WO2011159153A2 - Promoteur inductible et son utilisation - Google Patents

Promoteur inductible et son utilisation Download PDF

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Publication number
WO2011159153A2
WO2011159153A2 PCT/NL2011/050428 NL2011050428W WO2011159153A2 WO 2011159153 A2 WO2011159153 A2 WO 2011159153A2 NL 2011050428 W NL2011050428 W NL 2011050428W WO 2011159153 A2 WO2011159153 A2 WO 2011159153A2
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translation
jgi
lacbil
cc1g
mushroom
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PCT/NL2011/050428
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WO2011159153A3 (fr
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Robin Arthur Ohm
Luis Gaston Lugones
Herman Abel Bernard WÖSTEN
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Universiteit Utrecht Holding B.V.
Stichting Voor De Technische Wetenschappen
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Publication of WO2011159153A2 publication Critical patent/WO2011159153A2/fr
Publication of WO2011159153A3 publication Critical patent/WO2011159153A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the invention relates to a method of producing mushroom using a heat inducible promoter.
  • the invention further relates to a heat inducible promoter, a nucleic acid construct, a vector and a fungus or mushroom comprising a heat inducible promoter of the invention and to methods for producing such a mushroom or fungus or a substance of interest.
  • Background of the invention relates to a heat inducible promoter, a nucleic acid construct, a vector and a fungus or mushroom comprising a heat inducible promoter of the invention and to methods for producing such a mushroom or fungus or a substance of interest.
  • Formation of mushrooms is a highly complex developmental process.
  • agaric fruiting bodies such as those of Agaricus bisporus (see Kiies, 2000; Umar and van Griensven, 1997).
  • hyphae escape the substrate to grow into the air.
  • These hyphae form aggregates, which are called hyphal knots or nodules.
  • Within the knots hyphae aggregate forming a fruiting body initial.
  • the pileus trama and gills with a hymenium can be distinguished.
  • different cell types are formed, among which the basidia.
  • the basidia karyogamy and meioses take place, ultimately resulting in basidiospores.
  • That development of fruiting bodies is complex, is also exemplified by the fact that formation of the different tissues overlaps in time.
  • cells in the developing mushroom differ in diameter, length, the number of septa, nuclei and vacuoles as well as the molecular composition (e.g. the content of reserve carbohydrate).
  • A. bisporus contain two nuclei with a different mating type. Germination of these spores thus results in a self-fertile heterokaryotic mycelium, containing a variable number of both nuclear types.
  • the fertile stage of a majority of mushroom forming fungi results from a mating of two compatible strains with different mating type loci.
  • partners exchange nuclei. These nuclei do not fuse but are maintained in the hyphal compartment.
  • Such mycelia are therefore called heterokaryotic (in the case that each compartment contains one nucleus of each type it is called a dikaryon). They can form fruiting bodies under the appropriate environmental and nutritional conditions.
  • the mating type loci are the master regulators of fruiting body development.
  • the mating type loci of Schizophyllum commune and Coprinus cinereus and their role in development have been studied well (for a review see Kiies, 2000). Both S. ses and C. cinereus contain two mating type loci.
  • the A locus encodes homeodomain proteins. These proteins function by forming heterodimers with homeodomain proteins encoded in a compatible A locus. Some of these homeodomain proteins also seem to form functional homodimers.
  • the B locus encodes pheromones and receptors. These receptors can bind pheromones encoded by other alleles of the B locus.
  • Both the A and the B locus regulate distinct cellular processes involved in establishing the dikaryotic mycelium. However, they co-ordinately regulate fruiting body initiation. Clearly, the presence of compatible A and B mating type loci is not sufficient for fruiting. For instance, in C. cinereus aggregates formed by a dikaryon can develop into a fruiting initial or into sclerotia. Environmental conditions such as light and nutrient availability will determine which developmental program will be switched on.
  • the present invention provides a method for the production of a mushroom, mycelium, fungal cell, a fruiting body, a spore or a gene product of interest comprising the steps of: a) culturing a fungal cell, fruiting body, spore, mycelium or mushroom comprising a nucleic acid sequence coding for a gene product of interest wherein said nucleic acid sequence is operably linked to a heat inducible promoter functional in said mushroom, mycelium, fungal cell, fruiting body and/or spore; b) treating the mushroom, mycelium, fungal cell, fruiting body, or spore with heat during part of the culturing under a); and c) optional recovery of the mushroom, mycelium, fungal cell, fruiting body, spore or gene product of interest.
  • the method according to the invention comprises the step of transforming a mushroom, mycelium or a fungal cell with an expression vector comprising a nucleic acid sequence coding for a gene product of interest operably linked to the heat inducible promoter.
  • the method of the invention further comprises heat treatment of the mushroom, mycelium, fungal cell, fruiting body or spore.
  • the heat treatment is selected from the group consisting of 2 minutes to 2 hour placement in an incubator of 37-95 °C, contacting a needle with a temperature of 37-95°C for 0.01 to 120 seconds with the culture medium of the mushroom, the mycelium, the fruiting body, the fungal cell or the spore or into the mushroom, the mycelium, the fruiting body or a colony of fungal cells or spores.
  • Heat treatment can also be obtained by using light, preferably laser light, or another energy source.
  • the light source may expose the whole culture to the light (e.g the whole mushroom bed), or may expose a limited area of 10 cm 2 or less, 1 cm 2 or less, or 1 mm 2 or less.
  • the heat treatment is selected from the group consisting of: i) 2 minutes to 2 hour placement in an incubator of 37-95 °C;
  • a heat source preferably a needle with a temperature of 37- 95°C for 0.01 to 120 seconds with the culture medium of the mushroom, the mycelium, the fruiting body, the fungal cell or the spore or into the mushroom, the mycelium, the fruiting body or a colony of fungal cells or spores;
  • treatment i), ii) and/or iii) may be given once or at time intervals of 8 h, 16 h or 24 h.
  • the heat inducible promoter is preferably from a eukaryote or functional in a eukaryote, more preferably a fungus, even more preferably a Basidiomycete, even more preferably an Agaricales, even more preferably a Schizophyllaceae, even more preferably a Schizophyllum, most preferably Schizophyllum commune.
  • said promoter is derived from a eukaryote, more preferably a fungus, even more preferably a Basidiomycete, even more preferably from an Agaricales, even more preferably a Schizophyllaceae, even more preferably a Schizophyllum, most preferably Schizophyllum commune.
  • Said promoter may be derived from a native promoter by substituting, deleting and/or adding a nucleotide in order to improve the transcriptional activity of the promoter as later defined herein.
  • the heat inducible promoter is represented by:
  • the heat inducible promoter comprises the following nucleic acid sequence: GAAX1X2X3TCX4X5GX6X7 (SEQ ID NO:262), wherein X u X 2 are A, G, C, T; X 3 is T or G; X 4 is C, T, or G, X 5 is A, G or T, X 6 is A or T and X 7 is A or C.
  • the gene of interest is a gene which upon expression of the gene product of interest induces mushroom formation.
  • the gene of interest is a gene which upon expression of the gene inhibits mushroom formation.
  • the gene of interest is represented by a nucleotide sequence encoding a polypeptide that comprises an amino acid sequence: (a) that has at least 40 % amino acid identity or similarity with a sequence selected from SEQ ID NO: 8-207; and/or, (b) that has at least 50% amino acid identity or similarity with a sequence selected from SEQ ID NO: 208-215.
  • the invention provides a heat inducible promoter having a length of 100 - 1000 base pairs that is represented by a nucleic acid sequence comprising any of SEQ ID NO: 1-7 or having at least 60% identity with any of SEQ ID NO: l-7.
