Construction and Characterization of an Oxalic Acid Nonproducing Strain of Aspergillus niger

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Metabolic Engineering 2, 34�41 (2000)
Construction and CharacterizationStrain of Asp

Henrik Pedersen and

Center for Process Biotechnology, Department of Biotechnology

Carsten

Novo Nordisk A�S, DK-2

an

Jens N

Center for Process Biotechnology, Department of Biotechnology

Received May 12, 1999;

Aspergillus niger produces oxalic acid as a by-product whichcauses problems with downstream processing of industrial enzymes.To overcome this problem the oah gene encoding oxaloacetatehydrolase (EC 3.7.1.1) was disrupted in a glucoamylase-producingstrain of A. niger and the resulting strain was incapable of producingoxalic acid. The strain with the disrupted gene was compared withthe wild-type strain producing oxalic acid in batch cultivations. Thespecific growth rate of both strains was 0.20 h&1. The citric acidyields were identical, but the glucoamylase yield was only 500 inthe disruptant compared with the wild-type strain. Batch experimentswith 13C-labeled glucose as substrate were carried out to determinethe metabolic fluxes through the central metabolism. The two strainshad almost identical metabolic fluxes, which suggested that it waspossible to disrupt the oah gene without pleiotropic consequences.The flux through the pentose phosphate pathway was around 600of the glucose uptake for both strains, which suggested that asufficient supply of NADPH was available for biosynthesis. � 2000

Academic Press

INTRODUCTION

The filamentous fungus Aspergillus niger is known toproduce many industrially important organic acids, includ-ing gluconic, malic, and citric acid in high amounts(Bercovitz et al., 1990). As a by-product during citric acidproduction, A. niger also produces oxalic acid, which needsto be removed in the final purification of the product (Roehret al., 1992). A. niger strains used for production of

Article ID mben.1999.0136, available online at http:��www.idealibrary.com

1 Present address: Department of Biotechnology, Building 223, Techni-cal University of Denmark, 2800 Lyngby, Denmark. Fax: +45 45 88 41 48.E-mail: jn�ibt.dtu.dk.

341096-7176�00 �35.00Copyright � 2000 by Academic PressAll rights of reproduction in any form reserved.

of an Oxalic Acid Nonproducingrgillus niger

Bjarke Christensen

Technical University of Denmark, DK-2800 Lyngby, Denmark

Hjort

80 Bagsvaerd, Denmark

d

elsen1

Technical University of Denmark, DK-2800 Lyngby, Denmark

ccepted October 6, 1999

industrial enzymes also produce oxalic acid (Pedersen et al.,1999a), and Streptomyces cattleya produces oxalic acid dur-ing antibiotic production (Houck and Inamine, 1987). Theoxalic acid formed in a cultivation designed for citric acid,enzyme, or antibiotic production is a major problem since(1) it is toxic, (2) it represents a loss of carbon, and (3) itforms precipitates that cause problems in the downstreamprocessing.

Oxaloacetate hydrolase (EC 3.7.1.1), which cleavesoxaloacetic acid to oxalic acid and acetic acid, has beenshown to be the only pathway leading to oxalic acid forma-tion in A. niger (Kubicek et al., 1988). The oah gene encod-ing oxaloacetate hydrolase has been cloned from A. niger(Pedersen et al., 1999c). To overcome the problems of oxalicacid formation in A. niger, we disrupted the oah gene from aglucoamylase-producing A. niger strain. Both the wild-typestrain and the strain in which oah was disrupted were charac-terized in batch cultivations. To verify that the disruption ofthe oah gene from A. niger did not have drastic effects onmetabolism, the distribution of the metabolic fluxes throughthe central carbon metabolism was quantified using the con-cept of metabolite balancing (Vallino and Stephanopoulos,1993) combined with information from 13C-labeling pat-terns of intracellular metabolites (Marx et al., 1996).

MATERIALS AND METHODS

Strains and Plasmids

The A. niger strains used were the glucoamylase-produc-ing BO-1 (kindly donated by Novo Nordisk A�S) and the

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pyrG mutant JRoy3. Escherichia coli DH5: was used forroutine plasmid propagation (Woodcock et al., 1989).Vectors pUC19 (Yanisch-Perron et al., 1985) and pCRII(Invitrogen, Carlsbad, CA) were used. Plasmid pJRoy10,which is the pyrG gene from A. niger inserted in the pUC19vector, was used to construct the disruption plasmid pHP3.JRoy3 and pJRoy10 were kindly donated by J. Royer, NovoNordisk A�S.

