Fumaric acid is an intermediate in the tricarboxylic acid (TCA) cycle used by cells to produce energy and building blocks (Krebs, 1970). It is formed by the oxidation of succinic acid by the enzyme succinate dehydrogenase. Fumaric acid is further converted to l-malic acid by fumarase. Fumaric acid does not accumulate under normal growth conditions of cells and microorganisms. It may accumulate in mutant eukaryotic cells and in mammals under certain pathological conditions. Uniquely, this acid is synthesized and accumulated in the medium-to-high molar yields (i.e., about 150% of glucose) and concentrations (i.e., more than 100 g/L) by the filamentous fungusR. oryzae, under specific stress conditions. This unusual phenomenon appears to be a general situation where, following a specific stimulus, certain filamentous fungi accumulate intermediates of the TCA cycle as end products (e.g., citric and l-malic acids) (Goldberg et al., 1991, 2006; Magnuson and Lasure, 2004). Although fumaric acid was commercially produced on a large scale (see above, 15.1.2), the mechanism(s) by which it is produced are not fully understood and have been the subject of relatively few publications.
According to the present suggested mechanism, fumaric acid accumulation by R. oryzae occurs mainly by a cytosolic reductive pathway converting pyruvic to fumaric acid by the successive activities of pyruvate carboxylase, malate dehydro- genase, and fumarase (Osmani and Scrutton, 1985; Kenealy et al., 1986). In the following discussion we will try to answer some of the questions that arise from this general mechanism.
15.3.1 How Can the High Molar Yield of Fumaric Acid be Explained?
Although Felix Ehrlich was the first to show in 1911 that fumaric acid is produced byR. nigricans, it took 28 years until Foster and Waksman (1939) identified an over- producerRhizopusstrain, which was (probably) used shortly after in the industry for the production of fumaric acid on a large scale (see above, 15.2.1).
Fumaric acid (molar yield of approximately 100%; moles of acid produced per moles of glucose utilized×100), l-malic acid (15 mol%), and succinic acid (5 mol%) were the major acids formed during the fermentation. C4acid (fumaric, l-malic, and succinic acid) molar yields of 120–145%, with a maximal fumaric acid concentration of 107 g/L, were obtained after 4–5 days (Goldberg and Stieglitz, 1985, 1986; Kenealy et al., 1986; Ng et al., 1986). These high yields confirmed earlier data obtained by Rhodes et al. (1959, 1962).
How can we explain the unusually high molar yield of fumaric acid obtained by a Rhizopusstrain?
Back in 1949, it was demonstrated by Foster et al., withR. nigricansthat ethanol and carbon dioxide may serve as substrates for fumaric acid production. Experiments with [14C]ethanol indicated that the yield of fumaric acid from ethanol was so high that the TCA cycle was excluded as the sole mechanism in acid formation byRhizopus.
These results were taken as presumptive evidence for the operation of a C-2 plus C-2 condensation in fumaric acid synthesis (Foster et al., 1949), similar to the later- discovered sequence of reactions involved in the glyoxylate cycle in bacteria (Kenealy et al., 1986). However, this pathway cannot account for a molar yield exceeding 100%.
More recent data appear to rule out the glyoxylate bypass as a significant pathway in the bulk accumulation of fumaric acid by R. nigricansin media of high sugar concentration (Wegener and Romano, 1964; Romano et al., 1967).
According to Rhodes et al. (1959) the accumulation of fumaric acid maybe attributed to low activity or absence of fumarase. However, a high in vitroactiv- ity of fumarase was measured during the fumaric acid production stage (Osmani and Scrutton, 1985; Kenealy et al., 1986).
Alternatively, a C-3 plus C-1 carbon dioxide fixation mechanism was suggested (Romano et al., 1967; Overman and Romano, 1969). This mechanism involves the operation of pyruvate carboxylase yielding oxaloacetic acid, so that C4acids can be withdrawn for biosynthesis during the growth phase (Osmani and Scrutton, 1985).
Operation of this reaction in conjunction with reactions of the TCA cycle would yield only 1 mole of fumaric acid per mole of glucose utilized if the oxidative reactions of the pathway were used. However, if the reductive reactions of the TCA cycle (see below, 15.3.2) were used, 2 moles of fumaric acid per mole of glucose would be formed.
Direct proof that this reductive TCA pathway participates in fumaric acid pro- duction came from13C NMR experiments using [1-13C] and [U-13C] glucose as a carbon source forR. oryzae(Kenealy et al., 1986). The unexpectedly high fumaric acid molar yields can thus be explained in terms of pyruvate carboxylation as the initial reaction of a reductive TCA pathway (Figure 15.1).
Recently, Yu et al. (2012) obtained a stable high-yieldingR. oryzaeFM19 mutant by laser irradiation. The mutant produced fumaric acid approximately 1.56-fold higher than that of the wild type. Metabolic profiling showed that among the different changes in metabolic pathways, compared with the parental strain, the citrate cycle and the reductive pyruvate carboxylation pathways under aerobic conditions were simultaneously elevated, thus providing more ATP, reducing power (NADH), and TCA cycle intermediates, which is necessary to maintain cell metabolism and product synthesis (Kenealy et al., 1986).