  • the invention provides a heat inducible promoter represented by a nucleic acid sequence of 100 till 1000 bp present upstream of the start codon of a nucleic acid sequence encoding a heat shock protein having at least 60% identity or similarity with any of SEQ ID NO: 263-269.
  • the invention provides a heat inducible promoter having a length of 100 - 1000 base pairs, which is preferably a promoter from a basidiomycete or derived there from and/or functional therein, comprising the following nucleic acid sequence: GAAXiX 2 X 3 TCX 4 X 5 GX 6 X 7 (SEQ ID NO:262), wherein X u X 2 are A, G, C, T; X 3 is T or G; X 4 is C, T, or G, X 5 is A, G or T, X 6 is A or T and X 7 is A or C.
  • the invention provides a nucleic acid construct comprising a promoter according to the invention.
  • said nucleic acid construct further comprises a gene of interest operably linked to a heat inducible promoter.
  • the invention provides an expression vector comprising a nucleic acid construct according to the invention.
  • the invention provides a fungal cell, a mycelium, a spore, a fruiting body or a mushroom, comprising a nucleic acid construct or an expression vector according to the invention.
  • Inducible promoters can be used in biotechnological applications to produce proteins in a specific growth phase or in (part of) the vegetative or reproductive mycelium. Inducible promoters can also be used to study gene function at a particular condition, developmental stage or position in the colony. Many inducible promoters have been described. For instance, the promoter of hsp30 of Aspergillus oryzae, which encodes a heat-shock protein, was found to be highly induced simply by a short heat- treatment at 40 °C (Matsushita et al, 2009). Heat shock proteins are found throughout the domains of life. They function in protection of cells against stress such as that caused by high temperature.
  • heat shock protein promoters can be low, but upon heat stress these genes are rapidly activated by the transcription factor HSF (heat shock factor) and expression increases several orders of magnitude (Santoro, 2000).
  • HSF heat shock factor
  • Advantages of heat shock protein promoters over other inducible promoters such as the copper-inducible promoter of Histoplasma capsulatum, the pectin-inducible promoter of Penicillium griseoroseum, the thiamine- regulatable thiA promoter (pthiA) of Aspergillus oryzae, the benzoic acid inducible promoter of Aspergillus niger, and the inducible AlcA promoter of Aspergillus nidulans are that no additives to the medium are needed to induce the promoter. Thus, the composition of the medium is not changed upon induction of the promoter.
  • S. commune contains 7 genes of the small heat shock protein family hsp26/hsp42 that are homologous to hsp30 of A. oryzae. Three of these genes, hspl-3, were found not to be expressed at 25 °C. However, expression was detected at 42 °C.
  • the promoters of the 7 genes of the heat shock protein family hsp26/hsp42 can be used as an inducible system in S. commune.
  • the promoters of hspl-3 are used, as is further explained hereafter.
  • the promoters of hspl-7 correspond to SEQ ID NO: 1-7 herein. Promoter
  • a promoter preferably a heat inducible promoter having a length of 100 - 1000 base pairs (bp).
  • base pairs is used herein as to depict a double stranded polynucleotide, such as DNA, in which form a promoter, gene and/or nucleic acid molecule according to the invention are generally present in genomic DNA in their indigenous situation.
  • a polynucleotide, such as a promoter, gene and/or nucleic acid molecule can also be depicted by one of the two polynucleotide strands and can furthermore also be present in the form of a single stranded polynucleotide.
  • the promoter according to the invention is from a eukaryote and/or is functional in a eukaryote, more preferably from a fungus, more preferably from a Basidiomycete, more preferably from an Agaricales, more preferably from a Schizophyllaceae or a Agaricaceae, more preferably from a Schizophyllum or an Agaricus and most preferably from Schizophyllum commune or Agaricus bisporus.
  • the promoter according to the invention, or a nucleic acid molecule comprising said promoter is isolated from its indigenous situation.
  • the promoter is represented by a nucleic acid sequence comprising 100-1000 bp, or 200-800 bp, or 300-500 bp and which is found upstream of the start codon of a heat shock protein.
  • a heat shock protein Preferably such promoter sequence ends at the last nucleotide before the start codon.
  • the promoter is thus in its indigenous situation preferably located immediately upstream of the start codon of a heat shock protein.
  • Heat shock proteins are a class of functionally related proteins whose expression is increased when cells are exposed to elevated temperatures or other stress. This increase in expression is transcriptionally regulated. An up-regulation of the heat shock proteins is a key part of the heat shock response.
  • the heat shock protein is a heat shock protein from a eukaryote and/or functional in a eukaryote, more preferably from a fungus, more preferably from a Basidiomycete, more preferably from an Agaricales, more preferably from a Schizophyllaceae or a Agaricaceae, more preferably from a Schizophyllum or an Agaricus and most preferably from Schizophyllum commune or Agaricus bisporus.
  • the heat shock protein has at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity or similarity with any of SEQ ID NO: 263-269.
  • the promoter is represented by a nucleic acid sequence comprising any of SEQ ID NO: 1-7 or having at least 60% identity with any of SEQ ID NO: l-7.
  • the promoter is represented by the following nucleic acid sequence comprising: GAAX1X2X3TCX4X5GX6X7 (SEQ ID NO:262), wherein X l s X 2 are A, G, C, T; X 3 is T or G; X 4 is C, T, or G, X 5 is A, G or T, X 6 is A or T and X 7 is A or C.
  • the promoter is represented by a nucleic acid sequence comprising any of SEQ ID NO: l - 3 or having at least 60% identity with any of SEQ ID NO: 1 - 3, since there is no expression of these promoters at 25°C, whereas expression is induced at a temperature of 37°C or higher. Therefore there is no leak of expression at the preferred maintenance temperature of many mushroom forming fungi or mushrooms as further described later herein.
  • promoter refers to a nucleic acid fragment that functions to control the transcription of one or more genes or nucleic acids, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is related to the binding site identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
  • a promoter preferably ends at nucleotide -1 of the transcription start site (TSS).
  • a promoter of the invention is preferably said to be non-constitutive and/or preferably inducible. More preferably, said promoter is inducible by a change in temperature and/or is called a heat shock promoter and/or a heat inducible promoter; said are used interchangeably herein. It may mean that such promoter is active above a given temperature and inactive below a given temperature. One speaks also of the activity or transcriptional activity of said promoter. Within this context, "active" means that said promoter is able to induce the expression of a nucleotide sequence operably linked thereto in a given expression assay.
  • inactive means that said promoter is not able to induce the expression of a nucleotide sequence operably linked thereto in the same expression assay.
  • a promoter according to the invention exhibits in its inactive situation (i.e.
  • the promoter exhibits in its active form as determined under identical conditions (except for induction), preferably determined using the assay described in example 2 herein. Most preferably, the promoter according to the invention exhibits no transcriptional activity in its inactive situation.
  • Example 2 illustrates a way of testing the functionality (active/inactive) of the promoter.
  • This assay is preferred to determine transcriptional activity of a promoter according to the invention.
  • said promoter is inactive at a temperature below 36°C, 35°C, 34°C, 33°C, 32°C, 31°C, 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21°C, 20°C, 19°C, 18°C, 17°C, 16°C or 15°C or a temperature of 15 °C.
  • said promoter is active at a temperature of at least 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or a temperature of 42 °C.
  • said promoter is inactive at a temperature of 25°C and is active at a temperature of 42°C. Such a promoter is able to induce a heat shock.
  • Temperature can be determined using conventional means in the art, such as for example by using a conventional thermometer or a thermograph. Temperature can be determined in the medium or soil, in the air surrounding the fungi/mushroom/fruiting body/spore/mycelium, or within a fungus colony/mushroom/fruiting body/mycelium.
