Journal of Chemical Technology and Biotechnology

J Chem Technol Biotechnol 81:16011611 (2006)

Review Organic acids: old metabolites, new themes

Israel Goldberg,1 J Stefan Rokem1 and Ophry Pines2
1 Department

of Molecular Genetics and Biotechnology, The Hebrew University Hadassah Medical School, POB 12272, Jerusalem 91120, Israel 2 Department of Molecular Biology, The Hebrew University Hadassah Medical School, POB 12272, Jerusalem 91120, Israel

Abstract: Fumaric, L-malic and citric acids are intermediates of the oxidative tricarboxylic acid (TCA) cycle which in eukaryotes is localized in mitochondria. These organic acids are synthesized and accumulated in the medium to very high concentrations by lamentous fungi such as Aspergillus spp. and Rhizopus sp. This article reviews basic research on the unusual acid production capability and the associated metabolic pathways operating under dened stress conditions in these specic fungi. In particular, we describe and discuss the importance of the cytosolic reductive TCA pathway, which includes the cytosolic activities of pyruvate carboxylase, malate dehydrogenase and fumarase, for production of fumaric and L-malic acids. This article also describes the differences between fumaric acid, L-malic acid and citric acid production by different organisms (lamentous fungi, yeast, and higher eukaryotes), and the possible application of novel technologies (genetic engineering and bioinformatics) to fungal systems which may offer new industrial potential of lamentous fungi for the production of valuable metabolites. 2006 Society of Chemical Industry

Keywords: tricarboxylic acid (TCA) cycle; fumaric acid; L-malic acid; citric acid; lamentous fungi; Aspergillus sp.; Rhizopus oryzae

INTRODUCTION In 1817 the dry distillation of malic acid by Braconnet and independently by Vauquelin led to the discovery of two acids, which became known as maleic acid (the cis-form) and fumaric acid (the trans-form).1 Fumaric acid derives its name from the fact that the acid is found in plants that belong to the genus Fumaria, a common European herb. Fumaric acid (2-butenedioic acid trans; 1,2-ethylenedicarboxylic acid) is a symmetric dicarboxylic acid. In 1785 Scheele isolated an acid from unripe apples.1 This acid is still called malic acid after the Latin word malum meaning apple. Malic acid (2-hydroxybutanedioic acid, 2-hydroxysuccinic acid) is a dicarboxylic acid and has an asymmetric carbon and therefore it occurs in two isomers, L and D. Citric acid (2-hydroxypropane-l,2,3-tricarboxylic acid) was rst isolated and crystallized from lemon juice by Scheele in 1784. Citric acid is a symmetric tricarboxylic acid and derives its name from the Latin word citrus, the citron tree, the fruit of which resembles a lemon.2 The involvement of these acids in the tricarboxylic acid (TCA) cycle was discovered in 1937 by Krebs.3 This pathway is also named the Krebs cycle. Fumaric, L-malic and citric acids are synthesized and accumulated in the medium to high concentrations by lamentous fungi such as Aspergillus spp. and Rhizopus sp. Although the acids (especially citric acid) are commercially produced on a very large scale,

the mechanism(s) by which these acids are produced have been the subject of relatively few publications, as compared with other products of lamentous fungi (e.g. penicillin), maybe because the organic acids have had a less visible impact on human well-being.4 Even with citric acid, the most studied, understanding of the events leading to its accumulation is still incomplete.4,5 Fumaric, L-malic and citric acids do not accumulate under normal growth conditions. They may accumulate in mutant eukaryotic cells and in mammals under certain pathological conditions, while in lamentous fungi there is signicant accumulation of fumaric, L-malic and citric acids under specic stress conditions. Thus, after a specic stimulus, certain fungi accumulate intermediates of the TCA cycle as end products. The accumulation of fumaric and L-malic acids is carried out by a separate and unique pathway, the reductive TCA pathway which is localized in the cytosol.6 Citric acid, on the other hand, is the product of citrate synthase, an enzyme of the mitochondrial oxidative TCA cycle, yet cytosolic L-malic acid is an intermediate in citric acid biosynthesis. The cytosolic pathway of fumaric and L-malic acid production is discussed, and, where relevant, its contribution to citric acid production is mentioned. Yeast species such as Yarrowia lipolytica and other Candida strains have been used to produce citric acid since the 1960s, initially from n-alkanes and later

Correspondence to: Israel Goldberg, Department of Molecular Genetics and Biotechnology, The Hebrew University Hadassah Medical School, POB 12272, Jerusalem 91120, Israel E-mail: goldberg@md.huji.ac.il (Received 24 October 2005; revised version received 17 January 2006; accepted 17 January 2006) Published online 18 July 2006; DOI: 10.1002/jctb.1590

