Bioprocess Technology- Fundamentals and Applications Chapt 4_Metabolic basis

Reprint from: Enfors-Häggström, Bioprocess technology- Fundamentals and Applications,KTH, Stockholm, 2000

Chapter 4. METABOLIC BASIS OF PRODUCT FORMATION

The cellular metabolism is made up of a large number of reactions co-ordinated by enzymessubjected to various control mechanisms. Some of these reactions may lead to a desiredproduct. Knowledge of metabolism and physiology constitutes the basis for developingcontrol strategies in biotechnical processes. This chapter provides a framework of metabolicand physiological features relevant to microbial processes. Textbooks on biochemistry andmicrobial metabolism should be consulted for further details. Metabolism of cultured animalcells will be treated separately (chapter 17).4.1 Metabolic organisation

A simplified overview of the primary metabolism is shown in Fig. 4.1. Metabolism can bevisualised as composed of three compartments as indicated by the boxes for catabolism,anabolism and synthesis of macromolecules

.

Fig. 4.1. Primary metabolism - an overview.

The catabolic and anabolic compartments contain biochemical pathways whereas thesynthesis of macromolecules rather is the assembly of pre-made building blocks. A flow ofmaterial runs through the three compartments, eventually resulting in the production of newcells i.e. growth. Environmental changes such as changes in substrate concentration, pH andtemperature and the accumulation of waste products follow from this process.

Glycolysis:

Respiration:

ATP

Acetyl-CoA

NAD+

H O

2

TCA-cycle:

NADH

O2

ADP

CO

2

Fig 4.2. Aerobic energy metabolism- a simplified overview. The reduced energy substrate(glucose) is oxidised to pyruvate by NAD+ in a series of reactions called glycolysis. thisgenerates a small amount of the energy carrier ATP. Pyruvate is further oxidised to CO2 byco-enzymes (NAD+) in the TCA-cycle. The reduced coenzymes (NADH) are re-oxidised (NAD+ ) in the respiratory chain, where molecular oxygen is the ultimate receiver of theelectron. This oxidation is coupled to generation of a relatively large amount of ATP.

In anaerobic respiration, nitrate, sulphate or some other oxidised specie is used as electronacceptor instead of molecular oxygen. In fermentative energy metabolism, the NADH fromthe glycolysis is reoxidised by reduction of pyruvate to fermentation products like ethanol,lactate etc. (Fig 4.6-4.7)

The entrance of substrates into the catabolic compartment is the starting point for all theseactivities. The catabolic compartment contains the central metabolic pathways i.e. glycolysis,the hexose monophosphate shunt (HMS) and the pentose cycle, the tricarboxylic acid cycle(TCA) as well as respiration and the generation of the energy carrier ATP (Fig. 4.2). (Inaddition to the aerobic heterotrophic metabolism - other types of energy metabolism likeaerobic autotrophic, anaerobic fermentation, anaerobic respiration and photosynthesis occurin microorganisms.) NAD+/NADH are, with few exceptions, recirculated within thecatabolic compartment. Intermediate metabolites from the catabolic pathways constitute theprecursors for biosynthesis of building blocks in the anabolic compartment. Catabolism alsofurnishes the reducing power (NADPH) required for biosynthesis.

Anabolism is the synthesis of building blocks used for macromolecule synthesis. Here, thewell known pathways for amino acid and nucleotide synthesis are harboured as well as thesynthesis of fatty acids and sugar moieties. Finally, these building blocks are assembled intomacromolecular constituents of the cell like DNA, RNA, protein, membrane lipids and cellwalls.

