Section Molecular Genetics of Industrial Micro-organisms, Wageningen Agricultural University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands1
Author for correspondence: Jaap Visser. Tel: +31 317 484439. Fax: +31 317 484011. e-mail: office{at}algemeen.mgim.wau.nl
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ABSTRACT |
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Keywords: oxalic acid, citric acid, oxaloacetate acetylhydrolase, acetyl-CoA synthase, A. niger
Abbreviations: ACS, acetyl-CoA synthase (EC 6 . 2 . 1 . 1); OAH, oxaloacetate acetylhydrolase (EC 3 . 7 . 1 . 1)
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INTRODUCTION |
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Several pathways have been described for oxalic acid production. In A. niger (Hayashi et al., 1956 ; Müller, 1975
; Lenz et al., 1976
) and in a number of other fungi (Dutton & Evans, 1996
), as well as in some Streptomyces species (Houck & Inamine, 1987
), the current evidence favours production of oxalic acid by a Mn2+-dependent enzyme, oxaloacetate acetylhydrolase (EC 3 . 7 . 1 . 1) (OAH). In A. niger this enzyme is localized in the cytoplasm (Kubicek et al., 1988
), where it catalyses the following reaction:
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Cleland and & Johnson (1956 ) postulated that a second pathway should exist in A. niger which generates oxalate from pentoses via glycolate and glyoxylate as intermediates. This route requires operation of glyoxylate dehydrogenase as the final step to oxidize glyoxylate to oxalate, but attempts to measure glyoxylate-oxidizing enzymes in extracts of A. niger that produced oxalate were unsuccessful (Müller, 1975
).
The physiology of oxalic acid production has been studied to some extent (Kubicek, 1987 ), but some details are lacking, e.g. a proper investigation of the effect of pH has not been performed. Production of oxalate has been reported to be optimal in the pH range of 58 (Cleland & Johnson, 1956
; Lenz et al., 1976
; Kubicek et al., 1988
). In most cases information about oxalate biosynthesis has only been obtained under conditions also leading to the synthesis of other organic acids, in particular gluconic acid. In this study we followed another approach using an A. niger mutant lacking glucose oxidase.
In several processes employing A. niger, such as citric acid production or production of enzymes, oxalate may arise as an unwanted by-product for a number of reasons. First, it may decrease yield of the intended product. Another reason is that oxalate complicates recovery of the product because additional steps are required to remove the acid. Since oxalic acid is toxic, its removal is particularly important from products that have applications in food or medicine. In this report we describe a number of features of oxalic acid production by A. niger and, in addition, we present data on an A. niger mutant that is unable to produce oxalic acid and demonstrate its use in citric acid production.
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METHODS |
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Media and culture conditions.
Conidiospores were propagated on complete medium (Pontecorvo et al., 1953 ) solidified with 1·5% (w/v) agar and containing 50 mM glucose. Spores were harvested from the agar slopes with 0·05% (w/v) Tween 80. Cultures were inoculated with spores to a final concentration of 106 ml-1. For isolation and analysis of acu mutants mycelium was cultured on minimal medium (MM) (Pontecorvo et al., 1953
) containing 0·02% (v/v) of a trace metal solution (Vishniac & Santer, 1957
) and appropriate carbon sources. For plate tests MM was solidified with 1·5% (w/v) agar. Production of acids was studied in 3 litre jacketed stirred tank reactors (Applikon) using two different media. PM medium contained, per litre: 1·2 g NaNO3,0·5 g KH2PO4, 0·2 g MgSO4.7H2O, 0·5 g yeast extract, 0·04 ml of a trace metal solution (Vishniac & Santer, 1957
) and carbon source as indicated in the legends of tables and figures. Unless indicated otherwise the culture pH was 5. Culture pH was controlled by automatic addition of either 2 M HCl or 5 M NaOH. Cultures were sparged with 0·2 v.v.m. air, while addition of pure oxygen was used to keep the dissolved oxygen tension above 30% air saturation. A medium optimized for citric acid production (CAF) was described earlier (Ruijter et al., 1997
). In bioreactor cultures 0·5 ml 30% polypropylene glycol in alcohol was added per litre of medium as antifoam. Where necessary media were supplemented with, per litre, 4 µg biotin, 0·2 g arginine, 0·2 g phenylalanine, 0·1 mg pyridoxine, 1 mg nicotinamide, 0·2 g tryptophan, 0·2 g histidine, 0·365 g lysine and 1·22 g uridine. In all experiments A. niger was cultured at 30 °C.
Preparation of cell extracts and enzyme assays.