  • the heat induciblity of a promoter is tested using Northern blotting, qPCR, or a reporter system.
  • a promoter to be tested is cloned in front of the coding sequence of red fluorescent protein dTomato and is then introduced in the monokaryotic strain 4-8 of S. Commune (FGSC #9210).
  • Transformed S. ses is grown on solid minimal medium (Dons et al, 1979) for 5 days at 25°C. Then the 5 day old colonies are incubated for 1 hour at either 25°C, 37°C or 42°C, after which they are placed back at 25°C.
  • the promoter is said to be a heat inducible promoter.
  • more fluorescence means at least 2 times more, at least 4 times more, at least lOx, at least lOOx, at least lOOOx or at least lOOOOx more in the clones that were incubated at 42°C (and preferably also in the clones that were incubated at 37°C) as compared to the clones that were incubated at 25°C.
  • more fluorescence means an infinite induction (if there is no fluorescence at all at 25°C). More preferably, fluorescence intensity increases with increasing incubation temperature. Fluorescence can be measured using conventional means in the art, such as fluorescence microscopy using e.g. dsRED3 filters.
  • a promoter according to the invention has a length of
  • Said promoter may also have a length of 100-750, 100-500, 100-400, 100-250 base pairs.
  • the length of the promoter is not critical as long as it exhibits a transcriptional activity as defined above, preferably an inducible transcriptional activity, more preferably a transcriptional activity which is inducible by a change in temperature as defined above.
  • the origin of the promoter and the way the promoter has been identified are also not critical as long as it is functional in a desired fungal cell, mycelium, fruiting body, spore and/or a mushroom as identified herein. Functional means that this promoter may exhibit a transcriptional activity as defined herein, preferably an inducible transcriptional activity, more preferably a transcriptional activity which is inducible by a change of temperature or induce a heat shock as defined above.
  • the promoter is represented by any of SEQ ID NO: 1-7; i.e.
  • Said promoter also preferably exhibits a transcriptional activity as defined above, preferably an inducible transcriptional activity, more preferably a transcriptional activity which is inducible by a change in temperature as defined above. More preferably, the promoter is represented by any of SEQ ID NO: l - 3, i.e. SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 3or has at least 60% identity with at least one of SEQ ID NO: 1 - 3, for the same reason as explained earlier herein.
  • Each promoter sequence described herein by virtue of its identity percentage (at least 60%)) with a given nucleotide sequence respectively has in a further preferred embodiment an identity of at least 70%>, 75%>, 80%>, 82%>, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the given nucleotide sequence.
  • sequence identity is determined by comparing the whole length of the sequences as identified herein.
  • a promoter according to the invention may be a mutant, truncated or hybrid promoter derived from a promoter according to the invention.
  • nucleic acid construct comprising a promoter as defined in the previous section entitled "promoter”.
  • a nucleic acid construct of the invention further comprises a gene of interest. More preferably, a gene of interest is operably linked to a promoter of the invention.
  • a nucleic acid construct is defined as a nucleic acid molecule, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acids which are combined or juxtaposed in a manner which would not otherwise exist in nature.
  • a nucleic acid molecule is represented by a nucleotide sequence.
  • a nucleotide sequence present in a nucleic acid construct is operably linked to one or more control sequences, which direct the production of said polypeptide in a fungus or in a mushroom or in any suitable system, in vitro or in vivo, to produce the encoded gene product.
  • a promoter represented by a nucleotide sequence present in a nucleic acid construct is operably linked to another nucleotide sequence encoding a gene of interest.
  • a gene of interest codes for a polypeptide or for another gene product of interest (as defined elsewhere herein).
  • the promoter according to the invention may be natively associated with the gene of interest or may be foreign to the gene of interest, i.e. in its indigenous from the promoter is not natively associated with the gene of interest.
  • Polypeptide as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term “polypeptide” encompasses naturally occurring or synthetic molecules.
  • “Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the nucleotide sequence coding for the polypeptide of the invention such that the control sequence directs the production of the polypeptide of the invention in a fungal cell and/or in a mushroom.
  • Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to transcription, post-transcriptional modification, translation, post-translational modification and secretion.
  • Control sequence is defined herein to include all components, which are necessary or advantageous for the expression of a polypeptide. At a minimum, the control sequences include a promoter and transcriptional and trans lational stop signals.
  • an expression vector comprising a nucleic acid construct of the invention.
  • an expression vector comprises a nucleotide sequence of the invention, which is operably linked to one or more control sequences, which direct the production of the encoded polypeptide in a fungal cell and/or in a mushroom.
  • An expression vector may be seen as a recombinant expression vector.
  • An expression vector may be any vector which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of a nucleotide sequence encoding a polypeptide of the invention in a fungus and/or in a mushroom. Depending on the identity of the fungus/mushroom wherein this expression vector will be introduced and on the origin of the nucleotide sequence of the invention, the skilled person will know how to choose the most suited expression vector and control sequences.
  • Single or multiple copies of a nucleic acid construct according to the invention may be introduced into a fungal cell, a mycelium and/or a mushroom.
  • a nucleic acid construct may be maintained episomally and thus comprises a sequence for autonomous replication, such as an ARS sequence.
  • Suitable episomal nucleic acid constructs may e.g. be based on the yeast 2 ⁇ or pKDl (Fleer et al, 1991) plasmids.
  • a nucleic acid construct is integrated in one or more copies into the genome of a fungal cell, a mycelium and/or a mushroom.
  • a nucleic acid construct integrates into the genome of a fungus, a mycelium and/or a mushroom.
  • This type of nucleic acid construct may comprise a bacterial cloning vehicle, a nucleotide sequence encoding a gene product of interest and a selection marker.
  • a selection marker may confer antibiotic resistance or be an auxotrophic marker. Such markers are known to the skilled person.
  • Nucleic acid constructs comprising a bacterial cloning vehicle and a selection marker are for example disclosed in Schuren et al (1994) and Munoz-Rivas et al (1986). Alternatively, this type of nucleic acid construct may be synthesised using techniques such as for example PCR.
  • a nucleotide sequence encoding said polypeptide is introduced into an expression construct. If the expression level of a polypeptide of the invention is to be decreased, a nucleotide sequence encoding said polypeptide may be introduced into an inactivation construct.
  • An inactivation construct is known to the skilled person. Such construct may comprise a nucleotide sequence encoding a mutated polypeptide or containing the flanking sequences of a nucleotide sequence encoding said polypeptide. Such a construct should integrate at the endogenous locus of said polypeptide to replace the endogenous gene and inactivate it. Alternatively, the inactivation construct may contain a sequence inducing RNAi. RNAi techniques are known to the skilled person (De Jong et al, 2006).
  • Inactivation of the polypeptide may be due to the inactivation of the corresponding gene or nucleotide sequence.
  • RNAi like inactivation mRNA levels are reduced.
  • the inactivation construct may also result in mRNA levels similar to that observed in the wild-type.
  • the encoded mutated polypeptide has a decreased activity, wherein said decreased activity is assessed by comparison with the activity of the polypeptide the mutated polypeptide originates or derives from.
  • An activity of a polypeptide may be assessed using an assay known to the skilled person.
  • Such assay may include the introduction of said mutated polypeptide into a fungus and compare an activity of said expressed mutated polypeptide with corresponding activity of the polypeptide the mutated polypeptide originates from.
  • An activity of a mutated polypeptide may be compared with the activity of a control polypeptide. If a fungus is a Schizophyllum, a control activity of a control polypeptide may be an activity as present in the strain Schizophyllum commune strain 4-8 (FGSC #9210).
  • FGSC #9210 strain Schizophyllum
  • a mutated polypeptide has no detectable activity.