Construction of the Disruption Plasmid pHP3

Plasmid pHP1 (Pedersen et al., 1999c) was digested withNruI and BstEII and the 6.0-kb fragment was isolated.pJRoy10 was digested with NcoI, and the 5$ recessive endswere then filled in with the Klenow fragment of DNApolymerase I and digested with BstEII. The 2948-bp frag-ment was isolated and ligated to the pHP1 fragment.The ligation mixture was used to transform E. coli DH5:.A colony harboring the desired plasmid was identified, andthe plasmid was termed pHP3 (Fig. 1).

Southern Blot Analysis

Genomic DNA was digested with BglII and subjected toSouthern blot analysis. One blot was probed with a 219-bpfragment of the oah gene (Fig. 2A). An identical blot wasprobed with a 2.9-kb NcoI�BstEII fragment of pJRoy10that encodes pyrG (Fig. 2B).

Media and Culture Conditions

The Aspergillus strains were grown in a defined mediumcontaining: 15 g liter&1 glucose monohydrate, 7.3 g liter&1

(NH4)2 SO4 , 1.5 g liter&1 KH2PO4 , 1.0 g liter&1 MgSO4

} 7H2O, 1.0 g liter&1 NaCl, 0.1 g liter&1 CaCl2 } 2H2O,0.5 ml liter&1 sb2121 (antifoam, Schill 6 Seilacher,Hamburg, Germany), 7.2 mg liter&1 ZnSO4 } 7H2O, 1.3 mgliter&1 CuSO4 } 5H2O, 0.3 mg liter&1 NiCl2 } 6H2O, and6.9 mg liter&1 FeSO4 } 7H2O.

The 1-liter bioreactor was equipped with stirring, pH,and temperature control. The pH was kept constant byaddition of 4 M NaOH. The 1-liter bioreactor wasinoculated with approximately 109 spores per liter. Theagitation in the 1-liter bioreactor ranged from 50 rpm whenthe bioreactor was inoculated to 1100 rpm at the end of thecultivation. To avoid pellet formation caused by agglomera-tion of spores, the pH was set to 2.5 at the time of inocula-tion. The pH was gradually increased to 6.0 when the sporesgerminated. Growth in the 1-liter bioreactor lasted about35�40 h for cultivation of both BAN25 and BAN40.

Oxalic Acid Nonproducer

When the biomass concentration had reached 2�5 gliter&1, 10�20 ml of the culture was inoculated into 400 ml

35

of defined medium containing 1000 [1-13C]glucose as thecarbon source in a 450-ml bioreactor equipped with stirring,pH, and temperature control and measurement of the dis-solved oxygen tension. In the inoculation step somenaturally labeled glucose was transferred from the 1-literbioreactor and this also served as a carbon source. The airused for aeration was stripped of CO2 by passing it through4 M NaOH to prevent naturally labeled CO2 from enteringthe bioreactor. The pH was kept constant at 6.0 by additionof 4 M NaOH, and the dissolved oxygen tension was keptabove 700 saturation (air containing 210 O2 at 30%C).Growth in the 450-ml bioreactor lasted about 18 h forcultivation of both BAN26 and BAN41.

For analysis of transformants, they were grown in definedmedium containing: 20 g liter&1 glucose monohydrate, 15 gliter&1 NaNO3 , 1.5 g liter&1 KH2PO4 , 1 g liter&1 MgSO4

} 7H2O, 1 g liter&1 NaCl, 0.1 g liter&1 CaCl2 } 2H2O,7.2 mg liter&1 ZnSO4 } 7H2O, 1.3 mg liter&1 CuSO4 }5H2O, 0.3 mg liter&1 NiCl2 } 6H2O, and 6.9 mg liter&1

FeSO4 } 7H2 O in 96-well microtiter dishes for 48 h at 34%C,pH 6.0.

Sampling

Samples were taken throughout the exponential growthphase. For biomass measurements the samples were filtered,washed with 0.90 (w�v) NaCl, and dried at 105%C for 24 to48 h. The filtrate was used for measurement of glucoamylaseactivity.

