WO2022175440A1

WO2022175440A1 - Inactive heme polypeptides

Info

Publication number: WO2022175440A1
Application number: PCT/EP2022/054058
Authority: WO
Inventors: Jesper Vind; Lars Olsen
Original assignee: Novozymes A/S
Priority date: 2021-02-18
Filing date: 2022-02-18
Publication date: 2022-08-25
Also published as: AR124921A1

Abstract

The present invention relates to heme-containing polypeptide variants having a reduced or eliminated activity, and polynucleotides encoding the variants. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptide variants, such as the use of the polypeptide variants in food or feed.

Description

INACTIVE HEME POLYPEPTIDES

Reference to a Sequence Listing

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

Background of the Invention

Field of the Invention

The present invention relates to heme-containing polypeptide variants having a reduced or eliminated enzyme activity, and polynucleotides encoding the variants. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptide variants, such as the use of the polypeptide variants in food or feed.

Description of the Related Art

The global demand for food and feed products which are free of animal-derived components (e.g. milk or meat), such as vegetarian burgers, livestock-feed and pet-food, has witnessed a constant increase during the past years and decades. The increased demand is based on a change of lifestyle towards a more sustainable, vegan or vegetarian lifestyle, or due to religious or health-related reasons. Although a growing number of consumers prefer the absence of animal-derived components in their food, many consumers still desire the presence of a meat-like flavor or a meat-like color, as well as a certain protein content in their food. A moderate or high protein content is also desired for feed products to ensure a protein-rich nutrition of livestock or pets, especially when their owners decide to pursue feed products which are free of animal-derived products. Typical animal-free foods are often based on soy (e.g. tofu or tempeh), wheat gluten (seitan), pea or mycoprotein. To meet the consumers^' expectation, the food and feed manufacturers as well as their suppliers face the challenging task to provide a meat-like flavor/color without adding animal-derived components. It has been suggested that the blood-based hemoglobin protein and the muscle-based myoglobin protein are giving traditional meat-food the flavor which the consumer associates with a typical meat-flavor.

Previous attempts to imitate the meat-like flavor of vegetarian food products include the addition of several components, such as hydrolyzed wheat, hydrolyzed milk, yeast extracts or isolated hemoglobin, but also the addition of chemical compounds to imitate specific flavors, such as a meaty smokey bacon flavor by adding butyl levulinate, 3-methyl-2-butanethiol, 3-methyl-2- butenyl thioacetate, 2-methoxy phenol (guaiacol), 4-propyl 2,6-dimethoxy phenol, 2-isopropyl pyridine, 2-methyl 5- ethyl thiophene, or 5-methyl thiophene 2-carboxyaldehyde. As described in

W09704110, hemoglobin protein can be produced in recombinant host cells. However, the recombinant production of hemoglobin is presently neither very efficient nor sustainable, and the recombinant hemoglobin yields are fairly low when compared to other recombinantly produced proteins. Therefore, when produced in industrial scale the recombinant hemoglobin cannot meet the amounts which are demanded by the food and feed industry and the consumer.

The object of the present invention is to provide an alternative food or feed additive that can provide a meat-like flavor and/or meat-like color to the food or feed whilst also facilitating an adequate amino acid supply to the consumer.

Summary of the Invention

The present invention is based on the surprising and inventive finding that inactivated heme-containing enzymes can be obtained from recombinant host cells in order to provide a meat-like flavor and/or meat-like color in a food or feed. Surprisingly, the recombinant expression of said inactivated heme-containing enzymes was found to be convenient and efficient, wherein the heme-group of the inactivated enzyme, when added to food or feed, contributes to a meat like flavor and/or meat-like color.

The present invention provides isolated or purified heme-containing enzymes having reduced or eliminated enzymatic activity and polynucleotides encoding the heme-containing enzymes.

Peroxidases and peroxygenases, both classified as oxidoreductases / oxidases, are some of the key antioxidant enzymes and are widely distributed in nature. Peroxidases catalyze the oxidation of various electron donor substrates concomitant with the decomposition of H2O2. The enzymatic activity of peroxidases has been successfully used for biopulping and bio-bleaching in the paper and textile industries. Peroxygenases are promising catalysts for preparative oxyfunctionalization chemistry as they combine the versatility of P450 monooxygenases with simplicity of co-factor-independent enzymes. Both peroxidases and peroxygenases often comprise a heme-group contributing to the enzymatic activity.

The inventors successfully expressed heme-containing enzymes other than hemoglobin, wherein the heme-containing enzymes have been inactivated in order to prevent undesired enzymatic activity or side effects after the inactivated enzyme has been added to the food or feed, or after said food or feed containing the inactivated enzyme has been consumed. The heme- containing enzymes have been inactivated by introducing single amino acid mutations in the amino acid sequence of the heme-containing enzymes, wherein the enzyme is inactivated by at least one of (i) mutation of the amino acid coordinating the iron atom in the heme; (ii) restricting the access to active site by cysteine-bridges or bulky amino acids; or (iii) mutation of one or more amino acids involved in the catalysis. The inventors have surprisingly found that inactivated oxidoreductases / oxidases, such as peroxidases and peroxygenases, can be efficiently produced in recombinant cell systems without compromising the meat-like taste or meat-like color associated with the heme-group of the enzyme. Based on the results of the examples, the invention is expected to also work for other heme-containing enzymes.

Thus, in a first aspect the invention relates to a heme-containing enzyme variant of a heme-containing parent enzyme, said enzyme variant comprising at least one amino acid alteration, such as an amino acid substitution, amino acid deletion and/or amino acid insertion, whereby the enzymatic activity of the variant is reduced or eliminated, wherein the enzyme variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 2, SEQ ID NO: 35, SEQ ID NO: 4, SEQ ID NO: 36, SEQ ID NO: 6, or SEQ ID NO: 37.

In a second aspect, the present invention relates to nucleic acid constructs or expression vectors comprising a heterologous promoter operably linked to a polynucleotide encoding the enzyme variant of the first aspect of the invention.

In a third aspect, the present invention also relates to recombinant host cells comprising in their genome a nucleic acid construct or expression vector according to the second aspect of the invention.

In a fourth aspect, the present invention relates to methods for producing an inactivated heme-containing enzyme variant, comprising: a) providing a recombinant host cell according to the third aspect or a host cell producing an enzyme variant according to the first aspect; b) cultivating said host cell under conditions conducive for expression of the heme-containing enzyme variant, and optionally c) recovering the heme-containing enzyme variant.

In a fifth aspect, the present invention relates to a method of flavoring and/or coloring food or feed, the method comprising the steps of a) providing the food or feed, and b) adding the heme- containing enzyme variant according to the first aspect to the food or feed.

In a sixth aspect, the present invention relates to a food or feed product comprising an inactivated heme-containing enzyme variant according to the first aspect.

In a seventh and final aspect, the invention relates to the use of an inactivated heme- containing enzyme variant according to the first aspect for the flavoring and/or coloring of food or feed.

Brief Description of the Drawings

Figure 1 shows the absorption spectra of purified inactivated heme-containing enzymes.

Figure 2 shows detailed absorption spectra of purified inactivated-heme containing enzymes in the 450-650 nm range. Definitions

In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

Reference to “about” a value or parameter herein includes aspects that are directed to that value or parameter perse. For example, description referring to “about X” includes the aspect “X”.

Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Animal: The term “animal” refers to all animals except humans. Examples of animals are non-ruminants, and ruminants. Ruminant animals include, for example, animals such as sheep, goats, cattle, e.g. beef cattle, cows, and young calves, deer, yank, camel, llama and kangaroo. Non-ruminant animals include mono-gastric animals, e.g. pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers); horses (including but not limited to hotbloods, coldbloods and warm bloods), young calves; fish (including but not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish); and crustaceans (including but not limited to shrimps and prawns).

Animal feed: The term “animal feed” or “feed” refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal. Animal feed for a mono-gastric animal typically comprises concentrates as well as vitamins, minerals, enzymes, direct fed microbial, amino acids and/or other feed ingredients (such as in a premix) whereas animal feed for ruminants generally comprises forage (including roughage and silage) and may further comprise concentrates as well as vitamins, minerals, enzymes direct fed microbial, amino acid and/or other feed ingredients (such as in a premix).

Catalytic domain: The term “catalytic domain” or “active site” means the region of an enzyme containing the catalytic machinery of the enzyme and/or comprising the substrate binding domain of the enzyme. The catalytic domain and/or active site is responsible for the enzymatic activity of the protein. For heme-containing enzymes, the enzymatic activity can be reduced or eliminated by (i) mutation of the amino acid coordinating the iron atom in the heme, (ii) restricting the access to active site by introducing cysteine-bridges or one or more “bulky” amino acids (e.g. tryptophan, phenylalanine, tyrosine, proline, histidine, glutamine, leucine, isoleucine and methionine) in close proximity to the catalytic domain, or by (iii) mutation of one or more amino acids involved in the catalysis or comprised in the catalytic domain. In particular, phenylalanine residues in close proximity to the catalytic domain or active site of heme-containing enzymes play a major role for the electron transfer and enzymatic activity of the heme-containing enzymes. The reduction or elimination of the enzymatic activity is preferably carried out without losing the meat like flavor, which is predominantly facilitated by the heme-group of the enzyme. cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon, such as ATG, GTG, or TTG, and ends with a stop codon, such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Coloring: The term “coloring” means the color adjustment of food, feed or at least one of a food or feed component by adding a heme-containing polypeptide, preferably with inactivated enzymatic activity, so that the food, feed or at least one of the food or feed component appears predominantly dark red, red, light red, or in a red-related color to the eye of the consumer or feed/food producer. The red color is predominantly caused by the heme of the heme-containing polypeptide and can be controlled by varying the amount of heme-containing polypeptide added to the food, feed or at least one of the food or feed component.

