Production of commercial products by recombinant microorganisms

Citric acid
About 1 billion pounds of citric acid are produced worldwide (Table 9.1) by fermentation with the wild-type strains of a fungus, Aspergillus niger. Citric acid is used, for example, as a flavoring agent in food and drinks and to prevent oxidation and rancidity of fats and oils. In the very efficient fermentation process, up to 80% of the source sugar is converted into citric acid. Because this process, in operation since 1916, has been studied probably more than any other process in mold metabolism, according to one review, and could serve as a model for primary metabolite fermentation processes including glutamate fermentation.

 Citric acid, unlike ethanol, is not one of the typical waste products of energy metabolism, and is usually degraded completely into CO2 and water through the operation of the citric acid cycle.

Thus, the efficient conversion of sugars, such as glucose and sucrose, into citric acid by A. niger is rather unexpected, especially because mitochondria of A. niger contain all the enzymes of the citric acid cycle. The citric acid production occurs predominantly in the stationary phase, and indeed requires several unusual conditions.
(1) The medium should be strongly acidic, with the pH value between 1.6 and 2.2. (2) The sugar concentration should be very high (120 to 250 g/L). (3) The medium should be deficient inMn2+. (4) The medium should contain a high concentration of NH4+ ions.

 Extensive studies showed that under these conditions, glucose is fermented rapidly by the glycolytic pathway. Normally, the key controlling step, phosphofructokinase (PFK1), would be inhibited by citrate, to which the glycolytic product, pyruvate, is converted. However, this citrate inhibition is abolished by the presence of high concentrations of NH4+. Furthermore, very high concentrations of sugars lead to the increased level of fructose-2,6-bisphosphate, the most powerful activator of PFK1. These factors presumably contribute to increased flux through the glycolytic pathway, culminating in the runaway production of pyruvate.

 Some of the pyruvate molecules enter mitochondria and are converted into acetyl-CoA by the normal process catalyzed by pyruvate dehydrogenase. Acetyl- CoA then condenses with oxaloacetate to produce citrate. However, the flux is far higher than what can be handled by the rest of the citric acid cycle, especially because the isocitrate dehydrogenase is inhibited by glycerol, which is generated as a part of the hyperosmotic response to the high concentration of sugars. Thus, citrate is exported out of mitochondria via citrate/malate antiporter, concomitantly with the influx of malate, which is converted to oxaloacetate for the next round of citrate synthesis. The remainder of the pyruvate molecules generated through glycolysis is converted into oxaloacetate by pyruvate carboxylase, which is located in the cytosol in this group of fungi. The cytosolic oxaloacetate is then reduced to malate by cytosolic malate dehydrogenase, and malate enters mitochondria as described above.

Metabolic pathways involved in the production of citric acid by A. niger. Shaded circles represent specific transport proteins. Thus, one of the pyruvate molecules generated from glucose via glycolysis enters the mitochondrion through the ubiquitous pyruvate/H+ symporter and is converted into acetyl-CoA by pyruvate dehydrogenase. The other pyruvate molecule is converted into oxaloacetate (OAA) by the cytosolic pyruvate carboxylase. Citric acid leaves the cell via a specific carrier. It seems plausible that the carrier catalyzes the export of the protonated citric acid, thereby preventing the acidification of the cytosol from the protons that have leaked into the cytosol from a highly acidic medium.

 Until recently, however, there was almost nothing known about one area of this proposed scheme. That is the exit of citrate from the cytosol into the medium. Citrate, with its three negative charges, cannot cross membranes spontaneously, and its export into the medium is likely to be catalyzed by a transporter protein.

Furthermore, intracellular concentration of citrate is several millimolars, and the uphill export into the medium, which could contain more than 0.5 M of citrate, is likely to be catalyzed by an active, energy-consuming process.
In deed, export of citrate from A. niger was shown in 1997 to be inhibited by sodium azide and a proton conductor, carbonyl cyanide (mchlorophenyl)hydrazone (CCCP), a result consistent with the presence of an active exporter protein. (Interestingly, cultivation of A. niger in Mn2+-poor medium was necessary to activate this process, and this may be the major reason for the inhibitory effect of Mn2+ for citrate production).

