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WO1996041882A1 - Hydrophobines de champignons commestibles, genes, sequences nucleotidiques, fragments d'adn codant pour lesdites hydrophobines et leur expression - Google Patents

Hydrophobines de champignons commestibles, genes, sequences nucleotidiques, fragments d'adn codant pour lesdites hydrophobines et leur expression Download PDF

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Publication number
WO1996041882A1
WO1996041882A1 PCT/NL1996/000234 NL9600234W WO9641882A1 WO 1996041882 A1 WO1996041882 A1 WO 1996041882A1 NL 9600234 W NL9600234 W NL 9600234W WO 9641882 A1 WO9641882 A1 WO 9641882A1
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WIPO (PCT)
Prior art keywords
hydrophobin
nucleotide sequence
dna
fungus
gene
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PCT/NL1996/000234
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English (en)
Inventor
Leonardus Johannes Lambertus Donatus VAN GRIENSVEN
Petrus Wilhelmus Johannes De Groot
Petrus Johannes Schaap
Jacob Visser
Original Assignee
Proefstation Voor De Champignoncultuur
Rijkslandbouwuniversiteit Wageningen
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Application filed by Proefstation Voor De Champignoncultuur, Rijkslandbouwuniversiteit Wageningen filed Critical Proefstation Voor De Champignoncultuur
Priority to AU59141/96A priority Critical patent/AU5914196A/en
Publication of WO1996041882A1 publication Critical patent/WO1996041882A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/375—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Basidiomycetes

Definitions

  • the present invention relates to hydrophobins of edible fungi and to genes, nucleotide sequences and DNA-fragments encoding for said hydrophobins.
  • Hydrophobins are a only recently discovered class of highly hydrophobic proteins and/or peptides occurring in certain fungi. So far, their existence has been shown in for instance the fungi Schizophyllum commune, Aspergillus nidulans and Neurospora crassa, vide the list of references mentioned hereinbelow.
  • the hydrophobins from Schizophyllum commune are produced by the hyphae and/or fruit bodies. In emerging aireal hyphae, they accumulate at the hyphal surface to form SDS-insolvable complexes at the air/cell wall interface, which can only be destroyed by substances such as concentrated formic acid and trifluoroacetic acid. As such, the hydrophobins from S. ses make the aireal hyphae highly water-resistant and water repellent.
  • hydrophobins of Aspergillus nidulans and Neurospora crassa are produced by the conidiophores to form water resistant and water repellant coatings around the spores produced by the conidiophores.
  • ccg-2 from Neurospora crassa.
  • This gene encodes for a fungal hydrophobin, said hydrophobin being a low-molecular-weight hydrophobic protein that provides for spore wall hydrophobicity and therefore ultimately for spore dispersal.
  • hydrophobins from the above mentioned fungi also show remarkable properties in vitro as for instance those of surfactants or emulgators.
  • the hydrophobins from Schizophyllum commune form self-assembling membranes around oil droplets with a hydrophilic outer surface giving very stable emulsions.
  • Hydrophobin SC3P from Schizophyllum commune is reported to form an SDS-resistant complex at a water-gas interface that can for instance be used for the formation of air vesicles.
  • hydrophobins A major drawback of the abovementioned hydrophobins, and other hydrophobins of other fungi so far described in the art, is however that they are derived from fungi that are considered non- edible and are sometimes even toxic to humans. Therefore, these hydrophobins cannot be regarded as safe for human consumption and/or be safely used in for instance personal care products, which could be an important field of applications for hydrophobins.
  • a first object of the invention is therefore to provide hydrophobins that can be considered safe for use in such products.
  • hydrophobins could be derived from edible fungi, especially from the fruit bodies of edible mushrooms, such as Agaricus bisporus.
  • hydrophobins from edible fungi would require the production thereof in sufficient quantities, e.g. by means of recombinant DNA-technology. This would require identifying, isolating, cloning and expressing the genes coding for these hydrophobins.
  • Another object of the invention is the production of hydrophobins by expressing a gene, nucleotide sequence or DNA- fragment as described hereinabove. Further objects of the invention will be described hereinbelow. The above and other objects are achieved in that the genes and the nucleotide sequences coding for the hydrophobins from edible fungi, especially from edible mushrooms from the genera Agaricus, are provided for the first time. Also, the amino acid sequences of the peptides/proteins for which these genes and nucleotide sequences code have been established, which for the first time provides said hydrophobins.
  • the invention therefore relates to a hydrophobin from an edible fungus and/or to a gene, nucleotide sequence or DNA-fragment coding for a ripening form of such a hydrophobin.
  • the invention relates to the hydrophobins from fungi of the genus Agaricus, more particular to the hydrophobins of Agaricus bisporus, the common mushroom, and especially to the hydrophobins from the fruit bodies of Agaricus bisporus, as well as to genes, nucleotide sequences or DNA-fragments coding for a ripening form of such a hydrophobin.
  • the invention relates the nucleotide sequences and DNA-fragments derived from the hyp A, hyp B, hyp C and hyp D genes of Agaricus bisporus, the common mushroom, as well as to the amino acid sequences and the peptides/proteins - e.g. the hydrophobins - for which they code.
  • the hydrophobins of the invention will in general be a low molecular-weight cysteine rich protein, whith usually a hydrophobic H amino terminus and an internal hydrophobic domain. They will in general contain 50-150, especially 90-120 amino acids, and 6-10, usually about 8 conserved cysteine-residues, as is characteristic for all hydrophobins described so far in the above mentioned references. However, the invention is not limited to a specific amino acid chain length or a specific number of cysteine residues.
  • proteins and peptides with a similar structure and/or function as the hydrophobins will also be encompassed by the invention, as long as they are derived from an edible fungus and/or are not harmful to humans.
  • hydrophobins as used herein furthermore comprises all ripening forms of said hydrophobins, i.e. all the different forms in which the expressed proteins occur after expression of the genes, nucleotide sequences or DNA-fragments of the invention, such as the pre, prepro and pro forms thereof, as will be clear to a person skilled in the art.
  • hydrophobins of the invention are derived from edible mushrooms, they can be considered safe to humans or animals and can therefore be used both for human consumption, i.e. in food stuffs, as well as for products that come into contact with for instance the human skin, i.e. in personal care products.
  • Edible fungi and edible mushrooms are herein defined as fungi and mushrooms that can are not harmful and/or non-toxic to humans and/or animals and/or that can be eaten by humans and/or animals without deleterious effects.
  • fungi and mushrooms Preferably said fungi and mushrooms have the GRAS (Generally Regarded As Safe) status.
  • Such edible fungi and mushrooms are well known to a person skilled in the art, belonging to all classes of fungi, including the ascomycetes, basidomycetes and the fungi imperfecti, from which the basidomycetes are usually preferred because they generally from large edible fruit bodies.
  • Preferred examples are the edible fungi from the group comprising the genera Agaricus, Pleurotus, Lentinus.
  • the hydrophobins of the invention are derived from the part of the fungus that is usually consumed by human beings as food, for instance the fruit body. It is also to be understood that if not all parts of a fungus are edible, the invention is only related to the hydrophobins derived from the fungus that are not toxic and/harmful to humans or animals and that are preferably edible. Usually, these hydrophobins will be derived from the edible parts of the fungus, such as the fruit body, although the genes, nucleotide sequences or DNA-fragments coding for these hydrophobins may be derived from any part of the fungus.
  • the hydrophobins of the invention are preferably derived from fungi of the genus Agaricus, such as A. arvensis, A. brunescens, and A. bitorquis, and especially from Agaricus bisporus, the common mushroo .
  • the genes, nucleotide sequences and DNA-fragments coding for the hydrophobins of the invention are also derived from an edible fungus, more preferably from fungi of the genus Agaricus, and especially from Agaricus bisporus, the common mushroom. This assures that the hydrophobins for which they encode are indeed safe to use in foodstuffs and/or personal care products.
  • hyp A genes encoding for hydrophobins of Agaricus bisporus have been identified and have been named hyp A, hyp B, hyp C and hyp D repectively.
  • hyp A and hyp C are coded for on the same chromosome ( chromosome III and IV, which are indistinguishable by CHEF-analysis)
  • hyp B and hyp D are coded for on chromosome XII and chromosome I respectively.
  • a DNA sequence showing a great deal of homology with the Agaricus bisporus h pk nucleotide sequence of figure 2 is present 2,5 kb downstream of hypA_.
  • a DNA sequence showing a great deal of homology with the Agaricus bisporus hypB nucleotide sequence of figure 5 is present in the same organism.
  • a part of the amino acid sequences of ripening forms of polypeptides having hydrophobic properties according to Figure 2 and 5 not only code for a part of HYPA and HYPB but also code for at least parts of other polypeptides having hydrophobic properties that can be derived from the same organism.
  • the invention in a preferred aspect therefore relates to a nucleotide sequence or DNA-fragment derived from the hyp A gene of Agaricus bisporus.
