INTRODUCTION

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Global lactic acid production is estimated to be more than 100,000 tons per year, and approximately 75% of the lactic acid produced is used in the food industry as an acidulant for flavor or as an antimicrobial agent (26). More recent uses for lactic acid have been driven by ecological interest and include production of the nonchlorinated solvent ethyl lactate and the biodegradable plastic polylactic acid. Polylactic acid is a polymer whose properties are similar to those of polyolefins, and it could replace a significant portion of the polyethylene terephthalate-based polymers, which are produced at a rate of approximately 15 million tons per year worldwide (4, 14, 26). Lactic acid can be synthesized chemically, but such synthesis results in a mixture of D and L isomers. The products of microbiological fermentations depend on the organism used and also may include a mixture the two isomers or individual isomers in a stereospecific form. The desired stereospecificity of the product depends on the intended use; however, L-(+)-lactic acid is the form desired for most applications.

In 1936, Lockwood et al. found that in a chemically defined medium, Rhizopus oryzae was able to aerobically convert glucose to large amounts of L-(+)-lactic acid (18). Research on lactic acid production by Rhizopus spp. has continued primarily because of the ease of product purification and the ability of the fungus to utilize both complex carbohydrates and pentose sugars (35, 38). Production of lactic acid by Rhizopus cultures is often preferred to bacterial fermentations, because lactobacilli require that the growth medium be supplemented with complex components, such as yeast extract, which adds to the cost and complicates purification.

Rhizopus cells can produce more than 1.5 mol of lactic acid from 1 mol of glucose under aerobic conditions (19), and the remainder of the glucose is converted to mycelial mass, glycerol, fumarate, or ethanol. Previously, other authors have described (22, 24, 25, 39) the presence of two lactate dehydrogenase (LDH) enzymes in R. oryzae. An NAD⁺-dependent LDH (EC 1.1.1.27) that converts pyruvate to lactate but exhibits negligible activity for the reverse reaction is produced during early growth and synthesis of lactic acid. It is thought that after glucose is exhausted, a second LDH activity is involved in NAD⁺-independent oxidation of L-lactic acid to pyruvic acid.

In this study, my objective was to isolate and characterize the gene involved in synthesis of lactic acid by R. oryzae, so I focused on the NAD⁺-dependent conversion of pyruvate to lactate. In the process of cloning this gene, I identified a second gene that appears to encode another NAD⁺-dependent LDH that has not been described previously. This is the first description of ldh genes in a fungal species, and my results fill a significant void between information concerning higher eukaryotic (e.g., plant, mammalian, fish, etc.) ldh and prokaryotic ldh genes, which have been extensively studied.

	MATERIALS AND METHODS

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Preparation of genomic and cDNA libraries. R. oryzae NRRL 395 DNA was purified by cetyltrimethylammonium bromide extraction (2), partially digested with Sau3A, and used to construct a genomic library in Lambda Zap Express (Stratagene, La Jolla, Calif.) as recommended by the manufacturer. To prepare the cDNA library, R. oryzae spores were germinated for 24 h with shaking at 30°C in RZ medium, which contained (per liter) 100 g of glucose, 2 g of (NH₄)₂SO₄, 0.5 g of KH₂PO₄, 0.25 g of MgSO₄, 2.2 mg of ZnSO₄ · 7H₂O, and 0.5 mg of MnCl₂ · 4H₂O. Calcium carbonate chips (Malinckrodt Baker, Paris, Ky.) were added to control the pH, and the preparation was incubated for an additional 8 h before the mycelium was harvested in order to isolate RNA by a hot phenol method (2). A cDNA library was prepared by following the manufacturer's instructions for a Lambda Zap unidirectional cDNA synthesis kit (Stratagene).

