ASPERGILLUS ORYZAE 5-AMINOLEVULINIC ACID SYNTHASES AND NUCLEIC ACIDS ENCODING SAME

Abstract: The present invention relates to Aspergillus oryzae 5-aminolevulinic acid synthases and isolated nucleic acid fragments comprising nucleic acid sequences encoding the 5-aminolevulinic acid synthases as well as nucleic acid constructs, vectors, and recombinant host cells comprising the nucleic acid sequences. The invention also relates to methods of producing the 5-aminolevulinic acid synthases.

Description: ASPERGILLUS ORYZAE 5-AMINOLEVULINIC ACID SYNTHASES AND NUCLEIC ACIDS ENCODING SAME Background of the Invention Field of the Invention The present invention relates to Aspergillus oryzae 5-aminolevulinic acid synthases and isolated nucleic acid fragments comprising nucleic acid sequences encoding the 5-aminolevulinic acid synthases. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the 5- aminolevulinic acid synthases. Description of the Related Art Heme, a chelate complex of protoporphyrin IX and iron, serves as a prosthetic group of hemoproteins. Protoporphyrin IX consists of a porphyrin ring, substituted with four methyl groups, two vinyl groups, and two propionic acid groups, which acquires an iron atom to form heme. The biosynthesis of heme from glycine and succinyl-CoA involves eight enzymatic steps. The first enzyme in the biosynthetic pathway is 5-aminolevulinic acid synthase which catalyzes the condensation of glycine and succinyl-CoA to form 5- aminolevulinic acid. In the biosynthesis of heme in liver cells and differentiating erythrocytes, 5-aminolevulinic acid synthase is a key regulatory enzyme. The conversion of an apoprotein into a hemoprotein depends on the availability of heme provided by the heme biosynthetic pathway. The apoprotein form of the hemoprotein combines with heme to produce the active hemoprotein. The active hemoprotein acquires a conformation which makes the hemoprotein more stable than the apoprotein to proteolytic attack. If the amount of heme produced by a microorganism is less relative to the amount of the apoprotein produced, the apoprotein will accumulate and undergo proteolytic degradation lowering the yield of the active hemoprotein. In order to overcome this problem, Jensen showed that the addition of heme or a heme-containing material to a fermentation medium led to a significant increase in the yield of a peroxidase produced by Aspergillus oryzae (WO 93/19195). While heme supplementation of a fermentation medium results in a significant improvement in the yield of a hemoprotein, it is non-kosher, costly, and difficult to implement on a large scale. The cloning and sequencing of a 5-aminolevulinic acid synthase gene from Aspergillus nidulans (Bradshaw et al, 1993, Current Genetics 2233:501-507) have been disclosed. It is an object of the present invention to provide new 5-aminolevulinic acid synthases and genes encoding same. Summary of the Invention The present invention relates to substantially pure 5-aminolevulinic acid synthases obtained from Aspergillus oryzae and to isolated nucleic acid fragments comprising a nucleic acid sequence which encodes an Aspergillus oryzae 5-aminolevulinic acid synthase. The present invention further provides nucleic acid constructs, vectors, and recombinant host cells comprising a nucleic acid fragment of the present invention as well as methods for producing the 5-aminolevulinic acid synthases. Brief Description of the Figures Figure 1 shows a restriction map of plasmid pSE04. Figure 2 shows a restriction map of a 4.2 kb genomic fragment containing an Aspergillus oryzae 5-aminolevulinic acid synthase gene. Scale in kilobases (kb) is shown under the map. The arrow represents the location of the open reading frame of the gene. Figure 3 shows the nucleotide and deduced amino acid sequences of an Aspergillus oryzae 5-aminolevulinic acid synthase gene (SEQ ID NOS: 1 and 2, respectively). Potentially important transcriptional sites, CCAAT box and TATA box are underlined. The two conserved putative HRM motifs are boxed; the glycine loop involved in pyridoxal phosphate co-factor binding is circled and the important lysine is indicated with an asterisk. Figure 4 shows the conserved heme regulatory motifs in various 5- aminolevulinic acid synthase genes. The pentapeptide motifs are boxed. Figure 5 shows the alignment of the deduced amino acid sequences for 5- aminolevulinic acid synthases from Aspergillus oryzae, Aspergillus nidulans, Saccharomyces cerevisiae and human erythroid (SEQ ID NOS: 2, 16, 17 and 18, respectively). Conserved amino acids are boxed. Figure 6 shows a restriction map of plasmid pBANeό. Figure 7 shows a restriction map of plasmid pSE31. Figure 8 shows the construction of plasmid pJVi9. Figure 9 shows a restriction map of plasmid pJeRSό. Figure 10 shows a restriction map of plasmid pJRoC50. Detailed Description of the Invention The present invention, as mentioned above, relates to 5 -aminolevulinic acid synthases obtained from an Aspergillus oryzae strain. Strains of this species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), International Mycological Institute (IMI), Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL), and Institute for Fermentation in Osaka, Japan (IFO). In a preferred embodiment, the present invention relates to 5-aminolevulinic acid synthases obtained from Aspergillus oryzae or a mutant strain thereof. In a more preferred embodiment, the present invention relates to 5-aminolevulinic acid synthases obtained from Aspergillus oryzae IFO 4177 or a mutant strain thereof, e.g., the 5- aminolevulinic acid synthase having the amino acid sequence set forth in SEQ ID NO:2. The present invention also relates to 5-aminolevulinic acid synthases which are encoded by nucleic acid sequences which are capable of hybridizing under high stringency conditions (i.e., prehybridization and hybridization at 45°C in 5 X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide) with a probe which hybridizes with the nucleic acid sequence set forth in SEQ ID NO:l under the same conditions. The gene, or an oligonucleotide based thereon, can be used as a probe in Southern hybridization to isolate homologous genes of any Aspergillus species. In particular, such probes can be used for hybridization with the genomic or cDNA of the species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding 5-aminolevulinic acid synthase gene therein. Degenerate PCR primers (oligonucleotides) can be used with genomic DNA or cDNA segments to amplify 5- aminolevulinic acid synthase-specific gene segments. Identification and isolation of 5 -aminolevulinic acid synthase genes from a source other than those specifically exemplified herein can be achieved by utilization of the methodology described in the present examples, with publicly available Aspergillus strains. For purposes of the present invention, the term "obtained from" means that the 5 -aminolevulinic acid synthase is produced by a specific source, e.g., an Aspergillus strain, or by a cell in which a gene from the source encoding the 5-aminolevulinic acid synthase has been inserted. The invention also encompasses 5-aminolevulinic acid synthase variants which have at least about 80%, preferably about 85%, more preferably about 90%, and most preferably about 95% homology with the amino acid sequence set forth in SEQ ID NO:2, and which qualitatively retains the activity of the 5-aminolevulinic acid synthases described herein. The present invention is also directed to 5-aminolevulinic acid synthase variants which have an amino acid sequence which differs by three amino acids, preferably by two amino acids, and more preferably by one amino acid from the amino acid sequence set forth in SEQ ID NO:2. Each difference may be an insertion or deletion of an amino acid or the substitution of an amino acid residue by a different amino acid. Useful variants within the categories defined above include, for example, ones in which conservative amino acid substitutions have been made, which substitutions do not significantly affect the activity of the protein. By conservative substitution is meant that amino acids of the same class may be substituted by any other amino acid of that class. For example, the nonpolar aliphatic residues Ala, Val, Leu, and He may be interchanged, as may be the basic residues Lys and Arg, or the acidic residues Asp and Glu. Similarly, Ser and Thr are conservative substitutions for each other, as are Asn and Gin. The physical-chemical properties of the 5 -aminolevulinic acid synthases of the present invention may be determined using various techniques well known in the art including, but not limited to, SDS-PAGE, isoelectric focusing, and cross-reaction immunoidentity tests. The 5-aminolevulinic acid synthases of the present invention may be assayed using methods known in the art. The 5-aminolevulinic acid synthases of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, for example, Protein Purification, eds. J.