Industrial production of amino acids by fermentation and chemo-enzymatic methods 8. 1 Introduction 8.2 Essential and nonessential amino acid 3 Stereochemistry of amino acids 8. 4 Amino acid fermentation 8.5 Recovery of the amino acid from the fermentation broth 248 8.6 Case study: fermentative production of L-phenylalanine from glucose 8.7 Case study: The production of L-phenylalanine by enzymatic Summary and objectives Appendix 8. 1: Representation of and the nomenclature for stereo-isomers Appendix 8.2 Examples of industrial production of amino adds by enzymatic method A8.2.1 Enzymatic resolution of racemates A8. 2.2 Enzymatic asymmetric synthesis A8. 2.3 Oxidation-Reduction reactions
231 Industrial production of amino acids by fermentation and chemo-enzymatic methods 8.1 Introduction 8.2 Essential and nonessential amino acids 8.3 Stereochemistry of amino acids 8.4 Amino acid fermentation 8.5 Recovery of the amino acid from the fermentation broth 8.6 Case study: fermentative production of L-phenylalanine from glucose 8.7 Case study: The production of L-phenylalanine by enzymatic methods Summary and objectives Appendix 8.1: Representation of and the nomenclature for stereeisomers Appendix 8.2 Examples of industrial production of amino acids by A8.2.1 Enzymatic resolution of racemates enzymatic methods A8.2.2 Enzymatic asymmetric synthesis A8.2.3 Oxidation - Reduction reactions 232 234 236 240 248 253 262 272 273 277 277 286 289
232 Chapter 8 Industrial production of amino acids by fermentation and chemo-enzymatic methods 8.1 Introduction Amino acids have always played animportant role in the biology of life, in biochemistry and in (industrial) chemistry. There are several reasons why they are of commercial amino acids are the building blocks of proteins and they play an essential role in the regulation of the metabolism of living organisms. Large-scale essential chemical and microbial producton processes have been commercialised for a number no aads of essential amino acids The use of glutamic acid lysine and methionine as food and feed additives is well established nowadays. Secondly, current interest in developing on-proteino. peptide-derived chemotherapeutics has heightened the importance of rare and genic amino non-proteinogenic pure amino acids. For example, D-phenylglycine and acds D-p-hydroxy-phenylglycine are building blocks for the broad spectrum B-lactam antibiotics ampicillin and amoxycillin, respectively. The natural amino acid L-valine is used as feedstock in the fermentative production of the cyclic peptide cyclosporin A, which has immuno-suppressive activity and is used in human transplant surgery Thirdly, amino acids are versatile chiral (optically active)building blocks for a whole range of fine chemicals. In the last two decades, there has been a growing public precursors for awareness and concern with regard to the exposure of man and his environment to an fine chemical ever increasing number of chemicals. The benefits, however, arising from the use synthesis therapeutic agents, pesticides, food and feed additives, etc are enormous. hence there is still an ever increasing demand for more selective drugs and pesticides which are targeted in their mode of action, exhibit less toxic side-effects and are more environmentally acceptable. To this end a central role will be played by chiral compounds, as nature at the molecular level is intrinsically chiral. Consequently, this provides an important stimulus for companies to market chiral products as pure optical isomers. This in turn results in an increasing need for efficient methods for the industrial synthesis of optically active compounds Amino acids are, therefore, important as nutrients (food and feed ), as seasoning flavourings and starting material for pharmaceuticals, cosmetics and other chemicals They can be produced in a variety of ways(see Table 8.1) · chemical synthesis; isolation from natural materials(extraction) amino acid fermentations(using micro-organisms); chemo-enzymatic methods In this chapter we consider amino acid production by fermentation and by chemo-enzymatic methods. we first consider the stereochemistry of amino acids and the importance of chirality in chemical synthesis. General approaches to amino acid fermentation and recovery of amino acids from fermentation broths are then dealt with followed by a detailed consideration of the production of L-phenylalanine by direct fermentation. Later in this chapter, chemo-enzymatic methods of amino acid
