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 which are catalyzed by 5-aminolevulinic acid synthase (EC 2.3.1.37), porphobilinogen synthase (EC 4.2.1.24), porphobilinogen deaminase (EC 4.3.1.8), uroporphyrinogen III synthase (EC 4.2.11.75), uroporphyrinogen III decarboxylase (EC 4.1.1.37), coproporphyrinogen III oxidase (EC 1.3.3.3), protoporphyrinogen IX oxidase (EC 1.3.3.4), and ferrochelatase (EC 4.99.1.1). 5-Aminolevulinic acid synthase catalyzes the condensation of glycine and succinyl-CoA to form 5-aminolevulinic acid. Porphobilinogen synthase (also called 5-aminolevulinic acid dehydratase or 5-aminolevulinic acid dehydrase) catalyzes the condensation of two molecules of 5-aminolevulinic acid to form porphobilinogen. Porphobilinogen deaminase (also called hydroxymethylbilane synthase or uro I synthase) catalyzes the tetrapolymerization of pyrole porphobilinogen into preuroporphyrinogen. Uroporphyrinogen III synthase (also called uro III synthase or uro III cosynthase) catalyzes a rearrangement of the fourth ring of preuroporphyrinogen followed by cyclization to produce uroporphyrinogen III. Uroporphyrinogen III decarboxylase (also called uro D or uroporphyrinogen decarboxylase) catalyzes the decarboxylation of all four acetic acid side chains of uroporphyrinogen III to methyl groups to yield coproporphyrinogen III. Coproporphyrinogen III oxidase (also called coproporphyrinogenase) catalyzes the oxidative decarboxylation of two propionate groups at positions 2 and 4 on the A and B rings of coproporphyrinogen III to vinyl groups yielding protoporphyrinogen IX.

Protoporphyrinogen IX oxidase catalyzes a six electron oxidation of protoporphyrinogen IX to yield protoporphyrin IX. Ferrochelatase (also called ferrolyase, heme synthase, or protoheme ferrolyase) catalyzes the insertion of iron into the protoporphyrin to yield heme.

The catabolism of heme involves two enzymatic steps which are catalyzed by heme oxygenase (EC 1.14.99.3) and biliverdin reductase (EC 1.3.1.24). Heme oxygenase catalyzes the oxidative cleavage of heme to biliverdin. Biliverdin reductase catalyzes the reduction of biliverdin in the presence of NAD (P) H to bilirubin.

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 which acquires a conformation which makes the hemoprotein more stable against proteolytic attack than the apoprotein. 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.

It is an object of the present invention to provide improved methods for increasing production of hemoproteins in fungal strains to yield commercially significant quantities.

Summary of the Invention The present invention relates to methods for producing a hemoprotein, comprising: (a) cultivating a mutant of a parent fungal cell in a nutrient medium suitable for production of the hemoprotein, wherein (i) the mutant comprises one or more first nucleic acid sequences comprising a modification of one or more genes encoding one or more heme catabolic enzymes or a control sequence thereof and (ii) the mutant produces less of the one or more heme catabolic enzymes and more of the hemoprotein than the parent cell when cultivated under the same conditions; and (b) recovering the hemoprotein from the nutrient medium of the mutant cell.

The present invention also relates to methods for producing a hemoprotein wherein the mutant fungal cell further comprises (i) one or more second control sequences which direct the expression of one or more heme biosynthetic enzymes encoded by one or more second nucleic acid sequences endogenous to the fungal cell, wherein the one or more second control sequences are operably linked to the second nucleic acid sequences; and/or (ii) one or more copies of one or more third nucleic acid sequences encoding one or more heme biosynthetic enzymes.

The present invention also relates to methods for producing a hemoprotein wherein the mutant fungal cell further comprises one or more copies of a fourth nucleic acid sequence encoding the hemoprotein.

The present invention also relates to mutant fungal cells for producing hemoproteins and methods for producing the mutant fungal cells.