  • An activity has the same meaning as a transcriptional activity or being functional as earlier defined herein.
  • Suitable procedures for transformation of a fungus are well known to the skilled person. Such procedures may involve a process comprising protoplast formation, transformation of the protoplast, and regeneration of the cell wall in a manner known to the skilled person. Suitable transformation procedures for Aspergillus are described in Yelton et al (1984), whereas transformation of a mushroom forming fungus could be done using protoplasts using for instance procedures according to van Peer et al (2009).
  • the invention is not limited to a specific gene product of interest, such as for example a polypeptide.
  • a polypeptide in this section entitled nucleic acid construct, several polypeptides are identified. Depending on the method of the invention the identity of the polypeptide may be distinct.
  • a fungus is a fungus which is able to produce a mushroom, a polypeptide is encoded by a gene of interest as defined below.
  • inactivation of eight of these genes affected mushroom production. The production was either promoted or decreased, if present at all. Homologues of these putative regulatory genes can be found in other mushroom forming fungi.
  • genes which encode putative transcriptional regulators and whose expression change during mushroom formation may be used in combination with a promoter of the present invention in order to 1) enable commercial production of mushrooms that can not yet be produced in a commercial setting, 2) improve yield of mushrooms, 3) improve quality (e.g. shape and homogeneity of morphology), 4) improve predictability of the process of mushroom formation and/or 5) enable production of mushrooms on substrates that can not yet be used for commercial mushroom production.
  • nucleic acid construct comprising a nucleotide sequence encoding a polypeptide that comprises an amino acid sequence: (a) that has at least 40 % amino acid identity or similarity with an amino acid sequence selected from SEQ ID NO: 8-207 (Tables 1 and 8); and/or,
  • nucleotide sequence is operably linked to a promoter of the invention.
  • nucleic acid construct comprising a nucleotide sequence encoding a polypeptide that comprises an amino acid sequence that has at least 40% amino acid identity or similarity with a sequence selected from the group consisting of SEQ ID NO: 36; 184; 27; 63; 11; and/or that has at least 50% amino acid identity or similarity with a sequence selected from the group consisting of SEQ ID NO: 211; 214; 215; 209; 208.
  • nucleic acid construct comprising a nucleotide sequence encoding a polypeptide that comprises an amino acid sequence that has at least 40% amino acid identity or similarity with a sequence selected from the group consisting of SEQ ID NO: 62; 28; 61; and/or that has at least 50% amino acid identity or similarity with a sequence selected from the group consisting of SEQ ID NO: 213; 212; 210.
  • the promoter of the invention is capable of driving expression of the nucleotide sequence in a fungus and/or in a mushroom or in another system suitable to produce the product encoded by the nucleotide sequence.
  • Table 8 links the name of each polypeptide to a given SEQ ID NO corresponding to an amino acid sequence. Each of these polypeptides is suspected to be involved in the regulation of the production of a mushroom. These polypeptides are further identified in Table 1, Table 7 and Table 8 and are also named Transcription Factor (TF). These polypeptides are available in a public database (htt ://j gi.doe.gov/Scommune). Therefore using a promoter of the invention operably linked to a nucleotide sequence encoding a TF involved in mushroom formation allows to develop an inducible method for producing a mushroom.
  • TF Transcription Factor
  • Each amino acid sequence described herein by virtue of its amino acid identity or similarity percentage (at least 40% identity or similarity for SEQ ID NO's 8 - 207; at least 50% identity or similarity for SEQ ID NO's 208 - 215) with a amino acid sequence respectively has in a further preferred embodiment an identity of at least 42%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity with the given polypeptide.
  • sequence identity or similarity is determined by comparing the whole length of the sequences as identified herein.
  • an amino acid sequence described herein by virtue of its amino acid identity or similarity percentage (at least 40% identity or similarity for SEQ ID NO's 8 - 207; at least 50% identity or similarity for SEQ ID NO's 208 - 215, respectively) has an identity of at least 42%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID NO: 8-207 or has an identity of at least 52%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID NO: 208-215, respectively.
  • Each nucleotide sequence encoding a polypeptide as described herein may encode a fungal polypeptide, i.e. a polypeptide with an amino acid sequence that is identical to that of a polypeptide that naturally occurs in a fungal or a mushroom organism.
  • the functionality of such polypeptide depends on the relatedness (identity or similarity percentage) of the amino acid sequence compared to that of the corresponding identified SEQ ID NO.
  • a transcription factor or TF is preferably said to be functional when said TF has a detectable transcriptional activity during at least part of the life cycle of a mushroom.
  • a mushroom is a Schizophyllum. More preferably strain 4-8 (FGSC#9210) of Schizophyllum commune (Fungal Genetic Stock Center, Missouri, USA).
  • the presence of an activity, preferably a transcriptional activity is preferably assessed by inactivating a nucleotide sequence encoding said TF in said fungus or mushroom and analysing whether a mushroom will be produced compared to the mushroom production of a control mushroom wherein said nucleotide sequence has not been inactivated. If a mushroom is not produced or if less or more mushroom is produced, said TF is said to exhibit an activity, preferably a transcriptional activity, and therefore to be functional. Less or more mushroom are later defined herein.
  • a polypeptide of the invention may be a natural polypeptide or it may be a polypeptide that does not occur naturally.
  • a polypeptide that does not occur naturally may be a polypeptide encoded by a nucleic acid sequence that is mutated for example by using site directed mutagenesis or a mutation prone PCR.
  • a skilled person may select a TF that has a detectable transcriptional activity during at least part of the life cycle of a mushroom.
  • a mushroom is a Schizophyllum. More preferably strain 4-8 (FGSC#9210) of Schizophyllum ses.
  • the presence of an activity, preferably a transcriptional activity is preferably assessed by inactivating a nucleotide sequence encoding said TF in said fungus or mushroom and analysing whether a mushroom will be produced compared to the mushroom production of a control mushroom wherein said nucleotide sequence has not been inactivated.
  • This inactivation or down regulation may have been achieved by deletion of one or more nucleotides in the encoding gene. Alternatively, it may have been caused by an R Ai-like mechanism. It is also possible to introduce a mutation in the gene or nucleotide encoding said polypeptide. This may be done by introducing a replacement or inactivation vector into a fungus/mushroom by transformation. The skilled person knows how to construct such a vector. For example such vector may comprise flanking regions of a nucleotide sequence coding for a polypeptide with a selection marker gene present in between said flanking regions.
  • a mushroom is not produced or if less mushroom is produced, said TF is said to exhibit an activity, preferably a transcriptional activity and therefore said TF can be used as a gene product of interest in the invention.
  • the meaning of less mushroom is preferably at least 3%, 6%, 10% or 15% less of the mushroom than the parental mushroom the transformed mushroom derives from will produce when both types of cells (parental and transformed cells) are cultured under the same conditions. Also at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 150% less of said mushroom than the parental mushrooms are preferred.
  • a preferred parental mushroom is strain 4-8 (FGSC#9210) of Schizophyllum ses.
  • said TF is said to exhibit an activity, preferably a transcriptional activity and therefore said TF can be used as a gene product of interest in the invention.
  • the meaning of more mushroom is preferably at least 1%, 3%, 6%, 10% or 15% more of the mushroom than the parental mushroom the transformed mushroom derives from will produce when both types of cells (parental and transformed cells) are cultured under the same conditions. Also at least 20%>, 30%>, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 500%, 1000%, 10.000% or more of said mushroom than the parental mushrooms are preferred.
  • strain 4-8 (FGSC#9210) of Schizophyllum ses is a parental mushroom.
  • the increased mushroom production is detectable by eye.