Control sequences: The term “control sequences” means nucleic acid sequences necessary for expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native (/.e., from the same gene) or heterologous (/.e., from a different gene) to the polynucleotide encoding the polypeptide or native or heterologous to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide. Expression: The term “expression” means any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

Feed: The term “feed” means any type of raw or processed product suitable for domestic animal consumption, including (i) feed or feed concentrates high in energy value including fat, cereal grains and their by-products (barley, corn, oats, rye, wheat), high protein oil meals or cakes (soybean, canola, cottonseed, peanut), and by-products from processing of sugar beets, sugarcane, animals, and fish, (ii) roughages, including pasture grasses, hays, silage, root crops, straw and stover (cornstalks), (iii) feed supplements and (iv) pet food including cat food and dog food.

Flavoring: The term “flavoring” means the flavor adjustment of food, feed or at least one of a food or feed component by adding a heme-containing polypeptide, preferably with inactivated enzymatic activity, so that the food, feed or at least one of the food or feed component acquires a meat-like taste for the consumer or feed/food producer. The meat-like taste is predominantly caused by the heme of the heme-containing polypeptide and can be controlled by varying the amount of heme-containing polypeptide added to the food, feed or at least one of the food or feed component.

Food: The term “food” means any type of raw or processed product suitable for human or animal consumption, and includes dairy products and analogues, fats, oils, fat emulsions, edible ices including sherbet and sorbet, fruits and vegetables, seaweeds, nuts, seeds, confectionery, cereals and cereal products, pasta, tofu, soybean products, bakery wares, meat and meat products, fish and fish products including mollusks, crustaceans and echinoderms, eggs and egg products, sweeteners, salts, spices, soups, sauces, salads, protein products, protein shakes, vegetarian or vegan burgers, foodstuffs intended for particular nutritional uses, infant consumables, beverages, sport or energy or electrolyte drinks, dietary supplements, ready-to-eat savouries, vegetarian meals or beverages, or vegan meals or beverages. With regards to the invention, the inactivated heme-containing enzyme variant can be added to the food in order to add a meat-like flavor and/or meat-like color to the food, and/or to increase the protein content of the food.

Fragment: The term “fragment” means a polypeptide, a catalytic domain, or a heme- containing polypeptide module having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide or domain; wherein the fragment comprises a heme.

In some embodiments, a fragment contains at least 60 amino acid residues (e.g., amino acids 20 to 80 of SEQ ID NO: 8 or SEQ ID NO: 10), at least 80 amino acid residues (e.g., amino acids 10 to 90 of SEQ ID NO: 8 or SEQ ID NO: 10), or at least 100 amino acid residues ( e.g ., amino acids 10 to 110 of SEQ ID NO: 8 or SEQ ID NO: 10). In some embodiments, a fragment contains at least 150 amino acid residues (e.g., amino acids 20 to 170 of SEQ ID NO: 8 or SEQ ID NO: 10), at least 200 amino acid residues (e.g., amino acids 20 to 220 of SEQ ID NO: 8 or SEQ ID NO: 10), or at least 300 amino acid residues (e.g., amino acids 30 to 330 of SEQ ID NO: 8 or SEQ ID NO: 10).

In some embodiments, a fragment contains at least 100 amino acid residues (e.g., amino acids 50 to 150 of SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16), at least 150 amino acid residues (e.g., amino acids 50 to 200 of SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16), or at least 200 amino acid residues (e.g., amino acids 50 to 250 of SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16). In some embodiments, a fragment contains at least 250 amino acid residues (e.g., amino acids 50 to 300 of SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16), at least 300 amino acid residues (e.g., amino acids 50 to 300 of SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16), or at least 50 amino acid residues (e.g., amino acids 70 to 120 of SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16).

In some embodiments, a fragment contains at least 50 amino acid residues (e.g., amino acids 200 to 250 of SEQ ID NO: 18), at least 100 amino acid residues (e.g., amino acids 200 to 300 of SEQ ID NO: 18), or at least 150 amino acid residues (e.g., amino acids 150 to 300 of SEQ ID NO: 18). In some embodiments, a fragment contains at least 200 amino acid residues (e.g., amino acids 120 to 320 of SEQ ID NO: 18), at least 300 amino acid residues (e.g., amino acids 60 to 360 of SEQ ID NO: 18), or at least 330 amino acid residues (e.g., amino acids 20 to 350 of SEQ ID NO: 18).

In some embodiments, a fragment contains at least 50 amino acid residues (e.g., amino acids 1 to 50 of SEQ ID NO: 20), at least 70 amino acid residues (e.g., amino acids 10 to 90 of SEQ ID NO: 20), or at least 100 amino acid residues (e.g., amino acids 10 to 110 of SEQ ID NO: 20). In some embodiments, a fragment contains at least 150 amino acid residues (e.g., amino acids 5 to 155 of SEQ ID NO: 20), at least 200 amino acid residues (e.g., amino acids 10 to 210 of SEQ ID NO: 20), or at least 220 amino acid residues (e.g., amino acids 5 to 225 of SEQ ID NO: 20).

In some embodiments, a fragment contains at least 50 amino acid residues (e.g., amino acids 140 to 190 of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID

NO: 30, SEQ ID NO: 32 or SEQ ID NO: 34), at least 70 amino acid residues (e.g., amino acids

130 to 200 of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,

SEQ ID NO: 32 or SEQ ID NO: 34), or at least 90 amino acid residues (e.g., amino acids 110 to

200 of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ

ID NO: 32 or SEQ ID NO: 34). In some embodiments, a fragment contains at least 120 amino acid residues (e.g., amino acids 50 to 170 of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,

SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 or SEQ ID NO: 34), at least 130 amino acid residues ( e.g ., amino acids 100 to 230 of SEQ ID NO: 22), or at least 150 amino acid residues (e.g., amino acids 70 to 220 of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 or SEQ ID NO: 34).

Fusion polypeptide: The term “fusion polypeptide” is a polypeptide in which one polypeptide is fused at the N-terminus or the C-terminus of a polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779). A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

Heme: The term “heme” means an iron-containing compound of the porphyrin class which forms the non-protein part of e.g. hemoglobin and other heme-containing polypeptides. A heme is an organic, ring-shaped molecule which due to its special structure is capable of holding, or “hosting” an iron molecule. A heme is made from 4 pyrroles, which are small pentagon-shaped molecules made from 4 carbons and 1 nitrogen. Four pyrroles together form a tetrapyrrole. If the tetrapyrrole has substitutions on the side chains which allow it to hold a metal ion, it is called a porphyrin. Thus, a heme is an iron-holding porphyrin.

Heterologous: The term "heterologous" means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" means, with respect to a polypeptide or nucleic acid, that a control sequence, e.g., promoter, or domain of a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37.

Host cell: The term "host cell" means any microbial or plant cell into which a nucleic acid construct or expression vector comprising a polynucleotide of the present invention has been introduced. Methods for introduction include but are not limited to protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. In some embodiments, the host cell is an isolated recombinant host cell that is partially or completely separated from at least one other component with, including but not limited to, proteins, nucleic acids, cells, etc.

Hybrid polypeptide: The term “hybrid polypeptide” means a polypeptide comprising domains from two or more polypeptides, e.g., a binding module from one polypeptide and a catalytic domain from another polypeptide. The domains may be fused at the N-terminus or the C-terminus.

Hybridization: The term "hybridization" means the pairing of substantially complementary strands of nucleic acids, using standard Southern blotting procedures. Hybridization may be performed under medium, medium-high, high or very high stringency conditions. Medium stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours, followed by washing three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 55°C. Medium-high stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours, followed by washing three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 60°C. High stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours, followed by washing three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 65°C. Very high stringency conditions means prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours, followed by washing three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 70°C.

Isolated: The term “isolated” means a polypeptide, nucleic acid, cell, or other specified material or component that is separated from at least one other material or component with which it is naturally associated as found in nature, including but not limited to, for example, other proteins, nucleic acids, cells, etc. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted polypeptide.

Mature polypeptide: The term “mature polypeptide” means a polypeptide in its mature form following N-terminal processing (e.g., removal of signal peptide). In one aspect, the mature polypeptide is a peroxidase essentially consisting of, consisting of or comprising SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 35; a peroxygenase essentially consisting of, consisting of or comprising SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO:18 or SEQ ID NO: 36; or a peroxygenase essentially consisting of, consisting of or comprising SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34 or SEQ ID NO: 37.

Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature heme-containing enzyme variant having reduced or eliminated enzymatic activity. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 1089 of SEQ ID NO: 7, nucleotides 61 to 1089 of SEQ ID NO: 9, nucleotides 76 to 1161 of SEQ I D NO: 11 , nucleotides 76 to 1161 of SEQ I D NO: 13, nucleotides 76 to 1161 of SEQ ID NO: 15, nucleotides 76 to 1161 of SEQ ID NO: 17, nucleotides 226 to 942 of SEQ ID NO: 19, nucleotides 226 to 942 of SEQ ID NO: 21, nucleotides 226 to 942 of SEQ ID NO: 23, nucleotides 226 to 941 of SEQ ID NO: 25, nucleotides 226 to 942 of SEQ ID NO: 27, nucleotides 226 to 942 of SEQ ID NO: 29, nucleotides 226 to 942 of SEQ ID NO: 31 , or nucleotides 226 to 942 of SEQ ID NO: 33, or the cDNA sequence of any thereof.

Meat analogue: The term “meat analogue” means a meat-like substance made predominantly from plants. Synonyms for meat analogue are plant-based meat, vegan meat, meat substitute, mock meat, meat alternative, imitation meat, fake meat or faux meat. Meat analogues typically facilitate certain aesthetic qualities such as texture, flavor, appearance, or chemical characteristics of specific types of meat. Meat analogue also means a food made from vegetarian ingredients, preferably without animal products such as dairy. Many meat analogues are soy- based (e.g. tofu, tempeh) or gluten-based, but may also be made from pea protein or mycoprotein.

Native: The term "native" means a nucleic acid or polypeptide naturally occurring in a host cell.

Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.

Operably linked: The term “operably linked” means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.

Purified: The term “purified” means a nucleic acid or polypeptide that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.