2006 Society of Chemical Industry. J Chem Technol Biotechnol 02682575/2006/$30.00

I Goldberg, JS Rokem, O Pines

from other carbon sources such as glucose, acetate, molasses, alcohols, fatty acids and natural oils.7 The advantages of using yeasts in citric acid production as compared with lamentous fungi (e.g. A. niger) are: (1) yeasts are more tolerant to high glucose concentrations, (2) yeasts have high conversion rates of substrates to acids, (3) yeasts are less sensitive to metal ions, (4) yeasts are unicellular fungi allowing for better process control, and (5) yeasts are capable of utilizing a wider range of carbon sources.8 In contrast to A. niger (see the section on Accumulation of organic acids in response to stress), citric acid production in yeasts starts when the nitrogen source becomes limiting.9 However, during this process isocitric acid is the main by-product, constituting up to 50% of total acids produced. Other disadvantages of yeast citric acid fermentations are the lower concentration and productivity of citric acid formation and, in the case of Y. lipolytica, the main yeast species for citric acid production; it cannot utilize molasses and whey. Research is being carried out to partially eliminate the disadvantages.8 Presently, A. niger is almost exclusively used to produce citric acid.10 It should be noted that other intermediates of the TCA cycle, such as isocitric acid and -ketoglutaric acid, are the products of the transformations of citric acid by aconitase and isocitrate dehydrogenase, respectively. Therefore, it can be assumed that these acids share a similar biosynthetic pathway with citric acid. The remaining acid of the TCA cycle, succinic acid, is usually produced, as a main product, by bacterial strains grown under anaerobic conditions.11 Succinic acid as well as isocitric acid and -ketoglutaric acid are not dealt with in the present review. One of the reasons that various lamentous fungi accumulate fumaric, L-malic or citric acids in the medium is the presence of the enzyme pyruvate carboxylase (PYC) in the cytosol. This is in contrast to mammalian and other eukaryotic cells where this enzyme is exclusively situated in the mitochondria.12,13 Nevertheless, higher eukaryotes harbor in their cytosol the other two enzymes of the reductive TCA pathway (malate dehydrogenase (MDH), and fumarase (FUM)) that may be involved in the scavenging of unwanted acids in the cytosol, or have some other unknown functions.6 The accumulation of organic acids in mammals is highly pathological and results in metabolic diseases, such as fumaric aciduria in which fumaric acid accumulates.14 It appears that control of organic acid metabolism and accumulation may be vital for all eukaryotes. This paper reviews basic research on the unusual acid production capability of specic fungi under dened stress conditions and the associated metabolic pathways. The differences between fumaric acid, L-malic acid and citric acid production by different organisms (lamentous fungi, yeast, and higher eukaryotes) are described. For other aspects of this subject the reader is referred to additional reviews.4,15 17 We propose that, because of the
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high production capacity of these fungi, the marked increase in the use of health-related products, and the rapid advances of novel technologies (genetic engineering and bioinformatics), it is appropriate that these old metabolites should become the subject of new themes of research. Uses and market Fumaric acid is widely used in the food industry and is a valuable intermediate for preparing edible products such as L-malic acid and L-aspartic acid (for use in preparing aspartame). The bicarboxylic nature of fumaric acid makes it an important intermediate for polymer production.6 Fumaric acid is presently produced by isomerizing maleic acid (or maleic anhydride), obtained from benzene oxidation. In the early 1940s fumaric acid was made by fermentation on a commercial scale by Pzer (about 4000 tons per year) using a strain of the fungus Rhizopus arrhizus (later named R. oryzae).6,18,19 The biological production of fumaric acid was stopped when the chemical synthesis became economically more attractive.6 Malic acid has an asymmetric carbon and therefore has D-()- and L-(+)-isomers. L-Malic acid is the natural isomer, which occurs in many fruits and is produced by biological processes. DL-Malic acid, which is the other commercial form, is classied as generally recognized as safe (GRAS) by the US Food and Drug Administration and is available as a food-grade product conforming to Food Chemicals Codex (FCC) specications. Malic acid is mainly used in beverages, candy and food. Nonfood applications for malic acid include metal cleaning and nishing, textile nishing, electroless plating, pharmaceuticals, infusions in hospitals and paints. Presently, malic acid is produced mainly by chemical synthesis via hydration of maleic or fumaric acid at a high temperature and a high pressure, yielding the racemic mixture of D-()- and L-(+)-malic acid.6,16 Alternatively malic acid is manufactured by an enzymatic process, whereby fumaric acid is converted to L-malic acid (see the section on Production of L-malic acid). There is no known commercial process for the production of L-malic acid by one-step fermentation from glucose up to now. Malic acid has been an intermediate-volume chemical (annual world production of about 40 000 tons). It has great potential for becoming a very large volume, commodity-chemical intermediate produced from renewable carbohydrates since it is an oxygenated chemical. It can be used as a specialty chemical intermediate and a feedstock for biodegradable polymers, similar to polylactic acid (NatureWorks ) which is currently being produced on a large scale (about 140 000 tons year1 ) at Blair, Nebraska.20 Citric acid is a natural constituent of a variety of citrus fruits, pineapple, pear, peach and g, and today is the most important food acidulant. It has a variety of uses in food, pharmaceuticals and other industrial elds. More specically, it is used in candies,
J Chem Technol Biotechnol 81:16011611 (2006) DOI: 10.1002/jctb

