Industrial production of amino acids by fermentation and chemo-enzymatic methods 8. 4. 1 Wild strains Normally amino acid synthesis will just satisfy the metabolic demand. In some cases, when the amino acid occurs in both biosynthetic and energy production path overproduction of the amino acid can take place. This is the case, especial L-glutamic acid, with Corynebacterium, Brevibacterium, Microbacterium and Arthrobacter characteristic isms have some common features: Gram-positive, non-spore inuencing overproaucaon general characteristics have been found to influence the overproduction of L-glutamic nutritional requirement for biotin; intracellular levels of phospholipids; lack of, or low activity of, a-oxoglutarate dehydrogenase In the first two cases, the permeability of the cell membrane to L-glutamate is altered through changes in the fatty adid composition of the cell membrane. In the third case, the degradation of the amino acid is inhibited resulting in accumulation. Other amino acids produced by wild strains include L-valine, DL-alanine and L-proline Why are amino acid synthesised in only small amounts in wild strains? The small overproduction of amino acids by wild type strains in culture media is the result of regulatory mechanisms in the biosynthetic pathway. These regulatory mechanisms are feedback inhibition and repression These mechanisms are considered fully in other BIOTOL texts However, details of the mechanisms are not essential for an understanding of the material in this chapter. In th section we will, therefore, only briefly describe the mechanisms Feedback inhibition In the metabolic pathway to an amino acid several steps are involved. Each step is the result of an enzymatic activity. The key enzymatic activity(usually the first enzyme in the synthesis)is regulated by one of its products(usually the end product, eg the amino acid). If the concentration of the amino acid is too high the enzymatic activity is decreased by interaction of the inhibitor with the regulatory site of the enzyme (allosteric enzyme). This phenomenon is called feedback inhibition Repression In repression the enzyme concentration is regulated at the DNA level The genetic information for a certain enzyme is located on the genetic material in the cell, the DNA. In general this consists of several integrated regions working together (an operon)
Industrial production of amino acids by fermentation and chemo-enzymatic methods 241 8.4.1 Wild strains Normally amino acid synthesis will just satisfy the metabolic demand. In some cases, when the amino acid occurs in both biosynthetic and energy production pathways, overproduction of the amino acid can take place. This is the case, especially for L-glutamic acid, with Corynebacterium, Brm'bacterium, Microbacterium and Arthbacter. These micro-organisms have some common features: Gram-positive, non-spore forming, non motile, cocci or rod and biotin-requiring for growth. The following general characteristics have been found to influence the overproduction of L-glutamic acid: characteristic influmdng ove~duclian 0 nutritional requirement for biotin; 0 intracellular levels of phospholipids; 0 lack of, or low activity of, a-oxoglutarate dehydrogenase. In the first two cases, the permeability of the cell membrane to L-glutamate is altered through changes in the fatty acid composition of the cell membrane. In the third case, the degradation of the amino acid is inhibited, resulting in accumulation. Other amino acids produced by wild strains include L-valine, DL-alanine and L-proline. n Why are amino acid synthesised in only small amounts in wild strains? The small overproduction of amino acids by wild type strains in culture media is the result of regulatory mechanisms in the biosynthetic pathway. These regulatory mechanisms are feedback inhibition and repression. These mechanisms are considered fully in other BIOTOL texts. However, details of the mechanisms are not essential for an understanding of the material in this chapter. In this section we will, therefore, only briefly desaibe the mechanisms. Feedback inhibition In the metabolic pathway to an amino acid several steps are involved. Each step is the result of an enzymatic activity. The key enzymatic activity (usually the first enzyme in the synthesis) is regulated by one of its products (usually the end product, eg the amino acid). If the concentration of the amino acid is too high the enzymatic activity is decreased by interaction of the inhibitor with the eatery site of the enzyme (allosteric enzyme). This phenomenon is called feedback inhibition. Repression In repression the enzyme concentration is regulated at the DNA level. The genetic information for a certain enzyme is located on the genetic material in the cell, the DNA. In general this consists of several integrated regions working together (an operon)
