AT517831A4 - Process for the production of secondary metabolites - Google Patents

Abstract

The present invention relates to a method of producing at least one secondary metabolite in a genetically modified fungal cell, characterized in that the method comprises cultivating a genetically modified fungal cell capable of producing the at least one secondary metabolite, wherein the unmodified fungal cell comprises at least one Gene encoding at least one polypeptide comprising at least two motifs selected from the group consisting of the amino acid sequences AHNDX1ERKYRTNLKX2KI (SEQ ID NO: 3), KX3SKGX4X5LX6KATEYI (SEQ ID NO: 4), RX7LQAF (SEQ ID NO: 5), LFDPRGFC (SEQ ID No. 6) and variants thereof having an identity of at least 60%, wherein X1 is V or I, X2 is D or V, X3 is V or I, X4 is T or A, X5 is V or I, X6 is any amino acid and X7 is R or K, and the expression of this polypeptide in the genetically modified fungal cell is reduced compared to the unmodified fungal cell and / or the polyp eptid in the genetically modified fungal cell compared to the unmodified fungal cell comprises at least one mutation, whereby the formation of the at least one secondary metabolite is increased in the genetically modified fungal cell compared to the unmodified fungal cell.

Description

The present invention relates to methods for producing at least one secondary metabolite.

Mushrooms produce a large amount of chemical compounds. These compounds are very different in structure and effects, but share a fundamental characteristic. They are all synthesized in the secondary metabolism of fungi. The secondary metabolism complements the primary metabolism, which includes all biological processes necessary for the growth of the fungi. Fungi use secondary metabolism for various purposes, such as communication, competition, toxicity, pathogenicity and mycoparasitism. Some secondary metabolism products are used to fight infectious diseases (as antibiotics) and cancer (as immunosuppressants). In addition, they provide a rich source of new therapeutics and provide opportunities for pharmaceutical research. Numerous compounds have been identified in recent decades and are used in the biotechnology and pharmaceutical industries. Many other compounds can potentially be discovered. One obstacle is the fact that most of the genes of secondary metabolism are not active under laboratory conditions. One strategy to address this is the use of pleiotropic regulators of secondary metabolism. A well-studied example of this is the regulator LaeA (see, for example, WO 2011/161063). He finds himself in some mushrooms again. If LaeA is deleted there, a downregulation of the secondary metabolism occurs (see WO 2011/161063).

It is an object of the present invention to provide means and methods to regulate, in particular increase, the production of secondary metabolites in fungi.

The present invention therefore relates to a method of producing at least one secondary metabolite in a genetically modified fungal cell which comprises cultivating a genetically modified fungal cell capable of producing the at least one secondary metabolite, wherein the unmodified fungal cell comprises a gene encoding a polypeptide comprising at least two motifs selected from the group consisting of the amino acid sequences AHNDXiERKYRTNLKX2KI (SEQ ID NO: 3), KX3SKGX4X5LX6KATEYI (SEQ ID NO: 4), RX7LQAF (SEQ ID NO: 5), LFDPRGFC (SEQ ID NO: 6) and Variants thereof having an identity of at least 60%, wherein Χχ V or I, X2 is D or V, X3 is V or I, X4 is T or A, X5 is V or I, X6 is any amino acid and X7 is R or K, and Expression of this polypeptide is reduced in the genetically modified fungal cell as compared to the unmotivated fungal cell and / or the polypeptide in the genetically modified fungal cell i m comprises at least one mutation compared to the unmodified fungal cell, whereby the formation of the at least one secondary metabolite in the genetically modified fungal cell is increased in comparison to the unmodified fungal cell.

It has surprisingly been found that in fungal cells polypeptides which contain at least two motifs selected from the group consisting of the amino acid sequences AHNDX1ERKYRTNLKX2KI (SEQ ID No. 3), KX3SKGX4X5LX5KATEYI (SEQ ID No. 4), RX7LQAF (SEQ ID No. 5), LFDPRGFC (SEQ ID NO: 6) and variants thereof having an identity of at least 60%, are responsible for the regulation of secondary metabolism and thus for the production of secondary metabolites. If the activity or rate of expression of this polypeptide within the fungal cell is reduced, the amount of secondary metabolite formed by the fungal cell increases. Therefore, in the method according to the invention a genetically modified fungal cell is used, which has a reduced expression rate of the aforementioned polypeptide compared to the unmodified fungal cell. Alternatively, the polypeptide may also have one or more mutations, thereby altering its biological function in the fungal cell such that production of at least one secondary metabolite in the fungal cell is increased. Preferably, the increase in the amount of secondary metabolite produced is at least 10%, preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 100%, even more preferably at least 150%, even more preferably at least 200%, compared to unmodified fungal cells.

The unmodified fungal cell comprises at least two, preferably at least three, in particular four, motifs selected from the group consisting of the amino acid sequences AHNDX1ERKYRTNLKX2KI (SEQ ID No. 3), KX3SKGX4X5LX6KATEYI (SEQ ID No. 4), RX7LQAF (SEQ ID No. 5), LFDPRGFC (SEQ ID NO: 6) and variants thereof having an identity of at least 60%, wherein Χχ V or I, X2 is D or V, X3 is V or I, X4 is T or A, X5 is V or I, X6 is any amino acid and X7 is R or K. If the polypeptide according to the invention comprises all four motifs, these are preferably present in the polypeptide in the order AHNDX1ERKYRTNLKX2KI (SEQ ID No. 3), KX3SKGX4X5LX6KATEYI (SEQ ID No. 4), RX7LQAF (SEQ ID No. 5) and LFDPRGFC (SEQ ID No. 6) ) arranged. Even in the presence of less than four of these motifs, they may occur in this order in the polypeptide. If the peptide additionally comprises the motif GSX8X9IDX10SX11 (SEQ ID No. 7), where X8 is F or Y,

Xg D or E, Χχo is F or Y and Χχχ R or K, the polypeptide may have the motifs in the order GSX8X9IDX10SXxx (SEQ ID NO: 7), AHNDX1ERKYRTNLKX2KI (SEQ ID NO: 3), KX3SKGX4X5LX6KATEYI (SEQ ID NO: 4) , RX7LQAF (SEQ ID NO: 5) and LFDPRGFC (SEQ ID NO: 6).

According to a preferred embodiment of the present invention, the at least one polypeptide comprises at least one, preferably at least two, more preferably at least three, further motifs selected from the group consisting of the amino acid sequences GSX8X9IDXx0SXxx (SEQ ID NO. 7), PGLGX12GX13Y (SEQ ID NO. 8), REGLYSTPLSWEX14PQPGX15RMD (SEQ ID NO: 9), GRAPQLSQQQXieQQQQQQ (SEQ ID NO: 10), PPPEVPPXi7EGLYSTPLXi8WE (SEQ ID NO: 11), PSTEX19QSSRSLYSTPSX20GWE (SEQ ID NO: 12), TKAKPGEK-MAQLESISTSQQPLD (SEQ ID NO: 13), ALENSAQGX21DKFQPG (SEQ ID NO: 14) and variants thereof having an identity of at least 60%, wherein Xs is F or Y, X9 is D or E, X10 is F or Y, Χχχ R or K, X12 S or T, Χχ3 V, I or L, X14 R or K, Χχ5 I or L, X16 is any amino acid, X17 R or K, X18 S or T, X19 A or P, X2o L or M and X2X is any amino acid. The polypeptide to be regulated in the method according to the invention may comprise further motifs in addition to the aforementioned motifs.

