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 involvedBiotransformation 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