Chapter 8 If the information can be translated into messenger RNA, and this information can be transferred to the ribosome, the enzyme can be synthesised. If not, the key enzyme in a metabolic route to the wanted amino acid is not synthesised This might involve a apo-repressor repressor, consisting of an apo-repressor (ie a protein from a regulatory gene)and the and Co-repressor (normally the end product of the pathway) which binds to the operator co-repressor gene and prevents translation of the operon. Normally this is not an absolute effect. If the concentration of the end product is high the end product blocks the operator gene, so the m-RNA cannot be formed and the enzyme cannot be produced. The concentration of the enzyme is kept low in such a case. It is obvious s nanisns are not wanted. One way to overcome these regulations is to at in the case of overproduction of amino acids the above mentioned gulatory mecl make use of mutants 8.4.2 Mutant strains Two types of mutants have been used for amino acid overproduction: auxotrophic and regulatory mutants. In some cases, mutant strains have been further improved through DNA-recombination Auxotrophic mutants Auxotrophic mutants are mutants that miss one or more of the enzymes used in the biosynthetical pathway for one or more amino acids. In practice this means that the mutant needs one or more key yy metabolites which it cannot synthesise for growth in its growth medium. For example, consider Figure 8.4 erythrose--P+ phosphoenolpyruvate 1 3-deoxy-D-arabino-heptulosonate-7-phosphate chonsmate E3 prephenate anthranilate phenylalanine tyrosine tryptophan Figure 8.4 Skeleton pathway leading to L-phenylalanine, tyrosine and tryptophan in of the enzymes to synthesise tyrosine(E6 in Figure 84 Coes not produce at least one In the literature this is described as Atyr. This means that a small amount of tyrosine has to be added to the culture medium, just enough to support growth, because the micro-organism is not capable of producing tyrosine In the wild strain, El is controlled by feedback inhibition involving tyrosine. So, accumulation of tyrosine slows the rate phenylalanine of flow through the pathway. In the mutant strain, however, this does not occur because overproduction tyrosine is not produced, so overproduction of phenylalanine will occur. The genetic marker of this particular organism is reported in literature as Tyr
242 Chapter 8 If the information can be translated into messenger RNA, and this information can be transferred to the ribosome, the enzyme can be synthesised. If not, the key enzyme in a metabolic route to the wanted amino acid is not synthesised. This might involve a repressor, consisting of an apo-repressor (ie a protein from a regulatory gene) and the wrepressor (normally the end product of the pathway) which binds to the operator gene and prevents translation of the operon. Normally this is not an absolute effect. If the concentration of the end product is high the end product blocks the operator gene, so the m-RNA cannot be formed and the enzyme cannot be produced. The concentration of the enzyme is kept low in such a case. It is obvious that in the case of overproduction of amino acids the above mentioned regulatory mechanisms are not wanted. One way to overcome these regulations is to make use of mutants. 8.4.2 Mutant strains --repressor and mWr-r Two types of mutants have been used for amino acid overproduction: auxotrophic and regulatory mutants. In some cases, mutant strains have been further improved through DNA-recombination. Auxotrophic mutants Auxotrophic mutants are mutants that miss one or more of the enzymes used in the biosynthetical pathway for one or more amino acids. In practice this means that the mutant needs one or more key metabolites which it cannot synthesise for growth in its growth medium. For example, consider Figure 8.4. Figure 8.4 Skeleton pathway leading to Lphenylalanine, tyrosine and tryptophan in Escherichia coli. In the case of a tyrosine auxotrophic mutant, the mutant does not produce at least one of the enzymes to synthesise tyrosine (E6 in Figure 8.4). In the literature this is described as Atyr. This means that a small amount of tyrosine has to be added to the culture medium, just enough to support growth, because the micro-organism is not capable of producing tyrosine. In the wild strain, El is controlled by feedback inhibition involving tyrosine. So, accumulation of tyrosine slows the rate of flow through the pathway. In the mutant strain, however, this does not occur because tyrosine is not produced, so overproduction of phenylalanine will occur. The genetic marker of this particular organism is reported in literature as Tyr-. phmylalanine ove~*clim in Tyf
Industrial production of amino acids by fermentation and chemo-enzymatic methods 243 branched Auxotrophic mutants are used in the production of end products of branched pathways pathways, ie pathways leading to more than one amino acid at the same time. This is the case for L-lysine, L-methionine, L-threonine and L-isoleucine in brevibacterium Aaoum and Corynebacterium glutamicum. Regulatory mutants There are two possible sites that are genetically inactivated in regulatory mutants · the regulatory site; the functions of repressor and co-repressor These mutants lack feedback inhibition and are used for the production of many amino acids selection using Selection of these regulatory mutants is often done by using toxic analogues of amino toxic analogues acids; for example p-fluoro-DL-phenylalanine