  • a TF the absence of which results in more mushroom being produced, can be used in a method to produce mushrooms.
  • the advantage is that less mushroom can be produced by regulating the production of the TF using the promoter of the invention, thereby facilitating easy harvesting of the mushrooms and /or better conditions for the mushrooms to grow (e.g. more space and more nutrients per mushroom).
  • a mushroom develops that is not hindered by other, perhaps smaller, mushrooms in their vicinity that do not fully develop.
  • the inventors found that inactivation of SEQ ID NO: 28, 61, 62 led to more mushroom production.
  • Detection of mushroom is preferably done visually, such as for example by determining the number of mushrooms in a petri dish, a container with substrate or on a mushroom bed.
  • a fungus preferably a fungus which is able to form a mushroom, a mycelium, a fruiting body, a spore, or a mushroom comprising a nucleic acid construct or an expression vector as defined earlier herein.
  • Said fungus or mushroom comprising a nucleic acid construct or an expression vector as defined earlier herein are herein referred to as fungus according to the invention and mushroom according to the invention.
  • a fungus according to the invention may be any fungus.
  • Said fungus is preferably a fungus which is able to form or produce a mycelium/mushroom, more preferably, a filamentous fungus, a Basidiomycete or an Ascomycete as described elsewhere herein.
  • the choice of the fungus, mycelium and/or mushroom will to a large extent depend upon the source of the nucleic acid sequence of the invention. Depending on the identity of the fungus, the skilled person would know how to transform it with the construct or vector of the invention.
  • a mycelium is the vegetative part of a fungus, consisting of a mass of branching, thread-like hyphae. Fungal colonies composed of mycelia are found in soil and on or within many other substrates. A mycelium may be minute and forming a colony that is too small to see by eye, or it may be extensive. The mycelium is involved in absorption of nutrients from its environment by first secreting enzymes which break down biological polymers into smaller units (e.g. monomers) and subsequently absorbing the smaller units by facilitated diffusion and active transport.
  • a mushroom may be defined as a fleshy, spore-bearing fruiting body of a fungus, typically produced above ground on soil or on its food source.
  • a mushroom is defined as a fleshy, spore-bearing fruiting body of a fungus, typically produced above ground on soil or on its food source.
  • the standard for the name "mushroom” is the cultivated white button mushroom, Agaricus bisporus, hence the word mushroom is most often applied to those fungi (Basidiomycota, Agaricomycetes) that have a stem (stipe), a cap (pileus), and gills (lamellae, sing, lamella) on the underside of the cap, just as do store-bought white mushrooms.
  • Mushrooms may also have pores in stead of lamellae.
  • the word “mushroom” can also be used for a wide variety fungal fruiting bodies that produce sexual spores and that either or not have stems, and the term is used even more generally, to describe both the fleshy fruiting bodies of some Ascomycota and the woody or leathery fruiting bodies of some Basidiomycota. Forms deviating from the standard morphology usually have more specific names, such as “puffball”, “stinkhorn”, and “morel”, and gilled mushrooms themselves are often called “agarics” in reference to their similarity to Agaricus or their place Agaricales.
  • the term “mushroom” can also designate the entire fungus when in culture or the thallus (called a mycelium) of species forming the fruiting bodies called mushrooms, or the species itself.
  • a fruiting body (also known as sporocarp or fruit body) in fungi is a multicellular structure on which spore-producing structures, such as basidia in basidiomycetes, are born.
  • the fruiting body is part of the sexual phase of a fungal life cycle, with the rest of the life cycle being characterized by vegetative mycelial growth and asexual spore production.
  • a fruiting body of a basidiomycete is also known as a basidiocarp.
  • basidiocarp A significant range of different shapes and morphologies is found in basidiocarps, which features play an important role in the identification and taxonomy of fungi.
  • Fruiting bodies may be either epigeous (if they grow on the ground as with ordinary mushrooms) or hypogeous (if they grow underground).
  • Epigeous fruiting bodies that are visible to the naked eye, especially fruiting bodies of a more or less agaricoid morphology i.e., a fungal fruiting body characterized by the presense of a pileus [cap] that is clearly differentiated from the stipe [stalk], with lamellae [gills] on the underside of the pileus; often also refers to a basidiomycete species characterized by an agaric-type fruiting body
  • mushrooms while hypogeous fungi are usually called truffles or false truffles.
  • truffles lost the ability to disperse their spores via air currents, instead opting for animal consumption and subsequent dispersal of their spores.
  • a spore is a reproductive structure that is formed during a a-sexual or sexual process and that are adapted for dispersal and surviving for extended periods of time in unfavourable conditions. Spores form part of the life cycles of many bacteria, plants, algae, fungi and some protozoans. Spores may be dispersed by forcible ejection from their reproductive structures. This ejection ensures exit of the spores from the reproductive structures as well as travelling through the air over long distances. Many fungi thereby possess specialized mechanical and physiological mechanisms as well as spore-surface structures, such as hydrophobins.
  • spore or “spores” is used to mean a spore of a fungus, preferably of a fungus that is able to form a mushroom, more preferably of a basidiomycete (i.e. a basidiospore).
  • a fungus/mycelium/fruiting body/spore/mushroom according to the invention is preferably not (of) a yeast.
  • a fungus/mycelium/fruiting body/spore/mushroom is (of) a basidiomycete or an bscomycete, preferably a basidiomycete, preferably an Agaricales, more preferably a Schizophyllaceae and even more preferably a Schizophyllum. More preferred is a Schizophyllum ses. Even more preferably Schizophyllum commune strain 4-8 (FGSC#9210).
  • Agaricales are for example Agaricus bisporus, Pleurotus ostreatus and Lentius edodus.
  • Other preferred fungal organisms are fungi which are not able to produce a mushroom. Such fungi include filamentous fungi such as those belonging to Aspergillus, Trichoderma, Penicillium, Chrysosporium, Fusarium.
  • Preferred species include: Aspergillus fumigatus,
  • a fungus according to the invention is a fungus which is able to form or produce a mycelium/mushroom
  • said fungus comprises a nucleic acid construct comprising a promoter of the invention as earlier defined herein, wherein said promoter is operably linked with a nucleotide sequence that encodes for a polypeptide that is represented by an amino acid sequence having or comprising at least 40% amino acid identity or similarity with SEQ ID NO: 8-207 as identified earlier herein or for a polypeptide that is represented by an amino acid sequence having or comprising at least 50% amino acid identity or similarity with SEQ ID NO: 208-215 as identified earlier herein in the section entitled nucleic acid construct.
  • a mushroom that is attractive to be produced or which is suspected to be attractive to produce a gene product of interest may be due to commercial reasons.
  • Such fungus/my celium/fruiting body/spore/mushroom is attractive to be used in many different applications. These procedures would improve/enable mushroom production. Improvement or enabling commercial mushroom production may result from
  • our invention may enable to improve growth of mushrooms in a commercial setting, or to allow commercial production of species that can not yet be produced, thereby creating opportunities to produce edible fungi in a cheaper way or to produce (or improve production of) pharmaceuticals or proteins that are of interest for agriculture, food, feed or non-food or non-feed applications. These proteins may either or not originate from the mushroom forming fungus. Some possible applications of a fungus of the invention are below presented.
  • a gene product of interest e.g. preferably a polypeptide, that is expressed in a fungus, mycelium and/or a mushroom, as is described above, may be a heterologous polypeptide or an endogenous polypeptide, as is further defined below.
  • a polypeptide that is expressed in a fungus, mycelium and/or a mushroom is a heterologous polypeptide, for example when a polypeptide of the invention is used to improve the production of an edible mushroom.
