WO2017125602A1

WO2017125602A1 - Increased vegetative growth of fungi

Info

Publication number: WO2017125602A1
Application number: PCT/EP2017/051310
Authority: WO
Inventors: Herman Abel Bernard WÖSTEN; Luis Gaston Lugones; Jordi Franciscus PELKMANS; Freek Vincentius Wilhelmus APPELS
Original assignee: Stichting voor de Technische Wetenschappen STW
Current assignee: Stichting voor de Technische Wetenschappen STW
Priority date: 2016-01-21
Filing date: 2017-01-23
Publication date: 2017-07-27
Anticipated expiration: 2018-07-21

Abstract

The present invention relates to the field of microbiology, specifically the field of fungi, especially Basidiomycetes.

Description

Increased vegetative growth of fungi.

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Filamentous fungi grow by means of tip-growing hyphae that branch subapically. As a result, a network of hyphae is formed that is called mycelium. The hyphae feed on organic material such as plant waste. To this end, they penetrate their substrate while secreting enzymes that degrade the plant polymers into molecules that can be taken up to serve as nutrients. Hyphal penetration of the substrate is facilitated by turgor pressure generated by the cytoplasm and by the rigidity of the surrounding cell wall. The composition of fungal cell walls is dynamic and varies between species, strains, environmental conditions (Bowman and Free 2006), and developmental stage (Wessels 1994). In general, fungal cell walls consist of (glyco)proteins and polysaccharides. Glucan and chitin are the most abundant polysaccharides in the cell wall. Chitin provides rigidity to the cell wall. It is composed of β-1-4 linked N-acetylglucosamine and its microfibrils form inter-chain hydrogen bonds (Rudall and Kenchington 1978; Ruiz- Herrera 1992; Kameda et al. 2005). Chitin accounts for 1-2 % of the cell wall dry mass of Saccharomyces cerevisiae (Klis et al. 2002), while it can make up 30% of the cell wall of filamentous ascomycetes (de Nobel et al. 2000). Glucan is predominantly present in the cell wall as linear chains of (l-3)-P-linked glucose that provide elasticity (Klis et al. 2002). Other glucans like (l-6)-P-glucan, (l-3)(l-4)-P-glucan (Inoue et al. 1996; Douglas 2001), (l-3)(l-6)"P-glucan, and (l-3)-a-glucan (Beauvais et al. 2013; Latge and Beauvais 2014) may also be present within the cell wall. The cell wall of the vegetative mycelium of Schizophyllum commune, as an example of a basidiomycete, consists of glucose (67.6%), N-acetylglucosamine (12.5%), mannose (3.4%>), xylose (0.2%>), amino acids (6.4%)), and lipids (3.0%>) (Sietsma and Wessels, 1977). These monomers make up chitin, glucan, and proteins as well as other, not yet identified cell wall constituents. The outer layer of the cell wall is a water-soluble mucilage consisting of (l,3)-P-linked glucose units with branches of single (l,6)-P-linked glucose molecules at every third glucose along the chain (Wessels et al, 1972). This mucilage, known as schizophyllan, is also secreted into the culture medium. An alkali-soluble glucan consisting of (l ,3)-a- linked glucose units, known as S-glucan, is located beneath the mucilage and accounts for about half of the thickness of the water-insoluble portion of the wall. The inner layer of the cell wall consists of an alkali- insoluble glucan, known as R-glucan, and chitin (Wessels, 1994). The R-glucan was found to be a highly branched (l,3)(l,6)-P-glucan. Part of this highly insoluble glucan has structural similarity to schizophyllan. Most of the R-glucan is linked to chitin (Sietsma and Wessels, 1981) via basic amino acids and N- acetylglucosamine (Sietsma and Wessels, 1977).

Mycelium initially grows vegetative but at a certain moment asexual or sexual development is initiated. Thus, vegetative growth occurs when the mycelium is not yet differentiated to allow asexual or sexual development and / or when environmental conditions suppress these developmental processes. Strains in which genes (e.g. regulatory genes) have been inactivated may be unable to develop reproductive structures and may therefore always grow vegetative. It should be noted that during asexual or sexual development vegetative growth may continue.

Schizophyllum commune is an example of a model system to study sexual development in filamentous fungi and in particular that of basidiomycetes. The life cycle of S. commune starts with a monokaryotic (i.e. homokaryotic) mycelium that results from the germination of a basidiospore. Monokaryotic mycelia are sterile and always grow vegetative. A fertile dikaryon (i.e. heterokaryon) is formed upon fusion of two monokaryons with compatible mating types. Blue light is required to initiate fruiting in the dikaryotic mycelium (Perkins and Gordon 1969), whereas high C0₂ levels repress this developmental program (Niederpruem 1963; Raudaskoski and Viitanen 1982). So, in the dark and / or at high CO2 levels the dikaryon grows vegetative, while at ambient CO2 levels and in the light fruiting bodies are formed. Initiation of fruiting body formation by S. commune starts with asymmetrical colony growth. This means that vegetative growth is reduced; in some parts stronger than in other parts of the colony. Asymmetrical colony growth is followed by aggregation of aerial hyphae, and subsequent formation of primordia. These primordia can develop into fruiting bodies that form basidia within the hymenium. Karyogamy, meiosis, and one round of mitosis occur in the basidia, resulting in haploid, binucleate basidiospores.

The fruiting bodies of S. commune are an example of a mushroom. Mushrooms are the most conspicuous fungal structures. Mushrooms 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 typical example of a "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. Mushrooms may also have pores instead of lamellae. The word "mushroom" is also used for a wide variety of 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 "bracket", "puffball", "stinkhorn", and "morel", and gilled mushrooms themselves are often called "agarics" in reference to their similarity to Agaricus or their place Agaricales. Mushrooms are inter alia used as a food source and for their therapeutic compounds (Kiies and Liu 2000).

Regulation of mushroom formation has been studied in S. commune (Ohm et al., 2010b, 2011; 2013). The blue light receptor complex consists of Wc-1 that has a blue light sensing domain and the transcription factor Wc-2. Inactivation of wc-1 and / or wc-2 results in a blind phenotype. Dikaryotic colonies of the homozygous deletion strains grow symmetrically in blue light (similar to dark-grown wild-type dikaryons) and do not produce aggregates, primordia, and fruiting bodies. Deletion of the homeodomain gene hom2 and the DNA binding Bright domain protein gene bril shows a similar phenotype. In contrast, inactivation of the zinc finger transcription factor gene fst4 results in dikaryons that still grow irregular in the light under low C0₂ conditions but aggregates, primordia, and fruiting bodies are not produced. Strains in which the Cys2His2 zinc finger protein gene c2h2 has been inactivated are arrested at the aggregate stage (Ohm et al. 2011). On the other hand, deletion strains of fst3, gatl or homl form smaller fruiting bodies but in higher numbers. The zinc finger protein Fst3 was proposed to play a role in repression of outgrowth of fruiting bodies from primordia. On the other hand, Gatl, a GAT A type zinc finger protein, and Homl, a homeodomain protein, have been proposed to play a role in expansion of the fruiting body. Homologues of the S. commune transcription factors have been identified in Agaricus bisporus, Laccaria bicolor and Coprinopsis cinerea. Expression studies suggest the existence of a core regulatory program for fruiting body development in basidiomycetes (Ohm et al. 2010b; Morin et al. 2012; Plaza et al. 2014). Variations in expression of these genes would explain species-specific morphology and environmental sensing, i.e. phenotypical features during growth under fruiting conditions. In addition to understanding of species-specific morphology and environmental sensing, it would be of great value if modulators of vegetative growth would be known since all art cited here above relates to phenotypical features during growth under fruiting conditions.

SUMMARY OF THE INVENTION

In an aspect, the present invention provides for a fungus derived from a parental fungus, wherein said fungus exhibits:

- a decreased expression level of at least one polynucleotide encoding a polypeptide selected from the group consisting of: Wc-1, Wc-2, Hom2, Fst4 and Teal compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions, and/or has

- an increased expression level of at least one polynucleotide encoding a polypeptide selected from the group consisting of Bril and Homl compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions,

wherein the decreased and/or increased expression level results in increased vegetative growth of said fungus compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions. Preferably, the fungus according to claim 1, wherein the fungus is a filamentous fungus. Preferably, the fungus is an Ascomycete or a Basidiomycete, preferably a Basidiomycete. More preferably, the fungus is a mushroom forming Basidiomycete. Preferably, at least the expression level of a polynucleotide encoding Fst4 is decreased compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions, and wherein the expression level of at least one polynucleotide encoding a polypeptide selected from the group consisting of: Wc-1, Wc-2, Hom2, and Teal is decreased, and/or wherein the expression level of at least one polynucleotide encoding a polypeptide selected from the group consisting of Bril and Homl is increased. Preferably, said fungus comprising a polynucleotide encoding a compound of interest. The invention further provides a method of culturing a fungus according to any one of the preceding claims, comprising culturing said fungus under conditions conducive to the production of said fungus and, optionally, isolating and/or purifying said fungus. The invention further provides a method for the production of a fungus and/or a mushroom, comprising contacting a fungus according to the invention with a substrate and with a fungus able to produce mushrooms, wherein said fungus according to the invention grows more rapidly in the substrate. The fungus able to produce mushrooms may either or not fuse with the fungus according to the invention.

The invention further provides for a method for the production of a compound of interest comprising culturing a fungus according to the invention under conditions conducive to the production of the compound of interest and, optionally, isolating and/or purifying the compound of interest.

The invention further provides for a method for the degradation of organic material comprising contacting a fungus according to the invention with an organic material and applying conditions suitable for the degradation of the organic material to the fungus contacted with the organic material.

The invention further provides for a method for the degradation of inorganic material comprising, contacting a fungus according to the invention with an inorganic material and applying conditions suitable for the degradation of the inorganic material to the fungus contacted with the inorganic material.

The invention further provides for a method for the production of a composite material, comprising contacting a fungus according to the invention with an organic and/or inorganic material and applying conditions suitable for the production of the composite material consisting of fungal mycelium and the organic and/or inorganic material.

The invention further provides for a method for the production of fungal mycelium comprising contacting a fungus according to the invention with an organic material and applying conditions suitable for the production of the fungal mycelium. Preferably, the composite material or the fungal mycelium is a construction material, an isolation material, a packaging material, a textile, a container, a material or device used for medical applications, a material or device used in horticulture, or a decoration material.

The invention further provides for a composite material or a fungal mycelium obtained or obtainable by the method here above. DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, it has now been demonstrated that several fungal genes involved in fruiting body development or in a broader context in sexual or asexual development are also involved in the modulation of vegetative growth of fungi, even under non-fruiting conditions, and thereby in determining the mechanical properties of the mycelium. Previously, several regulatory genes (i.e. wc-1, wc-2, hom,2, fst4) have been described that are involved in mushroom development as well as asymmetrical growth during initiation of fruiting, (Ohm, 2010c; Ohm et al, 2013). However, it was unexpected that vegetative growth was also suppressed by these regulators under non-fruiting conditions. Accordingly, in a first aspect the present invention provides a fungus derived from a parental fungus, wherein said fungus exhibits:

wherein the decreased and/or increased expression level results in increased vegetative growth of said fungus compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions. Said fungus is herein referred to as a fungus according to the invention. Said polynucleotide is herein referred to as a polynucleotide according to the invention and said polypeptide is herein referred to as a polypeptide according to the invention.

Vegetative growth or vegetative propagation of a fungus is herein defined as any growth or propagation that does not involve the sexual or asexual stages of development. In filamentous fungi, hyphae are the main mode of vegetative growth, and are collectively called a mycelium.

Increased vegetative growth is herein defined as that more bio mass is produced by the fungus according to the invention in a specified time unit, such as but not limited to an hour, a day, two days, three days, four days, five days or a week, compared to the parental fungus said fungus according to the invention is derived from when both are cultured and assayed under identical conditions. The person skilled in the art knows how to measure the production of biomass. The produced biomass can e.g. be expressed in dry weight. The term "expression" is to be construed as to include any step involved in the production of the peptide or polypeptide including, but not limited to, transcription, post- transcriptional modification, translation, post-translational modification and, optionally, secretion.