Organic acids: old metabolites, new themes

jellies, jams, gelatin desserts, soft drinks, syrups, fruits and vegetable juices, wines, ciders, frozen fruits, dairy products, animal fats, oils, pharmaceuticals, cosmetics and toiletries.21 Commercial production of citric acid commenced around 1826 in England, by extracting the acid from Italian lemons. The rst industrial fermentation process using Aspergillus niger began in 1919 in Belgium.2 Commercially, citric acid is produced by surface, submerged and solid state fermentation techniques. Recovery of pure acid from fermentation broths is done primarily by precipitation with lime and by solvent extraction.21 In 1998, the global production of citric acid was about 879 000 tons4 and in 2004 it had increased to 1.4 million tons (G Graf, http://www.purchasing.com/article/CA624878.html). Over 200 g L1 of citric acid are made by industrial strains of A. niger.22 The size of the citric acid market is massive and continues to grow, largely because of expanding food and beverage markets in developing countries, and increasing use of citric acid salts (mainly the calcium salt) in health-related consumer products.4

THE BIOLOGY OF ORGANIC ACID PRODUCTION IN FUNGI Accumulation of organic acids in response to stress Stress appears to be a common requirement for the unusually high production and accumulation of organic acids by all producing organisms. The most important factor is nitrogen limitation; fungi starved of nitrogen produce mainly fumaric acid and/or L-malic acid, and not biomass, from glucose. In fermentations, only after full depletion of the limiting nitrogen source do the acids accumulate by secretion into the medium. In certain lamentous fungi fumaric or L-malic acid concentrations above 100 g L1 are obtained,23 26 highlighting the massive diversion of cellular carbon to a specic set of reactions of the reductive TCA pathway. In addition to nitrogen limitation, CaCO3 is required in the medium, in order to obtain high concentrations of the acid salt. CaCO3 ensures a pH of about 6.0 during fumaric and L-malic acid accumulation processes and provides CO2 as a substrate for the production of these acids (see the section on Pyruvate carboxylase). CaCO3 also causes precipitation of calcium salts of
Table 1. Production of citric and L-malic acids by A. niger and A. avus

the acids, thus pulling the reaction towards the acid end products. In contrast to the synthesis and accumulation of L-malic and fumaric acids by fungi, the requirements for citric acid accumulation are different. The critical parameters for citric acid production by A. niger (approximately 200 g L1 of citric acid from 240 g L1 of glucose, i.e. almost 1 mol L1 of acid) have been dened empirically and include: high carbohydrate concentration, low but nite manganese concentrations, high dissolved oxygen, constant agitation, and low pH.3,22 Citric acid is produced in acid form whereas fumaric and L-malic acids accumulate as calcium salts. Aspergillus strains differ in their ability to produce citric and L-malic acids; Aspergillus niger is a citric acid producer while A. avus produces mainly L-malic acid. In order to demonstrate the difference in stress conditions required to produce acids we show results of an experiment in which Aspergillus strains were incubated in two different production media (Table 1) (E Battat and I Goldberg, unpublished). The rst medium (PC) was designed to enhance mainly citric acid production. PC medium is characterized by a high nitrogen concentration and a low initial pH, and it does not contain ferrous ions. The second medium (PM) was designed to enhance mainly Lmalic acid production and is characterized by a low nitrogen concentration, a relatively high ferrous ion concentration, and it contained biotin and CaCO3 (ensuring an almost constant pH throughout the fermentation process). The concentration of the carbon source and other medium constituents were the same in both media. Aspergillus niger produced citric acid on PC medium (L-malic acid could not be detected under these conditions) and produced both citric and L-malic acids in PM medium. Aspergillus avus produced mainly L-malic acid on PM medium and did not produce citric acid in both media. Under the stress conditions of the PC medium, the limited growth of A. avus resulted in the conversion of glucose to gluconic acid and not to TCA cycle intermediates. However, in PM medium only negligible amounts of gluconic acid were produced by these strains (Table 1). This experiment demonstrates the importance of the specic limiting nutrient(s) for the selectivity of the acid produced by different fungal strains.

Acids produced after 160 h (g L1 ) Strain A. niger NRRL 599 A. avus ATCC 13697 A. niger NRRL 599 A. avus ATCC 13697 Medium PC PC PM PM Growth (g cell dry wt L1 ) 12.5 3.0 11.6 10.4 Citric 70.3 ND 7.0 ND
L-Malic

Succinic ND ND 2.6 7.9

Fumaric 0.013 0.006 0.59 1.10

Pyruvic ND ND 2.2 3.8

Gluconic 15.2 43.5 2.6 2.2

ND ND 21.6 48.6

PC Production medium for citric acid; PM production medium for L-malic acid. ND The specic acid was not detected.