Isolation of LDH. R. oryzae was grown under conditions similar to those used for cDNA library preparation. Mycelium was collected by filtration and was washed with ice-cold sodium phosphate buffer (100 mM, pH 7.0) before it was suspended in 1 volume of the same buffer containing glycerol (10%, vol/vol), dithiothreitol (0.15 mg/ml), phenylmethylsulfonyl fluoride (1 mM), pepstatin A (1 µg/ml), and leupeptin (0.5 µg/ml). The cell mass was broken by agitating it with 0.45-mm-diameter glass beads for 3 min at 4,000 rpm in a Braun (Frankfurt, Germany) homogenizer and was clarified by centrifugation at 12,000 × g for 30 min. The protein that precipitated between 35 and 55% ammonium sulfate saturation was collected and dissolved in BTP buffer (50 mM bis-Tris-propane [pH 6.8], 10% glycerol, 0.15 mg of dithiothreitol per ml). The crude protein was desalted and separated from high-molecular-weight polysaccharides by using a Sephadex G-150 column (2.5 by 30 cm) equilibrated in BTP buffer. Fractions containing activity were combined, concentrated with a Centriprep-20 concentrator (Amicon, Beverly, Mass.), and injected onto a SynChropak AX300 anion-exchange high-performance liquid chromatography (HPLC) column (SynChrom, Inc., Linden, Ind.) equilibrated in BTP buffer. The column was washed with BTP buffer until all unbound proteins were removed. LDH was eluted by using a linear 0 to 0.5 M NaCl gradient in the same buffer. Combined fractions were desalted and concentrated by washing in the Centriprep-20 concentrator. The protein was again resolved by anion-exchange chromatography by using a Bio-Gel TSK-DEAE-5-PW column (Bio-Rad, Hercules, Calif.) and the conditions described above. Pooled fractions were concentrated and separated by using a Sephacryl 300 column (1.5 by 115 cm) that was equilibrated in BTP buffer. The fractions that were collected were precipitated, resolved by denaturing polyacrylamide gel electrophoresis (PAGE), and transferred to a polyvinylidene difluoride membrane (6). The protein corresponding to the predominant semipure 36-kDa enzyme was eluted, digested, and sequenced by workers at the Protein/DNA Technology Center of Rockefeller University.

Analysis of LDH activity and zymograms. LDH activity was assayed spectrophotometrically by measuring the first-order change in absorbance at 340 nm as a result of oxidation of NADH or reduction of NAD⁺. Reductive LDH activity (pyruvate  lactate) was determined by monitoring the decrease in absorbance of 175 µM NADH in 0.1 M bis-Tris-propane (pH 6.8), after the reaction was started by adding sodium pyruvate to a final concentration of 4 mM. LDH oxidative activity (lactate  pyruvate) was determined by measuring the increase in absorbance with 200 mM lithium lactate-0.1 M Tris-Cl (pH 8.0), after the reaction was started with 750 µM (final concentration) NAD⁺. The pH values for reaction buffers were chosen based on the results of preliminary experiments in which I determined the optimal conditions that resulted in the maximum change in absorbance. All protein concentrations were adjusted to ensure that the change in absorbance followed first-order kinetics for at least 3 to 5 min. All assays were performed in triplicate, and 1 U of enzyme activity was defined as the amount of activity necessary to convert 1 µmol of NADH to NAD⁺ per min or 1 µmol of NAD⁺ to NADH per min. Protein concentrations were determined with a Bio-Rad protein assay kit.

Isolation of ldh genes. A method which involved an anchored PCR (2) performed with a degenerate primer was used to isolate an ldh gene fragment from R. oryzae. The degenerate primer 5'-(GC)(AT)(AG) TC(GAT) CC(GA) TG(CT) TCA CC-3' was designed to preferentially anneal to a region encoding the consensus amino acid motif GEHGDS, which is involved in substrate binding and proton transfer for NAD⁺-dependent L-LDH enzymes (10, 36). This primer was used with M13 reverse primer to amplify the upstream region of ldh from the cDNA library. PCR amplification was performed as described by Ausubel et al. (2), except that the following program was used: 25 cycles consisting of 95°C for 45 s, 52°C for 45 s, and 72°C for 90 s. A 600-bp fragment was recovered and TA cloned into pCRII/TOPO (Invitrogen, Carlsbad, Calif.). Sequence analysis confirmed that the fragment represented a partial ldh gene. A probe was prepared from the gel-purified fragment and used to isolate hybridizing clones from both genomic and cDNA libraries.