Glucoamylase Activity

Glucoamylase activity in culture filtrates was quantifiedspectrophotometrically using p-nitrophenyl-:-D-gluco-pyranoside (pNPG) as substrate. Analysis was performedon a Cobas Mira (Roche Diagnostics, Basel, Switzerland)analyzer with the following modified assay (Holm, 1986):A 50-+l volume of diluted sample or standard (0�25 U_

ml&1) was mixed with 100 +l pNPG (2 g liter&1) in 0.1 Macetate buffer, pH 4.3, and incubated at 37%C for 20 min.The reaction was stopped by addition of 95 +l 0.1 MNa2CO3 and the concentration of p-nitrophenol (*=405 nm) was used as a measure of the glucoamylase activityin the sample. The influence of yellow-colored by-productsin filtrates could be taken into account by calculating thechange in absorbance as 2 Abs=Abs20 min-150�245 }Abs0 min . The absolute amount of glucoamylase wascalculated assuming that all protein in the used standardwas glucoamylase and with a protein content of 25 +g

Metabolic Engineering 2, 34�41 (2000)Article ID mben.1999.0136

protein per unit glucoamylase (PESL protein assay,Boehringer-Mannheim, Mannheim, Germany).

Oxalic Acid Assay

Supernatants from growth in microtiter dishes wereanalyzed for oxalic acid using an oxalate kit (Sigma, St.Louis, MO).

Quantification of Sugars and Metabolites

Extracellular metabolites and carbohydrates wereanalyzed by an isocratic HPLC method using a HPX-87HAminex ion-exclusion column. The column was eluted at60%C with 5 mM H2SO4 at a flow rate of 0.6 ml min&1. Thedetectors were a Waters 410 refractive index detector and aWaters 486 tunable absorbance detector (Nissen et al.,1997).

Analysis of the 13C-Labeling Patterns of Amino Acids

All procedures were carried out according to the methodof Christensen and Nielsen (1999). The biomass used foranalysis was in exponential growth phase. To release aminoacids from biomass, crude biomass was hydrolyzed by treat-ment with 6 M HCl at 105%C for 18 h. The amino acids werederivatized by either ethylchloroformate�trifluoroaceticanhydride or N,N-dimethylformamide dimethyl acetal andanalyzed in a Hewlett�Packard HP G1723A gas chromato-graph�quadrupole mass selective detector (EI) operated at70 eV. The column was a JW-1701 (30 m, 250-+m i.d.,0.15-+m film thickness).

GC�MS measures the mass isotopomer distribution ofthe various ion clusters. To convert the mass isotopomerdistribution of an ion cluster into so-called summed frac-tional labelings (SFL), the following formula was used,where mi is the intensity of the (m+i)-mass isotopomer.

SFL=�n

i=0 i } mi

�nj=0 mj

(1)

The value calculated in Eq. (1) represents the sum of thefractional labelings in the carbon atoms contained in theion. For example, the summed fractional labeling of theglycine molecular ion can be calculated as follows, wherem j�k

i is the intensity of the (m+i) mass isotopomer contain-ing carbon atoms j through k of glycine. The fractionallabelings of C1 and C2 of glycine are denoted x(1) and x(2),respectively.

SFL(Gly12)=x(1)+x(2)=0 } m1�2

0 +1 } m1�21 +2 } m1�2

2


m1�20 +m1�2

1 +m1�22

(2)

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Since the values calculated using Eq. (1) represent sumsof fractional labelings, the upper limit for a fragment con-taining n carbon atoms is equal to n times 1000. It shouldbe noted that a correction step for the natural abundance ofatoms introduced by the derivatization was performed priorto the calculations given in Eq. (1) and Eq. (2) (seeWittmann and Heinzle, 1999).

Calculation of Metabolic Fluxes

The metabolic model that describes the centralmetabolism of A. niger is shown in Appendix A. The inputsto the model were (1) a biomass composition, which wastaken to be the same as for Aspergillus oryzae CF 1.1 grow-ing at a specific growth rate of 0.15 h&1 (Pedersen et al.,1999b), (2) the actual biomass yield on glucose, (3) thecitrate and oxalate yields, (4) the 13C-labeling patterns ofamino acids and glucose, and (5) the size of the inoculum.

Glucoamylase formation was not included in the model,since glucoamylase only represents about 60 of the totalprotein (intracellular+extracellular).