J Chem Technol Biotechnol 81:16011611 (2006) DOI: 10.1002/jctb

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I Goldberg, JS Rokem, O Pines

Acids are accumulated by the cytosolic reductive TCA pathway Historically, the high accumulation of fumaric and L-malic acids was thought to transpire through the glyoxylate cycle and via a part of the mitochondrial oxidative TCA cycle.18,19,27 There was even a suggestion that deciencies in certain TCA enzymes, such as fumarase, may result in the accumulation of specic intermediates such as fumaric acid.18 The rst indication that this cannot be the case came from the experimental acid molar yields obtained. Based on the glucose utilized, R. oryzae accumulated 145% of C4 acids (mainly fumaric acid but also L-malic and succinic acids).24,28,29 A similar situation was found for L-malic acid produced by A. avus; 134% of C4 acids, mainly L-malic acid.26,30 These experimentally-obtained yields are higher than the maximal theoretical acid molar yields calculated for the TCA cycle (67% for the oxidative cycle and 100% for the concomitantly operating glyoxylate cycle).14 Evidence for the possibility that an alternative pathway to the glyoxylate cycle and the oxidative TCA cycle is responsible for acid production came from two sources. Osmani and Scrutton31,32 discovered that certain lamentous fungi (Aspergillus nidulans and R. oryzae) harbor cytosolic pyruvate carboxylase, malate dehydrogenase, and fumarase. On the basis of the availability of these enzymes in the cytosol, these authors suggested the existence of an extramitochondrial pathway leading from pyruvic to L-malic or fumaric acid. Direct proof that the reductive TCA pathway participates in fumaric and L-malic acid production came from 13 C NMR experiments using [1-13 C] glucose as a carbon source for A. avus25 and R. oryzae,24 respectively. The unexpectedly high fumaric and L-malic acid molar yields can thus be explained in terms of pyruvate carboxylation as part of a reductive TCA pathway. But once notion of a cytosolic reductive TCA pathway is accepted, the question is why the experimentally-obtained yields of C4 organic acids (134145%, see above) are lower than the theoretical maximal yield of 200% calculated for this pathway.24 A plausible explanation for this is that the fungus must divert some of the pyruvate carbon to mitochondria and to the oxidative TCA cycle to obtain energy required for maintenance requirements and limited cellular growth.18 The discovery of a novel pathway for fumaric and L-malic acids production in lamentous fungi, which results in high acid molar yields, enables efcient fermentation processes for these acids to be designed (especially for L-malic acid). These improved processes could potentially enable fermentation-based fumaric and L-malic acids to be economically competitive with citric acid. The citric acid process is very efcient although its molar yield from glucose is lower than for these other acids. It is perhaps unexpected that the accumulation of citric acid (theoretically the formation of one mole of citric acid per mole glucose utilized) grown
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in high-glucose medium follows a similar metabolic pathway to that of fumaric and L-malic acids. Citric acid formation in efcient fermentations involves the glycolytic catabolism of glucose to two molar equivalents of pyruvate. One mole of pyruvate is converted to acetyl-CoA (by releasing one mole of carbon dioxide) and the other mole to oxaloacetate (by xing one mole of carbon dioxide onto the second mole of pyruvate). The two precursor molecules obtained are condensed to form citric acid. The oxaloacetate formed is not directly converted by the mitochondrial citrate synthase to citric acid (in A. niger pyruvate carboxylase is localized in the cytosol12 ), but is rst reduced to L-malic acid by the cytosolic MDH, thereby entering the mitochondria via a malatecitrate antiport.33 35 The existence of the pathway was demonstrated by showing that during growth in a high-glucose concentration (14%, w/v), most of the label from [1-13 C] glucose of citric acid is detected in carbons 2 and 4, and is thus due to the pyruvate carboxylase reaction.34 The similarity between the biosynthetic pathways involved in the accumulation of fumaric, L-malic and citric acids, by different fungal strains and different stress conditions, emphasizes the importance of carbon dioxide xation by a unique pyruvate carboxylase, which in these organisms is localized only in the cytosol. Enzymes of the cytosolic reductive TCA pathway Characterization of the enzymes of the reductive TCA pathway and the ability to accumulate fumaric, Lmalic and citric acids remains incomplete. There is little information regarding enzymes from lamentous fungi (the high acid-producers), in contrast to the wealth of biochemical, molecular, and physiological information available from yeast (low levels of acid production). With developments in molecular biology technologies, which presently can be implemented in lamentous fungi, knowledge on this important subject will hopefully expand. Pyruvate carboxylase Pyruvate carboxylase is a biotin-dependent tetrameric enzyme that catalyzes the carboxylation of pyruvic acid to oxaloacetic acid. The biotin prosthetic group is covalently bound to the polypeptide chain of the enzyme.36 Pyruvate carboxylase is a key enzyme in the cytosolic reductive TCA pathway rather than the mitochondrial oxidative TCA cycle, since it is situated at the branch point of pyruvate metabolism in the cytosol. Thus, it is expected that the expression of PYC genes would be strictly regulated. In general, pyruvate carboxylase in eukaryotic organisms is localized in mitochondria.13 However, in certain lamentous fungi and in the yeast Saccharomyces cerevisiae, the enzyme is situated exclusively in the cytosol and in fact lacks a mitochondrial-targeting peptide.12,37 The cytosolic localization appears to be important for the ability
J Chem Technol Biotechnol 81:16011611 (2006) DOI: 10.1002/jctb

