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). 148 153 156 164 165 168 179 181 186 187 148 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 149 Figure 6.1 The chemical structure of some members of the important classes of antibiotics. 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 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. 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 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 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 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. 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). Production and diversification of antibiotics 157 C~a~ek-Dox medium slow release of assimihbie carbohydrata corn steep liquor CSL conmined e!hy!amine Bph=M Initially, Czapek-Dox medium was used. This medium, containing mineral salts, sodium nitrate and sugar (usually sucrose or glucose), allowed rapid growth but only very small amounts (around lpg ml-') of penicillin were produced. Effectively what happened in these cultures was rapid growth with no penicillin production until virtually all the sugar had been used. At the same time, the pH dropped dramatically as the sugars were being metabolised, but rose sharply when the mycelium began to lyse. The culture was, therefore, only in the optimum pH range (pH 657.0) for penicillin production for a very short time. These observations are illustrated in Figure 6.4. The important question was, how could the period of penicillin production be extended? See if you can list two or three ways in which this might have been achieved. n There are several possibilities. One would be to ensure that carbohydrates were available at such a rate that they did not cause excessively rapid growth. This was achieved by using lactose in place of the more readily assimilated glucose or sucrose. The fungus hydrolyses this substrate only slowly, thereby releasing the more readily assimilated glucose and galactose at a slow rate. Thus the mycelium behaved as though it was semi-starved and the biomass produced penicillin over a much longer time period (day 2 to 7). Also under these conditions the pH was maintained nearer to the pH optimum of 6.6-7.0 for penicillin production. The result was an improvement in penicillin yield of about 5-10 fold. Further improvements were achieved by using ammonium acetate or ammonium lactate as nitrogen source in place of nitrate. This reduced the rise in pH observed at the end of the fermentation. Replacement of these nitrogen sources by complex nitrogen sources, such as casein hydrolysate, improved the long term availability of nitmgen and further stabilised the pH with concomitant improvements in yields. Thus by changing the energy, carbon and nitrogen sources significant advances in product yield were achieved as a consequence of slower growth and pH stabilisation. The subsequent advance was rather fortuitous and rested more with serendipity than with scientific logic. A search was made for cheaper more effective replacements for casein hydrolysate. Amongst the tested materials was corn steep liquor (CSL). CSL is a by-product of the manufacture of starch from maize kernals. Whole maize is incubated in warm water, at 50°C acidified with so2. Thermophilic bacteria hydrolyse proteins and other components of the kernals, thereby loosening the starch granules. These are removed, leaving behind the steep liquor which is used to treat further maize kernals. Ultimately, the liquor is too viscous to re-use and the liquor is concentrated and used as cattle feed. It was this material that was used for penicillin fermentation. Surprisin y, the yield of penicillin increased by a further 5-10 fold giving yields of 50-10 pg ml- . In part, the increase in yield could be attributed to the capacity of the CSL to buffer the pH and to facilitate the slow release of carbohydrate. However, by far the most important factor was that CSL provided a precursor that led to the production of a more stable and easier to isolate form of penicillin, what we now call penicillin G. In essence, what was happening was that the CSL supplied phenylethylamine. This had been produced from the amino acid phenylalanine by the action of the microflora in the CSL. Thus: 8' 158 Chapter 6 v v phenylalanine phenyl ethylmine The P. ilofatum took up the p-phenyl ethylamine, converted it to p-phenylacetate, which was subsequently attached to the &amino group of penicillanic acid to give benzyl penicillin (penicillin G). We can represent this prcxxss by: penicilli G easily Crystall& and improved Sbtili Previous penicillins had aliphatic groups attached to the &amino penicillanic acid moiety. Penicillin G has many advantages over the aliphatic derivatives, eg it is mom easily aystallised and it is more stable. n suggest an alternative way of producing penicillin G, without using CSL. The obvious way is to include bphenylacetic acid or kphenylethylamine in cultures. Indeed, when bphenylacetic acid was added to cultures grown in CSL, the yields of penicillin were enhanced further. Typical yields were 1W150 pg ml-'. The results obtained by the addition of kphenylacetic acid to cultures suggest a n method of producing a wide variety of penicillins. See if you can explain what this is. In principle, by adding derivatives of acetic acid to culture media, we mght be able to produce a wide range of penicillins. This strategy was adopted, eg inclusion of phenoxyacetic acid led to the production of penicillin V Production and diversification of antibiotics 158 oral Penicillin V has advantages over some other penicillins as it is stable at low pH and can We may describe the production of diverse penicillins using this strategy as directed biosynthesis using precursor feeding. We have listed some examples of penidllins in Table 6.2. administration be admini*& orally. Table 6.2 Examples of penicillins Suggest what precursors should be fed to cultures to produce each of the penicillins shown in Table 6.2. Read our response carefully as it contains some additional information. 1 160 Chapter 6 6.3.2 Use of deep cultures in the production of penicillin So far we have shown how, by manipulating the formulation of media, improvements in product yield and product diversification were achieved in the early years of penicillin production. We have deliberately selected the high points of these development activities. We will now turn our attention to another aspect of the development of penicillin production: the switch from surface to deep culture. IN tially, penicillin was produced in shallow earthenware "penicillin pots" that resembled bedpans used in hospitals. h4ilk bottles were then used. The problems with these approaches stemmed from the costs of the multiple inoculations that were needed and the costs of harvesting from multitudinous small cultures. Replacement of these small vessels by lqer tray-like vessels, however, was not entirely successful: the trays often warped during sterilisation. n See if you can list some advantages of using surface cultures. The main advantages of using shallow surface cultures are that there are few problems ensuring that the cultures remain aerated and, because of the large surface area and thin layer of medium, there are few problems with