Products produced in biotechnical processes may originate from any of the three metaboliccompartments in Fig. 4.1, or constitute the result of them all as in the production of bakersyeast or clean water in a sewage plant. The characteristics of product formation in themetabolic compartments will be described later in sections 4.6, 4.7 and 4.8. However, the

metabolism in the three individual compartments is regulated according to differentprinciples. It is important to understand the metabolic regulatory mechanisms since i) thesemechanisms must sometimes be circumvented to achieve overproduction of certainmetabolites, and ii) they furnish the basis for the microbial physiology which, in variousindirect ways, influence the performance of the whole process.4.2 Metabolic regulation

Figure 4.3 indicates the different regulatory principles within each metabolic compartment.These principles will be described in sections 4.3, 4.4 and 4.5. During growth, the metabolicactivities are regulated in a highly co-ordinated manner to ensure maximum economical useof substrates for maximum growth rates during the prevailing conditions. As visualised inFig. 4.3, it is the rate with which energy, precursors and building blocks flow through thesystem that determines the overall reaction rate (growth rate), which in turn is determined bythe growth rate supporting ability of the medium. Nature has evolved metabolic systems thatappear to have over capacity, in terms of enzyme capacity relative to substrate concentration.This means that the metabolic network often is subsaturated with substrate and responds to

increases in substrate concentration and substrate quality with an increase in reaction rate.

Fig. 4.3. Principles of regulation of metabolism.

The input rate of components into the anabolic compartment limits the biosynthesis rate.There is, however, not a very strong feed-back from anabolism to catabolism. Even if growthceases and anabolism is shut off, the catabolism, although reduced, is never shut off.Catabolism must always be running in order to furnish the cell with energy for maintenancepurposes. If the energy metabolism ceases or the energy charge falls below a critical valuethe cell will die. One exception to this is endospore formation or other forms of resting stageformation.

The rate of macromolecule synthesis is also limited by the input rate of building blocks,already determined by the events in the catabolic compartment. Although evolution hasadjusted the capacities in various metabolic sequences to each other there must also exist

mechanisms that compensate for unbalances in the flow of materials. To avoid wastefuloverproduction of building blocks the synthesis is regulated in relation to the consumptionfor macromolecular synthesis. These mechanisms are well understood and it is also easy toimagine that overall protein synthesis rate is limited by the availability of amino acids. Buthow is then the synthesis of the various macromolecular species adjusted to fit each other andthe growth rate? Recent research give by hand that a mechanism, since long known as the"stringent response" and originally thought of as a shock response to amino acid starvation,has a much broader range of action and, in fact, might be a growth rate regulating signal, asfurther described in section 4.5.

Which are then the metabolic bottlenecks? Generally, the transport of a key substrate into thecell is regarded as the growth rate limiting reaction. This is clearly recognised duringmicrobial growth in a minimal medium with glucose as the sole carbon and energy source.On the other hand, if the same microbe is cultivated in a complex medium containing pre-made building blocks (amino acids, nucleotides) and reaches a much higher growth rate it isno longer obvious which is the growth rate limiting reaction. In fact, during such conditionsthe growth rate may approach the maximum DNA replication rate, or rather the minimumtime between two consecutive initiations of replication.4.3 Catabolic regulation.

4.3.1 Aerobic energy metabolism in aerobic and facultative organisms.

The central metabolic pathways (HMS, glycolysis, TCA-cycle) are made up of constitutiveenzymes which always are present in the cell. (In facultative organisms like E. coli or S.cerevisiae transition from aerobic to anaerobic conditions shuts off respiration and the TCA-cycle.) Regulation of the activities of individual enzymes in these pathways, leading to co-ordination of rates between them and in response to the cellular energy demand are wellknown and will not be considered here.

Microorganisms can use many different substrates which feed into the central metabolism atvarious points. Each substrate has a specific transport system and specific initial catabolicenzymes that convert it to an intermediate of the central pathways. The various substrates aretransported into the cell and enter the metabolism with different easiness and are not equallyefficient for energy production. A microorganism in Nature is often simultaneously exposedto a variety of substrates that can be used as carbon and energy sources. To guaranty that themost efficient substrate is metabolised first, intricate mechanisms exist that regulate transportand initial catabolism. E. coli will be used as an example to illustrate such features.4.3.2 The phosphotransferase system of E. coli.