 If the export substrate is the protonated form, citric acid, perhaps its extrusion may decrease the proton concentration in the cytoplasm and thereby help the survival of A. niger in the strongly acidic environment, in which proton leakage from the medium will produce the acidification of the cytoplasm.
In this speculative scheme, the production of citrate by this organism under strongly acidic conditions will therefore be a process that is physiologically significant. Alternatively, if the transporter catalyzes the flux of citrate3, the accumulation of citric acid in the medium can be explained as the result of conversion of most of citrate3 into uncharged citric acid; this flux might be facilitated by the outside-positive membrane potential, if such a potential exists.

AMINO ACID: L-GLUTAMATE

The industrial production of an amino acid, l-glutamate, dates back to 1908, when the Japanese agricultural chemist K. Ikeda discovered that l-glutamate was responsible for the characteristic taste, much appreciated in Japan, of foods cooked with dried kelp (konbu). For the first 50 years, monosodium-L-glutamate (MSG) was manufactured by expensive chemical processes, based largely on the acid hydrolysis of proteins. This hydrolysis process was costly because glutamate had to be separated from all other amino acids in the hydrolyzate.

Significant amounts of MSG were also made by chemical synthesis. This process was expensive too, because it produced a mixture of D- and L-glutamate that had to be resolved to eliminate the disomer, which is tasteless.

 A revolutionary change was introduced in 1957, when scientists at Kyowa Hakko Co. discovered a soil bacterium that excreted large amounts of Lglutamate into the medium. Similar bacteria were soon isolated by several other companies, ushering in a new industrial technology: amino acid fermentation, the production of amino acids by microorganisms. Except for ethanol, some other organic solvents, and a few vitamins, glutamate was the first organic compound produced on an industrial scale by a microbial fermentation technique. MSG is used as a flavor enhancer in very large amounts . It was also the first amino acid to be produced by the fermentation process, using wild-type strains. Because amino acids are building blocks for the assembly of proteins, their biosynthesis is usually regulated tightly so that energy and carbon are not wasted for the synthesis of needless excesses of these compounds. Thus, the overproduction of glutamate by wild-type bacteria is surprising, and the mechanism, despite much study, is not yet totally clear.

 MSG overproduction (or fermentation) requires Corynebacterium glutamicum.

Various companies isolated glutamate-producing organisms named, for example, Brevibacterium and Arthrobacter, but it now appears that all these strains are close relatives of C. glutamicum. One striking observation made in the early days of development of glutamate fermentation technology was that all these strains were naturally auxotrophic for (requiring as growth factors) biotin, and the production of glutamate required starvation for biotin (Figure).

Influence of biotin concentration on the production of glutamic acid by C. glutamicum.

NEED FOR BIOTIN STARVATION

Why is biotin starvation necessary for the excretion of glutamic acid? The major function of biotin is to serve as a prosthetic group in acetyl-CoA carboxylase, the first enzyme of fatty acid synthesis, so it was hypothesized that the excretion might be related to an increased general permeability of the cell membrane, caused by an insufficiency of fatty acids. This notion appeared to be supported by the finding that other treatments affecting the integrity of the cell membrane for example, adding to the medium a detergent, Tween-60, or a low concentration of -lactam antibiotics (the latter treatment strains the cell membrane byweakening the peptidoglycan cell wall) or limiting the amount of exogenously fed oleic acid in unsaturated fatty acid auxotrophs also led to glutamic acid production, even in the presence of excess biotin. These observations were also of practical importance because they suggested ways to stimulate glutamic acid excretion while feeding the bacteria with inexpensive carbon sources (such as molasses) that happen to be rich in biotin.

 During the period when petroleum was inexpensive, petroleum hydrocarbons were also tried as a carbon source. When scientists found that unsaturated fatty acid auxotrophs do not produce glutamic acid when petroleum hydrocarbons are present (presumably because they are easily converted to unsaturated fatty acids by the organism), they tried an alternative way of weakening the cell membrane: They constructed conditional auxotrophs for glycerophosphate so that the synthesis of phospholipids from glycerophosphate became deficient under certain conditions. Such mutants also excreted glutamate glycerophosphate became limiting. when the supply of

The results described above seemed to support the hypothesis that a leaky cell membrane is responsible for the excretion of glutamic acid.

