furthermore, because of the large numbers of cells involved, their rapid rates of growth and the ability to transfer genetic material between often quite unrelated organisms, new varieties of disease-causing microorganisms arise quite frequently. Amongst the changes that are detected amongst disease-causing micro-organisms is the development of resistance to antibiotics. This resistance may, for example, depend upon the production of enzymes that destroy the antibiotic or on changes to structural components of cells which result in the antibiotic not being taken up by cells, or on its failure to interact with the target component. The widespread use of antibiotics will itself act as a selection mechanism leading to the proliferation of antibiotic resistant strains. Thus, when penicillin was first introduced, most diseasecausing Gram-positive bacteria were sensitive to this antibiotic. Now many such organisms are resistant to this antibiotic. In most cases, this resistance is based upon the production of an enzyme which hydrolyses either the p-lactam ring (p-lactamase) or the secondary amine linking the lactam ring to another moiety (penicillinase). In many instances the genes coding for the resistance factor are encoded in plasmids and are, therefore, readily transmitted from organism to organism. Many strains of bacteria now carry multiple antibiotic resistances. It is for these reasons that a search for new antibiotics must continue. resktance to antibiotics In this chapter, we will examine strategies for producing antibiotics. We have had to be selective and have chosen to confine discussion largely to the b-lactams, with particular emphasis on the diversification of the primary antibiotics using biotransformation. We have adopted this strategy in order to produce a manageable study, while enabling us to explain the main principles involved. We will bep by giving a brief overview of the stratqjes that may be employed to produce desirable antibiotics. Then we will give a brief review of the history of the production of penicillin. We will then examine the mode of action of &lactam antibiotics and briefly describe the biosynthetic pathways of p-lactam antibiotic production. Subsequently we will examine, in greater depth, the biotransformation of penicillins. A consideration of cephalsporin production will follow and will be compared with the production and diversification of penicillins. In the final part of this chapter we will briefly describe the new p-lactams. chapBr overview

Production and diversification of antibiotics 15 6.2 General strategies for the production of antibiotics secondary The major classes of antibiotics are secondary metabolic products of micro-organisms metabolites Many were discovered by empirically screening culture filtrates or cell extracts for antimicrobial activity. a range of techniques (examples are methods using techniques impregnated discs, porous cylinders, cut wells, see Figure 6. 2)have been used to carry out such screening Petn dish disc impregnated with culture filtrate b) porous cylinder method Petri dish lawn of test bactera porous cylinde c)gutter-plate method culture filtrate △A gutter cut into agar a filled with culture filtrate steaks of culture ( indicates growth) nutnent agar igure 6.2 Examples of techniques used to screen microbial cultures for antibiotic activity. In a), filter discs are impregnated with the test sample and placed on the surface of a nutrient agar filled Petri dish, which had been seeded with bacteria. Anti-microbial activity is detected by the inhibition of growth around the impregnated disc. b)Illustrates a simil ar approach except in this procedure that depends upon the diifusion of antibiotics through agar. In this case, a single sample is tested for antimicrobial activity against a range of organisms ∏ Examine Figure 6.2 carefully. Which test sample in a)appears to have the greatest

Production and diversification of antibiotics 153 6.2 General strategies for the production of antibiotics secondary metabolites screening eddques The major classes of antibiotics are secondary metabolic products of micro-organisms. Many were discovered by empirically scmening culture filtrates or cell extracts for antimicrobial activity. A range of techniques (examples are methods using, impregnated discs, porous cylinders, cut wells, see Figure 6.2) have been used to carry out such screening. Figure 6.2 Examples of techniques used to screen microbial cultures for antibiotic activity. In a), filter discs are impregnated with the test sample and placed on the surface of a nutrient agar filled Petri dish, which had been seeded with bacteria. Anti-microbial activity is detected by the inhibition of growth around the impregnated disc. b) Illustrates a similar approach except in this case the test substances is placed in the centre of a porous cylinder. Antimicrobial substances diffuse out of the cylinder and inhibit growth around the cylinder. c) Illustrates another procedure that depends upon the diffusion of antibiotics through agar. In this case, a single sample is tested for antimicrobial activity against a range of organisms. Examine Figure 6.2 carefully. Which test sample in a) appears to have the greatest n antimicrobial activity?

