Biotransformation of lipids 9.1 Introduction 9.2 The structure, roles and abundance of sterols and steroids 9.3 Selective degradation of the sterol side chain 298 9.4 Specific steroid interconversions and reactions 9.5 Transformation of other terpenoids 321 9.6 Chemical conversion of miscellaneous organic compounds 9.7 Production and use of fatty acids and their derivatives 9.8 Selection of production systems for the biotransformation of lipids 337 339
293 Biotransformation of lipids 9.1 Introduction 9.2 The structure, roles and abundance of sterols and steroids 9.3 Selective degradation of the sterol side chain 9.4 Specific steroid interconversions and reactions 9.5 Transformation of other terpenoids 9.6 Chemical conversion of miscellaneous organic compounds 9.7 Production and use of fatty acids and their derivatives 9.8 Selection of production systems for the biotransformation of lipids Summary and objectives 294 295 298 309 321 326 329 337 339
hapter 9 Biotransformation of lipids 9.1 Introduction In this chapter we will examine how cells and enzymes are used in the transformation molecular of lipids. The lipids are, of course, a very diverse and complex series of molecular alcohols, waxes, terpenes and steroids. It is usual to teach about these molecules, in a iochemical context, in more or less the order given above, since this represents a logical sequence leading from simple molecules to the more complex. Here, however, we have dopted a different strategy. Clearly, the technical and commercial aspects of industrial lipid transformation are both diverse and complex. We have, therefore, to be selective in what we can include in a ingle chapter. We have decided to begin the chapter by discussing the transformation of sterols and steroids since these transformations are illustrative of the potency of biocatalysts in bringing about selective and stereospecific chemical transformation of uite complex molecules. This part of this chapter is a logical extension of the issues iscussed in the previous chapter. We have also elected to focus on the technical rather than the commercial issues although attention is drawn to the importance of commercial criteria in the selection of strategies for production. The reader should be aware, however, that the production of steroids for pharmaceutical use and as contraceptives is a large market. Estimates of annual sales of these materials vary widely(WHO 1990 Year Book $3x10 annum"-$10 annum" )reflecting the difficulty of accessing details on such a diverse group of compounds After discussing the biological capability to transform steroids, we briefly examine the biotransformation of other terpenoids to ensure that the reader develops an awareness of the potential of biotechnology to modify or produce derivatives of a wide range of natural materials that are of tremendous potential, commercial value in the food and health care sectors. We also include a brief consideration of the use of biocatalysts to transform a range of other hydrocarbon compound The bulk of the chapter is therefore concerned with highly specific reactions arising to produce molecules of known structure. However, in a chapter on lipid transformation, we should not miss the use of general lipases to change the composition of triglycerides Although the replacement of one set of fatty acids in trig ycerides is, from a chemical/biochemical point of view, not as stimulating as the biotransformation of steroids, it is of major commercial value in the food industry. The biotransformation of triglycerides (and phospholipids) to produce food materials which have desirable organoleptic properties(eg melt in the mouth feel)potentially dwarfs the steroid market in terms of volume and turnover The placing of this topic towards the end of the chapter does not imply that it is of limited commercial value The final part of the chapter briefly explores the potential of using non-aqueous solve for lipid transformation
294 Chapter 9 Biotransformation of lipids 9.1 Introduction In this chapter we will examine how cells and enzymes are used in the transformation of lipids. The lipids are, of course, a very diverse and complex series of molecular entities including fatty acids, triglycerides, phospholipids, glycolipids, aliphatic alcohols, waxes, terpenes and steroids. It is usual to teach about these molecules, in a biochemical context, in more or less the order given above, since this represents a logical sequence leading from simple molecules to the more complex. Here, however, we have adopted a different strategy. Clearly, the technical and commercial aspects of industrial lipid transformation are both diverse and complex. We have, therefore, to be selective in what we can include in a single chapter. We have decided to begn the chapter by discussing the transformation of sterols and steroids since these transformations are illustrative of the potency of biocatalysts in bringing about selective and stereospecific chemical transformation of quite complex molecules. This part of this chapter is a logical extension of the issues discussed in the previous chapter. We have also elected to focus on the technical rather than the commercial issues although attention is drawn to the importance of commercial criteria in the selection of strategies for production. The reader should be aware, however, that the production of steroids for pharmaceutical use and as contraceptives is a large market. Estimates of annual sales of these materials vary widely (WHO 1990 Year Ekmk $3~10' annum-' - $10'' annum-') reflecting the difficulty of accessing details on such a diverse group of compounds. After discussing the biological capability to transform steroids, we