  • this aspect of the invention relates to a nucleotide sequence or DNA-fragment containing the nucleotide sequence ID NO 1 shown in figure 2, as well as for the amino acid sequence for which it encodes, also shown in figure 2.
  • the invention relates to a nucleotide sequence or DNA-fragment derived from the hyp B gene of Agaricus bisporus.
  • this aspect of the invention relates to a nucleotide sequence ID NO 3 or DNA-fragment containing said nucleotide sequence as shown in figure 6, as well as for the amino acid sequence for which it encodes, also shown in figure 6.
  • the invention relates to a nucleotide sequence or DNA-fragment derived from the hyp C gene of Agaricus bisporus.
  • this aspect of the invention relates to a nucleotide sequence ID no 2 or DNA-fragment containing said nucleotide sequence as shown in figure 5. as well as for the amino acid sequence for which it encodes, also shown in figure 5.
  • the invention preferably relates to a nucleotide sequence or DNA-fragment derived from the hyp D gene of Agaricus bisporus, as well as for the amino acid sequence for which it encodes.
  • nucleotide sequences of the invention show a remarkably low homology of less than about 40 % with the hydrophobin encoding genes of Schizophyllum commune mentioned hereinabove. This means that probes for the Schizophyllum-hydrophobin genes cannot and could not be used for identifying or isolating the above Agaricus genes.
  • hydrophobins of the invention derived from the abovementioned nucleotide sequences show a remarkably low identity and similarity with the known hydrophobins from Schizophyllum commune, Aspergillus nidulans and Neurospora crassa. This is in correspondence with both the fact that they originate from different fungi (belonging to the ascomycetes and the basidomycetes) as well as with their presence in different parts of the respective fungi (fruit body vs. aireal hyphae/spore coatings).
  • hydrophobins of the invention also show the remarkable properties of said known hydrophobins, such as their hydrophobic properties, self-assembling properties, surface active properties and emulgating properties.
  • Agaricus bisporus have been found to be very hydrophobic proteins that contain eight intramolecular sulfhydryl-bonds, that as such provide for a major part of the tertiary structure of the protein.
  • these hydrophobins have now been shown to be part of the edible fruit body, where they form the water resistant and water repellent outer layer thereof. As such, they are now considered major building blocks of the architecture of the fruit body, having an important role as a barrier-forming protein in its protection against water and against dehydration.
  • the hyp A and hyp B genes are for instance fruit body specific end show different expression levels during the various stages of Agaricus bisporus fruit body development.
  • the hyp B gene shows highest expression during the early stages of fruit body development whereas the hyp A gene shows high mRNA expression levels from the onset of fruiting at least untill the growth stage in which the fruit bodies are used for consumption.
  • the invention is however not restricted to a certain mechanism of action or structural purpose of the hydrophobins of the invention within the fungus or fruit body.
  • the invention not only provides for the hydrophobins as they naturally occur in edible fungi, but also for proteins and peptides that "essentially correspond" to the hydrophobins of the invention, i.e. that contain a large part of the protein structure or amino acid sequence of said hydrophobins.
  • proteins having the same amino acid sequence as the hydrophobins of the invention can be: proteins having the same amino acid sequence as the hydrophobins of the invention, however with a part of the peptide chain missing, preferably the N-terminal or -C00H- terminal part thereof. proteins having the same amino acid sequence as the hydrophobins of the invention, in which one or more amino acids have been deleted and/or replaced by other amino acids. proteins having the same amino acid sequence as the hydrophobins of the invention, in which part of the amino acid sequence has been inversed, i.e. is present in reversed order compared to the naturally occuring hydrophobin. - proteins having the same amino acid sequence as the hydrophobins of the invention, to which one or more amino acids have been added, or a combination thereof, as will be clear to a person skilled in the art.
  • a protein will be considered to essentially correspond to a hydrophobin of the invention when it contains at least 50% . preferably at least 70 and more preferably at least 90 of the amino acid sequence of the hydrophobin of the invention, as a closed sequence or as seperate fragments within the total amino acid sequence.
  • proteins or polypeptides preferably have essentially the same tertairy and/or quarternary structure, as well as the same properties as the naturally occuring hydrophobins. More preferably, they contain the same number of sulfhydryl bonds as the naturally occuring hydrophobins.
  • proteins or polypeptides may also be specifically engineered or tailored for obtaining desired and/or improved properties, such as improved hydrophobicity, improved stability and/or improved surfactant/ emulgator properties, i.e. by adding further cysteine residues, adding/replacing or deleting amino acids with hydrophilic or hydrophobic residues etc.
  • proteins and peptides can be prepared by a person skilled in the art in a manner known per se, for instance by deleting or adding one or more amino acids from/to the hydrophobins of the invention.
  • they will be produced by recombinant DNA- technology, i.e. by expressing a nucleotide sequence or DNA-fragment coding for such a peptide or protein, and these nucleotide sequences and DNA-fragments are also encompassed by the nucleotide sequences and DNA-fragments of the invention.
  • these nucleotide sequences and DNA-fragments will be "genetic variations" of the genes, nucleotide sequences and DNA-fragments of the invention, as further defined hereinbelow.
  • the invention therefore also relates to peptides and proteins that essentially correspond to the hydrophobins coded for by the hyp A, hyp B, hyp C and hyp D genes of Agaricus bisporus, i.e. with an amino acid sequence that essentially corresponds to one of the amino acid sequences shown in figures 1-3-
  • hydrophobin All these peptides and proteins that essentially correspond to the hydrophobins of the invention, in all their ripening forms as defined herein, are to be understood as being comprised by the term hydrophobin as used hereinbelow.
  • the invention also comprises homologues of the above genes, nucleotide sequences or DNA-fragments, i.e. sequences that code for the same amino acid sequence or peptide/protein but with a different nuleotide sequence due to the degeneracy of the genetic code, as will be clear to a person skilled in the art.
  • the invention expressly includes homologues of the nucleotide sequences shown in figures 1- 3>
  • the invention comprises genetic variants of these genes, nucleotide sequences or DNA-fragments, as mentioned hereinabove and/or as defined hereinbelow.
  • hydrophobins of the invention may advantageously be used in industrial processes, for example as emulgators, thickeners or surfactants, for example for giving hydrophilic properties to hydrophobic surfaces or for improving water-resistance of hydrophilic substrates.
  • they may also be used as surfactants or emulgators, or they may be used with advantage for improving the storage properties of various foodstuffs that are water sensitive, such as biscuits, candies and chocolate, or for lowering the water activity of foodstuffs that become easily infected by micro-organisms.
  • the hydrophobins of the invention can be used as emulgators for the preparation of both oil-in-water as well as water-in-oil emulsions, as well as higher emulsions, such as /O/ and/or 0/W/O emulsions, wherein the excellent stability of the hydrophobin complexes formed on the oil/water interfaces, especially against other surfactants such as SDS, may be of particular value.
  • Such emulsions may be part of pharmaceutical preparations, especially preparations for topical application such as ointments and creams. Also, they may be used in for instance shampoos and conditioners in which their property of forming a water repellent layer can be used for protecting the human skin or dehydration.
  • the invention therefore further relates to foodstuffs and compositions, especially pharmaceutical preparations and/or personal care preparations, comprising a hydrophobin of the invention, or a peptide or protein essentially corresponding thereto.
  • the hydrophobins of the invention may further advantagously be applied in the field of environmental technology, for instance in the cleaning of contaminated soil or water, especially in the soil or water contaminated with oil-like substances.
  • the biological compatability of the hydrophobins of the invention is a further advantage.
  • the present invention in a further aspect also relates to recombinant DNA material comprising a) at least part of a nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus, b) a genetic variant of a nucleotide sequence according to a); or c) a nucleotide sequence capable of hybridizing to either of the nucleotide sequences a) or b) .
  • the recombinant DNA material comprising a nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus can comprise a nucleotide sequence derivable from an organism that is homologous to the expression host cell into which cell said nucleotide sequence is incorporated or said nucleotide sequence can be heterologous to the expression host cell.
  • the expression of the nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus can be regulated by operably linking said nucleotide sequence to regulatory sequences that control a gene native to the edible fungus from which said nucleotide sequence has been derived.
  • the regulatory sequences can also be foreign i.e.
  • the regulatory regions can be regulatory regions of a hydrophobin gene or regulatory regions of other genes.
  • Another preferred embodiment of the invention is related to a cell capable of overexpression and secretion of a ripening form of a hydrophobin of an edible fungus, preferably a mature form.
  • Fig. 1 Restriction map of the genomic DNA of A. bisporus in the region comprising the h p and hyp C genes.
  • Fig. 2 Nucleotide sequence of cDNA derived from mRNA expressing the A. bisporus hypk gene and amino acid sequences derived therefrom.
  • SEQ ID NO: 1 Fig. 3: Nucleotide sequence of genomic DNA of the A. bisporus hypk and hypC genes.