A number of carbohydrate carbon and energy sources including glucose are transported intothe E. coli cell by the phosphoenolpyruvate: sugar phosphotransferase system (PTS), aso called "group translocation" transport system (Fig. 4.4). Phosphorylation of glucose toglucose-6-P occurs during transport through the inner cell membrane. The phosphate group isderived from phosphoenolpyruvate (PEP) which in turn is converted to pyruvate (PYR). Anumber of enzymes participate in the transfer of phosphate from PEP to glucose or to othercarbohydrates. Two of these enzymes (Enz I and Hpr) are common to all PTS-sugars while

each sugar has its specific membrane bound permease (Enz II) and some sugars also aspecific soluble enzyme which is the last link between Enz II and PEP. For glucose, thissoluble enzyme is called Enz IIIglc. Enz IIIglc has turned out to have a central regulatory rolefor the metabolism of non-PTS-sugars i.e. sugar substrates that are transported by othertransport systems.

Fig. 4.4 The phosphoenolpyruvate: sugar phosphotransferase system of E. coli. Enz I= Enzyme I; Hpr = Heat stable protein; Enz IIIglc = Enzyme III, soluble enzyme,specific for glucose; Enz IIglc = Enzyme II, membrane bound glucose specificpermease.

In E. coli the PTS-system transports among others glucose, fructose, mannose, mannitol. ThePTS-sugars are superior to other sugar substrates in sustaining rapid growth. In otherbacterial species other sugars can be transported by the PTS-system. The PTS-system isabundant in anaerobic as well as facultative bacteria. Strictly aerobic bacteria usepredominantly hexokinase for phosphorylation of hexoses in combination with other types oftransport systems (e.g. symports or active transport).

The metabolic consequence of the PTS-system is indicated in Fig.4.5. For every molecule ofglucose (or another PTS-sugar) transported into the cell - one molecule of PEP is inevitablyconverted to PYR and thus withdrawn from glycolysis and HMS.

If a product produced in a biotechnical process requires precursors from that part of themetabolism it means that the maximum theoretical yield from glucose may be less than fromother substrates, not transported by the PTS system. This is the case for certain amino acidslike the aromatic ones and those originating from oxaloacetate. (In E. coli the anapleroticreaction supplying oxaloacetate uses PEP, not PYR as is the case in eukaryotic cells.) In suchcases it might be worthwhile to investigate whether non-PTS-sugars (e.g. glycerol or xylose)can be used in the process.

Fig. 4.5 Interaction between PTS and initial metabolism.

4.3.3 Regulation of transport and initial catabolism by PTS.

All carbohydrate catabolic operons in E. coli, including those of the PTS-system, areregulated by induction of the substrate and by cAMP via its receptor protein. These operonsusually contain genes for a membrane bound permease and the initial catabolic enzymesnecessary for conversion of the substrate to an intermediate of the central metabolicpathways. For maximum transcription intracellular inducer and a certain concentration ofcAMP is required.

The PTS system regulates the transcription of catabolic operons by two mechanisms. Firstly,it influences the formation of cAMP by activating the adenylate cyclase when PTS sugars areexhausted. This is thought to be brought about by Enz IIIglc in its phosphorylated form.Adenylate cyclase activity is also regulated by the membrane potential. When glucose or anyother PTS-sugar is present cAMP levels are low because adenylate cyclase activity is verylow. Secondly, Enz IIIglc, in its non-phosphorylated form, hinders substrates to enter the cellby binding to the permeases, making them inactive. This phenomenon is called "inducerexclusion" and has been shown for lactose, maltose, melibiose and glycerol in E. coli.By these mechanisms (and others not described here) a hierarchy of substrates exist, thepurpose of which is to ensure consumption in the most favourable order. Knowledge of suchrelationships are important e.g. in processes where complex media are used and forunderstanding waste water treatment processes.

In E. coli and S. cerevisiae, the aerobic metabolism of glucose leads to excretion of thepartially oxidised products acetic acid and ethanol respectively. This is called overflowmetabolism. It occurs when the glucose concentration exceeds a critical value and is typicalfor facultative organisms that have part of their anaerobic enzyme set up active under aerobicconditions. Thus, many but not all microorganisms exhibit some sort of overflowmetabolism. This behaviour is often detrimental to a production process because productionof ethanol and acetic acid reduce the yield of biomass and the desired product and thecompounds are toxic to the organism. It can, however, be avoided by keeping the glucoseconcentration low with the fed-batch technique (chapter 7).

