298 hapter 9 we would need a multistage reaction and that the desired isomer would only be a small fraction of the final product. We would also be presented with the difficulty of isolating the desired isomer from its isomeric partners. We, therefore, conclud that, although technically feasible, this approach isnot a realistic commercial option. 3)Partial chemical synthesis is perhaps more realistic. If, for example, we wish to lightly modify the structure of a naturally occurring substance, then this might be possible using chemical processes. The problem here is to identify reagents and reactions which will be specific, both in terms of the site of attack on the natural product and the stereospecificity of the reaction Wemust anticipate, therefore, that hemical reactions may be used in some cases, but thisis not a universally applicable strategy 4)The natural systems that produce steroids do so in quantitatively small amounts. Although in principle the cells producing these might be isolated and cultivated in vitro, the quantities produced will still be small and the costs of cultivation are high This approach is, therefore, not generally commercially viable. You may have considered the option of transferring the genes, which encode for the enzymes involved, into an easy to cultivate system(for example a yeast or bacterium) and to ulties in isolating the necessary genes and the multiplicity of the enzyme ste eeded for steroid biosynthesis makes the development costs of this approac extremely high. In the longer term, this may become a realistic option, but is not, tly cially viable 5) The enzymatic transformation of natural products is by far the most attractive option. In this approach, it can be envisaged that sterols, which are relatively abundant, may be selectively modified to produce desired products. The diversity of enzyme activities, their reaction specificity, regiospecificity and stereospecificity areall features which could contribute to carrying out the desired changes. This does not mean, however that transformations using enzyme systems are simple Nevertheless, biotransformations have become of vital importance in the roduction of steroids In the following sections we will explain some applications of enzymes(and cells) in the transformation of sterols and steroids. you should realise however that for each process a decision has to be made whether to use an enzyme-mediated transformation or to use a chemical reaction. In many instances the biotransformation process is the most attractive but, as we will see later, this is not always the case 9.3 Selective degradation of the sterol side chain Re-examine the structures shown in Figure 9.1 and see if you can identify the fundamental difference between sterols and the steroid hormones Although there are many differences between these two groups of molecules, the fundamental difference between them is that the steroids do not possess the long side chain attached to position 17 that occurs in sterols. Thus, if we are to use sterols as the starting point for producing steroids, then we need to selectively remove this side chain
298 Chapter 9 we would need a multistage reaction and that the desired isomer would only be a small fraction of the final product We would also be presented with the difficulty of isolating the desired isomer from its isomeric partners. We, therefore, conclude that, although technically feasible, this approach is not a realistic commercial option. 3) Partial chemical synthesis is perhaps more realistic. If, for example, we wish to slightly modify the structure of a naturally OcCuRing substance, then this rmght be possible using chemical processes. The problem here is to identify reagents and reactions which will be specific, both in terms of the site of attack on the natural product and the stereospecificity of the reaction. We must anticipate, therefore, that chemical reactions may be used in some cases, but this is not a universally applicable strategy. 