  • SEQ ID NO: 2 Fig. 4: Restriction map of the genomic DNA of A. bisporus in the region compirising the hypB gene.
  • Fig. 1 Restriction map of the genomic DNA of A. bisporus in the region comprising the h p and hyp C genes.
  • Fig. 2 Nucleotide sequence of cDNA derived from mRNA expressing the A. bisporus hypk gene and amino acid sequences derived therefrom.
  • Fig. 3 Nucleotide sequence of genomic DNA of the A.
  • Fig. 5 Nucleotide sequence of cDNA derived from mRNA expressing the A. bisporus hypB gene and amino acid sequences derived therefrom.
  • SEQ ID NO: 3 Nucleotide sequence of genomic DNA of the A. bisporus hypB gene.
  • Fig. 7 Partial restriction enzyme map of the hybridizing phage clone 10 comprising the A. bisporus hyp ⁇ gene.
  • Fig. 8 Map of plasmid pIM3100 comprising cDNA encoding the
  • FIG. 9 Map of plasmid pIM3101 comprising cDNA encoding the A. bisporus hyp B gene.
  • Fig. 10 Map of plasmid pIM3104 comprising the A. bisporus hypk and hypC genes and regulatory sequences.
  • Fig. 11 Map of plasmid pIM3106 comprising the A. bisporus hypB gene and regulatory sequences.
  • Fig. 12 CHEF-analysis of hypB and hyp ⁇ genes.
  • lane 1 A. bisporus strain 39
  • lane 2 A.
  • bisporus strain 97 A probed with a 1 kb B ⁇ mHI- EcoRi fragment of phage 1 located 2 kb upstream of hypB .
  • B probed with the insert of pIM3101.
  • C probed with a 1.2 kb EcoRI-B ⁇ mHI fragment of ph ⁇ ge 10.
  • the present invention is directed at a recombinant DNA material comprising at least a part of a nucleotide sequence encoding a ripening form of a hydrophobin of en edible fungus and genetic variants thereof.
  • said recombinant DNA-material will comprise at least a part of a gene, nucleotide sequence or DNA-fragment as described hereinabove, or a genetic variant or a homolog due to the degenerecy of the genetic code thereof.
  • recombinant DNA material can comprise a DNA molecule, or a mixture of various DNA fragments/ molecules. It also includes for instance expression vectors and cloning vehicles, such as plasmids.
  • genetic variants includes hybrid DNA sequences comprising at least a part of a nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus optionally coupled to regulatory regions such as promoter, secretion and terminator signels IRAting from homologous or heterologous organisms.
  • genetic variants also includes DNA sequences encoding mutant hydrophobins and degenerate DNA sequences encoding polypeptides that essentially correspond to the hydrophobins of the invention as defined hereinabove. These genetic variants may be obtained in a manner known to a nam skilled in the art, for instance by edding or deleting one or more codons coding for one or more amino acids, either at the beginning or the end of the of the nucleotide sequence or somewhere inbetween, or by replacing one or more codons coding for an amino acid with one or more other codons coding for one or more other amino acids, for instance by means of point mutation.
  • these genetic variants show a homology of more than ⁇ %, preferably more than 80%, with the nucleotide sequence derived from the edible fungus.
  • the present invention also includes recombinant DNA material comprising at least a part of a nucleotide sequence capable of hybridizing under low stringency conditions to at least a part of the nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus and genetic variants thereof as described above which may differ in codon sequence due to the degeneracy of the genetic code or cross species variation.
  • ripening form refers to any of the different forms in which en enzyme m ⁇ y occur after expression of the associated gene. More in particular it refers to both the naturally and not naturalli' occurring mature foiT ⁇ of an enzyme that can result after cleavage of a "leader” peptide and also to any form of an enzyme still comprising a "leader” peptide in any form.
  • a "leeder peptide” can be a prepro peptide, a pre peptide or a pro peptide.
  • the recombinant DNA material according to the invention can comprise at least a pert of a nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus wherein said nucleotide sequence can be derived from any edible fungus, such as the fungi mentioned hereinabove.
  • a more concrete preferred embodiment of this aspect of the invention is recombinant DNA materiel comprising at least a part of e nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus with an amino acid sequence as shown in SEQ ID's 1, 2 or 3 and even more concretely a recombinant DNA material comprising at leest a pert of the nucleotide sequence.
  • a more concrete preferred embodiment of this aspect of the invention is recombinant DNA material comprising at least a part of a nucleotide sequence encoding a ripening form of a polypeptide having hydrophobic properties with an amino acid sequence as shown in Figures 2 or 5 and even more concretely a recombinant DNA materiel comprising et leest a pert of the nucleotide sequence encoding a polypeptide having hydrophobic properties as shown in Figure 3 or 6.
  • the genetic variants of the nucleotide sequence of Figure 3 or 6, including sequences encoding mutant polypeptides with hydrophobic properties and degenerate nucleotide sequences coding for polypeptides wherein the hydrophobic properties are retained are also part of the invention, as are nucleotide sequences capable of hybridizing to at least a part of the nucleotide sequences encoding a polypeptide having hydrophobic properties as shown in Figure 3 and 6 and genetic varients thereof (as described above), wherein said nucleotide sequences may differ in codon sequence due to the degeneracy of the genetic code or cross species variation.
  • the cDNA sequences of Figure 2 and 5t encoding polypeptides having hydrophobic properties are obtained by screening an Agaricus bisporus mixed primordia - fruit body cDNA library for highly expressed genes.
  • the genetic varients of the nucleotide sequences of SEQ ID's are also part of the invention, as are nucleotide sequences capable of hybridizing to at least a part of the nucleotide sequences encoding a hydrophobin of an edible fungus as shown in SEQ ID's 1, 2 and 3 and genetic variants thereof (as described above) , wherein said nucleotide sequences may differ in codon sequence due to the degeneracy of the genetic code or cross species varietions.
  • Sequences showing a great deal of homology with the nucleotide sequences of SEQ ID's 1, 2 or 3 derived from Agaricus bisporus may be present in other edible fungi, especially in other species from the genus Agaricus, such as the ones mentioned hereinabove.
  • Such a nucleotide sequence from another fungus can be selected due to the fact that at least a part of the nucleotide sequences of SEQ ID's 1, 2 or 3 as derived from Agaricus bisporus or a corresponding degenerate DNA sequence derived from the amino acid sequence of SEQ ID's 1 2 or 3 or derived from an equivalent amino acid sequence can hybridize with genetic material of said other fungus.
  • the hybridizing part of the genetic material of the other organism comprises at least a part of a nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus.
  • nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus from another organism.
  • nucleotide sequences and/or DNA- fragments especially those coding for hydrophobins thet are formed during the emergence of the fruit bodies and/or that form part of the -preferably edible- fruit bodies can also be obtained by the procedure put forward in the Examples with respect to Agaricus bisporus, said procedure being based on the abundance of mRNA and transcription thereof during said phase of the life cycle of the fungus.
  • the recombinent DNA meterial according to the invention can be used to express a nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus or the recombinant DNA materiel cen be used as a probe or a primer for detection or production of genetic material encoding at least a part of a ripening form of a hydrophobin in a fungus, preferably an edible fungus.
  • the recombinant DNA materiel eccording to the invention can comprise regul ⁇ tory regions, i.e. regulatory regions native to the fungus from which the hydrophobin is derived or regulatory regions foreign to the fungus from which the nucleotide sequence encoding the hydrophobin of an edible fungus is derived operably linked to seid nucleotide sequence, for instance bacterial, yeast, plant or animal regulatory regions.
  • the regulatory regions will usually be regul ⁇ tory regions thet regulete genes coding for other peptides then hydrophobins, es b ⁇ cteri ⁇ , yeasts, plants and animals are not known to contain and/or produce hydrophobins.
  • the recombinant DNA material according to the invention comprises regulatory regions from other fungi than the fungus from which the hydrophobin gene has been derived, said regulatory regions themselves regulating hydrophobin genes and/or regulating other genes in their respective netive fungus.
  • the selection of e desirable regulatory region will be obvious to one skilled in the art and will for example depend on the host cell into which the recombinant DNA material according to the invention is introduced. If a heterologous expression host is preferred, meaning that the nucleotide sequence encoding a hydrophobin of an edible fungus is derived from another strain of organism than the host cell (e.g. a different strain, variety, species, genus, family, order, class, division or kingdom) the regulatory region is preferably a regulatory region derived from an organism similar to or equal to the expression host. For example, if the expression host is a yeast cell, then the regulatory region will be derived from a yeast cell.
  • the regulatory region need not however necessarily be derived from the same strain or the same genus as the host cell, i.e. a yeast cell.
  • the selection of a yeest cell promoter in this instence is required to enable expression of the nucleotide sequence.
  • a regulatory region operably linked to a nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus in the recombinant DNA material according to the invention can be e.g. e constitutive promoter or en inducible promoter. Especielly suited ere constitutive promoters derived from genes encoding ei-zymes involved in the glycolytic pathway.