localised overheating. Despite these advantages, deep (submerged) cultures were still deemed to be the most viable route to satisfymg the market demand for penicillin. It was estimated that a surface culture equivalent to 2 hectares would be required to produce the same amount of penicillin as a deep culture equivalent to 5 x lo' Iitres. The desire to switch to deep cultures was thus driven by commercial consideration. In practice, P. notutum was found to be unsatisfactory for deep culture. It grew in large tightly packed pellets. This lead to oxygen starvation in the centre of the pellets. Alternative organisms were sought which would combine good growth characteristics (loose, confluent growth) with high penicillin yield. A strain of Penicillium chrysasemtm was selected. This strain, NRRL 1951, produced only 50 pg penicillin ml-'. Nevertheless, its growth characteristics made it desirable. Subsequently, variants of this strain (for example B25, X1612), produced by mutagenesis, gave higher yields. Strain X1612 produces above 400-500 pg penicillin mP. The development of the deep culture approach followed conventional routes, including optimising inoculum density, oxygen dispersal and contml of temperature, pH and foam. These aspects of process technology are dealt with elsewhere in the BIOTOL series (for example "Operational Modes of Bioreactors" and "Bioreador Design and Product Yield) so we will not deal with them in any detail here. You should appreciate that the development of the technology recognised the apparent distinction between the growth of the producing organism and the biosynthesis of the desired product. The concept arose that two phases in penicillin production could be distinguished. In the first phase, rapid growth of the organism took place, while in the second phase little growth occurred, but this phase was marked by penicillin production. So there was separaiion into a growth phase and a production phase which were later called the "trophophase" and "idiophase" by Bu'llock. Although the separation of these two phases may not be quite as distinct as may be implied by the use of these two terms, it provides the basis of contemporary penicillin production processes. In these processes, the culture is first cultivated under conditions which favour growth. Once the culture has fully grown, culture conditions are manipulated to favour penicillin production. Although these manipulations may be carried out in a single vessel, it is molp usual to Produdion and diversification of antibiotics 161 physically separate them into two vessels. Such two phase processes have become the norm for the production of secondary products. A stylid system is shown in E- 6.5. Figure 6.5 A stylised system for producing secondary metabolites such as penicillin. Although processes differ in detail, this generalised scheme is adopted for the production of secondary metabolites. Usually, the vessel used for biomass production is smaller than that used to produce the desired product Explain why this is so. The aim in most processes is to produce biomass as quickly as possible (that is use conditions which allow rapid growth), but to maintain cells in production as long as possible. If for example it took the cultures 3 days to grow, but they could be maintained in a productive state (that is in the idiophase) for 10 days, then in principle the ratio of vessel volumes could be 3:10, troph0phase:idiophase. Again this is a simplification, since in some processes the cultures leaving the growth phase may be concentrated before being transferred to the production phase, in order to establish very high biomass concentrations in the idiophase tank. Such processes may be operated in a batch-wise manner, the biomass produced in the trophophase being transferred en bloc into the idiophase. Alternatively, biomass may be transferred continually from the trophophase vessel into the idiophase tank. In this description we have made a clear distinction between growth and secondary product synthesis. You should, however, realise that the distinction is not quite so sharp in practice. Thus we might expect some, albeit a small amount, of secondary product formation in the trophophase and some growth of new cells replacing dead ones in the idiophase. Nevertheless, the separation of the process into two phases enables the optimisation of conditions for growth in one phase and the imposition of conditions which maximise production of antibiotic in the other. Before we leave this description of the production of peNcillin, we should point out that it is not essential that growth and production phases are physically separated. It is possible by using a preset feed pattern to carry out both processes in the same vessel. n batd-ior contkwous transfers 162 Chapter 6 In Figure 6.6 we have illustrated the production of penicillin in a single vessel. You will notice that sugars are fed into the vessel, first slowly because there is little biomass to support, but as this increases the rate of sugar input is also increased. Once growth is complete, the sugar feed rate is again reduced to a level which maintains the grown culture and allows penicillin production. Notice also the change in pH during growth and its maintenance at an optimum for penicillin biosynthesis in the production phase. single vesd Figure 6.6 Production of penicillin using pre-set feed patterns. Note that increase in biomass occurs over the first short phase, while the penicillin production phase is maintained for a much longer period. In Figure 6.6 we have used sugar feed rate as an example of a pre-set feed pattern. In practice we can adjust a wide variety of parametes during the incubation. Suggest some parameters that might be adjusted to enhance penicillin n production. You may for example have suggested that the supply of other nutrients, such as ammonia and Oz, may be adjusted to respond to the diff-t needs of growth and penicillin production. You may also have suggested that precursors of specific penicillins, such as f&phenylacetic acid, may be added after the growth phase is complete. You may have also considered altering physical parameters such as pH and temperature. The development of deep cultures for the production ofpicillin posed a number of important technological questions. A typical large (50 m ) bioreactor uses 05 tonne of sugar per day. The heat generated by the metabolism would, if not removed, cause the temperature to rise by almost 2°C per hour. Similarly, such a vessel would consume almost 05 tonne of oxygen per day. Thus important questions of how to remove the excess heat and supply the necessaryoxygen without causing foaming had tobe solved. sugar feed rata Pam Oxygen supPlv and heat mavd Production and diversification of antibiotics 163 This is as far as we want to take our discussion of the development of deep cultures for the production of penicillin in this chapter. It is an aspect of the production of secondary metabolites, such as penicillin, more appropriately dealt with in the technology texts in the BIOTOL series. The lessons we hope you will take from this brief description of the history of penicillin ‘taka home’ mews production are that: yields of secondary metabolites may be greatly influenced by the composition of the culture media and by physical parameters such as pH; growth of biomass and accumulation of secondary metabolite frequently occur in two separate phases (trophophase and idiophase) and the optimum conditions for each may be quite different; growth and production phases may be carried out in separate vessels or, by using preset feed rates and parameter control, in a single vessel; the exact form of the product may be influenced by components in the culture media and precursor feeding may be used to direct the biosynthesis of a specific product. Two figures showing changing parameters in surface cultures of Penicillium notaturn any given below. The medium used in each case was based on that of Czapek-Dox. In the upper figure, the substrate was glucose, while in the lower figure the substrate was an unspeuf~ed sugar X. Explain why the yield of penicillin was greater for X than it was with glucose. 