Mycelium was collected from a culture sample by filtration under vacuum, washed three times with approximately 50 ml 10 mM potassium phosphate buffer pH 7·0 and frozen in liquid nitrogen. For each sample approximately 0·5 g of the frozen mycelium was powdered using a micro-dismembrator (B. Braun Biotech) and suspended in 1 ml extraction buffer at 0 °C. For ACS, extraction was done in 100 mM potassium phosphate pH 7 containing 1 mM EDTA, whereas OAH was extracted in 50 mM potassium phosphate pH 7 containing 0·5 mM EDTA, 5 mM 2-mercaptoethanol, 5 mM MgCl2, 10% (v/v) glycerol. Following centrifugation at 15000 g for 5 min enzyme activities were assayed in the resulting supernatant. Enzyme assays were performed at 30 °C using a Shimadzu UV2501 spectrophotometer. Biochemicals were from Boehringer or Sigma. ACS activity was assayed in 75 mM triethanolamine pH 8·4 containing 7·5 mM MgCl2, 20 mM malate, 0·4 mM NAD+, 2·5 mM ATP, 0·17 mM CoA, 1 U citrate synthase ml-1, 100 U malate dehydrogenase ml-1, 20 mM acetate. OAH activity was measured using direct optical determination of oxaloacetate at 255 nm as described by Lenz et al. (1976 ).
Purification of OAH.
OAH was purified from strain NW131 cultured for 28 h on PM medium (see above) containing 2% (w/v) glucose at pH 6. Harvest and disruption of mycelium was done as described under Preparation of cell extracts and enzyme assays. Powdered mycelium (8 g) was suspended in 50 ml extraction buffer containing 50 mM Bistris pH 7·0, 5 mM MgCl2, 0·5 mM EDTA, 5 mM 2-mercaptoethanol and 10% (v/v) glycerol. The resulting suspension was centrifuged at 10000 g for 10 min at 4 °C. To the supernatant, (NH4)2SO4 was added to 40% saturation. Precipitation of protein was allowed to occur for 20 min at 4 °C with gentle mixing. To the supernatant obtained after centrifugation for 10 min at 10000 g and 4 °C, (NH4)2SO4 was added to obtain 50% saturation. Following 20 min incubation at 4 °C and another centrifugation step the precipitated protein, which contained OAH, was dissolved in 3 ml extraction buffer and applied to a Sephacryl S-300 (Pharmacia Biotech) column (90 cmx5 cm2) which was pre-equilibrated with extraction buffer. Fractions containing OAH activity were pooled and applied to a 1 ml Resource Q (Pharmacia Biotech) column. Following rinsing of the column with extraction buffer, adsorbed protein was eluted with a 00·5 M NaCl gradient in extraction buffer over 20 column volumes. Fractions containing OAH activity were pooled and rechromatographed on Resource Q applying a 00·5 M NaCl gradient over 40 column volumes. Fractions having OAH activity were stored at -70 °C.
Analytical methods.
Denaturing electrophoresis in 10% (w/v) polyacrylamide gels containing 0·1% (w/v) SDS was performed as described by Laemmli (1970 ) in a Mini-V system (Life Technologies). Molecular mass markers were phosphorylase b (92·5 kDa), BSA (68 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). For immunochemical detection, protein was blotted onto nitrocellulose filters and blots were then incubated with specific antisera, followed by staining with alkaline phosphatase labelled goat anti-mouse IgG as described by the manufacturer (Bio-Rad). Antibodies against A. niger OAH were prepared as described previously (Van der Veen et al., 1991
). Sugars and organic acids were analysed by HPLC using an Aminex HPX-87H (Bio-Rad) column eluted with 25 mM HCl at 50 °C and using UV (210 nm) and RI (refractive index) detection. Sugars and citric acid were, in addition, determined enzymically according to Bergmeyer (1985
). Fungal dry weight and protein were determined as described earlier (Ruijter et al., 1997
).
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RESULTS |
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In basidiomycetes, the type and concentration of nitrogen source is known to affect oxalate production (e.g. Micales, 1994 ; Kuan & Tien, 1993
). Using 2% glucose as a carbon source, an increase in the NaNO3 concentration from 6 to 60 mM resulted in an increase of molar yield from 0·5 to 0·54, with a concomitant increase in dry weight from 3·2 to 4·2 g l-1. Using 28 mM of NH4Cl instead of NaNO3 the molar yield was 0·5 (dry weight 5·5 g l-1). Thus, the type and concentration of the nitrogen source did not affect the molar yield of oxalic acid very much. These data are in accordance with the findings of Müller (1965
), who reported production of 4·36·3 g oxalate l-1 from 50 g glucose l-1 in buffered medium when the KNO3 content was varied between 12 and 100 mM.
The environmental factor that influenced production of oxalic acid most was pH (Fig. 1). Using goxC strain NW131 oxalic acid production was similar between pH 4 and 6 (approx. 190 mM oxalate from 110 mM glucose). Below pH 4 the amount of oxalic acid produced decreased. At pH 2, 4050 mM was still being produced from 110 mM glucose, but at pH 1·5 oxalic acid production was very low (<1 mM). Production of oxalic acid correlated qualitatively to the OAH activity measured in cell extracts (Fig. 1
). At culture pH 36 the OAH activity was 3·55 U mg-1, whereas below pH 3 the activity decreased markedly. At culture pH 1·5 OAH activity was not detectable.