Proximity: The term “proximity” means, in the folded protein state of the enzyme variant, a distance of less than 20 A, such as less than 15 A, less than 10A, or less than 5A between the C_a of the mutated amino acid residue and the closest heme center (or its iron atom), or a distance of less than 20 A, such as less than 15 A, less than 10A, or less than 5A between the C_a of the mutated amino acid residue and the C_a of the closest amino acid residue of the catalytic domain of the enzyme variant.

Recombinant: The term "recombinant," when used in reference to a cell, nucleic acid, protein or vector, means that it has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. Recombinant nucleic acids differ from a native sequence by one or more nucleotides and/or are operably linked to heterologous sequences, e.g., a heterologous promoter in an expression vector. Recombinant proteins may differ from a native sequence by one or more amino acids and/or are fused with heterologous sequences. A vector comprising a nucleic acid encoding a polypeptide is a recombinant vector. The term “recombinant” is synonymous with “genetically modified” and “transgenic”.

Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et ai, 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. , 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment) Soret peak: The term “Soret peak” or “Soret band” means an intense peak in the blue wavelength region of the visible spectrum, corresponding to a wavelength of maximum absorption (electromagnetic radiation) ranging around 400 nm in the blue region. For example, the "Soret peak" is used to describe the absorption of vividly-pigmented heme- containing moieties, such as various cytochromes.

Subsequence: The term “subsequence” means a polynucleotide having one or more (e.g., several) nucleotides absent from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a heme-containing polypeptide fragment having reduced or eliminated enzymatic activity.

Variant: The term “variant” means a heme-containing polypeptide having reduced or eliminated enzymatic activity comprising a man-made mutation, i.e., a substitution, insertion, and/or deletion (e.g., truncation), at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. The total number of amino acid substitutions in a variant can vary from one substitution to two, three four or five substitutions.

Wild-type: The term "wild-type" in reference to an amino acid sequence or nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally- occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild- type sequence).

Detailed Description of the Invention

Heme-containing enzyme variants having reduced or eliminated enzymatic activity

In a first aspect, the invention relates to a heme-containing enzyme variant of a heme- containing parent enzyme, said enzyme variant comprising at least one amino acid alteration, such as an amino acid substitution, amino acid deletion and/or amino acid insertion, whereby the enzymatic activity of the variant is reduced or eliminated, wherein the enzyme variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 2, SEQ ID NO: 35, SEQ ID NO: 4, SEQ ID NO: 36, SEQ ID NO: 6, or SEQ ID NO: 37. The reduced or eliminated enzymatic activity of the variant is reduced or eliminated compared to the enzymatic activity of the heme-containing parent enzyme.

In some embodiments, the parent enzyme is an oxidoreductase or an oxidase.

In some embodiments, the parent enzyme and/or enzyme variant is chosen from the list of a NADPH-cytochrome P450 oxidoreductase (EC 1.6.2.4); a cytochrome B (EC 1.10.2.2); a peroxidase (EC 1.11.1) such as a catalase (EC 1.11.1.6), a cytochrome-C peroxidase (EC 1.11.1.5) or peroxidases categorized as EC 1.11.1.7; a peroxygenase (EC 1.11.2), such as a haloperoxidase (EC 1.11.2.1); a plant peroxidase or a halo-peroxidase; a cytochrome P450 enzyme (EC 1.14.14.1), such as a P450 mono-oxygenase or a P450 di-oxygenase; a heme oxygenase (EC 1.14.99.3); a ferredoxin reductase (EC 1.18.1.3); a cytochrome bd-l oxidase (Cytochrome-D; EC 7.1.1.7); and a cytochrome c-oxidase (cytochrome A; EC 7.1.1.9; former EC 1.9.3.1).

In a preferred embodiment the at least one amino acid mutation comprises or consists of:

(i) at least one amino acid mutation of at least one amino acid located in proximity to the iron atom of the heme;

(ii) at least one amino acid mutation of at least one amino acid located in proximity to the catalytic domain of the enzyme, or

(iii) at least one amino acid mutation of at least one amino acid within the catalytic domain of the enzyme; wherein the at least one amino acid mutation comprises or consists of an amino acid insertion, an amino acid deletion, and/or an amino acid substitution, such as an amino acid insertion of an amino acid selected from the list of lysine, arginine, cysteine, tryptophan, phenylalanine, tyrosine, proline, histidine, glutamine, leucine, isoleucine and methionine, and/or an amino acid substitution by an amino acid selected from the list of lysine, arginine, cysteine, tryptophan, phenylalanine, tyrosine, proline, histidine, glutamine, leucine, isoleucine and methionine. In a more preferred embodiment the amino acid insertion and/or the amino acid substitution is an amino acid insertion of and/or amino acid substitution by an amino acid selected from the list of cysteine, tryptophan, phenylalanine, tyrosine, proline, histidine, glutamine, leucine, isoleucine and methionine.

The substitution of amino acids located in close proximity to the heme and/or catalytic domain by at least one cysteine or by “bulky” amino acids as tryptophan, phenylalanine, tyrosine, proline, histidine, glutamine, leucine, isoleucine and methionine, or by lysine or arginine is an efficient method to reduce or eliminate enzymatic activity. In some embodiments two, three, four or five amino acids are substituted in order to reduce or eliminate enzymatic activity.

In one embodiment the enzyme variant is a variant of a parent enzyme encoded by the genome of a fungal genus or species.

In one embodiment the enzyme variant is a variant of a parent enzyme encoded by the genome of a filamentous fungal cell, e.g., an Acremonium , Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell, in particular, an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, 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 sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

In an preferred embodiment the enzyme variant is a variant of a parent enzyme encoded by Coprinus cinereus or Humicola insolens.

In one embodiment the enzyme variant is a peroxidase or a peroxygenase, such as a peroxidase essentially consisting of, consisting of or comprising SEQ ID NO: 8 or SEQ ID NO:10; a peroxygenase essentially consisting of, consisting of or comprising SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18; or a peroxygenase essentially consisting of, consisting of or comprising SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, or SEQ ID NO: 34.

In one embodiment the variant is having reduced or eliminated peroxidase activity and having an amino acid sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, but less than 100% sequence identity, to SEQ ID NO: 2 or SEQ ID NO: 35, and comprising an alteration at a position corresponding to position 75 of SEQ ID NO: 2, preferably H75D, or to position 55 of SEQ ID NO: 35, preferably the alteration comprises or consists of H55D.

In one embodiment, the enzyme variant is comprising at least one amino acid substitution at a position corresponding to position 75 of the polypeptide of SEQ ID NO: 2, preferably comprising a substitution of the amino acid residue at the position corresponding to position 75 of SEQ ID NO: 2 with Asp (D) H75D or Trp (W) H75W, most preferably comprising a substitution of the amino acid residue at the position corresponding to position 75 of SEQ ID NO: 2 with Asp (D) H75D.

In one embodiment, the enzyme variant is comprising at least one amino acid substitution at a position corresponding to position 55 of the polypeptide of SEQ ID NO: 35, preferably comprising a substitution of the amino acid residue at the position corresponding to position 55 of SEQ ID NO: 35 with Asp (D) H55D or Trp (W) H55W, most preferably comprising a substitution of the amino acid residue at the position corresponding to position 55 of SEQ ID NO: 35 with Asp (D) H55D.

In another embodiment, the variant is having reduced or eliminated peroxygenase activity and having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 4, and comprising at least one alteration at a position corresponding to position 123, 127 and/or 249 of SEQ ID NO: 4, preferably the at least one alteration comprises or consists of I123W, V127L, V127W, and/or F249W.

In another embodiment, the variant is having reduced or eliminated peroxygenase activity and having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 36, and comprising at least one alteration at a position corresponding to position 98, 102 and/or 224 of SEQ ID NO: 36, preferably the at least one alteration comprises or consists of I98W, V102L, V102W, and/or F224W.

In one embodiment, the variant is comprising at least one amino acid substitution, at one or more positions corresponding to positions 98, 102, and 224 of the polypeptide of SEQ ID NO: 36, preferably the one or more substitution is selected from the group consisting of: a substitution of the amino acid residue at a position corresponding to position 98 of SEQ ID NO: 36 with Trp (W) I98W; a substitution of the amino acid residue at a position corresponding to position 102 of SEQ ID NO: 36 with Leu (L) V102L or Trp (W) V102W, preferably with Leu (L) V102L; and a substitution of the amino acid residue at a position corresponding to position 224 of SEQ ID NO: 36 with Trp (W) F224W.

In another embodiment the variant is having reduced or eliminated peroxygenase activity and having a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, but less than

100% sequence identity, to the polypeptide of SEQ ID NO: 6 or SEQ ID NO: 37, and comprising at least one alteration at a position corresponding to position 17, 151, 154, 158, and/or 162 of SEQ ID NO: 37, preferably the at least one alteration comprises or consists of C17H, L151C, I154L, G158A, G158S, G158W, G158C, and/or A162L

In one embodiment the variant is comprising at least one amino acid substitution, at one or more positions corresponding to positions 17, 151 , 154, 158, and 162 of the polypeptide of SEQ ID NO: 37, preferably the one or more substitution is selected from the group consisting of: a substitution of the amino acid residue at a position corresponding to position 17 of SEQ ID NO: 37 with His (H) C17H; a substitution of the amino acid residue at a position corresponding to position 151 of SEQ ID NO: 37 with Cys (C) L151C; a substitution of the amino acid residue at a position corresponding to position 154 of SEQ ID NO: 37 with Leu (L) I154L; a substitution of the amino acid residue at a position corresponding to position 158 of SEQ ID NO: 37 with Ala (A) G158A, Ser (S) G158S, Trp (W) G158W, Cys (C) G158C, preferably with Ala (A) G158A, Ser (S) G158S, or Trp (W) G158W, more preferably with Ala (A) G158A or Ser (S) G158S; and a substitution of the amino acid residue at a position corresponding to position 162 of SEQ ID NO: 37 with Leu (L) A162L.

In some embodiments, the present invention relates to isolated or purified heme- containing enzyme variants having a sequence identity of at least 60%, e.g., at least 65%, at least

70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least

85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least

92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least

99%, but less than 100% to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 35, SEQ ID NO: 4, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 6 having reduced or eliminated enzymatic activity. In one aspect, the polypeptides differ by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37.