The motifs are separated in the polypeptide by peptides which preferably comprise between 15 and 300, more preferably between 16 and 280, even more preferably between 18 and 260, amino acid residues. Between the motif with the amino acid sequence GSX8X9IDX10SX11 (SEQ ID NO: 7) and the motif with the amino acid sequence AHNDX1ERKYRTNLKX2KI (SEQ ID NO: 3), a peptide having 200 to 300, preferably 220 to 280, more preferably 230 to 260 more preferably having 238 to 252 amino acid residues. Between the motif with the amino acid sequence AHNDX1ERKYRTNLKX2KI (SEQ ID NO: 3) and the motif with the amino acid sequence KX3SKGX4X5LX5KATEYI (SEQ ID NO: 4), a peptide with 20 to 40, preferably 22 to 35, more preferably 25 to 31 , Amino acid residues are located. Between the motif with the amino acid sequence KX3SKGX4X5LX6KATEYI (SEQ ID NO: 4) and the motif with the amino acid sequence RRLQAF there may be a peptide having 15 to 20, preferably 17 to 19, more preferably 18, amino acid residues. Between the motif having the amino acid sequence RRLQAF and the motif having the amino acid sequence LFDPRGFC (SEQ ID NO: 6), there may be a peptide having from 15 to 25, preferably from 17 to 23, more preferably from 19 to 21, amino acid residues.

Includes a fungal cell, a polypeptide of the invention with four or five designs, this may include the following general sequences include: (SEQ ID NO. 15) AHNDXiERKYRTNLKX2KIX22KX3SKGX4X5LX6KATEYIX23RX7LQAFX24LFDPRGFC and GSX8X9lDXioSXiiX25AHNDXiERKYRTNLKX2KIX22KX3SKGX4X5LX6KATEYIX23RX 7LQAFX24LFDPRGFC (SEQ ID NO. 16) wherein X22 is a peptide having from 20 to 40, preferably with 22 to 35, more preferably with 25 to 31, amino acid residues, X23 a peptide with 15 to 20, preferably with 17 to 19, even more preferably with 18, amino acid residues, X24 a peptide with 15 to 25, preferably with 17 to 23 , even more preferably having 19 to 21, amino acid residues, and X25 is a peptide of 200 to 300, preferably 220 to 280, more preferably 230 to 260, even more preferably 238 to 252, amino acid residues. At the N-terminus there may be a peptide of up to 150 amino acid residues.

The polypeptide of the cell used according to the invention may also comprise motifs or variants of motifs which comprise at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, more preferably at least 90%, even more preferably at least 95% identical to the amino acid sequences AHNDX1ERKYRTNLKX2KI (SEQ ID NO: 3), KX3SKGX4X5LX5KATEYI (SEQ ID NO: 4), RX7LQAF (SEQ ID NO: 5), LFD-PRGFC (SEQ ID NO: 6), GSX8X9IDXi0SXii (SEQ ID No. 7), PGLGX12GX13Y (SEQ ID NO: 8), REGLYSTPLSWEX14PQPGX15RMD (SEQ ID NO: 9), GRAPQLSQQQigQQQQQQ (SEQ ID NO: 10), PPPEVPPXi7EGLYSTPLX18WE (SEQ ID NO: 11), PSTEX19QSSRSLYSTPSX20GWE (SEQ ID NO: 12), TKAKPGEK-MAQLESISTSQQPLD (SEQ ID NO: 13) and ALENSAQGX21DKFQPG (SEQ ID NO: 14). "Identity" refers to the degree of correspondence between two or more nucleic acid or amino acid sequences that can be determined by comparing the sequences The percentage of "identity" results from the percentage of identical regions in two or more sequences, taking into account gaps or other sequence peculiarities (ie% identity is the number of identical positions / total number of positions x 100). A particularly preferred method for determining identity is the BLAST X program of the National Center for Biotechnology Information (NCBI) (see, inter alia, Altschul S et al., J Mol Biol 215 (1990): 403-410). In this case, the BLOSUM62 algorithm with the parameters "Gap" "Existence": 11 and "Extension": 1 is preferably used.

Depending on the fungal cell or depending on the number of biosynthetic pathways for secondary metabolites in the fungal cell, a different number of secondary metabolites can be produced. Preferably, at least one, preferably at least two, more preferably at least three, even more preferably at least four, even more preferably at least five, even more preferably at least ten, secondary metabolites are produced by the method according to the invention. "Genetically modified fungal cell" as used herein refers to fungal cells that have been genetically engineered so that the gene encoding the inventive and claimed polypeptide having the at least two motifs has a different nucleic acid sequence compared to an unmodified fungal cell of the same strain To obtain a genetically modified fungal cell, the gene in the genome is mutated starting from an unmodified fungal cell.

An "unmodified fungal cell" or "unmodified fungal cell" as used herein forms the starting point for the production of a modified fungal cell according to the invention. The unmodified fungal cell comprises at least one gene coding for the polypeptide according to the invention and claimed and has a certain production rate of secondary metabolites. An unmodified fungal cell can be a fungal cell that occurs naturally in nature. However, an unmodified fungal cell can also be a fungal cell which has already been genetically or genetically engineered at any point in the genome or comprises extra-chromosomal nucleic acid molecules that naturally do not occur in the fungal cell. The mutations in the fungal cell genome may also relate to the gene encoding the polypeptide of the invention. This fungal cell is used as a starting line for further modifications as described herein and thus also constitutes an "unmodified fungal cell".

The term "secondary metabolite" includes not only an end product of a secondary metabolism but also intermediates.

The feature that the expression of a polypeptide in a genetically modified fungal cell is reduced compared to an unmodified fungal cell means that a fungal cell in a genetic modification synthesizes a smaller amount of the polypeptide of the invention having the motifs mentioned herein than a fungal cell of the same kind that is unmodified or unmodified. The reduction of the expression rate or the amount of polypeptide produced is at least 10%, preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, in particular 100%. The expression rate of this polypeptide can be determined, for example, by means of an mRNA determination (for example by means of PCR (polymerase chain reaction)) or polypeptide determination with the aid of polypeptide-specific antibodies (eg ELISA (enzyme-linked immunosorbent assay).) The expression rate can of course also be determined indirectly by: the amount of one or more fungal cell derived secondary metabolites is determined If the amount of secondary metabolites produced in the modified fungal cell increases compared to unmodified fungal cells, the introduction of a mutation into the gene encoding the polypeptide is successful.

The one-letter code for amino acid residues at amino acid sequences used herein corresponds to the commonly used:

The polypeptide comprising at least two motifs selected from the group consisting of the amino acid sequences AHNDX1ERKYRTNLKX2KI (SEQ ID NO: 3), KX3SKGX4X5LX5KATEYI (SEQ ID NO: 4), RX7LQAF (SEQ ID NO: 5), LFDPRGFC (SEQ ID NO: 6) and variants thereof having an identity of at least 60%, and Trichoderma reesei has the amino acid sequence SEQ ID NO: 1, is known from Trichoderma reesei as Xppl (xylanase promoter binding protein 1) (see Derntl C et Biotechnol Biofuels 8 (2015): 112). So far, it was only known that Xppl is responsible for the regulation of the expression of xylanases in fungal cells, but not for the regulation of biosynthetic pathways of secondary metabolites. Since Xppl has been described as a very specific regulator of xylanase expression - cellulases are not regulated by Xppl - it is surprising that down-regulation of Xppl leads to increased production of secondary metabolites. Therefore, the polypeptide having the at least two motifs may also be referred to as "Xppl".

In the method according to the invention, on the one hand, fungal cells can be used whose expression of the polypeptide described herein is reduced (for example by modification or replacement of the corresponding promoter, by deletion of the gene which codes for the polypeptide or parts thereof). On the other hand, it is also possible to reduce the activity of the polypeptide in that this e.g. modified by mutations. The polypeptide comprising the above-described motifs is a transcription factor. That the polypeptide controls the transcription of various genes of the secondary and possibly, the primary metabolism. Targeted mutations make it possible, for example, to alter the activity of the polypeptide in such a way that the biosynthesis of the secondary metabolites increases in comparison to fungal cells which express an unchanged polypeptide. By determining the levels of secondary metabolites produced in the fungal cells, it is possible to test whether a mutation of the polypeptide leads to the desired effect.