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Strain N573 did not produce acetate from fructose, whereas approximately 200 mM oxalate is produced (Fig. 3a). In contrast, strain NW254, carrying acuA5, transiently produced acetate (Fig. 3b
). During consumption of fructose, acetate slowly accumulated to a maximal concentration of 21 mM in the culture broth, but was reconsumed after exhaustion of the fructose. The rates of acetate consumption can be calculated from the data. Between 30 and 46 h of culturing, the acuA5 mutant produced 1·06 mmol oxalate (g dry wt)-1 h-1 and 0·12 mmol acetate (g dry wt)-1 h-1. With OAH producing equimolar oxalate and acetate, the difference is the acetate consumption by the mutant: 0·94 mmol (g dry wt)-1 h-1. Between 46 and 54 h there is net consumption of acetate [0·33 mmol (g dry wt)-1 h-1], but still production of oxalate and concomitantly acetate [0·57 mmol (g dry wt)-1 h-1]. The difference [0·9 mmol (g dry wt)-1 h-1] is again the acetate consumption rate, which closely matches the rate calculated for the period 3046 h. Since ACS activity was not detectable, the remaining acetate catabolism probably proceeds via another pathway, which is unknown at present.
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A recombinant strain was constructed that combined the acuA5 and prtF28 mutations. This strain did not produce any acetate from fructose, suggesting that the acetate produced by the acuA5 mutant is associated with oxalate production, i.e. formed by OAH.
Citric acid production by a prtF mutant
The finding that the prtF28 mutant produced a reasonable amount of citric acid from 2% (w/v) fructose at pH 5 prompted us to investigate citric acid production by this strain. For this purpose a recombinant, NW185, was constructed that lacked both glucose oxidase (goxC17) and OAH (prtF28). In a traditional citric acid production process (i.e. pH<2, no Mn2+) strain NW185 performed comparably to a strain not carrying the prtF mutation (NW131) using glucose as the substrate (Table 2). However, at pH 5 in a minimal medium containing a mixture of trace metals (PM), NW131 produced a large amount of oxalic acid in addition to citric acid, whereas NW185 produced only citric acid (Table 2
). The amount of citric acid produced by NW185 in PM at pH 5 is even slightly higher than observed in the traditional citric acid fermentation. We tested a number of different conditions (e.g. using glucose, fructose or sucrose as the substrate, regulation of pH at 5 or no pH regulation) and found that production was optimal from sucrose at pH 5. Under these conditions NW185 produced approximately 90 g citric acid from 140 g sucrose in 10 d (Fig. 4
). PM contains 1 µM Mn2+ (55 p.p.b.), which is well above the concentration recommended in citric acid fermentation (<1 p.p.b.; Mattey, 1992
). To test whether NW185 was sensitive to Mn2+ we again performed fermentation in PM at pH 5 using sucrose, but 50 µM MnCl2 (2·7 p.p.m.) was now added. Surprisingly, citric acid production was almost identical to that in the fermentation with 1 µM MnCl2 (Fig. 4
). Thus, it appears that with the prtF mutation citric acid can be produced at relatively neutral pH and production is then completely insensitive to the presence of Mn2+. Like a goxC strain, a goxC prtF strain does not produce any citric acid in a traditional citric acid fermentation medium to which 50 µM MnCl2 was added (data not shown). This implies that the requirement for Mn2+ deficiency is related to the specific conditions in a traditional citric acid fermentation and not to the prtF mutation. Only at relatively neutral pH is the absence or presence of Mn2+ no longer relevant.
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DISCUSSION |
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In traditional citric acid production processes two very important conditions to obtain a good fermentation are low pH (<2) and absence of Mn2+. Low pH is important mainly to prevent production of gluconic acid and oxalic acid. A low starting pH of the medium might easily be obtained with relatively clean substrates, such as glucose syrups, but is more difficult to achieve in crude media, such as molasses, which have a high buffering capacity at pH 56. A mutant lacking both glucose oxidase (goxC) and OAH (prtF) produced citric acid at relatively neutral pH and could therefore be particularly advantageous in production processes making use of molasses. An additional advantage is that it is no longer necessary to remove metal ions by, for example, potassium ferrocyanide treatment or cation-exchange treatment of the substrate, because, in the media tested, the mutant is completely insensitive to the presence of Mn2+. Although we have shown here the usefulness of an oxalate non-producing strain for citric acid production, it is clear that such a strain will also be valuable in other processes employing A. niger, such as production of enzymes. In particular, downstream processing may be simplified when oxalate is no longer produced. Besides applying mutagenesis and selection of oxalate-negative mutants, it is obvious that cloning of the oah gene and subsequent gene disruption provides a straightforward approach to eliminate oxalate production.
The main conclusions from the work described in this paper are: (1) The external pH was the main factor controlling oxalic acid production by A. niger. (2) An oxalate non-producing A. niger mutant lacked OAH, implying that OAH is the only enzyme involved in oxalate production in A. niger. (3) A strain lacking both glucose oxidase (goxC) and OAH (prtF) produced citric acid from sugar substrates in a regular synthetic medium at pH 5 and under these conditions production was completely insensitive to Mn2+.
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ACKNOWLEDGEMENTS |
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Received 1 February 1999;
revised 28 April 1999;
accepted 10 May 1999.