154 Chapter 6 The most obvious answer is a because the zone of inhibition is greatest around the disc impregnated with A. However, there are other possibilities. The size of the zone of inhibition depends on: the amount of antimicrobial agent present (ie its concentration) the rate at which it diffuses(this depends on its molecular mass); the sensitivity of the organism that has been used to seed the plate to the antimicrobial component. Thus, it could be for example that B produces an anti-microbial agent but this is ineffective against the test organism. The gutter plate method (Figure 6.2c) provides a method for testing samples for antimicrobial activity against a range of organism Which organism(s)seem to be a) most sensitive to the antimicrobial activity of the sample illustrated in Figure 6.2c? b)insensitive to the sample being tested in Figure 6.2c? The answer to a)is B The growth of this organism is prevented at quite some distance from the gutter. The answer to b)is a and D, neither of these organisms appear to be nhibited by the test substances. Once an antibiotic producer has been identified the next stage is to produce sufficient to humans?, is it effective against disease organisms, does it possess suitable characteristics(for example solubility, chemical stability)for use as a medicine need answering. Let us assume that a new, potentially useful antibiotic has been discovere The key questions then become, how can the desired material be produced in the most cost effective way? is it possible to produce variants of the antibiotic which have desirable properties, such as greater effectivity against infection, cheaper ways to produce it or increased stability? See if you can list some approaches that may be used to reduce costs/maximise lds of antibiotic production There are many approaches that may be used here. One approach is to screen relate changed by organisms to see if a higher yielding strain may be obtained. altenatively the culture conditions used to cultivate the antibiotic-producing strain may be modified with the objective of increasing antibiotic production. This may include manipulation of physical conditions such as pH and temperature, addition of precursors of the antibiotic or specific inhibition of particular metabolic activities. We might also use genetic You pulation(for example mutation or genetic engineering) to enhance product yield n will meet specific examples of these strategies in our discussions of penicillin production

154 Chapter 6 The most obviaus answer is A because the zone of inhibition is greatest around the disc impregnated with A. However, there are other possibilities. The size of the zone of inhibition depends on: 0 the amount of antimicrobial agent present (ie its concentration); the rate at which it diffuses (this depends on its molecular mass); the sensitivity of the organism that has been used to seed the plate to the antimicrobial component. Thus, it could be for example that B produces an anti-microbial agent but this is ineffective against the test organism. The gutter plate method (Figure 6.2~) provides a method for testing samples for antimicrobial activity against a range of organisms. a) most sensitive to the antimicrobial activity of the sample illustrated in Figure 62c? b) insensitive to the sample being tested in Figure 6.2c? The answer to a) is B. The growth of this organism is prevented at quite some distance from the gutter. The answer to b) is A and D, neither of these organisms appear to be inhibited by the test substances. Once an antibiotic producer has been identified, the next stage is to produce sufficient of the antibiotic toevaluate its potential for therapeutic use. Questions, such as, is it toxic to humans?, is it effective against disease organisms?, does it possess suitable characteristics (for example solubility, chemical stability) for use as a medicine?, need answering. Let us assume that a new, potentially useful antibiotic has been discovered. The key questions then become, how can the desired material be produced in the most cost effective way? is it possible to produce variants of the antibiotic which have desirable properties, such as greater effectivity against infection, cheaper ways to produce it or increased stability? new strains, wlture Conditions changed by See if you can list some approaches that may be used to reduce costs/maximise n yields of antibiotic production. There are many approaches that may be used here. One approach is to xmen related organisms to see if a higher yielding strain may be obtained. Alternatively, the culture conditions used to cultivate the antibiotic-producing strain may be modified with the objective of increasing antibiotic production. This may include manipulation of physical conditions such as pH and temperature, addition of precursors of the antibiotic or specific inhibition of particuiar metabolic activities. We might also use genetic manipulation (for example mutation or genetic engineering) to enhance product yield. You will meet specific examples of these strategies in our discussions of penicillin production

Production and diversification of antibiotics 155 Assume you have an antibiotic-producing organism. See if you can list some approaches that may enable you to use the same organism to produce a range of ferent, but related antibiotics different precursors which may lead to the uggested, for example, using slightly There are several production of slightly products. Alternatively, metabolic inhibition might be used or you may have considered using mutants. Another approach would be to isolate the antibiotic first and to modify it in vitro using chemical or biotransformation (enzymatic)methods. All of nese approaches have found practical applications. We will again use the p-lactams to illustrate these strategies elds of antibiotics produced by microbial cultures and for diversifying the natureof the products that are manufactured a)ImprovIng yield optimise physical condition manipulate media to maximise yield use genetic engineering procedures to over-come rate limiting step b)dIversifying products directed biosynthesis using precursor use mutations to diversify product diversified antibiotics use genetic engineenng strategy use in vitro biotransformation using whole cells and isolated enzymes chemical transformation Figure 6.3 Summary of the strategies available for improving yields and for diversifying the products made by antibiotic-producing micro-organisms Consideration of pencillin production serves to illustrate the success of these strategies Penicillin was produced at a concentration of about 1 ppm by the first penicillin producers that were isolated. By manipulation of culture conditions together with genetic manipulation, yields in excess of 10 g I(excess of 10,000 ppm) are routinely achieved. This development has also been paralleled by the diversification of the