  • An example of a recombinent DNA meteriel according to the invention comprising a strong constitutive promoter operably linked to the nucleotide sequence encoding a ripening form of a hydrophobin from an edible fungus is a recombinant DNA material wherein said promoter is the glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter.
  • gpdA glyceraldehyde-3-phosphate dehydrogenase
  • phosphoglycerate kin ⁇ se (pgk) promoter the phosphoglycerate kin ⁇ se (pgk) promoter, the pyruvate kinase (pki) promoter, TPI, the triose phosphate isomerase (tpi) promoter, the APC synthetase subunit g (oliC) promoter and the acetamidase (amdS) promoter.
  • pgk phosphoglycerate kin ⁇ se
  • pki pyruvate kinase
  • TPI the triose phosphate isomerase
  • tpi the triose phosphate isomerase
  • oliC APC synthetase subunit g
  • amdS acetamidase
  • Examples of recombinant DNA material according to the invention comprising inducible promoters operably linked to the nucleotide sequence encoding a ripening form of a hydrophobin from an edible fungus activity are recombinant DNA materials, wherein said inducible promoters are selected from the promoters of the following genes: xylanase A (xylA) , glucoamylese A (gl ⁇ A), cellobiohydrolase (cbh) , amylase (a y) , invertase (sue) and alcohol dehydrogenase alcA, TAKA amylase and amyloglucosidase (AGT) .
  • xylanase A xylanase A
  • gl ⁇ A glucoamylese A
  • cbh cellobiohydrolase
  • a y amylase
  • invertase invertase
  • yeast promoters are selected from the promoters of the following genes: alcohol dehydrogenase, lactase, 3-phosphoglycerate kinase, triose phosphate isomerase, ⁇ -D-galactose-phosphate uridyl trensferese (Gal7) and glycereldehyde-3-phosphete dehydrogen ⁇ se (GAPDH) .
  • Examples of recombinant DNA materiel according to the invention comprising bacteriel promoters oper ⁇ bly linked to the nucleotide sequence encoding a ripening form of hydrophobin from an edible fungus activity are recombinant DNA materials, wherein said bacterial promoters are selected from the promoters of the following genes: ⁇ -amylase, SP02 and extracellular proteases.
  • a heterologous expression host is a yeest or e becterial strain
  • a recombinant DNA materiel according to the invention comprising an uninterrupted (intronless) nucleotide sequence encoding a ripening form of a hydrophobin of en edible fungus is preferred.
  • This preference stems from the feet that the possibility that the heterologous host does not recognize splicing signals residing on the recombinant DNA material can thus be avoided.
  • Such an uninterrupted nucleotide sequence may be obtained from a cDNA library constructed from RNA isolated from cells expressing a nucleotide sequence encoding a ripening form of a polypeptide with hydrophobin from an edible fungus.
  • an uninterrupted nucleotide sequence may be obtained by applying one or more poly- merase chain reections using suiteble primers, so as to precisely remove the introns, using genomic DNA as a templete, as is known to a person skilled in the art.
  • the introns are removed and that the fungal leader sequence is replaced by a signal sequence suitable for yeast such as the signal sequence of the invertase gene ensuring correct processing and secretion of the mature polypeptide.
  • a signal sequence suitable for yeast such as the signal sequence of the invertase gene ensuring correct processing and secretion of the mature polypeptide.
  • the removal of introns is necessary for expression in becteria such as Bacillus subtilis.
  • the ⁇ - amylase signal sequence can be used as signal sequence.
  • a preferred embodiment of recombinant DNA materiel according to the invention comprises a selection marker.
  • a selection marker serves to discriminate host cells into which the recombinant DNA material has been introduced from cells that do not comprise said recombinant DNA material.
  • This selection marker provided with the appropriate regulatory sequences may reside on the same DNA fragment conteining the nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus or can be present on a separate fragment. In the latter case a co-transformation must be performed with the various components of the recombinant DNA materiel according to the invention.
  • the ratio of expression component (containing the nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus) / selection component (with the selection marker) can be adjusted in such a manner that a high percentage of the selected cells comprising the selection component have also incorporated the expression component.
  • the term re- combinant DNA material as used herein therefore comprises one or more recombinant DNA fragments, wherein the selection marker can be incorporated on the same recombinant DNA molecule as the nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus or on a different recombinant DNA fragment. Very often filamentous fungi are transformed through co-transformation.
  • any resulting pyrA * strain will obviously have incorporated some recombinant DNA materiel end will most probably also comprise the nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus.
  • selection systems for industrial microorganisms are those formed by the group of selection markers which do not require a mutation in the host organism.
  • fungal selection markers are the genes for acetamidase (amdS) , ATP- synthetase, subunit 9 (oliC) and benomyl resistance (benA).
  • amdS acetamidase
  • oliC ATP- synthetase
  • benomyl resistance benA
  • Another example of a fungal selection merker is the nitrate reductase system.
  • non-fungal selection markers are the g ⁇ l ⁇ resistance gene (yeest) , the ampicillin resistance gene ⁇ £• coli) and the neomycin resistance gene (Bacillus) , a gene conferring resistance to hygromycin (hph) or a gene conferring resistance to fleomycin (Ble).
  • Suitable trensformetion methods and suitable expression vectors provided with e.g. a suitable transcription promoter, suiteble transcription termination signals and suitable marker genes for selecting transformed cells are alre ⁇ dy known for m ⁇ ny orgenisms including different bacteriel, yeast, fungal and plant species. Reference may be made for yeast for example to Tagima et al.
  • Yeast 1, 67-77, 198 which shows expression of a foreign gene under control of the gal7 promoter inducible by galectose in yeest end for Bacillus subtilis for example in EP-A-0,157. ⁇ 1 describing a plasmid pNS48 containing the SP02 promoter as an expression vector.
  • the generel litereture For the possibilities in these and other organisms reference is made to the generel litereture.
  • Overexpression of a ripening form of a hydrophobin of an edible fungus may be achieved by the incorporation of recombinant DNA materiel eccording to the invention in an expression host, said recombinant DNA material comprising one or more regulatory regions (selected for example from promoter and terminator regions) which serve to increase expression levels of the polypeptide of interest from said expression host. If desired the polypeptide of interest can be secreted from the expression host. This can be achieved by incorporating recombinant DNA material according to the invention as described further comprising at least one signal sequence (e.g. a pre or prepro sequence) .
  • signal sequence e.g. a pre or prepro sequence
  • the present invention is not only directed at the recombinant DNA material comprising at least a part of a nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus in the various embodiments as described above but is also directed at a cell comprising at least a part of said recombinant DNA material, said cell being capable of expression of said nucleotide sequence.
  • Progeny of an expression host comprising recombinant DNA materiel according to the invention is also embraced by the present invention.
  • a cell according to the invention will be capable of overexpression of a nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus.
  • overexpression is defined as the expression of the ripening form of a hydrophobin of an edible fungus at levels above those ordinarily encountered under the same conditions in the native organism from which said polypeptide originates.
  • overexpression also covers the expression of the ripening form of e hydrophobin of en edible fungus in an organism other than the organism from which the nucleotide sequence comprised on the recombinant DNA materiel according to the invention can be derived, a so called heterologous organism.
  • the heterologous host organism does not normally produce such a ripening form of a hydrophobin of en edible fungus et appreciable levels and the heterologous organism is therefore only capable of such production after introduction of the recombinant DNA material according to the invention.
  • homologous expression host means that the non transformed expression host belongs to the same strain or species as the organism from which the nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus that is comprised on the recombinant DNA material according to the invention has been derived.
  • the overexpression can be further achieved by the introduction of the recombinant DNA material according to the invention into a host belonging to a strain other than the strain from which the nucleotide sequence encoding a ripening form of hydrophobin of an edible fungus was isolated a so-called heterologous host, such that the resulting expression host comprises a nucleotide sequence encoding a ripening form of hydrophobin of an edible fungus in increased gene copy numbers, becoming a so-celled multicopy transformant.
  • the methods generally known for obtaining multicopy transformants can be used.
  • the recombinant DNA material according to the invention therefore comprises eny embodiment required for obteining e multicopy transformant comprising multiple copies of the nucleotide sequence encoding a ripening form of hydrophobin of an edible fungus.
  • the overexpression can also be achieved by the introduction of the recombinant DNA material according to the invention in the various embodiments already described into a host cell such that the host cell comprises the nucleotide sequence encoding a ripening form of hydrophobin of an edible fungus under the control of a regulatory region other than the native regulatory region for the hydrophobin from an edible fungus gene in the organism from which said nucleotide sequence is derived, said other regulatory region preferably being more efficient than the native regulatory region.
  • the invention is also directed at recombin ⁇ nt DNA material in any of the various embodiments described further comprising a regulatory region other than the native regulatory region for the hydrophobin from an edible fungus gene in the organism from which said nucleotide sequence is derived.