164 Chapter 6 Now that we have provided you with an overview of the history of penicillin production, we will examine some more details of the biotransformation of &lactams. We will briefly outline the normal biosynthesis pathways that lead to their productim and then consider how these products may be diversified in uih to give a wider range of valuable compounds. We begin by briefly explaining how the giactam antibiotics am effective as therapeutic agents. 6.4 Antibacterial mode of action of p-lactam antibiotics The origin of the success of glactam antibiotics mainly results from the extreme low toxicity of these compounds with regard to human beings. In other words, &ladam antibiotics have a highly selective toxicity for bacteria since they do not interfere with human metabolism, but inhibit the formation of the cell wall of growing bacteria. In bacteria the cytoplasmic membrane is covered with a peptidoglycan layer, which determines cell shape and imparts the rigidity necessary to protect the bacterium from osmotic rupture. The peptidoglycan structure consists of alternating units of the amino sugars N-acetylglucosamine and N-acetylmuramic acid. The N-acetylmuramic acid units are linked to peptide chains. Many of these peptide chains are also cross-linked to each other. In StaphyIococcus au~eus, for example, the cross-linking is achieved when the amino group of a terminal glycine unit is inserted in the bond that links an alanylalanine unit of another chain (Figure 6.7). seleclive bw peptidoglycan Layer Figure 6.7 Formation of cross-linkage between individual peptide chains in the peptidoglycan layer of S. aureus. Production and diversification of antibiotics 165 This insertion is accomplished by an enzyme called transpeptidase. f3-Lactam antibiotics function as substrates for the transpeptidase, thereby establishing selective inhibition of bacterial cell wall synthesis. The structural similarity between g-lactam antibiotics and the alanylalanine unit is remarkable as can be seen in Figure 6.8. transpeplidase Figure 6.8 Pencillins are similar to the bacterial peptidoglycan terminal alanylalanine moiety. Because of this similarity, the enzyme transpeptidase recognizes plactam antibiotics as substrate. As a result of this the fMactam is incorporated in the peptide chain thereby making peptidepeptide cross-linking impossible. The occurrence of this phenomenon stops the construction of the bacterial cell wall. 6.5 Biosynthesis of penicillins and cephalosporins Use Figure 6.9 to help you follow the description given below. Penicillins and cephalosporins are products of biosynthetic pathways that have many identical enzymatic steps. It is generally accepted that the tripeptide, G(L-a-aminoadipy1)-Lcysteinyl-D-valine (LLD-Am), is the direct precursor to both penicillin and cephalosporin C. The first biosynthetic steps are two reactions that generate ACV from its constituent amino acids L-a-aminoadipic acid, L-cysteine and L-valine. L-a-aminoadipic acid and L-cysteine are condensed by the enzyme 'AC synthetase' and, in the next step, the resultant G(L-a-aminoadipy1)-Lcysteine is coupled with L-valine. In this step the configuration of L-valine is inverted to D-valine. The tripeptide LLD-ACV is then cyclised to isopenicillin N by an oxidative reaction involving the removal of four protons. The enzyme that catalyses this reaction is isopenicillin N synthetase or synthase (IPNS). many identiml ~ZY~~ SOeps 166 Chapter 6 Figure 6.9 Formation of isopenicillin N from its constituent amino adds. After condensation of Lu-aminoadipic acid with L-cysteine, L-valine is coupled. During this transformation, ?he configuration of the latter amino acid inverts to give D-valine. Production and diversification of antibiotics 1 67 Now use Figm 6.10 to follow the rest of the discussion. ~ ~~ Figure 6.10 Biosynthetic pathways from isopeniullin N to penicillin G and cephalosporin C. Some strains have the ability to convert deacetylcephalosporin C into cepharnycin C. 168 Chapter 6 Different strains of microorganisms are responsible for the production of either penicillins or cephalosporins. In penicillin-producing strains, an acyltransferase enzyme system is present which can remove the side chain from isopenicillin N to give 6-aminopenicillanic acid (&MA), and which can subsequently acylate 6APA to generate various penicillins, the most important ones being penicillin G and V(see section 6.3, Table 6.2). Cephalosporin-producing strains are characterid by the presence of the enzyme epimerase, responsible for the conversion of the L-a-aminoadipyl side chain in isopenicillin N, into the Da-aminoadipyl side chain in penicillin N. The next step is an oxidative ring expansion and involves the loss of two protons, it is catalysed by the enzyme expandase. Unaware of this phenomenon, chemists carried out the ring enlargement of the penicillin skeleton by nonenzymatic means, finding only much later that nature had been doing the same for a long time. Deacetoxycephalosporin C thus obtained is hydroxylated to give deacetylcephalosporin C, using the enzyme hydroxylase (mono-oxygenase). Finally, deacetylcephalosporin C is converted into cephalosporin C with the aid of an acyltransferase. epinerase ewmdase hydmxyhse acylbansferase From what you read in Section 6.3, does the enzyme I�"-acyltransferase exhibit a high degree of specificity? Given reasons for your answer. 1 I 6.6 Semi-synthetic penicillins penicillin G resistance In spite of its remarkable therapeutic usefulness and low toxicity, penicillin G appeared to have had its limitations when resistant strains of bacteria emerged. Thus it became of interest to consider producing variants of this molecule with different activities. Although a range of penicillins could be produced by directed biosynthesis using precursor feeding, this approach is limited by the toxicity of the precursors, the ability of the penicillin producing cells to take up the precursor and by the capability of the acyltransferase to transfer the acyl pups onto the 6-aminopenicillanic acid moiety. It was noted that many penicillin-resistant organisms produce enzymes that catalyse the hydrolyses of the amide links in penicillin. These enzymes are penicillin acylases and &lactamases depending upon the amide links they hydmlyse. penidin acyl- gLacramases Production and diversification of antibiotics 169 Figure 6.1 1 Hydrolysis of penicillin G by penicillin acylase and plactamase. Obviously, the production of penicillins that were not sensitive to this hydrolysis would be advantageous. Examine Figure 6.11 and see if you can suggest a strategy that might be adopted n to produce modified penicillin. The strategy we hope you identified is to first produce 6aminopenidanic acid, then attempt to add different moieties to the &amino group. This can be achieved either chemically or enzymatically. In the following section we will consider the conversion of penicillin G into 6-aminopenicillanic acid and follow this by examining how 6-aminopenicillanic acid may be converted into ampicillin and amoxidin. 