Ethanol

Fig 4.6. Energy metabolism of S. cerevisiae. At low rate of glycolysis all pyruvate is

completely oxidised to COvalue, part of the pyruvate is reduced to ethanol. If ethanol is present and the rate of2 in the TCA cycle. When the glycolysis rate exceeds a criticalglycolysis is below the critical value, ethanol is consumed and converted to acetylCoA viaacetate.

PDC: pyruvate decarboxylase, ADH : alcohol dehydrogenase, AldDH: aldehydedehydrogenase, ACoAS: Acetylcoenzyme A synthase, PDH: pyruvate dehydrogenase.As phenomena, the overflow metabolism of E. coli and S. cerevisiae is very similar althoughfrom a metabolic regulation point they may be very different. In none of the cases is theregulatory mechanisms behind the overflow metabolism known.

In S. cerevisiae, overflow metabolism, i.e. ethanol formation under aerobic conditions, startswhen the rate of glycolysis becomes higher than corresponding to an observed maximum rateof respiration. This overflow metabolism sets on when the glucose concentration exceeds

60% of the maximal growth rate. The so called bottleneck for the electron flow frompyruvate to the molecular oxygen is unknown. It is not the capacity of the respiration per se,but may be some reaction in the TCA-cycle (see Fig 4.6).

Ethanol production is a means to dispose of the surplus carbon when the metabolismdownstream the pyruvate can not keep up with the high rate of pyruvate formation forced onthe cell by a high glucose concentration. Concomitantly some of the pyruvate (about 10% ofethanol amount) is secreted as acetaldehyde and acetate. The additional glycolysiscontributed by the overflow metabolism provides extra ATP in addition to that obtained fromglucose going through to respiration. This extra energy supply is used for increasing thegrowth rate. A mathematical description of these reactions are given in the section of fed-batch technique ( chapter 7).

The overflow metabolism of yeast is sometimes referred to as the glucose effect. However, itmust be distinguished from the original meaning of the glucose effect, that was used as asynonym to the Crabtree effect, which is catabolite repression (i.e. on enzyme synthesislevel) of the respiration, that takes place during long-term exposure of the cell to high glucoseconcentration. The overflow metabolism is observed within seconds after exposure of the cellto high glucose concentration and it is therefore sometimes also referred to as the short-termglucose effect.

In E. coli the overflow metabolism is observed as conversion of pyruvate by pyruvatedehydrogenase to acetylCoA and further to actetate that is secreted to the medium (see Fig4.6, though observe that bacteria do not have mitochondria !). A maximum respiratory rate,that is reached before the maximum glucose uptake is reached, is characteristic also for E.coli. When the rate of glycolysis is low, acetate is resorbed by the cells, if present in themedium. The regulatory explanation to acetic acid formation by E. coli is even less clear thanthe ethanol formation by yeast. It may, in fact, have several causes. Firstly, there appears tobe a lack of regulation of the glucose uptake rate at high glucose concentrations. Enz IIglc(Fig 4.4) is believed not to respond to the normal regulatory signal (the membrane potential)when saturated with glucose. Secondly, at high glucose concentrations the α-ketoglutaratedehydrogenase activity is decreased, as it is under anaerobic conditions, thus interrupting theTCA-cycle. In analogy with the situation in yeast, acetic acid production may, in terms ofenergy production, be beneficial to the bacterium. Though, different strains of E. coli vary intheir ability to form acetic acid.4.3.5 Summary.

Catabolism is regulated at the point of entrance of various substrates into the centralmetabolic pathways. These pathways are made up of constitutive enzymes whose activitiesare regulated in relation to the energy demand and the properties of the substrate. Infacultative aerobic organisms overflow metabolism results in production of partially oxidisedproducts like ethanol and acetic acid. Understanding the regulation of catabolism and energyformation is especially important in biotechnical processes because these activities furnishthe basis for the rest of the metabolism (Figs. 4.1 and 4.3).