  • a fungus, mycelium and/or a mushroom has an "increased expression level of a polypeptide”
  • the "increased" expression level in this situation is construed to mean that there is a detectable expression of the heterologous polypeptide whereas a control fungus and/or mushroom does not have detectable expression of the heterologous polypeptide.
  • the increased expression of a gene product of interest occurs during and/or after heat treatment of the fungus/my celium/fruiting body/spore/mushroom.
  • Heat treatment of a fungus/mycelium/fruiting body/spore/mushroom is defined as subjecting the fungus/my celium/fruiting body/spore/mushroom or a culture comprising said fungus/mycelium/fruiting body/spore/mushroom to a higher temperature than the temperature wherein the mushroom or fungus is normally maintained.
  • Heat treatment may also mean subjecting a fungus/mycelium/fruiting body/spore/mushroom or a culture comprising said fungus/mycelium/fruiting body/spore/mushroom to a heat source.
  • Heat treatment may be either systemically, such as for example by placing the fungal cells/mushroom/mycelium/spore/fruiting body in an incubator or by using (laser) light, or it may be locally, such as for example by inserting a needle/rod/pole/stick/stave of the desired temperature into the mycelium/mushroom or by locally exposure to (laser) light.
  • the period of heat treatment depends on the temperature that is used and on the method that is used and can be determined by the skilled person in the art.
  • a needle may be seen as a heat source.
  • a needle, or a hot needle (or rod/pole/stick/stave) (e.g. held several minutes in boiling water or heated using heating elements) is used to locally treat a mycelium or a mushroom, preferably a mycelium.
  • Such needle may contact or may be inserted into the mycelium or mushroom for at least 0.01, 0.5, 1, 5, 10, 11 , 12, 13, 14, 15, 16, 17 seconds and up to 10 minutes.
  • a mycelium or a mushroom preferably a mycelium is preferably contacted with a needle with a temperature of 37-95°C for 0.01 to 120 seconds.
  • Said needle may also be called a hot needle
  • a needle may comprise or consist of a metal, plastic, ceramic.
  • a needle that has been held in boiling water for several minutes is thought to rapidly cool when exposed to the humid environment of the mushroom/mycelium/culture medium. It is probable that the needle will be fully back to the temperature of the environment after about a minute. However, if the needle is continued to be heated while being inserted, the maximum time may be reduced depending on the temperature used in order to prevent cells from dying. For example, Aspergillus niger cells die if they are placed in water of 65°C for 10 minutes. Further, and without wishing to be bound to any theory, it is thought that even a few seconds of insertion of said needle are sufficient to provide heat induction of the promoter.
  • the needle is inserted into or contacted with the mycelium or mushroom for less than 120, preferably 100, more preferably 80, 60, 40, 35, 30, 25 or 20, 17, 16, 15, 14, 13, 12, 11, 10, 5, 1, 0.5, 0.1, 0.01 seconds.
  • an array of needles is used to give a heat pulse in the culture medium in order to start the induction.
  • a light source may act as a heat source.
  • the light source may expose a limited area of the whole culture, i.e. 10 cm 2 or less, 1 cm 2 or less , or 1 mm 2 or less or large areas to the light (i.e. more than 10 cm 2 , more than 1 m 2 , more than 10 m 2 , more than 100 m 2 ).
  • the light of the light source may be exposed to the fungal cells, the mycelium, the fruiting body, the spore or the mushrooms for at least 0.01 , 0.5, 1, 5, 10, 1 1, 12, 13, 14, 15, 16, 17 seconds, up to 10 minutes, up to 1 h, 2h, 5h, and up to 16 h.
  • Another example is when the fungal cells, the mycelium, the fruiting body, the spore or the mushrooms or culture comprising said fungal cells, the mycelium, the fruiting body, the spore or the mushrooms are placed in an incubator, which may be done for at least 10, 20, 30, 40, 50, 60, 90, 120, 150 or 180 minutes, but preferably not longer than 16 hours, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 hours.
  • heat treatment is done for a period of 2 minutes to 4 hours, more preferably of 2 minutes to 2 hours, more preferably 5 minutes to 1.5 hours, most preferably 1 hour or 40, 50, 60 minutes.
  • the heat treatment is preferably done at a temperature of more than 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45°C.
  • Heat treatment is preferably done at a temperature of less than 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46°C. More preferably, heat treatment is done at a temperature of 37-95°C, more preferably 40-90°C, more preferably 42-70°C, more preferably 42-60°C, more preferably 42- 55°C, more preferably 42-50°C.
  • the fungal cells, the mycelium, the fruiting body, the spores or the mushroom are exposed to light or another energy source as a heat source, preferably laser light.
  • the light is of the visible spectrum (about 390-750 nm), of the infrared spectrum (about 0.7-300 ⁇ ).
  • Exposure time to light depends on the intensity of the light source and treatment could therefore take 0.01, 0.1, 0.5, 1, 5, 10, 11 , 12, 13, 14, 15, 16, 17 seconds and up to 10 minutes.
  • the treatment could take 10, 20, 30, 40, 50, 60, 90, 120, 150 or 180 minutes, but preferably not longer than 16 hours, 15, 14, 13, 12, 1 1, 10, 9, 8, 7, 6, 5 hours.
  • the fungal cells, the mycelium, the fruiting body, the spores or the mushroom are exposed to light for less than a minute, more preferable for less than 10 seconds, most preferably for less than a second.
  • a heat shock is preferably defined as being a transcriptional activation of the promoter of the invention as earlier defined herein.
  • a treatment with a needle or a light source with a narrow beam is preferred since by applying this treatment, one can specifically define where a mushroom will appear or will be formed and will grow. Accordingly, a method of the invention is wherein the heat treatment is selected from the group consisting of:
  • a heat source preferably a needle with a temperature of 37- 95°C for 0.01 to 120 seconds with the culture medium of the mushroom, the mycelium, the fruiting body, the fungal cell or the spore or into the mushroom, the mycelium, the fruiting body or a colony of fungal cells or spores;
  • treatment i), ii) and/or iii) may be given once or at time intervals of 8 h, 16 h or 24 h.
  • Increased expression of a gene product of interest is detectable, e.g. using RT- PCR or Northern blot, preferably between several minutes to an hour after heat treatment.
  • the inventors have observed that RNA of the gene product of interest sometimes has disappeared within an hour after heat treatment, whereas RNA of other genes was still present. This is related to the stability of the RNA.
  • the maintenance temperature depends on the fungus/mycelium/mushroom that is used and is for example between 15 and 30°C.
  • the method comprises the steps of culturing a fungal cell comprising a nucleic acid molecule which is represented by a nucleic acid sequence coding for a gene product of interest as defined above under circumstances that are conducive for the generation of the gene product of interest, wherein the nucleic acid sequence is operably linked to a heat inducible promoter which is functional in a eukaryote, preferably a basidiomycete.
  • the fungal cell is a mushroom forming fungal cell, preferably as defined earlier herein.
  • the heat inducible promoter is a promoter as defined earlier herein.
  • the present invention provides a method for the production of a mushroom, mycelium, fungal cell, a fruiting body, a spore or a gene product of interest comprising the steps of: a) culturing a fungal cell, fruiting body, spore, mycelium or mushroom comprising a nucleic acid molecule represented by a nucleic acid sequence coding for a gene product of interest, wherein said nucleic acid sequence is operably linked to a heat inducible promoter which is from a basidiomycete and/or which is functional in a basidiomycete; b) treating the mushroom, mycelium, fungal cell, fruiting body, or spore with heat during part of the culturing under a); and c) optional recovery of the mushroom, mycelium, fungal cell, fruiting body, spore or gene product of interest.