RESULTS AND DISCUSSION

Construction of an Oxalic Acid Nonproducing Strain

Protoplasts of the pyrG-deficient A. niger BO-1 derivativeJRoy3 were transformed with a 4.7-kb EcoRI�NotI frag-ment of pHP3 (see Fig. 1) and plated on agar containing aminimal medium with 1 M sucrose. The 275 transformantsobtained were reisolated twice and then tested for oxalicacid production in microtiter dishes. Eight transformantswere found not to produce oxalic acid. Six of those were

Pedersen et al.

FIG. 1. The pHP3 plasmid used to disrupt the oah gene.

subjected to Southern blot analysis together with two oxalicacid-producing transformants, BO-1 and JRoy3, as positivecontrols. The Southern blot probed with oah (Fig. 2A)revealed that the band corresponding to oah was absent in

FIG. 2. (A) A Southern blot probed with the 219-bp fragment of theoah gene. Lanes 1 to 10: genomic DNA cut with BglII. Lane 1, JRoy3(oxalic acid producer); lane 2, BO-1 (oxalic acid producer); lane 3, 10-22;lane 4, 10-11; lane 5, 9-13 (oxalic acid producer); lane 6, 9-1; lane 7, 8-8;lane 8, 4-1 (2oah); lane 9, 3-5 (oxalic acid producer); lane 10, 1-8. (B)A Southern blot probed with a 2.9-kb NcoI�BstEII fragment of pJRoy10containing the pyrG gene. Lane 1, JRoy3 (oxalic acid producer); lane 2,


BO-1 (oxalic acid producer); lane 3, 10-22; lane 4, 10-11; lane 5, 9-13(oxalic acid producer); lane 6, 9-1; lane 7, 8-8; lane 8, 4-1 (2oah); lane 9,3-5 (oxalic acid producer); lane 10, 1-8.

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the six oxalic acid-negative strains and present in the strainsproducing oxalic acid. When the transformation cassetteintegrates correctly, i.e., by homologous recombination,and disrupts the oah gene, the expected band sizes fromgenomic DNA digested with BglII and probed with pyrGare 2.90 and 2.46 kb. These band sizes are indeed foundexperimentally in the oxalic acid-negative strains (seeFig. 2B). A band of 1.88 kb was expected for BO-1, whichwas found experimentally (see Fig. 2B). It was thereforeconcluded that the examined oxalic acid-negative strainswere disrupted in the oah gene.

The attempt to disrupt the oah gene from A. niger wassuccessful and resulted in a strain incapable of producingoxalic acid. This finding confirms that the pathwaycatalyzed by oxaloacetate hydrolase (EC 3.7.1.1) is the onlypathway leading to oxalic acid formation in A. niger as pre-viously found by Kubicek et al. (1988).

Characterization of Oxalic Acid-Producing andNonproducing Strains

Figure 3 shows the oxalic acid and citric acid yields fromseveral batch cultivations with BO-1 at various pH values.The expression of the oah gene in A. niger BO-1 has pre-viously been shown to be influenced by pH in the growthmedium, with increasing expression with increasing pH(Pedersen et al., 1999c). Also, the oxalic acid yield has beenshown to be influenced by pH in the medium (Kubiceket al., 1988). The data in Fig. 3 seem to be concurrent withthese findings but the scatter of the data in Fig. 3 shows thatthe oxalic acid yield in batch cultivations is controlled byother factors (e.g., the dissolved oxygen tension) than pH inthe growth medium. Since we did not measure the dissolvedoxygen tension during the cultivations, it cannot be ruledout that the variations in the oxalic acid yields were caused

FIG. 3. Oxalic acid and citric acid yields from batch cultivations with


BO-1 at different pH values. Oxalic acid yield, [circles]; citric acid yield,[triangles].

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TAB

Physiological Paramet

Labeling of +max

Cultivation Strain glucose [h&1] [(

BAN25 BO-1 Naturally 0.17BAN26 BO-1 [1-13C]-Glucose 0.19BAN40 4-1 Naturally 0.23BAN41 4-1 [1-13C]-Glucose 0.18

Note. The yields and specific growth rates are calculated from at least f\ the standard deviation determined from 12 batch cultivations with the BBO-1 strain determined from 12 cultivations is 0.19\0.02 h&1. BAN25 anhours before transfer of biomass to BAN26 and BAN41, respectively.

a The low pH (2.5) throughout most of BAN25 explains why no oxalic apH 6.0 with [1-13C]-glucose as carbon source.