Biosynthetic pathways leading to the formation of building blocks (amino acids, nucleotides,constituents of cell walls) for macromolecule synthesis have a defined starting point and endin the final product. The maximum flux of material through these pathways is determined bythe rate with which precursors, energy and reducing power are supplied from the catabolism.To avoid wasteful overproduction of certain metabolites, accumulated end products inhibittheir own synthesis. Such phenomena are known as feed-back inhibition of enzyme activitiesand repression of enzyme synthesis. This type of regulation is typical for the biosyntheticpathways. To achieve overproduction of metabolites from the anabolic compartment it isnecessary to overcome the regulatory mechanisms that normally limit their production.Examples of such processes will be described below (section 4.7).

4.5 Regulation of synthesis of macromolecules

During growth in sufficient media (e.g. in a laboratory fermenter) the capacity of the proteinsynthesising machinery as well as the synthesis of individual proteins and othermacromolecules (membrane lipids, cell walls) is co-ordinated and adjusted to the growth rateas will be explained below. Microorganisms living in their natural habitats seldom experiencesuch situations but for short periods of time. The "normal" situation is rather characterised bya deficiency of essential nutrients. To survive transitions from feast to fast microorganismsexhibit additional mechanisms that adjust metabolism to special stress situations that mayarise. Regulatory systems with such properties affect several genes or groups of genes andare called "global regulatory systems" (Table 4.1).

Table 4.1 Global regulatory systemsMultigene systemEnvironmental stimulusNitrogen utilisationAmmonia limitationCarbon utilisationCarbon/energy limitationPhosphate utilisationPhosphate limitationStringent responseAmino acid/energy limitationHeat shock responseHeat, certain toxic agentsSOS responseUV and other DNA damaging agentsTranslation apparatusGrowth rate supporting ability of mediumOsmotic stress responseHigh osmolarityAnaerobic respirationPresence of electron acceptors other than oxygenAnaerobic fermentationAbsence of electron acceptorsAerobic responseAddition of oxygen

The regulation of genes encoding catabolic and anabolic enzymes, already discussed, are,together with all other protein encoding genes, under control of a general growth rateregulating signal that adjusts the overall protein synthesis rate to the availability of nutrients.This is effected by an increase in the cellular content of RNA polymerase, tRNA, ribosomesand protein elongation factors in relation to growth rate.

yet fully understood. However, the well known response to amino acid starvation, shown toexist in E. coli, S. typhimurium, B. subtilis (and exists probably in many other prokaryotes aswell), and named "the stringent response", may play a role in this context. Two smallmolecules, guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) areformed in response to amino acid deficiency. The pppGpp forming enzyme - pppGppsynthetase I (gene designation: relA) - is attached to ribosomes and the synthesis of pppGppis triggered when an uncharged tRNA binds to the complementary codon in the ribosomalsite A. The primary effects of the stringent response are inhibition of synthesis (transcription)of rRNA and tRNA. How (p)ppGpp mediates this response is still under debate. (p)ppGppcan however also be synthesised in response to carbon and energy deprivation. This responseis also well documented but not well characterised. It is clear though, that this route of ppGppsynthesis is independent of relA.

The physiological effects of the stringent response mediated through (p)ppGpp includereduction of the protein synthesis rate as a consequence of the reduced number of ribosomesand tRNA molecules in the cell. Furthermore, synthesis of phospholipids and cell walls arearrested concomitantly with a reduced flux in glycolysis. A number of genes are, on the otherhand, under positive stringent control i.e. the relative rate of the synthesis of these proteinsincrease. This holds for operons controlled by attenuation encoding amino acid biosyntheticenzymes. Some of the heat shock proteins including protease La (chapter 16) are also underpositive stringent control. This appears logical from a physiological point of view in that theproteases diminish unnecessary activities and at the same time supply amino acids but maybe an obstacle in processes for the production of recombinant proteins.