Detailed Description of the Invention The present invention relates to methods for producing a hemoprotein, comprising: (a) cultivating a mutant of a parent fungal cell in a nutrient medium suitable for production of the hemoprotein, wherein (i) the mutant comprises one or more first nucleic acid sequences comprising a modification of one or more genes encoding one or more heme catabolic enzymes or a control sequence thereof and (ii) the mutant produces less of the one or more heme catabolic enzymes and more of the hemoprotein than the parent cell when cultivated under the same conditions; and (b) recovering the hemoprotein from the nutrient medium of the mutant cell.

"Hemoprotein"is defined herein as any member of a group of proteins containing heme as a prosthetic group. The hemoprotein may be a globin, cytochrome, oxidoreductase, or any other protein containing a heme as a prosthetic group. Heme-containing globins include hemoglobin and myoglobin. Heme-containing cytochromes include cytochrome P450, cytochrome b, cytochrome cl, and cytochrome c. Heme-containing oxidoreductases inclue, but are not limited to, a catalase, oxidase, oxygenase, haloperoxidase, and peroxidase. In a preferred embodiment, the oxidoreductase is a catalase. In another preferred embodiment, the oxidoreductase is an oxidase. In another preferred embodiment, the oxidoreductase is an oxygenase. In another preferred embodiment, the oxidoreductase is a haloperoxidase. In another preferred embodiment, the oxidoreductase is a peroxidase. The hemoprotein may be native or foreign to the mutant fungal cell.

In a more preferred embodiment, the peroxidase is obtained from a Coprins, Arthromyces, or Phanerochaete strain. In an even more preferred embodiment, the peroxidase is obtained from Coprins cinereus, e. g., Coprins cinereus IFO 8371, Coprins macrorhizus, or Arthromyces ramosus. In another more preferred embodiment, the catalase is obtained from a Scytalidium, Aspergillus, or Humicola strain. In another even more preferred embodiment, the catalase is obtained from Scytalidium thermophilum, e. g., Scytalidium thermophilum CBS 117.65, Aspergillus niger, or Humicola insolens.

The mutant fungal cells of the present invention are cultivated in a nutrient medium suitable for production of the hemoprotein using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, small-scale 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 hemoprotein to be expressed and/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 using published compositions (e. g., in catalogues of the American Type Culture Collection). If the hemoprotein is secreted into the nutrient medium, the hemoprotein can be recovered directly from the medium. The signal peptide coding region for secretion of the hemoprotein in the fungal host cell may be obtained, e. g., from the Aspergillus oryzae TAKA amylase gene, Aspergillus niger neutral amylase gene, Rhizomucor miehei aspartic proteinase gene, Humicola lanuginosa cellulase gene, or Rhizomucor miehei lipase gene. If the hemoprotein is not secreted, it is recovered from cell lysats.

The resulting hemoprotein may be recovered by methods known in the art. For example, the hemoprotein may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. The recovered protein may then be further purifie by a variety of chromatographic procedures, e. g., ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like.

In one aspect of the present invention, a hemoprotein is produced in higher amounts by a fungal cell by modifying, e. g., disrupting or deleting, one or more first nucleic acid sequences encoding a heme catabolic enzyme or a control sequence thereof of a parent fungal cell, which results in a mutant fungal cell producing less of the heme catabolic enzyme and more of the hemoprotein than the parent cell when cultivated under the same conditions. The first nucleic acid sequence may be any sequence encoding a heme oxygenase or a biliverdin reductase.

The construction of mutant fungal strains that are deficient in heme catabolic enzyme activity may be conveniently accomplished by modification or inactivation of a first nucleic acid sequence necessary for expression of the heme catabolic enzyme in the cell. The first nucleic acid sequence to be modifie or inactivated may be, for example, a nucleic acid sequence encoding the heme catabolic enzyme or a part thereof essential for exhibiting heme catabolic enzyme activity, or a regulatory element of the nucleic acid sequence required for the expression of the heme catabolic enzyme from the coding sequence of the nucleic acid sequence. An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, i. e., a part which is sufficient for affecting expression of the heme catabolic enzyme. Other control sequences for possible modification are described herein and inclue, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, signal peptide sequence, and transcription terminator..

Modification or inactivation of the first nucleic acid sequence may be performed by subjecting the cell to mutagenesis and selecting for cells in which the heme catabolic enzyme producing capability has been reduced. The mutagenesis, which may be specific or random, may be performed, for exemple, by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed by incubating the cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and selecting for cells exhibiting reduced heme catabolic enzyme activity or production.