Organic acids: old metabolites, new themes

of these organisms to accumulate high concentrations of organic acids. The localization does not seem to inuence the catalytic properties of the enzyme.38 Pyruvate carboxylase has been implicated in the accumulation of high concentrations of fumaric and/or L-malic acids by lamentous fungi and yeast by using effectors of pyruvate carboxylase (such as biotin and avidin).24,39 Saccharomyces cerevisiae has two pyruvate carboxylase genes that exhibit a homology of 90% at the amino acid level and 85% at the nucleotide level.40 During fermentative growth, the absence of one or the other PYC gene had little effect on growth, whereas in the absence of both genes the organism required aspartate for growth on minimal medium.40 Even though the two proteins appear to be catalytically identical, the control of their gene expression is different, suggesting that the isoenzymes may have different functions in the cell.41

isoenzymes were observed. It was found that the presence of a specic electromorph of the MDH was related to the ability of Aspergillus strains (such as A. avus, A. oryzae, A. sojae and A. parasiticus) to produce relatively high amounts of L-malic acid as the predominant acid. It has been suggested that this technique can be used to nd suitable L-malic acid-producing strains (Y Peleg, E Battat, JS Rokem and I Goldberg, unpublished data). Fumarase Fumarase catalyzes the reversible hydration of fumaric acid to L-malic acid. Two classes of fumarases have been described.44 50 Class I are oxygen-labile homodimeric fumarase hydratases. Members of this class have been characterized in Escherichia coli,46 Euglena gracilis,51 and Bacillus stearothermophilus.52 In all other organisms, class II fumarases have been described. The fumarases of class II are stable homotetrameric enzymes. In E. coli three different genes for fumarase were described, two belonging to class I, FUMA and FUMB, and one belonging to class II, FUMC.46 The S. cerevisiae, rat, and human fumarases are encoded in each organism by a single nuclear gene, yet the enzymes are found both in the cytosol and mitochondria. A novel model postulates that in S. cerevisiae, which accumulates only up to 2 g L1 of L-malic acid but not fumaric acid, fumarase translation initiates only from the 5 proximal AUG codon and results in a single fumarase translation product. All the fumarase molecules synthesized are targeted and processed by the matrix protease in the mitochondria before distribution. The targeting is coupled to translation and involves insertion of fumarase precursors across the mitochondrial membranes, rapid folding and retrograde movement of most of the processed protein back into the cytosol.53 56 The precise mechanism of fumarase distribution in higher eukaryotes remains to be determined. The role of fumarase in L-malic acid accumulation has been most thoroughly studied in yeast and may shed light on the situation in other organisms (e.g. A. avus). The fact that S. cerevisiae accumulates L-malic and not fumaric acid can be explained by the activity of a cytosolic fumarase which, like the mitochondrial enzyme, catalyzes the conversion of fumaric to L-malic acid and not the reverse reaction.56 Thus, the cytosolic enzyme scavenges cytosolic fumaric acid, converting it to L-malic acid. Whereas this reaction can explain L-malic acid accumulation in yeast and probably in A. avus,26 an intriguing question that remains to be answered is the mechanism by which large amounts of fumaric acid are produced by the lamentous fungus R. oryzae. This organism, which is one of the few fumaric acidproducing organisms described in the literature, can accumulate more than 100 g L1 of fumaric acid as the main product.23,24,28 Two possibilities to explain
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Malate dehydrogenase Malate dehydrogenase catalyzes the NAD(H)dependent reversible conversion of L-malic acid to oxaloacetic acid. At least two forms of the enzyme are found in eukaryotic cells, a mitochondrial enzyme (MDH1) that functions in the oxidative TCA cycle and a cytosolic enzyme (MDH2) catalyzing the rst step in gluconeogenesis from pyruvate. In S. cerevisiae a third malate dehydrogenase (MDH3) is localized in peroxysomes.42 In A. avus, which produces high amounts of L-malic acid (113 g L1 from 120 g L1 glucose utilized),26,43 the activity of malate dehydrogenase increased 610-fold after 6 days in production medium, whereas the activity of fumarase changed only slightly.30 Thus, there is a strong correlation between MDH expression and L-malic acid production, indicating that this enzyme is a limiting factor in acid production. Electrophoretic analysis of A. avus cell extracts on cellulose acetate has established the presence of unique mitochondrial and cytosolic isoenzymes of MDH and fumarase. During the acid production stage changes in the isoenzyme pattern according to electrophoretic mobility were observed for MDH but not for fumarase. Cycloheximide completely inhibited L-malic acid production and there was no increase in the major isoenzyme of MDH, without affecting either the total activity of fumarase or its isoenzyme pattern.25 These results suggest that de novo protein synthesis is involved in the increase in activity of the major isoenzyme of MDH and that this isoenzyme is important for L-malic acid production and accumulation. A comparative study of different Aspergillus strains showed that all 54 strains tested produced L-malic acid. The MDH activity of these strains was characterized by cellulose acetate electrophoresis to detect the different isoenzymes mentioned above. Ten electrophoretic patterns (electromorphs) of MDH
J Chem Technol Biotechnol 81:16011611 (2006) DOI: 10.1002/jctb

