Production and diversification of antibiotics Czapek-Dox Initially, Czapek-Dox medium was used. This medium, containing mineral salts, medum sodium nitrate and sugar(usually sucrose or glucose), allowed rapid growth but only very small amounts (around lug mr ) of penicillin were produced. Effectively what happened in these cultures 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 6. penicillin production for a very short time. These observations are illustrated in Figure 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 slow release of There are several possibilities. One would be to ensure that carbohydrates were ydrate 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 ng ammonium acetate or ammonium lactate as nitrogen source in place of nitrate. This reduced the rise in pHobserved at the m end of the fermentation. Replacement of these nitrogen sources by complex nitrogen sources, such as casein hydrolysate, improved the long term availability of nitrogen 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 aa bsein 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 50C acidified with SO. Thermophilic bacteria hydrolyse proteins and other components of the kemal, 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. Surprisingly the yield of penicillin increased by a further 5-10 fold giving yields of 50-100 ]g 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 table and easier to isolate form of penicillin, what we now call penicillinG CSL contaned In essence, what was happening was that the CSL supplied p phenylethylamine. This P-pheny had been produced from the amino acid phenylalanine by the action of the microflor etyiamit in the Csl. thus
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
Co phenylalanine phenyl ethylamine The P notatum took up the p-phenyl ethylamine, converted it to p-phenylacetate, which penialing was subsequently attached to the b-amino group of penicillanic acid to give benzyl process by CH2-CH2-NH CH2 -CoOH nIne B-phenylacetic acid acid moiety CH3 penicillin G Previous penicillins had aliphatic groups attached to the 6-amino penicillanic acid moiety. Penicillin G has many advantages over the aliphatic derivatives, eg it is more easily crystallised and it is more stable Suggest an alternative way of producing penicillin G, without using CsL. The obvious way is to include p-phenylacetic acid or P-phenylethylamine in cultures. Indeed, when P-phenylacetic acid was added to cultures grown in CSL, the yields of icillin were enhanced further. Typical yields were 100-150 ug!. The results obtained by the addition of p-phenylacetic acid to cultures suggest a method of producing a wide variety of penicillins. See if you can explain what In principle, by adding derivatives of acetic acid to culture media, we might 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:
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 CH3 H一c NH→c一CH-0 COOH administ ral Penicillin V has advantages over some other penicillins as it is stable at low pH and can administered orally e may describe the production of diverse penicillins using this strategy as direct Tabsynthesis using precursor feeding. We have listed some examples of penicillins in H-C NH-R COOH C-CH2' C-CH2 OH H2 c一CH2-(HsCH C-c o-(cH-沖-2 COOH Table 6.2 EXamples of penicillins SAQ 6.2 Suggest what precursors should be fed to cultures to produce each of the nicillins shown in Table 6.2. Read our response carefully as it contains some dditional in formation
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 1 additional information
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 levelopment activities. We will now turn our attention to another aspect of the development of penicillin production: the switch from surface to deep culture. surface culture Initially, penicillin was produced in shallow earthenware " penicillin pots"that resembled bedpans used in hospitals. Milk bottles were then used. The problems with proaches stemmed from the costs of the multiple inoculations that were needed the costs of harvesting from multitudinous small cultures. Replacement of these vessels by larger tray-like vessels, however, was not entirely successful: the trays often warped during sterilisation 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 commercal Despite these advantages, deep(submerged) cultures were still deemed to be the most onsideration viable route to satisfying 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 10 litres. The desire to switch to deep cultures was thus driven by commercial consideration In practice, P notatum 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 ternative organisms were sought which would combine good growth characteristics Penicillium (loose, confluent growth)with high penicillin yield. A strain of Penicillium chrysogenum as selected.This strain, NRRL 1951, produced only 50ug 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 ug penicillin ml" The development of the deep culture approach followed conventional routes, including optimising inoculum density, oxygen dispersal and control 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 "Bioreactor 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 appaf the desired product.The nt distinction between the growth of the producing organism and the biosynthesis oncept 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 a加 growth occurred, but this phase was marked by penicillin production. So there was separation 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 ovides the basis of contemporary penicillin production processes. In these processes, the culture is first cultivated under conditions which favour growth. Once the culture processes has fully grown, culture conditions are manipulated to favour penicillin production Although these manipulations may be carried out in a single vessel, it is more usual to
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
Production and diversification of antibiotics 161 physically separate them into two vessels. Such two phase processes have become the orm for the production of secondary products. a stylised system is shown in Figure 6.5 cultures production of product down stream 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 sf salen dary hasel ito dual the des ired pr dsed for ain mah this is so atch or The aim in most processes is to produce biomass as quickly as possible(that is use ontnuous conditions which allow rapid growth), but to maintain cells in production as long as transfers possible. If for example it took the cultures 3 days to grow, but they could be maintained n a productive state(that is in the idiophase)for 10 days, then in principle the ratio of vessel volumes could be 3: 10, trophophase 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 batch-wise manner, the biomass produced in the trophophase being transferred en into the idiophase. Alternatively, biomass may be transferred continually from 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 ptimisation 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 penicillin, we should point out that it is not essential that growth and production phases are physically separated. It is possible by using a pre-set feed pattern to carry out both processes in the same vessel
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