-C. Janson and Lars Ryden, VCH Publishers, New York, 1989). As defined herein, a "substantially pure" 5 -aminolevulinic acid synthase is a 5 -aminolevulinic acid synthase which is essentially free of other non-5-aminolevulinic acid synthase proteins, for example, at least about 20% pure, preferably about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably at least about 95% pure, as determined by SDS-PAGE. The present invention also relates to nucleic acid fragments comprising a nucleic acid sequence which encodes a 5-aminolevulinic acid synthase of the present invention and to nucleic acid constructs comprising a nucleic acid fragment of the present invention. In a preferred embodiment, the nucleic acid sequence encodes a 5- aminolevulinic acid synthase obtained from Aspergillus oryzae. In a more preferred embodiment, the nucleic acid sequence encodes a 5 -aminolevulinic acid synthase obtained from Aspergillus oryzae IFO 4177, e.g., the nucleic acid sequence set forth in SEQ ID NO:l . The present invention also encompasses nucleic acid sequences which encode a 5- aminolevulinic acid synthase having the amino acid sequence set forth in SEQ ID NO:2, which differ from SEQ ID NO:l by virtue of the degeneracy of the genetic code. The nucleic acid sequences of the present invention encompass both the genomic sequence depicted therein as well as the corresponding cDNA and RNA sequences, and the phrase "nucleic acid sequence" as used herein will be understood to encompass all such variations including synthetic DNA. The present invention also relates to nucleic acid constructs comprising a nucleic acid fragment of the invention. "Nucleic acid construct" shall generally be understood to mean a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. In a preferred embodiment, the nucleic acid constructs are operably linked to regulatory regions capable of directing the expression of the 5 -aminolevulinic acid synthase in a suitable expression host. The present invention also provides recombinant vectors comprising a nucleic acid construct of the present invention. In a preferred embodiment, the nucleic acid sequence is operably linked to a promoter sequence. In another preferred embodiment, the vectors of the present invention further comprise a transcription termination signal and/or a selectable marker. The recombinant vectors of the invention are useful for the expression of an Aspergillus oryzae 5 -aminolevulinic acid synthase gene in active form. A useful vector contains an element that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in a host cell independent of the genome of the host cell, and preferably one or more phenotypic markers which permit easy selection of transformed host cells. The vector may also include control sequences such as a promoter, ribosome binding site, translation initiation signal, and, optionally, a selectable marker or various activator or repressor sequences. To permit the secretion of the expressed protein, nucleic acids encoding a signal sequence may be inserted prior to the coding sequence of the gene. For expression under the direction of control sequences, a 5 -aminolevulinic acid synthase gene to be used according to the present invention is operably linked to the control sequences in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. The vectors carrying a nucleic acid construct of the present invention may be any vector which can conveniently be subjected to recombinant DNA procedures. The choice of a vector will typically depend on the host cell into which the vector is to be introduced. The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. The vector system may be a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be integrated into the genome. In the vectors, the DNA sequence should be operably linked to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be obtained from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the nucleic acid construct of the invention, especially in a bacterial host, are the promoter of the lac operon of E. coll, the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis α-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens α-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes, the prokaryotic |S-lactamase promoter (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75:3727-3731) or the tac promoter (DeBoer et ai, 1983, Proceedings of the National Academy of Sciences USA 80:21-25). Further promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; and in Sambrook et al, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor, New York, 1989. In a yeast host, a useful promoter is the eno-1 promoter. For transcription in a fungal host, examples of useful promoters are those obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase or Aspergillus nidulans acetamidase. Particularly preferred promoters are the TAKA amylase, NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillus niger neutral α-amylase and Aspergillus oryzae triose phosphate isomerase), and glaA promoters. The vectors of the invention may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding a 5-aminolevulinic acid synthase of the present invention. Termination and polyadenylation sequences may be obtained from the same sources as the promoter. The vectors may further comprise a DNA sequence enabling the vectors to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO, pE194, pAMBl, and pIJ702. The vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs and the like. The selectable marker may be selected from the group consisting of, but not limited to, amdS, pyrG, argB, niaD, sC, trpC, bar, and hygB. Preferred for use in an Aspergillus cell are the amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus. Furthermore, selection may be accomplished by co-transformation, e.g., as described in WO 91/17243 where the selectable marker is contained in a separate vector. The vectors of the invention preferably also contain a signal peptide coding region, which codes for an amino acid sequence linked to the amino terminus of the heme biosynthetic enzyme, permitting the localization of the 5-aminolevulinic acid synthase to a particular cellular compartment. The signal peptide coding region may be native to the first nucleic acid sequence encoding the 5-aminolevulinic acid synthase or may be obtained from foreign sources. The 5' end of the coding sequence of the first nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the localized 5-aminolevulinic acid synthase. Alternatively, the 5' end of the coding sequence may contain nucleic acids encoding a signal peptide coding region which is foreign to that portion of the coding sequence which encodes the localized heme biosynthetic enzyme. The signal peptide coding region may be obtained from a Neurospora crassa ATPase gene (Viebrock et al. , 1982, EMBO Journal 1 :565-571) or from a Saccharomyces cerevisiae cytochrome c peroxidase gene (Kaput et al, 1982, Journal of Biological Chemistry 257: 15054-15058). However, any signal peptide coding region capable of permitting localization of the 5-aminolevulinic acid synthase in a filamentous fungal host of choice may be used in the present invention. To avoid the necessity of disrupting the cell to obtain the expressed 5- aminolevulinic acid synthase, and to minimize the amount of possible degradation of the expressed 5-aminolevulinic acid synthase within the cell, it is preferred that expression of the 5 -aminolevulinic acid synthase gene gives rise to a product secreted outside the cell. To this end, the 5-aminolevulinic acid synthases of the present invention may thus comprise a preregion permitting secretion of the expressed protein into the culture medium. If desirable, this preregion may be native to the 5 -aminolevulinic acid synthase of the invention or substituted with a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions. For example, the preregion may be obtained from a glucoamylase or an amylase gene from an Aspergillus species, an amylase gene from a Bacillus species, a lipase or proteinase gene from Rhizomucor miehei, the gene for the α-factor from Saccharomyces cerevisiae or the calf preprochymosin gene. Particularly preferred is the preregion for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, the maltogenic amylase from Bacillus NCIB 11837, Bacillus stearothermophilus α-amylase, or Bacillus licheniformis subtilisin. An effective signal sequence for fungal hosts is the Aspergillus oryzae TAKA amylase signal, the Rhizomucor miehei aspartic proteinase signal, or the Rhizomucor miehei lipase signal. The procedures used to ligate the nucleic acid construct of the invention, the promoter, terminator and other elements, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons of ordinary skill in the art (cf., for instance, Sambrook et al, supra). The present invention also relates to host cells comprising a nucleic acid construct or an expression vector of the invention which are advantageously used in the recombinant production of the 5-aminolevulinic acid synthases of the invention. The cell may be transformed with the nucleic acid construct of the invention, conveniently by integrating the construct into the host chromosome. This integration is generally considered to be an advantage as the sequence is more likely to be stably maintained in the cell. Integration of the construct into the host chromosome may be performed according to conventional methods, e.g., by homologous or non-homologous recombination. Alternatively, the cell may be transformed with an expression vector as described below in connection with the different types of host cells. The choice of host cells and vectors will to a large extent depend upon the 5- aminolevulinic acid synthase and its source. The host cell may be selected from prokaryotic cells, such as bacterial cells. Examples of suitable bacteria are gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyces lividans or Streptomyces murinus, or gram-negative bacteria such as E. coli. The transformation of the bacteria may, for instance, be effected by protoplast transformation or by using competent cells in a manner known per se. The host cell is preferably a eukaryote, such as a mammalian cell, an insect cell, a plant cell or preferably a fungal cell, including yeast and filamentous fungi. For example, useful mammalian cells include CHO or COS cells. A yeast host cell may be selected from a species of Saccharomyces or Schizosaccharomyces, e.g., Saccharomyces cerevisiae. Useful filamentous fungi may be selected from a species of Aspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Alternatively, a strain of a Fusarium species, e.g., Fusarium oxysporum or Fusarium graminearum, can be used as a host cell. Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023. A suitable method of transforming Fusarium species is described by Malardier et al, 1989, Gene 78:147-156 or in copending US Serial No. 08/269,449. In a particularly preferred embodiment, the expression of the 5-aminolevulinic acid synthase gene is achieved in a fungal host cell, such as Aspergillus. The 5- aminolevulinic acid synthase gene is ligated into a plasmid preferably containing the Aspergillus oryzae TAKA amylase promoter or the Aspergillus niger neutral amylase NA2 promoter and amdS or pyrG as the selectable marker. Alternatively, the selectable marker may be on a separate plasmid and used in co-transformation. The plasmid (or plasmids) is used to transform an Aspergillus species host cell, such as Aspergillus oryzae or Aspergillus niger in accordance with methods described in Yelton et al , 1984, Proceedings of the National Academy of Sciences USA 81 : 1470-1474. The present invention also relates to methods for producing a 5-aminolevulinic acid synthase of the present invention comprising (a) cultivating an Aspergillus oryzae strain in a nutrient medium to produce the 5-aminolevulinic acid synthase, and (b) recovering the 5-aminolevulinic acid synthase. The present invention also relates to methods for recombinantly producing a 5-aminolevulinic acid synthase of the present invention comprising (a) fermenting a host cell comprising a nucleic acid construct comprising a nucleic acid sequence encoding the 5- aminolevulinic acid synthase under conditions conducive to the production of the enzyme, and (b) recovering the 5-aminolevulinic acid synthase. If the expression system secretes the 5- aminolevulinic acid synthase into the fermentation medium, the enzyme can be recovered directly from the medium. If the recombinant 5-aminolevulinic acid synthase is not secreted, it is recovered from cell lysates. Any method of cultivation of a cell known in the art may be used which results in the expression or isolation of a 5-aminolevulinic acid synthetase of the present invention. For example, cultivation may be understood as comprising shake flask cultivation, small- or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the 5-aminolevulinic acid synthase to be expressed or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts using procedures known in the art (see, e.g., Bennett, J.W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, Academic Press, CA, 1991). Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). The 5-aminolevulinic acid synthases produced by the methods described above may be recovered from the fermentation medium by conventional procedures including, but not limited to, centrifugation, filtration, spray-drying, evaporation, or precipitation. The recovered protein may then be further purified by a variety of chromatographic procedures, e.g., ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like. The present invention is also directed to methods of using the 5-aminolevulinic acid synthases. The 5 -aminolevulinic acid synthases of the present invention may be used to convert glycine and succinyl-CoA to 5 -aminolevulinic acid which is useful as a herbicide. The 5-aminolevulinic acid synthases of the present invention may be also used to increase the yield of a hemoprotein produced by a host cell, where 5-aminolevulinic acid synthase is a rate-limiting step in the production of heme in the host cell, by overexpressing the nucleic acid sequence encoding the 5-aminolevulinic acid synthase in the host cell. The method comprises: (a) introducing into the host cell, which is capable of producing the hemoprotein, one or more copies of the nucleic acid sequence encoding the 5-aminolevulinic acid synthase, wherein the nucleic acid sequence is operably linked to regulatory regions capable of directing the expression of the 5-aminolevulinic acid synthase; (b) cultivating the cell in a nutrient medium suitable for production of the hemoprotein and the 5 -aminolevulinic acid synthase; and (c) recovering the hemoprotein from the nutrient medium of the cell. The present invention is further described by the following examples which should not be construed as limiting the scope of the invention. EXAMPLES Example 1: Aspergillus oryzae strain A1560 genomic DNA extraction Aspergillus oryzae strain A 1560 (IFO 4177) was grown in 25 ml of 0.5% yeast extract-2% glucose (YEG) medium for 24 hours at 32°C and 250 rpm. Mycelia were then collected by filtration through Miracloth (Calbiochem, La Jolla, CA) and washed once with 25 ml of 10 mM Tris-1 mM EDTA (TE) buffer. Excess buffer was drained from the mycelia which were subsequently frozen in liquid nitrogen. The frozen mycelia were ground to a fine powder in an electric coffee grinder, and the powder was added to 20 ml of TE buffer and 5 ml of 20% w/v sodium dodecylsulfate (SDS) in a disposable plastic centrifuge tube. The mixture was gently inverted several times to insure mixing, and extracted twice with an equal volume of phenol: chloroform :isoamyl alcohol (25:24:1 v/v/v). Sodium acetate (3 M solution) was added to a final concentration of 0.3 M followed by addition of 2.5 volumes of ice cold ethanol to precipitate the nucleic acids. The nucleic acids were then pelleted by centrifuging the tube at 15,000 x g for 30 minutes. The pellet was allowed to air dry for 30 minutes before resuspension in 0.5 ml of TE buffer. DNase-free ribonuclease A was added to a concentration of 100 μg/ml and the mixture was incubated at 37ϋC for 30 minutes. Proteinase K was then added at a concentration of 200 μg/ml and the mixture was incubated an additional hour at 37°C. Finally, the mixture was extracted twice with phenol:chloroform:isoamyl alcohol (25:24: 1 v/v/v) before precipitating the DNA with sodium acetate and ethanol as described earlier. The DNA pellet was dried under vacuum, resuspended in TE buffer, and stored at 4°C until further use. Example 2: Construction of plasmid pSE04 Genomic DNA was obtained from Aspergillus nidulans strain A26 (Fungal Genetics Stock Center, Kansas City, KS) using the same procedure described in Example 1. Plasmid pSE04 was constructed by ligation of PCR fragments from an amplification reaction containing Aspergillus nidulans A26 genomic DNA. The amplification reaction contained the following components: 50 ng of Aspergillus nidulans A26 genomic DNA, 100 μM each of dATP, dCTP, dGTP, and dTTP (Boehringer Mannheim, Indianapolis, IN), 50 pmoles of primers ALAS3d 5'-TTTATGATGGAGGCCCTTCTCCAGCAGTCTC-3' (SEQ ID NO:3) and ALAS4e 5'-CTATGCATTTAAGCAGCAGCCGCGACTGG-3' (SEQ ID NO:4), 2 units of Taq DNA polymerase (Perkin-Elmer Corp., Branchburg, NJ), and IX Taq DNA polymerase buffer (Perkin-Elmer Corp., Branchburg, NJ). The reaction was incubated in a Perkin-Elmer Thermal Cycler (Perkin-Elmer Corp., Branchburg, NJ) programmed for 30 cycles each at 95°C for 1 minute, 55°C for 1 minute, and 72°C for 90 seconds. The 2 kb PCR product was isolated by excision after electrophoresis using a 1.1% low melting temperature agarose gel (FMC, Rockland, ME) with 40 mM Tris-acetate-1 mM disodium EDTA (TAE) buffer, and subcloned into the pCRII vector (Invitrogen, San Diego, CA) according to the manufacturer's instructions to produce pSE04 (Figure 1). Example 3: Aspergillus oryzae strain A1560 DNA libraries and identification of ALA synthase (hemA) clones Aspergillus oryzae strain Al 560 genomic DNA libraries were constructed using the bacteriophage cloning vector λZipLox (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions using E. coli Y1090ZL cells as a host for plating and purification of recombinant bacteriophage and E. coli DHl OBzip for excision of individual pZLl-hemA clones. Total cellular DNA prepared as described in Example 1 was partially digested with r.sp509I and size-fractionated on a 1% agarose gel with 50 mM Tris-50 mM borate-1 mM disodium EDTA (TBE) buffer. DNA fragments migrating in the size range 4-7 kb were excised and eluted from the gel using Prep-a-Gene reagents (BioRad Laboratories, Hercules, CA). The eluted DNA fragments were ligated with EcoRI -cleaved and dephosphorylated λZipLox vector arms, and the ligation mixtures were packaged using commercial packaging extracts (Stratagene, La Jolla, CA). The packaged DNA libraries were plated and amplified in E. coli Y1090ZL cells. The unamplified genomic library contained l X 10 6 pfu/ml. Bacteriophage DNA from 7 x IO 4 plaques was transferred to duplicate circular Nytran Plus membranes (Schleicher & Schuell, Keene, NH) and probed with a digoxigenin (DIG)-labeled probe which was prepared by PCR amplification of Aspergillus nidulans hemA genomic DNA from plasmid pSE04 described in Example 2. The amplification reaction contained the following components: IX DIG probe synthesis