TAB

Derivatized Ion cluster measured incomponent mass spectrometer (m�z) Intermediat

Glucose 331 Glucose-6-PGlycine 175 GlycineGlycine 144 GlycineGlycine 85 GlycineSerine 174 SerineSerine 228 SerineSerine 256 SerineAlanine 116 PyruvateAlanine 99 PyruvateAlanine 158 PyruvateLeucine 158 Acetyl-CoA and (pyruvate)Valine 144 PyruvateValine 143 PyruvateValine 127 PyruvateValine 186 PyruvateAspartate 188 OxaloacetateAspartate 115 OxaloacetateAspartate 216 OxaloacetateIsoleucine 158 Oxaloacetate and (pyruvate)Threonine 175 OxaloacetateGlutamate 143 :-KetoglutarateGlutamate 230 :-KetoglutarateProline 142 :-KetoglutarateLysine 156 Acetyl-CoA and (:-ketoglutarHistidine 327 Ribose-5-P and (C1)Phenylalanine 192 Erythrose-4-P and (phosphoePhenylalanine 143 Phosphoenolpyruvateb

Oxalate 111 Oxalate

a Carbon atoms Nos. refer to the intermediate carbon positions that are pfrom two different intermediates. In these cases, the numbers in parenthesesused: glucose-6-P, glucose 6-phosphate; ribose-5-P, ribose 5-phosphate; er


yCoA, acetyl-coenzyme A.

b In the calculations, phosphoenolpyruvate and pyruvate were treated as o

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E 1

rs for the Cultivations

iomass yield on Citric acid yield on Oxalic acid yieldglucose biomass on biomass

dry wt)(g glc)&1] [mg(g dry wt)&1] [mg(g dry wt)&1]

0.51 38 0a

0.54 26 340.57 34 00.58 21 0

e measurements from the same cultivation. The average yield of citric acid-1 strain is 24\6 mg (g dry wt)&1. The average specific growth rate of theBAN40 were inoculated at pH 2.5 and the pH was increased to 6.0 three

cid was formed in this cultivation. BAN26 and BAN41 were carried out at

E 2

Wild type DisruptantCarbon atoms Nos.a (BO-1) (4-1)

1, 2, 3, 4, 5, 6 90.2 861, 2 7.2 9.41, 2 7.1 9.1

2 3.2 4.31, 2 4.2 5.12, 3 23.9 22.6

1, 2, 3 27.4 25.42, 3 27.0 27.22, 3 26.8 26.7

1, 2, 3 28.3 28.32, (2), (2), (3), (3) 74.1 72.4

2, 2, 3, 3 51.9 51.21, 2 3.3 4.2

2, 2, 3, 3 51.4 50.71, 2, 2, 3, 3 52.8 52.4

2, 3, 4 45.6 44.22 11.9 12.5

1, 2, 3, 4 56.5 56.32, 3, 4, (2), (3) 71.3 69.2

1, 2 19.3 20.61, 2 31.9 30.5

1, 2, 3, 4, 5 71.3 70.92, 3, 4, 5 56.4 54.9

te) 2, (2), (3), (4), (5) 78.2 76.61, 2, 3, 4, 5, (1) 51.5 39.2

olpyruvate) 1, 2, 3, 4, (2), (2), (3) 70.1 66.61, 2 �� 3.01, 2 19.0 ��

esent in the ion. Some of the measured components consist of carbon atomsrefer to the carbon atoms of the intermediate in parentheses. Abbreviationsthrose-4-P, erythrose 4-phosphate; C1, methylenetetrahydrofolate; acetyl-

Pedersen et al.

ne metabolite.

by differences in dissolved oxygen tension (Roehr et al.,1992). In cultivation of BAN25, the pH was 2.5 throughoutmost of the cultivation, which explains the lack of oxalicacid formation.