A decreased expression level of a polynucleotide encoding a polypeptide is herein defined as a decreased activity of the polypeptide in the fungus according to the invention compared to the activity of the polypeptide in the parental fungus said fungus according to the invention is derived from. It follows that an increased expression level of a polynucleotide encoding a polypeptide is herein defined as an increased activity of the polypeptide in the fungus according to the invention compared to the activity of the polypeptide in the parental fungus said fungus according to the invention is derived from. Higher or lower activity of the polypeptide may be due to a respective higher or lower specific activity of the polypeptide (i.e. a higher or lower activity for the same amount of polypeptide) or may be higher or lower activity due to a respective higher or lower amount of the polypeptide (i.e. the specific activity of the polypeptide is the same where the amount is different). A combination of both higher amount and higher specific activity or lower amount and lower specific activity is also within the scope of the invention. The person skilled in the art knows how to determine polypeptide activity. An increased expression level or a decreased expression level may be achieved by any means known to the person skilled in the art. Increased expression may be achieved by recombinant techniques such as but not limited to overexpression by using a stronger promoter and/or introducing multiple copies of the gene to be overexpressed. Overexpression may also be achieved by other methods such as for example by increasing mR A stability or by introducing introns. Increased expression may also be achieved by non-recombinant means, e.g. by classical mutagenesis of a parental fungus, preferably followed by screening for a mutant with increased expression level of the polypeptide. Likewise, decreased expression may be achieved by recombinant techniques such as but not limited to underexpression by using a weaker promoter or by removing one or multiple copies of the gene from the parental fungal cell. Decreased expression may also be achieved by other methods such as for example by decreasing mRNA stability, introduction of an inactivation construct and RNAi. Decreased expression may also be achieved by non-recombinant means, e.g. by classical mutagenesis of a parental fungus, preferably followed by screening for a mutant with decreased expression level of the polypeptide. Alternatively or in combination with any of the herein mentioned ways of obtaining a fungus according to the invention, one may cross natural or non-natural isolates of fungi with different degrees of expression of a polynucleotide according to the invention.

Preferably, increased expression level of a polynucleotide encoding a polypeptide in a fungus according to the invention means at least 10%, at least 20%>, 30%>, 40%>, 50%>, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or at least 1000% higher expression level as compared to the expression level of the corresponding polypeptide in the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions. Likewise, decreased expression level of a polynucleotide encoding a polypeptide in a fungus according to the invention means at least 10%, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or at least 100% lower expression level as compared to the expression level of the corresponding polypeptide in the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions. A "nucleic acid molecule" or "polynucleotide" (the terms are used interchangeably herein) is represented by a nucleotide sequence.

A "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. A "polypeptide" is represented by an amino acid sequence.

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-trans lational 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 translational stop signals. As used herein, the term "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. Within the context of the invention, a promoter preferably ends at nucleotide -1 of the transcription start site (TSS).

A promoter is preferably capable of driving expression of the nucleotide sequence in a fungus and/or in a mushroom. Preferred promoters include: promoters that are constitutively expressed such as that of glyceraldehyde-3-phosphate-dehydrogenase (gpd) of Schizophyllum commune (Harmsen et al, 1992) and inducible promoters. The polypeptide according to the invention, encoded by the polynucleotide according to the invention, is a polypeptide selected from the group consisting of Wc-1, Wc-2, Hom2, Fst4, Teal, Bril and Homl . The polynucleotide encoding the polypeptide preferably is a polynucleotide native (also referred to as wild-type) to the fungus according to the invention. Alternatively, the polynucleotide is heterologous to the fungus according to the invention. The polynucleotide may e.g. be a mutant, variant and/or may be codon optimized. The term "heterologous" in the context of a polynucleotide or a polypeptide as used herein refers to a polynucleotide or polypeptide that does not naturally occur in a host cell. With respect to a heterologous polynucleotide, the sequence has a portion which is not native to the cell in which it is expressed. With respect to a heterologous polypeptide, the sequence has a portion which is not native to the cell in which it is expressed.

The properties and activities of the polypeptides according to the invention are extensively described in the examples herein.

A polypeptide according to the invention may be Wc-1. Wc-1 has been studied in Schizophyllum commune and is part of the blue light receptor complex. A preferred Wc- 1 is a polypeptide that has at least 40% sequence identity with SEQ ID NO: 1 and/or comprises a domain that has at least 50% sequence identity with SEQ ID NO: 24, and has Wc-1 activity. A polypeptide according to the invention may be Wc-2. Wc-2 has been studied in Schizophyllum commune and is a transcription factor part of the blue light receptor complex. A preferred Wc-2 is a polypeptide that has at least 40% sequence identity with SEQ ID NO: 2 and/or comprises a domain that has at least 50% sequence identity with SEQ ID NO : 25 , and has Wc-2 activity.

A polypeptide according to the invention may be Hom2. Hom2 has been studied in Schizophyllum commune and is a transcription factor homeodomain protein and functions in early stages of mushroom development. A preferred Hom2 is a polypeptide that has at least 40% sequence identity with SEQ ID NO: 3 and/or comprises a domain that has at least 50% sequence identity with SEQ ID NO: 26, and has Hom2 activity.

A polypeptide according to the invention may be Fst4. Fst4 has been studied in Schizophyllum commune and is a zinc-finger transcription factor and functions in early stages of mushroom development . A preferred Fst4 is a polypeptide that has at least 40% sequence identity with SEQ ID NO: 4 and/or comprises a domain that has at least 50% sequence identity with SEQ ID NO: 27, and has Fst4 activity.

A polypeptide according to the invention may be Teal . Teal has been studied in Schizophyllum commune and is a TEA/ATTS domain transcription factor and functions downstream of the blue light receptor complex and of Hom2 and Fst4. A preferred Teal is a polypeptide that has at least 40%> sequence identity with SEQ ID NO: 5 and/or comprises a domain that has at least 50% sequence identity with SEQ ID NO: 28, and has Teal activity.

A polypeptide according to the invention may be Bril . Bril has been studied in Schizophyllum commune and is a transcription factor, a DNA binding Bright domain protein. A preferred Bril is a polypeptide that has at least 40% sequence identity with SEQ ID NO: 6 and/or comprises a domain that has at least 50% sequence identity with SEQ ID NO: 29, and has Bril activity.

A polypeptide according to the invention may be Homl . Homl has been studied in Schizophyllum commune and is a transcription factor homeodomain protein. A preferred Homl is a polypeptide that has at least 40% sequence identity with SEQ ID NO: 7 and/or comprises a domain that has at least 50%> sequence identity with SEQ ID NO: 30, and has Homl activity.

A polypeptide according to the invention preferably has at least 40% sequence identity with the respective amino acid sequence as described here above or comprises a domain that has at least 50% sequence identity with the respective amino acid sequence as described here above. More preferably, a polypeptide according to the invention has a sequence identity of at least 42%, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or most preferably 100% sequence identity with the respective amino acid sequence as described here above and or comprises a domain that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 95%, 96%o, 97%), 98%o, 99%) or most preferably 100% sequence identity with the respective amino acid sequence as described here above. Preferably, sequence identity is determined by comparing the whole length of the sequences as identified herein.

A polypeptide according to the invention preferably is a polypeptide native (also referred to as wild-type) to the fungus according to the invention. Alternatively, the polypeptide is heterologous to the fungus according to the invention and may e.g. be mutant or variant.

"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. In the art, "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 "Identity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988).

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 et al, 1984). BestFit, BLASTP, BLASTN, and FASTA (Altschul, et al, 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). 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 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff (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). Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons.

Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and iso leucine; 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-iso leucine, 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. Preferably, 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. A fungus according to the invention may be any fungus known to the person skilled in the art. Preferably, the fungus according to the invention is a filamentous fungus. A preferred filamentous fungus is a strain selected from the group of filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al, In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). Such filamentous fungal strains include, but are not limited to, strains of Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mortierella, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete, Pleurotus, Schizophyllum, Talaromyces, Rasamsonia, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma. A further preferred filamentous fungal host cell according to the present invention is from a genus selected from the group consisting of Acremonium, Aspergillus, Chrysosporium, Myceliophthora, Penicillium, Talaromyces, Rasamsonia, Thielavia, Fusarium and Trichoderma; more preferably from a species selected from the group consisting of Aspergillus niger, Acremonium alabamense, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus, Talaromyces emersonii, Rasamsonia emersonii, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium oxysporum, Mortierella alpina, Myceliophthora thermophila, Trichoderma reesei, Thielavia terrestris, and Penicillium chrysogenum.

A further preferred fungus is a fungus which produces a mushroom, preferably a mushroom that is attractive to be produced or which is suspected to be attractive to produce a substance or material of interest. Preferably, the fungus according to the invention is a Basidiomycete or an Ascomycete, preferably a Basidiomycete, more preferably a mushroom forming Basidiomycete, preferably a Polyporales or an Agaricales or a Schizophyllaceae, more preferably Schizophyllum. Further preferred is a Schizophyllum commune, more preferably Schizophyllum commune strain 4-8 (FGSC#9210). Preferred Agaricales are for example members of the genus Agaricus (e.g. Agaricus bisporus), Pleurotus (e.g. Pleurotus ostreatus), Lentinus (e.g. Lentinus edodus) and Armillaria (e.g. Armillaria bulbosa). Preferred Polyporales are species belonging to the genus Trametes (e.g. Trametes versicolor), and Phanerochaete (e.g. Phanerochaete chrysosporium) Preferably, in a fungus according to the invention, a modification in view of the parental fungus is derived from can be a homozygous or a heterozygous modification; preferably such modification is a homozygous modification. A fungus according to the invention may be monokaryotic, dikaryotic, homokaryotic, heterokaryotic, or haploid, diploid, or aneuploid.

Preferably, in the fungus according to the invention, at least the expression level of a polynucleotide encoding Fst4 is decreased compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions, and the expression level of at least one polynucleotide encoding a polypeptide selected from the group consisting of: Wc-1, Wc-2, Hom2, and Teal is decreased, and/or the expression level of at least one polynucleotide encoding a polypeptide selected from the group consisting of Bril and Homl is increased. A preferred fungus according to the invention is a fungus wherein at least the expression level of a polynucleotide encoding Fst4 and of a polynucleotide encoding Hom2 is decreased compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions. Preferred fungi according to the invention are the ones provided in the examples herein. A fungus according to the invention can conveniently be used for the production of a compound of interest. Accordingly, a preferred fungus according to the invention comprises a polynucleotide encoding a compound of interest. The fungus according to the invention may have been modified in order to be able to produce said compound of interest. The compound of interest may be any compound that can be produced by a fungus according to the invention. Such substance includes, but is not limited to, a protein, a polypeptide and a primary or secondary metabolite. A protein or polypeptide in this context may a pharmaceutical protein or polypeptide and/or a protein or polypeptide for interest for food, feed, or non-food, non-feed applications. Such compound may be native (endogenous) for the fungus according to the invention or may be foreign (heterologous) to the fungus according to the invention.

In an embodiment, a fungus according to the invention comprises a nucleic acid construct comprising a polynucleotide encoding the compound of interest to be produced. In this case, the compound of interest may be a polypeptide. Alternatively, a polypeptide encoded by the polynucleotide may be involved in the production or synthesis of a compound of interest. Also encompassed by the invention is a fungus which has been further modified by increasing/decreasing the expression level of a polypeptide known to be involved in the production of the compound of interest. In an aspect, the invention provides for a method for the production of a compound of interest comprising culturing a fungus according to the invention under conditions conducive to the production of the compound of interest and, optionally, isolating and/or purifying the compound of interest.

In a further aspect, the invention provides for a method of culturing a fungus according to the invention, comprising culturing said fungus under conditions conducive to the production of said fungus and, optionally, isolating and/or purifying said fungus. Methods to culture fungi such as a fungus according to the invention are known to the person skilled in the art. Culturing may be performed in any scale from lab scale (in a petri dish or flask with a volume of tens of microliters to several litres) to industrial scale surface culture and industrial scale submerged culture (several litres to a several hundred cubic metres).