4) The natural systems that produce steroids do so in quantitatively small amounts. Although in principle the cells producing these might be isolated and cultivated in mho, the quantities produced will still be small and the costs of cultivation are high. This approach is, therefore, not generally commercially viable. You may have considered the option of transferring the genes, which ende for the enzymes involved, into an easy to cultivate system (for example a yeast or bacterium) and to control the expression of these genes using strong promoters. Although this approach is theoretically possible using the techniques of genetic engineering, the difficulties in isolating the necessary genes and the multiplicity of the enzyme steps needed for steroid biosynthesis makes the development costs of this approach extremely high. In the longer term, this may become a realistic option, but is not, currently, commercially viable. 5) The enzymatic transformation of natural products is by far the most attractive option. In this approach, it can be envisaged that sterols, which are relatively abundant, may be selectively modified to produce desired products. The diversity of enzyme activities, their reaction specificity, regiospecificity and stereospeaficity are all features which could contribute to carrying out the desired changes. This does not mean, however, that transformations using enzyme systems are simple. Nevertheless, biotransformations have become of vital importance in the production of steroids. In the following sections we will explain some applications of enzymes (and cells) in the transformation of sterols and steroids. You should realise, however, that for each process a decision has to be made whether to use an enzyme-mediated transformation or to use a chemical reaction. In many instances the biotransformation process is the most attractive but, as we will see later, this is not always the case. 9.3 Selective degradation of the sterol side chain Re-examine the structures shown in Figure 9.1 and see if you can identify the n fundamental difference between sterols and the steroid hormones. Although there are many differences between these two groups of molecules, the fundamental difference between them is that the steroids do not possess the long side chain attached to position 17 that occurs in sterols. Thus, if we are to use sterols as the starting point for producing steroids, then we need to selectively remove this side chain
Biotransformation of lipids micro-organisms Fortunately many micro-organisms can be used to selectively remove the side chain of may selectively abundant, naturally occurring sterols such as cholesterol, B-sitosterol and compesterol side chain These organisms include members of the genera Nocardia, Pseudomonas, Mycobacterium Corynebacterium and Arthrobacter. They are capable of using sterols as their sole source of carbon. Unfortunately, the natural occurring organisms catabolise both the side chain and the ring structure of the sterols. The catabolism of these two components may occur simultaneously. Therefore, methods have to be found to prevent ring structure catabolism whilst allowing the degradation of the side chain. ee if you can identify two strategies for achieving this objective use of mutants In practice, several strategies have been used. In one, mutants are produced which defective in the enzymes involved in ring structure catabolism but still retain the enzymes involved in side chain catabolism Would such mutants grow on a)cholesterol b)testosterone? b)The mutants would probably not grow on testosterone as their is no side chain for them to catal We could use these differences to identify putative mutants with the desired metabolic block inhibition of aA second strategy is to find a way of inhibiting an enzyme involved early in specmc catabolism of the ring. One such enzyme is a 9a-hydroxylase (it hydroxylates carbon 9) nzime This enzyme has an absolute requirement for Fe" ions. By adding chelating agent which complex with these ions the enzyme can be inhibited ation of A third option is to modify the ring structure of the sterol so that it no longer serves as substrate a substrate for the ring-catabolising enzyme. In this approach, a chemical reaction is used to modify the ring-structure and the product is subsequently incubated with th catabolising organism. For example, hydroxylation at C-19 prevents ring cleavage Some examples in which modified sterols have been used for selective side chain degradation are given in Table 9. 1. this table also indicates the nature of the product formed and the organisms used. We would not expect you to remember all of the details of these substrates, products and organisms. We will, however, examine some examples detail to illustrate the dles involved