232 Chapter 8 essential ammo adds non-pcoteinogenic amino acids precursors for me chemical synthesis Industrial production of amino acids by fermentation and cherno-enzymatic methods 8.1 Introduction Amino acids have always played an important role in the biology of life, in biochemistry and in (industrial) chemistry. There are several reasons why they are of commercial interest. Firstly, amino acids are the building blocks of proteins and they play an essential role in the reguiation of the metabolism of living organisms. Largescale chemical and microbial production processes have been commercialised for a number of essential amino acids. The use of glutamic acid, lysine and methionine as food and feed additives is well established nowadays. Secondly, current interest in developing peptide-derived chemotherapeutics has heightened the importance of rare and non-proteinogenic pure amino acids. For example, D-phenylglycine and D-phydroxy-phenylglycine are building blocks for the broad spectrum f3-ladam antibiotics ampicillin and amoxycillin, respectively. The natural amino acid L-valine is used as feedstock in the fermentative production of the cyclic peptide cyclosporin A, which has immuno-suppressive activity and is used in human transplant surgery. Thirdly, amino acids are versatile chiral (optically active) building blocks for a whole range of fine chemicals. In the last two decades, there has been a growing public awareness and concern with regard to the exposure of man and his environment to an ever increasing number of chemicals. The benefits, however, arising from the use of therapeutic agents, pesticides, food and feed additives, etc are enormous. Hence there is still an ever increasing demand for more selective drugs and pesticides which are targeted in their mode of action, exhibit less toxic side-effects and are more environmentally acceptable. To this end a central role will be played by chiral compounds, as nature at the molecular level is intrinsically chiral. Consequently, this provides an important stimulus for companies to market chiral products as pure optical isomers. This in turn results in an increasing need for efficient methods for the industrial synthesis of optically active compounds. Amino acids are, therefore, important as nutrients (food and feed), as seasoning, flavourings and starting material for pharmaceuticals, cosmetics and other chemicals. They can be produced in a variety of ways (see Table 8.1): 0 chemical synthesis; 0 isolation from natural materials (extraction); amino acid fermentations (using micro-organisms); 0 chemo-enzymatic methods. In this chapter we consider amino acid production by fermentation and by chemoenzymatic methods. We first consider the stereochemistry of amino acids and the importance of chirality in chemical synthesis. General approaches to amino acid fermentation and recovery of amino acids from fermentation broths are then dealt with, followed by a detailed consideration of the production of L-phenylalanine by direct fermentation. Later in this chapter, chemo-enzymatic methods of amino acid
Industrial production of amino acids by fermentation and chemo-enzymatic methods fermentation are examined. We first consider general aspects of the approach, followed by more detailed case studies. We have again selected L-phenylalanine for detailed consideration, since this important amino acid can be produced by direct fermentation and by a variety of chemo-enzymatic methods. This allows comparisons between the two different approaches to be made, including a consideration of economic aspects of large scale production of the amino acid Two appendices are included at the end of this chapter The first is intended to serve as a reminder, for those of you who might need it, of the nomenclature and representation of stereoisomers. The second appendix contains descriptions of various chemo-enzymatic methods of amino acid production. This appendix has been constructed largely from the recent primary literature and includes many new advances in the field. It is not necessary for you to consult the appendix to satisfy the learning objectives of the chapter, rather the information is provided to illustrate the extensive range of methodology associated with chemo-enzymatic approaches to amino acid production. It is therefore available for those of you who may wish to extend your knowledge in this area. Where available, data derived from the literature are used to illustrate methods and to discuss economic aspects of large-scale production amino acld chemical extraction fermentation enzymatIc synthesls LLL -histidine(HCI L-phenylalanine (+) L-threonine L-tyrosine L-valine (+) Table 8. 1 Production methods of proteinogenic amino acids