I Goldberg, JS Rokem, O Pines

this phenomenon exist. The rst is that in R. oryzae the cytosolic fumarase is kinetically different from the mitochondrial isoenzyme, due to distinct posttranslational modications, or to specic conditions in the two compartments. The second possibility is that R. oryzae harbors two genes encoding two different fumarases, one in mitochondria, which catalyzes the conversion of fumaric to L-malic acid, and a cytosolic enzyme, which catalyzes the conversion of L-malic to fumaric acid. To distinguish between these possibilities an effort has been made to characterize the R. oryzae fumarase isoenzymes and clone the respective gene or genes. The yeast fumarase gene FUM1 was used as a probe to clone an homologous fumarase gene from R. oryzae. Accordingly, the alignment of the amino acid sequence of this R. oryzae fumarase with other class II fumarases indicated a high degree of sequence similarity (7580%).50 The gene designated fumR was shown to produce a single transcript, and as predicted from the DNA sequence, a protein with an apparent molecular mass of 50 kDa was detected, employing S. cerevisiae-fumarase antiserum. This protein did not appear to be responsible for the approximately sixfold increase in fumarase activity (as measured with L-malic acid as the substrate)25 upon transfer of cells into acid production medium. This is because the level of the protein as detected by western blotting changed only slightly upon this shift.50 Furthermore, antiserum against yeast fumarase only partially neutralized the fumarase activity in R. oryzae cell extracts, suggesting the existence of an additional non-homologous enzyme (E Battat, O Pines and I Goldberg, unpublished data). Thus, our preference is for the possibility of two different fumarase genes, which is based mainly on our unpublished observations. Additional suggestive evidence for the existence of a second distinct fumarase in this fungus comes from the analysis of fumarase activity in cell lysates. Fumarase in lysates of R. oryzae from medium B (growth medium) has a lower Km value for fumaric acid (0.78 mmol L1 ) than for Lmalic acid (2.9 mmol L1 ), similar to fumarase from lysates of S. cerevisiae.56 Fumarase activities (with L-malic acid as the substrate) in both these lysates were not inhibited by fumaric acid. In sharp contrast, fumarase activity measured in extracts prepared from R. oryzae cells incubated in medium C (production medium), but not with S. cerevisiae, was completely inhibited by 2 mmol L1 fumaric acid (E Battat and I Goldberg, unpublished data). Taken together, a plausible explanation for fumaric acid production by R. oryzae would be that upon transfer into medium C, a fumarase with unique characteristics is induced. L-Malic acids conversion to fumaric acid is enhanced by the induced fumarase and when the concentration of fumaric acid in the cell exceeds 2 mmol L1 , the reverse reaction to L-malic acid is fully inhibited. Thus, this property of the unique fumarase, whose existence is only hypothesized, can ensure that fumaric acid is further accumulated. Attempts are being made to
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isolate and characterize this unstable (half life of 2 h) cytosolic fumarase in R. oryzae.

MODERN BIOTECHNOLOGIES FOR OLD METABOLITES Recently, Magnuson and Lasure4 stated that in terms of productivity, fungal organic acid processes might offer the best examples of fungal commercial production processes. Yet this high productivity has been the subject of relatively few research papers. With citric acid the research is aimed mainly at process improvements due to the erce competition between producing companies. With L-malic acid the research is aimed at improvements of the enzymatic process and developing a novel one-step fermentation process from glucose. In the case of fumaric acid there have been only a few publications on R. oryzae subsequent to the termination of the industrial biological production.4 However, in recent years fungal research using novel molecular and cell biology technologies has increased markedly. The quantities of data from genomic and other omic studies of fungal systems offer new opportunities to reassess the industrial potential of lamentous fungi. The new technologies should make these old metabolites new themes for industrial research. Production of fumaric acid Fumaric acid is the least expensive of the foodgrade acids. Since the chemical process for acid production (fumaric acid is a symmetrical molecule) is cheaper than the biological one the chemical process has become the prevailing process of fumaric acid production in industry. Nonetheless, the biological process may replace the chemical process in the future owing to recent developments. In this regard one can recall that in the late 1970s and early 1980s DuPont was interested in developing a process for the production of Nylon based on glucose and not petroleum-based hydrocarbons. Thus, the biological production of fumaric acid by R. oryzae was considered,23,28 together with the subsequent chemical hydrogenation of fumaric acid to succinic acid and then to Nylon through tetrahydrofuran.28 DuPont terminated this project in 1981, although research continued in various university groups.57 Recently, similar results for fumaric acid fermentation were obtained using various neutralizing agents.58 Interest in Rhizopus oryzae has increased recently since it was found that this organism is capable of producing high levels of only one stereospecic isomer (the L-(+)-form) of lactic acid.4 Thus, it caters to the need for producing a food additive, as an acidulant and preservative. In addition, the novel use of biodegradable plastic (polylactic acid PLA or NatureWorks ) further promoted the application of lactic acid and dramatically increased its production.20,59
J Chem Technol Biotechnol 81:16011611 (2006) DOI: 10.1002/jctb