According to a preferred embodiment of the present invention, the polypeptide has a range which is at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, especially 100% with amino acid residues 118 to 157 and / or 394 to 494 of the amino acid sequence SEQ ID NO: 1. SEQ ID NO: 1 is derived from Trichoderma reesei and has the following amino acid sequence:

MAQALDISSSSGPGPLVALPERTLGPPASAITPPSTVQRSAWTSSPRAPAMDMFNFTTAASASA VASYAPAGGSNSNSNSNSGGNSGGLGDMNFSQMPQYSGRNPLQSMLSQTKEGPDAKRIKVESPH SALDSIDSWLQFDEDADKFGSFEIDFSKQYNTANQPRASTNMTPGLGSGLYANPTAPFREEDFL DDSAFDHSLSEDDEMFDNININDQLSKLGTLPSTEAQSSRSLFSTPSMGWEKPQQGVQPSAVMG DEIPRRMT SD SWDPIPQGTNYTLTPDERRRLLEIAMGPGRLASYVNP SRFNMGFGSTMQP SISQ EFGGFGAPKQGLSHMGMYQRNNSADTSASPLSQSRELKHKQPKVETPPKTVPTKRPSTLSETSS IGKEKVKSADRMAHNDVERKYRTNLKDKITELRNAVPALQQVAEGEPDAAQGIAKVSKGTVLTK ATEYIHQLEQRNKDIMTEHKQLMRRLQAFEALLNNSMRVDIMPSHSMTLFDPRGFC

According to a further preferred embodiment of the present invention, the polypeptide is at least 60%, preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, in particular 100%, identical to the amino acid sequence SEQ ID No. 1.

The at least one mutation of the polypeptide in the genetically modified fungal cell is preferably a deletion, substitution or insertion of at least one amino acid residue. Methods of producing mutated polypeptides by introducing mutations in the corresponding nucleic acid sequence are well known to those skilled in the art.

According to a preferred embodiment, the polypeptide comprises at least one mutation in at least one motif selected from the group consisting of the amino acid sequences AHNDX1ERKYRTNLKX2KI (SEQ ID NO: 3), KX3SKGX4X5LX5KATEYI (SEQ ID NO: 4), RX7LQAF (SEQ ID NO: 5), LFDPRGFC (SEQ ID NO: 6) and variants thereof having an identity of at least 60%.

According to a preferred embodiment, the polypeptide comprises at least one mutation in at least one motif selected from the group consisting of the amino acid sequences GSXsXglDXioSXn (SEQ ID No. 7), PGLGX12GX13Y (SEQ ID No. 8), REGLY-STPLSWEX14PQPGX15RMD (SEQ ID No. 9) , GRAPQLSQQQieQQQQQQ (SEQ ID NO: 10), PPPEVPPXi7EGLYSTPLXi8WE (SEQ ID NO: 11),

PSTEXi9QSSRSLYSTPSX20GWE (SEQ ID NO: 12), TKAKPGEKMAQLESISTSQQPLD (SEQ ID NO: 13), ALENSAQGX2iDKFQPG (SEQ ID NO: 14) and variants thereof having an identity of at least 60%.

The polypeptide which can be produced by the genetically modified fungal cell preferably has one or more mutations in one or more of the above-mentioned motifs as compared with the polypeptide of the unmodified fungal cell. In this case, individual amino acids may be substituted or the entire motif or parts thereof (comprising at least one, two, five or ten amino acid residues) may be deleted. These mutations should lead to an increase in the formation of secondary metabolites or corresponding precursor molecules thereof in the modified fungal cell compared to the unmodified fungal cell.

The reduction of the expression of the polypeptide in the genetically modified fungal cell is preferably achieved by modification of the gene or parts thereof.

Preferably, the promoter of the gene or the polypeptide coding region of the gene is mutated. The mutation at the promoter may include a deletion, insertion or substitution of at least one nucleotide. The naturally occurring in the gene promoter can also be replaced by another promoter. This promoter may be from the same fungal cell or from another organism, preferably also a fungal cell.

By modifications in the nucleic acid sequence of the gene encoding the polypeptide having the at least two motifs as defined herein, it is possible to reduce its transcription rate, thereby also less polypeptide is translated from the corresponding RNA. Preferably, with appropriate modification, the amount of polypeptide is at least 10%, preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferred at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, especially 100% reduced in comparison to the unmodified fungal cell. The amount of transcribed RNA encoding the polypeptide can be determined by known methods for RNA determination.

The expression of the polypeptide can be reduced or prevented, for example, if preferably at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%. even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, especially 100% of the coding region for the polypeptide are deleted or substituted.

For example, to reduce or completely inhibit expression of the polypeptide in the genetically modified fungal cell, the promoter region or portions thereof may be modified by, for example, deletion or substitution. By modifications in the promoter region, the transcription rate can be reduced or the transcription can be completely suppressed. In this case, at least 10%, preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, especially 100% of the promoter are deleted or substituted. Preferably, at least 10, preferably at least 20, more preferably at least 50, even more preferably at least 100, even more preferably at least 200, nucleotides are deleted or substituted upstream of the start codon, wherein the modification of the promoter is directly from the start codon of the polypeptide coding region can be done.

The gene encoding the polypeptide Xppl in Trichoderma reesei comprises the following nucleic acid sequence (SEQ ID NO: 2):