Industrial production of amino acids by fermentation and chemo-enzymatic methods 233 fennentation are examined. We first consider general aspects of the approach, followed by more detailed case studies. We have again selected L-phenylalanine for detailed consideration, since this important amino acid can be produced by direct fermentation and by a variety of chemo-enzymatic methods. This allows comparisons between the two different approaches to be made, including a consideration of economic aspects of large scale production of the amino acid. Two appendices are included at the end of this chapter. The first is intended to serve as a reminder, for those of you who might need it, of the nomenclature and representation of stereoisomers. The second appendix contains descriptions of various chemo-enzymatic methods of amino acid production. This appendix has been constructed largely from the recent primary literature and includes many new advances in the field. It is not necessary for you to consult the appendix to satisfy the learning objectives of the chapter, rather the information is provided to illustrate the extensive range of methodology associated with chemo-enzymatic approaches to amino acid production. It is therefore available for those of you who may wish to extend your knowledge in this area. Where available, data derived from the literature are used to illustrate methods and to discuss economic aspects of large-scale production. + amino ackl chemical extradlon fermentation enzymatic synthesls CatalySlS L-alanine + + L-arginine + + L-aspartic acid + + L-cystine + L-glutamic acid (Na) (+I + L-histidine (.HCI) + + L-isoleucine + + L-leucine + L-lysine (.HCI) + + L-methionine + L-phenylalanine (+) (+) + L-proline + (+I L-serine + + L-threonine + + L-tryptophan + + L-tyrosine + L-valine + (+) + L-cysteine Table 8.1 Production methods of proteinogenic amino acids
Chapter 8 8.2 Essential and nonessential amino acids An amino acid is defined as a compound that possesses both amino and carboxyl roups. Some amino acids are aminocarboxylic acids such as proline while others are sulphur containing amino acids, such as cysteine and methionine table 8.2). Over 10 amino acids have been isolated and identified from natural sources to date. The great majority of these naturally occurring amino acids have the amino group attached to the a-carbon carbon a to the carboxylic acid. With very few exceptions, the a-carbon also be ydrogen atom. The fourth bond of the a-carbon is joined to a group which has 100 variations. Thus, most of the naturally occurring amino acids differ only structure of the organic residue attached to the a-carbon An interesting and important fact is that almost all amino acids isolated from proteins L-configuration have the L-configuration at the a-carbon, although some amino acids isolated from microbiological sources are the mirror image isomers, ie in the D-configuration. We shall consider amino acid stereochemistry in more detail in section 8.3 Of the amino acids isolated from living material only about 20 are naturally occurring components of proteins. Some of these are shown in Table 8. 2. The remainder, non-proteinogenic amino acids, are found as intermediates or end products of One of the amino adids commonly found in protein hydrolysates is called cystine; it has the following structure HOOC- CH- CH2 -S-S- CH2- CH-COOH NH H2 of cysteine, where the thiol of two monomers spaced intervals in the polypeptide are j a disulphide bridge asic amino acid is cysteine and consequently the is not included here
234 Chapter 8 8.2 Essential and nonessential amino acids a-carbon Lconfiguration dimer An amino acid is defined as a compound that possesses both amino and carboxyl groups. Some amino acids are iminocarboxylic acids, such as proline while others are sulphur containing amino acids, such as cysteine and methionine (Table 8.2). Over 100 amino acids have been isolated and identified from natural sources to date. The great majority of these naturally occumng amino acids have the amino group attached to the carbon a to the carboxylic acid. With very few exceptions, the a-carbon also bears a hydrogen atom. The fourth bond of the a-carbon is pined to a group which has over 100 variations. Thus, most of the naturally occurring amino acids differ only in the structure of the organic residue attached to the a-carbon. An interesting and important fact is that almost all amino acids isolated from proteins have the L-configuration at the