Modification or inactivation of production of a heme catabolic enzyme may be accomplished by introduction, substitution, or removal of one or more nucleotides in the first nucleic acid sequence encoding the heme catabolic enzyme or a regulatory element required for the transcription or translation thereof. For example, one or more nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a change of the open reading frame. Such modification or inactivation may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art. Although, in principle, the modification may be performed in vivo, i. e., directly on the cell expressing the nucleic acid sequence to be modifie, it is preferred that the modification be performed in vitro as exemplified below.

An example of a convenient way to inactivate or reduce production is based on techniques of gene replacement or gene disruption. For example, in the gene disruption method, a nucleic acid sequence corresponding to the endogenous gene or gene fragment of interest is mutagenized in vitro to produce a defective nucleic acid sequence which is then transformed into the host cell to produce a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous gene or gene fragment. It may be desirable that the defective gene or gene fragment also encodes a marker which may be used for selection of transformants in which the gene encoding the heme catabolic enzyme has been modifie or destroyed.

Alternatively, modification or inactivation of the first nucleic acid sequence encoding a heme catabolic enzyme may be performed by established anti-sense techniques using a nucleotide sequence complementary to the heme catabolic enzyme encoding sequence. More specifically, production of the heme catabolic enzyme by a cell may be reduced or eliminated by introducing a nucleotide sequence complementary to the first nucleic acid sequence encoding the heme catabolic enzyme which may be transcribed in the cell and is capable of hybridizing to the heme catabolic enzyme MARNA produced in the cell. Under conditions allowing the complementary anti-sense nucleotide sequence to hybridize to the heme catabolic enzyme MARNA, the amount of heme catabolic enzyme translate is thus reduced or eliminated.

A nucleic acid sequence complementary to the first nucleic acid sequence may be obtained from any microbial source. The choice of the source of the nucleic acid sequence will depend on the mutant fungal cell, but preferred sources are fungal sources, e. g., yeast and filamentous fungi. Preferred filamentous fungal sources inclue, but are not limited to, species of Acremonium, Aspergillus, Fusarium, Humicola, Myceliophthora, Mucor, Neurospora, Penicillium, Phanerochaete, Thielavia, Tolypocladium, and Trichoderma.

Preferred yeast sources inclue, but are not limited to, species of Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, and Yarrowia. Furthermore, the nucleic acid sequence may be native to the fungal cell.

Alternately, the nucleic acid sequence complementary to the first nucleic acid sequence may be one or more of the following: 1. Heme oxygenase genes: a. human (Yoshida et al., 1988, European Journal of Biochemistry 171: 457- 461; Shibahara et al., 1989, European Journal of Biochemistry 179: 557-563); b. rat (Shibara et al., 1985, Proceedings of the National Academy of Sciences USA 82: 7865-7869; Muller et al., 1987, Journal of Biological Chemistry 262: 6795-6802); and c. Cyanidium caldarin (Cornejo and Beale, 1988, Journal of Biological Chemistry 263: 11915-11921); d. Synechocystis sp. (Cornejo et al., 1998, Plant Journal 15: 99-107); and e. chicken (Lu et al., 1998, Gene 207: 177-186).

2. Biliverdin reductase genes: a. human (Maines et al., 1996, European Journal of Biochemistry 235: 372-381; Komuro et al., 1996, Biochimca Biophysica Acta 1309: 89-99); b. rat (McCoubrey et al., 1995, Gene 160: 235-240); and c. Synechocystis sp. (Schluchter and Glazer, 1997, Journal of Biological Chemistry272 : 13562-13569).

In the methods of the present invention, the mutant fungal cell produces at least 10% less, preferably at least 25% less, more preferably at least 50% less, even more preferably at least 75% less, and most preferably at least 95% less of the heme catabolic enzyme than a corresponding parent cell when cultivated under the same conditions. Furthermore, the mutant fungal cell produces at least 10% more, preferably at least 25% more, more preferably at least 50% more, even more preferably at least 75% more, and most preferably at least 95% more of the hemoprotein than a corresponding parent cell when cultivated under the same conditions.