A fungus according to the invention can conveniently be combined with another fungus according to the invention or with a wild-type fungus; such wild-type fungus may e.g. be a fungus that is able to produce mushrooms. The fungi according to the invention may conveniently be used in the methods described in WO2011130247, which is herein incorporated by reference. Accordingly, in a further aspect, the invention provides for a method for the production of a fungus and/or a mushroom, comprising contacting a fungus according to the invention with a substrate and with a fungus able to produce mushrooms, wherein said fungus according to the invention grows more rapidly in the substrate (i.e. having increased vegetative growth as defined previously herein). The fungi used may either or not fuse with each other.

In an embodiment of the method according to this aspect of the invention, there is provided a method for the production of mushrooms, comprising:

- seeding a lower nutrient substrate layer with a first filamentous fungal inoculant which is a fungus according to the invention, so that said substrate becomes colonized with the inoculant according to the invention,

- overlaying said nutrient substrate with an upper water-holding substrate layer that has been inoculated with a second filamentous fungal inoculant, and allowing fruiting bodies, preferably mushrooms, to form, wherein the increased vegetative growth of the first filamentous fungus inoculant results in increased growth of the fruiting bodies and wherein the second filamentous fungal inoculant preferably is a wild-type fungus able to produce fruiting bodies, preferably mushrooms.

In an embodiment of the method according to this aspect of the invention, there is provided a method of modulating the activity of a protein in a fruiting body, preferably a mushroom, of a filamentous fungus comprising:

- seeding a lower nutrient substrate layer with a first filamentous fungal inoculant which is a fungus according to the invention, so that said substrate becomes colonized with the inoculant, wherein said first filamentous fungal inoculant comprises and expresses a heterologous polynucleotide construct,

- overlaying said nutrient substrate with an upper water-holding substrate layer that has been inoculated with a second filamentous fungal inoculant, and allowing fruiting bodies, preferably mushrooms, to form,

wherein the polynucleotide construct from the first filamentous fungal inoculant construct provides modulation of the activity of a protein in the formed fruiting bodies, wherein the protein is a heterologous or an endogenous protein, and wherein the second filamentous fungal inoculant preferably is a wild-type fungus able to produce fruiting bodies, preferably mushrooms.

In an embodiment of the method according to this aspect of the invention, there is provided a method of modulating the activity of a protein in a filamentous fungus comprising:

- overlaying said nutrient substrate with an upper water-holding substrate layer that has been inoculated with a second filamentous fungal inoculant, and allowing the second filamentous fungal inoculant to grow and colonize the substrate,

wherein the polynucleotide construct from the first filamentous fungal inoculant construct provides modulation of the activity of a protein in the mycelium of the second filamentous fungal inoculant, wherein the protein is a heterologous or an endogenous protein, and wherein the second filamentous fungal inoculant preferably is a wild-type fungus able to produce fruiting bodies, preferably mushrooms. Herein a fungal inculant may be any part of the fungus that can used as an inoculant, such as but not limited to mycelium or a spore In a further aspect, the invention provides for a method for the degradation of organic material comprising contacting a fungus according to the invention with an organic material and applying conditions suitable for the degradation of the organic material to the fungus contacted with the organic material. The organic material may be any material that can be used as a nutrient source by the fungus, such as but not limited to material originating from vegetative or reproductive parts of plants, animals, fungi, bacteria or protists such as algae; materials of plants such as saw dust e.g. from oak, beech, birch; cotton; bamboo; straw such as that of rice or wheat; coffee; tea; grape; molasses and silage. The organic material may be used as such or may be pre-treated such as by enzymes, chemicals and/or heat to improve the availability of the nutrients to the fungus according to the invention. The person skilled in the art is aware of pretreatment methods and of conditions suitable for the degradation of the organic material.

In a further aspect, the invention provides for a method for the degradation of an inorganic material comprising contacting a fungus according to the invention with an inorganic material and applying conditions suitable for the degradation of the inorganic material to the fungus contacted with the inorganic material. The inorganic material may be any inorganic material that can be used as a nutrient source by the fungus or that can be at least partly degraded or can be modified by the fungus according to the invention, such as but not limited to a plastic (such a polycarbonate, a polyethylene and a polypropylene), an aromatic hydrocarbon, a polyaromatic hydrocarbon, a dioxin and a furan. The inorganic material may be used as such or may be pre-treated such as by enzymes, chemicals and/or heat to improve the availability of the nutrients to the fungus according to the invention. The person skilled in the art is aware of pretreatment methods and of conditions suitable for the degradation of the inorganic material.

Within the scope of the invention is a combination of the two aspects here above; i.e. a method for the degradation of an inorganic material as well as an organic material.

In a further aspect, the invention provides for a method for the production of a composite material, comprising contacting a fungus according to the invention with an organic and/or inorganic material and applying conditions suitable for the production of the composite material consisting of fungal mycelium and the organic and/or inorganic material. In the method for the production of a composite material, a mold may be used to shape the composite material and/or to control the conditions for the production of the composite material. Preferably, the composite material or the fungal mycelium is a construction material, an isolation material, a packaging material, a textile, a container, a material or device used for medical applications, a material or device used in horticulture, or a decoration material. The invention further provides for a composite material obtained or obtainable by the method according to this aspect. The invention further provides for a composite material comprising a fungus or a fungal mycelium according to the invention.

In a further aspect, the invention provides for a method for the production of fungal mycelium comprising contacting a fungus according to the invention with an organic material and applying conditions suitable for the production of the fungal mycelium. In the method for the production of a fungal mycelium, a mold may be used to shape the fungal mycelium and/or to control the conditions for the production of the fungal mycelium. Preferably, the fungal mycelium is used as a construction material, an isolation material, a packaging material, a textile, a container, a material or device used for medical applications, a material or device used in horticulture, or a decoration material; either or not with chemical (e.g. acid treatment), biological (e.g. enzyme treatment) or physical (e.g. heat treatment or using high pressure) pre- or post-treatments. The invention further provides for a fungal mycelium obtained or obtainable by the method according to this aspect. In this document and in its claims, 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. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The word "about" or "approximately" when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1% of the value. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

Table 1. Sequences

SEQ Gene / Sequence

ID Polypeptide

NO

1 Wc-1 See sequence listing

2 Wc-2 See sequence listing

3 Hom2 See sequence listing

4 Fst4 See sequence listing

5 Teal See sequence listing

6 Bril See sequence listing

7 Homl See sequence listing

8 PCR primer GGCCTAATAGGCCTAGAATGCGCTCTCCGTC

A2519514ufw

9 PCR primer GGCCTCGCAGGCCAGGGAGGATGACGCAAAG

A2519514urv

10 PCR primer GGCCTGCGAGGCCGTCCGTGTTCTTGGATAC

A2519514dfw

11 PCR primer GGCCTATTAGGCCGTGCGTTGTTTCGTTTCC

A2519514drv

12 PCR primer TCCACGCTGGCTGAATAG

2519514 ufcfw

13 PCR primer TCGATGTGAGGTACTGTC

2519514 dfcrv

14 PCR primer TAAGCCGTGTCGTCA

Nourdelrev

15 PCR primer CCGGGAATTCCAGAT

sc3tersqf

16 PCR primer CCGACTTCGATATCACTC

2519514 ifw

17 PCR primer TCGGCGATGCAAGAAGTC

2519514 irv

18 PCR primer CATGGCTAGCAGGTGATGCAGCGCGACGATAG

teal comp fw

19 PCR primer GGATCCTTAGATCATGAAAGCGCCGCC

teal comp rv

20 PCR primer GGCCTAATAGGCCCTGTCACGCACCAGTACG A2703923ufw

21 PCR primer GGCCTCGCAGGCCGGGCGAACGTGAGATAAG

A2703923urv

22 PCR primer GGCCTGCGAGGCCGCTGTGGACGGTCTTAAC

A2703923dfw

23 PCR primer GGCCTATTAGGCCCATTGCACGAGTCCATTC

A2703923drv

24 Wc-1 sensory LLEKTPDFVHWSLKGSFLYVAPTVKRVLGYDPSELVGKGLSDICH domain PADWPLMRELKEASSTGTESSGVTSASAGASKASAQQDPSMPRTV

DLLFRAQMKTGRYVWMECRGRLHVEPGKGRKAIMLSGR

25 Wc-2 DNA FTKRKRWADLLVTELADAI ILVLGVPNPKILYCGAAVEELLGWRDT binding domain DVIDLDLTELM

26 Hom2 DNA DYRTFFPYQPNEVKHRRRTTAVQLKVLEGIFKTETKPNAALRNKLA binding domain VQLEMTARGVQVWFQNRRAKEKLKASK

27 Fst4 DNA LSPRRLALLLMVLSIGSLVDLKRPLGYLSAEAYHHLARASVCEIPL binding domain MEEPDFDTVHALFFMIWYHLIFSDNRKALGYAWNLLGFVAKLVQGV

HRETSGSSKLIPEESERRRNIFWELLNLDYRMSLTLGRPPSIS

28 Teal DNA SVTGRKAWKTMKGKGEAVWPPHLEKALLKGLAAYEPDTKSTKALNR binding domain FPRRNRFISEYIYQRTGEWRTAKQVSSRLQQLK

29 Bril DNA RRKIEYVPFAREVDTFGGRDLAALEKYAEEARRRPIRDFNDWGNID binding domain VDHLIMSLRSRVATELSYALTTLSMLSAMR

30 Homl DNA KKKRKRADANQLRVLNDVYMRTAFPSTEERHQLAKQLDMSPRSVQI binding domain WFQNKRQAMRSTNRQ

FIGURE LEGENDS

Figure 1. Biomass of 6-day-old dark-grown agar cultures of the wild-type dikaryon (H4-8) and dikaryotic transcription factor deletion strains using glucose as carbon source.

Figure 2. Biomass of 6-day-old dark-grown cultures of the wild-type dikaryon (H4- 8) and dikaryotic transcription factor deletion strains that had been grown on agar medium (A) or as liquid shaken cultures (B) using glucose as a carbon source. Figure 3. Biomass of 6-day-old dark-grown agar cultures of the wild-type dikaryon (H4-8) and dikaryotic transcription factor deletion strains using 4% xylose (A), 3.4% sucrose (B), and 1% pectin (C) as carbon source. Figure 4. Fruiting body development of the wild-type dikaryon (A,D), AbrilAbril (B, E), and AhomlAhoml (C, F) after 7 (A-C) and 15 (D-F) days of growth.

Figure 5. Principal component analysis of expression profiles of wild-type (H4-8) and deletion strain dikaryons during aggregation (A) and fruiting body formation (B).

Figure 6. Venn diagrams showing overlapping differentially expressed transcription factor genes in Afst3Afst3, AhomlAhoml, and AgatlAgatl (A) and Awc-2Awc-2, Ahom2Ahom2, and Afst4Afst4 (B) when compared to the wild-type aggregate and fruiting body stage, respectively.

Figure 7. Expression of wc-1 (a), wc-2 (b), hom2 (c),fst4 (d), c2h2 (o),fst3 (f), gatl (g), homl (h), bril (i), teal (j), and c2h2d (k) in dikaryotic transcription factor deletion strains when compared to the wild-type during aggregation (A) and fruiting body formation (B).

Figure 8. Aerial growth of dikaryotic wild-type colonies (H4-8) (A) and Ateal (B, C, D). Strain Ateal produced less dense and high aerial hyphae when transferred to light (B) and only forms small clusters of fruiting bodies (C and D). Arrow indicates the transition from dense aerial hyphae production to thinner aerial hyphae production upon light induction.

Figure 9. Regulatory model of activation and repression of vegetative growth and initiation and maturation of fruiting body formation in S. commune. Transcription factor genes control both vegetative growth and fruiting body development (A) and influence each other's expression levels (B).

Figure 10. S. commune wild-type, Ahom2, and Afst4 were grown at 30 °C as a thin layer in a Petri-dish in the light or in the dark at ambient or high C0₂ for 3 days. Medium was introduced underneath the colony and growth of the floating mycelium was prolonged for 5 days. Initial Young's modulus in MPa (A), Maximum tensile strength at breaking in MPa (B), and elongation at breaking in % (C) of mycelia were determined and are depicted. Asterisks indicate * p < 0.05, ** p < 0.005 and *** p < 0.0005.

