《化学合成中生物技术的革新》（英文版） 5 The large scale production of organic acids by mIcro-organIsms.pdf_大学文库

116 Chapter 5 The large scale production of organic acids by micro-organisms Overview In this chapter we shall look at the use of micro-organisms to produce organic acids of ommercial importance. Although all of the examples to be mentioned are relatively imple chemically, they are interesting in that they are metabolically diverse. Some are genuine end products of metabolism, while others are compounds considered to be entral metabolites in all living cells. These central metabolites are normally present in relatively small, constant amounts. However, some micro-organisms can b persuaded"to produce enormous yields of these metabolites The first part of this chapter the Introduction, will identify some of the organic acids roduced by micro-organisms and highlight those which are of commercial interest. The products considered in this chapter are metabolites of the tricarboxylic acid (tCa) cycle or oxidative derivatives of glucose. Since most of the biological commerical processes involve interference with the metabolism of micro-organisms, we present a section discussing relevant pathways together with the control mechanisms involved The industrial production of the most commercially important organic acid, citric adid, is then considered in depth. Finally, we outline the biochemistry, formation and downstream processing of other TCA cycle intermediates(malic acid, fumaric acid) and oxidation products of glucose(itaconic acid, gluconic acid and its derivatives) 5.1 Introduction organic acid We must first indicate exactly what is meant by the term'organic acid in the context of efiniton this Chapter. Inevitably it will be far more restricted in scope than the literal definition, which essentially means'any organic compound which is acidic adids whig ve one or more examples in each of the categories below of organic 1) Acids of non-carbohydrate origin which are produced by all living systems 2) Acids continuously produced by all living systems 3) Acids of carbohydrate origin which are constantly produced by living systems and are not considered as waste products. 4)Acidic examples of continuously produced waste products Acceptable answers to part 1)include amino acids and fatty acids or specific examples of each, such as glycine or stearic acid respectively. The obvious answer for part 2)is the central metabolite Pyruvate, though all of the acids of the TCA cycle would be appropriate Answers to part 3)include the principal acid of the hexose monophosphate

116 Chapter 5 The large scale production of organic acids by m icro-org an isms Overview In this Chapter we shall look at the use of mim~~rganisms to produce organic acids of commercial importance. Although all of the examples to be mentioned are relatively simple chemically, they are interesting in that they are metabolically diverse. Some are genuine end products of metabolism, while others are compounds considered to be central metabolites in all living cells. These central metabolites are normally present in relatively small, constant amounts. However, some micro-organisms can be "persuaded" to produce enormous yields of these metabolites. The first part of this Chapter, the Introduction, will identdy some of the organic acids produced by micro-organisms and highlight those which are of commercial interest. The products consided in this chapter are metabolites of the tricarboxylic acid UCA) cycle or oxidative derivatives of glucose. Since most of the biological coITLmerical processes involve interference with the metabolism of micro-organisms, we present a section discussing relevant pathways together with the control mechanisms involved. The industrial production of the most commercially important organic acid, citric acid, is then considered in depth. Finally, we outline the biochemistry, formation and downstream processing of other TCA cycle intermediates (malic acid, fumaric acid) and oxidation products of glucose (itaconic acid, gluconic acid and its derivatives). 5.1 Introduction organic acid definmon We must first indicate exactly what is meant by the tern 'organic acid' in the context of this Chapter. Inevitably it will be far more restricted in scope than the literal definition, which essentially means 'any organic compound which is acidic'. n acids which are produced by living cells? 1) Acids of non-carbohydrate origin which are produced by all living systems. 2) Adds continuously produced by all living systems. 3) Acids of carbohydrate origin which are constantly produced by living systems and are not considered as waste products. 4) Acidic examples of continuously produced waste products. Amptable answers to part 1) include amino acids and fitly acids or specific examples of each, such as glycine or stearic acid mpectively. The obvious answer for part 2) is the central metabolite pyruvate, though all of the acids of the TCA cycle would be appropriate. Answers to part 3) include the principal acid of the hexose monophosphate Can you give one or more examples in each of the categories below of organic