CAGATGGTGAAGACGGGCAGAAAGGGGAGCGAAAGCTGAAAAATACTTAAGCAGCAGCTGTTCC

CCGTACGTTCGTGGAGATGGCAAGAGCAGCCGGCCGCGGGGAGTCTGGAGCAGGAGCCGCAGCT

GCGTAGCTGCCGGTTGCTGCTGCCCATCTGCCCAGGTTCGTTTGCGCCGGATCCGTCCAAATAT

CTCCTGATAAATGCGGCCACTTTTCCCTCATGCTCGTCCTTTTTTATTCCCCGAAGCGCAGCAA

ACGAGCAGCAAGAGCAAAGAGCAGAGCAGAAAGTCGCCTCATCTCGGGCGCCGCCTTGCCTAAC

GGGGTAGCAATAAGCCCATCGACACCGACGGCGGGCGGCGGGCTGGCCAATCAGCGGCGCGCGG

TGCTGTTCCTTTGGCCCTTGGAGCCTTGGGGAATCATACGATGTCTGAGCAGCCAATTCTTGCA

TTCTTCCCACATGCATGGCATCCCATGGCGCAGGGTCCCCCCCCCTTCTCCCCTCTCCGCTCTC

TTCTCCTCTCTTCTTGGCTGAGGTGCTCTGCTCCCTCTGCTCGGCCAGCTAAGACAACCAGCGC

TGTGGCCCGGCAAGACAGCCACTCTTGTCATTCGCCAGGGGGGTTGGAGGAGGGCGCCACGGCT

CCGCGGAAAGTCAGGCCATTTTCATCAACAGCCGCAGACACTGGCCCAGAGAACTATCCTTATC

CTGGACGCGCGCACGTCGAAAATTTTCATTTTTGCTCGCCCAGGCGCGCCTGCAATTCGCCATG

CGGGCTGCTCGATACCCACGCAGACGGCAAATGACAGGCCATTCTGGCTCACAGACGCCGCCGT

CCCGGCTCTCGCCGAGGTGGAAAAGGGTCATCTGGGTTCGTTGCGCTGAGCTCTAGCTAGGCCA

GAAGACACAGCCGAGACATGCCTATTCGACGTGCCTACGGGGCGGAGGCAGAGGGCAGAGGACA

GGAGGGCAGGAGGACATCCCAACAAAGGCTCCCCATGCCGGAAACGCAAAGAGAAGCATCGGAG

AGCTGCGAAGCACCCGCCGAAACAGCCACTACCTTACCCAGCAAGAGCAGCAGCTCTCTTGAAT

GGCCTGACTGGCGACACCGCGGGTCCCTAGCGCCGGGGCCTTGCCTGGATGGCACCGGTCCCTG

GAGCGGGATCCTCGTCGCTGGTGCTCGACGGGCTGTGGTGCTGCTGTGCGTCTTGCGCTGCCGG

CTGGGCTGGGCTGGGCTGGGCTGGATCTGAGCTGCTTGCAGCGCCGTTCCCTCAGGCTTCGGAA

TCGCAGCCGTCGCGGCGTTGCCCGAACAGCCACCCGGAGCTTGGCCTGCCTCTGCGAGGTGCTA

ATGCTGAAACTGGTCGACCCTGTATATTACCCGTGATGCTCTGGCACCGCAGCTCCAGACCCAG

ACCCGGGTCTAGGTTCCCAATATCGATCCTGTAGCATTGATATCGAACGGTACTTGCTGGGTAC

TACCTCCCACTCTTGCTGCTCGGTACCCTGCCCTGTCTCACCTGTGCATGCACAGAAGCACCAT

CACTATCCATTGACTTCCAATTTTGCATCTGCATCCGTCGGCCTATCTTGCCGGCATCTACTTC

TACTTCTACTTCTACGTCTCCGCCTGCACTCGCATTGACCTCTTTTTGCATCTACATCTGCGGC

AGAGCTGTGCGCTATCTACCTTACCTCGTCTTGAACCCCCCTGATCCGAGCATCGTATCCCTCC

ATAACCACCTCGGCTCGCTACCTCACCTCCTTCGTGATAGGTATTCAATCGTGGTGTCTCTTGC

TCATCTGCATCTGGAATTCTTCCCTTTCTCCTCTTGACATCTTCTACCTATTATCGCGCTCTTC

CTCCTCCGTCTGCAACCCCCCAGACGCCGCCGAACCCGATCTGTGGCTGCCATTAGCCTTTCGG

CGCGCATTCCTCTACGCCACCCGGTTTGGGTCCCCCTTCACCCCCAGCACATATTCCGACAACC

CTGTGTCTGCAGCCGCATGGCACAAGCCCTCGACATTTCCTCGTCGTCAGGACCCGGACCCCTC

GTCGCCCTGCCCGAGCGAACTCTCGGCCCCCCAGCCTCGGCCATCACGCCTCCCTCGACCGTCC

AGCGCTCGGCCTGGACGTCGTCGCCTCGCGCGCCCGCCATGGACATGTTCAACTTCACAACCGC

TGCCTCGGCATCCGCAGTCGCCTCGTATGCCCCCGCCGGCGGCAGCAATAGCAATAGCAATAGC

AATAGCGGCGGCAACAGCGGCGGCCTGGGCGACATGAACTTCTCTCAGATGCCGCAGTACTCTG

GCCGCAATCCCCTGCAGTCGATGCTCTCCCAGACCAAGGAGGGCCCCGATGCCAAGCGGATAAA

GGTCGAGTCGCCGCACTCGGCGCTGGATTCGATCGATTCATGGCTTCAGTTTGACGAGGATGCC

GACAAGTTTGGCAGCTTCGAGATTGACTTTTCCAAACAGTACAACACGGCCAACCAGCCAAGGT

GAGCTGCGCTCCTGAACCGCCAATCTGGTGTTTCCCGCTCTCTAACA.CATTGCGA.CA.TTAAGA.G

CTTCCACAAACATGACGCCGGGACTGGGCAGCGGTCTCTACGCCAATCCCACCGCCCCCTTCAG

AGAAGAAGACTTTCTCGACGACAGCGCCTTTGACCACTCTCTGTCCGAAGACGACGAGATGTTC

GACAACATCAACATCAATGACCAGCTGTCCAAGCTTGGCACGCTGCCCTCGACCGAAGCTCAGT

CATCCAGGAGTCTCTTCTCCACGCCGTCGATGGGCTGGGAGAAGCCTCAGCAAGGCGTCCAGCC

CAGCGCCGTCATGGGCGACGAGATCCCGAGGCGCATGACTAGCGATTCCTGGGACCCGATTCCG

CAGGGCACCAATTACACCCTCACGCCGGACGAGCGCCGCCGACTGCTCGAAATCGCCATGGGCC

CCGGCAGATTGGCGTCCTACGTCAACCCCAGCCGGTTCAACATGGGCTTCGGATCTACGATGCA

GCCCAGCATAAGCCAGGAGTTTGGCTTCGGCTTCGGCGCTCCGAAACAGGGGCTGAGCCATATG

GGCATGTACCAGAGGAACAACTCGGCCGACACGAGCGCATCCCCCCTCTCGCAGAGTCGGGAAT

TGAAACACAAGCAGCCCAAGGTCGAGACGCCGCCGAAGACGGTGCCCACAAAGCGGCCCAGCAC

GCTTTCGGAGACGAGCAGCATCGGCAAAGAAAAGGTCAAGTCCGCCGACCGCATGGCGCACAAC

GACGTTGAGCGCAAGTATCGAACCAATCTCAAGGACAAGATTACCGAGCTGCGAAATGCGGTGC

CGGCGCTGCAGCAGGTGGCTGAAGGGGAGCCGGACGCTGCGCAGGGCATTGCCAAAGTGAGCAA

GGTGAGTTGGAAAACAGCATGCCCCATCTTGTCAGTGACTCGTGAAGACGGCATCACTGACTCT

GATCCTATAGGGCACCGTATTGACAAAAGCGACTGAATACATTCATCAGCTCGAGCAACGCAAC

AAGGATATCATGACGGAGCATAAGCAGCTTATGCGACGACTGCAAGCCTTCGAAGCACTTCTCA

ACAACTCGATGAGAGTCGACATCATGCCCAGCCATAGCATGACGCTCTTCGACCCGAGAGGATT CTGTTGA, with the promoter region underlined, the exons in bold, and the introns in italics and underlined printed between the exons.

According to another preferred embodiment of the present invention, the fungal cell is a fungal cell of the class Sordariomycetes, in particular of the family Hypocreaceae, Clavici-pitaceae, Glomerellaceae or Nectriaceae.

The fungal cell is preferably a fungal cell of the genus Trichoderma, Claviceps, Colletotrichum or Fusarium.