The stringent response functions as a shock response when the organism is deprived of anamino acid immediately adjusting the metabolism to the new situation. The stringentresponse functions also as a general mechanism that continuously senses the availability ofamino acids during different growth conditions and respond to these differences by adjustingthe basal level of (p)ppGpp in the cell. The basal level of (p)ppGpp is 10-20 times lower thanduring amino acid deficiency but it varies inversely to the growth rate. Thus, it is believedthat (p)ppGpp can act as a fine regulator of the macromolecule synthesis rate during normalgrowth conditions, but also as a complete inhibitor of the synthesis of stable RNA species(tRNA, rRNA) when an amino acid is totally unavailable.

4.6 Products from catabolism

4.6.1 Aerobic heterotrophic energy metabolism.

Products from the catabolic compartment in aerobic organisms include intermediatemetabolites from the central pathways or derivates thereof. An important example is citricacid production by Aspergillus niger. Other TCA-related substances have also beenconsidered for production in biotechnical processes. Normally such products are notoverproduced unless they are end products in overflow metabolism but are used up asprecursors for biosynthesis or converted to carbon dioxide. During citric acid productiongrowth cannot occur because this process consumes all the carbon source and yields very

little energy. Another important product is acetic acid (vinegar) obtained from Acetobactersp. Strictly aerobic acetic acid producing bacteria obtain energy from the partial oxidation ofethanol yielding acetic acid as an end product. This means that the product is spontaneouslyproduced as is the end products of anaerobic fermentations described below.4.6.2 Anaerobic fermentative energy metabolism.

A number of products that are, or have been produced in biotechnical processes are endproducts of anaerobic fermentative energy metabolism. Examples of such products includeethanol, lactic acid, butanediol and acetone-butanol.

Products of this type are spontaneously produced both in growing and non-growing cells. Therate of production increases with growth rate but the yield coefficient varies inversely to thegrowth rate. The principles of these fermentations are outlined in Fig. 4.7. The mixed acidfermentation of E. coli is also shown (Fig 4.8) because it will be triggered if oxygenlimitation occur in any process utilising E. coli (e.g. as host for a plasmid encoded product).Anaerobic fermentative energy metabolism gives a very low energy yield compared toaerobic. ATP is produced only by substrate level phosphorylation. Fermentative organismsobtain 2 ATP/glucose in glycolysis and 1-2 ATP further in other reactions associated withacid production (e.g. acetic or butyric acid). A high flux through glycolysis is required toobtain sufficient energy for growth resulting in excretion of large amounts of end products. Infacultative organisms the rate of glycolysis increases during a shift from aerobic to anaerobicconditions. This was originally observed in yeast by Pasteur and has since then been calledthe Pasteur effect. Today this phenomenon can be explained in terms of regulation of thephosphofructokinase, the rate limiting enzyme of glycolysis. This enzyme responds tochanges in the ATP concentration, or rather to the energy charge. During anaerobicconditions the energy charge tends to fall resulting in an increased enzyme activity.

The second point of importance in fermentative energy metabolism is the cofactor balance. Inglycolysis two moles of NADH are produced per mole of glucose. The cofactors must beregenerated to maintain the flux in glycolysis. In yeast and homofermentative lactic acidbacteria this is accomplished by the reduction of acetaldehyde to ethanol and pyruvate tolactate respectively (Fig. 4.7). In other cases it may be more complicated as in the mixed acidfermentation of E. coli (Fig 4.8).

Fig. 4.7 Left: Fermentation of glucose to ethanol. Right: Fermentation of glucose to lacticacid. Lactic acid bacteria (Lactobacillus spp; Lactococcus spp.) use the PTS system totransport lactose. Intracellular lactose-P is hydrolysed into galactose-P and glucose whichsubsequently is phosphorylated by hexokinase as depicted above.

Fig. 4.8 Outline of the mixed acid fermentation used by, among others, E. coli.4.6.3 Anaerobic respiration.