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

Unless stated otherwise, the practice of the invention will employ standard

conventional methods of molecular biology, virology, microbiology or biochemistry. Such techniques are described in Sambrook et al. (1989) Molecular Cloning, A

Laboratory Manual (2^nd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA; and in Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK); Oligonucleotide Synthesis (N. Gait editor); Nucleic Acid Hybridization (Hames and Higgins, eds.).

Example 1. Transcription factors of Schizophyllum commune that link vegetative growth and mushroom formation

Abstract

Transcription factors have been identified that are involved in mushroom formation in Schizophyllum commune. The DNA binding Bright domain protein Bril and the homeodomain protein Homl are involved in late stages of mushroom development, while the blue light receptor transcription factor Wc-2, the homeodomain protein Hom2, and the zinc-finger transcription factor Fst4 function in early stages of mushroom development. Here it is shown that Bril and Homl also stimulate vegetative growth, while biomass formation is repressed by Wc-2, Hom2, and Fst4. The AbrilAbril and the Ahoml homl strains formed up to 0.6 fold less biomass when compared to the wild- type. In contrast, Awc-2Awc-2, Ahom2Ahom2, and Afst4Afst4 strains formed up to 2.8 fold more bio mass. R A sequencing showed that repression of vegetative growth correlates with decreased expression of genes involved in carbohydrate metabolism. The TEA/ATTS domain transcription factor gene teal was also downregulated in the Awc- 2Awc-2, Ahom2Ahom2 and Afst4Afst4 strains. The AtealAteal strain produced 1.3 fold more biomass than the wild-type and was severely affected in fruiting body development. Together, these data show that transcription factors Wc-2, Hom2, Fst4, and Teal link mushroom initiation and repression of vegetative growth. Introduction

Mushrooms are the most conspicuous fungal structures. They are used as a food source or for their therapeutic compounds (Kiies and Liu 2000). The formation of mushrooms involves a complex developmental program. Coprinopsis cinerea and Schizophyllum commune are the model systems to study this program (Kiies and Navarro-Gonzalez 2015). The life cycle of S. commune starts with a monokaryotic mycelium that results from the germination of a basidiospore. A fertile dikaryon is formed upon fusion of two monokaryons with compatible mating types. Blue light is required to initiate fruiting in the dikaryotic mycelium (Perkins and Gordon 1969), whereas high C0₂ levels repress this developmental program (Niederpruem 1963; Raudaskoski and Viitanen 1982). Initiation of mushroom formation starts with asymmetrical colony growth, followed by aggregation of aerial hyphae, and subsequent formation of primordia. These primordia can develop into fruiting bodies that form basidia within the hymenium. Karyogamy, meiosis, and one round of mitosis occur in the basidia, resulting in haploid, binucleate basidiospores.

Regulation of mushroom formation has been studied in S. commune. The blue light receptor complex consists of Wc-1 that has a blue light sensing domain and the transcription factor Wc-2. Inactivation of wc-1 and / or wc-2 results in a blind phenotype (Ohm et al. 2013). Dikaryotic colonies of the homozygous deletion strains grow symmetrically in blue light (similar to dark-grown wild-type dikaryons) and do not produce aggregates, primordia, and fruiting bodies. Deletion of the homeodomain gene hom2 and the DNA binding Bright domain protein gene bril shows a similar phenotype (Ohm et al. 2011). In contrast, inactivation of the zinc finger transcription factor gene fst4 results in dikaryons that still grow irregular in the light under low CO2 conditions but aggregates, primordia, and fruiting bodies are not produced (Ohm et al. 2011). Strains in which the Cys2His2 zinc finger protein gene c2h2 has been inactivated are arrested at the aggregate stage (Ohm et al. 2011). On the other hand, deletion strains of fst3, gatl or homl form smaller fruiting bodies but in higher numbers (Ohm et al. 2011). The zinc finger protein Fst3 was proposed to play a role in repression of outgrowth of fruiting bodies from primordia. On the other hand, Gatl, a GATA type zinc finger protein, and Homl, a homeodomain protein, have been proposed to play a role in expansion of the fruiting body. Homologues of the S. commune transcription factors have been identified in Agaricus bisporus, Laccaria bicolor and C. cinerea. Expression studies suggest the existence of a core regulatory program for fruiting body development in basidiomycetes (Ohm et al. 2010b; Morin et al. 2012; Plaza et al. 2014). Variations in expression of these genes would explain species-specific morphology and environmental sensing.

In this study, it is shown that Wc-1, Wc-2, Hom2, and Fst4 not only initiate mushroom formation but also repress vegetative growth. On the other hand, Bril and Homl were shown to stimulate biomass formation. Whole genome expression analysis indicates that repression of vegetative growth is the result of down-regulation of genes involved in carbohydrate metabolism. Expression analysis also revealed that the TEA/ATTS domain transcription factor gene teal is down regulated in Awc-lAwc-1, Awc-2Awc-2, Ahom2Ahom2, and Afst4Afst4. Inactivation of teal resulted in increased vegetative growth, and severely reduced formation of fruiting bodies. These data indicate that Teal functions downstream of the blue light receptor complex, Hom2 and Fst4.

Experimental procedures

Culture conditions and strains

The compatible S. commune strains H4-8 (matA43matB4l; available inter alia at the fungal [http://www.fgsc.net/], accession number FGSC 9210) (Ohm et al. 2010b) and H4-8b (matA4lmatB43) (Ohm et al. 2010a), their derived dikaryotic deletion strains Awe- 1 Awe- 1, Awc-2Awc-2, Ahom2Ahom2, Afst4Afst4, Ac2h2Ac2h2, Afst3Afst3, AhomlAhoml, AbrilAbril, AgatlAgatl (Ohm et al. 2010b; 2011, 2013), as well as Ahom2Ahom2Afst4Afst4 and Ahom2Ahom2Afst3Afst3 were used in this study. The Aku80 H4-8 strain (de Jong et al. 2010) was used for gene inactivation. Strains were grown in the dark or in the light (1200 lux white LED light; Conrad Electronic, Hirschau, Germany) at 25 °C on minimal medium (MM) containing 1% glucose and 1.5% agar, if applicable (van Peer et al. 2009). Liquid shaken cultures were inoculated with a mycelial homogenate (van Wetter et al. 2000) and grown at 250 rpm in 250 ml Erlenmeyers containing 100 ml MM. Agar cultures were inoculated with a point inoculum taken from the periphery of a 7-day-old colony. To assess growth on other carbon sources, glucose was replaced for 4% xylose, 3.4% sucrose, or 1% pectin.

Gene inactivation

Deletion vectors for teal (Protein ID 2519514; http://genome.jgi-psf.org/Schco3) and c2h2d (Protein ID 2703923) were constructed using pDelcas that contains a nourseothricine and a phleomycin resistance cassette (Ohm et al. 2010a). Upstream and downstream flanks of teal and c2h2d were cloned at either site of the nourseothricine resistance cassette. To this end, the flanks were amplified by PCR using Taq polymerase and H4-8 chromosomal DNA as template. The 906 bp upstream flank and the 946 bp downstream flank of teal were amplified using the primer combination A9519514ufw/A9519514urv and A9519514dfw/A9519514drv, respectively (Table 1). Primer pair combinations A2703923ufw/A2703923urv and A2703923dfw/A2703923drv were used to amplify the 897 bp upstream and 975 bp downstream flank of c2h2d, respectively (Table 1). The PCR products were cloned into pGEM-T Easy (Promega, Madison, USA). The upstream flanks were retrieved from the resulting constructs using Van91I and introduced into the Van91I site of pDelcas, resulting in pDel-2519514-UF and pDel-2703923-UF. The downstream flanks were retrieved from the pGEM-T easy derived constructs using Sfil and introduced into the Sfil site ofpDel_2519514-UF and pDel_2703923-UF. This resulted in the knock-out constructs pDelcas-2519514 and pDelcas-2703923.

Transformation

Deletion constructs were introduced in H4-8Aku80. Transformation was done as described (van Peer et al. 2009). 1 · 10⁷ protoplasts were incubated with 20 μg vector DNA and regenerated overnight without antibiotic. Selection took place for 4 days at 30 °C on MM plates containing 8 μg ml^"1 nourseothricin. Transformants were transferred to a second selection plate containing 20 μg ml^"1 phleomycin to distinguish between homologous and ectopic integrations. Gene deletion was confirmed by PCR using primers outside the flanks and inside the nourseothricin cassette. Primer pairs A2519514ufcfw/nourdelrev and A2519514dfcrv/sc3tersqf were used to screen for teal deletion (Table 1), while primer pairs c2h2D UFCFW/nourdelrev and c2h2D DFCRV/sc3tersqf were used to confirm c2h2d deletion (Table 1). Biomass of colonies

Colonies were grown as liquid shaken cultures or on agar medium on a PC-membrane (diameter 76 mm, pore size 0.1 μιη; Osmonics, GE Water Technologies). Mycelium of liquid cultures was separated from the medium using Miracloth filter (Merck Millipore, Billerica, USA). Mycelium was freeze-dried and weighed. Statistical analyses was done with an independent sample t-test (p-value < 0.05) using IBM SPSS 20.

Whole genome expression analysis

The wild type dikaryon and strains Awe- 1 Awe- 1, Awc-2Awc-2, Ahom2Ahom2, Afst4Afst4, Ac2h2Ac2h2, Afst3Afst3, AhomlAhoml, AbrilAbril, and AgatlAgatl were grown for 5 days in the dark at 22°C, after which they were transferred to the light. Colonies (biological duplicates) were harvested at the moment the wild-type dikaryon formed aggregates (day 8) or fruiting bodies (day 12). H4-8 colonies were also harvested at the moment they were transferred to the light (day 5) and when they had formed primordia (day 10). Mycelium was frozen in liquid nitrogen and homogenized using the TissueLyser II (Qiagen, Dusseldorf, Germany). RNA was extracted using TriZol (Life technologies, Carlsbad, USA) and purified using the NucleoSpin RNA kit (Macherey- Nagel, Duren, Germany). Quality of RNA was checked using the Bio Analyzer and sent to ServiceXS (Leiden, the Netherlands) for Illumina Next Generation Sequencing. RNA-Seq Analysis Pipeline

The RNA-Seq pipeline used the STAR aligner (Dobin et al. 2013) to align the 100 bp paired end reads to the S. commune v3.0 genome (http://genome.jgi- psf.org/Schco3/Schco3.home.html). The size of introns was limited to a maximum of 1500 bp based on the largest intron sizes in the genome annotation.

Abundance estimation and differential expression were performed by Cufflinks version 2.1.1 (Trapnell et al. 2012a), and Cuffdiff (Trapnell et al. 2012b). Enrichments of GO terms were analysed within sets of differentially expressed genes. Proteins annotated to contain a DNA-binding or regulatory protein domain were defined as transcription factors (Ohm et al. 2010b).

Results

Genes involved in mushroom development also control vegetative growth

Dikaryotic colonies of wild-type H4-8, Awc-lAwc-1, Awc-2Awc-2, Ahom2Ahom2, AbrilAbril, Afst4Afst4, Ac2h2Ac2h2, Afst3Afst3, AhomlAhoml, and AgatlAgatl were grown for 6 days on PC membranes on glucose MM in the dark. The Ahom2Ahom2 strain formed 1.4-fold more biomass when compared to H4-8, while AbrilAbril and AhomlAhoml formed 0.4 and 0.6-fold less biomass, respectively (Figure 1). Biomass formation of the other strains was not significantly different from H4-8. Strains Ahom2Ahom2Afst3Afst3 and Ahom2Ahom2Afst4Afst4 also formed more biomass than the wild-type. Biomass of both strains was similar to that of Ahom2Ahom2 in the case of the agar cultures (Figure 2A). Notably, the Ahom2Ahom2Afst4Afst4 strain formed 1.7 fold more biomass in liquid shaken cultures when compared to Ahom2Ahom2 (Figure 2B). These data show that bril and homl stimulate vegetative growth, while hom2 and fst4 repress biomass formation of dikaryotic strains when glucose is used as a carbon source.