  • Such a host cell can be either homologous or heterologous.
  • the host cell can comprise one or more copies of the nucleotide sequence encoding a ripening form of hydrophobin of an edible fungus comprised on the recombinant DNA materiel according to the invention.
  • chromosom ⁇ l integr ⁇ tion always takes place in successful transformations. No plasmid DNA is maintained.
  • yeast both plasmids and integrated DNA can be maintained satisfactorily.
  • Plasmids have the advantege that they exist normally in the cell in multiple copies which also means that a certain gene located on such a plasmid exists in the cell in multicopy form which may result in a higher expression of the proteins encoded by the genes.
  • the disadvantage of plasmids is that they can be unstable resulting in e possible loss of the plasmids from the cells at a certain stage.
  • the loss of a plasmid can be prevented by using a plasmid comprising at least one stretch of nucleotides capable of hybridizing with chromosomal DNA of the non-transformed host cell enabling said vector to integrate stably into the chromosome of said host cell after transformation.
  • a stretch of homologous DNA that is already present in multiple copies in the chromosomal DNA will lead to multicopy insertion of the vector DNA resulting in integrated multimeric DNA comprising one or more copies of the nucleotide sequence encoding a ripening form of a hydrophobin from an edibe fungus.
  • Another prerequi ⁇ it for a vector resulting in integrated DNA in the chromosomal DNA is that the vector does not comprise ⁇ functional replicon as the vector must be uneble to maintain itself in the host cell unless it is integrated.
  • the stretch of nucleotides en ⁇ bling integration is prefer ⁇ bly deriveble from DNA thet comprises et least part of ⁇ non-recessed portion of the chromosome of a non-transformed host cell (in this instance the term "derivable from” implies that the stretch of nucleotides in the vector according to the invention must show enough homology with the chromosomal DNA to enable hybridization for an integration event to occur) .
  • the integration of the vector will subsequently take place in said non-essential portion of the chromosome of the host cell and will not lead to the loss of essential function of the host cell. It is preferable for the integration to take place in a non-essential selectable gene of the chromosome of the non-transformed host cell. This can be subsequently a selection criterium for transformed host cells.
  • a preferred embodiment of the invention is directed at e cell comprising recombin ⁇ nt DNA meterial according to the invention in any of the embodiments described, wherein said cell is capeble of secreting a ripening form in particular capable of secreting a mature form of a polypeptide with hydrophobin from an edible fungus as encoded by said recombinant DNA material. It is often desirable for the ripening form of a hydrophobin of an edible fungus to be secreted from the expression host into the culture medium as said polypeptide may be more easily recovered from the medium than from the cell. Preferably the meture form of the hydrophobin from en edible fungus will be secreted into the culture medium.
  • the term "secretion" in the subject invention comprises the polypeptide crossing e cell well or ⁇ cell membr ⁇ ne.
  • the polypeptide can pass such a cell wall or membrane into the culture medium but can also remain att ⁇ ched to said cell wall or cell membrane.
  • the polypeptide can also pass a cell membrane into the periplasmic space end not into the culture medium.
  • the processing c.q. secretion route to be followed by the ripening form of a hydrophobin of an edible fungus will depend on the selected host cell and the composition of the recombinant DNA material according to the invention. Most preferably, however, the polypeptide will be secreted into the culture medium.
  • hydrophobins secretion thereof may not always be possible, and intracellular excretion of excretion at or near the cell wall or membrane, or on the outside thereof, followed by destruction of the cell, for example by lysis or sonication, and isolation of the hydrophobins in a manner known per se is also included by the invention.
  • the excellent stability of the hydrophobin complexes, especially against SDS and other surfactants, may be used with advantege.
  • the cell according to the invention can comprise recombin ⁇ nt DNA m ⁇ teriel in eny of the verious embodiments described further comprising DNA encoding the n ⁇ tive le ⁇ der sequence (pre or prepro) of the hydrophobin of an edible fungus.
  • the cell according to the invention can comprise recombinant DNA material further comprising DNA encoding for foreign leader sequences (pre or prepro) instead of the native leader sequences.
  • the invention is also directed at recombinant DNA material comprising DNA encoding the mature hydrophobin of an edible fungus coupled to DNA encoding a leeder sequence foreign to the hydrophobin of en edible fungus.
  • an increese in the expression of e hydrophobin of en edible fungus cen result in the production of polypeptide levels beyond those the expression host is cep ⁇ ble of processing and secreting resulting in a build up of polypeptide product within the host cell creating a bottle neck in the transport of the polypeptide through the cell membrane or cell wall.
  • the present invention is also directed et ⁇ cell comprising recombinant DNA material in any of the verious embodiments described comprising heterologous signel sequences to provide for the most efficient secretion of the hydrophobin from en edible fungus from the chosen expression host and the invention is also directed at said recombinant DNA material.
  • a heterologous secretion signal sequence may be chosen such that it is derived from the seme strein as the organism from which the other regulatory regions of the nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus have been derived, preferably from the same gene.
  • the signal of the highly secreted amyloglucosidase protein m ⁇ y be used in combin ⁇ tion with the amyloglucosidase promoter itself as well as in combination with other promoters.
  • heterologous secretion signal sequences are those originating from the glucoamylase A or xylanase
  • a gene for fungi the invertase gene for yeast and the ⁇ -amylase gene for Bacillus.
  • Hybrid secretion sequences may also advantageously be used within the context of the present invention.
  • terminators of transcription are not considered to be critical elements for the overexpression of genes.
  • a termin ⁇ tor of transcription may be selected from the same gene as the promoter or alternatively the homologous terminator may be employed. In fact any terminator can be employed.
  • Factors such as size (molecular weight) the possible need for glycosylation or the desirability of the secretion over the cell membrane or cell wall or into the medium of the hydrophobin from en edible fungus pl ⁇ y an important role in the selection of the expression host.
  • nucleotide sequence encoding a hydrophobin of an edible fungus will be used either with or without introns occurring in said DNA sequence either with its own promoter and/or trenscription termin ⁇ tion signels or originating from another gene and either with its own leader sequence or with a signal sequence originating from another gene.
  • the invention knows no special limits with respect to the neture of the cells comprising recombinent DNA meteri ⁇ l ⁇ ccording to the invention.
  • Cells according to the invention may be important as agents for multiplying the recombinant
  • DNA materiel or as agents for producing a ripening form of a hydrophobin of an edible fungus are preferred.
  • Those expression hosts capeble of overexpression of e nucleotide sequence encoding a ripening form of a hydrophobin of en edible fungus are preferred.
  • an expression host cell capable of secretion of a ripening form of hydrophobin of an edible fungus is preferred.
  • the expression hosts are preferably selected from the group consisting of bacteriel cells, fungal cells, yeast cells and plant cells.
  • Preferred examples of eminently suited host cells are a) fungal cells, in particular filamentous fungal cells, such as a fungal cell from the group comprising the genera Aspergillus. Trichode ⁇ na.
  • Neurospora. Penj ⁇ illium and Mucor Examples of particular species that are suitable as host cell are fungal cells of one of the species Aspergillus niger Aspergillus awamori. Aspergillus pryzae, Aspergillus soiae. Aspergillus tvbigensjs, Asper illus ⁇ cule ⁇ tus, AspergilluR aponicus. Trichoderma reesei and Trichoderma viride: b) yeast cells, for example of the genera Saccharomvces. luweromvces. Hensenule and Pichia. in particular yeast cells of one of the species Saccheromvces cerevisiae.
  • Lactobacillus and Streptococcus such as bacteri ⁇ of the species Bacillus subtilis or Bacillus licheniformis.
  • the host cell to be selected for recombin ⁇ nt DNA meteriel ⁇ ccording to the invention will emongst others depend on the epplic ⁇ tion for which the resulting hydrophobin of en edible fungus is destined.
  • a preferred cell according to the invention is a foodgrade cell. This preference stems from the fact that products of such foodgrade cells can be used in processes for producing foodstuffs.
  • Bacteri ⁇ from the genus Bacillus are very suitable as expression host cells bec ⁇ use of their cepability to secrete proteins into the culture medium.
  • a host selected from the group of yeasts or fungi may be preferred.
  • some proteins are either poorly secreted from the yeast cell or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these and other instances ⁇ fungal host organism can be selected.
  • GRAS gener ⁇ lly regerded as safe.
  • eukaryotic hosts have been found to have a high productivity of secreted active polypeptides.
  • fungal hosts are very often used in industrial processes, particularly suitable examples of a host cell are therefore Aspergillus niggr and Aspergillus niger var. aw mori.
  • These perticuler species of Aspergillus h ⁇ ve previously been demonstr ⁇ ted to be excellent host cells for industrially producing enzymes.
  • a person skilled in the art is ⁇ ble to obt ⁇ in multicopy transformants of these species.