The BO-1 strain and the oah disruptant 4-1 were com-pared in batch cultivations. Table 1 shows specific growthrates, biomass yields, and citrate and oxalic acid yields fortwo batch cultivations with BO-1 and 4-1, respectively. Dis-ruption of the oah gene did not influence the maximumspecific growth rate in batch cultivations. The yield of citricacid is also not affected by disruption of the oah gene. Theglucoamylase yield in the disruptant 4-1 is about 500 of theglucoamylase yield in BO-1 and about 800 of the gluco-amylase yield in JRoy3 (data not shown). It is unlikely thatthe disruption of oah has caused the decrease in theglucoamylase yield since the glucoamylase yield in BO-1 isindependent of whether there is oxalic acid production ornot (Pedersen et al., 1999a).

FIG. 4. Metabolic fluxes in the central metabolism of A. niger BO-1 an


dnumbers to the left are for BO-1 and the numbers to the right (italic) arP, phosphate; acetyl-CoA, acetyl-coenzyme A; ext, extracellular; PP-pathwa

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4-1 during batch cultivation (BAN26 and BAN41). For each reaction, the

The 13C-labeling patterns of several metabolites from thecentral metabolism of both BO-1 and 4-1 are shown inTable 2. The derived net metabolic fluxes through the cen-tral metabolism of the two strains are shown in Fig. 4. Themetabolic fluxes in the central metabolism of BO-1 and 4-1were almost identical, which shows that the disruption ofoah has not altered the metabolism significantly, except forthe production of oxalic acid. The main differences betweenthe flux distributions in the two strains are in the productionof oxalic acid. The flux through the pentose phosphatepathway is 58 and 670 of the glucose uptake for BO-1 and4-1, respectively. For a recombinant A. oryzae grown in achemostat at a specific growth rate of 0.15 h&1, the fluxthrough the pentose phosphate pathway was calculatedusing metabolite balancing to be 470 of the glucose uptake(Pedersen et al., 1999b). Schmidt et al. (1998), based on 13Ctracer experiments combined with NMR analysis, haveestimated the pentose phosphate pathway flux to be 530 of


e for 4-1. Fluxes are normalized with respect to the glucose uptake rate.y, pentose phosphate pathway.

the glucose uptake in A. oryzae grown in a chemostat at aspecific growth rate of 0.1 h&1. Using metabolite balancing,Henriksen et al. (1996) found a pentose phosphate pathwayflux of 610 of the glucose uptake rate for Penicilliumchrysogenum grown in a chemostat at a specific growth rateof 0.1 h&1. The formation of biomass requires 100 to120 mol NADPH per 100 mol glucose when the biomassyield is 0.47 to 0.55 (g dry wt) (g glucose)&1 (calculatedfrom the biomass composition used in this study). A fluxthrough the pentose phosphate pathway of around 600 ofthe glucose uptake, as found in this study, produces enoughNADPH to support the requirement of NADPH forbiomass components, and other NADPH-generating reac-tions do not need to occur (except for reactions generatingmitochondrial NADPH).

The summed fractional labeling of carbon atom numbers1 and 2 in oxaloacetate is 22.80 and the summed fractionallabeling of carbon atom numbers 3 and 4 is 33.70 in BO-1,when the labeling pattern of aspartate is used. When thelabeling pattern of threonine is used, the summed fractionallabeling of carbon atom numbers 1 and 2 in oxaloacetate is19.30. The summed fractional labeling of the two carbonatoms in oxalic acid is 19.00, which suggests that threonineand oxalic acid have the same precursor, oxaloacetate. It istherefore concluded that oxalic acid is formed by cleavage ofoxaloacetate by oxaloacetate hydrolase. If oxalic acid wasto originate from glyoxylate as in Sclerotium rolfsii(Balmforth and Thomson, 1984), the labeling would havematched the labeling in carbon atom numbers 3 and 4 inoxaloacetate, which is clearly not the case. It should benoticed that the labeling pattern of oxaloacetate obtainedfrom threonine matches the labeling of oxalic acid to ahigher degree than the labeling pattern of oxaloacetateobtained from aspartate. This can be explained by com-partmentalization of oxaloacetate in A. niger, sincethreonine and oxalate are only produced in the cytosol(Jones and Fink, 1982; Kubicek et al., 1988), whereas aspar-tate may be produced both in the cytosol and in themitochondria (Pedersen et al., 1999b).

From this work we conclude that it is possible to generateA. niger strains that do not produce oxalic acid. This findingmay be of industrial relevance, since oxalic acid representsa major problem in some types of downstream processes.The flux distribution in the central carbon metabolism of anoxalic acid-producing strain is almost identical to that of anonproducing strain. This shows that it is possible to dis-rupt the oah gene without affecting other parts of themetabolism. The fluxes through the pentose phosphatepathway are slightly higher for this study than for previousstudies of protein-producing filamentous fungi, which can


be due to differences in strain, growth conditions, and theway the fluxes were determined.