a-carbon, although some amino acids isolated from microbiological sources are the mirror image isomers, ie in the Dconfiguration. We shall consider amino acid steremhemistry in more detail in section 8.3. Of the amino acids isolated from living material, only about 20 are naturally Occurring components of proteins. Some of these are shown in Table 8.2. The remainder, non-proteinogenic amino acids, are found as intermediates or end products of metabolism. One of the amino acids commonly found in protein hydrolysates is called cystine; it has the following structure: HOOC- CH-CH,-S-S-CH,-CH-COOH (CYSCYS) I I NH2 NH2 cystine It is clearly a dimer of cysteine, where the thiol groups have been oxidised to form a disulphide linkage. The dimer actually results because of two monomers at widely spaced intervals in the polypeptide are joined together by a disulphide bridge. Thus the basic amino acid is cysteine and consequently, the dimer is not included here
Industrial production of amino acids by fermentation and chemo-enzymatic methods 2H COH L-alanine CO2H rtic acid tophan HsCS CO2H NH2 L-methionine L-glutamine L-phenylalanine (L-phe Table 8. 2 Structure of some proteinogenic a-amino acids All living species are able to synthesise amino acids. Many species, however, are deficient in their ability to synthesise within their own metabolic system all the amino esential acids necessary for life. The eight amino acids with this special significance for the human species are called essential amino acids, these are ·L- valine L-leucine L-threonine · L-methionine; L-phenylalanine; L-tryptopha They are essential not because they are the only amino acids required for human functioning, but because they are essential in the diet of the human species
Industrial production of amino acids by fermentation and chemo-enzymatic methods 235 Table 8.2 structure of some proteinogenic a-amino acids. All living species are able to synthesise amino acids. Many species, however, are deficient in their ability to synthesise within their own metabolic system all the amino acids necessary for life. The eight amino acids with this special significance for the human species are called essential amino acids, these are: L-valine; 0 L-leucine; L-isoleucine; 0 L-threonine; 0 L-methionine; L-phenylalanine; 0 L-tryptophan; L-lysine. They are essential not because they are the only amino acids requred for human functioning, but because they are essential in the diet of the human species. essential amino acids
Chapter 8 SAQ 8. 1 1)Name three sulphur containing amino acids 2)Name five of the eight essential amino acids. 3)Name two amino acids that contain a heterocyclic ring. 4)Name the amino acid with the simplest structur 5) Name the amino acid considered to be a dimer 6)Name an amino acid that is produced industrially only by enzymatic 7) Name an amino acid that is produced industrially only be chemical 8.3 Stereochemistry of amino acids The French physicist Biot discovered during the early nineteen th century, that a number of naturally occurring organic compounds rotate the plane of polarisation of an incident beam of polarised light. In the latter part of the nineteenth century it was found that many pairs of compounds seemed to have an identical structure and identical physical properties, such as melting point and solubility. Compounds in each pair were differentiated by the fact that even in solution they rotated polarised light in equal amounts but in opposite direction. Such compounds are called optical isomers and are symmetric arrangement of groups around a tetrahedral carbon atom. The gomes? described as being optically active. Optical activity requires, and is explained by an tetrahedral properties of a tetrahedron are such that if there are four different substituents attached to a carbon atom, the molecule does not contain a plane of symmetry, and there are two kinds of geometrical arrangements which the molecule can have. These two arrangements(configurations)are different in that it is not possible to simultaneously superimpose all the atoms of one figure on the like atoms of the other The two configurations are, in fact non-superimposable mirror images An illustrative example is given in Figure 8.1