In another aspect of the present invention, the mutant cell further comprises one or more second control sequences which direct the expression of one or more heme biosynthetic enzymes encoded by one or more second nucleic acid sequences endogenous to the mutant fungal cell, wherein the one or more second control sequences are operably linked to the second nucleic acid sequences.

The control sequences and/or the nucleic acid sequences can be introduced into the mutant fungal cell by methods well known in the art. For example, the sequences may be introduced and integrated into the host genome by homologous or non-homologous recombination where one or more copies of the sequences are integrated into a single target sequence and/or multiple target sequences. Alternatively, the sequences may be introduced and maintained as a non-integrated expression vector, e. g., a self-replicating extrachromosomal plasmid. A standard procedure in the art for introducing a nucleic acid sequence into a fungal cell involves protoplast formation, transformation of the protoplasts, and regeneration of the cell wall of the transformed protoplasts in a manner known per se (see EP 238 023 and Malardier et al., 1989, Gene 78: 147-156). The cell is preferably transformed with an integrative vector comprising a nucleic acid construct which contains the control sequences and/or nucleic acid sequences encoding the heme biosynthetic enzymes where the construct is conveniently integrated into the host genome of the filamentous fungal cell, preferably the chromosome (s). The term"nucleic acid construct"is defined herein to mean a nucleic acid molecule, either single-or double-stranded, which is isolated from a naturally occurring gene or which has been modifie to contain segments of nucleic acids which are combine and juxtapose in a manner which would not otherwise exist in nature.

The second nucleic acid sequence may be any nucleic acid sequence encoding a heme biosynthetic enzyme including a 5-aminolevulinic acid synthase, porphobilinogen synthase, porphobilinogen deaminase, uroporphyrinogen synthase, uroporphyrinogen decarboxylase, coproporphyrinogen oxidase, protoporphyrinogen oxidase, or ferrochelatase, wherein the second nucleic acid sequence is endogenous to the parent fungal cell. The term "endogenous"is defined herein as originating from the parent fungal cell.

The term"control sequences"is meant herein to include all components which direct the expression of the coding sequence of the second nucleic acid sequence in the mutant fungal cell under conditions compatible with the control sequences. Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. The control sequences may be native to the second nucleic acid sequence encoding the heme biosynthetic enzyme, may be obtained from other sources, or may be a combination of native and foreign control sequences. The foreign control sequences may simply replace or be added to the natural control sequences in order to obtain enhanced production of the desired heme biosynthetic enzyme relative to the natural control sequence normally associated with the coding sequence. Such control sequences inclue, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. For expression under the direction of control sequences, the second nucleic acid sequence 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 of the second nucleic acid sequence is achieved under conditions compatible with the control sequences. The term"coding sequence"as defined herein is a sequence which is transcribed into MARNA and translate into a heme biosynthetic enzyme when placed under the control of the above mentioned control sequences. The boundary of the coding sequence is generally determined by the ATG start codon located just upstream of the open reading frame at the 5'end of the MARNA and a transcription terminator sequence located just downstream of the open reading frame at the 3'end of the MARNA. A coding sequence can inclue, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences. The term"operably linked"is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the production of a polypeptide.

The second control sequence may be an appropriate promoter sequence, a nucleic acid sequence which is recognized by the mutant fungal cell for expression of the second nucleic acid sequence. The promoter sequence contains transcription and translation control sequences which mediate the expression of the heme biosynthetic enzyme. The promoter may be any promoter sequence which shows transcriptional activity in a mutant fungal cell of choice and may be obtained from genes either native or foreign to the mutant fungal cell.

Examples of suitable promoters for directing the transcription of the second nucleic acid sequence in filamentous fungal cells are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protase, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protase (WO 96/00787), NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof.

Examples of suitable promoters for directing the transcription of the second nucleic acid sequence in yeast cells are promoters obtained from the genes encoding Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL 1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.

Other useful promoters for yeast cells are described by Romanos et al., 1992, Yeast 8: 423- 488.

The second control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the mutant fungal cell to terminate transcription. The terminator sequence is operably linked to the 3'terminus of the second nucleic acid sequence encoding the heme biosynthetic enzyme. The terminator sequence may be native or foreign to the second nucleic acid sequence encoding the heme biosynthetic enzyme. Any terminator which is functional in a mutant fungal cell of choice is likely to be useful in the present invention.