According to a particularly preferred embodiment of the present invention, the fungal cell is selected from the group consisting of Trichoderma reesei, Trichoderma harzianum, Trichoderma virens, Trichoderma atroviride, Tolypocladium ophio-glossoides, Ophiocordyceps unilateralis, Hirsutella minnesoten-sis, Metarhizium robertsii, Metarhizium anisopliae, Metarhizium majus , Neonectria ditissima, Nectria haematococca, Villosiclava virens, Colletotrichum suhlineola, Colletotrichum fioriniae, Colletotrichum graminicola Claviceps purpurea, Stachybotrys chartarum, Stachybotrys chlorohalonata, Fusarium avenaceum, Colletotrichum gloeosporioides, Fusarium langsethiae, Fusarium pseudograminearum, Fusarium graminearum, fujikuroi, Fusarium oxysporum, Verticillium longisporum, Fusarium verticillioides, Colletotrichum orbiculare, Thielaviopsis punctulata, Madurella mycetomatis, Verticillium dahlia, Phaeoacremonium minimum, Thielavia terrestris, Magnaporthe oryzae, Neurospora tet-raspe rma, Myceliophthora thermophila, Neurospora crassa, Metarhizium acridum, Magnaporthiopsis poae, Sordaria macrospora, Po-dospora anserine, Metarhizium guizhouense, Chaetomium globosum, Metarhizium brunneum, Gaeumannomyces graminis, Sporothrix schenckii, Sporothrix brasiliensis, Ophiostoma piceae, Colletotrichum higginsianum, Metarhizium album and Diaporthe ampeli-na, preferably Trichoderma reesei, Trichoderma atroviride, Claviceps purpurea and Fusarium graminearum, more preferably Trichoderma reesei. All of these fungi have in their unmodified form a gene encoding at least one polypeptide comprising at least two motifs selected from the group consisting of the amino acid sequences AHNDX1ERKYRTNLKX2KI (SEQ ID NO: 3), KX3SKGX4X5LX6KATEYI (SEQ ID NO: 4), RX7LQAF (SEQ ID No. 5), LFD-PRGFC (SEQ ID No. 6) and variants thereof having an identity of at least 60% and optionally a further motif selected from the group consisting of the amino acid sequences GSX8X9IDX10SX11 (SEQ ID No. 7), PGLGX12GX13Y ( SEQ ID NO: 8), REG-LYSTPLSWEX14PQPGX15RMD (SEQ ID NO: 9), GRAPQLSQQQXieQQQQQQ (SEQ ID NO: 10), PPPEVPPXi7EGLYSTPLXi8WE (SEQ ID NO: 11), PSTEX19QSSRSLYSTPSX20GWE (SEQ ID NO: 12), TKAKPGEKMAQLESISTSQQPLD (SEQ ID NO: 11) 13), ALENSAQGX21DKFQPG (SEQ ID NO: 14) and variants thereof having an identity of at least 60%. All of the above-mentioned unmodified fungal species are capable of a polypeptide having the motifs AHNDX1ERKYRTNLKX2KI (SEQ ID NO: 3), KX3SKGX4X5LX6KATEYI (SEQ ID NO: 4), RX7LQAF (SEQ ID NO: 5) and LFDPRGFC (SEQ ID NO: 6) express. The motifs GSXsXglDXioSXn (SEQ ID NO: 7), PGLGX12GX13Y (SEQ ID NO: 8), REGLYSTPLS-WEX14PQPGX15RMD (SEQ ID NO: 9), GRAPQLSQQQXieQQQQQQ (SEQ ID NO: 10), PPPEVPPXi7EGLYSTPLXi8WE (SEQ ID NO: 11), PSTEX19QSSRSLYSTPSX20GWE (SEQ ID NO: 12), TKAKPGEKMAQLESISTSQQPLD (SEQ ID NO: 13), ALENSAQGX21DKFQPG (SEQ ID NO: 14) and variants thereof having an identity of at least 60% are present in polypeptides of various fungal families, fungal genera and fungal species, respectively.

The motifs GSX8X9IDX10SXii (SEQ ID NO: 7) and PGLGX12GX13Y (SEQ ID NO: 8) are preferably in polypeptides of the fungal cells defined herein Trichoderma reesei, Trichoderma harzianum, Trichoderma virens, Trichoderma atroviride, Claviceps purpurea, Metarhizium album, Villosiclava virens, Metarhizium robertsii, Metarhizium anisopliae, Metarhizium majus, Metarhizium brunneum, Metarhizium guizhouense, acridum Metarhizium, Toly-pocladium ophioglossoides, Hirsutella minnesotensis, Ophiocordy- ceps unilateralism Stachybotrys chlorohalonata, Stachybotrys chartarum, Fusarium graminearum, Fusarium pseudograminearum, Fusarium langsethiae, verticillioides, Fusarium fuji -kuroi, Fusarium oxysporum, Fusarium favenaceum, Nectaria hae-matococca, Neonectria ditissima, Thielaviopsis punctulata, Vericotium dahlia, Verticillium logisporum, Colletotrichum orbi-culare, Colletotrichum gloeosporioides, Colletotrichum higginsi-anum, Colletotrichum graminicola, Colletotrichum sublineola and Colletotrichum fioriniae present.

The motif REGLYSTPLSWEX14PQPGX15RMD (SEQ ID NO: 9) is preferably in polypeptides of the fungal Ophiostoma piceae, Sporothrix brasiliensis, Sporothrix schenckii, Gaeumannomyces graminis, Magnaporthiopsis poae, Magnaporthe oryzae, Diaporthe ampelina, Phaeoacremonium minimum, Madurella mycetomatis, Podospora anserine, Thielavia terrestris, Myceliophthora thermophila and Chaetomium globosum.

The motif GRAPQLSQQQisQQQQQQQ (SEQ ID NO: 10) is preferably present in polypeptides of the type of fungal cells Sordaria macrospora, Neurospora crassa and Neurospora tetrasperma defined herein.

The motif PPPEVPPXi7EGLYSTPLXi8WE (SEQ ID NO: 11) is preferably in polypeptides of the type of fungal cells Stachybotrys chlorohalonata, Stachybotrys chartarum, Fusarium graminearum, Fusarium pseudograminearum, Fusarium slowethiae, Fusarium verticillioides, Fusarium fujikuroi, Fusarium oxysporum, Fusarium favenaceum, Nectaria haematococca, Neonectria ditissima, Thielaviopsis punctulata, Verticillium dahlia, Verticillium logisporum, Colletotrichum orbiculare, Colletotrichum gloeosporioides, Colletotrichum higginsianum, Colletotrichum graminicola, Colletotrichum sublineola, Colletotrichum fioriniae, Sordaria macrospora, Neurospora crassa and Neurospora tetrasperma.

The motif PSTEX19QSSRSLYSTPSX20GWE (SEQ ID NO: 12) is preferably present in polypeptides of the type of fungal cells of the genus Trichoderma defined herein, preferably Trichoderma reesei, Trichoderma harzianum, Trichoderma virens and Trichoderma atro-viride.

The motif TKAKPGEKMAQLESISTSQQPLD (SEQ ID NO: 13) is preferably present in polypeptides of the type of the fungal cells defined herein Metarhizium album, Metarhizium robertsii, Metarhizium anisopliae, Metarhizium majus, Metarhizium brunneum, Metarhizium guizhouense and Metarhizium acridum.

Various secondary metabolites can be produced by the method of the invention using suitable modified fungal cells as described herein. The at least one secondary metabolite is therefore preferably selected from the group consisting of polyketides, alkaloids, terpenes, melanines, pteridines, phenylpropanoids, flavonoids, and non-ribosomal peptides, polyketides being particularly preferably prepared.

Fungal cells are known to be capable of producing secondary metabolites. The fungal cells produce different substances depending on the cell. By providing the modifications of the invention, it is possible that the amount of secondary metabolites produced by the fungal cells can be increased. It is also possible to modify fungal cells so that they are able to produce secondary metabolites that these cells could not naturally produce. For this purpose, additional nucleic acid molecules can be introduced into the fungal cell which code polypeptides and proteins which catalyze these new or alternative biosynthetic pathways. Therefore, it is particularly preferred that the genetically modified fungal cell comprises at least one nucleic acid molecule coding for at least one heterologous protein. In Watanabe CM et al. (Chem Biol 3 (2013): 463-469), Abrar M, et al. (Crit Rev Food, Nutr 53 (2013): 862-874), Ortei I et al. (J Biol Chem 284 (2009): 6650-6660), Ozcengiz G et al. (Biotech Adv 31 (2013): 287-311), Ward TJ et al. (PNAS 99 (2002): 9278-9283), Ei-senman HC et al. (Appl Microbiol Biotech 93 (2012): 931-940) and Nesic K et al. (Rev Envir Contamin Toxicol 228 (2014): 101-120) disclose secondary metabolites and their metabolic pathways which can be controlled by the method of the present invention.