A number of microorganisms that normally derive energy from aerobic respiration can usenitrate as electron acceptor when oxygen is lacking (see Fig 4.2). Thus, they are facultativelyanaerobic. The most common type of anaerobic respiration is denitrification that is commonamong Pseudomonas and many other bacteria. The denitrification includes a stepwisereduction of nitrate to gaseous nitrogen :

NO3- ----> NO2- ----> NO ----> N2O ----> N2

Each step in the denitrification is coupled to ATP synthesis. The different enzymes (nitratereductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase, respectively)are differently sensitive to oxygen, which may result in incomplete denitrification and releaseof the intermediates, especially nitrite and nitrous oxide. Nitrate reduction to nitrogen gas isan important reaction in the removal of nitrogen from waste water (Chapter 19).

A small group of facultative and obligate anaerobic bacteria can use sulphate as electronacceptor and obtain energy by electron transport phosphorylation. Sulphate is reduced tohydrogen sulphide. This reaction is important because i) it contributes to the corrosion of ironand ii) hydrogen sulphide may accumulate in anaerobic digesters because sulphate is reducedin preference to carbonate, which otherwise would have yielded methane gas.

4.7 Products from anabolism

Classical examples are amino acids like lysine and glutamic acid. Vitamins and nucleotidesbelong also to this group. The aromatic amino acids tyrosine, phenylalanine and tryptophanhave long been extremely difficult to produce directly from glucose but genetic methods haveeventually made this possible.

Anabolic metabolites are never overproduced by normal microorganisms. To obtainproduction worthwhile for an industrial process the metabolic regulatory mechanisms mustbe overcome. The strong desire to produce amino acids forced research to understand thesemechanisms and to circumvent them. The principles of this regulation and the methodsdeveloped which made economical production possible will be illustrated by using thearomatic amino acids as an example.

Figure 4.9 outlines the biosynthetic pathway for tyrosine, phenylalanine and tryptophan. Thepathway starts with the condensation of erythrose-4-P and phosphoenolpyruvate to DAHP.This step is catalysed by three isoenzymes, the DAHP-synthases, which all contribute to theflux through the pathway. A series of successive reactions lead to chorismate, the lastcommon intermediate. Three individual branches then lead to the respective amino acid. Thesynthesis is regulated at the points indicated in the figure by feed-back inhibition of enzymeactivity (allosteric regulation) and repression of enzyme synthesis (transcriptional regulation).Each amino acid regulates its own branch and one of the three isoenzymes in the first step ofthe common pathway. This mechanism ensures that the common pathway will not be turnedoff as long as there is a need for one of the amino acids. Similar mechanisms exist in otherbranched biosynthetic pathways.

Classical and newer methods for abolishing the regulatory mechanisms involve: i)Auxotrophic mutants, i.e. a mutant with a block (an enzyme is deleted or otherwiseunfunctional) somewhere in a biosynthetic pathway. The regulatory enzymes are notinhibited in the auxotrophic mutant because the end product is not synthesised. Such a mutantmay overproduce the intermediate in the pathway before the metabolic block or increase theflux to other end products of a branched pathway. ii) Feed-back resistant enzymes, i.e.mutant regulatory enzymes that are insensitive to allosteric inhibition by the end product or torepression of synthesis (mutation in the repressor gene) or to both. Such a mutant willoverproduce the end product of that pathway because there are no longer any mechanismsthat recognises the cells real demand for it. iii) Gene amplification, i.e. genes encoding ratelimiting enzymes, usually the regulatory ones, are inserted on a multicopy plasmid to ensurethat enzyme levels are not rate limiting.

Fig. 4.9 Biosynthesis of the aromatic amino acids (shikimic acid pathway). Feed-backinhibition is indicated by backward arrows from the end products. PEP =phosphoenolpyruvate; E4P = erythrose-4-phosphate; DAHP = 3-deoxy-D-arabino-heptulosonate 7-phosphate; DHQ = 3-dehydroquinate; DHS = 3-dehydroshikimate; EPSP =5-enolpyruvoylshikimate-3-phosphate. aroG = DAHP synthase (phe); aroF = DAHP synthase(tyr); aroH = DAHP synthase (trp); pheA = the bifunctional enzyme chorismate mutaseprephenate dehydratase; tyr A = the bifunctional enzyme chorismate mutase prephenatedehydrogenase; trpE = anthranilate synthase.