6.6.1 Conversion of penicillin G into 6-aminopenicillanic acid In Figure 6.11 we indicated that penicillin acylases selectively hydrolysed the secondary amide link, releasing 6-aminopenicillanic acid (6-MA). Although these enzymes could be used to produce 6-APA from peNcillin G, initially, the vulnerability and hi processes. As can be seen in Figure 6.12, penicillin G contains two amide functionalities, of which the plactam linkage is extremely susceptible to basic and nucleophilic attack. Therefore, cleavage of the phenylacetyl side chain could not be performed using classical base hydrolysis. The problem of selectivity was resolved by taking advantage of the fact that the amide bond to be hydrolysed is secondary rather than tertiary. penicilli @as= of enzymatic deacylation were important reasons to search for alternative, h- emical 1 70 Chapter 6 Figure 6.12 Penicillin G contains two amide linkages (circled in a ). The amide linkage to the side chain is secondary and exists in two forms (shown in b). Key factor in addressing this problem was the application of silyl chemistry in order to protect the penicillin C-3 carboxyl in situ, giving high yields at low cost. The process is illustrated in Figure 6.13. Examine this figure carefully so that you remember, at least in outline, how the chemical conversion takes place. Furthermore, the occurrence of various undesirable side-reactions, so easily met within penicillin chemistry, was successfully avoided by performing reactions with phosphorus pentachloride and alcohol at low temperatures. Thus an inexpensive one-stage process to &MA, now one of the world's largest selling B-lactam intermediates, was developed. siw chemistry Figure 6.13 Deacylation of penicillin G using phosphorous pentachloride. Production and diversification of antibiotics 171 water-immiscible solvents Enzymatic approach Eventually, a growing concern for environmental safety pmmpted researchers to reinvestigate the possible application of the previously described enzymatic deacylation. Application of enzymes in industrial synthesis had long been viewed as being highly impractical since enzymes are unstable at extreme reaction conditions (organic solvents, pH and temperature), sometimes quire very expensive cefactors, and are often difficult to recover from complex reaction mixtures. However, as research progresses, it becomes more and more clear that these drawbacks are not always commonplace. As we have illustrated elsewhere in this text, nowadays enzymatic catalysis is known to be very practical in several non-aqueous solvents. Enzymes that function at extreme pH and temperatures have been isolated and certain enzymes, particularly hydrolytic ones, do not need co-factors. Enzymes have become potentially useful catalysts for a wide range of chemical transformations. However, practical industrial application is dictated by economic factors. Costsffectiveness is still the most important consideration and one always needs to question whether the enzyme of choice can indeed fulfil its catalytic properties. In many examples, enzyme regeneration is not practically applicable and thedore the enzyme needs to be available at low cost in order to compete with traditional chemicals. In other instances, regeneration is a relatively simple concept and the enzyme can indeed be referred to as a catalyst. If this is the case, the cost of the enzyme is of almost no consequence. Simple enzyme regeneration techniques can be applied when the product of a given transformation is soluble in a water-immiscible solvent Since the enzyme is soluble in water, the reaction can be carried out in a twephase system consisting of water and a water-immiscible solvent. Transfoxmation can only occur at the contacting surfaces of the two phases, and it is for this reason that severe mechanical stirring is often required in order to achieve a sufficient degree of mixing. When the desired transformation has been effected, separation of the phases gives an uncontaminated solution of the enzyme in water which is easily reusable. When the reaction product is soluble in water, enzyme regeneration is difficult to achieve, since the enzyme is often lost during isolation of the product. One way to overcome this problem is application of immobilised enzyme systems. The enzyme is either covalently or ionically attached to an insoluble carrier material or is entrapped in a gel. Depending on the size of the particles used, a simple filtration and washing procedure can be used to separate the immobilised enzyme from the dissolved product. A well-known example of this technique is the industrial production of 6-APA. Here we will focus on the biochemical aspects. The techniques of isolating enzymes, the process of enzyme immobilisation and the behaviour of immobilised enzyme reactors are discussed in detail in the BIOTOL text 'Technological Applications of Biocatalysts", so will not deal with these aspects in detail here. In outline, however, once the desired enzyme is isolated, it is attached to a carrier material. In order to ascertain sufficient accessibility of the enzyme, a bifunctional spacer molecule is attached to the carrier: 172 Chapter 6 functiomsation Thus, the active functions of the carrier material, which is usdy a naturally ocaming or synthetically prepared polymer, can be of almost any naturr. Introduction of the spacer molecules (functionalisation) is also the phase determining the activity of the immobilised enzyme preparation; the more spacer molecules per unit area, the more enzyme molecules can be attached. Of course, steric hindrance exerts a limit on this number. After the carrier has been functionalid, excess reagent is removed by filtration and washing and the enzyme can be attached to the support. The immobilised enzyme thus obtained is usually storpd in an aqueous medium in order to avoid dehydration, which may lead to irreversible deactivation of the enzyme. Just before use, the beads containing the enzyme are collected by filtration, washed, and added to the aqueous solution of substrate. Once the desired conversion has been effected, the beads are removed by filtration, washed, and either stored or reused directly afterwards. GAPA is produced from penicillin G or penicillin V. We remind you that during fermentation leading to these products, a precursor is added which determines the formation of product. When phenylacetic acid is used as precursor, penicillin G is formed. Likewise, when phenoxyacetic acid is used as pTecursor, peNcillin V is formed. Different enzymes show different specificities. Those of bacterid origin have, in general, higher affinity for benzyl penicillin and hence hydrolyse penicillin G morr readily than they do penicillin V. In contrast, the penicillin acylases produced by actinomycetes and fungi are generally more active with phenoxymethyl penicillin and therefore more readily hydrolyse penicillin V. We might conveniently regard these two types of enzymes as penicillin G acylase and peNcillin V acylase respectively. The reactions they catalyse are illustrated in Figure 6.14. aflinityof acylaw for antbiolics Figure 6.14 Enzymatic side chain cleavage of penicillins. 