40

APPENDIX A

Reactions in the Metabolic Model

Note: Reactions of the type A+B � B+A is a way ofexpressing reversible reactions. These reactions affect thelabeling distribution, but not the mass balances. Reaction20 is included to describe complete scrambling at thefumarate-succinate stage of the TCA cycle.

Glucose Uptake1. Glucose � glucose-6-P

Embden Meyerhof Parnas Pathway

2. Glucose-6-P � fructose-6-P3. Fructose-6-P+glucose-6-P � glucose-6-P+

fructose-6-P4. Fructose-6-P � glyceraldehyde-3-P+

glyceraldehyde-3-P5. Glyceraldehyde-3-P � pyruvate

Pentose Phosphate Pathway

6. Glucose-6-P � ribose-5-P+carbon dioxide7. Ribose-5-P+ribose-5-P � sedoheptulose-7-P+

glyceraldehyde-3-P8. Sedoheptulose-7-P+glyceraldehyde-3-P+

ribose-5-P+ribose-5-P � ribose-5-P+ribose-5-P+sedoheptulose-7-P+glyceraldehyde-3-P

9. Sedoheptulose-7-P+glyceraldehyde-3-P �fructose-6-P+erythrose-4-P

10. Fructose-6-P+erythrose-4-P+sedoheptulose-7-P+glyceraldehyde-3-P � sedoheptulose-7-P+glyceraldehyde-3-P+fructose-6-P+erythrose-4-P

11. Ribose-5-P+erythrose-4-P � fructose-6-P+glyceraldehyde-3-P

12. Fructose-6-P+glyceraldehyde-3-P+ribose-5-P+erythrose-4-P � ribose-5-P+erythrose-4-P+fructose-6-P+glyceraldehyde-3-P

Formation of Acetyl-CoA in the Cytosol

13. Citrate � acetyl-CoA+oxaloacetate

Anaplerotic Reactions

14. Pyruvate+carbon dioxide � oxaloacetate15. Oxaloacetate+pyruvate+carbon dioxide �

oxaloacetate+pyruvate+carbon dioxide

Formation of Oxalate

16. Oxaloacetate � oxalate+acetyl-CoA

TCA Cycle

17. Oxaloacetate+pyruvate � citrate+carbon dioxide

Pedersen et al.

18. Citrate � :-ketoglutarate+carbon dioxide19. :-Ketoglutarate � fumarate+carbon dioxide

20. Fumarate+fumarate � oxaloacetate+oxaloacetate21. Oxaloacetate � fumarate

Specialities around Glyceraldehyde-3-P

22. Glyceraldehyde-3-P � serine23. Serine � glycine+C124. Glycine � carbon dioxide+C125. Serine+glycine+C1 � serine+glycine+C126. Oxaloacetate � glycine+acetyl-CoA

Drain of Intermediates to Biomass

27. Glucose-6-P � carbohydrates28. Fructose-6-P � chitin+mannitol29. Ribose-5-P � protein+RNA+DNA30. Erythrose-4-P � protein31. Glyceraldehyde-3-P� protein+lipid+RNA+DNA32. Pyruvate � protein33. Oxaloacetate � protein+RNA+DNA34. :-Ketoglutarate � protein35. Acetyl-CoA � protein+lipid+chitin36. Serine � protein37. Glycine � protein38. C1 � protein+lipid+RNA+DNA

Secretion to the Medium

39. Citrate � citrate (extracellular)40. Oxalate � oxalate (extracellular)

Abbreviations used: P, phosphate; acetyl-CoA, acetylcoenzyme A; C1, methylenetetrahydrofolate.

REFERENCES

Balmforth, A. J., and Thomson, A. (1984). Isolation and characterizationof glyoxylate dehydrogenase from the fungus Sclerotium rolfsii.Biochem. J. 218, 113�118.

Bercovitz, A., Peleg, Y., Battat, E., Rokem, J. S., and Goldberg, I. (1990).Localization of pyruvate carboxylase in organic acid-producingAspergillus strains. Appl. Environ. Microbiol. 56, 1594�1597.