Not only can secondary metabolites be produced by the method of the invention, but it can also be used to identify new secondary metabolites and biosynthetic pathways. In this case, secondary metabolites produced according to the invention modified fungus cells can be isolated and their

Structure (e.g., by MS / MS or NMR). The secondary metabolites produced by the fungal cells modified according to the invention are compared with those secondary metabolites which produce a corresponding unmodified fungal cell. This can be used to determine which secondary metabolite synthesis pathway in the fungal cell by reducing the expression or activity of the polypeptide with the at least two motifs selected from the group consisting of the amino acid sequences AHNDX1ERKYRTNLKX2KI (SEQ ID No. 3), KX3SKGX4X5LX6KATEYI (SEQ ID No. 4), RX7LQAF (SEQ ID NO: 5), LFDPRGFC (SEQ ID NO: 6) and variants thereof are up-regulated. The involved enzymes and other proteins can be determined indirectly by means of RNA analyzes.

The present invention will be explained in more detail with reference to the following figures and examples, but without being limited thereto.

Fig. 1 shows the course of yellowing (absorption at 370 nm) in culture supernatants of the wild-type strain (black squares, solid lines), the Xppl deletion strain (black diamonds, dashed lines) and the Xppl overexpression strain (white squares, dotted lines) Carboxymethylcellulose (CMC) (A), lactose (B) and D-glucose (C). The data comes from three independent biological replicates. The error bars indicate the standard deviation.

Fig. 2 shows the quality of the samples analyzed by RNASeq. (A) The wild-type strain (Etmus53) and the Xppl deletion strain (Δχρρΐ) were cultured in triplicates on CMC and the mycelium harvested after 48 hours and weighed. (B) The individual samples were clustered according to the RNASeq analysis according to the measured amounts of individual transcripts.

FIG. 3 shows a comparison of the proportions of the differently expressed genes (UEG) and of all annotated genes per KOG (Eu-Karyotic Orthologous Groups) or per KOG class from the respective total. The ratio of percentages is given as a base 2 logarithm. KOG or KOG classes above 0.2 are bold (as likely to be regulated gene groups).

4 shows all LELs from the COG "carbohydrate transport and metabolism" including the detailed results from the RNA

Seq analysis (average number of transcripts per sample, difference between wild-type and Xppl deletion strain as logarithm base 2, p-value according to Benjamini & Hochberg).

Fig. 5 shows the results of the biolog analysis. The Xppl deletion strain and wild-type strain were cultured in triplicates on 95 different carbon sources and growth measured by measuring absorbance at 750 nm. (A) shows the average values of the wild type strain (black bars) and the Xppl deletion strain (hatched bars) on a representative selection of carbon sources (in terms of carbon source type as well as growth rate) after 72 hours. The error bars indicate the standard deviation. In (B) and (C), the average values on all carbon sources after 72 h and 96 h, respectively, of the Xppl deletion strain were plotted against the wild type strain and a trend line inserted.

Fig. 6 shows all genes coding for polyketide synthases (PKS) including the detailed results from the RNASeq analysis (average number of transcripts per sample, difference between wild-type and Xppl deletion strain as base 2 logarithm, Benjamini p-value & Hochberg). Fat-printed PKS-encoding genes were considered differently expressed in the evaluation of the RNASeq analysis.

Fig. 7 shows the expression rates of the PKS-encoding genes 73621 (A), 73618 (B), 65116 (C), 65172 (D), 60118 (E) and 81964 (F) in the growth course on CMC of the Xppl deletion strain (Rauten ) and wild-type strain (squares). The two strains were cultured in triplicates and the relative transcript levels were determined by means of qPCR, the reference value used being in each case the value of the wild-type strain after 36 h. The error bars indicate the standard deviation.

Fig. 8 shows the basic data of cultivation in modified medium (no citrate) with D-glucose. The wild-type strain (black squares), the Xppl deletion strain (black diamonds) and the Xppl overexpression strain (white squares) were cultured in "24-well-plates." The course of the biomass (A), the yellowing of the supernatant (B) and the expression rates of the PKS coding genes 73621 (C) and 65172 (D) are given, the values are from three biological replicates, the error bars indicate the standard deviation, the relative transcript amounts were determined by qPCR, the reference value being used in each case of the wild-type strain after 48 h.

9 shows the number of low-molecular substances which were formed by T. reesei in the culture supernatants after 72 h and 96 h, respectively, in modified medium (no citrate) with D-glucose in the wild-type strain (Etmus53), the Xppl deletion Strain (Δχρρΐ) and the Xppl overexpression strain (OExppl).

Fig. 10 shows a pedigree of the orthologist of Xppl. EXAMPLES:

material and methods

Mushroom trunks and cultivation conditions

The T. reesei strains QM6aAtmus53 (Steiger M et al., Appl Environ Microbiol. 77 (2011): 114), the Xppl deletion strain QM6aEtmus53Axppl (QMoAxppl) (Derntl C et al., Biotechnol Biofuels 8 (2015): 112) and the Xppl overexpression strain (QMaoOppl) (Derntl C et al., Biotechnol Biofuels 8 (2015): 112) was cultured on malt extract (MEX) agar plates at 30 ° C. Hygromycin B was added as needed at a final concentration of 113 U / ml. For comparative growth T. reesei was pre-cultured on plates with Mandels-Andreotti medium (MAM) with 1% glycerol at 30 ° C. Comparably large pieces of overgrown agar were placed as inoculum on MAM plates containing 1% D-glucose and incubated at 30 ° C. Photos were taken from the bottom of the plates. For the cultivation in carboxymethyl cellulose (CMC) T. reesei was incubated in 60 ml MAM with 1% CMC (Carl Roth GmbH, Germany) in 1 L Erlenmeyer flask without shaking at 30 ° C. For the cultivation in D-glucose and lactose, T. reesei was shaken in 60 ml MAM with 1% D-glucose or lactose in 250 ml Erlenmeyer flasks at 180 ° C. at 180 rpm. Mycelium and culture supernatants were separated by filtration over Miracloth (EMD Millipore, Germany). The mycelia were stored in liquid nitrogen. For small scale culture, T. reesei was incubated in 1.5 ml of modified MAM with 1% U-12C or U-13C-labeled D-glucose in 24 well plates without shaking at 30 ° C. The MAM was modified to be buffered by a sodium phosphate buffer instead of a phosphate-citrate buffer. The culture supernatant was stopped with 30% (v / v) acetonitrile.

RNAseq RNA transcript analysis was isolated using the RNeasy Plant Mini Kit (Qiagen) and the quality of the samples checked on an Agilent 2100 Bioana-lyzer. The RNA library was generated using a TruSeq Stranded mRNA Sample Prep Kit and Poly (A) Enrichment (Illumina, Switzerland) and quantitated using an Illumina Library Quantification Kit (ΚΑΡΑ Biosystems). The mixed libraries were read on a NextSeq500 instrument (Illumina) with sequence lengths of 75 nt. For the bioinformatic evaluation, the sequencing results were compared with the reference genome of T. reesei using TopHat and Bowtie 2 software (http://genome.jgi-psf.org/Trire2/Trire2.home.html) and finally quantified with HTSeq. The counted transcripts were statistically evaluated using the DESeq2 software package. Both strains tested were grown for this analysis in three biological replicates.

Biolog microarray technique

The global carbon assimilation profile was investigated using Biolog FF MicroPlate (Biolog, Inc., USA). The protocol described in Druzhinina IS et al. (Appl. Environ. Microbiol. 72 (2006): 2126-2133), with the following exceptions: The inoculum was grown on MEX plates and the assay plates were incubated at 30 ° C. Fungal growth was measured at 18, 24, 30, 36, 42, 48, 66, 72, and 90 hours in biological triplicates.