Preferred terminators for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protase.

Preferred terminators for yeast host cells are obtained from the genes encoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

The second control sequence may also be a suitable leader sequence, a nontranslated region of a MARNA which is important for translation by the mutant fungal cell. The leader sequence is operably linked to the 5'terminus of the second nucleic acid sequence encoding the heme biosynthetic enzyme. The leader sequence may be native or foreign to the second nucleic acid sequence. Any leader sequence which is functional in a mutant fungal cell of choice is likely to be useful in the present invention.

Preferred leaders for filamentous fungal cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase and Aspergillus oryzae triose phosphate isomerase.

Suitable leaders for yeast cells are obtained from the genes encoding Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The second control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3'terminus of the second nucleic acid sequence and which, when transcribed, is recognized by the mutant fungal cell to add polyadenosine residues to transcribed MARNA. The polyadenylation sequence may be native or foreign to the second nucleic acid sequence encoding the heme biosynthetic enzyme. Any polyadenylation sequence that is functional in a mutant fungal cell of choice is likely to be useful in the present invention.

Particufarly preferred polyadenylation sequences for filamentous fungal cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, and Aspergillus niger alpha- glucosidase. Useful polyadenylation sequences for yeast cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The second control sequence may also be 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 heme biosynthetic enzyme to a particular cellular compartment. The signal peptide coding region may be native or foreign to the second nucleic acid sequence encoding the heme biosynthetic enzyme. The 5'end of the coding sequence of the second 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 heme biosynthetic enzyme. 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 heme biosynthetic enzyme in a mutant fungal cell of choice may be used in the present invention.

An effective signal peptide coding region for filamentous fungal cells is the signal peptide coding region obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Rhizomucor miehei aspartic proteinase gene, Humicola lanuginosa cellulase, or Rhizomucor miehei lipase.

Useful signal peptides for yeast cells are obtained from the genes encoding Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinas, and Saccharomyces cerevisiae invertase. Other useful signal peptide coding regions are described by Romanos et al., 1992, supra.

The second control sequence may also be a propeptide coding region which codes for an amino acid sequence positioned at the amino terminus of a mature biochemically active polypeptide. The resultant polypeptide is known as a proenzyme or a propolypeptide (or a zymogen in some cases). Proenzymes are generally inactive and can be converted to mature active polypeptides by catalytic or autocatalytic cleavage of the propeptide from the proenzyme. A biochemically active polypeptide is defined herein as a heme biosynthetic enzyme which is produced in active form which performs the biochemical activity of its natural counterpart. The propeptide sequence may be native or foreign to the second nucleic acid sequence encoding the heme biosynthetic enzyme. The nucleic acid sequence encoding a propeptide may be obtained from the genes encoding Saccharomyces cerevisiae alpha- factor and Myceliophthora thermophila laccase.

In another aspect of the present invention, the mutant fungal cell further comprises one or more copies of one or more third nucleic acid sequences encoding one or more heme biosynthetic enzymes as described in WO 97/47746. The third nucleic acid sequence (s) may be any nucleic acid sequence encoding a heme biosynthetic enzyme including a 5- aminolevulinic acid synthase, porphobilinogen synthase, porphobilinogen deaminase, uroporphyrinogen synthase, uroporphyrinogen decarboxylase, coproporphyrinogen oxidase, protoporphyrinogen oxidase, or ferrochelatase. The third nucleic acid sequence (s) may be obtained from any microbial source. The choice of the source of the third nucleic acid sequence will depend on the mutant fungal cell, but preferred sources are fungal sources, e. g., yeast and filamentous fungi. Preferred filamentous fungal sources inclue, but are not limited to, species of Acremonium, Aspergillus, Fusarium, Humicola, Myceliophthora, Mucor, Neurospora, Penicillium, Phanerochaete, Thielavia, Tolypocladium, and Trichoderma. Preferred yeast sources inclue, but are not limited to, species of Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, and Yarrowia. Furthermore, the third nucleic acid sequences may be native to the mutant fungal cell.