  • Such production includes colonization of a substrate (with or without a casing layer) followed by a phase where fruiting bodies are produced.
  • the production may be carried out at a commercial scale with an optimal production level and/or quality level (e.g. more homogeneity in size and outgrowth).
  • This method is preferably carried out using a fungus able to produce or form a mushroom, wherein said mushroom comprises a nucleic acid construct comprising a promoter of the invention as earlier defined herein, wherein said promoter is operably linked with a nucleotide coding for a polypeptide represented by an amino acid sequence having at least 50% amino acid identity or similarity with SEQ ID NO: 8-207 as identified earlier herein or having at least 40% amino acid identity or similarity with SEQ ID NO:208-215 as earlier defined herein in the section entitled nucleic acid construct.
  • said promoter is operably linked to a nucleotide sequence encoding a polypeptide that comprises an amino acid sequence that has at least 40% amino acid identity or similarity with a sequence selected from the group consisting of SEQ ID NO: 36; 184; 27; 63; 11; and/or that has at least 50% amino acid identity or similarity with a sequence selected from the group consisting of SEQ ID NO: 211; 214; 215; 209; 208; and/or said promoter is operably linked to a sequence selected from the group consisting of SEQ ID NO: 62; 28; 61; and/or that has at least 50% amino acid identity or similarity with a sequence selected from the group consisting of SEQ ID NO: 213; 212; 210; as earlier defined herein in the section entitled nucleic acid construct.
  • culturing a fungal cell, fruiting body, spore, mycelium or mushroom is herein understood to mean growing and/or maintenance of a fungal cell, mycelium or mushroom in medium, e.g. culture medium.
  • the temperature depends on the fungal cell, mycelium or mushroom that is cultured, but is typically between 15 and 30°C.
  • treating with heat is herein used interchangeably with “heat treatment” and is intended to mean exposure of the fungal cell, mycelium, fruiting body, spore or mushroom to a temperature that is higher than the growth/maintenance temperature and is preferably between 37° and 97°C as is further defined elsewhere herein.
  • the term "during part of the culturing” is herein understood to mean as during part of the time of the culturing for the durations as indicated above in the section entitled "Fungal cell/mycelium/mushroom/fruiting body/spore”. Part is preferably at least 0.01% of the duration of the culturing step a), or at least, 0.05%, 0.1%>, 0.5%>, 1%, 1.5%, 2%, 5% or more. It is preferred that after heat treatment the fungal cell/mycelium/mushroom/fruiting body/spore is maintained at the maintenance temperature that is suitable for the specific species. Heat treatment during part of the culturing can be performed without interruption the process of culture, but can also be performed during an interruption of the culture.
  • a method of the invention comprises the step of transforming a mushroom, mycelium or a fungal cell with an expression vector comprising a nucleic acid sequence coding for a gene product of interest operably linked to a heat inducible promoter.
  • a method of the invention further comprises heat treatment of the mushroom, mycelium, fungal cell, fruiting body or spore as further discussed earlier herein.
  • the heat treatment is selected from the group consisting of at least 2 minutes to 4 hour, more preferably 2 minutes to 2 hour, placement in an incubator of 37 - 95 °C, inserting a needle with a temperature of 37 - 95 °C for 0.5 to 120 seconds into culture medium of the mushroom, the mycelium, the fruiting body, the fungal cell or the spore or into the mushroom, the mycelium, the fruiting body or a colony of fungal cells or spores.
  • the gene of interest is a gene which upon expression of the gene product of interest induces mushroom formation.
  • the gene of interest is a gene which upon expression of the gene inhibits mushroom formation.
  • the gene of interest is represented by a nucleotide sequence encoding a polypeptide that comprises an amino acid sequence: (a) that has at least 40 % amino acid identity or similarity with a sequence selected from SEQ ID NO: 8-207, preferably as defined earlier herein; and/or,
  • inces mushroom formation is herein understood to encompass both direct and indirect induction. It can be used interchangeably with the term “stimulation of mushroom formation” and it is used to indicate that the number of mushrooms is increased as compared to a control (e.g. the same species not comprising that gene of interest and/or the same species comprising that gene of interest but not subjected to heat treatment).
  • the invention also relates to a method for producing a gene product of interest or a substance of interest using a fungal cell, a mycelium or a mushroom of the invention.
  • a fungus/mushroom may have been modified in order to be able to produce said gene product of interest or substance of interest.
  • a gene product of interest may be any gene product that could be produced by a fungus/mycelium/mushroom/fruiting body.
  • Such substance includes a nucleic acid sequence, a protein, a polypeptide, a metabolite.
  • a protein or polypeptide in this context may be a pharmaceutical protein or polypeptide and/or a protein or polypeptide for interest for food, feed, or non-food, non-feed applications.
  • Such gene product of interest may be endogenous for a fungus/mushroom or not.
  • the method comprises the steps of: culturing of a fungus/mycelium/mushroorn/fruiting body/spore of the invention that is able to produce a substance of interest under circumstances that are conducive for the generation of the substance of interest; and optional recovery of the substance of interest.
  • the circumstances that are conducive for the generation of the gene product of interest comprise a heat treatment as defined earlier herein.
  • the assessment of the production level of the polypeptide may be performed at the mPvNA level by carrying out a Northern Blot, qPCR or an array analysis and/or at the polypeptide level by carrying out SDS PAGE or a Western blot. All these methods are well known to the skilled person.
  • a wild type or native fungus/mycelium/mushroom/fruiting body/spore does not produce any detectable amounts of the polypeptide and/or does not exhibit any detectable activity of said polypeptide under maintenance conditions of the fungus/mushroom/mycelium/fruiting body/spore.
  • a native or wild type fungus/mycelium/mushroom does not produce or produces substantially no polypeptide under maintenance conditions.
  • a fungus/mycelium/mushroom/fruiting body/spore comprises a nucleic acid construct comprising a nucleotide encoding a protein/polypeptide to be produced.
  • a substance of interest may be such protein/polypeptide.
  • said protein/polypeptide may be involved in the production or synthesis of such substance of interest.
  • Each feature of said nucleic acid construct has been earlier defined herein. It is also encompassed by the present invention to use a fungus/mushroom which has been further modified by increasing/decreasing the expression level of a protein/polypeptide known to be involved in the method of production of said substance.
  • nucleic acid or polypeptide molecule refers to a nucleic acid or polypeptide as natively expressed in a fungus/mushroom/mycelium/fruiting body/spore, preferably in a wild type state.
  • heterologous is used as opposite of “endogenous”.
  • heterologous when used with respect to a nucleic acid or polypeptide molecule refers to a nucleic acid or polypeptide from a foreign fungus/mushroom/mycelium/fruiting body/spore which does not occur naturally as part of a given fungus/mushroom/mycelium/fruiting body/spore (genome or DNA or RNA from said fungus) or which is found in a fungus/mushroom or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature.
  • Heterologous nucleic acids or proteins are not endogenous to the fungus/mushroom/mycelium/fruiting body/spore into which they are introduced, but have been obtained from another fungus/mushroom/mycelium/fruiting body/spore or synthetically or recombinantly produced.
  • nucleic acids encode proteins or polypeptides that are not normally produced by the fungus in which the DNA is transcribed or expressed, similarly exogenous RNA codes for proteins not normally expressed in the fungus/mushroom/mycelium/fruiting body/spore in which the exogenous RNA is present.
  • heterologous protein or polypeptide can be composed of homologous elements arranged in an order and/or orientation not normally found in a fungus/mushroom/mycelium/fruiting body/spore in which it is transferred, i.e. the nucleotide sequence encoding said protein or polypeptide originates from the same species but is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins.