Organic acids: old metabolites, new themes

The renewed interest in the fungus R. oryzae is developing via different technologies. The initial project is aimed at the sequencing of the genome of R. oryzae through the Fungal Genome Initiative (FGI) at the Whitehead Center for Genome Research at MIT (see http://www.genome.gov/10002154).60 The availability of the genomic sequence may lead to strain and process improvements for L-lactic acid and fumaric acid as well as for many other potential products. The second approach relies on extrachromosomal plasmid replication in high-copy number and it was developed because this fungus rarely integrates DNA following transformation. The new technology is based on CaCl2 /PEG- or Agrobacterium-mediated DNA transfer systems that have been developed for R. oryzae.61 These systems ensure the stability of the genetic information introduced and make gene targeting and gene replacement possible.61 Interest in R. oryzae has shifted to its ability to produce large amounts of L-lactic acid, with fumaric acid as the main byproduct of metabolism with the latter arising from the cytosolic reductive TCA pathway described above.62 Reduction of byproduct formation by adjustment of the concentrations of medium components, e.g. phosphate,62 or through increasing lactate dehydrogenase activity by transformation with ldhA gene fragments might permit lactic acid fermentation to scavenge available pyruvate more effectively.63 The above approaches may result in better understanding of the metabolic pathways and the control mechanisms involved in acid, and especially fumaric acid, biosynthesis and accumulation. These technologies will enable the ux of carbon from glucose to fumaric acid to increase and improve the fermentation process to be economically competitive with the chemical process. Production of L-malic acid Presently, malic acid is produced mainly by chemical synthesis via hydration of maleic or fumaric acids at high temperatures and high pressures, yielding a racemic mixture of D-() and L-(+) isomers.6,16 Another route of malic acid manufacture is via an enzymatic process in which fumaric acid is converted to L-malic acid by immobilized bacterial cells (such as Brevibacterium ammoniagenes) containing the enzyme fumarase. The yield of L-malic acid reaches about 70% of the theoretical; the unconsumed fumarate is recycled. The enzymatic reaction is carried out at a neutral pH and results in L-malic acid salts. Thus, downstream processing involves separation of the unreacted substrate as well as isolation of the free acid.64 In order to increase the productivity of fumaric acid conversion to L-malic acid, a strain of Saccharomyces cerevisiae engineered to overproduce fumarase was used and immobilized.65,66 In these cells fumarase activity was 80 mmol g1 (wet cell weight), permitting conversion yields of 8090% with
J Chem Technol Biotechnol 81:16011611 (2006) DOI: 10.1002/jctb

a productivity of about 2 g L1 h1 .66 Good results for fumaric acid conversion to L-malic acid were obtained with agarose-immobilized cells, although the bioconversion rates decreased in a linear fashion with the diameter of the agarose beads, demonstrating mass transfer limitations controlling bioconversion rates.65 More recently, a conversion value of almost 100%, above the equilibrium value of ca 84% and higher than that for the industrial process (70%), was achieved with the above mentioned S. cerevisiae in a bioreactor which was divided into three compartments by two supported liquid membranes.67 The construction of the bioreactor ensured a unidirectional ow of the substrate from the Feed to the Reaction (middle) compartments and of the product from the Reaction to the Product compartments, with the inorganic counter-ion moving in the opposite direction. This process resulted in the production of the free acid, in contrast to the industrial process which results in L-malic acid salts. A one-step fermentation leading from glucose to L-malic acid is a possible process by which L-malic acid could be produced at a competitive cost when compared with citric acid. The reason for this is the operation of the reductive TCA pathway in the production of L-malic acid, resulting in the higher theoretical yield of L-malic acid as compared with citric acid. For example, from 1 mole of glucose utilized and 2 moles of CO2 originating from CaCO3 present in the medium, 2 molar equivalents (268 g L1 ) of L-malic acid can be obtained, as compared with only 1 mole (192 g L1 ) for citric acid. Various amounts of L-malic acid are accumulated in a minimal medium containing glucose, various salts, and CaCO3 by cultivating different Aspergillus strains.6,16 In A. avus a maximal amount of 113 g L1 (from 120 g L1 glucose utilized) L-malic acid or 128% on a molar basis, and a productivity of 0.59 g L1 h1 were achieved. However, this strain produced signicant quantities of succinic acid (a molar yield value of about 20% from glucose) and, to a much lesser extent, fumaric acid (a molar yield value of 13% from glucose). The molar yield of C4 acids obtained was 155%.26 The production of succinic and fumaric acids reduces the L-malic acid yield from the glucose utilized.26 In another study the production of L-malic acid from glucose was carried out with Aspergillus sp. N1-14 and 106 g L1 of L-malic acid and an overall productivity of 0.88 g L1 h1 were achieved.68 The genome sequencing project for A. oryzae was launched in 2001 by a whole genome shotgun approach69,70 and was completed recently.71 The 37 Mb genome was predicted to contain a total of 12 074 genes encoding proteins with a length greater than 100 amino acids.70,71 The knowledge of the genome sequences may aid in improving L-malic acid production and in preparing non-aatoxigenic strains.4 When the biological production of L-malic acid becomes more attractive economically than the
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I Goldberg, JS Rokem, O Pines