Christensen, B., and Nielsen, J. (1999). Isotopomer analysis using GC�MS.Metab. Eng. 1, 282�290.

Henriksen, C. M., Christensen, L. H., Nielsen, J., and Villadsen, J. (1996).Growth energetics and metabolic fluxes in continuous cultures ofPenicillium chrysogenum. J. Biotechnol. 45, 149�164.


41

Holm, K. A. (1986). Automatic spectrophotometric determination ofamyloglucosidase activity using p-nitrophenyl-a-D-glucopyranosideand a flow injection analyser. Analyst 111, 927�929.

Houck, D. R., and Inamine, E. (1987). Oxalic acid biosynthesis and oxalateacetylhydrolase activity in Streptomyces cattleya. Arch. Biochem.Biophys. 259, 58�65.

Jones, E. W., and Fink, G. R. (1982). Regulation of amino acid andnucleotide biosynthesis in yeast. In ``The Molecular Biology of theYeast Saccharomyces. Metabolism and Gene Expression'' (J. N.Strathern, E. W. Jones, and J. R. Broach, Eds.), pp. 181�299, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY.

Kubicek, C. P., Schreferl-Kunar, G., Wo� hrer, W., and Ro� hr, M. (1988).Evidence for a cytoplasmic pathway of oxalate biosynthesis inAspergillus niger. Appl. Environ. Microbiol. 55, 633�637.

Marx, A., de Graaf, A. A., Wiechert, W., Eggeling, L., and Sahm, H. (1996).Determination of the fluxes in the central metabolism of Corynebac-terium glutamicum by nuclear magnetic resonance spectroscopy com-bined with metabolite balancing. Biotechnol. Bioeng. 49, 111�129.

Nissen, T. L., Schulze, U., Nielsen, J., and Villadsen, J. (1997). Fluxdistributions in anaerobic, glucose-limited continuous cultures ofSaccharomyces cerevisiae. Microbiology 143, 203�218.

Pedersen, H., Beyer, M., and Nielsen, J. (1999a). Glucoamylase productionin batch, chemostat and fed-batch cultivations by Aspergillus niger.Appl. Microbiol. Biotechnol., in press.

Pedersen, H., Carlsen, M., and Nielsen, J. (1999b). Identification ofenzymes and quantification of metabolic fluxes in the wild type and ina recombinant Aspergillus oryzae strain. Appl. Environ. Microbiol.65(1), 11�19.

Pedersen, H., Hjort, C., and Nielsen, J. (1999c). Cloning and characteriza-tion of oah, the gene encoding oxaloacetate hydrolase from Aspergillusniger. Mol. Gen. Genetics, in press.

Roehr, M., Kubicek, C. P., and Kominek, J. (1992). Industrial acidsand other small molecules. In ``Aspergillus, Biology and IndustrialApplications'' (J. W. Bennett and M. A. Klich, Eds.), pp. 91�153,Butterworth�Heinemann, Stoneham, U.S.A.

Schmidt, K., Marx, A., de Graaf, A. A., Wiechert, W., Sahm, H., Nielsen,J., and Villadsen, J. (1998). 13C tracer experiments and metabolitebalancing for metabolic flux analysis: Comparing two approaches.Biotechnol. Bioeng. 58, 254�257.

Vallino, J. J., and Stephanopoulos, G. (1993). Metabolic flux distributionsin Corynebacterium glutamicum during growth and lysine overproduc-tion. Biotechnol. Bioeng. 41, 633�646.

Wittmann, C., and Heinzle, E. (1999). Mass spectrometry for metabolicflux analysis. Biotechnol. Bioeng. 62, 739�750.

Woodcock, D. M., Crowther, P. J., Doherty, J., Jefferson, S., DeCruz, E.,Noyer-Weidner, M., Smith, S. S., Michael, M. Z., and Graham, M. W.(1989). Quantitative evaluation of Escherichia coli host strains fortolerance to cytosine methylation in plasmid and phage recombinants.Nucleic Acids Res. 17, 3469�3478.

Yanisch-Perron, C., Vieira, J., and Messing, J. (1985). Improved M13phage cloning vectors and host strains: Nucleotide sequences of theM13mp18 and pUC19 vectors. Gene 33, 103�119.


Construction and Characterization of an Oxalic Acid Nonproducing Strain of Aspergillus niger

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