15.3.2 Where in the Cell is the Localization of the Reductive Reactions of the TCA Cycle?
Evidence for the carbon dioxide fixation mechanism for fumaric acid production came from two sources. Osmani and Scrutton (1985) discovered that R. oryzae
Citrate
Acetyl CoA
Fumarate L-malate Oxaloacetate
Pyruvate
Oxaloacetate Pyruvate
L-malate Fumarate
Citrate Isocitrate αα-ketoglutarate Glucose
Succinate
FUM FUM
CIT
MDH MDH
PYC
PDH
Mitochondrion Cytosol
Extracellular
FIGURE 15.1 The reductive reactions of the tricarboxylic acid (TCA) cycle. CIT, citrate synthase; FUM, fumarase; MDH, malate dehydrogenase; PDH, pyruvate dehydrogenase; PYC, pyruvate carboxylase.
harbors 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 oxaloacetic acid which is converted to l-malic and then to fumaric acid.
Electrophoretic studies of fumarase were carried out inR. oryzae. The analyses revealed two fumarase isoenzymes, one localized solely in the cytosol and the other found both in the cytosol and in the mitochondrial fraction. The activity of the cytoso- lic isoenzyme of fumarase was higher during the acid production stage than during growth. Addition of cycloheximide-inhibited fumaric acid production and decreased the activity of the cytosolic isoenzyme of fumarase. These results suggested thatde novosynthesis is required for the increase in the activity of the cytosolic isoenzyme and that such an increase in activity is essential for fumaric acid accumulation (Peleg et al., 1989b).
But once the notion of a cytosolic reductive TCA pathway is accepted, the question is why the experimentally obtained yields of C4organic acids (120–145%, see above, 15.3.1) are lower than the theoretical maximal yield of 200% calculated for this pathway. A plausible explanation for this is that the fungus must divert some of the pyruvic acid carbon to mitochondria and to the oxidative TCA cycle to obtain energy required for maintenance requirements and limited cellular growth (Figure 15.1).
15.3.3 What is the Role of Cytosolic Fumarase in Fumaric Acid Accumulation inRhizopusStrain?
The operation of the cytosolic reductive TCA pathway for fumaric acid production inR. oryzaeraises the question as to the unique role of the cytosolic fumarase in this fungus.
It was known that the fumarase enzymes in eukaryotic organisms such asSaccha- romyces cerevisiae, rat, and human are encoded in each organism by a single nuclear gene, yet the enzymes are found both in the cytosol and mitochondria. It was found that inS. cerevisiae(which accumulates up to 2 g/L of l-malic acid but not fumaric acid) fumarase translation initiates only from the 5′proximal AUG codon (Sass et al., 2001). Thus, mitochondrial and cytosolic isoforms of yeast fumarase are derivatives of a single translation product and have identical amino acids and kinetic properties (lowKm value for fumaric acid). Whereas these findings can explain l-malic acid accumulation in yeast, an intriguing question that remains to be answered is the mechanism by which large amounts of fumaric acid are produced by the eukaryotic fungusR. oryzae.
To shed more light on this question, the yeast fumarase gene FUM1 was used as a probe to clone a homologous fumarase gene fromR. oryzae. Accordingly, the alignment of the amino acid sequence of thisR. oryzaefumarase with other eukaryotic (class II) fumarases indicated a high degree of sequence similarity (75–80%). The gene designated fumRwas 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, employingS. cerevisiae–fumarase antiserum. However, this protein did not appear to be responsible for the approximately sixfold increase in fumarase activity upon transfer ofRhizopuscells into acid production medium. This is because the level of the protein as detected by western blotting changed only slightly upon this shift (Peleg et al., 1989b; Friedberg et al., 1995). Furthermore; antiserum against yeast fumarase only partially neutralized the fumarase activity inR. oryzaecell extracts, suggesting the existence of an additional nonhomologous enzyme (E. Battat, O. Pines, and I. Goldberg, unpublished data cited in Goldberg et al., 2006).
ThefumRgene (100% identity with the above gene) was cloned first and over- expressed inR. oryzae. All transformants showed significant increase in fumarase activity during both growth and fumaric acid production stages. However, fumarase overexpression inR. oryzaeyielded more malic acid, instead of fumaric acid, in the fermentation because the overexpressed fumarase catalyzed the hydration of fumaric acid to malic acid (Zhang and Yang, 2012).
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. oryzaefrom medium B (growth medium) has a lower Km value for fumaric acid (0.78 mM) than for l-malic acid (2.9 mM), similar to fumarase from lysates ofS. cerevisiae(Pines et al., 1996). 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 fromR. oryzae cells incubated in medium C (production medium), but not withS. cerevisiae, was completely inhibited by 2 mM fumaric acid (E. Battat and I. Goldberg, unpublished data cited in Goldberg et al., 2006).