The large scale production of organic acids by micro-organisms 117 pathway, 6-phosphogluconate, and the acid intermediate of glycolysis, 1 3-diphosphoglycerate. Answers to part 4)include an enormous number of acids since all living systems produce acid end products. However, in the context of this chapter the waste products produced by bacteria growing anaerobically are particularly relevant. these include: lactate oancea d in great quantity by the lactic acid bacteria; lactate, acetate, formate and succinate produced by the enterobacteriaceae butyrate and acetate produced by Clostridium species There are many more correct answers to each part of the question. To simplify matters, all of the answers are compounds which are acidic because they contain the carboxyl group(COOH). This chapter does not consider any organic acids which are organie compounds made acidic by the presence of, for example, phosphate or sulphate groups. Further, to warrant discussion in this Chapter, an organic acid has to satisfy the following criteria: there has to be a micro- organisms which will produce it in commercially significant there has to be a demand for the compound industrially the overall costs of producing and extracting the acid have to be economic In metabolic terms there are three clearly distinguishable types of compound to deal with. Firstly, compounds which are obviously waste products-end products of one or more pathways which would normally be excreted from the cell (for example lactic cid). Secondly, compounds which are end products of pathways but which are not waste products and whose synthesis is normally very carefully controlled (for example amino acids). Thirdly, compounds which are intermediates of pathways and hence not normally considered as end products or wastes at all (for example citric acid) 近比 seem easier if wean des it organic acids y micro-organisms would, on the face than non-waste compounds such as central metabolites. It is merely a technical problem to encourage certain bacteria to produce a waste product such as lactate as this mea pound is normally excreted into the surrounding medium. The removal of spent medium regularly and harvesting of lactate could allow continuous production of lactate. However production of metabolites such as amino acids is more complex and to obtain sufficient quantities of intermediate compounds such as citric acid is even more of a problem. The key problem is how to encourage a micro-organism to produce a vast excess of an organic acid whose synthesis is normally controlled very efficiently manipulation of at relatively low concentration. a detailed discussion of this problem will occur later, metabolic but we can generalise here and identify the four main ways in which metabolic pathways pathways can be manipulated by altering the environmental conditions, eg temperature, pH, medium composition pecially the elimination of ions and cofactors considered essential for particular by disrupting a pathway using substrate analogues; by mutation- giving rise to mutant organisms which may only use part of a metabolic pathway or regulatory mutants, by genetic engineering. It should be noted that apart from a passing reference to natural selection of wild-types with enhanced specific properties, the genetics of organic acid producing micro-organisms is beyond the scope of this chapter

The large scale production of organic acids by micro-organisms 117 pathway, 6-phosphogluconate, and the acid intermediate of glycolysis, 1,Wphosphoglycerate. Answers to part 4) include an enomus number of acids since all living systems produce acid end products. However, in the context of this chapter the waste products produced by bacteria growing anaerobically are particularly relevant These include: lactate, produced in great quantity by the lactic acid bacteria; lactate, acetate, formate and succinate produced by the EnterobacteriaCaae; butyrate and acetate produced by Clostridium species. There are many more correct answers to each part of the question. To simplify matters, all of the answers are compounds which are acidic because they contain the carboxyl group (-COOH). This chapter does not consider any organic acids which are organic compounds made acidic by the presence of, for example, phosphate or sulphate groups. Further, to warrant discussion in this Chapter, an organic acid has to stisfy the following criteria: 0 there has to be a micrmrganisms which will produce it in commercially significant quanti ties; 0 there has to be a demand for the compound industrially; 0 the overall costs of producing and extracting the acid have to be economic. In metabolic terms there are three clearly distinguishable types of compound to deal with. Firstly, compounds which are obviously waste products - end products of one or more pathways which would normally be excreted from the cell (for example lactic acid). Secondly, compounds which are end products of pathways but which are not waste products and whose synthesis is normally very carefully controlled (for example amino acids). Thirdly, compounds which are intermediates of pathways and hence not normally considered as end products or wastes at all (for example citric acid). Production of large quantities of organic acids by micrmrganisms would, on the face of it, seem easier if we are dealing with acids which are genuine waste products rather than non-waste compounds such as central metabolites. It is merely a technical problem to encourage certain bacteria to produce a waste product such as lactate as this compound is normally excreted into the surrounding medium. The removal of spent medium regularly and harvesting of lactate could allow continuous production of lactate. However, production of metabolites such as amino acids is more complex and to obtain sufficient quantities of intermediate compounds such as citric acid is even more of a problem. The key problem is how to encourage a micrmrganism to produce a vast excess of an organic acid whose synthesis is normally controlled very efficiently at relatively low concentration. A detailed discussion of this problem will occur later, but we can generalis here and identify the four main ways in which metabolic by altering the environmental conditions, eg temperature, pH, medium composition (especially the elimination of ions and cofactors considered essential for particular enzymes); by disrupting a pathway using substrate analogues; by mutation - giving rise to mutant organisms which may only use part of a metabolic pathway or regulatory mutants; 0 by genetic engineering. It should be noted that apart from a passing reference to natural selection of wild-types with enhanced specific properties, the genetics of organic acid producing micmrganisms is beyond the scope of this chapter. end Pro*& and intermediates manipulation of memblic pathways pathways can be manipulated:

The large scale production of organic acids by micro-organisms 119 You should note that the figure in SAQ 5. 1 is a simple outline as fermentations generall have more steps than indicated; for example many have a multiple purification step. If the product were the whole cell for example in single cell protein processes)then intracellular compound then some stage of cell ary. if the required product were ification of the cell biomass would be leakage would be essential 5.1.2 Organic acids relevant to this Chapter Table 5.1 shows the organic acids relevant to this Chapter together with the usual substrate, the micro-organisms employed to produce them and finally the potential end uses Such acids are often grouped into two broad categories, those which are members of or related to the tCa cycle and secondly those which are oxidation products of glucose the carbon source for the latter group is usually quite specific, either glucose itself or a polysaccharide which yields glucose easily. The carbon source for the former group can be much more diverse and/or example: glucose or its poly (molasses, starches); byproducts of industry(methanol, methane); waste products of ndustry (sulphite waste liquor from paper manufacture) or plant waste (lignins, cellulose derivatives). Acetate lactate and some amino acids however do not readily fit into either of the group organic acid substrate producer end use(s) micro-oro citric acid s) Aspergillus niger Flavouring for beverages and confectionery. Pharmaceutical food syrups, resins, dye mordants, antifoaming agents, sequestering ager malic acid ucose Lactobacillus brevis Food and drink manufacture Rhizopus delemar Used in the plastics industry and to a lesser extent. in the food industry ediate for organic illus spp syntheses, eg acrylic resins gluconic acid glucose Aspergillus niger Pharmaceutical industry and as a washing and softening agent preventing a build up of ethanol lactic acid lactose Lactic acid bacteria Dairy industry as a preservative/flavour enhancer Table 5. 1 Example of organic acids produced commercially by micro-organisms; organic acie considered in this chapter are labelled with. A related acid, a-oXoglutaric acid, is easy to produce microbiologically but has no current end use; succinic acid is produced chemically. Amino acids are beyond the scope of this chapter a detailed study of the amino acids is beyond the scope of this Chapter However, industrial production of amino acids is considered in Chapter 8 of this text

The large scale produdion of organic acids by microorganisms 119 You should note that the figure in SAQ 5.1 is a simple outline as fermentations generally have more steps than indicated; for example many have a multiple purification step. If the product were the whole cell (for example in *e cell protein processes) then purification of the cell biomass would be necessary. If the required product were an intracellular compound then some stage of cell breakage would be essential. 5.7.2 Organic acids relevant to this Chapter Table 5.1 shows the organic acids relevant to this Chapter together with the usual substrate, the miao-organisms employed to produce them and finally the potential end uses. Such acids are often grouped into two broad categories, those which are members of or related to the TCA cycle and secondly, those which are oxidation pducts of glucose. The carbon source for the latter group is usually quite specific, either glucose itself or a polysaccharide which yields glucose easily. The carbon source for the former group can be much more diverse and/or complex, for example: glucose or its polymers (molasses, starches); byproducts of industry (methanol, methane); waste products of industry (sulphite waste liquor from paper manufacture) or plant waste (lignins, cellulose derivatives). Acetate, lactate and some amino acids however do not readily fit into either of the groups. organic acid substrate producer end use(s) micro-organism citric acid sugar@) Aspergillus niger malic acid glucose Lactobacillus brevis fumaric acid glucose Rhizopus delemr itaconic acid glucose Aspergillus terreus gluconic acid glucose Aspegillus nger other Aspergillus spp Gluconobacter suboxidans acetic acid ethanol Acetobacter aceti lactic acid lactose Lactic acid bacteria Flavouring for beverages and confectionery. Pharmaceutical food syrups, resins, dye mordants, antifoaming agents, sequestering agents. Food and drink manufacture Used in the plastics industry and, to a lesser extent, in the food industry Intermediate for organic syntheses, eg acrylic resins Pharmaceutical industry and as a washing and softening agent preventing a build up of scale. Retards setting of building materials. Vinegar, food industry Dairy industry as a presewativenlavour enhancer Table 5.1 Example of organic acids produced commercially by micro-organisms; organic acids considered in this chapter are labelled with *. A related acid, a-oxoglutaric acid, is easy to produce microbiologically but has no current end use; succinic acid is produced chemically. Amino acids are beyond the scope of this chapter. A detailed study of the amino acids is beyond the scope of this Chapter. However, industrial production of amino acids is considered in Chapter 8 of this text