Biomass formation of Awc-2Awc-2, Ahom2Ahom2 and Afst4Afst4 was assessed on xylose, pectin, and sucrose (Figure 3). The Afst4Afst4 strain formed 2.8-fold more biomass on xylose when compared to H4-8 (Figure 3A). Moreover, Awc-2Awc-2, Ahom2Ahom2, and Afst4Afst4 formed more biomass on sucrose (1.2, 1.4, and 1.6-fold, respectively) and pectin (1.3, 1.4, and 1.6-fold, respectively) when compared to H4-8 (Figure 3BC). Together, these data show that Wc-2, Hom2 and Fst4 repress vegetative growth on different carbon sources.

Reduced vegetative growth of the AbrilAbril and AhomlAhoml strains may slow down fruiting body development. Therefore, fruiting was monitored after 7 days (Ohm et al. 2011) and 15 days of culturing. The AhomlAhoml strain formed more but smaller mushrooms both after 7 days and 15 days (Figure 4). In contrast, AbrilAbril had not formed fruiting bodies after 7 days but did so after 15 days showing that fruiting in this strain is delayed and not abolished as reported previously (Ohm et al. 2011). Genome-wide expression analysis

RNA composition of wild-type H4-8 was determined during vegetative growth in the dark, after transfer to the light, and during aggregate, primordium, and fruiting body formation. Expression of wc-1, wc-2, hom2,fst4,fst3, gatl, and bril changed less than 2-fold during development when compared to the vegetative mycelium grown in the dark (Table 2). In contrast, c2h2 and homl expression increased gradually with a maximum fold change of 4.7 and 2, respectively, during the fruiting body stage.

Expression profiles of wild type, Awc-lAwc-1, Awc-2Awc-2, Ahom2Ahom2, Afst4Afst4, Ac2h2Ac2h2, Afst3Afst3, AgatlAgatl, AhomlAhoml, and AbrilAbril strains were compared at the moment the wild-type formed aggregates (day 8) and fruiting bodies (day 12). Principal component analysis of the RNA profiles of 8-day-old colonies revealed a first and second component explaining 38 and 29% of the variation, respectively. Two distinct clusters were observed (Figure 5 A). The first cluster consisted of Awe- 1 Awe- 1, Awc-2Awc-2, and Ahom2Ahom2 that are all affected in early stages of fruiting body development. The second cluster consisted of AgatlAgatl and Afst3Afst3 that are affected in late stages of development. The other deletion strains did not cluster but rather showed a gradual change in expression. Principal component analysis of the RNA profiles of 12-day-old colonies revealed a first and second component explaining 72 and 7% of the variation, respectively. In this case, Awe- 1 Awe- 1, Awc-2Awc-2, Ahom2Ahom2, and Afst4Afst4 clustered, whereas the other strains clustered with the wild-type (Figure 5B).

The number of up- and down-regulated genes were between 86 and 1392 and 131 and 1463, respectively, when expression of the 8- and 12-day-old cultures of the deletion strains was compared with the wild type (Table 3). Enriched functional categories in the upregulated genes of 8-day-old colonies of Awc-lAwc-1, Awc-2Awc-2, Ahom2Ahom2, and Afst4Afst4 were mainly linked to carbohydrate metabolism, when compared to the 8-day-old wild-type colonies (Table 4). Downregulated functional groups were linked to energy transfer in these strains. Functional groups involved in energy transfer and carbohydrate metabolism were upregulated in 12-days-old colonies of Awc-lAwc-1 and Awc-2Awc-2 (Table 5). Downregulated genes were enriched for nucleosome, catalytic and oxidoreductase activity for Awc-lAwc-1 and Awc-2Awc-2. The functional group ATPase activity was also overrepresented in the downregulated genes of Awc-2Awc-2. In 12-day-old colonies of Ahom2Ahom2 functional groups involved in translation, energy transfer, and carbohydrate metabolism were overrepresented in the upregulated genes, while functional groups involved in transport and energy transfer were overrepresented in the downregulated genes. In 12-days-old colonies of Afst4Afst4 functional groups involved in carbohydrate processes and energy transfer were enriched in the upregulated genes. Groups involved in oxidoreductase activity, metabolic process and nucleosome, amongst others, were enriched in the downregulated genes. Functional categories involved in energy transfer and carbohydrate metabolism were upregulated in 8-day-old colonies of Ac2h2Ac2h2, while processes involved in cytoplasm, ATP binding, nucleosome and peptidase activity were enriched in the downregulated genes. In 12-day-old colonies of Ac2h2Ac2h2 upregulated genes were enriched in functional groups related to energy transfer, carbohydrate metabolic process, and chitin catabolic process. Downregulated genes were enriched in groups involved in cell wall, catalytic activity, tryptophan and fatty acid synthesis. No functional groups were overrepresented in the upregulated genes in 8-day-old and 12-day-old colonies of AhomlAhoml but functional groups related to chitinase activity, carbohydrate metabolism and energy transfer were enriched in the downregulated genes. 12-day-old AhomlAhoml colonies showed overrepresentation of functional groups related to amino acid synthesis, oxidoreductase, and carbohydrate metabolism activity in the down regulated genes. The functional categories that were enriched in the upregulated genes of 8-day-old colonies of Afst3Afst3 and AgatlAgatl were mainly involved in energy transfer, tryptophan metabolism, and cell wall processes. Genes involved in energy transfer and carbohydrate metabolism were enriched in the down-regulated genes. Groups involved in carbohydrate metabolism, methyltransferase, and leucine synthesis were enriched in the upregulated genes in 12-days-old colonies of Afst3Afst3. Downregulated genes were mainly enriched in cell wall processes and chitinase activity. Functional groups were upregulated for sphingo lipid metabolism in 12-days-old colonies of AgatlAgatl . Downregulated genes were mainly enriched for translation and cell wall. No functional groups were overrepresented in the upregulated genes of 8-day-old and 12-day-old colonies of the AbrilAbril strain. In contrast, functional groups related to metabolic process, carbohydrate metabolism, transcription and cell wall were overrepresented in the downregulated genes of 8-day-old colonies. Downregulated genes in 12-day-old AbrilAbril colonies were mainly enriched for carbohydrate metabolism, hydrolase activity and transcription repressor activity. Between 1 and 42 transcription factor genes were exclusively > 2-fold differentially expressed in one of the deletion strains when compared to the wild-type (Figure 6). Expression of homl and c2h2 had decreased in 12-day-old Awc-l wc-1 colonies. In 12- day-old colonies of Awc-2Awc-2 hom2 expression was increased, while c2h2 was downregulated. Expression of c2h2, gatl, and homl was downregulated in 12-day-old Ahom2Ahom2 colonies. In 8-day-old Afst4Afst4 colonies expression of homl was downregulated and in 12-day-old colonies hom2 expression increased, while c2h2 and homl expression decreased. In contrast, homl levels were upregulated in 8-day-old Afst3Afst3 colonies. Similarly, expression of homl and additionally c2h2 was increased in 8-day-old colonies of AgatlAgatl. In 8-day-old AbrilAbril colonies gatl expression was increased, while hom2 was increased in 12-day-old colonies (Figure 7).

Gene teal is involved in fruiting body development and represses vegetative growth Transcription factor gene teal (protein ID 2519514) showed > 2-fold decreased expression in Awe- 1 Awe- 1, Awc-2Awc-2, Ahom2Ahom2, AbrilAbril, and Afst4Afst4 when compared to the wild-type during aggregation (8-day-old colonies), while it was upregulated > 2-fold in Afst3Afst3, AhomlAhoml, and AgatlAgatl. A > 2-fold differential expression of teal was not observed when the wild-type had formed fruiting bodies (12-day-old colonies), explained by reduced expression of teal in 12-day-old wild-type colonies. Gene c2h2d (proteinID 2703923) was also downregulated in Awc- lAwc-1, Awc-2Awc-2, Ahom2Ahom2, and Afst4Afst4, while it was upregulated >2-fold in AgatlAgatl during aggregation (Figure 7). Furthermore, expression of c2h2d was decreased >2-fold in 12-day-old colonies of Awc-lAwc-1, Awc-2Awc-2, Ahom2Ahom2, Afst4Afst4, Afst3Afst3, and AgatlAgatl. Gene c2h2d has a predicted C2H2 DNA binding domain and teal is a predicted TEA/ATTS transcription factor. Expression of teal and c2h2 in the wild-type strain peaked during primordia and fruiting body formation, respectively (Table 2).

Deletion constructs were made for c2h2d and teal. No deletion strains were obtained for c2h2d after screening 100 transformants. In contrast, deletion of teal in the H4-8Aku80 was successful. PCR analysis confirmed inactivation of teal in one of the transformants. The transformant was crossed with the compatible wild-type H4-8b and siblings were selected with a deleted teal gene and an intact ku80 gene. One of these siblings was crossed with a H4-8 strain to obtain a Ateal strain with compatible mating types. These monokaryons were then crossed to obtain the ls.tealls.teal strain. The Isteallsteal strain showed a 1.3-fold increase in biomass when compared to the wild-type. This increase was similar to that of Ahom2Ahom2 (Figure 1). Transfer to the light did not induce irregular vegetative growth as observed in the wild-type. Interestingly, newly formed light-exposed mycelium did not produce aerial hyphae (Figure 8B). This resulted in a distinct border between the dark-grown mycelium and mycelium grown in the light. Fruiting body formation was almost completely abolished. Instead of the typical ring of fruiting bodies, only local clusters of fully-developed mushrooms were formed (Figure 8CD). These clusters were made at random positions in the colony; they were not restricted to the center, or the periphery. Together, these data show that teal represses vegetative growth and promotes fruiting body formation in the wild-type dikaryon.

Discussion

The transcription factor genes wc-2, hom2, fst4, bril, c2h2, fst3, gatl, and homl have been reported to be involved in fruiting body formation in S. commune (Ohm et al. 201 Ob; 2011; 2013). We here showed that the homeodomain domain protein Hom2, the zinc finger transcription factor Fst4 and the blue light complex transcription factor Wc-2 are not only involved in early stages of fruiting body development but also repress vegetative growth. In contrast, the DNA binding BRIGHT domain protein Bril and the homeodomain protein Homl stimulated vegetative growth. The latter protein is involved in late stages of mushroom development, while Bril was shown not to be essential for fruiting, although in its absence fruiting is delayed. Based on phenotypic analysis, whole genome expression analysis of the deletion strains, and inactivation of gene teal we propose a modified model for mushroom development (Figure 9).

The Abril bril strain does not fruit after the standard growth period of 10 days (Ohm et al. 2011). However, we here showed that fully developed mushrooms had formed after 4 weeks. This shows that Bril is not required for fruiting. Delayed mushroom development may be the result of reduced growth speed. Lower biomass formation may well be explained by the fact that functional categories metabolic process, carbohydrate metabolism, catalytic activity, transcription, and cell wall are down-regulated in the deletion strain in 8-day-old colonies. 12-day-old colonies showed down-regulation of functional categories carbohydrate metabolism, hydrolase activity, and transcription repressor activity. As a consequence of reduced biomass formation, a quorum sensing pathway may become activated at a later moment delaying the switch to fruiting body formation (Wosten and Willey 2000). Notably, Bril deletion has an effect on expression of teal and of gatl. The repression of teal may be a direct or an indirect effect due to its stimulatory effect on gatl expression. Together, Bril stimulates vegetative growth and has an effect on expression of the transcription factors teal and gatl that are involved in mushroom formation (Figure 9).

Genes hom2 and wc-2 are involved in the switch from vegetative growth to fruiting. Inactivation of these genes abolishes early stages of fruiting body formation (Ohm et al. 2011; 2013) but also increased the vegetative growth rate. Strain Awc-2 wc-2 formed more biomass on sucrose and pectin when compared to the wild-type, while Ahom2Ahom2 formed more biomass on glucose, sucrose, and pectin. Expression of hom2 and wc-2 is rather constant during development, suggesting post-transcriptional regulation of these genes. In the case of Wc-2, this may be accomplished by the interaction with the blue light sensor Wc- 1 (Ohm et al. 2013). Preliminary results indicate that Hom2 activity is regulated by phosphorylation of its Pka sites (data not shown). Increased growth rate was associated with enrichment of upregulated genes associated to carbohydrate metabolism in 8-day-old colonies, and of functional groups involved in carbohydrate metabolism and energy transfer in 12-day old colonies. Deletion of wc-2 and hom2 resulted in a > 2-fold downregulation of c2h2 in 12-day-old colonies. Downregulation was also observed in 8-day-old colonies, although the effect was less pronounced. Together, this confirms that Wc-2 and Hom2 stimulate c2h2 expression (Ohm et al. 2011; 2013; Figure 9).