briefly examine the biotransformation of other terpenoids to ensure that the reader develops an awareness of the potential of biotechnology to modify or produce derivatives of a wide range of natural materials that are of tremendous potential, commercial value in the food and health care sectors. We also include a brief consideration of the use of biocatalysts to transform a range of other hydrocarbon compounds. The bulk of the chapter is therefore concerned with highly speafic reactions arising to produce molecules of known structure. However, in a chapter on lipid transformation, we should not miss the use of general lipases to change the composition of triglycerides. Although the replacement of one set of fatty acids in trigycerides is, from a chemical/biochemical point of view, not as stimulating as the biotransformation of steroids, it is of mapr commercial value in the food industry. The biotransformation of triglycerides (and phospholipids) to produce food materials which have desirable organoleptic properties (eg melt in the mouth feel) potentially dwarfs the steroid market in terms of volume and turnover. The placing of this topic towards the end of the chapter does not imply that it is of limited commercial value. The final part of the chapter briefly explores the potential of using non-aqueous solvents for lipid transformation. bids i~~~ea wide^$^^! types
Biotransformation of lipids Despite the technical emphasis of this chapter, we have included some examples 9.2 The structure, roles and abundance of sterols and steroids Some examples of sterols and steroids are given in Figure g.1. Also included in this Figure are some examples of bile salts. You should realise that the structures shown are steroidal ring only a few of the many hundreds of compounds which occur in nature. All of these stucture compounds include the steroidal ring structure which is numbered as shown belor D B Substituents are designated as in the a configuration if they are below the plane of the steroidal nucleus, and as p if above the plane Thu whilst 3B hydroxy Examine Figure 9.1 and see if you can distinguish between the roles of the sterols (at the top of the figure)and the C18, Cig, Ca and Ca4 steroidal compounds In general, the sterols perform a structural function, for example as components of the lipid layers of membranes. The C18, C19 and C21 steroids mainly perform an endocrine function. In other words they are hormones. The bile salts( C24-steroids) fulfil a functional role in digestion in animals
Biotransformation of lipids 295 Despite the technical emphasis of this chapter, we have included some examples of industrial processes. 9.2 The structure, roles and abundance of sterols and steroids Some examples of sterols and steroids are given in Figure 9.1. Also included in this Figure are some examples of bile salts. You should realise that the structures shown are only a few of the many hundds of compounds which occur in nature. All of these compounds include the steroidal ring structure which is numbered as shown below. steroidal ring smare - Substituents are designated as in the a configuration if they are below the plane of the steroidal nucleus, and as 0 if above the plane: Thus is 3a hydroxy - whilst is 38 hydroxy - Examine Figure 9.1 and see if you can distinguish between the roles of the sterols n (at the top of the figure) and the C18, (219, Cn and CN steroidal compounds. In general, the sterols perform a structural function, for example as components of the lipid layers of membranes. The Cis, Ci9 and C21 steroids mainly perform an endocrine function. In other words they are hormones. The bile salts (Czlr-steroids) fulfil a functional role in digestion in animals
Chapter 9 Bearing this in mind, which of the three groups are likely to occur a)in greatest amounts, b)in lowest concentration in biological sy You should have predicted that the sterols are present in greatest quantity in biological stems. your knowledge of biology should have enabled you to identify the steroid hormones as being present in lowest concentrations since hormone, in general, are effective at very low concentrations sterols cholesterol bile salts COOH COOH (c2 24"steroids) cholic acid lithocholic acid steroid hormones Cn-steroids CH3 CH,OH c二 progesterone corticosterone cortisol C1 and C1a steroids H estradiol 17-p stradiol-17B Figure 9.1 Some examples of sterols, bile salts and steroids
296 Chapter 9 Bearing this in mind, which of the three groups are likely to occur a) in greatest n amounts, b) in lowest concentration in biological systems? You should have predicted that the sterols are present in greatest quantity in biological systems. Your knowledge of biology should have enabled you to identify the steroid hormones as being present in lowest concentrations since hormone, in general, are effective at very low concentrations. Figure 9.1 Some examples of sterols, bile salts and steroids