Chapter 5 5.2 Metabolic pathways and metabolic control mechanisms 5.2.1 Revision of the reactions of the tricarboxylic acid(TCA)cycle a detailed revision of the TCA cycle is necessary to ensure an understanding of the mechanisms and reasons governing the choice of process conditions for encouraging production of any selected TCA cycle intermediate or related compound. Descriptions of the cycle can be found in many text books, for example in the open learning BIOTOL text entitled Principles of Cell Energetics. Chapter 7 of that text describe in great detail the TCa and glyoxylate cycles, both of which are relevant to this Chapter. The most relevant parts of the cycle are the control mechanisms and processes involved in the intermediary metabolism; these are influenced and exploite efforts to upset the balance of normal metabolism leading to overproduction of the desired organic acid Let us first examine Figure 5.1. It is worth spending some time on this in order to understand the rationale of the remainder of this Chapter glycolysis Glycolysis(the Embden Meyerhof pathway)is a ten enzyme pathway which is summarised in Figure 5.1. During the course of this pathway, glucose is cleaved to two pyruvate molecules-a process involving the utilisation of two aTP(generating two ADP)but later there is formation of four ATP from four ADP. thus a net yield of two ATP is achieved. Another consequence of glycolysis is the reduction of two molecules of nicotinamide adenine dinucleotide (NAD)to NADH H. Although the exact mechanism of the individual reactions of glycolysis is not really necessary for this Chapter, we must bear in mind the fact that during the process energy and reducing power are formed. The two reactions indicated by *are two further enzymatic steps which, along with three reactions of the TCA cycle, constitute the glyoxylate cycle In the reaction that bridges glycolysis and the TCA cycle, for each pyruvate degraded to acetyl CoA, one Coz is released and a further nad is reduced to nadh+ A variety of starting materials other than glucose or its derivatives is possible for use by some micro-organisms; the four shown in Figure 5.1 are all initially converted to acetyl CoA for entry into the central metabolic pathways

120 Chapter 5 5.2 Metabolic pathways and metabolic control mechanisms 5.2.1 Revision of the reactions of the tricarboxylic acid (TCA) cycle A detailed revision of the TCA cycle is necessary to ensure an understanding of the mechanisms and reasons governing the choice of process conditions for encouraging production of any selected TCA cycle intermediate or related compound. Descriptions of the cycle can be found in many text books, for example in the open learning BIOTOL text entitled 'Principles of Cell Energetics'. Chapter 7 of that text describe in great detail the TCA and glyoxylate cycles, both of which are relevant to this Chapter. The most relevant parts of the cycle are the control mechanisms and processes involved in the intermediary metabolism; these are influenced and exploited in efforts to upset the balance of normal metabolism leading to overproduction of the desired organic acid. Let us first examine Figure 5.1. It is worth spending some time on this in order to understand the rationale of the remainder of this Chapter. Glycolysis (the Embden Meyerhof pathway) is a ten enzyme pathway which is summarised in Figure 5.1. During the course of this pathway, glucose is cleaved to two pyruvate molecules - a process involving the utilisation of two ATP (generating two ADP) but later there is formation of four ATP from four ADP. Thus a net yield of two ATP is achieved. Another consequence of glycolysis is the reduction of two molecules of nicotinamide adenine dinucleotide (NAD') to NADH i H'. Although the exact mechanism of the individual reactions of glycolysis is not really necessary for this Chapter, we must bear in mind the fact that during the process energy and reducing power are formed. The two reactions indicated by * are two further enzymatic steps which, along with three reactions of the TCA cycle, constitute the glyoxylate cycle. In the reaction that bridges glycolysis and the TCA cycle, for each pyruvate degraded to acetyl &A, one COZ is released and a further NAD+ is reduced to NADH + H+. gtycobsis A variety of starting materials other than glucose or its derivatives is possible for use by some micro-organisms; the four shown in Figure 5.1 are all initially converted to acetyl CoA for entry into the central metabolic pathways