  • polypeptide production it is possible to use the expression host cell to produce polypeptide and to subsequently either isolate the polypeptide from the culture medium or use the medium containing the polypeptide as such after removal of the cells. It is even possible to use the cells themselves to produce the polypeptide in situ in the process for which the hydrophobin of an edible fungus is required.
  • a host strain that is to be used directly can only be used if it is ⁇ food grade host strain.
  • yeast strains that have been genetically manipulated in ⁇ ccordence with the present invention c ⁇ n be used directly.
  • the expression host cell c ⁇ n be selected to avoid such problems.
  • the subject invention is also directed at a ripening form of e polypeptide with hydrophobin from en edible fungus activity wherein said ripening form is obtainable by expression of the recombinant DNA materiel according to the invention.
  • the invention is preferably directed at a m ⁇ ture form of a polypeptide with hydrophobin from an edible fungus ⁇ ctivity ⁇ s no further tre ⁇ tment of s ⁇ id polypeptide is necess ⁇ ry before using seid polypeptide in a desired process.
  • the invention is directed ⁇ t a ripening form of a polypeptide as encoded by a part of the amino acid sequence of Figure 1, 2 or 3-
  • a ripening form of a hydrophobin of an edible fungus said ripening form being encoded by ⁇ part of any equivalent amino acid sequence encoding a polypeptide with an equivalent terti ⁇ ry structure h ⁇ ving hydrophobin from an edible fungus also forms part of the invention.
  • the invention is ⁇ lso directed at a process for producing a ripening form of a hydrophobin of an edible fungus comprising the culture of a cell as previously described in the specification and optionally isolation of the resulting ripening form of a hydrophobin of an edible fungus.
  • the expression of the polypeptide with hydrophobin from an edible fungus activity can be effected by culturing expression host cells thet h ⁇ ve been tr ⁇ nsformed with the recombinant DNA material comprising a nucleotide sequence encoding a ripening form of a hydrophobin of an edible fungus in a conventional nutrient fermentation medium.
  • the fermentation medium can comprise an ordinary culture medium containing ⁇ c ⁇ rbon source, ⁇ nitrogen source, an organic nitrogen source and inorganic nutrient sources.
  • the selection of the appropriate medium may be based on the choice of expression host and/or based on the regulatory requirements of the recombinant DNA material. Such media are well-known to those skilled in the art.
  • the medium may, if desired, contain addition ⁇ l components f ⁇ vouring the tr ⁇ nsformed expression host over other potenti ⁇ lly cont ⁇ min ⁇ ting microorg ⁇ nisms. In the c ⁇ se of production of the hydrophobin of en edible fungus for food processing such ⁇ dditional components ere necess ⁇ rily also food grade.
  • the cells After fe ⁇ nentetion the cells can be removed from the fermentation broth by means of centrifugation or filtr ⁇ tion. Depending on whether the host cell hes secreted the hydrophobin of an edible fungus into the medium or whether said polypeptide is still connected to the host cell in some way either in the cytoplasm, in the periplasmic space or att ⁇ ched to or in the membrane or cell wall, the cells can undergo further treatment to obtain the polypeptide.
  • recovery of the polypeptide can for example be accomplished as described in US 4,894,340 or US 4,632,905 by rupturing the cells for example by high pressure disruption, sonication, enzymatic digestion or simply by cell autolysis followed by subsequent isolation of the desired product.
  • the polypeptide can be separated from the cell mass by various means. In one such method the cells are disrupted by the protease ficin and subjected to ultrafiltration. The polypeptide is subsequently precipitated with an organic solvent such as methanol or acetone.
  • the polypeptide can also be separ ⁇ ted from the cell mess by suspending the microorg ⁇ nism in e brine solution sufficient to p ⁇ rtition the polypeptide into the brine solution (for example 20% (w/v) NaCl). It is suggested that the brine solution creates osmotic pressure sufficient enough to partition a polypeptide into the brine solution.
  • e brine solution sufficient to p ⁇ rtition the polypeptide into the brine solution (for example 20% (w/v) NaCl). It is suggested that the brine solution creates osmotic pressure sufficient enough to partition a polypeptide into the brine solution.
  • the same methods heretofore employed to liber ⁇ te and produce solutions of other intracelluler enzymes c ⁇ n be employed.
  • the polypeptide isol ⁇ ted from microbi ⁇ l cells is based purified by convention ⁇ l precipit ⁇ tion end chrometogrephic methods. Such methods include amongst others methanol, ethanol, acetone and ammonium sulf ⁇ te precipitetion end ion exchange and hydroxy apatite chromatogr ⁇ phy.
  • the excellent stebility of the hydrophobin complexes especielly ⁇ gainst SDS and other surfactents, may be used with advantege in isolating the hydrophobins both from the cells in which they are produced, from a culture medium in which they have been secreted, which may ⁇ lso cont ⁇ in cell fragments and debris.
  • the cultures were ⁇ llowed to grow st ⁇ tionary for 14 days at 24 * C and were harvested by filtration over nylon gauze.
  • the mycelium w ⁇ s immedi ⁇ tely frozen in liquid nitrogen end stored ⁇ t -7 ⁇ "C.
  • Mycelium of Ag ⁇ ricus bisporus Horst Ul was allowed to colonize compost using standard cultivation conditions. A top layer (c ⁇ sing) w ⁇ s added to induce fruit body formation. The primordia and pins were picked from the first flush, immediately frozen in liquid nitrogen and stored at -70°C. The deep frozen tissue was grinded in a precooled Waring blender and total RNA was isolated from this tissue using a stand ⁇ rd method (section 1.1.1.). The obteined total RNA was subsequently used as source for the isolation of polyA * RNA by a stand ⁇ rd method (Sambrook et al., (1989).
  • RNA w ⁇ s subsequently used ⁇ s ⁇ source for the generation of complement DNA (cDNA) using a ⁇ -ZAP-cDNA synthesis kit (Stratagene, La Jolla) .
  • cDNA complement DNA
  • ⁇ -ZAP-cDNA synthesis kit Stratagene, La Jolla
  • bisporus stage 7 mushrooms were picked from the first flush, inmediately frozen in liquid nitrogen end stored ⁇ t -7 ⁇ "C.
  • the A. bisporus ⁇ -ZAP-cDNA library was used to generate 200 random cDNA clones using a stand ⁇ rd protocol.
  • the insert of each individual clone was amplified using polymerase chain reaction (PCR) ⁇ mplificetion using a Perkin-Elmer Cetus Thermal Cycler and a program of 20 thermal cycles, each consisting incubations of 1 min. at 94°C, 1 min. at 62 * C and 1 min. at 72 * C preceded by an incub ⁇ tion of 4 min. 95 * C and followed by an incubation of min. at 72"C.
  • PCR polymerase chain reaction
  • the oligonucleotides used were stand ⁇ rd SK and T7 primers which are able to anne ⁇ l directly adjacent to the cDNA insert on either side.
  • the reactions were performed in the precence of 200 ⁇ M each of dATP, dCTP, dTTP and dGTP, 1 units Taq polymerase (Boehringer Mannheim), 10 mM Tris/HCl (pH 8-3), 1.5 ⁇ M MgCl 2 , 50 mM KC1, 0.1 mg/ml gelatin and 50 pmol of each primer in a total volume of 25 ul, using plasmid DNA obtained from an A. bisporus cDNA clone as template.
  • the DNA templates were isoleted by boiling a part of an Escherichia coli colony bearing the desired cDNA containing plasmid in H 2 0 for 5 min. followed by removal of the cell debri by centrifugation.
  • Each individual PCR product was radio ⁇ ctively labeled using the method of Feinberg end Vogelstein (1983) and used as a probe in Northern an ⁇ lyses of 5 ug end 0.05 ug of total RNA isolated from mycelium (section 1.1.1.), 5 ug and 0.05 ug of totel RNA isoleted from a mixture of primordia and small fruit bodies (section 1.1.2) and 5 ug of total RNA isolated from stage 7 fruit bodies (section 1.1.3) spotted on Hybond N membrane (Amersham) .
  • Hybridization was performed o/n in a stand ⁇ rd solution of 6x SSC, 0.5% SDS end 5x Denherdts solution and 100 ⁇ g/ml single stranded herring sperm DNA at 65 C followed by stringent ⁇ tanderd wash procedures.
  • Two complement DNA inserts which g ⁇ ve very high hybridizing sign ⁇ ls with totel RNA isoleted from a mixture of pins and primordia and stage 7 mushrooms but gave low or not detectible hybridizing signals with total RNA isolated from vegetatively growing mycelium were analyzed further by sequencing and Southern analysis.
  • the matching genes were called hypA and hypB after the hydrophobic properties of the inferred polypeptide sequence they encode.
  • HYPA hypothalamic hormone
  • the cDNA probes were labeled according to method of Feinberg and Vogelstein and approximately 25xl0 3 plaques were screened in duplo with the cDNA probes according to stenderd methods (Sembrook et al., 1989) using Escherichia coli LE392 as plating b ⁇ cteri ⁇
  • the totel length of the inserts cont ⁇ ined within the analyzed plaques is equivalent to about 10 times the size of the Agericus bisporus genome (Sonnenberg et el., 1221). Hybridizetion end w ⁇ sh conditions were as describe ⁇ in in section 1.1.4.