  • heterologous nucleic acid or protein Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to a fungus/mushroom/mycelium/fruiting body/spore in which it is expressed is herein encompassed by the term heterologous nucleic acid or protein.
  • heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other.
  • a fungal cell, mycelium or mushroom according to the invention may further over-express one or more other gene products of interest under a promoter of the invention or under a promoter that is not a promoter of the invention.
  • Said other gene products of interest may either be endogenous or heterologous.
  • a fungal cell, mycelium or mushroom according to the invention may further comprise an inactivated endogenous gene (product). This inactivation or down regulation may have been achieved by deletion of one or more nucleotides in the encoding gene. Alternatively, it may have been caused by an R Ai-like mechanism.
  • sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (nucleotide, polynucleotide) sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.
  • similarity between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. In a preferred embodiment, identity or similarity is calculated over the whole SEQ ID NO as identified herein.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al, Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al, J. Mol. Biol. 215:403-410 (1990).
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al, J. Mol. Biol. 215:403-410 (1990).
  • the well-known Smith Waterman algorithm may also be used to determine identity.
  • Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89: 10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4.
  • a program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, WI. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).
  • amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine.
  • Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place.
  • the amino acid change is conservative.
  • Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; He to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.
  • medium or "culture medium” are used interchangeably herein.
  • (Culture) medium should not be construed narrowly, but encompasses any medium on which a fungus/mushroom/spore/mycelium/fruiting body can develop/grow.
  • the medium preferably is a solid minimal medium (Dons et al. 1979). However, it may also be soil.
  • the verb "to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • FIG. 1 A. Consensus sequence found in all 7 promoters of the predicted heat shock genes. B. Locations of the identified sequences on the promoters.
  • FIG. 1 Fluorescence induced by the promoter of the gene encoding heat shock protein 3 (hsp3). At 25 °C (A, E) and 30 °C (B, F) no fluorescence was visible. At 37 °C (C, G) weak fluorescence and at 42 °C (D, H) clear fluorescence was observed. (A- D) represent light microscopy pictures. (E-H) represent fluorescence microscopy pictures of the same areas as depicted in (A-D). Figure 3. Local induction of dTomato expression from the hsp3 promoter using a hot needle. Light (above) and fluorescence (below) microscopy of a wild-type S. ses colony (A) and a colony expressing dTomato from the hsp3 promoter (B).
  • hsp3 heat shock protein 3
  • FIG. 4 Colonies were grown continuously at 25 °C (A, B, C) or incubated at 42 °C for 1 hour a day (D, E, F). Wild type forms mushrooms (A, D), the Awc2Awc2 strain does not form mushrooms (B, E), the Awc2Awc2 strain complemented with hsp3 P rom-wc2 only forms mushrooms at 42 °C (C, F).
  • CBS 340.81 monokaryotic strain 4-40
  • CBS 341.81 monokaryotic strain 4-40
  • CBS 341.81 monokaryotic strain 4-40
  • CBS 341.81 monokaryotic strain 4-40
  • CBS 341.81 monokaryotic strain 4-40
  • CBS 341.81 A 7-day-old colony grown on solid minimal medium (MM; Dons et al, 1979) at 30°C in the dark was homogenized in 200 ml MM using a Waring blender for 1 min at low speed. 2 ml of the homogenized mycelium was spread out over a polycarbonate membrane that was placed on top of solidified MM. Vegetative monokaryotic mycelium was grown for 4 days in the light.
  • the dikaryon was grown for 2 and 4 days in the light to isolate mycelium with stage I aggregates and stage II primordia, respectively. Mature mushrooms of 3 days old were picked from dikaryotic cultures that had grown for 8 days in the light. R A was isolated as described (van Peer et al, 2009). MPSS was performed essentially as described (Brenner et al, 2000) except that after DpnII digestion Mmel was used to generate 20 bp tags. Tags were sequenced using the Clonal Single Molecule Array technique (Illumina, Hayward, CA, US). Programs were developed in the programming language Python to analyze the data. Tag counts were normalized to transcripts per million (TPM).
  • TPM values of tags originating from the same transcript were summed to assess their expression levels. If the gene of a putative TF did not contain a known 5' or 3 ' UTR, then 200 bp of genomic DNA was added to the respective end of the coding sequence of the gene. MPSS expression analysis agreed with expression studies that have been performed in the past (for a review see Wosten, H.A.B. & Wessels, 2006). Identification of interesting expression profiles
  • a TF is considered to be potentially involved in regulation of fruiting body formation when it is either up or down regulated in one of the developmental stages compared to the sterile monokaryon.
  • Table 1 A Transcription factor genes (first column) that are up regulated at least 2 times compared to monokaryons during some stage o fruiting body formation. Expression of the transcription factor genes was assessed by MPSS and is expressed in tags per millio Homoloqu k lih ( Me t onoa ry on gi s of the transcription factors in L. bicolor and C. cinereus are indicated in the last two columns.
  • Table 1C Transcription factor genes (first column) that are up regulated at least 4 times compared to monokaryons during some stage o fruiting body formation. Expression of the transcription factor genes was assessed by MPSS and is expressed in tags per millio Homoloques of the transcription factors in L. bicolour and C. cinereus are indicated in the last two columns.
  • Transcription factor genes (first column) that are up regulated at least 10 times compared to monokaryons during some stage o fruiting body formation. Expression of the transcription factor genes was assessed by MPSS and is expressed in tags per millio Homoloques of the transcription factors in L. bicolor and C. cinereus are indicated in the last two columns.
  • Transcription factor genes (first column) that are down regulated at least 10 times compared to monokaryons during some stag of fruiting body formation. Expression of the transcription factor genes was assessed by MPSS and is expressed in tags per millio Homoloques of the transcription factors in L. bicolor and C. cinereus are indicated in the last two columns.
  • a knock-out was made of the putative transcription factor gene fst3 (proteinID 257422).
  • vector pDelcas was used as described in Ohm et al. (2010). Primers that were used to create the knock out construct are indicated in Table 2.
  • This knock out construct called pRO097 consists of the flanking regions of the coding sequence of fst3 in between which the nourseothricin resistance cassette is situated. The phleomycin resistance cassette is present elsewhere in the construct (for details see Ohm et al., 2010). Transformation of S. ses strain H4-8 was done as described (van Peer et al., 2009). Regeneration medium contained no antibiotic, whereas selection plates contained 20 ⁇ g ml "1 nourseothricin.
  • a knock-out was made of the putative transcription factor gene fst4 (proteinID 66861).
  • vector pDelcas was used as described in Ohm et al. (2010). Primers that were used to create the knock out construct are indicated in Table 2.
  • the knock out construct called pR0191 consists of the flanking regions of the coding sequence of fst4 in between which the nourseothricin resistance cassette is situated. The phleomycin resistance cassette is present elsewhere in the construct (for details see Ohm et al, 2010). Transformation of S. ses strain H4-8 was done as described (van Peer et al, 2009). Regeneration medium contained no antibiotic, whereas selection plates contained 20 ⁇ g ml "1 nourseothricin.

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Abstract

L'invention se rapporte à une méthode de production d'un champignon utilisant un promoteur inductible par la chaleur. L'invention se rapporte en outre à un promoteur inductible par la chaleur, un acide nucléique hybride, un vecteur et un fongus ou champignon comprenant un promoteur inductible par la chaleur selon l'invention et à des méthodes de production d'un tel champignon ou fongus ou d'une substance intéressante.
PCT/NL2011/050428 2010-06-15 2011-06-14 Promoteur inductible et son utilisation WO2011159153A2 (fr)

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US4797359A (en) * 1983-05-10 1989-01-10 Board Of Regents, The University Of Texas System Heat shock regulated production of selected and fused proteins in yeast
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