The third nucleic sequence (s) may be one or more of the following: 1.5-Aminolevulinic acid synthase genes: a. Saccharomyces cerevisiae (Urban-Grimal et al., 1986, European Journal of Biochemistry 156: 511-519); b. Aspergillus nidulans (Bradshaw et al., 1993, Current Genetics 23: 501-507); c. Rhodobacter sphaeroides (Tai et al., 1988, Gene 70: 139-152); d. Rhodobacter capsulatus (Hornberger et al., 1990, Molecular General Genetics 211: 371-378); e. Escherichia coli (Drolet et al., 1989, Molecular General Genetics 216: 347- 352); and f Aspergillus oryzae (WO 97/47736).

2. Porphobilinogen synthase genes: a. Saccharomyces cerevisiae (Myers et al., 1987, Journal of Biological Chemistry 262: 16822-16829); b. Staphylococcus aureus (Kafala and Sasarman, 1994, Canadian Journal of Microbiology 40: 651-657); c. Rhodobacter sphaeroides (Delaunay et al., 1991, Journal of Bacteriology 173: 2712-2715); d. Escherichia coli (Echelard et al., 1988, Molecular General Genetics 214: 503- 508); e. Bacillus subtils (Hansson et al., 1991, Journal of Bacteriology 173: 2590- 2599); and f Aspergillus oryzae (WO 97/47753).

3. Porphobilinogen deaminase genes a. Saccharomyces cerevisiae (Keng et al., 1992, Molecular General Genetics 234:233-243); b. human (Yoo et al., 1993, Genomics 15: 221-229; Raich et al., 1986, Nucleic Acids Research 14: 5955-5968); c. Escherichia coli (Thomas and Jordan, 1986, Nucleic Acids Research 14: 6215-6226); and d. Bacillus subtils (Petricek et al., 1990, Journal of Bacteriology 172: 2250- 2258).

4. Uroporphyrinogen III synthase genes: a. Saccharomyces cerevisiae (millet and Labbe-Bois, 1995, Yeast 11: 419- 424); b. Bacillus subtils (Hansson et al., 1991, Journal of Bacteriology 173: 2590- 2599); and c. Escherichiacoli (Jordanetal., 1987, Nucleic Acids Research. 15: 10583).

5. Uroporphyrinogen III decarboxylase genes: a. Saccharomyces cerevisiae (Garey et al., 1992, European Journal of Biochemistry 205: 1011-1016); and b. human (Romeo et al., 1986, Journal of Biological Chemistry 261: 9825- 9831).

6. Coproporphyrinogen III oxidase genes: a. human (Martasek et al., 1994, Proceedings of the National Academy of Sciences USA 911: 3024-3028); b. Escherichia coli (Troup et al., 1994, Journal of Bacteriology 176: 673-680); and c. Saccharomyces cerevisiae (Zaagorec et al., 1986, Journal of Biological Chemistry 263: 9718-9724).

7. Protoporphyrinogen IX oxidase genes: a. human (Taketani et al., 1995, Genomics 29: 698-703); b. Bacillus subtils (Dailey et al., 1994, Journal of Biological Chemistry 269: 813-815); and c. Escherichia coli (Sasarman et al., 1993, Canadian Journal of Microbiology 39: 155-161).

8. Ferrochelatase genes: a. Saccharomyces cerevisiae (Labbe-Bois, 1990, Journal of Biological Chemistry 265 72878-72883); b. bovine (Shibuya et al., 1995, Biochimica Biophysica Acta 1231: 117-120); c. Bradyrhizobium japonicum (Frustaci and O'Brian, 1993, Applied Environmental Microbiology 59: 2347-2351); d. Escherichia coli (Frustaci and O'Brian, 1993, Journal of Bacteriology 175: 2154-2156); and e. Bacillus subtils (Hansson and Hederstedt, 1992, Journal of Bacteriology 174: 88081-88093).

In a more preferred embodiment, the third nucleic acid sequence is obtained from a species of Aspergillus. In an even more preferred embodiment, the third nucleic acid sequence is obtained from Aspergillus ficuum, Aspergillus foetidus, Aspergillus japonicus, Aspergillus niger, Aspergillus nidulans, or Aspergillus oryzae. In another more preferred embodiment, the third nucleic acid sequence is obtained from a species of Saccharomyces.