Transcript differentiation and expression of ldhA and ldhB. R. oryzae was grown in RZ medium containing 1.5% glycerol and 0.5% Trypticase peptone for 18 h with shaking at 30°C. The mycelium was then filtered and transferred to fresh RZ medium containing 1% (wt/vol) glucose, 1% (wt/vol) xylose, 1% (wt/vol) trehalose, 1% (wt/vol) Trypticase peptone, 1% (wt/vol) glycerol, 1% (wt/vol) ethanol, or 1% (wt/vol) sodium lactate. The preparations were incubated for an additional 5.5 h, and then RNA was isolated from the cultures. Northern analysis was performed by using 10 µg of RNA from each culture. Additionally, this RNA was used along with oligo(dT) primers to synthesize first-strand cDNA with the Superscript System as recommended by the manufacturer (Life Technologies, Gaithersburg, Md.). PCR amplification was performed as described above by using this cDNA as the template and primers specific for the ldhB sequence (5'-GCAGACGCAGCCAGTGTAAGCA-3' and 5'-CAACGGCTGCCCACCAATC-3').

Complementation of Escherichia coli. E. coli DC1368(thr-1 leu-6 thi-6 lacY tonA22 rpsL ldhA::kan pfl::Cam) was provided by D. P. Clark (Southern Illinois University, Carbondale). This strain, which lacks a functional LdhA and pyruvate-formate lyase (Pfl), is not able to grow anaerobically because it cannot regenerate NAD⁺ fermentatively (20). Plasmid pBluescript II KS() (Stratagene) containing either a genomic 1.8-kb HindIII fragment containing ldhA or a 3.2-kb HindIII fragment containing ldhB was introduced into E. coli DC1368. Transformants were tested to determine whether they grew anaerobically in rich broth (3) supplemented with cysteine-HCl (50 mg/ml) and glucose (0.4%). Culture tubes were flushed with nitrogen gas and sealed with rubber stoppers. Transformants containing only the vector served as negative controls.

Molecular analyses. Northern and Southern analyses were performed by using the Genius system (Boehringer Mannheim, Indianapolis, Ind.) as recommended by the manufacturer. RNase protection assays (RPA) were performed with RPA-III (Ambion, Austin, Tex.) by using conditions used for nondenaturing 5% PAGE. Gel-purified, full-length ldhA was used for random primed labeling with digoxigenin and as a probe for Southern analysis. A 263-bp region of ldhA between BamHI and EcoRV restriction sites was used to prepare digoxigenin-labeled RNA by T3 transcription. RNA-labeled probes were used for both RPA and Northern analyses.

Nucleotide sequence accession numbers. The GenBank accession numbers for the ldhA and ldhB sequences are AF226154 and AF226155, respectively.

	RESULTS

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Isolation of ldh genes. A 600-bp fragment was amplified by using anchored PCR performed with a degenerate primer and the cDNA library as the template. This fragment appeared to be a novel ldh fragment when the data were analyzed by performing Blast comparisons (1). Using this partial ldh gene as a probe, I isolated seven overlapping clones from the cDNA library that differed by only 5 to 10 nucleotides at the 5' end and by the location of polyadenylation. There were high degrees of amino acid sequence similarity between the predicted translation product of this gene, designated ldhA, and L-LDH proteins from other species. Comparisons between the genomic and cDNA sequences of ldhA showed that introns were not present in this gene. The 5' ends of all of the cDNA fragments isolated were within 5 to 10 nucleotides of the upstream start codon. A total of 69 amino acids were sequenced from the amino terminus and three internal fragments of the purified protein. All of these amino acids were identical to amino acids in the 320-amino-acid sequence predicted on the basis of translation of ldhA.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Relationship of LDH subunits from numerous hosts. A most parsimonious tree for 26 LDH amino acid sequences is shown. Levels of amino acid substitution are expressed as percentages (bar = 10%). Most of the nodes have levels of bootstrap support of 99 to 100%; the only exceptions are the nodes labeled a (78 to 80%) or b (44 to 60%). Data for Streptococcus thermophilus (13), Streptococcus bovis (37), Streptococcus mutans (5), Lactococcus lactis (17), Lactobacillus plantarum (30), Lactobacillus sake (accession no. U26688), Lactobacillus casei (15), Bacillus megaterium (34), Bacillus stearothermophilus (40), Bacillus caldolyticus (40), Thermus aquaticus (23), Deinococcus radiodurans (21), corn (9), rice (16), tomato (8), human A (32), human B (27), pigs A and B (31), bovine A (12), mouse A, chicken A (11), dogfish A (28), and lamprey (29) have been published previously.