In another embodiment the enzyme variant having reduced or eliminated enzyme activity is selected from the group consisting of:

(a) a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, but less than 100% sequence identity, to SEQ ID NO: 2 or SEQ ID NO: 35, and comprising an alteration at a position corresponding to position 55 of SEQ ID NO: 35.

(b) a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 4 or SEQ ID NO: 36, and comprising an alteration at a position corresponding to position 98, 102 and/or 224 of SEQ ID NO: 36;

(c) a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 6 or SEQ ID NO: 37, and comprising an alteration at a position corresponding to position 17, 154 151 , 158, and/or 162 of SEQ ID NO: 37;

(d) a polypeptide encoded by a polynucleotide that hybridizes under medium stringency conditions with the full-length complement of the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or the cDNA sequence thereof;

(e) a polypeptide encoded by a polynucleotide having at least 60%%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 , SEQ ID NO:

3, or SEQ ID NO: 5, or the cDNA sequence thereof;

(f) a polypeptide derived from a mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37 by substitution, deletion or addition of one or several amino acids in the mature polypeptide of SEQ ID NO: 2, SEQ ID NO:

4, SEQ ID NO: 6, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37; and

(g) a heme-containing fragment of the polypeptide of (a), (b), (c), (d), (e) or (f) that has reduced or eliminated enzymatic activity.

An isolated or purified heme-containing polypeptide variant with a reduced or eliminated enzymatic activity comprising a catalytic domain selected from the group consisting of:

(a) a catalytic domain having at least 60%, e.g., at least 65%, at least 70%, at least

75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least

94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, but less than 100% sequence identity to amino acids 51 to 55 of SEQ ID NO: 35 and/or amino acids 45 to 60 of SEQ ID NO: 35;

(b) a catalytic domain encoded by a polynucleotide that hybridizes under medium stringency conditions with the full-length complement of nucleotides 211 to 225 of SEQ ID NO: 1 and/or nucleotides 193 to 240 of SEQ ID NO: 1 ;

(c) a catalytic domain encoded by a polynucleotide having at least 60%, e.g., at least

65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least

99% sequence identity, but less than 100% sequence identity to nucleotides 211 to 225 of SEQ

ID NO: 1 and/or to nucleotides 193 to 240 of SEQ ID NO: 1 ; (d) a catalytic domain derived from amino acids 51 to 55 of SEQ ID NO: 35 and/or amino acids 45 to 60 of SEQ ID NO: 35 by substitution, deletion or addition of one or several amino acids in the amino acids 51 to 55 of SEQ ID NO: 35 and/or amino acids 45 to 60 of SEQ ID NO: 35; and

(e) a fragment of the catalytic domain of (a), (b), (c) or (d), that has reduced or eliminated enzymatic activity.

94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, but less than 100% sequence identity to amino acids 85 to 90 of SEQ ID NO: 37 and/or amino acids 155 to 165 of SEQ ID NO: 37;

(b) a catalytic domain encoded by a polynucleotide that hybridizes under medium stringency conditions with the full-length complement of nucleotides 478 to 495 of SEQ ID NO: 5 and/or nucleotides 688 to 720 of SEQ ID NO: 5;

99% sequence identity, but less than 100% sequence identity to nucleotides 478 to 495 of SEQ ID NO: 5 and/or nucleotides 688 to 720 of SEQ ID NO: 5;

(d) a catalytic domain derived from amino acids 85 to 90 of SEQ ID NO: 37 and/or amino acids 155 to 165 of SEQ ID NO: 37 by substitution, deletion or addition of one or several amino acids in the amino acids 85 to 90 of SEQ ID NO: 37 and/or amino acids 155 to 165 of SEQ ID NO: 37; and

(a) a catalytic domain having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, but less than 100% sequence identity to amino acids 210 to 225 of SEQ ID NO: 36 and/or amino acids 200 to 230 of SEQ ID NO: 36; (b) a catalytic domain encoded by a polynucleotide that hybridizes under medium stringency conditions with the full-length complement of nucleotides 703 to 750 of SEQ ID NO: 3 and/or nucleotides 673 to 765 of SEQ ID NO: 3;

(c) a catalytic domain encoded by a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, but less than 100% sequence identity to nucleotides 703 to 750 of SEQ ID NO: 3 and/or nucleotides 673 to 765 of SEQ ID NO: 3;;

(d) a catalytic domain derived from amino acids 210 to 225 of SEQ ID NO: 36 and/or amino acids 200 to 230 of SEQ ID NO: 36 by substitution, deletion or addition of one or several amino acids in the amino acids 210 to 225 of SEQ ID NO: 36 and/or amino acids 200 to 230 of SEQ ID NO: 36; and

In one embodiment, the variant has an reduced enzymatic activity of below 1%, below 2%, below 3%, below 4%, below 5%, below 6%, below 7%, below 8%, below 9%, below 10% relative to the enzymatic activity of the parent enzyme not comprising the at least one amino acid alteration.

In one embodiment, the enzymatic activity is measured with an ABTS assay, preferably the ABTS assay according to Example 3.

In one embodiment, the purified enzyme variant has a melting temperature similar to the melting temperature of myoglobin. In one embodiment the melting temperature is determined by differential scanning calorimetry.

In one embodiment, the purified enzyme variant shows a Soret peak with a maximum at 410 to 425 nm, preferably at 415 to 420 nm, such as at around 415 nm, or as at around 420 nm.

In one embodiment the Soret peak is determined in phosphate buffer with pH 9.

In some embodiments, the present invention relates to isolated or purified heme- containing enzyme variants having reduced or eliminated enzymatic activity encoded by polynucleotides that hybridize under medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with the full-length complement of the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or the cDNA thereof (Sambrook et ai, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).

The polynucleotide of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5 or a subsequence thereof, as well as the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ

ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37, or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding heme-containing enzymes from strains of different genera or species according to methods well known in the art. Such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a heme-containing enzyme variant having unaltered, reduced or eliminated enzymatic activity. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or another suitable carrier material. In order to identify a clone or DNA that hybridizes with SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 or a subsequence thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that the polynucleotides hybridize to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1 , SEQ ID NO: 3 or SEQ ID NO: 5; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5; (iii) the cDNA sequence thereof; (iv) the full-length complement thereof; or (v) a subsequence thereof; under medium to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.

In some embodiments, the present invention relates to isolated heme-containing enzyme variants having reduced or eliminated enzymatic activity encoded by polynucleotides having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to the mature polypeptide coding sequence of SEQ ID NO: 1 , SEQ ID NO: 3 or SEQ ID NO: 5 or the cDNA sequence thereof.

The polynucleotide encoding the inactivated heme-containing enzyme variant preferably comprises, consists essentially of, or consists of nucleotides 61 to 1089 of SEQ ID NO: 7, nucleotides 61 to 1089 of SEQ ID NO: 9, nucleotides 76 to 1161 of SEQ ID NO: 11 , nucleotides

76 to 1161 of SEQ ID NO: 13, nucleotides 76 to 1161 of SEQ ID NO: 15, nucleotides 76 to 1161 of SEQ ID NO: 17, nucleotides 226 to 942 of SEQ ID NO: 19, nucleotides 226 to 942 of SEQ ID NO: 21, nucleotides 226 to 942 of SEQ ID NO: 23, nucleotides 226 to 941 of SEQ ID NO: 25, nucleotides 226 to 942 of SEQ ID NO: 27, nucleotides 226 to 942 of SEQ ID NO: 29, nucleotides 226 to 942 of SEQ ID NO: 31, or nucleotides 226 to 942 of SEQ ID NO: 33, or the cDNA sequence of any thereof.

In some embodiments, the present invention relates to an inactivated heme-containing enzyme variant derived from a mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37 by substitution, deletion or addition of one or several amino acids in the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37. In some embodiments, the present invention relates to variants of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions. In one aspect, the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10. In an embodiment, the polypeptide has an N-terminal extension and/or C-terminal extension of 1-10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. The amino acid changes may be of a major nature, that is non-conservative amino acid substitutions or insertions that significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly histidine tract, an antigenic epitope or a binding module.

Essential amino acids in a heme-containing enzyme can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for reduced or eliminated enzymatic activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et ai, 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et ai, 1992, Science 255: 306-312; Smith et ai., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et ai, 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide. Essential amino acids for SEQ ID NO: 2 or SEQ ID NO: 35 are at least amino acids at a position corresponding to position 55 of SEQ ID NO: 35. Essential amino acids for SEQ ID NO: 4 or SEQ ID NO: 36 are at least amino acids at a position corresponding to position 98, 102 and/or 224 of SEQ ID NO: 36. Essential amino acids for SEQ ID NO: 6 or SEQ ID NO: 37 are at least amino acids at a position corresponding to position 17, 151 , 154, 158 and/or 162 of SEQ ID NO: 37.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et ai, 1991, Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et ai, 1986, Gene 46: 145; Ner et ai, 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et ai., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

The inactivated heme-containing enzyme variant may be a hybrid polypeptide or a fusion polypeptide.

The polypeptides of the present invention have a meat-like flavor and meat-like color and, when added to a feed or food, or a feed or food component, contribute to a meat-like tasting experience for the consumer while also providing an adequate amino acid source. Since the enzymatic activity of the polypeptides of the present invention are reduced or inactivated, the heme-containing polypeptides do not interfere negatively with the food or feed or its components.

Sources of heme-containing enzymes

A heme-containing enzyme variant with reduced or inactivated enzymatic activity of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the enzyme encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

In another aspect, the enzyme variant is a polypeptide obtained from a Basidiomycota or Ascomycota, e.g., a polypeptide obtained from Coprinus cinereus or Humicola insolens, respectively.

It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents. Strains of these 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 (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

The polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Catalytic Domains

In some embodiments, the present invention also relates to catalytic domains of a heme- containing polypeptide with reduced or eliminated enzymatic activity having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and less than 100% to amino acids 65 to 80 of SEQ ID NO: 2 or amino acids 45 to 60 of SEQ ID NO: 35. In one aspect, the catalytic domains comprise amino acid sequences that differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from amino acids 65 to 80 of SEQ ID NO: 2 or amino acids 45 to 60 of SEQ ID NO: 35.