Gene fst4 is constitutive ly expressed during the S. commune life cycle. Like Hom2 and Wc-2 it is involved in the switch from vegetative growth to fruiting. Strain Afst4Afst4 grows irregular in the light like the wild-type but does not aggregate. It formed more biomass than the wild-type on xylose, sucrose, and pectin but not on glucose. This indicates that Fst4 and Hom2 represent different parts of the repression pathway of vegetative growth. In liquid shaken cultures with glucose as carbon source these pathways may merge explaining why Ahom2 Ahom2 Afst4Afst4 formed more biomass than Ahom2Ahom2. Strain Afst4Afst4 showed enrichment of carbohydrate metabolism in the upregulated genes of 8-day-old and 12-day-old-colonies similar to that observed in Awc-2Awc-2 and Ahom2Ahom2. The fact that fst4 expression is not affected in Awc- 2Awc-2 and Ahom2Ahom2 strengthens the hypothesis that Fst4 and Hom2 represent different pathways. Gene fst4 stimulates c2h2 like honi2 and wc-2 do. This indicates that Fst4, Hom2, and Wc-2 input are channeled into the fruiting pathway via c2h2 (Figure 9B).

Transcription factor gene teal was downregulated in Awe- 1 Awe- 1, Awe- 2 Awe- 2, Ahom2Ahom2, AbrilAbril, and Afst4Afst4 when compared to the aggregating wild-type (8-day-old colonies), while it was upregulated in Afst3Afst3, AhomlAhoml, and AgatlAgatl. This indicates it is upregulated during early stages of development while it is repressed during late stages of mushroom formation. This agrees with the expression profile in the wild-type. The AtealAteal strain formed more biomass on glucose when compared to the wild-type. Moreover, it was severely affected in mushroom formation. Only local clusters of fully developed mushrooms were formed in the deletion strain. This phenotype may be explained by a reduced sensitivity of a signaling pathway leading to a developmental switch from "off to "on".

Expression of c2h2 increased >2-fold when R A profiles of 5- and 8-day-old colonies were compared. The increased expression during the aggregation stage and further increased expression in primordia and fruiting bodies agrees with the phenotype of Ac2h2Ac2h2 forming aggregates but not primordia and fruiting bodies (Ohm et al. 2011; Figure 9). C2H2 did not affect biomass formation implying that it is downstream of the switch between vegetative growth and mushroom development.

Deletion of fst3, gatl or homl results in more, but smaller mushrooms (Ohm et al. 2011). In addition, Homl and Gatl are involved in mushroom tissue formation (Ohm et al. 2011). Gene fst3 is constitutively expressed and its expression is not affected by any of the other transcriptional regulators. This suggests that Fst3 is subject to post- transcriptional regulation. The fact that a higher number of genes are differentially expressed in 8-day-old colonies (wild-type forming aggregates) when compared to 12- day-old colonies (wild-type forming fruiting bodies) (i.e. 882 and 217 genes, respectively) suggest that Fst3 exerts its effect already early in mushroom development. Upregulated functional groups were involved in energy transfer and cell wall processes in 8-day-old colonies, while downregulated functional groups were involved in carbohydrate metabolism. In 12-day-old colonies these groups were regulated in the opposite direction. Expression of gatl was highest during fruiting body formation. It is repressed by Bril in 8-day-old colonies, while it is activated by Hom2 during fruiting body formation. Colonies of 8 days old were enriched for groups involved in energy transfer and cell wall processes in upregulated genes, while groups involved in carbohydrate metabolism were downregulated. Groups involved in translation and cell wall were enriched in downregulated genes in 12-day-old colonies. Expression of homl gradually increases upon progression of fruiting. Expression analysis showed that Wc-1, Hom2 and Fst4, and probably Wc-2, stimulate expression of Homl . Notably, Homl stimulates formation of biomass of the vegetative mycelium. This suggests that Homl operates at two distinct stages of development (Figure 9). It may well be that Homl functions in mushroom formation by stimulation of biomass formation. This would explain the reduced size of the fruiting bodies in homl/Shoml . Notably, both Fst3 and Gatl repress homl expression. Functional groups involved in chitinase activity, carbohydrate metabolism, and energy transfer were enriched for downregulated genes in 8-day-old colonies. Functional groups involved in amino acid synthesis were enriched for downregulated genes in 12-day-old colonies.

The model of development of S. commune may apply to mushroom forming basidiomycetes as well. Hom2, Homl, Fst3, Fst4, C2H2, and Gatl are basidiomycete- specific regulatory proteins (Todd et al. 2014). Homologues of these genes were identified in L. bicolor and A. bisporus (Ohm et al. 2010b; Morin et al. 2012). The homologues for fst4,fst3, c2h2 and homl were upregulated during sexual development in two A. bisporus varieties (Morin et al. 2012), while homologues of hom2,fst4, c2h2, fst3, gatl and homl showed similar expression expression profiles in L. bicolor showed similarity to S. commune (Morin et al. 2012). Expression patterns of c2h2, fst3, homl and gatl were also found to be similar in C. cinerea (Plaza et al. 2014).

This is the first time a direct link has been shown between repression of vegetative growth and induction of sexual reproduction. Previously, a link has been shown between vegetative growth and asexual development in Aspergillus (Krijgsheld et al. 2013). This link involves trimeric G-protein signaling. The activity of the Ga-subunit of Aspergillus is regulated by the FlbA protein (Yu et al. 1996). Inactivation of this gene results in a strain that cannot initiate asexual development. Notably, S. commune has a homologue of flbA called thn. Inactivation of this gene results in a strain unable to form fruiting bodies (Fowler and Mitton 2000). This suggests that similar signaling pathways are involved in the decision to stop vegetative growth and to invest in reproduction in ascomycetes and basidiomycetes. We also showed that genes involved in fruiting may stimulate vegetative growth. These genes are involved in late stages of development. Table 2. Temporal expression of the blue light sensor gene wc-1 and transcription factor genes involved in fruiting body development in the wild-type dikaryon. Values are expressed in FKPM. Green shaded boxes indicate a significant >2-fold up-regulation when compared to dark grown vegetative mycelium.

Veg Veg

Aggregate Primordia Fruitin

Myc Ind

wc-1 37.30 40.06 54.83 51.97 62.23

wc-2 38.29 46.19 39.88 61.66 47.47

hom.2 107.64 135.84 115.99 104.15 58.63

teal 9.39 14.73 18.88 62.40 10.48

fst4 112.88 159.43 140.70 178.65 119.08

c2h2 22.06 29.61 47.48 87.84 105.36

fst3 103.23 97.82 102.70 1 19.53 115.64

gatl 75.01 65.00 72.79 61.87 126.09

homl 114.38 108.69 149.72 200.48 234.15

bril 30.03 34.22 30.56 34.99 29.95

c2h2d 3.72 8.76 16.57 39. 1 7 54.94

Table 3. Number of genes significantly up- and downregulated compared to wildtype at the moment the wild-type had formed aggregates (AG) and fruiting bodies (FB).

Upregulated Downregulated

AG FB AG FB

Awe 1 Awe 1 415 1195 431 1298

Awc2Awc2 375 1392 500 1421

Ahom2Ahom2 494 1267 652 1462

Afst4Afst4 1267 1150 1226 1330

Ac2h2Ac2h2 668 306 644 327

Afst3Afst3 400 86 482 131

AhomlAhoml 194 317 247 400

AgatlAgatl 480 662 556 991 Table 4. Enrichment of GO terms in up- and downregulated genes of 8-day-old colonies of Awc-l wc-1 and transcription factor deletion strains Awc-2Awc-2, Ahom2Ahom2, Afst4Afst4, Ac2h2Ac2h2, Afst3Afst3, AhomlAhomI, AgatlAgatl, and AbrilAbril when compared to the aggregating wild-type strain.

Awc-lAwc-1

Up-regulated genes in mutant strain Down-regulated genes in mutant strain GO term Functional annotation GO term Functional annotation

0005975 carbohydrate metabolic process oxidoreductase activity

hydrolase activity, hydro lyzing O- 0004553 0015171 monooxygenase activity glycosyl compounds

amino acid transmembrane

0008643 carbohydrate transport 0006865

transporter activity

0008643 L-arabino se isomerase activity 0016491 electron transport

0005351 sugar:hydrogen symporter activity 0005618 amino acid transport

hydrolase activity, acting on

0016810 0006118 metabolic process

carbon-nitrogen (but not peptide)

glutathione transferase

0005506 iron ion binding 0005199

activity

oxidoreductase activity, acting on structural constituent of cell

0016614 0004194

CH-OH group of donors wall

branched chain family amino acid

0009082 0006810 cell wall

biosynthetic process

0008812 choline dehydrogenase activity

Awc-2Awc-2

Up-regulated genes in mutant strain Down-regulated genes in mutant strain

GO term Functional annotation GO term Functional annotation

0005975 carbohydrate metabolic process 0016491 oxidoreductase activity

hydrolase activity, hydro lyzing O- 0004553 0004497 monooxygenase activity glycosyl compounds amino acid transmembrane

0005506 iron ion binding 0043039

transporter activity

0004497 monooxygenase activity 0006118 electron transport

0050381 unspecific monooxygenase activity 0006865 amino acid transport 0020037 heme binding 0008152 metabolic process

oxidoreductase activity, acting on glutathione transferase

0016614 0004364

CH-OH group of donors activity

structural constituent of cell

0008812 choline dehydrogenase activity 0005199

wall

branched chain family amino

0009082 0005618 cell wall

biosynthetic process

0006066 alcohol metabolic process 0050162 oxalate oxidase activity

\hom2\hom2

0005975 carbohydrate metabolic process 0004497 monooxygenase activity hydrolase activity, hydro lyzing O

0004553 0006810 transport

glycosyl compounds

0030246 carbohydrate binding 0020037 heme binding

0016491 oxidoreductase activity 0006118 electron transport

0003824 catalytic activity 0005215 transporter activity

unspecific monooxygenase

0005215 transporter activity 0050381

activity

0008152 metabolic process 0005506 iron ion binding

amino acid transmembrane

0016021 integral to membrane 0015171

transporter activity

0008422 beta-glucosidase activity 0006865 amino acid transport

oxidoreductase activity,

0006118 electron transport 0016614 acting on CH-OH group of donors \fst4\fst4

0005975 carbohydrate metabolic process 0005524 ATP binding

0008733 L-arabinose isomerase activity 0003677 DNA binding

0005351 sugar:hydrogen symporter activity 0000166 nucleotide binding

0008643 carbohydrate transport 0008152 metabolic process

0005215 transporter activity 0005737 cytoplasm

nucleoside-triphosphatase

0006810 transport 0017111

activity

hydrolase activity, hydro lyzing O-

0004553 0016491 oxidoreductase activity

glycosyl compounds

0016021 integral to membrane 0003824 catalytic activity

0030246 carbohydrate binding 0016887 ATPase activity

amino acid transmembrane

0015171 0005643 nuclear pore

transporter activity

\c2h2\c2h2

0005506 iron ion binding 0005737 cytoplasm

0004497 monooxygenase activity 0005524 ATP binding

0006118 electron transport 0008152 metabolic process

hydrolase activity, hydro lyz

0004553 0003824 catalytic activity

glycosyl compounds

0020037 heme binding 0016491 oxidoreductase activity

cellular protein metabolic

0008733 L-arabinose isomerase activity 0044267

process

0050381 unspecific monooxygenase activity 0000786 nucleosome

0005351 sugar:hydrogen symporter activity 0006334 nucleosome assembly threonine endopeptidase