6-Aminopenicillanic acid, a valuable intermediate for the production of various semi-synthetic penicillins, can be obtained through enzyme-mediated hydrolysis of the phenylacetyl group of penicillin G or the phenoxyacetyl group of penicillin V. The active site of the enzyme recognises the aromatic side chain and the amide linkage, rather than the penidtiin nucleus. Chemical entitles other than penicillins are therefore often good substrates, as long as they contain the aromatic acetamide moiety. Production and diversification of antibiotics 173 A variety of enzyme sources have been used. Some examples are given in Table 63. Organlsm Bacillus megaterium Bonsra phmbia Escherichia coli Kluyvera citrophila Pseudomonas melanogenum Table 6.3 Some sources of penicillin acylases used for the large scale production of GAPA. From the description given previously, see if you can identify a likely murres of i) penicillin G acylase and ii) penicillin Vacylase, from the examples given in Table 6.3. n To answer this you would need to have a little knowledge of the taxonony of the organisms listed in Table 6.3. You probably could identify BaciIlus megdm*um, Escherichia coli, Muyoem cifrophila and Pseudonumas melamgenum as bacteria and should have predicted, therefore, that these are likely to produce penicillin G acylases. You would have been mmct except for the enzyme from P. mehgmm, it has a rather different specificity. We will deal with this in section 6.62 The enzyme from E. di has found particular industrial application. In some cases whole cells of E. Cali are entrapped in polyacrylamide gels or the isolated enzyme is entrapped in cellulose triacetate fibres or immobilised onto Sephadex beads or polymethacrylate min. Immobilised enzyme preparations may, with careful handling, be used for over a hundred batches. Yields of over 90% 6-APA with a purity of over 95% are routinely achieved. The most commonly used source of penicillin V acylase is the fungus BoDista piumbia. Incubation of phenoxymethyl penicillin (penicillin V) with this enzyme produces a yield of about 90-92% 6-APA. What product other than 6-APA is produced from the enzymatic hydrolysis of n penicillin G? Has this product any value? If so explain how it may be used. You should have identified that the acyl moiety, phenylacetic acid, is the second product arising from the hydrolysis of penicillin G. This could be recovered from the reaction mixture and reused as a precursor for penicillin G production. In practice, an integrated process is used. We can represent this in the following way: 1 74 Chapter 6 In our description we have mentioned that either immobilised whole organisms which produce penicillin acylases or purified enzymes may be used to hydrolyse penicillins. See if you can list two advantages and two disadvantages of using whole cells cornpad with using purified enzymes (you should d that the advantages and disadvantages of using whole cells and enzymes in chemical synthesis were considered in Chapter 2). n There are several advantages and disadvantages you might have listed. The principle advantages are: 0 using whole cells requires less preparation of the catalytic entity. AU that needs to be done is for the cells to be grown, harvested and immobilised. With purified enzymes, substantial post-harvesting processing needs to be carried out; with whole cells, the enzyme is held intracellularly and is, to some extent, protected from denaturation arising from the physical and chemical conditions of the reaction mixture; 0 chemical attachment of the cells to matrix used for immobilisation may be made betwen components of the cells (for example the cell walls), rather than directly to the enzyme. There is, therefore, likely to be less damage to the enzyme; 0 if entrapment is used for immobilisation, cells are larger and a more open matrix may be used. This, in turn, will offer less resistance to the diffusion into the matrix; although not relevant to acylases, you may also have mentioned that for enzymes requiring co-factors, these co-factors will be present within the cells and need not be added. advantages of *le dls Production and diversification of antibiotics 1 75 The principle disadvantages are: using whole cells means that the substrate has to penetrate both the immobilisation matrix and the cells in order to come into contact with the enzyme. Cell walls and membranes may be a considerable barrier and, therefore, the rates of reactions may which pumps the substrate into the cells, then the rate will be governed by the rate of diffusion into the immobilisation matrix. We should also consider the diffusion of products away from the enzyme. If these are retained inside the cells, then their accumulation may result in a slowing of the overall reaction (remember, the hydrolytic reaction is reversible); inevitably, using whole cells means that the amount of catalyst (the penicillin acylase) per unit volume of mixture will be much lower than that achievable using purified enzyme. The penicillin acylase will represent only a small fraction of the cells used; disadvantage or whole ~IIS be slowed substantially. Of course, if the cells have an active transport mecham ‘sm 0 using whole cells may result in the production of a greater variety of end products. Cells will contain enzymes other than the acylases that may catalyse the transformation of the substrate or the products of the acylases. The latter two points usually tip the balance in favour of using p&ed enzymes. Ideally the enzyme should be easy to isolate. The penicillin acylase from &Icillus megaterium is, for example, an extracelluar enzyme and can be readily absorbed into bentonite. Before we leave our discussion of preparing 6-aminopenicillanic acid for use as a starting material in the manufacture of semi-synthetic penicillins, we should point out that similar processes are used in the manufacture of semi-synthetic cephalosporins. Here the key intermediate is 7-aminodeacetoxycephalosporanic acid (7-ADCA). We have drawn outline schemes comparing the production of semi-synthetic penicillins and cephalosporins in Figure 6.15. You will see that the two schemes are very similar. 