is an analogue of phenylalanine Mutants that have no feedback inhibition or repression to the amino acid are also resistant to the feedback inhibitors or inhibit the incorporation of the amino acid into the protei pa analogue amino acid. They are therefore selected for and can be used to overprodt the amino acid Some amino acid analogues function as false co-repressors The best amino acid producers are organisms that are both auxotrophic and regulator mutants What advantage do regulatory mutants have, when compared to auxotrophic mutants for amino acid fermentation? Unlike auxotrophic mutants, regulatory mutants can be grown in inexpensive, complex media and they do not require careful control of growth conditions. DNA-recombinant micro-organisms and combinations Another way to enhance the production of an amino acid is to make DNA-recombinant technology, often in combination with the mutations described In this way the negative features of the micro-organisms are avoided plain this, we will consider a well known fermentation of L-phenylalanine Escherichia coli. We have already seen the metabolic pathway leading to the prod of L-phenylalanine in Figure 8.4 Conversion of erythrose-4-P and phosphoenol pyruvate to 3-deoxy-D-arabinoseheptulosonic acid (DAHP)is catalysed by DAHP synthetase In by E coli there are three isoenzymes of the enzyme; these are known as aroF (regulated by phenylalanine each case, regulation is both at the level of enzyme formation(repression) and enuf as and tryptophan activity(feedback inhibition). Another site regulated through phenylalanine cont the expression of the structural genes pheA) for chorismate mutase-prephrenate dehydratase(E3 and E5 in Figure 8.4). The mechanism is based on the phenylalanine mediated repressor from the phea regulator gene that binds to the pheA operator site The effect of this regulation(on the pheA level)and feedback inhibition(on the aroG level)is low levels of the enzyme chorismate mutase-Prephrenate dehydratase and low activities of the enzyme DAHP synthetase. This results in low levels of phenylalanine
Industrial production of amino acids by fermentation and chemo-enzymatic methods 243 branched pa*wars selection using toxic anabgues regulation by and tryptophan tyrosine. phenylatanine Auxotrophic mutants are used in the production of end products of branched pathways, ie pathways leading to more than one amino acid at the same time. This is the case for L-lysine, L-methionine, L-threonine and Lisoleucine in Brertibacterium flmm and Corynebacterium glutamicum. Regulatory mutants There are two possible sites that are genetically inactivated in regulatory mutants: the regulatory site; the functions of repressor and wrepressor. These mutants lack feedback inhibition and are used for the production of many amino acids. Selection of these regulatory mutants is often done by using toxic analogues of amino acids; for example p-fluoro-DL-phenylalanine is an analogue of phenylalanine. Mutants that have no feedback inhibition or repression to the amino acid are also resistant to the analogue amino acid. They are therefore selected for and can be used to overproduce the amino acid. Some amino acid analogues function as false co-repressors, false feedback inhibitors or inhibit the incorporation of the amino acid into the protein. The best amino acid producers are organisms that are both auxotrophic and regulatory mutants. What advantage do regulatory mutants have, when compared to auxotrophic n mutants, for amino acid fermentation? Unlike auxotrophic mutants, regulatory mutants can be grown in inexpensive, complex media and they do not require careful control of growth conditions. DNA-recombinant micro-organisms and combinations Another way to enhance the production of an amino acid is to make use of DNA-recombinant technology, often in combination with the mutations already described. In this way the negative features of the miaworganisms are avoided. To help explain this, we will consider a well known fermentation of L-phenylalanine using Escherichia coli. We have already seen the metabolic pathway leading to the production of L-phenylalanine in Figure 8.4. Conversion of erythrose-4-P and phosphoenol pyruvate to 3-deoxy-D-arabinoseheptulosonic acid (DAW) is catalysed by DAHP synthetase. In E.coli there are three isoenzymes of the enzyme; these are known as mF (rrgulated by tyrosine), moG (regulated by phenylalanine) and mH (regulated by tryptophan). In each case, regulation is both at the level of enzyme formation (repression) and enzyme activity (feedback inhibition). Another site regulated through phenylalanine controls the expression of the structural genes (pheA) for chorismate mutaseprephrenate dehydratase (E3 and E5 in Figure 8.4). The mechanism is based on the phenylalanine mediated repressor from the pheA regulator gene that binds to the pheA operator site. The effect of this regulation (on the pheA level) and feedback inhibition (on the moG level) is low levels of the enzyme chorismate mutase-prephrenate dehydratase and low activities of the enzyme DAHP synthetase. This results in low levels of phenylalanine