236 Chapter 8 1) Name thee sulphur containing amino acids. 2) Name five of the eight essential amino acids. 3) Name two amino acids that contain a heterocyclic ring. 4) Name the amino acid with the simplest structure. 5) Name the amino acid considered to be a dimer. 6) Name an amino acid that is produced industrially only by enzymatic 7) Name an amino acid that is produced industrially only be chemical catalysis. synthesis. 8.3 Stereochemistry of amino acids The French physicist Biot discovered during the early nineteenth century, that a number of naturally occumng organic compounds rotate the plane of polarisation of an incident beam of polarised light. In the latter part of the nineteenth century, it was found that many pairs of compounds seemed to have an identical structure and identical physical properties, such as melting point and solubility. Compounds in each pair were differentiated by the fact that even in solution they rotated polarised light in equal amounts but in opposite direction. Such compounds are called optical isomers and are described as being optically active. Optical activity requires, and is explained by an asymmetric arrangement of groups around a tetrahedral carbon atom. The geometric properties of a tetrahedron are such that if there are four different substituents attached to a carbon atom, the molecule does not contain a plane of symmetry, and there are two kinds of geometrical arrangements which the molecule can have. These two arrangements (configurations) are different in that it is not possible to simultaneously superimpose all the atoms of one figure on the like atoms of the other. The two configurations are, in fact non-superimposable mirror images. An illustrative example is given in Figure 8.1. Optical isomers mra~~edra~ cabon a~m
Industrial production of amino acids by fermentation and chemo-enzymatic methods HOC YAY CO2H HO2C Figure 8. 1 Non-superimposable mirror images metric Such molecules result when the four groups attached to the carbon atom are all diffe arbon and a molecule of this kind is said to be asymmetric, or to contain an asymmetric car Molecules that are not superimposable on their mirror images are chiral. If two enantiomers compounds are related as non-superimposable mirror images, they are called enantiomers I cany you explain why the amino acid alanine is optically active, whereas glycine We can see from Table 8. 2 that the a-carbon of alanine is asymmetric (four different groups attached), whereas that of glycine is not. Optical activity requires an asymmetric carbon atom racemic If two enantiomers are mixed together in equal amounts the result is a racemic mixture mxtures We meet a number of enantiomeric items in daily life. The left hand, for example, is the mirror image of the right hand and they are not superimposable(see Figure 8.1). This becomes obvious if we try to put a right glove on a left hand. Similarly, a pair of shoes is an enantiomeric relationship while the stock in a shoe store constitutes a racemic mixture Representation of and the nomenclature for stereo-isomers are given in Appendix 8. 3. 1 Importance of chirality If we consider natural synthetic processes, enzymes are seen to exert complete control over the enantiomeric purity of biomolecules(see Figure 8.2). They are able to achieve this because they are made of single enantiomers of amino acids. The resulting enantiomer of the enzymes functions as a template for the synthesis of only one enantiomer of the product. Moreover, the interaction of an enzyme with the two enantiomers of a given substrate molecule will be different. Biologically important molecules often show effective activity as one enantiomer the other is at best ineffective or at worst detrimental
Industrial production of amino acids by fermentation and chemo-enzymatic methods 237 I I Figure 8.1 Non-superimposable mirror images. Such molecules result when the four groups attached to the carbon atom are all different and a molecule of this kind is said to be asymmetric, or to contain an asymmetric carbon. Molecules that are not superimposable on their mirror images are chiral. If two compounds are related as non-superimposable mirror images, they are called Can you explain why the amino acid alanine is optically active, whereas glycine n is not (refer to Table 8.2)? We can see from Table 8.2 that the a-carbon of alanine is asymmetric (four different groups attached), whereas that of glycine is not. Optical activity requires an asymmetric carbon atom. If two enantiomers are mixed together in equal amounts the result is a racemic mixture. We meet a number of enantiomeric items in daily life. The left hand, for example, is the mirror image of the right hand and they are not superimposable (see Figure 8.1). This becomes obvious if we try to put a right glove on a left hand. Similarly, a pair of shoes is an enantiomeric relationship while the stock in a shoe store constitutes a racemic mixture. Representation of and the nomenclature for stereo-isomers are given in Appendix 1. 