Biotransformation of lipids 299 miaOorgE4lliSmS may selectively degrade he side cham use of mutants modification of Ihe substrate Fortunately many micro-organisms can be used to selectively remove the side chain of abundant, naturally OcCuRing sterols such as cholesterol, &sitosterol and compesterol. These organisms include members of the genera Nmdia, Pseudomonas, Mywhzderium, Corynebacterium and Arthrobacter. They are capable of using sterols as their sole source of carbon. Unfortunately, the natural occurring organisms catabolise both the side chain and the ring structure of the sterols. The catabolism of these two components may occur simultaneously. Therefore, methods have to be found to prevent ring structure catabolism whilst allowing the degradation of the side chain. n See if you can idenhfy two strategies for achieving this objective. In practice, several strategies have been used. In one, mutants are produced which are defective in the enzymes involved in ring structure catabolism but still retain the enzymes involved in side chain catabolism. n Would such mutants grow on a) cholesterol b) testosterone? a) We would anticipate that such mutants would grow, albeit slowly, on cholesterol as they could still derive carbon and energy from catabolising the side chain. b) The mutants would probably not grow on testosterone as their is no side chain for them to catabolise. We could use these differences to identdy putative mutants with the desired metabolic block. A second strategy is to find a way of inhibiting an enzyme involved early in the catabolism of the ring. One such enzyme is a Sa-hydroxylase (it hydroxylates carbon 9). This enzyme has an absolute requirement for Fez+ ions. By adding chelating agents which complex with these ions, the enzyme can be inhibited. A third option is to modify the ring structure of the sterol so that it no longer serves as a substrate for the ring-catabolising enzyme. In this approach, a chemical reaction is used to modify the ring-structure and the product is subsequently incubated with the catabolising organism. For example, hydroxylation at C-19 prevents ring cleavage. Some examples in which modified sterols have been used for selective side chain degradation are given in Table 9.1. This table also indicates the nature of the products formed and the organisms used. We would not expect you to remember all of the details of these substrates, products and organisms. We will, however, examine some examples in more detail to illustrate the principles involved
300 Chapter 9 Substrate Product Mcr。 organIs8m 19-hydroxysterols, estrone Nocardia restrictus ATCC 14887 3 hydroxy-19nor△35 Nocardia sp ATCC 19170 sterols Arthrobacter simplex IAM 166 Corynebacterum sp M 9 hydroxy-△4-steo lin, equilenin, estrone Mycobacterium sp 3-ht Corynebacterium simplex sterols 6B, 19-oxidostenone 6B, 19-oxido-4-androstene-3, Nocardia sp ATCC 19170 Mycobacteria 19-oxidosterols 5a-bromo-6B, 19- 5a-bromo-6B, 19-0xidoandros- Nocardia sp ATCC 19170 ne 3.17-dione 5a, 5a-cyclosterols 3a, 5a-cycloandrostane-17-one Mycobactenum ph a,5αcyco6β,19ox a-cyclo-6B, 19-oxido- sterol-3-oximes 4-androstene-3 17-dione (after hydrolysis) 4-hydroxycholestenone 3B-hydroxy-5a-androstane-4, Mycobactenum phle 4a-dihydroxy-5a-andro 25D-spirost-4-ene-3-one 1. 4-androstadiene-3.16-dione. Fusanum solani 20a-hydroxy-4-pregnene-3, 16- Verticillium theobromae a,11B, 20a-trihydroxy-5a- ponasterone A rubrosterone Fusanum lini9593 Table 9.1 The use of modified sterols to allow selective cleavage of the side chain(based Martin, CKA Sterols in Biotechnology Volume 6a Edited by Kieslich K 1984 Verlag Chemie 9.3.1 Use of modified sterols First, let us briefly examine the route of side chain degradation in micro-organisms. The pathway is illustrated in Figure 9.2