1 76 Chapter 6 Figure 6.15 Produdion of semi-synthetic penicillins (left) and cephalosporins (right), from enzymatically obtained intermediates 6-APA and 7-ADCA respectively. We have learnt that the penicillin acylases show some -city towards the acyl pups attached to the APA moiety. For example, we have distinguished between penicillin G acylases and penicillin V acylases. Some of these enzymes will also work with cephalosporins. What does this tell you about their specificity? n Production and diversification of antibiotics In Clearly these enzymes are not highly speaiic, since they do not distinguish between the ring structures of the penicillins and those of the cephalosporins. Another important enzymatic process in the production of 7-ADCA, for use in the production of semi-synthetic cephalosporins, is the hydrolysis of ~-&n-halOsporaniC acid (7-ACA) by the enzyme acetyl esterase. This process, again using immobilisation techniques, is illustrated in Figure 6.16. The deacylatd product can be used, for example, as an intermediate in the production of the important oral cephalosporin cefuroxhe. We will return to cephalosporin antibiotics later in this inmobili acetyl mbrasB cehhe Chapter. Figure 6.16 Production of 7-aminodeacetylcephalosporanic acid from 7-ACA using an immobilised acetyl esterase. Following enzymatic removal of the acetyl group from 7-ACA, a 3-hydroxymethyl cephalosporin is obtained that can serve as intermediate in the production of cefuroxirne. The introduction of immobilised enzymes has several advantages over the chemical deacylation of blactam. List as many as you can. (You may need to refer back to Figures 6.13-6.16 and the associated text 178 Chapter 6 6.6.2 Conversion of 6-aminopenicilianic acid into ampicillin and amoxicillin In section 6.6.1, we described how enzymatic methods have come to dominate the production of the important intermediates used in the manufacture of semi-synthetic f.%lactams. In principle, the hydrolytic penicillin acylases may be used in the reverse direction to add acyl groups to GAPA. For example, a twestep enzymatic pmess has been described for the preparation of ampicillin (D-(-)u-aminobenzylpenicillin; structure shown in Figure 6.17). Figure 6.17 Structures of amoxidllin (R=OH) and ampiallin (R=H). In this process, penicillin G is first hydrolysed to 6-APA with the acylase derived from Muyvera citrophila at a slightly alkaline pH (pH 75). Subsequently the 6-APA is incubated with an acylase derived from Psmdomanas mdanogenum and with DL-phenylglycine methyl ester at pH 55. This produces ampicillin in reasonable yields only because of the specificity of the P. melamgmm enzyme. This enzyme does not react with penicillin G nor phenylacetic acid. Efficient synthetic methodologies for N-acylation of 6-APA have been developed. Ampicillin, for instance, can be prepared conveniently by acylation with phenylglycyl chloride hydrochloride under Schotten-Baumann conditions in an aqueous medium. As circumstances require, either ampicillin anhydrate, ampicillin trihydrate or ampicillin sodium can easily be produced on an industrial scale. Also illustrated in Figure 6.17 there is another important antibiotic, amoxicillin. Both amoxicillin and ampicillin can be made enzymatically or chemically. Although enzymes are available that can be applied very well for the conversion of 6-APA into a variety of semi-synthetic penicillins, economic reasons are still impeding large scale applications. A different approach has been used for the synthesis of amoxicillin (Figure 6.18). Based on the application of the inexpensive Dane salt of 4-hyhxyphenylglycine, a process has been developed giving the required compound in almost quantitative yield. mpidli amoxid'" Production and diversification of antibiotics 179 ~ Figure 6.18 Chemical synthesis of amoxicillin from 6-aminopenicillanic acid via the Dane Salt of 4-hydroxyphenylglycine. We do not expect you to remember the details of this chemistry. What we do, however, hope you realise that just because a process may be carried out enzymatically, this may not be the route chosen by industry. Commercial consideration may still favour the chemical approach. In other words, biotechnological processes have to compete commercially with alternative approaches if they are to be implemented. chemical aWm* 6.7 Cephalosporin diversification 6.7.1 Cephalosporin C: discovery, fermentation and production A highlight in the search for new f3-lactam antibiotics was the finding at Oxford (UK) in 1953 by Abraham and Newton that a fungus of the genus Cephlosportum, which had been discovered by Brotzu in Sardinia (Italy), produced a number of antibiotics, among them cephalosporin C (Figure 6.19). Interest was aroused when it appeared that this antibiotic was of potential medical importance, after it had shown activity against 180 Chapter 6 Gram-negative bacteria, had displayed resistance to hydrolysis by certain penicillinases and had the low toxicity of the penicillins. ionsxchange ChwalDaraphY narmw substrate specific9 of chemical side cham deavage After a strain improvement and development programme similar to, but more complicated than that of penicillin, the D-a-aminoadipyl side chain containing cephalosporin C was obtained by large scale fermentation. However, cephalosporin C could not be isolated as easily as penicillin G or V. Due to its amphoteric nature it is soluble at any pH in the fermentation broth. Several costly isolation procedures involving ionexchange chromatography have been developed, as a result of which cephalosporin C is much more expensive than penicillin G. 6.7.2 Conversion of cephalosporin C into 7-arninocephalosporanic acid It was almost immediately recognised that the deacylated product, 7-aminocephalosporanic acid (7-ACA, Figure 6.16), would be of similar importance as was BAPA in the development of new penicillins. However, 7-ACA, the cephalosporin equivalent of BAPA, could not be found in fermentations of CephaZospin acremunium. In Figure 6.15 we have shown that penicillin acylase hydrolyses the acyl residue from natural cephalosporins. Up to a point this is true. These acylases will, however, only work with a limited range of acyl residues. It now seems that nature does not provide for acylases or transacylases that have the capacity to remove or change the Da-aminoadipyl side chain of cephalosporin C efficiently in a single step. Widespread search for such an enzyme still remains unsuccessful. Fortunately, 7-ACA became readily available after the discovery that cephalosporin C could be converted into 7-ACA by chemical side chain cleavage. Initially, reaction of cephalosporin C with nitrosyl chloride led to a rearranged product that could be easily hydrolysed to give 7-ACA, as outlined in Figure 6.19. However, this method suffered from low yields and eventually a better approach was developed using silyl prokction, reaction with phosphorous pentachloride and subsequent alcoholysis. It is well worth mentioning that this chemistry, originally uncovered for cephalosporin C, was successfully applied for many years for the production of 6-APA as outlined before (see Figure 6.13). After the successful isolation of 7-ACA, numerous approaches to obtain new antibiotically active entities were developed. Apart from acylation of the iT-amino function, which is a logical approach in view of similar modifications of &MA, a remarkable innovation was accomplished when the acetoxy group at the C-3 position was xvplaced with various substituents. The smallest of these substituents, hydrogen, gave entry into the very important class of deacetoxyephalosporin intemediates. Production and diversification of antibiotics 181 Figure 6.19 Chemical conversion of cephalosporin C into 7-ACA. We will leave the story of cephalosporin here, since much of the subsequent modifications depend more upon synthetic chemistry than upon biotechnology. It is for example possible to convert deacetoxycephalosporin, exomethylenecephain and demethylcephalosporin derivatives using synthetic chemical prdures. If you wish to follow up this aspect of antibiotic production in more detail, we would mommend Sebek K. 0 "Antibiotics" in Biotechnology - Volume 6a, edited by Kieslich, K. 1984. Verlag Chemie, Weinheim. 