0050660 FAD binding 0004298

activity

proteasome endopeptidase 0008643 carbohydrate transport 0004299

activity

Afst3Afst3

0005199 structural constituent of cell wall 0005506 iron ion binding

0005618 cell wall 0004497 monooxygenase activity 0016491 oxidoreductase activity 0020037 heme binding

carbohydrate metabolic

0008152 metabolic process 0005975

process

0050660 FAD binding 0006118 electron transport

hydrolase activity, oxidoreductase activity, acting on

0016614 0004553 hydro lyzing O-glycosyl

CH-OH group of donors

compounds

indoleamine 2,3-dioxygenase unspecific monooxygenase

0033754 0050381

activity activity

oxidoreductase activity, acting on

0016717 chitinase activity

paired donors, with oxidation

0020037 heme binding 0008843 endochitinase activity 0005215 transporter activity 0006032 chitin catabolic process

AhomlAhoml

NONE 0004568 chitinase activity

0008843 endochitinase activity

0004497 monooxygenase activity

0016491 oxidoreductase activity carbohydrate metabolic

0005975

process

hydrolase activity,

0004553 hydro lyzing O-glycosyl compounds

00081 15 sarcosine oxidase activity 00061 18 electron transport

0006032 chitin catabolic process

aromatic compound

0006725

metabolic process

AgatlAgatl

Up-regulated genes in mutant strain Down-regulated genes in mutant strain

GO term Functional annotation GO term Functional annotation

carbohydrate metabolic

0005199 structural constituent of cell wall 0005975

process

0005618 cell wall 0005506 iron ion binding

0016491 oxidoreductase activity 0020037 heme binding

0008152 metabolic process 00061 18 electron transport

0004497 monooxygenase activity 0004497 monooxygenase activity

0050660 FAD binding 0005524 ATP binding

3 -oxoacyl- [acyl-carrier-

0020037 heme binding 0004316

protein] reductase activity hydrolase activity, indoleamine 2,3-dioxygenase

0033754 0004553 hydro lyzing O-glycosyl activity

compounds

oxidoreductase activity, acting on unspecific monooxygenase

0016614 0050381

CH-OH group of donors activity

0006118 electron transport 0005215 transporter activity

AbrilAbril Up-regulated genes in mutant strain Down-regulated genes in mutant strain GO term Functional annotation GO term Functional annotation

NONE 0008152 metabolic process carbohydrate metabolic

0005975

process

0003824 catalytic activity

0000786 nucleosome

0005215 transporter activity

structural constituent of cell

0005199

wall

0006334 nucleosome assembly

0005618 cell wall

0006810 transport

0016491 oxidoreductase activity

Table 5. Enrichment of GO terms in up- and downregulated genes of 12-day-old colonies of Awc-l wc-1 and transcription factor deletion strains Awc-2Awc-2, Ahom2Ahom2, Afst4Afst4, Ac2h2Ac2h2, Afst3Afst3, AhomlAhomI, AgatlAgatl, and AbrilAbril when compared to the fruiting wild-type strain.

Awc-lAwc-1

0005506 iron ion binding 0008152 metabolic process

0020037 heme binding 0016491 oxidoreductase activity

0004497 monooxygenase activity 0000786 nucleosome

0006118 electron transport 0006334 nucleosome assembly

0050381 unspecific monooxygenase activity 0003824 catalytic activity

0005975 carbohydrate metabolic process 0006810 transport hydrolase activity, hydro lyzing O-

0004553 0016020 membrane

glycosyl compounds

0005524 ATP binding 0004497 monooxygenase activity 0005215 transporter activity 0016021 integral to membrane

hydrolase activity, acting on

0016810 00061 18 electron transport

carbon-nitrogen (but not peptide)

Awc-2Awc-2

0020037 heme binding 0000786 nucleosome

0005506 iron ion binding 0006334 nucleosome assembly 0004497 monooxygenase activity 0003677 DNA binding

0050381 unspecific monooxygenase activity 0003824 catalytic activity

0005975 carbohydrate metabolic process 0008152 metabolic process

0006118 electron transport 0006260 DNA replication

hydrolase activity, hydro lyzing O

0004553 0042624 ATPase activity, uncoupled glycosyl compounds

0005215 transporter activity 0016491 oxidoreductase activity 0005524 ATP binding 0042623 ATPase activity, coupled

RNA-dependent ATPase

0006032 chitin catabolic process 0008186

activity

\hom2\hom2

0003735 structural constituent of ribosome 0006810 transport

0005840 ribosome 0005215 transporter activity

0006412 translation 0008152 metabolic process

0005975 carbohydrate metabolic process 0016020 membrane

0005506 iron ion binding 0016021 integral to membrane 0020037 heme binding 0016491 oxidoreductase activity hydrolase activity, hydro lyzing O-

0004553 0003824 catalytic activity

glycosyl compounds

0005622 intracellular 0006865 amino acid transport

amino acid transmembrane

0004497 monooxygenase activity 0015171

transporter activity

0006118 electron transport monooxygenase activity

\fst4\fst4

0005975 carbohydrate metabolic process 0016491 oxidoreductase activity 0005215 transporter activity 0008152 metabolic process

0006508 proteolysis 0003824 catalytic activity

0005506 iron ion binding 0000786 nucleosome

0020037 heme binding 0030170 pyridoxal phosphate binding 0050381 unspecific monooxygenase activity 0006334 nucleosome assembly

hydrolase activity, hydro lyzing O-

0004553 0006118 electron transport

glycosyl compounds

0004497 monooxygenase activity 0006810 transport

0006118 electron transport 0008483 transaminase activity

structural constituent of cell

0008236 serine-type peptidase activity 0005199

wall

\c2h2\c2h2

0005506 iron ion binding 0003824 catalytic activity

0020037 heme binding 0008152 metabolic process

0004497 monooxygenase activity 0030170 pyridoxal phosphate binding structural constituent of cell

0006118 electron transport 0005199

wall

0050381 unspecic monooxygenase activity 0005618 cell wall

flavonoid 3 '-monooxygenase trehalose biosynthetic 0016711 0005992

activity process

hydrolase activity, hydro lyzing O-

0004553 0005488 Binding

glycosyl compounds

hydrolase activity, hydro lyzing O- 3 -oxoacyl- [acyl-carrier-

0005975 0004316

glycosyl compounds protein] reductase activity indoleamine 2,3-

0006032 carbohydrate metabolic process 0033754

dioxygenase activity purine nucleotide

0005618 chitin catabolic process 0006164

biosynthetic process

Afst3Afst3

0004565 beta-galactosidase activity 0004568 chitinase activity

0009341 beta-galactosidase complex 0008843 endochitinase activity 0008168 methyltransferase activity 0005576 extracellular region

hydrolase activity,

0005975 carbohydrate metabolic process 0004553 hydro lyzing O-glycosyl compounds

3-isopropylmalate dehydratase

0009316 0006032 chitin catabolic process

complex

3-isopropylmalate dehydratase structural constituent of cell

0003861 0005199

activity wall

0004035 alkaline phosphatase activity 0005618 cell wall

zinc ion transmembrane transporter

0005385 0030246 carbohydrate binding

activity unspecific monooxygenase

0006829 zinc ion transport 0050381

activity

inorganic phosphate carbohydrate metabolic

0005315 ~ 0005975

transmembrane transporter activity process

AhomlAhoml

NONE 0003824 catalytic activity

0016491 oxidoreductase activity

3 -oxoacyl- [acyl-carrier-

0004316

protein] reductase activity

0008152 metabolic process

0006526 arginine bio synthetic process 0016021 integral to membrane

L-iditol 2-dehydrogenase

0003939

activity

mating-type factor

0004932

pheromone receptor activity acetylglutamate kinase

0003991

activity

glutamate N-

0004358

acetyltransferase activity

AgatlAgatl

serine C-palmitoyltransferase

0017059 0005840 ribosome

complex

serine C-palmitoyltransferase structural constituent of

0004758 0003735

activity ribosome 0006412 translation

0005622 intracellular

0005839 proteasome core complex structural constituent of cell

0005199

wall

0005618 cell wall

0008152 metabolic process

0006118 electron transport

0006526 arginine biosynthetic process

AbrilAbril

carbohydrate metabolic

NONE 0005975

process

hydrolase activity,

0004553 hydro lyzing O-glycosyl compounds

0016491 oxidoreductase activity

glutathione transferase

0004364

activity

0005618 cell wall

0003824 catalytic activity

0004568 chitinase activity

structural constituent of cell

0005199

wall

0008843 endochitinase activity

transcription repressor

0016564

activity Example 2: Fungal mycelium as a biomaterial Abstract

Filamentous fungi colonize organic material such as plant waste by means of hyphae that grow at their tips and that branch subapically. As a result, a hyphal network is formed that is called mycelium and that has a fabric-like appearance. Here, material properties of mycelium of the mushroom forming fungus Schizophyllum commune were determined in relation to light conditions and the genetic background. Mycelium of liquid standing cultures of the wild-type strain H4-8b grown in the light or in the dark in sealed Petri dishes showed a Young's modulus of 801-897 MPa. Maximum tensile strength was between 12.1 and 13.5 MPa, while elongation at breaking ranged between 3.1 and 3.8 %. Mycelium of the Ahom2 strain showed similar mechanical strength as the wild-type in the dark and in the light as well as a similar Young's modulus in the dark. The Young's modulus of Ahom2 was however higher when compared to the wild-type strain in the light. Elongation of Ahom2 ranged between 1.3 and 2.0 %. The Afst4 strain showed the highest Young's modulus and maximum strength in the light and in the dark with values of 1467-2015 MPa and 19.8 and 24.5 MPa, respectively. The elongation at breaking of the Afst4 strain was between 1.7 and 2.1 %. Together, these data show that the transcription factor genes hom2 and fst4 that are involved in fruiting can also affect mechanical properties of the vegetative mycelium. The resulting Young's moduli are similar to those of thermoplastics showing the potential of fungal mycelium as a sustainable biomaterial. Overall, it has clearly been demonstrated that the fungi according to the invention have superior mechanical properties as compared to wild-type fungi.

Introduction

Filamentous fungi grow by means of hyphae that grow at their tips and that branch subapically. As a result, a network of hyphae is formed that is called mycelium. The hyphae feed on organic material such as plant waste. To this end, they penetrate their substrate while secreting enzymes that degrade the plant polymers into molecules that can be taken up to serve as nutrients. Hyphal penetration of the substrate is facilitated by the turgor pressure generated by the cytoplasm and by the rigidity of the surrounding cell wall. The composition of fungal cell walls is dynamic and varies between species, strains, environmental conditions (Bowman and Free 2006), and developmental stage (Wessels 1994).

Schizophyllum commune is a model for mushroom forming fungi (Ohm et al. 2010b). Its life cycle starts with a monokaryotic (i.e. homokaryotic) mycelium that results from the germination of a basidiospore. Monokaryotic mycelia are sterile and always grow vegetative. A fertile dikaryon is formed upon fusion of two monokaryons with compatible mating types. Blue light is required to initiate fruiting in the dikaryotic (i.e heterokaryotic) mycelium (Perkins and Gordon 1969), whereas high C0₂ levels repress this developmental program (Niederpruem 1963; Raudaskoski and Viitanen 1982). So, in the dark and / or at high CO2 levels the dikaryon grows vegetative, while at ambient CO2 levels and in the light fruiting bodies are formed. Initiation of fruiting body formation by S. commune starts with asymmetrical colony growth, followed by aggregation of aerial hyphae, and subsequent formation of primordia. These primordia can develop into fruiting bodies that form basidia within the hymenium. Karyogamy, meiosis, and one round of mitosis occur in the basidia, resulting in haploid, binucleate basidiospores.