24 Chapter 8 To achieve overproduction of phenylalanine, the micro-organism should be derepressed at the phe a level and free of inhibition at the aroG level. Both genes are located on the chromosomal DNA of the micro-organism and, by means of amino acid analogues such as p-fluoro-DL-phenylalanine it is possible to make (phenylalanine) feedback resistant mutants of Ecoli(pheA and aroFR mutants). The following procedure can be used use of pa sna tht genes are somat d fce m bnAromosomal DNA and put into a plasmid (ircular ral gene in the plasmid is altered by using a strong promoter(several very strong promoters are nowadays used, eg the lac promoter) the total effect (ie overproduction of phenylalanine by deregulation) can be enhanced by using more than one pl in the id in multicopy recombinant n a good production strain, the plasmid should be present in a stable way and should ∏ The procedure described above is just one way to get to a good phenylalanine The most obvious alternative approach is to deregulate the aroF gene, which is subject to tyrosine regulation. This, of course, could be achieved by dNa-recombinant techniques or by mutation. SAQ 8.3 Provide brief explanations for the following: 1)wild strains of E. coli are not used for L-phenylalanine production by direct fermentation 2) Auxotrophic mutants of E. coli are particularly useful for the production of L-phenylalanine by direct fermentation. 3)Regulatory mutants improve the rate of L-phenylalanine overproduction by 4)An E coli mutant, Try, is likely to enhance L-phenylalanine overproduction. 8.4.3 Methods of fermentation The growth of micro-organisms used in the production of amino acids is done in a well balanced environment. The conditions required are a controlled pH of the fermentation medium (approximately neutral); rich growth media; highly aerobic conditions; sterile conditions
244 Chapter 8 To achieve overproduction of phenylalanine, the micro-organism should be drmpressed at the pheA level and free of inhibition at the ad level. Both genes are located on the chromosomal DNA of the micro-organism and, by means of amino acid analogues such as pfluomDLphenylalanine it is possible to make (phenylalanine) feedback resistant mutants of E.di (pheA and mp mutants). The following procedure can be used: the genes are isolated from the chromosomal DNA and put into a plasmid (circular regulation of the pkAR structural gene in the plasmid is altered by using a strong promoter (several very strong promoters are nowadays used, eg the lac promoter); 0 the total effect (ie overproduction of phenylalanine by deregulation) can be enhanced by using more than one pheAFR genes in the plasmid, in a so called multicopy recombinant. leedbadc resistant mutants d use of plasma Dm extrachromosomal piece of DNA); In a good production strain, the plasmid should be pmnt in a stable way and should not be lost from the micro-organism after a few generations. The pdure described above is just one way to get to a good phenylalanine n production strain. Briefly outline another way to achieve the goal. The most obvious alternative approach is to deregulate the mF gene, which is subject to tyrosine regulation. This, of course, could be achieved by DNA-recombinant techniques or by mutation. Provide brief explanations for the following 1) Wild strains of E. coli are not used for L-phenylalanine production by direct 2) Auxotrophic mutants of E. coli are particularly useful for the production of fermentation. L-phenylalanine by direct fermentation. E. wZi. 3) Regulatory mutants improve the rate of L-phenylalanine overproduction by 4) An E. coli mutant, Tv-, is likely to enhance L-phenylalanine overproduction. 8.4.3 Methods of fermentation The growth of micro-organisms used in the production of amino acids is done in a well balanced environment. The conditions required are: a controlled pH of the fermentation medium (approximately neutral); richgrowthmedia; highly aerobic conditions; sterile conditions. fermenta~on conditions
Industrial production of amino acids by fermentation and chemo-enzymatic methods 245 Slight changes in the fermentation conditions can greatly affect amino acid production. These variations are sometimes caused by changes in sterilising conditions, agitation and aeration, temperature, PH, pressure and liquid level. This is why the parameters have to be controlled in a proper way using precise equipment. We can differentiate between three possible methods of amino acid fermentation batch fermentation fed-batch fermentation continuous fermentation 1)Batch fermentation Batch fermentation is the most widely used method of amino acid production. Here the fermentation is a closed culture system which contains an initial, limited amount of nutrient. After the seed inoculum has been introduced the cells start to grow at the expense of the nutrients that are available. a short adaptation time is usually necessary Cag phase)before cells enter the logarithmic growth phase(exponential phase) Nutrients soon become limited and they enter the stationary phase in which growth has (almost)ceased. In amino acid fermentations, production of the amino acid normally starts in the early logarithmic phase and continues through the stationary phas hort For