  • Plaques which scored positive for hybridizetion to the probes on duplic ⁇ te sets of filters were purified ⁇ ccording to standard methods and of each gene and according to stenderd methods.
  • For e ⁇ ch gene was DNA isolated of four of those positive pleques.
  • the inserts of four positive clones were analyzed by Southern hybridization of single and combined digestion with the restriction enzymes EcoRl, BamHI,Hindlll, Sail and Bglll using the hypA cDNA (SEQ ID No: 1.) as ⁇ probe. Combination of the resulting date led to the identification of 1.5 kb EcoRI fragment, an ⁇ dj ⁇ cent 1.8 kb EcoRI-Hindlll fragment and an overlapping 4.2 kb Bglll fragment which all hybridized with the cDNA of the Agaricus bisporus hypA gene (Fig. 1).
  • sequence of the relevant parts of the 1.8 kb BamHI-EcoRI and the 2.9 kb BamHI-EcoRI fregment w ⁇ s determined by sequencing p ⁇ rts of the inserts of pIM3106 end pIM3107 and derived subclones in both directions as described in section 1.2.1.2. using stenderd and dedicated synthetic primers (Fig. 6, sequence listing no. 4). Additional sequence information was obtained from parti ⁇ l sequencing of the relevant p ⁇ rts of the insert of pIM310 ⁇ using dedic ⁇ ted synthetic primers.
  • Genomic DNA of the homok ⁇ ryotic streins 39 and 97 was ⁇ nelyzed by Southern hybridizetion of single end combined digestion with the restriction enzymes EcoRI, BamHI, Hindlll, Sail and Bglll using the hypB cDNA (SEQ ID No: 3.) as a probe.
  • Comperison of the hybridizetion pettern of this blot with the genomic DNA to the hybridization pattern of the Southern blot with the positive hypB ⁇ clones (section 1.2.2.1) showed one or two extra hybridizing bands in each lane of the blot with the genomic DNA, suggesting the possible existence of a second hypB like gene, hypD, in Agaricus bisporus.
  • transformation systems can be used to transform A. bisporus cells with the complete A. bisporus hypA and hypB genes using their own promoters or other availeble strong promoters of A. bisporus, such as the ⁇ vaileble promoter of the constitutive and highly expressed glyceraldehyde-3-phosphate dehydrogenase (gpd) gene (Harmsen et al., 1992).
  • gpd glyceraldehyde-3-phosphate dehydrogenase
  • the A. bisporus hypA or the hypB regulatory elements can also be used for expression of other A. bisporus proteins or heterologous proteins in A. bisporus fruit bodies i.e. proteins that influence the morphology and/or yield and nutrition value of the basidoc ⁇ rp, proteins that improve the quality of the fruit body, proteins that enhance the flavour or proteins that improve the shelf life of the crop.
  • proteins that influence the morphology and/or yield and nutrition value of the basidoc ⁇ rp proteins that improve the quality of the fruit body, proteins that enhance the flavour or proteins that improve the shelf life of the crop.
  • bisporus hypB gene is highly expressed e ⁇ rly during development of the b ⁇ sidiocerp, while the hypA gene is highly expressed during fruit body elongation and matur ⁇ tion.
  • the hypB promoter will be used, however when the desired protein is preferably expressed during fruit body elongation or maturation the most suitable promoter will be the hypA promoter.
  • the fusion constructs replacing the promoters of the gene encoding the desired protein by the selected A. bisporus hyp promoter can be made using stenderd cloning techniques, PCR or synthetic oligonucleotides.
  • A. bisporus HYPA and HYPB in Aspergillus species such as Aspergillus niger CBS 120.49 or Aspergillus niger var. awamori CBS 115»51 or related species
  • functional fusions can be m ⁇ de using regul ⁇ tory sequences of highly expressed Aspergillus genes such ⁇ s the gpd promoter and terminator and the A. bisporus hypA or the hypB gene.
  • the fusions can be m ⁇ de using PCR or synthetic oligonucleotides.
  • Such constructions can be integrated in the A. bisporus genome in single or multi copies using standard a Aspergillus cotransformation techniques.
  • Aspergillus niger NW128 (cspAl, goxC17, pyrA6, nicAl) can be used.
  • the cspAl mutation short conidiophores
  • the gox ut ⁇ tion no production of glucose oxid ⁇ se
  • the pyrAl mutation (requirement for uridine) can be utilized for the introduction of multiple copies of the hypA and hypB genes
  • the nicAl mutation (rement for nicotinamide) facilit ⁇ tes the biologic ⁇ lly cont ⁇ ined h ⁇ ndling of the str ⁇ in (Witteveen et al., 1990).
  • Aspergillus niger strain NW128 can be co-transformed with mixtures of two different DNA fragments in various ratios using stenderd techniques (e.g Goosen et al., 1987).
  • One of these fragments will be the 3-8 kb Xbal fr ⁇ gment of Aspergillus niger N4 ⁇ O, comprising the entire pyrA gene end function ⁇ l promoter (Goosen et al., 1987).
  • the other fragment will be the hyp fusion gene.
  • DNA sequences encoding functional signal sequences derived from A. bisporus hypA or hypB genes encoding proteins that ere efficiently secreted c ⁇ n be used.
  • functional signal sequences optimized for A are used.
  • niger can be added in a functional fusion before the region encoding the mature HYP protein mentioned above using PCR or synthetic oligonucleotides.
  • the levels of expression of heterologous proteins can be further improved by ⁇ djustment of the codon us ⁇ ge to the preferred codon usege of A. niger.
  • vectors c ⁇ n be constructed in which sequences encoding the mature A. bisporus HYPA and HYPB proteins are placed under the control of known yeast promoters. Since yeasts cannot recognize all the introns of the A. bisporus hypA and hypB genes, the constructs should be made from the complete cDNAs of hypA end hypB (plasmids pIM3100 and pIM3101) which can be used as starting materiel for the construction of function ⁇ l fusions between yeast promoters and functional sequences such as yeest transl ⁇ tional stert signals and part of the A.
  • the fusions can be made using PCR or synthetic oligonucleotides.
  • Such constructions can be incorporated in autonomously replicating yeest vectors or c ⁇ n be integrated in the yeest genome in single or multi copies using the ⁇ ppropi ⁇ te yeest vectors.
  • DNA sequences encoding functional yeest signal sequences derived from yeest genes encoding proteins thet ere efficiently secreted c ⁇ n be added in a functional fusion before the region encoding the mature HYPA or HYPB using PCR or synthetic oligonucleotides.
  • the levels of expression can be further improved by adjustment of the codon usage of the A. bisporus hypA and hypB genes according to the codon preferences known for yeasts.
  • the invention rel ⁇ tes to regul ⁇ tory regions of the hydrophobin genes mentioned herein ⁇ bove.
  • this espect of the invention is rel ⁇ ted to the reguletory regions end sequences of the hydrophobin genes of Agericus bisporus, more in perticuler to the promoters of the hyp A, hyp B, hyp C and hyp D genes of Agaricus bisporus.
  • a very desirable orgenism for the production of desired polypeptides would be the common mushroom Ag ⁇ ricus bisporus, because it is a food grade organism of GRAS status, the fruit bodies of which are edible. Also, a lot is known about the cultiv ⁇ tion. production and the processing of mushrooms, knowledge that could be used directly for the production of desired polypeptides in these organisms. Finally, mushrooms can be cultivated cheaply and economically on a large scale, even on industrial scale. It is therefore an object of the present invention to provide regulatory regions, reguletory sequences and/or promoters that can be used for the expression of homologous or heterologous genes in fungi, especially edible fungi, in particular fungi from the genus Agaricus and more in particular in Agaricus bisporus.
  • the regulatory regions of the hydrophobin genes of edible fungi especially the regulatory regions of said genes which are activ ⁇ ted in vivo during the emergence or form ⁇ tion of the fruit body, and more specifically, the regulatory regions of the hydrophobin genes of fungi from the genus Ag ⁇ ricus c ⁇ n with advantage be used for expression of homologous or heterologous genes.
  • the invention therefore relates to said regulatory regions.
  • the invention reletes to the reguletory regions end sequences of the hydrophobin genes of Ag ⁇ ricus bisporus, more in perticuler to the promoters of the hyp A, hyp B, hyp C end hyp D genes of Agaricus bisporus.
  • the regul ⁇ tory regions of the invention will usu ⁇ lly comprise ⁇ t leest ⁇ promoter, optionally an operon.
  • These regulatory regions which have now been identified and characterized for the first time, are in vivo activated only during the emergence of the fruit bodies. Therefore, the genes controlled by these regulatory regions are only expressed during the formation of or in the fruit bodies, so that the desired polypeptides for which said genes encode are (only) secreted into the fruit body, which can then be harvested and processed for the isolation of the desired polypeptide, which process is generally easy to perform with mushrooms.