Regulated expression of ldhA and ldhB. The similarity between the deduced LdhA protein sequence and the purified NAD⁺-dependent LDH sequence was evidence that LdhA plays a role in lactic acid synthesis. It was not clear that ldhB was expressed or functional. The high degree of similarity between ldhA and ldhB made it difficult to differentiate the transcripts of the two genes. Northern analysis of glucose-grown cultures always resulted in detection of only one transcript, although it was unlikely that I could have distinguished between ldhA and ldhB. PCR amplification with the universal LDH primers always yielded ldhA DNA when the template was cDNA from cultures that were actively producing lactic acid in a glucose-containing medium.

View larger version (101K): [in this window] [in a new window]   FIG. 2.   Northern analysis of ldh transcript accumulation after glycerol-peptone-grown R. oryzae was transferred to media containing new carbon sources and incubated for 5.5 h. Lane 1, glucose; lane 2, xylose; lane 3, trehalose; lane 4, peptone; lane 5, glycerol; lane 6, ethanol; lane 7, lactate; lane 8, glycerol-peptone. The gel used for transfer to the membrane is shown at the bottom. The locations of molecular weight standards (in kilobases) are indicated on the left. Due to the similarity of ldhA and ldhB, the labeled ldhA riboprobe detected both transcripts.

View larger version (37K): [in this window] [in a new window]   FIG. 3.   RPA for ldh accumulation in a bubble column apparatus. RNA was purified at various times during fermentation and hybridized to the ldhA riboprobe for RPA analysis. The conditions used for the analysis were such that the probe protected both ldhA and ldhB. The control was yeast RNA hybridized to the probe. The numbers at the top indicate the times (in hours) that the RNA were obtained.

Complementation of E. coli. Both ldhA and ldhB genes could restore the fermentative ability of E. coli DC1368 mutants so transformants grew anaerobically. All of the ldh transformants produced 10 to 25 mM lactic acid, and the orientation of the ldh gene in relation to the lacZ promoter had little effect on growth or lactate production. No growth or lactic acid production occurred under anaerobic conditions with transformants containing only the pBluescript plasmid.

	DISCUSSION

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This is the first description of ldh genes isolated from a fungus. Protein sequence comparisons with other LDH revealed the unique phylogeny of these genes. One of the genes, ldhA, encodes an NAD⁺-dependent L-LDH which is involved in lactic acid production by R. oryzae. There have been several previous reports that have described the role of this protein, although most of the previous work was performed with partially purified enzyme preparations because the short half-life of the enzyme prevented further purification (22, 24, 25). I overcame the instability of the enzyme by including proteinase inhibitors, dithiothreitol, and glycerol in my preparations. Yu and Hang (39) claimed that their enzyme was very stable in phosphate buffer, but it is difficult to interpret the results of these authors, since the total activity of their purified enzyme was only 4.6 U and the specific activity was 15.4 U/mg of protein.