chemical synthesis of DL-malic acid, we can expect that this may enlarge the market for this acid. L-Malic acid can then be used as raw material for the production of a novel biodegradable polymer (polymalic acid),72 in a similar manner to the use of L-lactic acid for production of polylactic acid (PLA).20,59 Production of citric acid The lamentous fungus A. niger is the main microorganism used for large-scale fermentative production of citric acid and various enzymes. Therefore, it is not surprising that most of the knowledge for improvements in producing strains, medium components, and fermentation conditions are not revealed by the industry.35 However, the academic and other published research is aimed at the better understanding of the biosynthesis, the accumulation, and the control mechanisms involved in acid production. A large Dutch fermentation and chemical company, DSM, in collaboration with other partners, recently announced the completion of the sequencing and annotation of the A. niger genome.73 The 35.9 Mb unique sequence obtained was assembled into 19 supercontigs that represent the eight A. niger chromosomes. A strong functional prediction could be made for approx. 45% of the orfs. EC numbers as well as many other functional classications were assigned to over 3000, out of 14 400 identied genes.73,74 It should be recognized that A. nigers genome sequence is not available to the public due to commercial considerations. It can be expected that in the near future public institutes will sequence the A. niger genome, maybe not because of its ability to produce citric acid but because it causes pulmonary aspergilloma (http://www.Aspergillus.man.ac.uk). In lamentous fungi it is still difcult to obtain the high levels of expression required for commercial purposes, for some homologous proteins and most heterologous proteins. The industrial application of A. niger, particularly as a host for heterologous protein expression, has had limited success. One factor limiting the development of strains suitable for protein production, or strains having amplied enzyme activities for citric acid production, is that few plasmid vectors are available for A. niger. Recently, an approach to speed up strain and process development was reported in which integrating and autonomously replicating plasmid vectors were described for gene and protein expression in A. niger.75 The authors have demonstrated the suitability of a set of vectors for genome manipulation by targeted gene replacement. Another approach by which to gain valuable basic knowledge in order to improve the production process is the development of a metabolic model for this fungus.74 It started with the development of a mathematical model76 of the carbohydrate metabolism and a metabolic ux balance model77 in A. niger under conditions of citric acid accumulation. Christensen and Nielsen78 have developed the metabolic network
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analysis for quantifying metabolic uxes using a 13 C label in the carbon source and subsequent measurements of amino acid patterns by GCMS. The data obtained could be used to calculate cellular carbon uxes more accurately.78 This model was later modied and rened by adding an approach of reciprocal labeling.79 In this approach, a uniformly 13 C-labeled substrate as the primary carbon source and a naturally-labeled co-substrate are investigated (in citric acid fermentation the latter could be the accumulated polyols). Metabolites derived from a naturally-labeled co-substrate, in this case amino acids, can then be identied by their relatively lower content of 13 C and information on the degradation pathway can be deduced.35,79 Recently, the topology of the central carbon metabolism of A. niger was identied and the metabolic network was reconstructed by integrating genomics, biochemical and physiological information available for this microorganism and other related fungi (e.g. A. nidulans). The reconstituted network may serve as a valuable database for annotation of genes identied in future genome sequencing projects of Aspergilli.74 An example of the application of the stoichiometric model for the in silico assessment of the metabolic capabilities of A. niger to produce metabolites other than citric acid has been described recently.74 Simulated results showed that when a mutant of this fungus disrupted for pyruvate decarboxylase was grown under microaerobic conditions at specic growth rates close to the optimal, it produced succinic acid at a yield of 0.47 mole succinate per mole glucose. However, when a double mutant, disrupted in both pyruvate decarboxylase and ATP:citrate oxaloacetatelyase was grown under the same conditions, the yield of succinic acid was at least 1.12 mole per mole glucose.74 These results suggest that the genes encoding the mentioned enzymes may be potential targets for metabolic engineering and show that this organism can be used as a platform fungus for the production of other, and perhaps more valuable, metabolites in addition to citric acid.

PERSPECTIVES Fumaric, L-malic and citric acids are considered bulk chemicals and research of their synthesis and regulation was, until recently, not in the mainstream of biological research. This is perhaps surprising as these metabolites are present at the heart of metabolism of almost every cell, being intermediates of a central pathway. The strong competition between many bulk chemical-producing companies may now act as a driver for research in the eld of acid production by lamentous fungi. This should proceed in two directions: the rst, optimization of the existing production processes and the second, development of novel fermentations for the production of more valuable acids. The recent developments in genomics
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Organic acids: old metabolites, new themes

and metabolomics may facilitate the research in both areas. For the rst time, scientists both in industry and research institutes can investigate the high ability of these organisms in product (acid) formation. Unlocking the biochemical networks and the regulatory mechanisms may provide an opportunity for their manipulation by amplifying or deleting critical steps in metabolism to improve acid production or to overproduce novel metabolites.73,80,81 The new information available will allow the application of systems biology in fungi, despite the present lack of databases for metabolite concentrations in cells.82 Thus, scientists will probably be able to design efcient cell factories (platform fungi) through metabolic engineering. This may be a major breakthrough in the scientic methodology that can restore and increase the importance of these acids and, thus, these old metabolites are becoming new themes for research.

13 14

15

16

17 18

19

20

ACKNOWLEDGEMENTS The authors would like to thank Mr E Battat for the data presented in Table 1, and those on the kinetic properties of fumarases.

21 22

23

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