In an even more preferred embodiment, the third nucleic acid sequence is obtained from Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis.

In a preferred embodiment, the third nucleic acid sequence is operably linked to one or more third control sequences as described in WO 97/47746. The third control sequences may be native to the third nucleic acid sequences encoding the heme biosynthetic enzymes or may be partially or wholly obtained from foreign sources. The foreign control sequences may simply replace the natural control sequences in order to obtain enhanced production of the desired heme biosynthetic enzyme relative to the natural control sequence normally associated with the coding sequence. The third control sequences can be any of the control sequences exemplified above in connection with the second control sequences.

In another aspect of the present invention, the mutant fungal cell comprises one or more copies of one or more second control sequences and one or more copies of one or more third nucleic acid sequences. Preferably, the third nucleic acid sequences are operably linked to one or more third control sequences.

The second control sequences, the third nucleic acid sequences and/or the third control sequences may be contained in the same nucleic acid construct, or they may be contained in different nucleic acid constructs. Each nucleic acid construct may comprise integrational elements for directing integration by homologous recombination into the genome of the fungal host at a precise location. To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the mutant fungal cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. On the other hand, each nucleic acid construct may be integrated into the genome of the mutant fungal cell by non-homologous recombination.

The nucleic acid constructs may be inserted into a suitable vector or the third nucleic acid sequences may be inserted directly into a vector which already contains the control sequences using molecular biology techniques known in the art. The vectors may be any vector which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of a nucleic acid sequence. The choice of a vector will typically depend on the compatibility of the vector with the mutant fungal 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 the mutant fungal cell, is integrated into the 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 introduced into the genome of the fungal cell.

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. Suitable selectable markers for filamentous fungal cells inclue, but not limited to, amdS, pyrG, argB, niaD, sC, trpC, bar, and hygB. Suitable markers for yeast cells inclue, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP 1, and URA3. 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 procedures used to ligate the nucleic acid constructs, 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., Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor, New York, 1989).

In the methods of the present invention, the mutant fungal eell may further comprise one or more copies of one or more fourth nucleic acid sequences encoding a hemoprotein (s).

The fourth nucleic acid sequence (s) may be contained in the same vector as the second control sequences, the third nucleic acid sequences and the third control sequences, or they may be contained in different vectors. Preferably, the fourth nucleic acid sequences are operably linked to fourth control sequences. The control sequences exemplified above in connection with the second control sequences are also applicable to the fourth control sequences.

In the methods of the present invention, the nutrient medium may further comprise a source of heme, analogs thereof or one or more heme biosynthetic pathway intermediates.

See Product Brochure of Porphyrin Products Inc. (Logan, UT) for list of heme analogs and pathway intermediates. For example, when a nucleic acid sequence encoding one of the enzymes in the heme biosynthetic pathway is introduced into the mutant fungal cell, one or more pathway intermediates in one or more preceding steps may become rate-limiting. In such a case, 6ne can supplement the culture medium with these one or more pathway intermediates. In order for these pathway intermediates to be introduced into the cell, one can use an enzyme which is capable of semi-permeabilizing the cell membrane, e. g., NOVOZYM 234'"'' (Novo Nordisk A/S).

In the methods of the present invention, the nutrient medium may further comprise a source of iron. Alternatively, the nutrient medium may further comprise any other metal ion that can induce porphyrin synthesis. See, e. g., Mamet et al., 1996, BioMetals, 9: 73-77.

The present invention also relates to mutant fungal cells for producing a hemoprotein which comprise a modification of one or more first nucleic acid sequences encoding one or more heme catabolic enzymes or a control sequence thereof, wherein the mutant cell produces less of the one or more heme catabolic enzymes and more of the hemoprotein than the parent cell of the mutant cell when cultivated under the same conditions.

The mutant fungal cells of the present invention may further comprise one or more second control sequences which direct the expression of one or more heme biosynthetic enzymes encoded by one or more second nucleic acid sequences endogenous to the cell and/or one or more copies of one or more third nucleic acid sequences encoding one or more heme biosynthetic enzymes. The sequences may be integrated into the genome of the mutant fungal cell or may be contained in a self-replicating extrachromosomal vector.