8.3.1 Importance of chirality If we consider natural synthetic processes, enzymes are seen to exert complete control over the enantiomeric purity of biomolecules (see Figure 8.2). They are able to achieve this because they are made of single enantiomers of amino acids. The resulting enantiomer of the enzymes functions as a template for the synthesis of only one enantiomer of the product. Moreover, the interaction of an enzyme with the two enantiomers of a given substrate molecule will be different. Biologically important molecules often show effective activity as one enantiomer, the other is at best ineffective or at worst detrimental. asymmetric carbon enantiomem enantiomers. racemic mixtures enantiomeric PJw
238 enantiomers n enzyme receptor Figure 8.2 Enzyme interaction with two enantiomers of a given substrate molecule In some cases the unwanted enantiomer can perturb other biological processes and cause catastrophic side effects. The use of enantiomerically pure compounds thus action permits more specific drug action and the reduction in the amount of drug involved in its metabolism before secretion can be avoided Numerous examples of the different biological effects of enantiomers are available. One of the enantiomers of limonene smells of lemons, the other of oranges; one of carvone smells of caraway, the other of spearmint. These differences obviously have important
230 Chapter 8 Figure 8.2 Enzyme interaction with two enantiomers of a given substrate molecule. In some cases the unwanted enantiomer can perturb other biological processes and cause catastrophic side effects. The use of enantiomerically pure compounds thus permits more specific drug action and the reduction in the amount of drug administered. Even in the cases where the other enantiomer is inactive, the work involved in its metabolism before secretion can be avoided. Numerous examples of the different biological effects of enantiomers are available. One of the enantiomers of limonene smells of lemons, the other of oranges; one of carvone smells of caraway, the other of spearmint. These differences obviously have important sped:iz
Industrial production of amino acids by fermentation and chemo-enzymatic methods consequences for the perfume and flavour industries. Both enantiomers of sucrose are equally sweet, but only the naturally occurring D-enantiomer is metabolised, making the synthetic L-enantiomer a potential dietary sweetener In the protection of crops from insects, one enantiomer of a compound may be a repellant while the other is an attractant, and the racemic mixture is ineffective. One enantiomer of penicillamine(D-)exhibits antiarthritic properties but the other is highly toxic(Figure 8.3). The teratogenic effects of thalidomide were induced by one enantiomer, the other exhibited the beneficial effects against moming sickness. Different optical enantiomers of amino acids also have different properties L-asparagine, for example, tastes bitter while D-asparagine tastes sweet(see Figure 8.3) demonstrate the importance of the use of homochiral compounde ( figure a 3).When achiral l-phenylalanine is a constituent of the artificial sweetener aspartame (Figure 8.3).When compounds one uses D-phenylalanine the same compound tastes bitter. These examples clearly 1)penicillamine H3C CH3 HOC NH2 extreme toxicity 2)asparagine CH2-CH-C-NH-CH sweet Figure 8.3 Examples of diferent biological effects of enantiomers. s and R refer to a particular system of nomenclature used to describe chiral carbon. (see Appendix A8. 1) SAQ 8.2 List possible advantages of using enantiomerically pure compounds as drugs, as opposed to using racemic mixtures
Industrial production of amino acids by fermentation and chemo-enzymatic methods 239 consequences for the perfume and flavour industries. Both enantiomers of sucrose are equally sweet, but only the naturally occurring D-enantiomer is metabolid, making the synthetic L-enantiomer a potential dietary sweetener. In the protection of mps from insects, one enantiomer of a compound may be a repellant while the other is an attractant, and the racemic mixture is ineffective. One enantiomer of penicillamine (D-) exhibits antiarthritic properties but the other is highly toxic (Figure 8.3). The teratogenic effects of thalidomide were induced by one enantiomer, the other exhibited the beneficial effects against morning sickness. Different optical enantiomers of amino acids also have different properties. L-asparagine, for example, tastes bitter while D-asparagine tastes sweet (see Figure 8.3). L-Phenylalanine is a constituent of the artificial sweetener aspartame (Figure 8.3). When one uses D-phenylalanine the same compound tastes bitter. These examples clearly demonstrate the importance of the use of homochiral compounds. hvnce Of hanochlral compo,,,,^ ~ ~~~ ~~ Figure 8.3 Examples of different biological effects of enantiomers. S and R refer to a particular system of nomenclature used to describe chiral carbon. (see Appendix A8.1) List possible advantages of using enantiomerically pure compounds as drugs, as opposed to using racemic mixtures
