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