mix (Boehringer Mannheim, Indianapolis, IN), 100 μM each of dATP, dCTP, dGTP, and dTTP, 50 pmoles of primer ALAS3d and primer ALAS4e described in Example 2, 2 units of Taq DNA polymerase, and IX Taq DNA polymerase buffer. The reaction was incubated in a Perkin-Elmer Thermal Cycler programmed for 30 cycles each at 95°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes. Denatured probe was added to the hybridization buffer at a concentration of 2 ng/ml and incubated overnight with prehybridized membranes. Prehybridization and hybridization was conducted at 42°C in 5 X SSC, 0.1% sarkosyl, 0.02% SDS, 1% Genius blocking agent (Boehringer Mannheim, Indianapolis, IN), and 30% formamide. Membranes were washed twice in 5 X SSC-0.1% SDS followed by two washes in 2 X SSC-0.1% SDS. Each wash was performed for 15 minutes at room temperature. The washed membrane was exposed to Kodak X-OMAT AR film for approximately 2 hours at room temperature followed by development using a Konica QX-70 automatic film processor according to the manufacturer's instructions. Primary plaques were purified and screened a second time. Five clones were identified and excised into pZL derivatives according to the manufacturer's instructions (Bethesda Research Laboratories, Inc., Gaithersburg, MD). The pZL derivatives were designated E. coli DH5α pSEl 1, pSE13, pSE15, pSE17, and pSE20. These clones were found to overlap and span a 4.2 kb region for which the restriction map is shown in Figure 2. Example 4: Southern hybridization of Aspergillus oryzae strain A1560 genomic DNA with a 5-aminoIevulinic acid synthase (hemA) probe Aspergillus oryzae strain A 1560 genomic DNA (10 μg) prepared as described in Example 1 was restriction digested with either BamHl or EcoRl. The fragments were separated by electrophoresis on a 1% agarose-TBE gel. DNA was transferred to a Nytran Plus membrane in 0.4 N NaOH using a TurboBlot apparatus (Schleicher & Schuell, Keene, NH) according to the manufacturer's instructions. The membrane was prehybridized for 2 hours at 42°C in 5 X SSC, 0.1% sarkosyl, 0.02% SDS, 1% Genius blocking agent (Boehringer Mannheim, Indianapolis, IN), and 50% formamide in a Hybaid oven (Labnet, Woodbridge, NJ). Hybridization was accomplished with a DIG-labeled hemA probe generated by PCR amplification as described in Example 3, except the hemA clone pSE17 was used as a template with primer hemA5' 5'-TCATTTAAATGATGGAGTCTCTTCTCC-3' (SEQ ID NO:5) and primer hemA3' 5'-TCTTAATTAATCAGCTCACATGCGGG-3' (SEQ ID NO:6). DIG-labeled hemA probe (1 ng probe/ml of solution) was added to fresh hybridization buffer and incubated with the membrane overnight at 42°C. Subsequently, the membrane was washed twice for 15 minutes each at room temperature in 5 X SSC-0.1% SDS followed by two washes under the same conditions in 2 X SSC-0.1% SDS. The washed membrane was exposed to Kodak X-OMAT AR film for approximately 2 hours at room temperature followed by development using a Konica QX-70 automatic film processor according to the manufacturer's instructions. Southern blot hybridization of Aspergillus oryzae genomic DNA with the Aspergillus oryzae hemA probe showed the presence of hybridization signals consistent with a single gene copy number. A 1.7 kb band observed in the BamUl lane was predicted from the restriction map (Figure 2). Example 5: Characterization of Aspergillus oryzae A1560 S-aminolevulinic acid synthase (hemA) gene E. coli DH5α pSEl 7 described in Example 3 was subjected to DNA sequencing according to the following procedure. DNA sequencing was performed with an Applied Biosystems Model 373A Automated DNA Sequencer (Applied Biosystems, Inc., Foster City, CA) on both strands using the primer walking technique with dye-terminator chemistry (Giesecke et al, 1992, Journal of Virol. Methods 38:47-60) using the M13 reverse (-48) and Ml 3 forward (-20) primers (New England Biolabs, Beverly, MA) and primers unique to the DNA being sequenced. The nucleotide sequence of the cloned gene revealed an open reading frame of 191 1 nucleotides as shown in Figure 3 (SEQ ID NO: l). The coding sequence does not contain any introns which was confirmed by cDNA cloning and sequence analysis which is in contrast to the Aspergillus nidulans hemA gene which contains one intron at its 5' end (Bradshaw et al, 1993, Current Genetics 23:501-507). The 5' untranslated sequence contains several pyrimidine-rich and AT-rich regions as in other fungal genes (Gurr et al , 1987, In Kinghorn, J.R. (ed.), Gene Structure in Eukaryotic Microbes, pp. 93-139, IRL Press, Oxford), a CCAAT sequence at position -249, and a putative TATA box located at position -35. The CCAAT sequence is a consensus binding site for transcriptional regulators which modulate transcription in response to oxygen, such as the Hap2/3/4 transcriptional regulatory complex in yeast and humans (Olesen and Guarente, 1990, Molecular and Cellular Biology 12:2302- 2314). This regulatory complex is also conserved in mammals, and a CCAAT-binding activity has been identified in Aspergillus nidulans (Davis et al, 1993, Genetica 90:133-145). The importance of this sequence in the Aspergillus oryzae hemA gene is not known and, due to limited sequence information, has not been confirmed in the Aspergillus nidulans hemA 5' region (Bradshaw et al, 1993, supra). Transcriptional regulation of the Aspergillus oryzae hemA gene in response to oxygen is not currently known, but the Aspergillus nidulans hemA gene does not appear to be transcriptionally regulated even under conditions of oxygen limitation (Bradshaw et al, 1993, supra). Interestingly, the yeast HEM1 gene is also constitutively expressed, but its expression is controlled by a balance between positive and negative regulatory sites (Keng and Guarente, 1987, Proceedings of the National Academy of Sciences USA 84:9113-9117). An (AC) 35 repeat motif occurs in the 3' untranslated region. Similar repeats have also been observed in subtelomeric, intron, and promoter regions of mammalian and yeast genes and have no known function, although they have been implicated in gene amplification events (Passananti et al, 1987, EMBO Journal 6:1697-1703). The deduced amino acid sequence of the Aspergillus oryzae strain A 1560 gene product is shown in Figure 3 (SEQ ID NO:2). The nucleotide sequence encodes a predicted protein of 636 amino acids with a molecular weight of 68 kDa. Since this enzyme is located in the mitochondria, the N-terminus is predicted to contain a mitochondrial leader sequence. In fact, the first 35 amino acids are rich in serine, threonine, lysine, and arginine residues consistent with a function as a mitochondrial leader. A potential heme regulatory motif (HRM) occurs in the presumed mitochondrial leader sequences of both the Aspergillus nidulans and Aspergillus oryzae hemA sequences (Figure 4). HRMs localized to leader sequences are believed to prevent import of 5-aminolevulinic acid synthase proteins into the mitochondria in mouse via direct interactions with heme (Lathrop and Timko, 1993, Science 259:522-525; Zhang and Guarente, 1995, EMBO Journal 14:313-320). A second potential HRM also occurs in the beginning of the putative mature protein sequence. It is probable that the HRMs play a role in the regulation of 5 -aminolevulinic acid synthase activity. Interestingly, the Saccharomyces cerevisiae 5-aminolevulinic acid synthase protein sequence does not contain any putative HRMs and does not appear to be a key regulatory step in yeast heme biosynthesis (Labbe-Bois and Labbe, In Daley, Harry A., ed., Biosynthesis of Heme and Chlorophylls, 1990, McGraw Hill Publishers, New York, pp 235-285). Overall, the deduced amino acid sequence as shown in Figure 5 shares 81% identity with the Aspergillus nidulans hemA gene (SEQ ID NO: 16), 57% identity with the Saccharomyces cerevisiae HEMl gene (SEQ ID NO: 17; Urban-Grimal, 1986, European Journal of Biochemistry 156:51 1-519), and 51% identity with the human erythroid heml (ALAS2) gene (SEQ ID NO: 18; Bishop, 1990, Nucleic Acids Research 18:7187-7188) which were determined using the Applied Biosystems GeneAssist program (blosum62.mat matrix). However, the highest degree of conservation occurs in the C-terminal two- thirds of the protein which contains the catalytic domain. Furthermore, the lysine and glycine-loop, important for catalytic activity and pyridoxal phosphate co-factor binding in other 5-aminolevulinic acid synthase enzymes (Ferreira et al , 1995, Journal of Bioenergetics and Biomembranes 27:151- 159; Ferreira, 1995, Protein Science 4:1001-1006) are also highly conserved. Example 6: Construction of plasmid pSE31 Plasmid pSE31 was constructed by directional cloning of PCR-amplified Aspergillus oryzae hemA DNA into pBANeό (Figure 6). The PCR amplification reaction was performed using DNA from hemA clone E. coli DH5α pSE17 described in Example 3 where the reaction contained the following components: 50 ng of pSE17, 2 units of Vent DNA polymerase (New England Biolabs, Beverly, MA), IX Vent DNA polymerase buffer (New England Biolabs, Beverly, MA), 400 μM each of dATP, dCTP, dGTP, and dTTP (Boehringer Mannheim, Indianapolis, IN), and 50 pmoles of primer hemA5 ' 5 '-TCATTTAAATGATGGAGTCTCTTCTCC-3 ' (SEQ ID NO:5) and primer hemA3' 5'-TCTTAATTAATCAGCTCACATGCGGG-3' (SEQ IDNO:6). The reaction was incubated in a Perkin-Elmer Thermal Cycler programmed for 30 cycles each at 95°C for 1 minute, 55°C for 1 minute, and 72°C for 90 seconds. Primer hemA5' contains a Swal site (underlined) and primer hemA3' contains a Pad site (underlined) which were used for cloning into pBANeό digested with Swal and Pad to produce pSE31 (Figure 7). Example 7: Construction of Aspergillus oryzae strain JRoC50.3.18A Aspergillus oryzae strain JRoC50.3.18A containing plasmid pJR0C50 was constructed as follows. Coprinus cinereus IFO 8371 peroxidase cDNA fragments were prepared by PCR using specific oligonucleotide primers shown below (Saiki et al, 1988, Science 239:487-491) constructed on the basis of the amino acid sequence of the Coprinus macrorhizus peroxidase (Baunsgaard et al. , 1993, European Journal of Biochemistry 213 :605- 611): 1. 