Biotransformation of lipids Although representatives of all of the classes of sterols and steroids are essential to steroids have humans, the biological (pharmacological)activities of the C18, C19 and cz steroids make pharmac vaa these potentially very useful as therapeutic agents. It has long been realised that variations on the structures of naturally occurring steroids lead to products with greatly modified biological activities. Thus we can visualise the situation in which steroids may be modified to produce substances which have enhanced or reduced activities. This has far reaching implications in the healthcare sector. For example, natural and modified corticosteroids have applications as anti-inflammatory agents and may be used where the immune response needs to be moderated. Similarly the ability of C1g and Ci8 steroids to modulate reproductive capabilities makes them useful as fertility agents and as ∏ Which of the groups of compounds shown in Figure 9.1 is(are)likely to be of greatest commercial (and social) value? steroids are of Again, the answer should be fairly obvious. The potential therapeutic value of the great steroid hormones makes these of tremendous commercial value. the commercial commercial valu market for these is of the order of hundreds of millions of dollars per year. There is no comparable market for sterols and bile salts. We are faced with the interesting situation, herefore, that sterols are relatively abundant in natural sources but of relatively low commercial value, whilst steroids occur naturally at very low concentrations but are of great commercial value. Although there are tremendous variations amongst different products, steroids with desirable properties command market prices that are(ten toone thousand fold) greater than their sterol counterparts Bearing in mind the relative abundance of sterols and steroids and their chemical structures, which of the following strategies for producing steroids is most likely to be commercially successful? 1)Extraction from animals. 2) Total chemical synthesis 3)Partial chemical synthesis starting from a natural product. 4) Total biosynthesis 5)Enzymatic transformation of natural products Below we have considered each of these strategies in turn. 1)Although animals produce steroids, the low concentrations of these compounds does not make these commercially (nor ethically)attractive sources of these substances. Furthermore, they could only serve as sources of naturally occurring steroids. Thus we would not have selected this option y The steroid ring structure is complex and contains many chiral carbons(for example at positions 5, 8, 9, 10, 13, 14 and 17) thus many optical isomers are possible. (The actual number of optical isomers is given by 2 where n =the number of chiral carbons). From your knowledge of biochemistry you should have realised that only ne of these optical isomers is likely to be biologically active. Synthesis of such a complex chemical structure to produce a single isomeric form is extremely difficult, especially when it is realised that many chemical reactions lead to the formation of racemic mixtures. Thus, for complete chemical synthesis, we must anticipate that
Biotransformation of lipids 297 Although representatives of all of the classes of sterols and steroids are essential to humans, the biological (pharmacological) activities of the CW, CI~ and Cn steroids make these potentidy very usef~l as therapeutic agents. It has long been realid that variations on the structures of naturally occurring steroids lead to products with greatly modified biological activities. Thus we can visualise the situation in which steroids may be modified to produce substances which have enhanced or reduced activities. This has far reaching implications in the healthcare sector. For example, natural and modified corticosteroids have applications as anti-inflammatory agents and may be used where the immune response needs to be moderated. Similarly the ability of CM and CI~ steroids to modulate reproductive capabilities makes them useful as fertility agents and as contraceptives. Which of the groups of compounds shown in Figure 9.1 is (are) likely to be of n greatest commeraal (and social) value? Again, the answer should be fairly obvious. The potential therapeutic value of the steroid hormones makes these of tremendous commercial value. The commercial market for these is of the order of hundreds of millions of dollars per year. There! is no comparable market for sterols and bile salts. We are faced with the interesting situation, therefore, that sterols are relatively abundant in natural sources but of relatively low commeraal value, whilst steroids occur naturally at very low concentrations but are of great commeraal value. Although there are tremendous variations amongst different products, steroids with desirable properties command market prices that are (ten to one thousand fold) greater than their sterol counterparts. Sbdds have @mamutical value steroids are of gFt commeraal ValUe Bearing in mind the relative abundance of sterols and steroids and their chemical structures, which of the following strategies for producing steroids is most likely to be commercially successful? n 1) Extraction from animals. 2) Total chemical synthesis. 3) Partial chemical synthesis starting from a natural product. 4) Total biosynthesis. 5) Enzymatic transformation of natural products. Below we have considered each of these strategies in turn. 1) Although animals produce steroids, the low concentrations of these compounds does not make these commercially (nor ethically) attractive sources of these substances. Furthermore, they could only serve as sources of naturally occurring steroids. Thus we would not have selected this option. The steroid ring structure is complex and contains many chiral carbons (for example at positions 5,8,9,10,13,14 and 17) thus many optical isomers are possible. (The actual number of optical isomers is given by 2" where n = the number of chiral carbons). From your knowledge of biochemistry you should have realised that only one of these optical isomers is likely to be biologically active. Synthesis of such a complex chemical structure to produce a single isomeric form is extremely difficult, especially when it is realised that many chemical reactions lead to the formation of racemic mixtures. Thus, for complete chemical synthesis, we must anticipate that
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