240 Chapter 8 8. 4 Amino acid fermentation wild strains Many micro-organisms accumulate amino acids in culture media. Indeed wild strains have proved to be effective producers of amino acids like alanine, glutamic acid and valine Since amino acids are used as essential components of the microbial cells and their biosynthesis is regulated to maintain an optimal level, they are normally synthesised in limited amounts and are subject to negative feedback control. The main problem using wild strains is, therefore, the production of minor amounts of amino acids at an early tage in the fermentation, giving rise to feedback control To achieve overproduction of amino acids the follo improvement of the uptake of the raw material(starting materiaL); hindrance of the side reactions stimulation of the enzymes that are involved in the synthesis inhibition of the degradation of the desired amino acid; stimulation of excretion of the amino acid that is produce ategies for The most successful way to achieve overproduction is to make use of mutants. Another rproducton way to overcome feedback regulation is to make use of a kind of semi-fermentation process called precursor addition fermentation; this will be considered later in this chapte Amino acids produced by fermentation on an industrial scale are listed in table 8.3 Ino acids tonnes/year ppllcatlons ca8,000 westener) synthesis of alan glutamic acid ca.270,00 flavours, pharmaceuticals ca.90,000 ca.8,000 aspartame(sweetener) ca.500 dietary ca.100 pharmaceuticals, dietary Table 8.3 Amino acids industrially produced by fermentation ∏ Examine the list of procedures to achieve overproduction(shown above)and identify which ones could be achieved by mutation of a wild strain. Since all the procedures listed involve enzymes they all could be achieved by mutation This emphasises the potential of using mutation for amino acid production
240 Chapter 8 8.4 Amino acid fermentation wild strains Many micro-organisrns accumulate amino acids in culture media. Indeed, wild strains have proved to be effective producers of amino acids like alanine, glutamic acid and valine. Since amino acids are used as essential components of the microbial cells and their biosynthesis is regulated to maintain an optimal level, they are normally synthesised in limited amounts and are subject to negative feedback control. The main problem using wild strains is, therefore, the production of minor amounts of amino acids at an early stage in the fermentation, giving rise to feedback control. To achieve overproduction of amino acids the following procedures can be used: 0 improvement of the uptake of the raw material (starting material); 0 hindrance of the side reactions; tzgz mw 0 stimulation of the enzymes that are involved in the synthesis; 0 inhibition of the degradation of the desired amino acid; 0 stimulation of excretion of the amino acid that is produced. The most successful way to achieve overproduction is to make use of mutants. Another way to overcome feedback rrgulation is to make use of a kind of semi-fermentation process called precursor addition fermentation; this will be considered later in this chapter. Amino acids produced by fermentation on an industrial scale are listed in Table 8.3. smegies br overpcoddm amino aclds tonneelyear aspartic acid ca. 8,000 glutamic acid lysine phenylalanine threonine tryptophan ca. 270,000 ca. 90,000 ca. 8,000 ca. 500 ca. 100 applications aspartame (sweetener) enzymatic synthesis of alanine and phenylanine flavours, pharmaceuticals dietary aspartame (sweetener) dietary pharmaceuticals, dietary Table 8.3 Amino adds industrially produced by fermentation. Examine the list of p'ocedures to achieve overproduction (shown above) and n idenbfy which ones could be achieved by mutation of a wild strain. Since all the procedures listed involve enzymes they all could be achieved by mutation. This emphasises the potential of using mutation for amino acid production