6.8 Alternative strategies for product diversification In this chapter, by using the examples of p-lactams we have briefly examined how microbial cultures may be used to produce sufficient antibiotics to meet market demands. We have also explained how enzymes (or cells) may be used to biotransform, and thereby diversify, antibiotics. By outlining the history of penicillin production, we explained how analysis and manipulation of culture regimes may be used to enhance the yields of antibiotics (and other secondary products). These studies led to the concept of directed biosynthesis by precursor feeding. This is not the only route by which new antibiotics may be produced in fermentation bxuth. See if you can identify ways in which new antibiotics or antibiotic-related compounds may be produced. n 182 Chapter 6 One alternative strategy is to use metabolic inhibitors. Although this approach has not been used with the fHactams, it is worthwhile bearing in mind as a possibility. It has been used in the production of tetracyclines. Streptumyces aurqficiens naturally produces chlortetracycline. This has the structure metabolic inhibitors chbrtetracydine By including an inhibitor of the chlorination step in the fermentation broth, the main product formed is tetracvcline hydrogen at position 5 use ofambgs Notice that, in this case hydrogen, not chlorine, is present at position 5. Similarly, by adding analogs of L-methionine, the methylation of C6 is inhibited, resulting in the formation of 6demethylchlortetracycline. The analogs that may be used include D-methionine and ethionine. An alternative strategy for pducing new derivatives by directed biosynthesis is to produce mutants in which particular pathways may be blocked or a new pathway mated. Again, we will use a specific example to illustrate this approach. Many strains of StreprOmyces peucetius produce daunomycin. These strains often carry a permanently repressed (silent) gene that codes for the enzyme duanomycin 14-hydroxylase. If this is reactivated by mutation, the daunomycin is further metabolised to produce a new antibiotics, 14-hydroxydaunomycin (adnamycin). More frequently, however, mutation is used to block a particular pathway. Sfreptontyces @dim produces neomycin. 2-Deoxystreptamine is an intermediate in the biosynthetic pathway leading to the production of neomycin (see Figure 6.20). useofrn*nts adnanyan manyan Production and diversification of antibiotics 183 Figure 6.20 Stylised representation of neomycin biosynthesis. Mutants can be isolated which are unable to produce 2deoxystreptamine. n HOW may such mutants be used to produce new neomycin andogs? The obvious solution is to feed these cells with compounds that have structures similar to 2-deoxyjtreptamine, which would become incorporated into the neomycin. If for example, strrptamine is included in the fermentation bmth, this is incorporated into the neomycin produced. Thus: 184 Chapter 6 streptatnine as precursor By this approach, a wide variety of structural analogues of 2deoxystreptamine have been used to produce a range of neomycin-like antibiotics. These structural analogues include methylated and halogenated derivatives of 2deoxystreptamine. 1) Assume that you have a mutant of a neomycin producing organism that is blocked in the synthesis of 2deoxystreptamine. You cultivate this organism in the presence of a) 6-O-methyldeoxystreptamine b) 3-N-methyldeoxystreptamine c) 5-0-methyldeoxystreptamine In each case, state whether or not a neomycin-like analogue is likely to be produced and give its probable structure. part 1. 2) Explain what assumption you have made in coming to your conclusion to We complete this section by reminding you that we have now identified three strategies of using directed biosynthesis to produce diversified products. These are: 0 precursor feeding (eg for producing penicillin G, V etc); metabolic inhibition (eg for producing various tetracyclines); mutation (eg for producing various neomycins). Production and diversification of antibiotics 185 In the next section, we will expand on the possibilities of using in oitru biotransformation to diversify products. 6.8.1 Further biotransformations of antibiotics In our discussion of the diversification of the &ladams, we explained how acylases and acylating enzymes may be used in the production of modified (semi-synthetic) fl-ladams. However, the potential of using enzymes to modify organic molecules is much wider than this. You should be able to recall from your earlier studies a much wider range of n reaction types that might be used. Try to write a list of these before reading on. The sorts of reactions we hope you would list include: oxidations; reductions; 0 aminations/deaminations; 0 esterifications / de-esterifications; 0 methylations/demethylations; nucleotidylations; 0 phosphorylations/dephosphorylations; 0 isomerisations; 0 hydrations / dehydrations; glycosylations. You may well have included many others. Many of these have been demonstrated with a range of antibiotics and antibiotic precursors, although relatively few have been applied commercially. We have included a list of published examples in the form of an Appendix at the end of this chapter. We do not expect you to remember the details of this Appendix. It has been included as an illustration of the potential to use enzymes to modify organic molecules like antibiotics. It should be anticipated that, as enzyme technology develops and the search for new antibiotics continues, an inmasing number of enzymebased transformation will find commeraal application. potenliat lor Using 186 Chapter 6 Summary and objectives In this chapter, we have examined some aspects of the application of biotechnology to the production of antibiotics. We began by briefly describing the range of antibiotics and their modes of action. We described the general strategies used for the production of antibiotics, with particular emphasis on the approaches used to improve yields and to diversify the products synthesised in antibiotic fermentations. We used the 0-lactams in particular to illustrate the principles involved, with special emphasis on directed biosynthesis by precursor feeding and in mtro modification of fermentation products. We explained how growth and antibiotic synthesis occurs in the phases called the 'trophophase' and 'idiophase' and the implications of this in process design. We also explained the competition between chemical and biological approaches to the production of so called semi-synthetic antibiotics. The use of metabolic inhibitors and mutants to achieve biosynthesis tailored to produce alternative products was also examined. At the end of the chapter we explored briefly the further potential of using biocatalytic systems to modify antibiotics. Now that you have completed this chapter you