  • the rest of the fungel orgenism will st ⁇ y ⁇ live and intact to form new fruit bodies producing the desired polypeptides.
  • the regulatory regions of the invention provide a very abundant expression of the product encoded the gene which they cntrol. Genes controlled by said regulatory sequences usuelly account for more than about 4-6% of the mRNA present in the fruit bodies.
  • the reguletory regions of the invention c ⁇ n be used to express any desired gene or nucleotide sequence, both homologous or heterologous, in a suiteble fungus.
  • this fungus is a food grade fungus, more preferably an edible fungus, and most preferred a fungus with GRAS stetus.
  • the fungus used as expression host is also preferably the fungus from which the reguletory region w ⁇ s ariolly derived.
  • the reguletory regions of the invention c ⁇ n ⁇ lso be used ⁇ s heterologous reguletory systems in other organisms, especi ⁇ lly in other fungi, in which c ⁇ se they can provide a more abund ⁇ nt expression of the controlled genes, compered to the homologous reguletory regions of the fung ⁇ l expression host.
  • the invention rel ⁇ tes to the promoters of the hyp A, hyp B, hyp C and hyp D genes of Agaricus bisporus.
  • the sequence listings of the regulatory regions/promoters of the Agaricus bisporus hyp A, hyp B and hyp C genes are shown in the sequence ID's for these respective genes as mentioned hereinabove.
  • the hyp A promoter has a TATA-box, shown in the sequence lsiting of pIM 3104.
  • the hyp A promoter ⁇ lso cont ⁇ ins en essent ⁇ il element of the enhencer more then 290 bp upstre ⁇ m of the tr ⁇ nsletion st ⁇ rt.
  • the TATA box is positiones about 130 bp before the tr ⁇ nsletion st ⁇ rt codon.
  • the TATA-box of the hypB promoter is shown in the sequence ID of pIM 3104.
  • the regul ⁇ tory regions of the invention thet control the hydrophobin genes ere usuelly positioned so as to operably control the expression of seid hydrophobin genes, es will be cle ⁇ r to person skilled in the art. In general, this means that the regulatory regions will be found upstream of the structural genes coding for the hydrophobins.
  • Homologous or heterologous genes for which it is desired that they are controlled by the regulatory regions of the invention, can be put under the control of the regulatory regions by operably linking them to said regulatory regions, as will be clear to a person skilled in the art. This can for instance be done by inserting said homologous or heterologous gene in a recombinant DNA fragment containing said regulatory region, in a manner known per se, and then introducing said recombinant DNA fragment containing the regulatory region and the nucleotide sequence encoding for the polypeptide or protein into the fungus that is used es the expression host. As pl ⁇ smids are generally not maintained in fungi, introducing this recombin ⁇ nt DNA meterial is preferably done by incorporation in the genomic DNA of the fungus.
  • the invention is therefore not limited to eny specific method of isol ⁇ ting/identifying the reguletory regions of the invention, for operably linking s ⁇ id regul ⁇ tory regions to homologous or heterologous genes to be expressed, or for tr ⁇ nsforming a selected expression host with the genetic meteriel thus obteined.
  • the genes can then be expressed by inducing the regul ⁇ tory gene with e suiteble inducer.
  • the preferred way of expressing the genes coding for the desired polypeptide or proteins is simply by cultivation of the fungi, whereby expression will take place during formation of the fruit body, and the proteins will simply be secreted in or into the fruit body.
  • the desired proteins or polypeptides can then simply be obtained by hervesting fruit bodies end isoleting the polypeptides or proteins in a m ⁇ nner known per se.
  • the invention further comprises all homologous and genetic variants of the regulatory regions of the invention as defined hereinabove, so long as they are still able to control the expression of a gene operably linked thereto.
  • these homologous or genetic variants can provide for decreased, the same or increased expression of the gene compered to the original regulatory sequence from which it was derived.
  • These homologous and genetic varients mey ⁇ lso have other advant ⁇ gous properties, such ⁇ s thermo-inducibility, "run ⁇ w ⁇ y” espression or continuous expression of the controled genes.
  • the term genetic veriant explicitely includes regulatory regions which are only a part of the original regulatory region from which it was derived, i.e. obtained by deletion of one or more nucleotides, both at the ' or 3' termini or somewhere inbetween, so long as these parts of the original regulatory regions still control the expression of a structural gene operably linked thereto.
  • the hyp promoters c ⁇ n be used to express in fruit bodies for instance in sense or antisense direction the clones A. bisporus m ⁇ nnitol dehydrogenese gene or glucose-6-phosphate dehydrogenese gene (Wood et al., 1991). the A. bisporus methallothionein genes (Nishiyama et al., 1991) or the cloned putative tyrosinase genes.
  • the promoters can be used to express e.g. a co ⁇ t protein of ⁇ dsRNA virus and generate in this way resistance through cross- protection (Harmsen et al., 1989. Harsem et al., 1991).
  • These hyp promoters can also be used to express heterologous proteins A. bisporus in fruit bodies such as homones for instance adreon- corticotropin, melonocyte stimuleting hormone, urog ⁇ strone or insulin or growth f ⁇ ctors such as epidermal growth factor insulin- like growth factor interleukin, such ⁇ s interleukin-1 or -2, inter- ferons such as humen interferon end proteinese inhibitors such es ⁇ l-entitrypsin and immunoglobulins such as the light or heavy chains of immunoglobulin D, E or G. References cited in the application:

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Abstract

L'invention concerne une hydrophobine, une protéine ou un polypeptide correspondant sensiblement à l'hydrophobine, dérivée d'un champignon commestible, de préférence classé GRAS, et, en particulier du genre Agaricus, par exemple Agaricus bisporus. L'invention concerne également des gènes, des séquences nucléotidiques ou des fragments d'ADN codant pour une forme murissante d'une telle hydrophobine, ainsi qu'un matériel d'ADN de recombinaison contenant ces gènes, ces séquences nucléotidiques ou ces fragments d'ADN. L'invention concerne en outre un procédé pour produire une forme murissante d'une hydrophobine, d'une protéine ou d'un polypeptide correspondant sensiblement à l'hydrophobine, dérivée d'un champignon commestible, consistant à exprimer un tel gène, une telle séquence de gène ou un tel fragment d'ADN, par exemple par mise en culture d'une cellule contenant ce matériel génétique. Selon un autre aspect, l'invention concerne une région régulatrice d'un gène d'hydrophobine d'un champignon commestible, un matériel d'ADN de recombinaison contenant cette région, ainsi qu'un procédé pour exprimer des gènes dans un champignon, de préférence un champignon commestible, dans lequel l'expression de ces gènes est commandée par l'action de cette région régulatrice. Ce procédé est, de préférence, mis en oeuvre en utilisant des espèces du genre Agaricus, en particulier leurs sporophores.
PCT/NL1996/000234 1995-06-12 1996-06-11 Hydrophobines de champignons commestibles, genes, sequences nucleotidiques, fragments d'adn codant pour lesdites hydrophobines et leur expression WO1996041882A1 (fr)

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WO2000048620A1 (fr) * 1999-02-19 2000-08-24 University Of Virginia Patent Foundation Peptides de levure membranaires et leurs anticorps
WO2001014521A1 (fr) * 1999-08-20 2001-03-01 Valtion Teknillinen Tutkimuskeskus Methode visant a reduire la formation de mousse pendant la culture d'un microorganisme
US7338779B1 (en) 1999-08-20 2008-03-04 Valtion Teknillinen Tutkimuskeskus Method for decreasing the foam formation during cultivation of a microorganism
WO2001057528A1 (fr) * 2000-02-04 2001-08-09 Applied Nanosystems, B.V. Procede pour le traitement de surface d'un objet avec une solution contenant de l'hydrophobine
WO2001057076A1 (fr) * 2000-02-04 2001-08-09 Applied Nanosystems B.V. Procede de purification d'une hydrophobine presente dans une solution contenant de l'hydrophobine
WO2003010331A2 (fr) 2001-07-23 2003-02-06 Applied Nanosystems B.V. Procede de liaison d'un compose a la surface d'un capteur
FR2833490A1 (fr) * 2001-12-14 2003-06-20 Oreal Utilisition cosmetique d'au moins une hydrophobine pour le traitement des matieres keratiniques et compositions mises en oeuvre
WO2003053383A3 (fr) * 2001-12-14 2004-01-22 Oreal Utilisation cosmetique d’au moins une hydrophobine pour le traitement des matieres keratiniques
WO2004039985A3 (fr) * 2002-10-31 2004-07-01 Horticulture Res Internat Expression sélective dans des champignons filamenteux
WO2005033316A3 (fr) * 2003-09-16 2005-10-06 Basf Ag Secretion de proteines a partir de levures
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