My findings concerning expression of the reductive NAD⁺-dependent enzyme, LdhA, in glucose-containing medium correlate well with the findings of Pritchard (24, 25). Enzyme activity was greatest in the presence of high concentrations of glucose and quickly disappeared after the glucose was depleted. I also found that both activity and transcripts were present in media containing xylose or trehalose. These results were not surprising, since R. oryzae is known to produce lactic acid when it is grown in the presence of xylose (35) and I measured lactate accumulation when trehalose was used as the carbon source (data not shown). Even though I did not detect lactic acid production in cultures grown in media containing ethanol, glycerol, and lactate, reductive LDH activity was detected by spectrophotometric assays; however, it was not detected by zymogram analysis. The results of incorporating pyrazole, an inhibitor of alcohol dehydrogenase, into the spectrophotometric assay preparations led me to believe that the difference was probably a result of pyruvate decarboxylase and alcohol dehydrogenase activities that interfered with the spectrophotometric analysis (unpublished data). Thus, I think that LdhA activity is probably negligible or absent in the presence of gluconeogenic substrates. The small amount of ldhA transcripts may indicate that there are both transcriptional and translational control mechanisms.

The function of ldhB is not clear, although this gene appears to be glucose repressed and ldhB expression occurs concomitantly with the appearance of an NAD⁺-dependent LDH that can catalyze oxidization of lactate to pyruvate. I originally hypothesized that LdhB was involved in lactic acid production during anaerobic stress, in a manner similar to that in other eukaryotes that produce multiple LDH isozymes (7, 8), but I did not detect the ldhB transcript under anoxic conditions when glucose was present (unpublished data). This enzyme activity also was present when fermentation exhausted the available glucose. I believe that this LDH is distinct from an enzyme that was previously described as an NAD⁺-independent LDH and is also produced following the disappearance of glucose (24). This NAD⁺-independent enzyme catalyzed oxidation of lactate to pyruvate by using dichlorophenol indolephenol, ferricyanide, or cytochrome c, but not oxygen, as an electron acceptor and could not use either NAD⁺ or NADH as a cofactor. I have also detected this NAD⁺-independent activity but have not been able to demonstrate it by zymogram analysis. This NAD⁺-independent LDH could be a membrane-bound flavoprotein coupled to the respiratory chain and may be required for aerobic growth on lactate.

Although I detected oxidative NAD⁺-dependent LDH activity (lactate  pyruvate) for the protein associated with ldhB, this does not mean that the protein has an oxidative function in vivo, as both ldhA and ldhB restore fermentative growth to E. coli DC1368 pfl ldh mutants. The ability of ldhB to complement these mutants was not expected, since I did not find any reductive activity associated with LdhB. It is possible that my assays were not sensitive enough to detect this conversion and that LdhB can catalyze both oxidative and reductive reactions.

During fermentation, slight decreases in lactic acid production may occur when glucose is exhausted, and these decreases are followed by increases in lactic acid levels. Pritchard (24) attributed the decreases in lactic acid levels to the NAD⁺-independent LDH but was not able to explain the increases in lactate levels. I hypothesize that LdhB is responsible for this phenomenon, since it is expressed during this phase of growth and it can produce lactic acid in E. coli DC1368. I suggest that the source of carbon for the increases in lactic acid levels is a storage sugar, such as trehalose. Although I found only ldhA transcripts in trehalose-grown cultures, the high concentrations of sugar that I used may not be representative of the concentrations found in mycelia.

In summary, my results suggest that at least three different LDH are produced by R. oryzae. Two of these enzymes, LdhA and LdhB, require the cofactor NAD⁺, while the third enzyme is probably a mitochondrial NAD⁺-independent LDH that is used for oxidative utilization of lactate. The results of transcriptional and enzyme analyses show that ldhA is probably responsible for most of the lactic acid produced by this fungus. I have only begun to examine the molecular mechanisms that control transcription and translation of this gene, although it is known that the ldhA transcript is present even with nonfermentable carbon sources, such as glycerol and ethanol. My results suggest that the function of the second gene, ldhB, may be associated with conversion of storage sugars to lactic acid, but further work is required to substantiate this hypothesis.