economical reasons the fermentation time should be as short as possible with a high fermentaton yield of the amino acid at the end. a second reason not to continue the fermentation in ime preferable the late stationary phase is the appearance of contaminant-products, which are often difficult to get rid off during the recovery stage In general, a relatively short lag phase helps to achieve this. The lag phase can be shortened by using a hig her concentration of seed inoculum. The seed is produced by growing the production strain in flasks and smaller fermenters. The volume of the seed inoculum is limited as a rule of tumb normally 10% of the fermentation volume, to prevent dilution problems 2)Fed-batch fermentation Fed-batch fermentations are batch fermentations which are fed continuously or intermitantly, with medium without the removal of fluid. In this way the volume of the culture increases with time low residual One of the advantages of the fed-batch fermentation is the fact that the residual substrate substrate concentration may be maintained at a very low level. This may rest ult concen taton removal of catabolite repressive effects and avoidance of toxic effects of medium OrDO Another advantageous effect is on the oxygen balance. The feed rate of the carbon oxygen demand source (mostly glucose) can be used to regulate cell growth rate and oxygen imitation, 3)Continuous fermentation es of In general, there are several advantages of continuous fermentation as compared contnuous batch fermentations. These include higher productivity, operation for a very long period of time, and lower installation and maintenance costs. The maintenance costs are particularly important In batch cultures, oxygen demand, pHcontrol requirements and amount of cooling required, changes throughout the whole fermentation run, whilst in continuous fermentations these factors are constant
Industrial produdion of amino acids by fermentation and chemo-enzymatic methods 245 methods of amino add fermentation short fermentation time preferable bw residual substrate concentration control of oxygen demand advantages of continuous fermentation Slight changes in the fermentation conditions can greatly affect amino acid produdion. These variations are sometimes caused by changes in sterilising conditions, agitation and aeration, temperature, pH, pressure and liquid level. This is why the parameters have to be controlled in a proper way using precise equipment. We can differentiate between three possible methods of amino acid fermentation: batch fermentation; fed-batch fermentation; continuous fermentation. 1) Batch fermentation Batch fermentation is the most widely used method of amino acid production. Here the fermentation is a closed culture system which contains an initial, limited amount of nutrient. After the seed inoculum has been introduced the cells start to grow at the expense of the nutrients that are available. A short adaptation time is usually necessary (lag phase) before cells enter the logarithmic growth phase (exponential phase). Nutrients soon become limited and they enter the stationary phase in which growth has (almost) ceased. In amino acid fermentations, production of the amino acid normally starts in the early logarithmic phase and continues through the stationary phase. For economical reasons the fermentation time should be as short as possible with a high yield of the amino acid at the end. A second reason not to continue the fermentation in the late stationary phase is the appearance of contaminant-products, which are often difficult to get rid off during the recovery stage. In general, a relatively short lag phase helps to achieve this. The lag phase can be shortened by using a higher concentration of seed inoculum. The seed is produced by growing the production strain in flasks and smaller fermenters. The volume of the seed inoculum is limited, as a rule of turd normally 10% of the fermentation volume, to prevent dilution problems. 2) Fed-batch fermentation Fed-batch fermentations are batch fermentations which are fed continuously, or intermitantly, with medium without the removal of fluid. In this way the volume of the culture increases with time. One of the advantages of the fed-batch fermentation is the fact that the residual substrate concentration may be maintained at a very low level. This may result in a removal of catabolite repressive effects and avoidance of toxic effects of medium components. Another advantageous effect is on the oxygen balance. The feed rate of the carbon source (mostly glucose) can be used to regulate cell growth rate and oxygen limitation, especially when oxygen demand is high in the exponential growth phase. 3) Continuous fermentation In general, there are several advantages of continuous fermentation as compared to batch fermentations. These include higher productivity, operation for a very long period of time, and lower installation and maintenance costs. The maintenance costs are particularly important. In batch cultures, oxygen demand, pH control requirements and amount of cooling required, changes throughout the whole fermentation run, whilst in continuous fermentations these factors are constant