The mutant fungal cells of the present invention may further comprise one or more copies of one or more fourth nucleic acid sequences encoding a hemoprotein (s), wherein the one or more fourth nucleic acid sequences are operably linked to fourth control sequences which direct the expression of the hemoprotein (s) in the cell, where the fourth nucleic acid sequence (s) encoding the hemoprotein (s) is integrated into the genome of the cell or is contained in a self-replicating extrachromosomal vector.

The present invention further relates to methods for producing a mutant fungal cell, comprising modifying, e. g., disrupting or deleting, one or more first nucleic acid sequences encoding a heme catabolic enzyme or a control sequence thereof of a parent fungal cell, wherein the mutant fungal cell produces less of the heme catabolic enzyme and more of a hemoprotein than the parent cell when cultivated under the same conditions.

The choice of a fungal cell in the methods of the present invention will to a large extent depend upon the sources of the control sequences, the nucleic acid sequences encoding the heme biosynthetic enzymes, and the hemoprotein.

In a preferred embodiment, the fungal cell is a yeast cell."Yeast"as used herein inclues ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). The ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is comprise of four subfamilies, Schizosaccharomycoideae (e. g., genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e. g., genera Kluyveromyces, Pichia, and Saccharomyces). The basidiosporogenous yeasts include the genera Leucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeast belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae (e. g., genera Sorobolomyces and Bullera) and Cryptococcaceae (e. g., genus Candida). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9,1980. The biology of yeast and manipulation of yeast genetics are well known in the art (see, e. g., Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B. J., and Stopani, A. O. M., editors, 2nd edition, 1987; The Yeasts, Rose, A. H., and Harrison, J. S., editors, 2nd edition, 1987; and The Molecular Biology of the Yeast Saccharomyces, Strathern et al., editors, 1981).

In a more preferred embodiment, the yeast cell is a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

In a most preferred embodiment, the yeast cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. In another most preferred embodiment, the yeast cell is a Kluyveromyces lactis cell. In another most preferred embodiment, the yeast cell is a Yarrowia lipolytica cell.

In another preferred embodiment, the fungal host cell is a filamentous fungal cell.

"Filamentous fungi"include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are characterized by a mycelial wall compose of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative. In a more preferred embodiment, the filamentous fungal host cell is an Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma cell.

In an even more preferred embodiment, the filamentous fungal cell is an Aspergillus cell. In another even more preferred embodiment, the filamentous fungal cell is an Acremonium cell. In another even more preferred embodiment, the filamentous fungal cell is a Fusarium cela. In another even more preferred embodiment, the filamentous fungal cell is a Humicola cell. In another even more preferred embodiment, the filamentous fungal cell is a Mucor cell. In another even more preferred embodiment, the filamentous fungal cell is a Myceliophthora cell. In another even more preferred embodiment, the filamentous fungal cell is a Neurospora cell. In another even more preferred embodiment, the filamentous fungal cell is a Penicillium cell. In another even more preferred embodiment, the filamentous fungal cell is a Thielavia cell. In another even more preferred embodiment, the filamentous fungal cell is a Tolypocladium cell. In another even more preferred embodiment, the filamentous fungal cell is a Trichoderma cell.

In a most preferred embodiment, the filamentous fungal cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most preferred embodiment, the filamentous fungal cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, or Fusarium venenatum cell. In an even most preferred embodiment, the filamentous fungal parent cell is a Fusarium venenatum (Nirenberg sp. nov.). In another most preferred embodiment, the filamentous fungal cell is a Humicola insolens or Humicola lanuginosa cell. In another most preferred embodiment, the filamentous fungal cell is a Mucor miehei cell. In another most preferred embodiment, the filamentous fungal cell is a Myceliophthora thermophila cell. In another most preferred embodiment, the filamentous fungal cell is a Neurospora crassa cell. In another most preferred embodiment, the filamentous fungal cell is a Penicillium purpurogenum cell. In another most preferred embodiment, the filamentous fungal cell is a Thielavia terrestris cell.

In another most preferred embodiment, the Trichoderma cell is a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride 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.

Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470- 1474. A suitable method of transforming Fusarium species is described by Malardier et al., 1989, supra or in WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

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 appende claims. In the case of conflit, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.