anabolism Anabolism is the building up or biosynthesis, of complex molecules such as protein, nucleic acids and polysaccharides, from raw materials originating from intra-or extracellular sources. The biosyntheses are energy (aTP)requiring processes. Catabolism and anabolism have to be carefully regulated and are inevitably intimately t are the three areas where the processes of catabolism and anabolism are Catabolism produces ATP, reducing power and intermediates. Anabolism requires all three, thus these are the three main links. No living cells can store large amounts of ATP. There is a finite amount of adenine distributed between AMP, ADP and ATP. Thus if the cell has a relatively high concentration of atP, the concentrations of AMP and/or ADP must be lowered. The balance alters like a"see-saw", as one goes up the other must come down. In addition the total amount of nad/NADH and NadP/NAdPH in the cell is constant. What is the advantage of the see-saw type of change to the ratio of the The answer is that such a system is far more sensitive to small changes in concentration of the respective compounds. The cell is recognising a change of the ratio of compounds, rather than the rise or fall of a single compound Although cells cannot store ATP, they must always have a minimum amount available to keep them alive. Thus a constant level of ATP must be maintained indicating that catabolism and anabolism occur constantly under normal conditions ∏ One compound controls the overall metabolism of the cell regulating and balancing anabolism and catabolism. Can you name it? The answer in practice is aTP though you would have been theoretically correct if you had said ADP and AMP. Indirectly, NAd or NADH are also compounds which regulate the anabolic/catabolic balance The metabolic control is exercised on certain key regulatory enzymes of enzymes called allosteric enzymes. These are enzymes whose catalytic activity is hrough non-covalent binding of a specific metabolite at a site on the protein the catalytic site. Such enzymes may be allosterically inhibited by aTP or all activated by ATP (some by ADP and /or AMP). Thus atp is the effective controller of metabolism but because AMP ADP + AtP is constant, it is really the ratio of adenine nucleotides which is important. This ratio is energy charge termed the adenylate charge or energy charge and is expressed as Energy charge 0.5 [ADP]+ [ATPI [AMP]+[ADPI+ [ATPI

122 Chapter 5 anabolism albsteric enzymes Anabolism is the building up or biosynthesis, of complex molecules such as protein, nucleic acids and polysaccharides, from raw materials originating from intra- or extracellular sources. The biosyntheses are energy (ATP) requiring processes. Catabolism and anabolism have to be carefully regulated and are inevitably intimately linked. What are the three areas where the processes of catabolism and anabolism am n linked? Catabolism produces ATP, reducing power and intermediates. Anabolism requires all three, thus these are the three main links. No living cells can store large amounts of ATP. There is a hite amount of 'adenine' distributed between AMP, ADP and ATP. Thus if the cell has a relatively high concentration of ATP, the concentrations of AMP and/or ADP must be lowered. The balance alters like a "see-saw", as one goes up the other must come down. In addition the total amount of NAD+/NADH and NADP+/NADPH in the cell is constant. What is the advantage of the see-saw type of change to the ratio of the n concentrations? The answer is that such a system is far more sensitive to small changes in concentration of the respective compounds. The cell is recognising a change of the ratio of compounds, rather than the rise or fall of a single compound. Although cells cannot store ATP, they must always have a minimum amount available to keep them alive. Thus a constant level of ATP must be maintained indicating that catabolism and anabolism occur constantly under normal conditions. One compound controls the overall metabolism of the cell regulating and n balancing anabolism and catabolism. Can you name it? The answer in practice is ATP though you would have been theoretically corred if you had said ADP and AMP. Indirectly, NAD+ or NADH are also compounds which regulate the anabolic/catabolic balance. The metabolic control is exercised on certain key regulatory enzymes of a pathway called allosteric enzymes. These are enzymes whose catalytic activity is modulated through noncovalent binding of a specific metabolite at a site on the protein other than the catalytic site. Such enzymes may be allosterically inhibited by ATP or allosterically activated by ATP (some by ADP and/or AMP). Thus ATP is the effective controller of metabolism but because AMP + ADP + ATP is constant, it is really the ratio of adenine nucleotides which is important. This ratio is termed the adenylate charge or energy charge and is expressed as: 0.5 [ADP] + [ATP] [AMP] + [ADP] + [ATPI Energy charge =