The catalytic domain preferably comprises, consists essentially of, or consists of amino acids 65 to 80 of SEQ ID NO: 2 or amino acids 45 to 60 of SEQ ID NO: 35; or is a fragment thereof having reduced or eliminated enzymatic activity.

In some embodiments, the present invention also relates to catalytic domains encoded by polynucleotides that hybridize under medium stringency conditions with the full-length complement of nucleotides 193 to 240 of SEQ ID NO: 1 (Sambrook et al., 1989, supra).

In some embodiments, the present invention also relates to catalytic domains encoded by polynucleotides having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to nucleotides 190 to 240 of SEQ ID NO: 1.

In some embodiments, the present invention relates to a catalytic domain derived from amino acids 65 to 80 of SEQ ID NO: 2 or amino acids 45 to 60 of SEQ ID NO: 35 by substitution, deletion or addition of one or several amino acids in the amino acids 65 to 80 of SEQ ID NO: 2 or amino acids 45 to 60 of SEQ ID NO: 35. In some embodiments, the present invention also relates to catalytic domain variants of amino acids 65 to 80 of SEQ ID NO: 2 or amino acids 45 to 60 of SEQ ID NO: 35 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions. In one aspect, the number of amino acid substitutions, deletions and/or insertions introduced into the sequence of amino acids 65 to 80 of SEQ ID NO: 2 or amino acids 45 to 60 of SEQ ID NO: 35 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 8, 9, or 10.

In some embodiments, the present invention also relates to catalytic domains of a heme- containing polypeptide with reduced or eliminated enzymatic activity having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and less than 100% to amino acids 160 to 165 of SEQ ID NO: 6 and/or amino acids 230 to 240 of SEQ ID NO: 6, or to amino acids 85 to 90 of SEQ ID NO: 37 and/or amino acids 155 to 165 of SEQ ID NO: 37. In one aspect, the catalytic domains comprise amino acid sequences that differ by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from amino acids 160 to 165 of SEQ ID NO: 6 and/or amino acids 230 to 240 of SEQ ID NO: 6, or to amino acids 85 to 90 of SEQ ID NO: 37 and/or amino acids 155 to 165 of SEQ ID NO: 37.

The catalytic domain preferably comprises, consists essentially of, or consists amino acids 160 to 165 of SEQ ID NO: 6 and/or amino acids 230 to 240 of SEQ ID NO: 6, or to amino acids 85 to 90 of SEQ ID NO: 37 and/or amino acids 155 to 165 of SEQ ID NO: 37; or is a fragment thereof having reduced or eliminated enzymatic activity.

In some embodiments, the present invention also relates to catalytic domains encoded by polynucleotides that hybridize under medium stringency conditions with the full-length complement of nucleotides 478 to 495 of SEQ ID NO: 5 and/or nucleotides 688 to 720 of SEQ ID NO: 5 (Sambrook et ai, 1989, supra).

In some embodiments, the present invention also relates to catalytic domains encoded by polynucleotides having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to nucleotides 478 to 495 of SEQ ID NO: 5 and/or nucleotides 688 to 720 of SEQ ID NO: 5.

In some embodiments, the present invention relates to a catalytic domain derived from amino acids 160 to 165 of SEQ ID NO: 6 and/or amino acids 230 to 240 of SEQ ID NO: 6, or to amino acids 85 to 90 of SEQ ID NO: 37 and/or amino acids 155 to 165 of SEQ ID NO: 37 by substitution, deletion or addition of one or several amino acids in the amino acids 160 to 165 of

SEQ ID NO: 6 and/or amino acids 230 to 240 of SEQ ID NO: 6, or to amino acids 85 to 90 of SEQ

ID NO: 37 and/or amino acids 155 to 165 of SEQ ID NO: 37. In some embodiments, the present invention also relates to catalytic domain variants of amino acids 160 to 165 of SEQ ID NO: 6 and/or amino acids 230 to 240 of SEQ ID NO: 6, or to amino acids 85 to 90 of SEQ ID NO: 37 and/or amino acids 155 to 165 of SEQ ID NO: 37 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions. In one aspect, the number of amino acid substitutions, deletions and/or insertions introduced into the sequence of amino acids 160 to 165 of SEQ ID NO: 6 and/or amino acids 230 to 240 of SEQ ID NO: 6, or to amino acids 85 to 90 of SEQ ID NO: 37 and/or amino acids 155 to 165 of SEQ ID NO: 37 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 8, 9, or 10.

In some embodiments, the present invention also relates to catalytic domains of a heme- containing polypeptide with reduced or eliminated enzymatic activity having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and less than 100% to amino acids 235 to 250 of SEQ ID NO: 4 and/or amino acids 225 to 255 of SEQ ID NO: 4, or to amino acids 210 to 225 of SEQ ID NO: 36 and/or amino acids 200 to 230 of SEQ ID NO: 36. In one aspect, the catalytic domains comprise amino acid sequences that differ by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from amino acids 235 to 250 of SEQ ID NO: 4 and/or amino acids 225 to 255 of SEQ ID NO: 4, or to amino acids 210 to 225 of SEQ ID NO: 36 and/or amino acids 200 to 230 of SEQ ID NO: 36.

The catalytic domain preferably comprises, consists essentially of, or consists amino acids 235 to 250 of SEQ ID NO: 4 and/or amino acids 225 to 255 of SEQ ID NO: 4, or to amino acids 210 to 225 of SEQ ID NO: 36 and/or amino acids 200 to 230 of SEQ ID NO: 36; or is a fragment thereof having reduced or eliminated enzymatic activity.

In some embodiments, the present invention also relates to catalytic domains encoded by polynucleotides that hybridize under medium stringency conditions with the full-length complement of nucleotides 703 to 750 of SEQ ID NO: 3 and/or nucleotides 673 to 765 of SEQ ID NO: 3 (Sambrook et ai, 1989, supra).

In some embodiments, the present invention also relates to catalytic domains encoded by polynucleotides having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% to nucleotides 703 to 750 of SEQ ID NO: 3 and/or nucleotides 673 to 765 of SEQ ID NO: 3.

In some embodiments, the present invention relates to a catalytic domain derived from amino acids 235 to 250 of SEQ ID NO: 4 and/or amino acids 225 to 255 of SEQ ID NO: 4, or to amino acids 210 to 225 of SEQ ID NO: 36 and/or amino acids 200 to 230 of SEQ ID NO: 36 by substitution, deletion or addition of one or several amino acids in the amino acids 235 to 250 of

SEQ ID NO: 4 and/or amino acids 225 to 255 of SEQ ID NO: 4, or to amino acids 210 to 225 of

SEQ ID NO: 36 and/or amino acids 200 to 230 of SEQ ID NO: 36. In some embodiments, the present invention also relates to catalytic domain variants of amino acids 235 to 250 of SEQ ID

NO: 4 and/or amino acids 225 to 255 of SEQ ID NO: 4, or to amino acids 210 to 225 of SEQ ID

NO: 36 and/or amino acids 200 to 230 of SEQ ID NO: 36 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions. In one aspect, the number of amino acid substitutions, deletions and/or insertions introduced into the sequence of amino acids 235 to 250 of SEQ ID NO: 4 and/or amino acids 225 to 255 of SEQ ID NO: 4, or to amino acids 210 to 225 of SEQ ID NO: 36 and/or amino acids 200 to 230 of SEQ ID NO: 36 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 8, 9, or 10.

In another aspect, a polypeptide comprising a catalytic domain of the present invention may further comprise a carbohydrate binding module.

Polynucleotides

The present invention also relates to isolated polynucleotides encoding a heme-containing enzyme variant with reduced or eliminated enzymatic activity according to the first aspect.

The techniques used to isolate or clone a polynucleotide are known in the art and include isolation from genomic DNA or cDNA, or a combination thereof. The cloning of the polynucleotides from genomic DNA can be affected, e.g., by using the polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis etal., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used. The polynucleotides may be cloned from a strain of a Basidiomycota (e.g. Coprinus cinereus) or a Ascomycota (e.g. Humicola insolens) or a related organism and thus, for example, may be a species variant of the polypeptide encoding region of the polynucleotide.

Modification of a polynucleotide encoding an enzyme variant of the present invention may be necessary for synthesizing polypeptides substantially similar to the variant. The term “substantially similar” to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., variants that differ in specific activity, thermostability, pH optimum, or the like. The variants may be constructed on the basis of the polynucleotide presented as the mature polypeptide coding sequence of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, or the cDNA sequence thereof, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence and result in a reduced or eliminated enzymatic activity. Fora general description of nucleotide substitution, see, e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. In a second aspect, then invention relates to a nucleic acid construct or expression vector comprising a heterologous promoter operably linked to a polynucleotide encoding the enzyme variant of the first aspect.

The polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing transcription of the polynucleotide of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene ( amyQ ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene ( penP ), Bacillus stearothermophilus maltogenic amylase gene ( amyM ), Bacillus subtilis levansucrase gene ( sacB ), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis crylllA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trc promoter (Egon et ai, 1988, Gene 69: 301-315), Streptomyces coelicolor agarase gene ( dagA ), and prokaryotic beta-lactamase gene (Villa- Kamaroff et ai, 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et ai, 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are described in "Useful proteins from recombinant bacteria" in Gilbert et ai, 1980, Scientific American 242: 74- 94; and in Sambrook et ai, 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of the polynucleotide of the present invention in a filamentous fungal host cell are promoters obtained from the genes for

Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase ( glaA ), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae those phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I,

Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, and Trichoderma reesei translation elongation factor, as well as the NA2-tpi promoter (a modified promoter from an Aspergillus neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus those phosphate isomerase gene; non-limiting examples include modified promoters from an Aspergillus niger neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus nidulans or Aspergillus oryzae those phosphate isomerase gene); and mutant, truncated, and hybrid promoters thereof. Other promoters are described in U.S. Patent No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae those phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423- 488.

The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3’-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.

Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease ( aprH ), Bacillus licheniformis alpha-amylase ( amyL ), and Escherichia coli ribosomal RNA ( rrnB ).

Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, and Trichoderma reesei translation elongation factor.

Preferred terminators for yeast host cells are obtained from the genes for 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 etai, 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene. Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue etal., 1995, J. Bacteriol. 177: 3465-3471).

The control sequence may also be a leader, a non-translated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5’-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans those phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes for 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 control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3’-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

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

Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell’s secretory pathway. The 5’-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5’-end of the coding sequence may contain a signal peptide coding sequence that is heterologous to the coding sequence. A heterologous signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a heterologous signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha- amylase, Bacillus stearothermophilus neutral proteases ( nprT , nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiol. Rev. 57: 109- 137.

Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease ( aprE ), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide would be operably linked to the regulatory sequence. Expression Vectors

The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vector that 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. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like 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.

Examples of bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

Selectable markers for use in a filamentous fungal host cell include, but are not limited to, adeA

(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB (phosphoribosyl- aminoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase), bar

(phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5’-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicus bar gene. Preferred for use in a Trichoderma cell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system as described in WO 2010/039889. In one aspect, the dual selectable marker is a hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on the polynucleotide’s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to 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 host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUE3110, pE194, pTA1060, and rAMb1 permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1 , ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et ai, 1991, Gene 98: 61-67; Cullen et ai, 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sam brook et al., 1989, supra).

Host Cells

The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a heme-containing enzyme variant of the present invention. A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.

In a third aspect, the invention relates to a recombinant host cell comprising in its genome the nucleic acid construct or expression vector of the second aspect.

In some embodiments, the polypeptide is heterologous to the recombinant host cell.

In some embodiments, at least one of the one or more control sequences is heterologous to the polynucleotide encoding the polypeptide.

In some embodiments, the recombinant host cell comprises at least two copies, e.g., three, four, or five, of the polynucleotide of the present invention.

The host cell may be any microbial or plant cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryotic cell or a fungal cell.

The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells. The bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

The introduction of DNA into a Bacillus cell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), competent cell transformation (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell may be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et ai, 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell may be effected by protoplast transformation, electroporation (see, e.g., Gong et ai, 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, e.g., Mazodier et ai, 1989, J. Bacteriol. 171: 3583-3585), or transduction (see, e.g., Burk e etal., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell may be effected by electroporation (see, e.g., Choi et ai, 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cell may be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etai., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.

The host cell may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et ai, In, Ainsworth and Bisby’s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). 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, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kiuyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

In one embodiment the host cell is a Pichia pastoris host cell or a Komagataella phaffii cell. The fungal host cell may be a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et a!., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed 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.

The filamentous fungal host cell may be an Acremonium , Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, 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 sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

In a preferred embodiment, the recombinant host cell is an Aspergillus oryzae 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 and Trichoderma host cells are described in EP 238023, Yelton et ai, 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and 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 ai, 1983, J. Bacteriol. 153: 163; and Hinnen et ai, 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide.

In a fourth aspect, the invention relates to a method of producing an inactivated heme- containing enzyme variant, comprising: a. Providing a recombinant host cell producing an enzyme variant according to the first aspect, or a host cell according to the third aspect; b. cultivating said host cell under conditions conducive for expression of the heme- containing enzyme variant; and optionally c. recovering the heme-containing enzyme variant.

In one aspect, the cell is an Aspergillus cell. In another aspect, the cell is an Aspergillus oryzae cell. In another aspect, the cell is an Aspergillus niger cell. In another aspect the cell is a Bacillus cell, such as a Bacillus subtilis cell. In another aspect the cell is a Pichia pastoris cell or a Komagataella phaffii cell.

In a preferred embodiment the enzyme variant of the invention is secreted into the cultivation medium by the host cell.

The host cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the polypeptide 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. 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). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that are specific for the polypeptides. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide The polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the fermentation medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a whole fermentation broth comprising the polypeptide is recovered.

The polypeptide 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), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.

Method of flavoring and/or coloring

In a fifth aspect, the invention relates to a method of flavoring and/or coloring food or feed, the method comprising the steps of a) providing the food or feed, and b) adding the heme-containing enzyme variant according to the first aspect to the food or feed.

In a preferred embodiment the provided food or feed is a meat-analogue.

In another embodiment the provided feed is an animal feed.

The ratio of added enzyme variant to the food/feed or its components is adjusted depending on the degree of desired meat-like flavor and/or meat-like color of the feed or food. The addition of the enzyme variant can be carried out manually or automatically, e.g. based on scaling the enzyme variant before adding if provided in powder form. The enzyme variant can also be provided as stock solution when being added to the food or feed or its components. Depending on the food or feed, the relative amount of added enzyme variant can vary.

In one embodiment the feed or food product is comprising the enzyme variant in a range selected from the list of 0,01 - 1 % (w/w), 0,02 - 1 % (w/w), 0,03 - 1 % (w/w), 0,04 - 1 % (w/w),

0,05 - 1 % (w/w), 0,06 - 1 % (w/w), 0,07 - 1 % (w/w), 0,08 - 1 % (w/w), 0,09 - 1 % (w/w), 0,1 -

1 % (w/w), 0,1 - 1 % (w/w), 0,2 - 1 % (w/w), 0,3 - 1 % (w/w), 0,4 - 1 % (w/w), 0,5 - 1 % (w/w),

0,1 - 2 % (w/w), 0,2 - 2 % (w/w), 0,3 - 2 % (w/w), 0,4 - 2 % (w/w), 0,5 - 2 % (w/w), 0,6 - 2 %

(w/w), 0,7 - 2 % (w/w), 0,8 - 2 % (w/w), 0,9 - 2 % (w/w), 1 - 2 % (w/w), 0,1 - 3 % (w/w), 0,2 - 3

% (w/w), 0,3 - 3 % (w/w), 0,4 - 3 % (w/w), 0,5 - 3 % (w/w), 0,6 - 3 % (w/w), 0,7 - 3 % (w/w), 0,8

- 3 % (w/w), 0,9 - 3 % (w/w), 1 - 3 % (w/w), 0,1 - 4 % (w/w), 0,2 - 4 % (w/w), 0,3 - 4 % (w/w),

0,4 - 4 % (w/w), 0,5 - 4 % (w/w), 0,6 - 4 % (w/w), 0,7 - 4 % (w/w), 0,8 - 4 % (w/w), 0,9 - 4 %

(w/w), 1 - 4 % (w/w), 0,1 - 5 % (w/w), 0,2 - 5 % (w/w), 0,3 - 5 % (w/w), 0,4 - 5 % (w/w), 0,5 - 5

% (w/w), 0,6 - 5 % (w/w), 0,7 - 5 % (w/w), 0,8 - 5 % (w/w), 0,9 - 5 % (w/w), 1 - 5 % (w/w), 1 - 2

% (w/w), 1 - 3 % (w/w), 1 - 4 % (w/w), 1 - 5 % (w/w), 1 - 6 % (w/w), 1 - 7 % (w/w), 1 - 8 % (w/w), 1 - 9 % (w/w), 1 - 10 % (w/w), 5 - 10 % (w/w), 5 - 11 % (w/w), 5 - 12 % (w/w), 5 - 13 % (w/w), 5 - 14 % (w/w), 5 - 15 % (w/w), 5 - 16 % (w/w), 5 - 17 % (w/w), 5 - 18 % (w/w), 5 - 19 % (w/w), 5 - 20 % (w/w), 10 - 15 % (w/w), 10 - 16 % (w/w), 10 - 17 % (w/w), 10 - 18 % (w/w), 10 - 19 % (w/w), 10 - 20 % (w/w), 10 - 21% (w/w), 10 - 22 % (w/w), 10 - 23 % (w/w), 10 - 24 % (w/w), 10

- 25 % (w/w), 10 - 30 % (w/w), 10 - 35 % (w/w), 10 - 40 % (w/w), 15 - 45 % (w/w), or 15 - 50 % (w/w).

Food or feed product

In a sixth aspect, the invention relates to a food or feed product comprising an inactivated heme-containing enzyme variant according to the first aspect.

In a preferred embodiment the food or feed is a meat analogue.

In one embodiment the feed or food product is comprising the enzyme variant in a range selected from the list of 0,01 - 1 % (w/w), 0,02 - 1 % (w/w), 0,03 - 1 % (w/w), 0,04 - 1 % (w/w), 0,05 - 1 % (w/w), 0,06 - 1 % (w/w), 0,07 - 1 % (w/w), 0,08 - 1 % (w/w), 0,09 - 1 % (w/w), 0,1 - 1 % (w/w), 0,1 - 1 % (w/w), 0,2 - 1 % (w/w), 0,3 - 1 % (w/w), 0,4 - 1 % (w/w), 0,5 - 1 % (w/w), 0,1 - 2 % (w/w), 0,2 - 2 % (w/w), 0,3 - 2 % (w/w), 0,4 - 2 % (w/w), 0,5 - 2 % (w/w), 0,6 - 2 % (w/w), 0,7 - 2 % (w/w), 0,8 - 2 % (w/w), 0,9 - 2 % (w/w), 1 - 2 % (w/w), 0,1 - 3 % (w/w), 0,2 - 3 % (w/w), 0,3 - 3 % (w/w), 0,4 - 3 % (w/w), 0,5 - 3 % (w/w), 0,6 - 3 % (w/w), 0,7 - 3 % (w/w), 0,8

- 3 % (w/w), 0,9 - 3 % (w/w), 1 - 3 % (w/w), 0,1 - 4 % (w/w), 0,2 - 4 % (w/w), 0,3 - 4 % (w/w), 0,4 - 4 % (w/w), 0,5 - 4 % (w/w), 0,6 - 4 % (w/w), 0,7 - 4 % (w/w), 0,8 - 4 % (w/w), 0,9 - 4 % (w/w), 1 - 4 % (w/w), 0,1 - 5 % (w/w), 0,2 - 5 % (w/w), 0,3 - 5 % (w/w), 0,4 - 5 % (w/w), 0,5 - 5 % (w/w), 0,6 - 5 % (w/w), 0,7 - 5 % (w/w), 0,8 - 5 % (w/w), 0,9 - 5 % (w/w), 1 - 5 % (w/w), 1 - 2 % (w/w), 1 - 3 % (w/w), 1 - 4 % (w/w), 1 - 5 % (w/w), 1 - 6 % (w/w), 1 - 7 % (w/w), 1 - 8 % (w/w), 1 - 9 % (w/w), 1 - 10 % (w/w), 5 - 10 % (w/w), 5 - 11 % (w/w), 5 - 12 % (w/w), 5 - 13 % (w/w), 5 - 14 % (w/w), 5 - 15 % (w/w), 5 - 16 % (w/w), 5 - 17 % (w/w), 5 - 18 % (w/w), 5 - 19 % (w/w), 5 - 20 % (w/w), 10 - 15 % (w/w), 10 - 16 % (w/w), 10 - 17 % (w/w), 10 - 18 % (w/w), 10 - 19 % (w/w), 10 - 20 % (w/w), 10 - 21% (w/w), 10 - 22 % (w/w), 10 - 23 % (w/w), 10 - 24 % (w/w), 10

The food material which is to be combined with the heme-containing enzyme variant of the invention may be any raw material which is to be included in the food product or it may be any intermediate form of the food product which occurs during the production process prior to obtaining the final form of the food product. It may be any individual raw material used and/or any mixture thereof and/or any mixture thereof also including additives and/or processing aids, and/or any subsequently processed form thereof.