Production and diversification of antibiotics 155 Assume you have an antibiotic-producing organism. See if you can list some approaches that may enable you to use the same organism to produce a range of different, but related antibiotics. There are several possibilities you may have suggested, for example, using slightly different precursors which may lead to the production of slightly different end products. Alternatively, metabolic inhibition might be used or you may have consid& using mutants. Another approach would be to isolate the antibiotic first and to modify it in zifm using chemical or biotransformation (enzymatic) methods. AH of these approaches have found practical applications. We will a+ use the &lactams to illustrate these strategies. In Figure 6.3, we have provided a su~~unary of the possible strategies for improving yields of antibiotics produced by microbial cultures and for diversifying the nature of the products that are manufactured. n strasgiesfor ?Proving pHsand diversifying pro&& Figure 6.3 Summary of the strategies available for improving yields and for diversifying the products made by antibiotic-produang rnicro-organisms. Consideration of pencillin production serves to illustrate the success of these strategies. Penicillin was produced at a concentration of about 1 ppm by the first penicillin producers that were isolated. By manipulation of culture conditions together with genetic manipulation, yields in excess of 10 g 1-' (excess of l0,ooO ppm) are routinely achieved. This development has also been paralleled by the diversification of the

156 Chapter 6 product and a wide variety of penicillins are now available. For these reasons, together with the fact that the history of penicillin production includes most of the important innovations now taken for granted in newer fermentation, it is worthwhile briefly reviewing the history of penicillin production. SAQ 6.1 The structure of penicillin G is drawn below in a different form from that illustrated for B-lactams in Figure 6.1 CH3 COOH Il Make a comparison of the structure of penicillin g and amoxycillin and briefly explain a strategy that might be used to diversify penicillins 6. 3 A brief history of penicillin production .3.1 Surface cultures and product diversification mya aung Penicillins, like most antibiotics, are secondary products whose synthesis is not directy linked to growth. The enzymes that produce secondary products are normally repressed or inhibited under conditions which favour rapid growth In the early work on penicillin, Penicillium notatum was grown as a floating mycelium on about 2 cm depth of liquid medium. The mycelium absorbed nutrients from the medium and penicillin was excreted into the medium. The mycelium and spent medium are readily separated Figure 6. 4 Stylised representation of changing parameters and penicillin production in cultures of Penicilium notatum, grown as a surface culture on Czapek-Dox medium(adapted from Hockenhull DJ-D"Production of Antibiotics by Fermentationin Essays in Applied Microbiology edited by Norris J R& Richmond M H 1981. John Wiley Sons Ltd Chichester)

156 Chapter 6 product and a wide variety of penicillins are now available. For these reasons, together with the fact that the history of penicillin production includes most of the important innovations now taken for granted in newer fermentation, it is worthwhile briefly reviewing the history of penicillin production. The structure of penicillin G is drawn below in a different form from that illustrated for &lactams in Figure 6.1. Make a comparison of the structure of penicillin G and amoxycillin and briefly explain a strategy that might be used to diverse penicillins. 6.3 A brief history of penicillin production 6.3.1 Surface cultures and product diversification Penicillins, like most antibiotics, are secondary products whose synthesis is not dkctly linked to growth. The enzymes that produce secondary products are normally qressed or inhibited under conditions which favour rapid growth. In the early work on penicillin, Penicillium notatum was grown as a floating mycelium on about 2 an depth of liquid medium The mycelium absorbed nutrients from the medium and penicillin was excreted into the medium. The mycelium and spent medium are readily separated. mating Wmlium Figure 6.4 Stylised representation of changing parameters and penicillin production in cultures of Penidllum notaturn grown as a surfaca culture on Czapek-Dox medium (adapted from Hockenhull DJ-D "Production of Antibiotics by Fermentation" in Essays in Applied Microbiology edited by Nonls J R & Richmond M H 1981. John Wiley 8 Sons Ltd Chichester)