5 '-GCGCGAATTCGTNGGNATNGGNATNAA(CT)CA(CT)GG-3 ' (SEQ ID NO:7) 2. 3'-TACAGNTT(GA)AC(GA)GGNGGCCTAGGCG-5' (SEQ ID NO:8) 3. 5'-GCGAATTCACNCCNCA(GA)GTNTT(CT)GA(CT)AC-3' (SEQ ID NO:9) 4. 3'-GGNAA(GA)GGNCCNCT(CT)AA(GA)CCTAGGCG-5' (SEQ ID NO: 10) 5. 5'-GCGCGAATTCTGGCA(GA)TCNAC-3' (SEQ ID NO:l l) 6. 5'-GCGCGAATTCTGGCA(GA)AGNATG-3' (SEQ ID NO: 12) 7. 3'-CGNTACCGNTT(CT)TACAGCCTAGG-5' (SEQ ID NO: 13) PCR was performed using the Gene Amp Kit and apparatus (Perkin Elmer Cetus, Norwalk, CT) in accordance with the manufacturer's instructions with the exception that the reaction was conducted at 28°C for the first 3 cycles in order to obtain better hybridization to the first strand cDNA (prepared from mRNA obtained from Coprinus cinereus strain IFO 8371) and subsequently at 65°C for 30 cycles of PCR. The primers were combined as follows: 1 with 2; 3 with 4; 5 with 7; 6 with 7; 1 with 4; and 3 with 7. The PCR fragments were extended with an EcoRI site at the 5'- end and a BamHl site at the 3 '-end. The reactions were analyzed on a 1% agarose-TBE gel where bands of the expected size were found in all the reactions. To verify that the bands corresponded to peroxidase-specific sequences, the gel was subjected to Southern blotting and hybridized to an oligonucleotide probe with the following sequence which is positioned between primers 3 and 4: 5'-GT(CT)TC(GA)AT(GA)TAGAA(CT)TG-3' (SEQ ID NO: 14) The probe was found to hybridize to bands of approximately 130 bp, 420 bp, 540 bp, and 240 bp, thus confirming that the DNA bands observed corresponded to peroxidase sequences. DNA from the various PCR reactions was digested with Eco RI and BamHl and cloned into the plasmid pUC19 (New England BioLabs, Beverly, MA). Colonies containing the correct PCR fragments were identified by hybridization using the oligonucleotide probe (SEQ ID NO.T4) described above. DNA from positive colonies was analyzed by restriction mapping and partial DNA sequence analysis as described by Sanger et al (1977, Proceedings of the National Academy of Sciences USA 74:5463-5467). A 430 bp fragment from one of the clones, obtained by using primers 1 and 4, was used to screen a Coprinus cinereus cDNA library as described below. Total RNA was extracted from homogenized Coprinus cinereus strain IFO 8371 mycelia, collected at the time of maximum peroxidase activity according to the methods described by Boel et al. (1984, EMBO Journal 3:1097-1102) and Chirgwin et al (1979, Biochemistry 18:5294-5299). Poly(A)-containing RNA was obtained by two cycles of affinity chromatography on oligo(dT)-cellulose as described by Aviv and Leder (1972, Proceedings of the National Academy of Sciences USA 69:1408-1412). cDNA was synthesized by means of a cDNA Synthesis Kit (Invitrogen, San Diego, CA) according to the manufacturer's instructions. Approximately 50,000 E. coli recombinants from the Coprinus cinereus cDNA library were transferred to Whatman 540 paper filters. The colonies were lysed and immobilized as described by Gerger et al. ( 1979, Nucleic Acids Research 7:2115-2135). The filters were hybridized with the 32 P-labelled 430 bp peroxidase- specific probe in 0.2 X SSC- 0.1% SDS. Hybridization and washing of the filters was conducted at 65 °C followed by autoradiography for 24 hours with an intensifier screen. After autoradiography, the filters were washed at increasing temperatures followed by autoradiography for 24 hours with an intensifier screen. In this way, more than 50 positive clones were identified. Miniprep plasmid DNA was isolated from hybridizing colonies by standard procedures (Birnboim and Doly, 1979, Nucleic Acids Research 7:1513-1523), and the DNA sequences of the cDNA inserts were determined by the Sanger dideoxy procedure (Sanger et al, 1977, Proceedings of the National Academy of Sciences USA 74:5463-5467). One of the colonies was selected and the vector was designated pCiP. The peroxidase cDNA fragment was excised from the vector by cleavage with BamHVXhol and was purified by agarose gel electrophoresis, electroeluted and made ready for ligation reactions. The cDNA fragment was ligated to BamWIIXhol digested pHD414 to generate pJVi9 wherein the cDNA was under transcriptional control of the TAKA promoter from Aspergillus oryzae and the AMG™ (Novo Nordisk A/S, Bagsvaerd, Denmark) terminator from Aspergillus niger as shown in Figure 8. The cDNA encoding the Coprinus cinereus peroxidase was excised from plasmid pJVi9 as a BamWl-Xhόl fragment and cloned into plasmid pJeRSό (Figure 9) to produce plasmid pJRoC50 (Figure 10) which contains pyrG as a selectable marker, the TAKA promoter, and the amdS terminator. Transformants of Aspergillus oryzae strain HowB425 were made using 5 μg of purified plasmid pJRoC50 as described below with the following changes. The agar overlay was omitted and the protoplasts were plated directly on Minimal Medium plates. The transformation was conducted with protoplasts at a concentration of 2x10 7 protoplasts per ml. One hundred μl of protoplasts were placed on ice with 5 μg DNA for 30 minutes. One ml of SPTC (40% PEG 4000, 0.8 M sorbitol, 0.05 M Tris pH 8.0, 0.05 M CaCl 2 ) was added and the protoplasts were incubated at 34°C for 20 minutes. The transformation was plated directly onto plates containing Minimal medium. The Minimal medium (pH 6.5) was composed of 6 g of NaNO 3 , 0.52 g of KC1, 1.52 g of KH 2 PO 4 , 1 ml of trace metals, 1 g of glucose, 500 mg of MgSO 4 -7H 2 O, 342.3 g of sucrose, and 20 g of Noble agar per liter. The trace metals solution (1000X) was composed of 22 g of ZnSO 4 -7H 2 O, 1 1 g of H 3 BO 3 , 5 g of MnCl 2 -4H 2 O, 5 g of FeSO 4 -7H 2 O, 1.6 g of CoCl 2 -5H 2 O, 1.6 g of (NH 4 ) 6 Mo 7 O 24* and 50 g of Na 4 EDTA per liter. Plates were incubated 5-7 days at 34°C. Transformants were transferred to plates of the same medium and incubated 3-5 days at 37°C. Sixty-six transformants were assayed for peroxidase activity using the following enzyme assay: 180 μl of substrate buffer {20 ml of 0.1 M potassium phosphate-0.01% Tween-80 pH 7.0, 250 μl of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) solution (22 mg/ml), and 2 μl of 30% hydrogen peroxide} were added to 20 μl of culture supernatant which was diluted 1 :900, quickly followed by measurement of the absorbance at 405 nm at 25°C using a Molecular Devices Thermomax Microplate Reader (Molecular Devices, Sunnyvale, CA). Measurements were recorded every 10 seconds over a 2 minute period with mixing and V max values were calculated using the SOFTmax program (Molecular Devices, Sunnyvale, CA). The peroxidase units (POXU) per ml were estimated using a standard curve constructed with a known amount of Cinereus coprinus peroxidase as a standard. A POXU was defined as the amount of enzyme that catalyzes the conversion of 1.0 μmole per minute of 0.88 mM H 2 O 2 , 1.67 mM ABTS, 0.1 M phosphate pH 7.0 at 30°C. The four transformants expressing the highest levels were spore purified by streaking spores and picking isolated colonies using the same plates under the same conditions described above. Final evaluations were performed in shake flasks where approximately 5 X10 6 spores of each transformant were inoculated into 25 ml of MY25 medium containing 1% yeast extract, 2.5% maltose, 0.2% urea, and IX MY salts pH 6.5. IX MY salts was composed of 2 g of MgSO 4 -7H 2 0, 2 g of K 2 PO 4 , 10 g of KH 2 PO 4 , 2 g of citric acid, 0.5 ml of trace metals solution and 1 ml of 10% CaCl 2 -2H 2 0 per liter. The trace metals solution was composed of 13.9 g of FeSO 4 -7H 2 O, 8.5 g of MnSO 4 -H 2 O, 14.28 g of ZnSO 4 -7H 2 O, 1.63 g of CuSO 4 , 0.24 g of NiCl 2 -6H 2 O, and 3.0 g of citric acid per liter. Hemin was added to a final concentration of 0.01 mg/ml from a fresh 10 mg/ml stock prepared in 50 mM NaOH. The shake flasks were incubated at 34°C and 200 rpm for 7 to 8 days. The best peroxidase producer was designated JRoC50.3.18A. Example 8: Transformation of Aspergillus oryzae JRoC50.3.18A with pSE31 Aspergillus oryzae strain JRoC50.3.18A was transformed with pSE31 in order to determine whether overexpression of the hemA gene increased peroxidase production. The transformation was conducted with protoplasts at a concentration of 2x10 7 protoplasts per ml. One hundred μl of protoplasts were incubated at 34°C with 10 μg DNA and 200 μl of 60% PEG 4000-10 mM HEPES-10 mM CaCl 2 solution for 30 minutes. Three ml of SPTC (40% PEG 4000, 0.8 M sorbitol, 0.05 M Tris pH 8.0, 0.05 M CaCl 2 ) were added and the protoplasts were plated directly onto COVE transformation plates (per liter: 0.52 g of KC1, 0.52 g of MgSO 4 -7H 2 0, 1.52 g of KH 2 PO 4 , 1 ml of trace metals solution as described in Example 7, 342.3 g of sucrose, 25 g of Noble agar, 10 ml of 1 M acetamide, and 10 ml of 3 M CsCl) for amdS transformations. Plates were incubated 5-7 days at 34°C. Transformants were transferred to plates of the same medium and incubated 3-5 days