300 Chapter 9 Substrate Produd Mlcro-organlsm 1 9-hydroxysterols, 1 9-norsterols, 3-hydro~y-19-nor-A’~~~~- sterols 19-hydro~y-A~~~-steroIs, 9-hydro~y-l9-nor-A’~~*~~’- sterols Sp,lO-oxidostenones, 3p-acetoxyda-chloro(fluoro)- Sp, 19-oxidosterols 3p-acetoxy-5a-bromo-Gp, 1 9- Dxidosterols 5a,5a-cyclosterols 3a,5a-cyclo-6p, 1 O-oxidojterols sterol-3-oximes I-hydroxycholestenone masterone A estrone Nocardia restrictus ATCC 14887 Nocardia sp ATCC 191 70 Arthrobacter simplex I AM 166( Corynebacterium sp M ycobaderia Corynebacterium simplex Nocardia mbra Sp, 19-oxido-4-androstene-3, Nocardia sp ATCC 191 70 17dione Mycobacteria 5a-bromo-Gfi,19-oxidoandros- Nocardia sp ATCC 191 70 tane3,17dione 3s5a-cycloandrostane-17-one Mycobacterium phlei 3a,5a-cyclo-6p, 1 9-oxido-an- Arthrobacter spp drostane-17-0ne Corynebacteria 4-androstene-3,17-dione Mymbacterium sp (after hydrolysis) 38 hydroxy-5a-androstane-4, Mycobactenurn phlei 17dione 3a-hydroxy-5a-androstane-4, 17dionq 3p,4u4i hydroxy-5a-androstane-17-One 1 ,4-androstadiene-3,1 Gdione, Fusarium solani 20a-hydroxy-4pregnene-3,16- Verticillium theobromae dione Stachylklium bicolor 3a,l1 p,20a-trihydroxy-5apregnane-16-one rubrosterone Fusarium lini 9593 equilin, equilenin, estrone Mycobacterium sp Table 9.1 The use of modified sterols to allow selective cleavage of the side chain (based on Martin, CKA Sterols in Biotechnology Volume 6a Edited by Kieslich K 1984 Verlag Chemie, Wein heim). 9.3.1 Use of modified sterols First, let us briefly examine the mute of side chain degradation in micro-organisms. ’Ihe pathway is illustrated in Figure 92
Biotransformation of lipids CH,OH b)C-27 hydroxy-sterol c〓0 c)C-27 aldo-sterol COOH CH3 CH2COOH COOH e)C-24 carboxylic acid d)C-27 carboxylic acid CH,COOH COOH CH,CH,COOH f c-22 carboxylic acid g)C-17 keto-derivative Figure 9.2 Generalised metabolic sequences of sterol side chain degradation by side chain First carbon 27 is hydroxylated and oxidised to a carboxylic acid. The resulting acid is degradaton then cleaved to release propionic and acetic acids and a second propionic acid. The final reaction in this sequence results in the formation of a keto group at C-17
Biotransformation of lipids 301 Figure 9.2 Generalised metabolic sequences of sterol side chain degradation by micro-organisms. Fit carbon 27 is hydroxylated and oxidised to a carboxylic acid. The resulting acid is then cleaved to release propionic and acetic acids and a second propionic acid. The final reaction in this sequence results in the formation of a keto pup at C-17. side chain degdaam
302 Chapter 9 hydroxylation If a hydroxyl group is introduced into position C-19 then complete breakdown of the examples shown in Figure 9.3, the incubation of 19-hydroxysterols with Nocardia restricTus ATCC 14887 or Nocardia sp ATCC 19170 leads to the production of estrone. In these cases, you will notice that ring a has become modified but the ring structure is not broken. The yields of estrone using these substrates and organisms are of the order of HOCH H 2- CH3 d Figure 9. 3 The figure shows the degradation of the side chain of sterols which have substitutions at C-19. Removal of the C-19 methyl group(eg 19 norcholesta-1, 3, 5 (10) riene-3-ol)also prevents ring breakdown. Note, however, hydroxylation of C-19 does not prevent all ring modifications a:19-hydroxycholesterone, b: 19-hydroxysitosterone, c: 3B-acetoxy-19-hydroxy-5-cholestene Other modifications which restrict ring cleavage are the formation of 6B, 19-oxido derivatives and 3a, 5a cyclo-derivatives. The structures of some of these are given in
302 Chapter 9 If a hydroxyl group is introduced into position C-19 then complete breakdown of the ring structure is prevented, although it may be sub- to some modification. In the exampl? shown in Figure 9.3, the incubation of 19-hydroxysterols with Nocardia restn'ctus ATCC 14887 or Nocardia sp ATCC 19170 leads to the production of estrone. In these cases, you will notice that ring A has become modified but the ring structure is not broken. The yields of estrone using these substrates and organisms are of the order of 30%. hydroxylation ate19 ~~ ~~~~ ~~ Figure 9.3 The figure shows the degradation of the side chain of sterols which have substitutions at C-19. Removal of the C-19 methyl group (eg 19 norcholesta-l13,5 (1 0) triene-3-01) also prevents ring breakdown. Note, however, hydroxylation of C-19 does not prevent all ring modifications. a: 19-hydroxychoIesterone, b: 1 9-hydroxyslosterone, c: 3p-acetoxy-l9-hydroxy-5cholestene, d: estrone. Other modifications which restrict ring cleavage are the formation of 6fi,19-oxido derivatives and 3a, 5a cyclo-derivatives. The structures of some of these are given in Figure 9.4