The food product may be made from at least one raw material that is of plant origin, for example a vegetable tuber or root, such as but not limited to the group consisting of potato, sweet potato, yams, yam bean, parsnip, parsley root, Jerusalem artichoke, carrot, radish, turnip, and cassava potato; cereal, soya, such as but not limited to the group consisting of wheat, rice, corn, maize, rye, barley, buckwheat, sorghum and oats; coffee; or cocoa. Also food products made from more than one raw material are included in the scope of this invention, for example food products comprising both wheat (e.g., in the form of wheat flour) and potato.

In one embodiment the food or feed product is vegetable-based, such as a vegetable- based burger or a meat-analogue. The vegetable-based food material may be any food material based on vegetables. It may be derived from a vegetable tuber or root such as but not limited to the group consisting of potato, sweet potato, yams, yam bean, parsnip, parsley root, Jerusalem artichoke, carrot, radish, turnip, and cassava.

The present invention also relates to animal feed compositions and animal feed additives comprising one or more heme-containing enzyme variants of the invention. In an embodiment, the animal feed or animal feed additive comprises a formulating agent and one or more heme- containing enzyme variants of the invention. In a further embodiment, the formulating agent comprises one or more of the following compounds: glycerol, ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, kaolin and cellulose.

Animal feed compositions or diets have a relatively high content of protein. Poultry and pig diets can be characterised as indicated in Table B of WO 01/58275, columns 2-3. Fish diets can be characterised as indicated in column 4 of this Table B. Furthermore, such fish diets usually have a crude fat content of 200-310 g/kg.

An animal feed composition according to the invention has a crude protein content of 50- 800 g/kg, and furthermore comprises at least one heme-containing enzyme variants as claimed herein.

Furthermore, or in the alternative (to the crude protein content indicated above), the animal feed composition of the invention has a content of metabolisable energy of 10-30 MJ/kg; and/or a content of calcium of 0.1-200 g/kg; and/or a content of available phosphorus of 0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or a content of methionine plus cysteine of 0.1-150 g/kg; and/or a content of lysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolisable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine, and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO 01/58275 (R. 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25, i.e. Crude protein (g/kg)= N (g/kg) x 6.25. The nitrogen content is determined by the Kjeldahl method (A.O.A.C., 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington DC).

Metabolisable energy can be calculated on the basis of the NRC publication Nutrient requirements in swine, ninth revised edition 1988, subcommittee on swine nutrition, committee on animal nutrition, board of agriculture, national research council. National Academy Press, Washington, D.C., pp. 2-6, and the European Table of Energy Values for Poultry Feed-stuffs, Spelderholt centre for poultry research and extension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen & looijen bv, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids in complete animal diets is calculated on the basis of feed tables such as Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the invention contains at least one vegetable protein as defined above. Preferably the animal feed composition is free of animal protein.

In still further particular embodiments, the animal feed composition of the invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or 0-20% whey.

In one embodiment the animal feed comprises vegetable proteins. In particular embodiments, the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w). Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example, materials from plants of the families Fabaceae ( Leguminosae ), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal, rapeseed meal, and combinations thereof.

In a particular embodiment, the vegetable protein source is material from one or more plants of the family Fabaceae, e.g., soybean, lupine, pea, or bean. In another particular embodiment, the vegetable protein source is material from one or more plants of the family Chenopodiaceae, e.g. beet, sugar beet, spinach or quinoa. Other examples of vegetable protein sources are rapeseed, and cabbage. In another particular embodiment, soybean is a preferred vegetable protein source. Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum.

Animal diets can e.g. be manufactured as mash feed (non-pelleted) or pelleted feed.

Typically, the milled feed-stuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question. Enzymes can be added as solid or liquid enzyme formulations. For example, for mash feed a solid or liquid enzyme formulation may be added before or during the ingredient mixing step. For pelleted feed the (liquid or solid) heme-containing enzyme variants/enzyme preparation may also be added before or during the feed ingredient step. Typically a liquid heme-containing enzyme variants/enzyme preparation comprises the heme-containing enzyme variants of the invention optionally with a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelleting step, such as by spraying the liquid formulation onto the pellets. The enzyme may also be incorporated in a feed additive or premix.

Alternatively, the heme-containing enzyme variants can be prepared by freezing a mixture of liquid enzyme solution with a bulking agent such as ground soybean meal, and then lyophilizing the mixture.

In an embodiment, the animal feed or animal feed additive comprises one or more additional enzymes. In an embodiment, the animal feed comprises one or more microbes. In an embodiment, the animal feed comprises one or more vitamins. In an embodiment, the animal feed comprises one or more minerals. In an embodiment, the animal feed comprises one or more amino acids. In an embodiment, the animal feed comprises one or more other feed ingredients. In another embodiment, the animal feed or animal feed additive comprises the polypeptide of the invention, one or more formulating agents and one or more additional enzymes. In an embodiment, the animal feed or animal feed additive comprises the polypeptide of the invention, one or more formulating agents and one or more microbes. In an embodiment, the animal feed comprises the polypeptide of the invention, one or more formulating agents and one or more vitamins. In an embodiment, the animal feed or animal feed additive comprises one or more minerals. In an embodiment, the animal feed or animal feed additive comprises the polypeptide of the invention, one or more formulating agents and one or more amino acids. In an embodiment, the animal feed or animal feed additive comprises the polypeptide of the invention, one or more formulating agents and one or more other feed ingredients.

In a further embodiment, the animal feed or animal feed additive comprises the polypeptide of the invention, one or more formulating agents and one or more components selected from the list consisting of: one or more additional enzymes; one or more microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.

Uses

In a seventh aspect, the invention relates to the use of an inactivated heme-containing enzyme variant according to the first aspect for the flavoring and/or coloring of food or feed.

A heme-containing enzyme variant of the invention may also be used in animal feed or human food. In a preferred embodiment the food or feed is a meat analogue.

In one embodiment the enzyme variant is used as a component in a feed or food product, said feed or food product is comprising the enzyme variant in a range selected from the list of 0,01 - 1 % (w/w), 0,02 - 1 % (w/w), 0,03 - 1 % (w/w), 0,04 - 1 % (w/w), 0,05 - 1 % (w/w), 0,06 -

1 % (w/w), 0,07 - 1 % (w/w), 0,08 - 1 % (w/w), 0,09 - 1 % (w/w), 0,1 - 1 % (w/w), 0,1 - 1 % (w/w), 0,2 - 1 % (w/w), 0,3 - 1 % (w/w), 0,4 - 1 % (w/w), 0,5 - 1 % (w/w), 0,1 - 2 % (w/w), 0,2 -

2 % (w/w), 0,3 - 2 % (w/w), 0,4 - 2 % (w/w), 0,5 - 2 % (w/w), 0,6 - 2 % (w/w), 0,7 - 2 % (w/w),

0,8 - 2 % (w/w), 0,9 - 2 % (w/w), 1 - 2 % (w/w), 0,1 - 3 % (w/w), 0,2 - 3 % (w/w), 0,3 - 3 %

(w/w), 0,4 - 3 % (w/w), 0,5 - 3 % (w/w), 0,6 - 3 % (w/w), 0,7 - 3 % (w/w), 0,8 - 3 % (w/w), 0,9 - 3 % (w/w), 1 - 3 % (w/w), 0,1 - 4 % (w/w), 0,2 - 4 % (w/w), 0,3 - 4 % (w/w), 0,4 - 4 % (w/w), 0,5 - 4 % (w/w), 0,6 - 4 % (w/w), 0,7 - 4 % (w/w), 0,8 - 4 % (w/w), 0,9 - 4 % (w/w), 1 - 4 % (w/w), 0,1 - 5 % (w/w), 0,2 - 5 % (w/w), 0,3 - 5 % (w/w), 0,4 - 5 % (w/w), 0,5 - 5 % (w/w), 0,6 - 5 % (w/w), 0,7 - 5 % (w/w), 0,8 - 5 % (w/w), 0,9 - 5 % (w/w), 1 - 5 % (w/w), 1 - 2 % (w/w), 1 - 3 % (w/w), 1 - 4 % (w/w), 1 - 5 % (w/w), 1 - 6 % (w/w), 1 - 7 % (w/w), 1 - 8 % (w/w), 1 - 9 % (w/w), 1 - 10 % (w/w), 5 - 10 % (w/w), 5 - 11 % (w/w), 5 - 12 % (w/w), 5 - 13 % (w/w), 5 - 14 % (w/w), 5 - 15 % (w/w), 5 - 16 % (w/w), 5 - 17 % (w/w), 5 - 18 % (w/w), 5 - 19 % (w/w), 5 - 20 % (w/w), 10 - 15 % (w/w), 10 - 16 % (w/w), 10 - 17 % (w/w), 10 - 18 % (w/w), 10 - 19 % (w/w), 10 - 20 % (w/w), 10 - 21% (w/w), 10 - 22 % (w/w), 10 - 23 % (w/w), 10 - 24 % (w/w), 10 - 25 % (w/w), 10 - 30 % (w/w), 10 - 35 % (w/w), 10 - 40 % (w/w), 15 - 45 % (w/w), or 15 - 50 % (w/w).