at 34°C. The transformants were then purified by streaking spores and picking isolated colonies using the same plates under the same conditions. Example 9: Peroxidase production by hemA transformants The transformants from Example 8 were inoculated into individual wells at approximately 1X10 5 spores per well of a 24-well microtiter plate containing 1 ml of quarter strength MY25 medium composed of 0.25% yeast extract, 0.63% maltose, and 0.05% urea pH 6.5, and IX MY salts (see Example 7). The microtiter plates were incubated at 34°C and 100 rpm in a humidity chamber for 5 days. Peroxidase production levels were determined using the enzyme assay described in Example 7. The results of the microtiter plate tests demonstrate that the average POXU/ml of hemA transformants was 1.4-fold greater than the average of the vector only transformants, with the best hemA transformant showing a 1.6-fold increase in peroxidase production. A minority (39%) of the hemA transformants show peroxidase levels similar to the majority of the vector only controls. PCR amplification using 50 ng of genomic DNA isolated as described in Example 1 from each transformant was performed as described in Example 2 except the primers hemA3' (see Example 4) and primer 5'-TCTCTTCCTTCCTGAATCCTC-3' (SEQ ID NO: 15) were used. This analysis showed that the hemA transformants contain the expression cassette. Eleven of the best hemA transformants obtained above were cultivated in shake flasks to better evaluate the effects on peroxidase production. For shake flask evaluations, approximately 5 X10 6 spores of each transformant were inoculated into 25 ml of MY25 medium containing 1% yeast extract, 2.5% maltose, 0.2% urea, and IX MY salts pH 6.5 (see Example 7). The shake flasks were incubated at 34°C and 200 rpm for 7 to 8 days. Peroxidase assays were performed as described above. The results demonstrated that five transformants, SE01 - 15 , SE01 -20, SE01 -26, SE01-28 and SE01 -32, produced peroxidase levels which were greater than the vector alone control strains, with three transformants expressing peroxidase at a level 1.9-fold greater than the average control peroxidase levels. The remaining six hemA transformants showed peroxidase levels which were comparable to control levels. Transformant SE01-28 and a control strain SE05-18 (pBANeό vector alone transformant) were grown in 2 liter fermentations using a standard fed-batch protocol which has high maltose syrup as carbon source. The batch and feed were supplemented with FeCl 3 to approximately 0.4 mM. Positive dissolved oxygen tension was maintained in both cultures with feed added at a rate of approximately 2 grams saccharide per liter per hour from day three to day eight. This level was reached in a step-wise manner over days two and three. Biomass in both cultures were approximately equal for the duration of the fermentation. A 2-fold increase in peroxidase activity was observed with SE01-28 over the control strain SE05-18. There was also a 2-fold increase in the polypeptide level for SE01-28 relative to the control strain SE05-18. The overall results demonstrated that overexpression of the hemA gene resulted in a 2-fold increase in peroxidase yield. The data indicated further that hemA may represent a key regulatory point during heme biosynthesis in filamentous fungi which upon genetic manipulation can improve hemoprotein production in the absence of hemin supplementation. DEPOSIT OF MICROORGANISMS The following strain has been deposited according to the Budapest Treaty in the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Laboratory, 1815 University Street, Peoria, Illinois 61604, USA. Strain Accession Number Deposit Date E. coli DH5α (pSE17) NRRL B-21563 April 22, 1996 The strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposit represents a substantially pure culture of each deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action. The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. SEQUENCE LISTING (1) GENERAL INFORMATION (i) APPLICANT: (A) NAME: Novo Nordisk Biotech, Inc. (B) STREET: 1445 Drew Avenue (C) CITY: Davis, California (D) COUNTRY: United States of America (E) POSTAL CODE (ZIP) : 95616-4880 <F) TELEPHONE: (916) 757-8100 (G) TELEFAX: (916) 758-0317 (ii) TITLE OF THE INVENTION: Aspergillus Oryzae 5-Aminolevulinic Acid Synthases And Nucleic Acids Encoding Same (iii) NUMBER OF SEQUENCES: 18 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Novo Nordisk Of North America, Inc. (B) STREET: 405 Lexington Avenue - 64th Fl . (C) CITY: New York (D) STATE: NY (E) COUNTRY: USA (F) ZIP: 10174 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS (D) SOFTWARE: FastSEQ for Windows Version 2.0 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 60/019,399 (B) FILING DATE: 10-JUN-1996 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Lambiris, Elias J (B) REGISTRATION NUMBER: 33,728 (C) REFERENCE/DOCKET NUMBER: 4808.204-WO (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 212-878-9652 (B) TELEFAX: 212-878-9655 (C) TELEX: (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4157 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 : ACCATTGACT CTCAAGCTAT GGATCGTGCT CACCGTCTCG GCCAGACAAG ACAGGTCACG 60 GTGTATCGCC TGATTACTCG CGGCACCATT GAGGAGCGTA TTCGCAAGCG AGCTTTGCAG 120 AAGGAGGAAG TGCAGCGTGT CGTCATCTCA GGTGGCGCAG CTGGTGGGGT TGACTTCAAT 180 ACTCGCAACC GCGAGAGCCG AACCAAGGAC ATCGCCATGT GGCTGGCAGA TGATGAACAG 240 GCGGAGCTTA TTGAGCAAAA GGAGAAGGAA GCGCTGGACC GAGGCGAAGT GTTTGGCGCT 300 AGTAAAGGCG GGAAGAAGGC TGCTCAGAAG AGAAAGAGAG ATATCACGCT GGATGATATG 360 TATCATGAAG GTATGTGAAT CTGATCAAAG CTCTTCGTTC CGGGGAGGCT TCTGGAAATA 420 GTACTAACCG CGTCAATCTA TAGGCGAAGG GAACTTTGAC GATGCCAGTG CAAAGCCATC 480 AGGAGCGGCC ACTCCTGTGT CGACTGCAGA GAATTTAGGC ACCCCATCCT CCACGCCAGT 540 TCCTAAACGA GGACGTGGAA GGGGGACAGG AAAGGGCACG TCTAAAAGAG CCAAAACTAC 600 CAAGGAGAGA TTACGTCTCA TTGATGGCGA CGGAGGCTTA GGGCCTAGTT GATTTAATCG 660 ATCTGTGCCT CAATAATGGA CACGGCTGGT TATGGTCATG GCGTTCAGAG ATTGCATTTC 720 TTTCCCACCC TTTATCTTTC TTTCTTTCCT CTTAAACCCC TCTTTTTTGT TTTTCTTTTT 780 ATCGGACTTT ACTTGTGGGC AGCTTACGTT CTGCCTTGTA TTAACAGCAT ATATTCCTGA 840 TTCCTGATGT ACGAAGCGAT TTAAGAGTCA TTGAAGACGA AGGATGAAAC CCGTGGTAAT 900 CAGCCGATAA TGGCAAAGAG AAGGAGAAGA AAAAAATCAA GTGCGAGTTT TGAAATTGAT 960 GGCAAGATAG ACATTGTATC CTGTACCTGT TCTTGGGCTG TGACGGGGGG GGTGAAATTG 1020 ACGGTCATCA CCCGGCTATT ATTACTATTG TTGTACTGTA CATCCGGATC CTGCTGGTCT 1080 GTATCTAGTT AGGGCAATAT TCCCCGTCGC CAGGCCTCTT GGGTTATGAA TGATTTCATA 1140 GGTGAAGTTT CGTATCCGTA CGCACCGAGA GATTTCTTAG TATTACTTGT ATTATGAAAA 1200 TGCACTTGCC GAGTTAAGTC CGCCGGCCAA TCACGGCGGA GGATATGGTA AGCCGAAAAG 1260 TCTCGCCGAA GTCCCCGACT TACTCTTACT GGAAGTGGCT TAGTGCCCTC AGCGCCCCCT 1320 CGCCCTCAGT CCATCAGCCA GATTGACTCT TATTTCTCTC TCCTCTTCGC CGCGGGTGAC 1380 ATATCCCTCT CCTTCTCCCT CTCCCTCTTG ACAACATTTC ATCTTCGCTT CCTTTTGTGA 1440 TATAGTCAGT TTCGCTATCC ATTGAAGCAT CACTCATGGA GTCTCTTCTC CAGCAGTCCC 1500 GGGCGATGTG CCCGTTCCTT AAGCGCACAT CTCCATCTTC TCTGCGTACG CTGGCAACCG 1560 CGACTCGACC TAGCACTAGT TCCGGTGGAG GCACTATGTC TAATCTCCAG GTCATTGCCC 1620 GTCGCTGCCC TGTCATGAGC AAGGCTCTGG CCGTGCAGAG CGCTCGCATG GCCGGTACCA 1680 AAAGATTCAC CTCATGTGCT GCCGGCATCA CCGGTCTCGG CAACAAGCAT TGCCGTGCTC 1740 CTACTGGGAA GAGAACCCTG CACTCCACCT CCGGTAACGG CGCCAATGTG AGCGCAGAGA 1800 TCTACAAGAA CACCCAGCGA GATCCCGCCG GTTTCTCGAA GATCAAGACC CCTGCCAATG 1860 CTACCGCCGC TGCCGCTACG TCTGGCCCTC GTCCAGAGGC TCCCGTGGCG AAGCCTTTCA 1920 ACTACAATTC TTTCTACAAC ACCGAATTGG AAAAGAAACA CAAGGACAAG TCGTATCGCT 1980 ATTTCAACAA CATCAATCGT CTCGCTCAGG AGTTTCCCCG GGCTCACACC ACATCTGCCG 2040 AGGAACGTGT GACGGTCTGG TGCTCGAACG ATTATCTCGG CATGGGCCGC AACCCCGAGG 2100 TTCTGGCCAC CATGCATAAG ACATTGGACA CCTACGGAGC CGGTGCGGGA GGTACTCGCA 2160 ACATTTCAGG TCACAATCAA CATGCCGTGA GCCTGGAGAA CACCCTGGCC AAATTGCACG 2220 GCAAGGAGGC GGCATTAGTC TTCAGCTCAT GCTTCGTGGC TAACGATGCC ACCCTCGCAA 2280 CCCTGGGTAG CAAGTTGCCC GACTGTGTTA TTCTGTCCGA TAGCCTGAAT CATGCATCGA 2340 TGATTCAGGG TATTCGCCAT TCAGGCGCCA AGAAAATGGT TTTCAAGCAT AATGATCTGG 2400 TCGACCTTGA GGCCAAGTTG GCAGCTCTAC CTCTTCATGT CCCCAAGATT ATTGCATTCG 2460 AATCAGTTTA TAGCATGTGC GGATCTATTG CCCCAATTGA GAAGATCTGT GATCTTGCAG 2520 ACAAGTACGG TGCCATTACT TTCCTGGATG AAGTCCACGC TGTGGGAATG TACGGACCTC 2580 ACGGAGCAGG TGTGGCAGAG CACCTTGACT ATGACATCTA TGCTTCCCAA GATACGGTCA 2640 ACCCGCGCAG TACTAAGGGA ACCGTGATGG ACCGAATCGA TATTATCACC GGTACTCTGG 2700 GCAAGGCCTA CGGATGTGTC GGGGGCTACA TTGCTGGATC CGCTGCGATG GTTGACACCA 2760 TCCGCTCCCT CGCCCCTGGC TTCATCTTCA CCACGTCCTT GCCGCCCGCC ACCATGGCTG 2820 GTGCAGACAC TGCTATCCAG TACCAGGCTC GTCACCAGGG CGACCGCGTC CTGCAGCAGT 2880 TGCACACCCG CGCGGTCAAA GCAGCTTTCA AGGAGTTGGA TATTCCTGTA ATTCCCAACC 2940 CCTCCCATAT CATTCCGCTC CTGGTTGGGG ATGCCGAGGT TGCTAAGAAG GCCTCGGACA 3000 AGCTTCTGGA GGAGCATGGA ATTTATGTAC AAGCCATCAA CTACCCAACC GTGCCTCGGG 3060 GTGAAGAGCG GCTTCGTATC ACGCCCACCC CGGGACATAT CAAGGAGCAC CGCGACCACC 3120 TGGTGCAAGC CGTCCAAACA GTCTGGAACG AACTGGGCAT CAAACGCACC AGCGATTGGG 3180 AAGCGCAAGG CGGCTTCGTC GGCGTGGGTG TCGATGGCGC CGAGGCTGAG AACCAGCCGA 3240 TTTGGAATGA TGTGCAGCTG GGGCTGAAGG AAAACGAAGC CATTGAGGCT GCTGTGGAAC 3300 GCGAGTTTGC CGAGGCCCCC ATGCGGACCG CCACCCGTCC TGCCGCGGCT GCTGCTTCGT 3360 CAATCCCGGT GGGTGTGGCT GCCTGAAGTG GCTGCCCGCA TGTGAGCTGA AATCGACGTG 3420 GAATTCTATA CACACACACA CACACACACA CACACACACA CACACACACA CACACACACA 3480 CACACACACA CACACACACT AACACACACT ATGTTATAAA TTCCACATCC ACTCCTTTGT 3540 CCCTTGTTGG ACGTAATTGG TATTTGGACT ATTAGTTAGA ACCAGTCAGT CGTTACCATG 3600 TGTTTCGGTT CGACTCGAAA TCTGACATGT TGTCTGCCCC CATGCCACTT CATCTCCTCC 3660 GTAACCGCAG GGCTTCAAAT ACACTGCCCA GTAATTGTAG TCAATATAGC