It should be mentioned that with another Rhizopus strain, Ding et al. (2011) showed in cell extracts, in accordance to previous findings that lowering the urea concentrations in the medium from 2.0 to 0.1 g/L caused an increase of 300% in the cytosolic fumarase activity, accompanied with an increase in fumaric acid production.
However, the activity of fumarase (measured with fumaric acid as the substrate) was not inhibited by fumaric acid.
Based on these results, Goldberg et al. (2006) suggested two possibilities to explain these findings. The first is that inR. oryzaethe cytosolic fumarase is kinetically differ- ent from the mitochondrial isoenzyme, due to distinct posttranslational modifications, or to specific conditions in the two compartments. The second possibility is thatR.
oryzaeharbors 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. Upon transfer into medium C, a fumarase with unique characteristics is induced. l-malic acid’s conversion to fumaric acid is enhanced by the induced fumarase and when the concentration of fumaric acid in the cell exceeds 2 mM, the reverse reaction to l-malic acid is fully inhibited. Thus, this property of the unique fumarase, whose existence was then only hypothesized, can ensure that fumaric acid is accumulated.
This question was further studied by Song et al. (2011) who isolated the fumR gene (GenBank accession number GU013473) by reverse transcription (RT)-(PCR) during the period of maximal fumaric acid accumulation byR. oryzae. The fumR gene was then expressed in recombinantEscherichia coliBL21(DE3) and resulted in the accumulation of a fusion protein. SDS-PAGE analysis confirmed the recombinant fumarase to have an apparent subunit molecular mass of 50 kDa. The specific activity of FUMR in crude extracts was 4.13 U/mg and was 63.52 U/mg for the purified enzyme, using l-malate (50 mM) as a substrate. TheKmvalues of the purified FUMR protein for l-malic acid and fumaric acid were 0.46 and 3.07 mM, respectively. The activity of FUMR catalyzing hydration of fumaric to l-malic acid was completely inhibited by 2 mM fumaric acid.
These elegant results, which were similar in part to those obtained in crude extracts ofR. oryzae, provided a direct proof for the unique enzymatic properties of FUMR.
The lowerKmvalue of FUMR for l-malic acid suggested its preference for conversion of l-malic acid to fumaric acid. Coupled with the observed inhibition of the reverse reaction from fumaric to l-malic acid by concentrations of fumaric acid exceeding 2 mM, would ensure the accumulation of fumaric acid inR. oryzae. However, as Gajewski et al. (1985) and Meussen (2012) pointed out, theΔG0′of the conversion of l-(+)-malic acid to fumaric acid is 3.6 kJ/mol, indicating that at equilibrium, the l-(+)-malic acid concentration is higher than the fumaric acid concentration.
Therefore, a dicarboxylic acid transporter with a high selectivity for fumaric acid may also be required for fumaric acid production inR. oryzae.
Thus, the cytosolic FUMR may be an important step for the accumulation of high concentrations of fumaric acid inR. oryzae. This conclusion can be better confirmed if fumaric acid production will increase as a result of the overexpression of FUMR inR. oryzae(Song et al., 2011; Zhang and Yang, 2012).
In order to further study the possible mechanisms that can explain fumarase distribution inR. oryzae, the deduced amino acid sequences of the two fumarase genes mentioned above were compared (GenBank accession number X78576—Friedberg et al., 1995, and GU013473—Song et al., 2011). The results confirmed that the later fumRhad a deletion of 15-amino acid sequence in the N-terminal region. It was
assumed that the difference in the amino acid sequences may cause the different characterization and distribution of the fumarase isozymes inR. oryzae(Song et al., 2011).
As mentioned above,R. oryzaeis one of the few fumaric acid producer organisms described in the literature which can accumulate more than 100 g/L of fumaric acid as the main product. Thus, it is not surprising to note that the cytosolic fumarase in R. oryzaeappears to have unique characteristics that differentiate it from previously reported cytosolic fumarases from other eukaryotic organisms. For example, the cytosolic fumarase inS. cerevisiaeexhibited a 17-fold higher affinity for fumaric acid than for l-malic acid, while theKmfor l-malic acid was very high. This indicated that the cytosolic fumarase, like the mitochondrial enzyme, catalyzed the conversion of fumaric to l-malic acid and not the reverse reaction, which explains the fact that this yeast accumulates small amounts of l-malic acid but not fumaric acid (Peleg et al., 1990; Pines et al., 1996). A similar conclusion was drawn for the mechanism responsible for l-malic acid accumulation (fumaric acid comprises only up 2% of l-malic acid) inAspergillus flavus (Peleg et al., 1988; Battat et al., 1991). While these findings strengthen the important role of the cytosolic fumarase in fumaric acid production byR. oryzae, more research is needed to ascertain if indeed the difference in 15-amino acids is responsible for the role of this enzyme in this unique fungus.