As an example, a combination of these methods can yield an organism with the followinggenetic properties that overproduces the aromatic amino acid phenylalanine: tyr, trp,pXX/aroF, pheAFBR(FBR = feed back resistant). This organism is auxotrophic for tyrosineand tryptophan (deletions of the trp E operon and tyr A operon) a property which increasesthe flux of metabolites to phenylalanine. The strain must however be supplied with theseamino acids in order to grow. Furthermore this microorganism harbours a multicopy plasmidwhich contain genes for the tyrosine inhibited DAHP-synthase and a feed-back resistant(both allosterically and transcriptionally) prephenate dehydratase. During growth with addedtyrosine and tryptophan there will be no real overproduction of phenylalanine becausetyrosine inhibits synthesis of the plasmid encoded DAHP-synthase(tyr) and phenylalanineregulates the chromosomal DAHP-synthase(phe). When the added tyrosine (and tryptophan)is exhausted from the medium growth stops but DAHP-synthase(tyr) will be produced as aresult of relief of inhibition. This leads to enhanced production of phenylalanine sincephenylalanine itself cannot inhibit DAHP-synthase(tyr) and the chorismate mutaseprephenate dehydratase is a mutant enzyme, insensitive to regulation by phenylalanine.

4.8 Macromolecular products

Macromolecules like proteins (e.g. hydrolytic enzymes or recombinant proteins) orpolysaccharides are important products in the biotechnical industry. It is not possible to giveany general rules regarding the conditions for their production because of the different natureand metabolic affiliation of the various macromolecular species. This circumstancedistinguishes macromolecular products from metabolites derived from catabolic or anabolicpathways. For example, extracellular hydrolytic enzymes like amylases or proteases arespontaneously produced by microorganisms in response to environmental stimuli likepresence or absence of certain nutrients. Such enzymes are in fact part of the catabolicmachinery as is the intracellular enzyme β-galactosidase. The latter is of course under controlof induction and cAMP. To obtain overproduction of such an enzyme it is necessary togenetically modify the regulatory mechanisms e.g. by creating a constitutive mutant that isindependent of induction. This may also be combined with the use of a multicopy plasmidthat harbours the gene as is common practise in production of recombinant proteins. Theproduction of recombinant proteins is further governed by the chosen promoter ruling outnormal regulatory mechanisms. It is important to consider the effects of a too high copynumber or a too strong promoter on the cellular physiology because it may lead to exhaustionof precursors and energy which in turn may trigger some of the starvation responses (Table4.1). Production of recombinant proteins will further be described in chapter 16.

4.9 Secondary metabolism

The preceding sections of this chapter has dealt with the primary metabolism and productsthereof. A great number of industrially important products such as antibiotics are, however,derived from the secondary metabolism. Secondary metabolism, common in plants andmicroorganisms, is characterised by not being essential for growth. The purpose of it orbenefit from it for the organism is still under debate. Usually, secondary metabolites areproduced during the stationary phase. This depends on their formation being repressed duringgrowth by different mechanisms. These include: 1) Carbon catabolite inhibition andrepression i.e. good carbon end energy sources repress formation of enzymes in the

secondary metabolite pathways. 2) Nitrogen metabolite repression i.e. easily metabolisednitrogen sources like ammonium ions likewise repress secondary metabolite producingenzymes. 3) Repression of pathways or individual enzymes by inorganic phosphate.Metabolic regulatory mechanisms like these necessitate the use of special cultivationconditions like fed batch techniques to obtain reasonable production. The synthesis ofsecondary metabolites is further regulated by feed-back inhibition by the end product or byregulation of the synthesis of precursors used for its formation. To overcome such obstacles itis necessary to improve strains by mutation.

Secondary metabolism is not separated from the primary metabolism but, on the contrary,intimately connected to it because precursor molecules and building blocks are derived bothfrom catabolic and anabolic pathways. This network is illustrated in Fig. 4.10, which at thesame time provides a rationale for classification of antibiotics according to their origin in the

primary metabolism.

Fig. 4.10 Connection between primary and secondary metabolism. Classification of secondarymetabolites.

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