should be able to: list a wide variety of classes of antibiotics and, in outline, described their modes of action; explain how antibiotic-producing micro-organisms may be identified; explain, using penicillin production as an example, how culture conditions may be manipulated to improve the yields of antibiotics produced by fermentation; explain, by using suitable examples, how directed biosynthesis may be achieved using precursor feeding; describe how enzymes may be used to produce 6aminopenicillanic acid from penicillin G and V and explain how this product may be used in the production of semi-synthetic penicillins; apply the principles of using preclursor feeding mutants and metabolic inhibitors to producing specific end products; explain, using suitable examples, metabolic inhibitors and mutation may be used to redirect biosynthesis; demonstrate an awareness of the potential to use enzymes to modify antibiotics. Production and diversification of antibiotics 1 87 A pendix 6.1 Examples of biotransformation of antibiotics &ita abstracted from Sebek, 0. K. "Antibiotics" in Liotechnoloqy - Volume 6a, edited by Kieslich, K. 1984 Verlag Chemie, Weinheim). Substrate Reaction Product Micm-organism Ampicillh, Ca&nicillin Cephalosporin C Cephaloglycine, Cephaloridine, Cephalothin Benzyl- and Hydrolysis Corresponding Escherichia coli Phenoxymethylpenicillins, B-lactarn ring Streptomyces dhs cleavage products Pseudomonas aemginosa Enterobacter cloacae Streptomyces sp. Benzyl-, phenoxyrnethyl- and other penicillins N-Acetylde hydroxy- thienarnycin (PS-5) Cephalosporin C Cephalothin Nocardicin C 6-APA + phenylacetic acid + phenoxyacetic acid + carboxylic acid and esters + phenyglycine esters 7-ACA, ?-ADCA, and their organic acid esters N-Deacylation 6-APA and the Escherichia coli corresponding acyl Fusarium semitemm side chains Penicillium chrysogenum Asper.giIIus oryzae Nine different bacteria Deacetylated PS-5 Strqtomyces olivaceus (NS-5) ODeacylation 3-Deacetylcephalo- Various bacteria and sporin actinomycetes 3-Deacetylcephalo- Eschericia coli thin 3-Arninonocardicinic Pseudomonas and a-arninoadipic schuy/killiensis acids Acylation Benzylpenicillin Escherichia coli Alcaligenes faecalis Phenoxyrnethyl- Alcaligenes faecalis penicillin Penicillins with the Kluyvera Citrophila corresponding acyl Pseudomonas sides chains melanogenum Ampicillin Kluyvera citrophila Corresponding Kluyvera citrophila cephalosporins Xanthomonas citm (cephalexin, and other cephaloglycine) pseudomonads 188 Chapter 6 Substrate Reaction Product Micro+rganism Amlnogiycoskles Mannosidostreptomycin Validamycins Validamycins A and D Validarnycin B Validamycins C, E and F Gentarnicins C, CI and Cla Kanamycin A Kanamycin B Tobramycin Gentamicin CI and Cla Sisomicin Tobramycin Lividomycins A and B Paromomycin Ribostarnycin Amikacin Butirosins Amikacin Butirosin Butirosin A Hydrolysis Streptomycin Streptomyces griseus Validamycin A and Validoxylamine yeasts Validoxylamine A Pseudomonas denitrihns and other micro-organisms H ydroxyvalidarni ne Pseudomonas denitrifkans Validmycin A Endomycopsis spp and Various bacteria and Candkja intermedia Acylation 3-N-Acetyl Escherichia coli derivatives of the Klebsiella penumoniae respective Pseudomonas aeruginosa substrates Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Escherichia coli Klebsiella pneumoniae 2’-N-Acetyl Providema spp. derivatives of the respective substrates Proridencia spp. Providema spp. 6‘-N-Acetyl Pseudomonas aeruginosa derivatives of the respective substrates Pseudomonas aeruginosa Phosphorylation 3’-OPhosphorylated Staphflmus aureus derivatives of the respective substrates Escherichia coli Pseudomonas aeruginosa Escherichia coli Pseudomonas aeruginosa Production and diversification of antibiotics 189 Substrate Reaction Product Microorganism Kanamycin A Neamine Neamine Neomycin Ribostamycin Gentamicins A, B, Cia, c2 Kanamycin B Sisornicin Tobramycin Dihydrostreptomycin Streptomycin Gentarnicins Cla and Cp Methylation KDemethylclindarnycin Anthracycllnes Adriamyci n Reduction Daunomycin Daunomycinol and its aglycone Dau nomyci none Nogalamycin Rubeornycin A Aurarnycinone Hydroxylation Pseudomonas aeruginosa Escherichia coli Pseudomonas aeruginosa Escherichia coli Pseudomonas aeruginosa Escherichia coli Staphylococcus aureus 3"- O-Phosphorylated Pseudomonas aeruginosa derivatives of the respective substrates Gentamicins Ca, and CI KDemethyl-K hydoroxymethyl clindamycin 7-Deoxyadria- mycinone and 7-Deoxyadria- mycinol aglycone Daunornycinol 7-Deoxydaunorny- cinone 7-Deoxydauno- mycinol aglycone 13-Dihydrodauno- rnycinone 7-Deoxynogalarol Rubeomycin B 1 1 -OH-Auramy- cinone and 9-methyl-1 O-OH- daunomycin Escherichia coli Pseudomonas aeruginosa Mkromonospora purpurea Streptomyces linwlnensis Streptomyces steffisbug ensis Corynebacterium qui Mucor spinosus Streptomyces steffisbug ensis Streptomyces steffisbug ensis Streptomyces Streptomyces galilaeus Streptomyces aureofaciens COerUleOtUbidUS Streptomyces nogalater Rhodotomla glutinis Streptomyces weruleombidus Daunomyci n 14-OH-Daunomycin Streptomyces peucetius (adriamycin) var. caesius 190 Chapter 6 Substrate Reaction Product MicrPorganism E- lsorhodomydnone e-P yrromycinone Daunomycinone derivatives Mecrolldes 1 4- OAcetyl-8- Oacyl- lankacidin C Lankacidin A Lankacidinol A Various 8-014-odl acylated Lankacidin C derivatives Leucornycines AT and A3 Magnamycins A and B Middamycin Leucomycin A5 Mariiornycin 111 A231 87-Methyl ester and polyvinylpyrrolidone Josarnycin Maridomycin I Narbom ycin Narbonolide Ketone formation Lan kacidi no I Midecarnycin AI Demet hylation Deacylation Hydroxylation 1 -0H-13-Dihydrodauno Streptomyces mycin (and its Nformyl derivative) Corresponding demethylated Carminornycinone derivatives Corresponding 8-OAcyllankaddins Lankacidin C 1 4-OAcylated lankacidin C Corresponding 4”-0 deisovaleryl derivatives Leucomycin V (Cdebutyrylleu- cornyan) 4’-ODepropionyl- maridornycin I1 18-Dihydromarido- rnycin 111, 4”-Depropionyl- maridomycin Ill and Sepedonium ahrjsospermurn Beauveria sulfurescens Streptomyces roche/ Streptomyces rochei, Aspergillus niger Aspergillys sojae Trametes sanguinea Streptomyces roche/ Cunninghamella el6gans and other fungi Streptomyces sp. Adinoplanes missouriensis Bacillus megatenurn Streptomyces pristinaespiralis streptomyces sp 1 8-Dihydro4“depopio nylmaridomycin 111 16-Hydroxylated (and Streptomyces chartreusis N-demethylated) produds 3”-OH-Josarnyci n Streptomyces oliwceus 3”-OH-Maridomycin I Picromycin Streptomyces narbonensis Picronolide Streptomyces zaomyceticus Lankacidin A Streptomyces rochei var. voiubilis Midecarnycin A3 Streptomyces mycarofacjens Production and diversification of antibiotics 191 substrate Reaction Product Miim-organism Expoxidation Carbomycin B Leucomycin A3 us-Propenylphosphonic acid Albocycline Carbomycin A Carbomycin A Carbomycin B Mariiomycin 111 Midecamycin A3 Tylosin carbomycin A Maridomycin II Fosfom yci n Reductions 2,3-Di hydroalbocycline Carbomycin A P1 Maridomycin II Leucomycin A3 18-Dihydromarido- mycin 111 Midecarnycin AI Relomycin Streptomyces h yg rosqicus Penicillium spnulosum Streptomyces venezuelae Streptomyces halstedii Streptomyces lutea and others Streptomyces hygmscqkus Streptomyces sp. Streptomyces mycarofaciens Nocardia corallina