AGTTAACTAA 3720 CCTTCACCAA TTTCCTAATA ACAATAGAAG GGGCCATACA CGCAGTACCA AAGATCACCT 3780 ACCTCCGATC AATATCCGAA CCTCAGGCTA CATACATCAA GTCGCATTAA TCGATTCCGA 3840 CCTCTGTTTA TCCCTGAAAA TAACTAAGAT CATGATCTAC GTTTGGTAAG TGGGACACCT 3900 ACCTACACTG GGAGGTATTG AATAAAGGCA TCATTCATAT AGTCACAAGA TGCCAGGGCC 3960 AATTCATGAT ATGGATAGCT ACTTCCAAAC ATAATTCAGA GGTATCATTC TGCTCTTCAG 4020 ACAGTTCTTC TCGAAGATCA GTAGGAGCCA GTTTTGACCA TTAACTTGTA ATGTAATTGC 408C GATTGTAGTA GATCCGAGAT CCATTCACTT TCTAAGGGTT AATTGATTCA TTTTACTGAT 4140 ACCTCACCCA CCATATT 4157 (2) INFORMATION FOR SEQ ID NO:2 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 636 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 : Met Glu Ser Leu Leu Gin Gin Ser Arg Ala Met Cys Pro Phe Leu Lys 1 5 10 15 Arg Thr Ser Pro Ser Ser Leu Arg Thr Leu Ala Thr Ala Thr Arg Pro 20 25 30 Ser Thr Ser Ser Gly Gly Gly Thr Met Ser Asn Leu Gin Val He Ala 35 40 45 Arg Arg Cys Pro Val Met Ser Lys Ala Leu Ala Val Gin Ser Ala Arg 50 55 60 Met Ala Gly Thr Lys Arg Phe Thr Ser Cys Ala Ala Gly He Thr Gly 65 70 75 80 Leu Gly Asn Lys His Cys Arg Ala Pro Thr Gly Lys Arg Thr Leu His 85 90 95 Ser Thr Ser Gly Asn Gly Ala Asn Val Ser Ala Glu He Tyr Lys Asn 100 105 110 Thr Gin Arg Asp Pro Ala Gly Phe Ser Lys He Lys Thr Pro Ala Asn 115 120 125 Ala Thr Ala Ala Ala Ala Thr Ser Gly Pro Arg Pro Glu Ala Pro Val 130 135 140 Ala Lys Pro Phe Asn Tyr Asn Ser Phe Tyr Asn Thr Glu Leu Glu Lys 145 150 155 160 Lys His Lys Asp Lys Ser Tyr Arg Tyr Phe Asn Asn He Asn Arg Leu 165 170 175 Ala Gin Glu Phe Pro Arg Ala His Thr Thr Ser Ala Glu Glu Arg Val 180 185 190 Thr Val Trp Cys Ser Asn Asp Tyr Leu Gly Met Gly Arg Asn Pro Glu 195 200 205 Val Leu Ala Thr Met His Lys Thr Leu Asp Thr Tyr Gly Ala Gly Ala 210 215 220 Gly Gly Thr Arg Asn He Ser Gly His Asn Gin His Ala Val Ser Leu 225 230 235 240 Glu Asn Thr Leu Ala Lys Leu His Gly Lys Glu Ala Ala Leu Val Phe 245 250 255 Ser Ser Cys Phe Val Ala Asn Asp Ala Thr Leu Ala Thr Leu Gly Ser 260 265 270 Lys Leu Pro Asp Cys Val He Leu Ser Asp Ser Leu Asn His Ala Ser 275 280 285 Met He Gin Gly He Arg His Ser Gly Ala Lys Lys Met Val Phe Lys 290 295 300 His Asn Asp Leu Val Asp Leu Glu Ala Lys Leu Ala Ala Leu Pro Leu 305 310 315 320 His Val Pro Lys He He Ala Phe Glu Ser Val Tyr Ser Met Cys Gly 325 330 335 Ser He Ala Pro He Glu Lys He Cys Asp Leu Ala Asp Lys Tyr Gly 340 345 350 Ala He Thr Phe Leu Asp Glu Val His Ala Val Gly Met Tyr Gly Pro 355 360 365 His Gly Ala Gly Val Ala Glu His Leu Asp Tyr Asp He Tyr Ala Ser 370 375 380 Gin Asp Thr Val Asn Pro Arg Ser Thr Lys Gly Thr Val Met Asp Arg 385 390 395 400 He Asp He He Thr Gly Thr Leu Gly Lys Ala Tyr Gly Cys Val Gly 405 410 415 Gly Tyr He Ala Gly Ser Ala Ala Met Val Asp Thr He Arg Ser Leu 420 425 430 Ala Pro Gly Phe He Phe Thr Thr Ser Leu Pro Pro Ala Thr Met Ala 435 440 445 Gly Ala Asp Thr Ala He Gin Tyr Gin Ala Arg His Gin Gly Asp Arg 450 455 460 Val Leu Gin Gin Leu His Thr Arg Ala Val Lys Ala Ala Phe Lys Glu 465 470 475 480 Leu Asp He Pro Val He Pro Asn Pro Ser His He He Pro Leu Leu 485 490 495 Val Gly Asp Ala Glu Val Ala Lys Lys Ala Ser Asp Lys Leu Leu Glu 500 505 510 Glu His Gly He Tyr Val Gin Ala He Asn Tyr Pro Thr Val Pro Arg 515 520 525 Gly Glu Glu Arg Leu Arg He Thr Pro Thr Pro Gly His He Lys Glu 530 535 540 His Arg Asp His Leu Val Gin Ala Val Gin Thr Val Trp Asn Glu Leu 545 550 555 560 Gly He Lys Arg Thr Ser Asp Trp Glu Ala Gin Gly Gly Phe Val Gly 565 570 575 Val Gly Val Asp Gly Ala Glu Ala Glu Asn Gin Pro He Trp Asn Asp 580 585 590 Val Gin Leu Gly Leu Lys Glu Asn Glu Ala He Glu Ala Ala Val Glu 595 600 605 Arg Glu Phe Ala Glu Ala Pro Met Arg Thr Ala Thr Arg Pro Ala Ala 610 615 620 Ala Ala Ala Ser Ser He Pro Val Gly Val Ala Ala 625 630 635 (2) INFORMATION FOR SEQ ID NO:3 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TTTATGATGG AGGCCCTTCT CCAGCAGTCT C 31 (2) INFORMATION FOR SEQ ID NO:4 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CTATGCATTT AAGCAGCAGC CGCGACTGG 29 (2) INFORMATION FOR SEQ ID NO: 5 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 : TCATTTAAAT GATGGAGTCT CTTCTCC 27 (2) INFORMATION FOR SEQ ID NO:6 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TCTTAATTAA TCAGCTCACA TGCGGG 26 (2) INFORMATION FOR SEQ ID NO:7 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GCGCGAATTC GTNGGNATNG GNATNAAYCA YGG 33 (2) INFORMATION FOR SEQ ID NO: 8 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 : GCGGATCCGG NGGRCARTTN GACAT 25 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 : GCGAATTCAC NCCNCARGTN TTYGAYAC 28 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: GCGGATCCRA AYTCNCCNGG RAANGG 26 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GCGCGAATTC TGGCARTCNA C 21 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GCGCGAATTC TGGCARAGNA TG 22 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GGATCCGACA TYTTNGCCAT NGC 23 (2) INFORMATION FOR SEQ ID NO: 14 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GTYTCRATRT AGAAYTG 17 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: TCTCTTCCTT CCTGAATCCT C 21 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 649 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16 : Met Glu Ala Leu Leu Gin Gin Ser Arg Ala Met Cys Pro Phe Leu Lys 1 5 10 15 Arg Ser Ser Pro Asn Thr Leu Arg Ser Leu Ala Thr Ala Thr Arg Pro 20 25 30 Ser Thr Ser Pro Gly Gly Gly Thr Met Thr Asn Leu Gin Arg He Ala 35 40 45 Arg Arg Cys Pro Val Met Ser Lys Ala Leu Ala Val Gin Ser Ala Arg 50 55 60 Met Thr Gly Thr Lys Arg Phe Thr Ser Ser Ala Ala Gly Val Pro Gly 65 70 75 80 Ala Gly Ala Gly Thr Pro Lys Pro Thr Arg Gly Ser Pro Gly Lys Arg 85 90 95 Ala Leu His Ser Thr Gly Gly Asn Gly Ala Asn Met Ser Thr Glu Phe 100 105 110 His Lys Gly Ala Gin Gin He His Pro Gly Leu Ser Asn Ala Thr Arg 115 120 125 Ser His Val Gly Ala Ser Ala Thr Val Ser Gly Pro Thr Pro Arg Ala 130 135 140 Pro Val Ala Ala Pro Phe Asp Tyr Asp Ala Phe Tyr Asn Ala Glu Leu 145 150 155 160 Gin Lys Lys His Gin Asp Lys Ser Tyr Arg Tyr Phe Asn Asn He Asn 165 170 175 Arg Leu Ala Gin Glu Phe Pro Arg Ala His Thr Ala Ser Lys Asp Glu 180 185 190 Lys Val Thr Val Trp Cys Ser Asn Asp Tyr Leu Gly Met Gly Arg Asn 195 200 205 Pro Glu Val Leu Ala Thr Met His Lys Thr Leu Asp Thr Tyr Gly Ala 210 215 220 Gly Ala Gly Gly Thr Arg Asn He Ser Gly His Asn Gin His Ala Val 225 230 235 240 Ser Leu Glu Asn Thr Leu Ala Lys Leu His Gly Lys Glu Ala Ala Leu 245 250 255 Val Phe Ser Ser Cys Phe Val Ala Asn Asp Ala Thr Leu Ala Thr Leu 260 265 270 Gly Ser Lys Met Pro Asp Cys Val He Leu Ser Asp Ser Leu Asn His 275 280 285 Ala Ser Met He Gin Gly He Arg His Ser Gly Arg Lys Lys Met Val 290 295 300 Phe Lys His Asn Asp Leu Val Asp Leu Glu Thr Lys Leu Ala Ser Leu 305 310 315 320 Pro Leu His Val Pro Lys He He Ala Phe Glu Ser Val Tyr Ser Met 325 330 335 Cys Gly Ser He Ala Pro He Glu Ala He Cys Asp Leu Ala Asp Lys 340 345 350 Tyr Gly Ala He Thr Phe Leu Asp Glu Val His Ala Val Gly Met Tyr 355 360 365 Gly Pro His Gly Ala Gly Val Ala Glu His Leu Asp Tyr Glu He Tyr 370 375 380 Ala Ser Gin Asp Thr Ala Asn Pro Leu Ser Thr Lys Gly Thr Val Met 385 390 395 400 Asp Arg He Asn He He Thr Gly Thr Leu Gly Lys Ala Tyr Gly Cys 405 410 415 Val Gly Gly Tyr He Ala Gly Ser Ala Ala Leu Val Asp Thr He Arg 420 425 430 Ser Leu Ala Pro Gly Phe He Phe Thr Thr Ser Leu Pro Pro Ala Thr 435 440 445 Met Ala Gly Ala Asp Thr Ala He Arg Tyr Gin Ala Arg His Gin Gin 450 455 460 Asp Arg He Leu Gin Gin Leu His Thr Arg Ala Val Lys Gin Ser Phe 465 470 475 480 Lys Asp Leu Asp He Pro Val He Pro Asn Pro Ser His He Val Pro 485 490 495 Leu Leu Val Gly Asp Ala Glu Leu Ala Lys Gin Ala Ser Asp Lys Leu 500 505 510 Leu Glu Glu His Gly He Tyr Val Gin Ala He Asn Tyr Pro Thr Val 515 520 525 Pro Arg Gly Glu Glu Arg Leu Arg He Thr Pro Thr Pro Gly His Thr 530 535 540 Gin Glu Leu Arg Asp His Leu Val Glu Ala Val Asn Thr Val Trp Asn 545 550 555 560 Asp Leu Gly He Lys Arg Ala Ser Asp Trp Lys Ala Met Gly Gly Phe 565 570 575 Val Gly Val Gly Val Glu Ala Ala Glu Leu Glu Asn Gin Pro He Trp 580 585 590 Thr Asp Ala Gin Leu Asn Met Arg Pro Asp Glu Thr Leu Glu Ala Ala 595 600 605 Val Glu Arg Glu Phe Gin Ala Ala Val Pro Gly Met Lys Ala Gly Gly 610 615 620 Ala Lys Ala Lys Pro Val Gly Ser He Ala Ala Asn Pro He Gly Ala 625 630 635 640 Ser He Pro Val Ala Ala Ala Ala Glx 645 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 548 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Met Gin Arg Ser He Phe Ala Arg Phe Gly Asn Ser Ser Ala Ala Val 1 5 10 15 Ser Thr Leu Asn Arg Leu Ser Thr Thr Ala Ala Pro His Ala Lys Asn 20 25 30 Gly Tyr Ala Thr Ala Thr Gly Ala Gly Ala Ala Ala Ala Thr Ala Thr 35 40 45 Ala Ser Ser Thr His Ala Ala Ala Ala Ala Ala Ala Ala Ala Asn His 50 55 60 Ser Thr Gin Glu Ser Gly Phe Asp Tyr Glu Gly Leu He Asp Ser Glu 65 70 75 80 Leu Gin Lys Lys Arg Leu Asp Lys Ser Tyr Arg Tyr Phe Asn Asn He 85 90 95 Asn Arg Leu Ala Lys Glu Phe Pro Leu Ala His Arg Gin Arg Glu Ala 100 105 110 Asp Lys Val Thr Val Trp Cys Ser Asn Asp Tyr Leu Ala Leu Ser Lys 115 120 125 His Pro Glu Val Leu Asp Ala Met His Lys Thr He Asp Lys Tyr Gly 130 135 140 Cys Gly Ala Gly Gly Thr Arg Asn He Ala Gly His Asn He Pro Thr 145 150 155 160 Leu Asn Leu Glu Ala Glu Leu Ala Thr Leu His Lys Lys Glu Gly Ala 165 170 175 Leu Val Phe Ser Ser Cys Tyr Val Ala Asn Asp Ala Val Leu Ser Leu 180 185 190 Leu Gly Gin Lys Met Lys Asp Leu Val He Phe Ser Asp Glu Leu Asn 195 200 205 His Ala Ser Met He Val Gly He Lys His Ala Asn Val Lys Lys His 210 215 220 He Phe Lys His Asn Asp Leu Asn Glu Leu Glu Gin Leu Leu Gin Ser 225 230 235 240 Tyr Pro Lys Ser Val Pro Lys Leu He Ala Phe Glu Ser Val Tyr Ser 245 250 255 Met Ala Gly Ser Val Ala Asp He Glu Lys He Cys