Transcript analysis by quantitative PCR (qPCR) 0.01 - 0.03 g mycelium were homogenized using a FastPrep FP120 BIO101 ThermoSavant digestion apparatus (Qbiogene, USA) in 1 mL peqGOLD TriFast DNA / RNA / protein purification system reagent (PEQLAB Biotechnologie, Germany). The RNA was isolated according to the manufacturer's instructions and finally the concentration was measured on a NanoDrop 1000 system (Thermo Scientific). Synthesis of the cDNA from the mRNA was performed by the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific).

The qPCRs were performed on a Mastercycler ep realplex 2.2 system (Eppendorf, Germany) in triplicates. The 25 μL PCR assays included 12.5 μL 2χIQ SYBR Green Mix (Bio-Rad Laboratories, USA), 100 nM forward and reverse primers, and 2.5 μL 1: 100 diluted cDNA. PCR and relative transcript rate calculations based on the reference genes actl and sarl were performed as described in Steiger MG et al. (J Biotechnol 145 (2010): 30-37). The following primers were used:

Metabolitmessungen

Equal volumes of all stopped, U-13C labeled supernatants were mixed. The stopped, U-12C labeled supernatants were individually mixed 1: 1 with the U-13C pool. These samples were prepared as described in Kluger B et al. Anal Bioanal Chem 405 (2013): 5031-5036, separated on a UHPLC system (Accela, Thermo Fisher Scientific, USA) and measured using a downstream LTQ Orbitrap XL system (Thermo Fisher Scientific). Ionization (positive) occurred at an electrospray interface. A reversed-phase XBridge C18 analytical column (150x2.1 mm id, 3.5 pm particle size) (Waters, USA), connected downstream of a C18 4x3 mm security cartridge (Phenomenex, USA), was charged with methanol (hereinafter A) and water (hereinafter B), each with 0.1% formic acid, as the eluent. The flow rate was set to 250 pL / min. The chromatography program included the following steps: constant 10% B for 2 minutes, gradient to 100% B for 30 minutes, 5 minutes 100% B. The column was then re-equilibrated with 10% B for 8 minutes. The Orbitrap mass spectrometer was operated in full scan mode (m / z 100-1000). Evaluation of the MS data was carried out as described in Bue-schl et al. (Metabolomics 10 (2014): 754-69).

Example 1: Deletion of Xppl leads to an early and increased secretion of a yellow pigment

The industrially used cellulase-producing fungus T. reesei secretes a typical, yellow pigment during growth. For earlier publication, an Xppl deletion strain and the wild type strain were cultured on CMC (Derntl C et al., Biotechnol Biofuels 8 (2015): 112). It was found that the cultures of the Xppl deletion strain became yellow earlier and more intensely than those of the comparison strain. In a repeat of the experiment, the two strains were cultured together with an Xppl overexpression strain for CMC, lactose and D-glucose. The absence of Xppl resulted in earlier and more intense yellowing of the supernatant on all three sources of carbon (Figure 1A-C). In addition, overexpression of Xppl resulted in later yellowing and less intense staining on lactose and D-glucose (Figures 1B and 1C).

Example 2: Xppl influences the expression of genes of primary and secondary metabolism

To study the effects of Xppl on the T. reesei transcriptome, an RNAseq analysis was performed. For this, the Xppl deletion strain and the wild type strain were cultured on CMC for 48 hours. The strains showed equal growth on this carbon source (Figure 2A). The individual samples were processed comparably well (in each case 50,209,455 to 57,345,532 individual sequences without significant difference between the strains). The sequences obtained were according to the reference genome of Γ. reesei (http://genome.jgi-psf.org/Trire2/Trire2.home.html) assigned to individual transcripts (assignment rates from 93.1% to 93.7%). In total, 9,129 different transcripts were identified. The six samples were clustered according to the measured number of individual transcripts. This reflects the two strains (Figure 2B). For a more detailed analysis, the individual transcripts were compared and statistically evaluated for different amounts in the two strains. The associated genes were considered differently expressed if the calculated p-value (according to Benjamini & Hochberg) was below 0.05. By means of this evaluation 995 differently expressed genes (UEG) were found. Comparably many genes were expressed less or more strongly in the Xppl deletion strain (in fact, 490 genes were upregulated and 505 genes downregulated).

Next, the LELs were classified according to their assigned KOG. Since not all genes could be assigned to a KOG, this list is shorter (only 669 entries) than the LEL list (995 entries). In order to assess which biological functions depend on Xppl, it was compared which KOGs in the RNASeq are represented with a higher proportion than would be statistically expected. The relative proportion of LEL of a given KOG at all assigned LELs was compared with the relative proportion of all KOG genes in all the assigned genes. This analysis shows that more genes from the KOG "metabolism" in the Xppl deletion strain are regulated than would be statistically expected (Figure 3). More specifically, six of the nine KOG classes are affected, including the class "biosynthesis, transport and degradation of secondary metabolites" (Figure 3). The other five classes can be summarized as primary metabolism. Especially genes of the class "carbohydrate transport and metabolism" show a striking picture: Almost all genes of this class, which are upregulated, are transporters, while the remaining genes (coding for eg hydrolases and enzymes from glycolysis) are downregulated (FIG. 4) This result suggests an attenuation of the primary metabolism in the Xppl deletion strain.

Example 3: Xppl Supports Fungal Growth

The results of the RNASeq analysis suggest that Xppl regulates the primary metabolism. To further investigate this, the carbon assimilation behavior of the Xppl deletion strain and the wild-type strain was determined in a "Biolog

Growth was measured on 95 different carbon sources, and only carbon sources on which both strains grew better than on water (control) were considered for the analysis. Deletion strain (Figure 5A) A Wilcoxon test on data pairs (averages for both strains) showed a significant difference (p <0.001) between the two strains for the two time points tested (48 and 72 hours) The statistically significant growth differential of the two strains indicates that the lower growth rate of the Xppl deletion strain is an inherent property, demonstrating that Xppl supports T. reesei growth again suggesting that Xppl activates the primary metabolism.

Example 4: Xppl Regulates Gene Expression of Polyketide Synthases

FIG. 6 lists the results from the RNASeq analysis for all PKSs. In order to investigate more closely the regulatory influence of Xppl on the expression of the corresponding genes, the relative transcript levels of those PKSs that are most heavily regulated by RNASeq (-1> change Log2> +1) were determined over time using qPCR. Interestingly, the most highly regulated genes encoding PKS (protein IDs 73621 and 73618, FIGS. 7A and 7B) are not represented in the LEL list because no p-values could be determined in the evaluation of RNASeq (FIG. 6). For protein ID 65116, no difference between the two strains could be determined by this method (Figure 7C). Protein ID 65172 appears to be expressed earlier in the Xppl deletion strain (Figure 7D). The method gives no clear results for the proteins ID 60118 and 81964 (FIGS. 7E and 7F).