The cell wall of the vegetative mycelium of Schizophyllum commune, as an example of a basidiomycete, consists of glucose (67.6%), N-acetylglucosamine (12.5%), mannose (3.4%)), xylose (0.2%>), amino acids (6.4%>), and lipids (3.0%>) (Sietsma and Wessels, 1977). These monomers make up chitin, glucan, and proteins as well as other, not yet identified cell wall constituents. The outer layer of the cell wall is a water-soluble mucilage consisting of (1 ,3)-P-linked glucose units with branches of single (1 ,6)-P-linked glucose molecules at every third glucose along the chain (Wessels et al, 1972). This mucilage, known as schizophyllan, is also secreted into the culture medium. An alkali- soluble glucan consisting of (l,3)-a- linked glucose units, known as S-glucan, is located beneath the mucilage and accounts for about half of the thickness of the water-insoluble portion of the wall. The inner layer of the cell wall consists of an alkali- insoluble glucan, known as R-glucan, and chitin (Wessels, 1994). The R-glucan was found to be a highly branched (1 ,3)(1 ,6)-P-glucan. Part of this highly insoluble glucan has structural similarity to schizophyllan. Most of the R-glucan is linked to chitin (Sietsma and Wessels, 1981) via basic amino acids and N-acetylglucosamine (Sietsma and Wessels, 1977).

The rigidity of cell walls combined with the capacity to produce large networks in low value waste streams makes mycelium of interest as a biomaterial. We here studied physical properties of mycelium of wild-type S. commune and of strains in which either fst4 or hom2 have been inactivated. Data show that transcription factor genes that are involved in fruiting can also affect mechanical properties of the mycelium. Material and Methods

Strains and culture conditions

S. commune wild-type strain H4-8b (Ohm et al, 2010a) and its derivative strains Afst4 (Ohm et al, 2010b) and Ahom2 (Ohm et al, 2011) were used in this study. Strains were grown on minimal medium (MM) (Dons et al. 1979) either or not solidified with 1.5 % agar. A quarter of a 7-day-old colony grown on agar medium was homogenized in 50 ml MM for 30 s at low speed using a Waring Blender (Waring Laboratory, Torrington, England). Static liquid cultures were inoculated by taking up 400 mg wet weight mycelial homogenate in a final volume of 6 ml MM and spreading it into a confluent layer in a 9 cm Petri dish. Cultures were grown at 30 °C in the light (450 Lux from fluorescent tubes, Osram, L36W/840 Lumilux Cool White, Munich, Germany) or in the dark. Cultures were sealed with a double layer of Parafilm and a layer of scotch tape to create high C0₂ conditions. After 3 days of incubation, 30 ml MM was applied underneath the mycelial mat. Growth was prolonged for 5 days at 30 °C using conditions described above. Tensile measurements

- Young's modulus or elastic modulus: A number that depicts the elastic deformation of an object when a force is applied to it

Maximum tensile strength: The amount of stress that a material can withstand before breaking

- Elongation at breaking: the elongation of an object (in %) at the moment the maximum tensile strength is reached and the object breaks

Stress (mechanical): The force (N) that is applied to an object divided by the area (m²) of the object to which the force is applied. (Stress is in MPa, lPa=lN/M²) Strain (mechanical): The relative change in length (tl) caused by the deformation to the original length (tO)

Static liquid cultures were dried at room temperature. Tensile measurements were performed on 5 rectangles (12 x 4 mm) of each biological duplicate. Thickness of the mycelium was measured by High Accuracy length gauge (Heidenhain MT1281, Traunreut, Germany). Tensile measurements were performed using a Dynamic Mechanical Analyzer (DMA) Q800 TA instrument (TA Instruments, New Castle, DE, USA), equipped with an 18 N capacity load cell. The elasticity modulus (i.e. Young's modulus) (in MPa) was obtained by dividing the mechanical stress by the mechanical strain. The maximum strength (in MPa) was obtained from force per unit area, while elongation at breaking point (in %) was obtained by calculating the strain (in mm) at the moment of breaking compared to the original length of the samples (in mm). Statistical analysis

Statistical analysis was performed with the software package IBM SPSS statistics 22.0 (IBM Corporation, Armonk, New York). Mechanical properties of mycelia were analysed by Independent T Tests (asterisks indicate * p < 0.05, ** p < 0.005 and *** p < 0.0005).

Results

The wild-type strain H4-8b and its derivatives Afst4 and Ahom2 were grown as a thin layer in a Petri-dish in the light or in the dark at high C0₂. After 3 days liquid medium was introduced underneath the colony. Growth of the floating mycelium was prolonged for 5 days, after which the mycelial mats were dried. Mycelium of liquid standing cultures of the wild-type strain H4-8b grown in the light or in the dark in sealed Petri dishes showed a Young's modulus of 801-897 MPa (Figure 10). Maximum tensile strength was between 12.1 and 13.5 MPa, while elongation at breaking ranged between 3.1 and 3.8 %. Mycelium of the Ahom2 strain showed a similar maximum strength in the dark and in the light (with a minimum of 11.4 MPa when grown in the dark) and a similar Young's modulus in the dark. However, its Young's modulus was higher when compared to the wild-type strain in the light. Elongation of Ahom2 was between 1.3 and 2.0 % and thereby lower as that of the wild-type. The Afst4 strain showed the highest Young's modulus and maximum strength in the light and in the dark with values of 1467-2015 MPa and 19.8 and 24.5 MPa. The elongation at breaking of the Afst4 strain was between 1.7 and 2.1 % being lower than the wild-type. Discussion

The oil-based economy should be transformed into a sustainable circular system to halt climate change, pollution and depletion of natural resources. This includes the use of sustainable and renewable energy and materials. The use of bio-based materials is part of this change. This includes a wide range of resources such as those of microbes, plants, macro-algae, shellfish and other animals. Waste streams can also be converted into bio materials. For instance, biodegradable plastics can be made from a wide variety of organic materials such as starch, cellulose and lactic acid. We here used mycelium as a new source of sustainable biomaterials. Mycelium-based materials could replace existing non-sustainable materials or even enable development of novel materials.

The Young's modulus and strength of materials is highly variable. For instance, elastomers (e.g. synthetic rubber) are characterized by a Young's modulus < 100 MPa and a maximum strength of 1-50 MPa, while steel and ceramics have an Young's modulus of 10-800 GPa and a strength of 100-2000 MPa (Ashby, 2005). Wood and thermoplastics (e.g. polyethylene, polypropylene, polyvinyl chloride, polystyrene, nylon, and Teflon) are characterized by intermediate Young's moduli and strength. These values are 1-10 GPa and 30-50 MPa for wood and 500-5000 MPa and 10-100 MPa for thermoplastics, respectively. The Young's modulus of mycelium as obtained in this study was in the range of 801-2015 MPa, while its strength ranged between 11.4-24.5 MPa depending on strain and growth conditions. Thus, mycelium has a Young's modulus and strength properties similar to that of thermoplastics.

Mycelium was grown in the light or the dark in sealed Petri dishes. Light did not impact elasticity and maximum strength of the wild-type mycelium. In the case of Ahom2 we did observe an effect for the Young's modulus, the maximum strength, and the elongation at breaking. These values were higher when the mycelium had grown in the light. Notably, the highest Young's modulus and maximum strength were obtained with strain Afst4. Also in this case the Young's modulus was higher in the light when compared to the dark. Yet, elongation at breaking and the maximum strength was similar when growth in the dark and light were compared.

Together, we have provided evidence that mycelium could replace non- sustainable materials. Fungi grow on low value waste streams and grow optimally at a temperature of 15-30 °C. The latter implies that heating of the growth facilities is minimum, if needed at all, to grow the fungal materials. We have shown that we can create mycelium materials with different properties by changing the genetic make-up and light conditions during growth. Moreover, CO2 levels, inoculum density, carbon source, pH, and growth temperature during growth may impact the mechanical properties of the mycelium. The latter three environmental conditions have been shown to impact cell wall composition of S. cerevisae and Candida albicans (Aguilar-Uscanga and Francois, 2003; Ene et al, 2012). On top of that, material properties of mycelium could be altered by posttreatment with plasticizers or crosslinker, thus increasing the palette of mycelium-based materials.

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Claims

1. A fungus derived from a parental fungus, wherein said fungus exhibits a decreased expression of a polynucleotide encoding Fst4 compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions, and

- a decreased expression level of at least one polynucleotide encoding a polypeptide selected from the group consisting of: Wc-1, Wc-2, Hom2 and Teal compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions, and/or has

wherein the decreased and/or increased expression level results in increased vegetative growth of said fungus compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions.

2. A fungus according to claim 1, wherein the fungus is a filamentous fungus.

3. A fungus according to claim 1 or 2, wherein the fungus is an Ascomycete or a Basidiomycete, preferably a Basidiomycete.

4. A fungus according to claim 3, wherein the fungus is a mushroom forming Basidiomycete.

5. A fungus according to any one of the preceding claims, wherein at least the expression level of a polynucleotide encoding Fst4 and of a polypeptide encoding Hom2 is decreased compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions.

6. A fungus according to any one of the preceding claims, or

a fungus derived from a parental fungus, wherein said fungus exhibits: - a decreased expression level of at least one polynucleotide encoding a polypeptide selected from the group consisting of: Wc-1, Wc-2, Fst4, Hom2 and Teal compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions, and/or has

wherein the decreased and/or increased expression level results in increased vegetative growth of said fungus compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions,

wherein said fungus comprising a polynucleotide encoding a compound of interest.

7. A method of culturing a fungus according to any one of the preceding claims or a fungus derived from a parental fungus, wherein said fungus exhibits:

- a decreased expression level of at least one polynucleotide encoding a polypeptide selected from the group consisting of: Wc-1, Wc-2, Fst4, Hom2 and Teal compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions, and/or has

wherein the decreased and/or increased expression level results in increased vegetative growth of said fungus compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions, comprising culturing said fungus under conditions conducive to the production of said fungus and isolating and/or purifying said fungus, or

culturing said fungus on an industrial scale under conditions conducive to the production of said fungus and, optionally, isolating and/or purifying said fungus.

8. A method for the production of a fungus and/or a mushroom, comprising contacting a fungus according to any one of claims 1 - 6 or a fungus derived from a parental fungus, wherein said fungus exhibits:

wherein the decreased and/or increased expression level results in increased vegetative growth of said fungus compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions, with a substrate and with a fungus able to produce mushrooms, wherein said fungus according to any one of claims 1 - 6 exhibits increased vegetative growth in the substrate.

9. A method for the production of a compound of interest comprising culturing a fungus according to any one of claims 1 - 6 or a fungus derived from a parental fungus, wherein said fungus exhibits:

wherein the decreased and/or increased expression level results in increased vegetative growth of said fungus compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions, under conditions conducive to the production of the compound of interest and isolating and/or purifying the compound of interest.

10. A method for the degradation of organic material comprising contacting a fungus according to any one of claims 1 - 6 with an organic material and applying conditions suitable for the degradation of the organic material to the fungus contacted with the organic material.

11. A method for the degradation of inorganic material comprising, contacting a fungus according to any one of claims 1 - 6 with an inorganic material and applying conditions suitable for the degradation of the inorganic material to the fungus contacted with the inorganic material.

12. A method for the production of a composite material, comprising contacting a fungus according to any one of claims 1 - 6 or a fungus derived from a parental fungus, wherein said fungus exhibits:

wherein the decreased and/or increased expression level results in increased vegetative growth of said fungus compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions, with an organic and/or inorganic material and applying conditions suitable for the production of the composite material consisting of fungal mycelium and the organic and/or inorganic material.

13. A method for the production of fungal mycelium comprising contacting a fungus according to any one of claims 1 - 6 with an organic material and applying conditions suitable for the production of the fungal mycelium.

14. A method according to claim 12 or 13 , wherein the composite material or the fungal mycelium is a construction material, an isolation material, a packaging material, a textile, a container, a material or device used for medical applications, a material or device used in horticulture, or a decoration material.

15. A composite material or a fungal mycelium obtained or obtainable by the method according to any one of claims 12 - 14.

16. A composite material comprising:

- a fungus according to any one of claims 1 - 6, or,

- a fungus derived from a parental fungus, wherein said fungus exhibits:

wherein the decreased and/or increased expression level results in increased vegetative growth of said fungus compared to the parental fungus said fungus is derived from when both are cultured and assayed under identical conditions, or,

- a fungal mycelium obtained or obtainable by the method according to any one of claims 12 - 14.

17. A composite material according to claim 16, wherein composite material is a construction material, an isolation material, a packaging material, a textile, a container, a material or device used for medical applications, a material or device used in horticulture, or a decoration material.