Asp Leu Ala Asp 260 265 270 Lys Tyr Gly Ala Leu Thr Phe Leu Asp Glu Val His Ala Val Gly Leu 275 280 285 Tyr Gly Pro His Gly Ala Gly Val Ala Glu His Cys Asp Phe Glu Ser 290 295 300 His Arg Ala Ser Gly He Ala Thr Pro Lys Thr Asn Asp Lys Gly Gly 305 310 315 320 Ala Lys Thr Val Met Asp Arg Val Asp Met He Thr Gly Thr Leu Gly 325 330 335 Lys Ser Phe Gly Ser Val Gly Gly Tyr Val Ala Ala Ser Arg Lys Leu 340 345 350 He Asp Trp Phe Arg Ser Phe Ala Pro Gly Phe He Phe Thr Thr Thr 355 360 365 Leu Pro Pro Ser Val Met Ala Gly Ala Thr Ala Ala He Arg Tyr Gin 370 375 380 Arg Cys His He Asp Leu Arg Thr Ser Gin Gin Lys His Thr Met Tyr 385 390 395 400 Val Lys Lys Ala Phe His Glu Leu Gly He Pro Val He Pro Asn Pro 405 410 415 Ser His He Val Pro Val Leu He Gly Asn Ala Asp Leu Ala Lys Gin 420 425 430 Ala Ser Asp He Leu He Asn Lys His Gin He Tyr Val Gin Ala He 435 440 445 Asn Phe Pro Thr Val Ala Arg Gly Thr Glu Arg Leu Arg He Thr Pro 450 455 460 Thr Pro Gly His Thr Asn Asp Leu Ser Asp He Leu He Asn Ala Val 465 470 475 480 Asp Asp Val Phe Asn Glu Leu Gin Leu Pro Arg Val Arg Asp Trp Glu 485 490 495 Ser Gin Gly Gly Leu Leu Gly Val Gly Glu Ser Gly Phe Val Glu Glu 500 505 510 Ser Asn Leu Trp Thr Ser Ser Gin Leu Ser Leu Thr Asn Asp Asp Leu 515 520 525 Asn Pro Asn Val Arg Asp Pro He Val Lys Gin Leu Glu Val Ser Ser 530 535 540 Gly He Lys Gin 545 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 587 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Met Val Thr Ala Ala Met Leu Leu Gin Cys Cys Pro Val Leu Ala Arg 1 5 10 15 Gly Pro Thr Ser Leu Leu Gly Lys Val Val Lys Thr His Gin Phe Leu 20 25 30 Phe Gly He Gly Arg Cys Pro He Leu Ala Thr Gin Gly Pro Asn Cys 35 40 45 Ser Gin He His Leu Lys Ala Thr Lys Ala Gly Gly Asp Ser Pro Ser 50 55 60 Trp Ala Lys Gly His Cys Pro Phe Met Leu Ser Glu Leu Gin Asp Gly 65 70 75 80 Lys Ser Lys He Val Gin Lys Ala Ala Pro Glu Val Gin Glu Asp Val 85 90 95 Lys Ala Phe Lys Thr Asp Leu Pro Ser Ser Leu Val Ser Val Ser Leu 100 105 110 Arg Lys Pro Phe Ser Gly Pro Gin Glu Gin Glu Gin He Ser Gly Lys 115 120 125 Val Thr His Leu He Gin Asn Asn Met Pro Gly Asn Tyr Val Phe Ser 130 135 140 Tyr Asp Gin Phe Phe Arg Asp Lys He Met Glu Lys Lys Gin Asp His 145 150 155 160 Thr Tyr Arg Val Phe Lys Thr Val Asn Arg Trp Ala Asp Ala Tyr Pro 165 170 175 Phe Ala Gin His Phe Phe Glu Ala Ser Val Ala Ser Lys Asp Val Ser 180 185 190 Val Trp Cys Ser Asn Asp Tyr Leu Gly Met Ser Arg His Pro Gin Val 195 200 205 Leu Gin Ala Thr Gin Glu Thr Leu Gin Arg His Gly Ala Gly Ala Gly 210 215 220 Gly Thr Arg Asn He Ser Gly Thr Ser Lys Phe His Val Glu Leu Glu 225 230 235 240 Gin Glu Leu Ala Glu Leu His Gin Lys Asp Ser Ala Leu Leu Phe Ser 245 250 255 Ser Cys Phe Val Ala Asn Asp Ser Thr Leu Phe Thr Leu Ala Lys He 260 265 270 Leu Pro Gly Cys Glu He Tyr Ser Asp Ala Gly Asn His Ala Ser Met 275 280 285 He Gin Gly He Arg Asn Ser Gly Ala Ala Lys Phe Val Phe Arg His 290 295 300 Asn Asp Pro Asp His Leu Lys Lys Leu Leu Glu Lys Ser Asn Pro Lys 305 310 315 320 He Pro Lys He Val Ala Phe Glu Thr Val His Ser Met Asp Gly Ala 325 330 335 He Cys Pro Leu Glu Glu Leu Cys Asp Val Ser His Gin Tyr Gly Ala 340 345 350 Leu Thr Phe Val Asp Glu Val His Ala Val Gly Leu Tyr Gly Ser Arg 355 360 365 Gly Ala Gly He Gly Glu Arg Asp Gly He Met His Lys He Asp He 370 375 380 He Ser Gly Thr Leu Gly Lys Ala Phe Gly Cys Val Gly Gly Tyr He 385 390 395 400 Ala Ser Thr Arg Asp Leu Val Asp Met Val Arg Ser Tyr Ala Ala Gly 405 410 415 Phe He Phe Thr Thr Ser Leu Pro Pro Met Val Leu Ser Gly Ala Leu 420 425 430 Glu Ser Val Arg Leu Leu Lys Gly Glu Glu Gly Gin Ala Leu Arg Arg 435 440 445 Ala His Gin Arg Asn Val Lys His Met Arg Gin Leu Leu Met Asp Arg 450 455 460 Gly Leu Pro Val He Pro Cys Pro Ser His He He Pro He Arg Val 465 470 475 480 Gly Asn Ala Ala Leu Asn Ser Lys Leu Cys Asp Leu Leu Leu Ser Lys 485 490 495 His Gly He Tyr Val Gin Ala He Asn Tyr Pro Thr Val Pro Arg Gly 500 505 510 Glu Glu Leu Leu Arg Leu Ala Pro Ser Pro His His Ser Pro Gin Met 515 520 525 Met Glu Asp Phe Val Glu Lys Leu Leu Leu Ala Trp Thr Ala Val Gly 530 535 540 Leu Pro Leu Gin Asp Val Ser Val Ala Ala Cys Asn Phe Cys Arg Arg 545 550 555 560 Pro Val His Phe Glu Leu Met Ser Glu Trp Glu Arg Ser Tyr Phe Gly 565 570 575 Asn Met Gly Pro Gin Tyr Val Thr Thr Tyr Ala 580 585

Claims: What is claimed is: 1. A substantially pure 5-aminolevulinic acid synthase which: (a) is obtained from an Aspergillus oryzae strain; (b) has an amino acid sequence which is at least 80% homologous with the amino acid sequence set forth in SEQ ID NO:2; or (c) is encoded by a nucleic acid sequence which is capable of hybridizing under conditions of high stringency with a probe which hybridizes with the nucleic acid sequence set forth in SEQ ID NO: l under the conditions. 2. A 5-aminolevulinic acid synthase according to claim 1 which is obtained from Aspergillus oryzae IFO 4177 or a mutant strain thereof. 3. A 5 -aminolevulinic acid synthase according to claim 1 which has an amino acid sequence which is at least 80% homologous with the amino acid sequence set forth in SEQ ID NO: 2, preferably at least 85 % homologous with the amino acid sequence set forth in SEQ ID NO:2, more preferably at least 90% homologous with the amino acid sequence set forth in SEQ ID NO:2, and most preferably at least 95% homologous with the amino acid sequence set forth in SEQ ID NO:2. 4. A 5-aminolevulinic acid synthase according to claim 1 which has an amino acid sequence set forth in SEQ ID NO:2. 5. A 5-aminolevulinic acid synthase according to claim 1 which has an amino acid sequence which differs by no more than three amino acids from the amino acid sequence set forth in SEQ ID NO:2, preferably by no more than two amino acids from the amino acid sequence set forth in SEQ ID NO:2 and most preferably by one amino acid from the amino acid sequence set forth in SEQ ID NO: 2. 6. A 5-aminolevuIinic acid synthase according to claim 1 which is encoded by a nucleic acid sequence which is capable of hybridizing under conditions of high stringency with a probe which hybridizes with the nucleic acid sequence set forth in SEQ ID NO: l under the conditions. 7. An isolated nucleic acid fragment comprising a nucleic acid sequence which encodes a 5-aminolevulinic acid synthase of claim 1. 8. A nucleic acid fragment according to claim 7, wherein the nucleic acid sequence encodes a 5-aminolevulinic acid synthase obtained from Aspergillus oryzae IFO 4177. 9. A nucleic acid fragment according to claim 8, wherein the nucleic acid sequence is set forth in SEQ ID NO: l . 10. A nucleic acid fragment according to claim 7, wherein the 5-aminolevulinic acid synthase has an amino acid sequence which is at least 80% homologous with the amino acid sequence set forth in SEQ ID NO: 2, preferably at least 85% homologous with the amino acid sequence set forth in SEQ ID NO:2, more preferably at least 90% homologous with the amino acid sequence set forth in SEQ ID NO:2, and most preferably at least 95% homologous with the amino acid sequence set forth in SEQ ID NO:2. 11. A nucleic acid fragment according to claim 7, which is capable of hybridizing under conditions of high stringency with a probe which hybridizes with the nucleic acid sequence set forth in SEQ ID NO: l under the conditions. 12. A nucleic acid construct comprising a nucleic acid fragment of claim 7 operably linked to regulatory regions capable of directing the expression of the 5 -aminolevulinic acid synthase in a suitable expression host. 13. A recombinant vector comprising a nucleic acid construct of claim 12. 14. A vector according to claim 13, wherein the nucleic acid sequence is operably linked to a promoter sequence. 15. A vector according to claim 14, further comprising a transcription termination signal. 16. A vector according to claim 14, further comprising a selectable marker. 17. A recombinant host cell comprising the nucleic acid construct of claim 12. 18. A host cell according to claim 17, wherein the nucleic acid construct is contained on a vector. 19. A host cell according to claim 17, wherein the host cell is a bacterial cell. 20. A host cell according to claim 17, wherein the host cell is a fungal cell. 21. A host cell according to claim 20, wherein the fungal cell is a filamentous fungal cell. 22. The host cell according to claim 21 , wherein the filamentous fungal cell is a cell of a species of Acremonium, Aspergillus, Fusarium, Humicola, Myceliophthora, Mucor, Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma. 23. A host cell according to claim 20, wherein the fungal cell is a yeast cell. 24. A host cell according to claim 23, wherein the yeast cell is a strain of Saccharomyces or Schizosaccharomyces. 25. A host cell according to claim 17, wherein the nucleic acid construct is integrated into the host cell genome. 26. A method for producing a 5 -aminolevulinic acid synthase of claim 1 comprising (a) cultivating an Aspergillus oryzae strain to produce the 5-aminolevulinic acid synthase; and (b) recovering the 5-aminolevulinic acid synthase. 27. A method for producing a 5 -aminolevulinic acid synthase of claim 1 comprising (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleic acid sequence encoding the 5-aminolevulinic acid synthase under conditions conducive to expression of the 5-aminolevulinic acid synthase; and (b) recovering the 5-aminolevulinic acid synthase.

Inventors: CHERRY JOEL R, ELROD SUSAN L, CHERRY, JOEL, R., ELROD, SUSAN, L.
Applicants: NOVO NORDISK BIOTECH INC, NOVO NORDISK BIOTECH, INC.
Application year: 1997
Main classification: C12N9
IPC codes: C12N1, C12N9, C12N15, C12R1