 In the early 1990s, it was shown that C. glutamicum and its relatives produce specific exporters that function solely to excrete amino acids, such as lysine, that they also overproduce. These efflux transporters are thought to be useful when the cytoplasm becomes flooded with amino acids, possibly as a consequence of the rapid uptake and intracellular hydrolysis of peptides from the medium. The intracellular glutamate concentration is unusually high among amino acids, around 200 mM, in contrast to those of other amino acids, which are usually around a few millimolars or less. The involvement of a specific glutamate efflux transporter presumably explains the export of only glutamate, if its activity is activated by alterations leading to the straining of the cell membrane. However, this glutamate exporter still remains unidentified in spite of the knowledge of the complete genome sequence of C. glutamicum.

IS THE CITRIC ACID CYCLE TRUNCATED?

Another factor that was thought to be important in the early days of glutamate fermentation was the presence of a truncated citric acid cycle. It was felt that the secretion of very large amounts of glutamate must involve unusual features in metabolic pathways, which lead to its extensive accumulation.

The metabolic precursor of glutamic acid is -ketoglutaric acid, an intermediate in the citric acid cycle.
When the enzymes of the citric acid cyclewere first examined, glutamic acid producers were reported to contain all the enzymes of the cycle except ketoglutarate dehydrogenase. More recently, -ketoglutarate dehydrogenase was shown to be present. However, it is difficult to decide, from enzyme activities determined in vitro, how much substrate flux the enzyme is catalyzing in intact cells.

 In this connection, it is important that recent elucidation of detailed substrate flux , determined by growing the organism with 1-13C-glucose followed by the NMR based determination of the labeling pattern of products, showed that the section of the cycle after the -ketoglutarate dehydrogenase step had full activity in nonsecreting cells , and even in glutamate-secreting cells it still had about half the activity of the flux of the section preceding -ketoglutarate; that is, the citric acid cycle is not truncated in C. glutamicum, and the change in the flow of substrates through the citric acid cycle, observed during glutamate secretion, may be a result, rather than the cause, of this process. In the simplest hypothesis, we may assume that the trigger of the whole process is the activation of the glutamate exporter by straining of the membrane. This will lower the cytosolic concentration of glutamate, shifting the steady-state fluxes around -ketoglutarate in the citric acid cycle. When only insufficient amounts of oxaloacetate are generated at the end of the cycle because of the siphoning off of a large amount of -ketoglutarate, the replenishing (or anaplerotic) reactions, generating C4 compounds from C3 compounds (pyruvate and phosphoenolpyruvate) become important. Indeed, the overproduction of pyruvate carboxylase, which catalyzes this reaction, was reported to improve the yield of glutamic acid.

 Regardless of the nature of the trigger, glutamate fermentation results in the consumption of NADPH (generated by the isocitrate dehydrogenase step)by glutamate dehydrogenase. In fact, glutamate fermentation is known to require oxygen but not very high concentrations of dissolved oxygen, which tend to favor the excretion of -ketoglutarate rather than glutamate.

It is also obvious that the process demands high concentration of NH3.

Oxygen tension and ammonia concentration are controlled precisely in the industrial process.

POSSIBLE PHYSIOLOGICAL ROLE OF THE GLUTAMATE PRODUCTION

Scientists used to assume that citric acid or glutamate fermentation occurred because of a complete block at relevant steps in general metabolism. Metabolic flux studies now show that such blocks are unlikely to exist and that massive excretion of primary metabolites may occur as a result of modest adjustments in the central metabolic pathway. This new knowledge of the immense flexibility of the major metabolic pathways is extremely important in the emerging field of metabolic engineering which deals with the alterations of the fluxes of the major pathways, often required for the production of not only the usual primary metabolites but also novel products. The processes leading to the production of primary metabolites, especially those involving fungi(such as citric acid fermentation),used to beand sometimes still are explained as over flow metabolism.

 This is an old idea in which microbial cells are thought to consume excess carbon sources (the overflow) when cell growth is limited by another nutrient for example, by the paucity of divalent cations in the A. niger citric acid fermentation process.
We now know, however, that catabolism is linked tightly to anabolism through many regulatory processes. Thus, the overproduction of metabolites is not simply an overflow phenomenon but must have physiological and ecological relevance. Such a physiological purpose of the glutamate excretion response is difficult to assess. However, C. glutamicum and its relatives, being natural biotin auxotrophs, are likely to become starved for biotin in their habitat, presumably soil. Blocked synthesis of membrane lipids will then cause mechanical strains in the cytoplasmic membrane because the intracellular osmotic pressure is usually much higher than the osmotic pressure of the extracellular medium.