In another embodiment, the present invention provides a method for preparing an animal feed composition comprising adding one or more heme-containing enzyme variants of the present invention to one or more animal feed ingredients. In another embodiment, the present invention provides a method for preparing a food composition comprising adding one or more heme- containing enzyme variants of the present invention to one or more food ingredients.

The heme-containing enzyme variant preparation can be (a) added directly to the feed or food, or (b) it can be used in the production of one or more intermediate compositions such as feed or food additives or premixes that is subsequently added to the feed or food (or used in a treatment process).

The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

Examples

Two peroxygenases (Per27 from H. insolens and wt392 from C. cinereus) and one peroxidase (CIP from C. cinereus) were chosen as templates for the mutation work. The variants were made and expressed in Aspergillus oryzae, by standard mutation technics (see Examples 1 - 2). Expression was seen on SDS-page, and activity was tested by a classic peroxidase assay using ABTS as substrate (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) (see Example 3). Nucleotide sequences encoding the enzyme variants of the present invention have been sequenced using next-generation sequencing as described in WO2017/147294 (Novozymes A/S).

Example 1 : Construction of enzyme variants by site-directed mutagenesis

Site-directed variants are constructed of the enzyme variants as shown in Table 1, comprising specific substitutions. The variants are made by traditional cloning of DNA fragments

(Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) using PCR together with properly designed mutagenic oligonucleotides that introduced the desired mutations in the resulting sequence.

Mutagenic oligos are designed corresponding to the DNA sequence flanking the desired site(s) of mutation, separated by the DNA base pairs defining the insertions/deletions/substitutions, and purchased from an oligo vendor such as Life T echnologies.

In order to test the enzyme variants, the mutated DNA comprising a variant are integrated into a competent A. oryzae strain by homologous recombination, fermented using standard protocols (yeast extract-based media, 3-4 days, 30°C), and purified by chromatography. Table 1.

Example 2: Recombinant expression and purification of the enzyme variants

The strains expressing an enzyme variant were inoculated in 5 shake flask each containing 200 ml MDU-2BP and added protoporhyrin IX (final concentration 100 mg/L). The strain was grown at 30°C for 4 days at 200 rpm. The culture broth was sterile-filtered before starting the purification. Sterile filtered culture broth was subject for the ABTS assay.

The filtered culture broth was reduced to 100-200 ml_ avoiding protein precipitation by using ultra-filtration. 5 mM Tris buffer pH 8 was added until 1 L, and then the volume was again reduced to 100-200 ml_ using ultra-filtration. This step was repeated until the conductivity of the sample matched the conductivity of buffer A of ion exchange chromatography: 25 mM Tris pH 8. The volume of sample was finally reduced to 100 ml_ using ultra-filtration. A Q-sepharose column was used for ion exchange chromatography. The column was equilibrated with 25 mM Tris pH 8 buffer. Flow rate was 10 mL/min. A gradient 0-100% of 25 mM Tris with 0.5 M NaCI buffer pH 8 buffer was applied. Fractions with high absorbance at 280 and 420 nm were loaded to SDS-PAGE gel. Expression of the enzyme variants was identified/verified as band on SDS-PAGE gel electrophoresis.

Example 3: ABTS Assay

Heme-containing parent enzymes of the present invention oxidize ABTS (2,2'-azino- bis(3-ethylbenzthiazoline-6-sulfonic acid) in the presence of hydrogen peroxide and the produced green color is quantified spectrophotometrically at 405 nm. Inactivated variants of the parent enzymes are thus identified by reduced or eliminated oxidation levels.

The reaction mixture contained 0.5 mM ABTS, 50 mM phosphate buffer pH 7, 0.005 mg/ml_ of purified enzyme variant, 0.5 mM hydrogen peroxide, and water ad 0.2 ml.

The reaction was started by adding the enzyme variant supernatant to the other ingredients used in the assay. A SpectraMax microtitre plate reader from Molecular Devices was applied to monitor the change in absorbance at 405 nm in a 96 well microtitre plate at room temperature. Blanks prepared without addition of enzyme were included.

The increase in absorbance was recorded over 5 minutes and the results are shown in Table 2. Variants were identified, which had expression and no detectable, or significantly reduced ABTS activity. As can be seen in Table 2, enzyme activity of the variants, measured by ABTS activity, was reduced to 3 - 6 % compared to the enzyme activity measured by ABTS activity of the respective wt control. Table 2. ABTS oxidation.

Example 4: Absorption spectra of purified inactivated heme containing enzymes is similar to spectra of myoglobin and other heme containing proteins

Four of the inactivated heme enzymes (SEQ ID NO: 12, 14, 30 and 31) were purified by cation exchange chromatography and size exclusion chromatography to obtain samples for characterization of the protein color and spectroscopic properties.

The purity of the inactivated heme enzymes was verified by SDS-PAGE (data not shown) and absorption spectra of the proteins diluted in phosphate buffer pH 9 were recorded from 250nm to 650nm using a NanoDrop™ spectrophotometer. The absorption spectra of the four purified samples are shown in Fig 1. The characteristic Soret peak of the heme group is for all variants detectable with maximum at 415nm for SEQ ID NO: 12 and 14 and at 420nm for SEQ ID NO: 30 and 31. Also, as can be seen in Fig. 2, the ligand and redox dependent absorption peaks at 500- 600nm are visible in the spectra (ref. K.C. Nam & D.U. Ahn, Journal of Food Science. Vol. 67, no 2, 2002)

The absorption spectra are very similar to the myoglobin and other heme proteins and thus the color profile of these inactivated heme proteins will likely be similar to meat myoglobin. Furthermore, the melting temperature as determined by differential scanning calorimetry showed a similar melting temperature to myoglobin and this points to a similar color transition upon cooking (data not shown).

The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects 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. In the case of conflict, the present disclosure including definitions will control.

Claims

1. A heme-containing enzyme variant of a heme-containing parent enzyme, said enzyme variant comprising at least one amino acid alteration, such as an amino acid substitution, amino acid deletion and/or amino acid insertion, whereby the enzymatic activity of the variant is reduced or eliminated, wherein the enzyme variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 2, SEQ ID NO: 35, SEQ ID NO: 4, SEQ ID NO: 36, SEQ ID NO: 6, or SEQ ID NO: 37.

2. The enzyme variant according to claim 1 , wherein the at least one amino acid mutation comprises or consists of:

(iii) at least one amino acid mutation of at least one amino acid within the catalytic domain of the enzyme; wherein the at least one amino acid mutation comprises or consists of an amino acid insertion, an amino acid deletion, and/or an amino acid substitution, such as an amino acid insertion of an amino acid selected from the list of lysine, arginine, cysteine, tryptophan, phenylalanine, tyrosine, proline, histidine, glutamine, leucine, isoleucine and methionine, and/or an amino acid substitution by an amino acid selected from the list of lysine, arginine, cysteine, tryptophan, phenylalanine, tyrosine, proline, histidine, glutamine, leucine, isoleucine and methionine.

3. The enzyme variant according to any of claims 1 to 2, wherein the enzyme variant is a variant of a parent enzyme encoded by the genome of a fungal genus or species.

4. The enzyme variant according to any of claims 1 to 3, the variant having reduced or eliminated peroxidase activity and having an amino acid sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, but less than 100% sequence identity, to SEQ ID NO: 35, and comprising an alteration at a position corresponding to position 55 of SEQ ID NO: 35, preferably the alteration comprises or consists of H55D.

5. The enzyme variant according to any of claims 1 to 3, the variant having reduced or eliminated peroxygenase activity and having a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least

99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO:

36, and comprising at least one alteration at a position corresponding to position 98, 102 and/or 224 of SEQ ID NO: 36, preferably the at least one alteration comprises or consists of I98W, V102L, V102W, and/or F224W.

6. The enzyme variant according to any of claims 1 to 3, the variant having reduced or eliminated peroxygenase activity and having a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least

37, and comprising at least one alteration at a position corresponding to position 17, 151, 154, 158, and/or 162 of SEQ ID NO: 37, preferably the at least one alteration comprises or consists of C17H, L151C, I154L, G158A, G158S, G158W, G158C, and/or A162L.

7. The enzyme variant according to any of the previous claims, the variant having reduced enzymatic activity, such as an enzymatic activity reduced to below 1%, below 2%, below 3%, below 4%, below 5%, below 6%, below 7%, below 8%, below 9%, or below 10% of the enzymatic activity of the parent enzyme not comprising the at least one amino acid alteration.

8. The enzyme variant according to claim 7, wherein the enzymatic activity is measured with an ABTS assay, preferably the ABTS assay according to Example 3.

9. A nucleic acid construct or expression vector comprising a heterologous promoter operably linked to a polynucleotide encoding the enzyme variant of any one of claims 1 to 8.

10. A recombinant host cell comprising in its genome the nucleic acid construct or expression vector of claim 9.

11. A method of producing an inactivated heme-containing enzyme variant, comprising: a. Providing a recombinant host cell producing an enzyme variant according to any one of claims 1-8, or a host cell according to claim 10; b. cultivating said host cell under conditions conducive for expression of the heme- containing enzyme variant; and optionally c. recovering the heme-containing enzyme variant.

12. A method of flavoring food or feed, the method comprising the steps of a) providing the food or feed, and b) adding the heme-containing enzyme variant according to any one of claims 1 to 8 to the food or feed.

13. A food or feed product comprising an inactivated heme-containing enzyme variant according to any of claims 1 to 8.

14. The use of an inactivated heme-containing enzyme variant according to any of claims 1 to 8 for the flavoring and/or coloring of food or feed.