 Perhaps the important point here is that the intracellular concentration of glutamate is about two, sometimes three, orders of magnitude higher than those of other amino acids.
Efflux of glutamate, thus a major anionic component of the cytosol, can then be seen as a response designed to reduce the strain in the membrane by decreasing the osmotic pressure difference across the membrane.

The realignment of metabolic fluxes caused by the excretion of glutamate and the decrease in growth rate will then lead to the characteristic flux pattern seen. For C. glutamicum, in addition to relieving the membrane stress through the export of glutamate, the glutamate production process produces additional ATP through the oxidation of pyruvate to acetylCoA and in addition produces a much less acidic product (0.66 mole of glutamate per one mole of glucose) in comparison with pure fermentation (two moles of lactate per one mole of glucose), thus keeping the environment closer to the neutral pH.

 The operation of the glutamate production system as a shifted yet balanced and interconnected central metabolic response is illustrated in the example of an ATP synthase mutant of C. glutamicum.
In this strain that showed only 25% activity of ATP synthase (or H+ATPase), secretion of glutamic acid was totally abolished, when the biotin starvation conditions were moderate; instead, a large amount of lactic acid was excreted. It appears that with low ATP synthase activity, not enough ATP can be made from the NADH obtained in the altered flow through the citric acid cycle, so glycolysis (which generates ATP directly) becomes enhanced instead, resulting in the excretion of lactate. It thus appears that C. glutamicum produces glutamate as a waste product of catabolism under a specific set of environmental conditions. This suggests that the enzyme responsible for the process, glutamate dehydrogenase, may not be regulated as tightly as are the obligatorily biosynthetic enzymes.

REGULATION OF THE ENZYMES INVOLVED IN GLUTAMATE SYNTHESIS

In most microorganisms, the major pathway for glutamate synthesis is the GS-GOGAT pathway: (1) l-Glutamate + NH3 + ATPl-glutamine + ADP + Pi (GS: glutamine synthetase)

(2) l-Glutamine + -ketoglutarate + NADPH + H+ 2 lglutamate + NADP+

(GOGAT: glutamine oxoglutarate aminotransferase) Sum(1) + (2): -ketoglutarate + NH3 + ATP + NADPH + H+ l-glutamate + ADP +Pi + NADP+.

 Although this pathway consumes one ATP for every molecule of lglutamate synthesized, it is preferred over the alternative pathway described below because it has a low Michaelis constant (Km; much lower than 1 mM) for NH3 and thus can efficiently utilize ammonia.

The second pathway is the glutamate dehydrogenase pathway: -Ketoglutarate+NH3 +NAD(P)H+H+ l-glutamate+NAD(P)+ +H2O.
Because of the rather high Km value (usually around 1 mM) for NH3, this pathway becomes significant only when NH3 concentrations are high.

In most organisms, mutational inactivation of this enzyme does not make the mutants auxotrophic for glutamate, a finding that confirms the relatively minor role of this pathway for glutamate synthesis in most species.

 Because the GS-GOGAT pathway is so much more important, its enzymes are strongly regulated, as is typical of obligatorily biosynthetic enzymes; on the other hand, regulation of the glutamate dehydrogenase activity tends to be rather loose. In C. glutamicum, the regulation of glutamate dehydrogenase is exceptionally loose; 50% inhibition of the enzyme requires the addition of 0.2 M glutamate to the cell extract, whereas similar inhibition of Alkaligenes eutrophus glutamate dehydrogenase requires only 20 mM. These considerations suggest that the overproduction of glutamic acid in C. glutamicum is the result of a fortuitous combination of several factors. Over evolutionary time, the organisms of this group have developed a peculiar combination of enzymes, presumably equipping them to deal with ecological conditions perhaps related to those described above.

 It appears that the fermentation conditions used in the laboratory and in industry are precisely those that led to stimulation of the synthesis, and excretion, of glutamic acid.