Example 5: Absence of Xppl Promotes the Secretion of Low Molecular Substances

To investigate the influence of Xppl on the global secretion of nondermolecular substances, the Xppl deletion strain, the Xppl overexpression strain, and the wild type strain were cultured in parallel on U-12C and U-13C labeled D-glucose, respectively. All low molecular weight substances from the supernatants were then purified as described in Bueschl C et al. (Metabolomics 10 (2014): 754-69). This method is based on the fact that the labeled D-glucose is the only carbon source available. For this experiment, the medium had to be modified accordingly. In a preliminary experiment, the three strains were cultured in the modified medium and then the growth, the course of yellowing and the expression rates of the genes coding for the PKS 73621 and 65172 were measured. As in previous experiments, the Xppl deletion strain grew worse (Figure 8A) and secreted more of the yellow pigment (Figure 8B) than the wild-type strain. The overexpression strain showed the expected opposite behavior (Figures 8A and 8B). The expression rate of the gene coding for the PKS 73621 also corresponds to the previous experiments and expectations (FIG. 8D). For the gene coding for the PKS 65172, increased expression in the Xppl deletion strain could be detected under these conditions (FIG. 8D). For the actual experiment, the low molecular weight substances were separated in the culture supernatants after 72 and 96 hours by liquid chromatography and measured in a mass spectrometer as in Bueschl C et al. (Metabolomics 10 (2014): 754-69). Significantly more individual substances were found in the Xppl deletion strain at both time points than in the two other strains (namely 31 after 72 h and 91 after 120 h, FIG. 9). Substantially fewer substances were found in the overexpression strain than in the two other strains (namely 43 after 72 h and 127 after 120 h, FIG. 9).

Example 6: A number of ascomycetes contain ortologists from Xppl

To identify orthologs from Xppl, a BLAST analysis was performed. The proteins found were entered in a cladogram (FIG. 10). Orthologues can be found, inter alia. also in the biocontrol active fungus Trichoderma atroviride, the plant pathogen Fusarium graminearum, and the pharmaceutically interesting ergot fungus Claviceps purpurea with E values of 0.0, 2e-70 and 8e-71 or sequence similarities of 86%, 60% and 55%. A "multiple alignment" results in two highly conserved regions: one C-terminal around the DNA binding domain (A394 - C505) and a second in the middle part (K120 - G337), without function prediction.

SEQUENCE LISTING <110> Vienna University of Technology <120> Procedure <130> 47436 <160> 32 <170> Patent version 3.5 <210> 1 <211> 504

<212> PRT <213> Trichoderma reesei <400> 1

Met Ala Gin Ala Leu Asp Ile Ser Ser Ser Ser Gly Pro Gly Pro Leu 1. 5 10 15

Val Ala Leu Pro Glu Arg Thr Leu Gly Pro Pro Ala Ser Ala lie Thr 20 25 30

Pro Pro Ser Thr Val Gin Arg Ser Ala Trp Thr Ser Ser Pro Arg Ala 35 40 45

Pro Ala Met Asp Met Phe Asn Phe Thr Thr Ala Ala Ser Ala Ser Ala 50 55 60

Val Ala Ser Tyr Ala Pro Ala Gly Gly Ser Asn Ser Asn Ser Asn Ser 65 70 75 80

Asn Ser Gly Gly Asn Ser Gly Gly Leu Gly Asp Met Asn Phe Ser Gin 85 90 95

Met Pro Gin Tyr Ser Gly Arg Asn Pro Leu Gin Ser Met Leu Ser Gin 100 105 110

Thr Lys Glu Gly Pro Asp Ala Lys Arg Ile Lys Val Glu Ser Pro His 115 120 125

Ser Ala Leu Asp Ser He Asp Ser Trp Leu Gin Phe Asp Glu Asp Ala 130 135 140

Asp Lys Phe Gly Ser Phe Glu l Asp Phe Ser Lys Gin Tyr Asn Thr 145 150 155 160

Ala Asn Gin Pro Arg Ala Ser Thr Asn Met Thr Pro Gly Leu Gly Ser 165 170 175

Gly Leu Tyr Ala Asn Pro Thr Ala Pro Phe Arg Glu Glu Asp Phe Leu 180 185 190

Asp Asp Ser Ala Phe Asp His Ser Leu Ser Glu Asp Asp Glu Met Phe 195 200 205

Asp Asn lie Asn lie Asn Asp Gin Leu Ser Lys Leu Gly Thr Leu Pro 210 215 220

Ser Thr Glu Ala Gin Ser Ser Arg Ser Leu Phe Ser Thr Pro Ser Met 225 230 235 240

Gly Trp Glu Lys Pro Gin Gly Val Gin Pro Ser Ala Val Met Gly 245 250 255

Asp Glu Ile Pro Arg Arg Met Thr Ser Asp Ser Trp Asp Pro Pro Pro 260 265 270

Gin Gly Thr Asn Tyr Thr Leu Thr Pro Asp Glu Arg Arg Arg Leu Leu 275 280 285

Glu Ile Ala Met Gly Pro Gly Arg Leu Ala Ser Tyr Val Asn Pro Ser 290 295 300

Arg Phe Asn Met Gly Phe Gly Ser Thr Met Gin Pro Ser Ile Ser Gin 305 310 315 320

Glu Phe Gly Gly Phe Gly Ala Pro Lys Gin Gly Leu Ser His Met Gly 325 330 335

Met Tyr Gin Arg Asn Asn Ser Ala Asp Thr Ser Ala Ser Pro Leu Ser 340 345 350

Gin Ser Arg Glu Leu Lys His Lys Gin Pro Lys Val Glu Thr Pro Pro 355 360 365

Lys Thr Val Pro Thr Lys Arg Pro Ser Thr Leu Ser Glu Thr Ser Ser 370 375 380

Ile Gly Lys Glu Lys Val Lys Ser Ala Asp Arg Met Ala His Asp Asp 385 390 395 400

Val Glu Arg Lys Tyr Arg Thr Asn Leu Lys Asp Lys Ile Thr Glu Leu 405 410 415

Arg Asn Ala Val Pro Ala Leu Gin Gin Val Ala Glu Gly Glu Pro Asp 420 425 430

Ala Ala Gin Gly Ile Ala Lys Val Ser Lys Gly Thr Val Leu Thr Lys 435 440 445

Ala Thr Glu Tyr Ile His Gin Leu Glu Gin Arg Asn Lys Asp Ile Met 450 455 460

Thr Glu His Lys Gin Leu Met Arg Arg Leu Gin Ala Phe Glu Ala Leu 465 470 475 480

Leu Asn Asn Ser Met Arg Val Asp Ile Met Pro Ser His Ser Met Thr 485 490 495

Leu Phe Asp Pro Arg Gly Phe Cys 500 <210> 2 <211> 3655

<212> DNA <213> Trichoderma reesei <400> 2 cagatggtga agacgggcag aaaggggagc gaaagctgaa aaatacttaa gcagcagctg 60 ttccccgtac gttcgtggag atggcaagag cagccggccg cggggagtct ggagcaggag 120 ccgcagctgc gtagctgccg gttgctgctg cccatctgcc caggttcgtt tgcgccggat 180 ccgtccaaat atctcctgat aaatgcggcc acttttccct catgctcgtc cttttttatt 240 ccccgaagcg cagcaaacga gcagcaagag caaagagcag agcagaaagt cgcctcatct 300 cgggcgccgc cttgcctaac ggggtagcaa taagcccatc gacaccgacg gcgggcggcg 360 ggctggccaa tcagcggcgc gcggtgctgt tcctttggcc cttggagcct tggggaatca 420 tacgatgtct gagcagccaa ttcttgcatt cttcccacat gcatggcatc ccatggcgca 480 gggtcccccc cccttctccc ctctccgctc tcttctcctc tcttcttggc tgaggtgctc 540 tgctccctct gctcggccag ctaagacaac cagcgctgtg gcccggcaag acagccactc 600 ttgtcattcg ccaggggggt tggaggaggg cgccacggct ccgcggaaag tcaggccatt 660 ttcatcaaca gccgcagaca ctggcccaga gaactatcct tatcctggac gcgcgcacgt 720 cgaaaatttt catttttgct cgcccaggcg cgcctgcaat tcgccatgcg ggctgctcga 780 tacccacgca gacggcaaat gacaggccat tctggctcac agac GCCGCC gtcccggctc 840 tcgccgaggt ggaaaagggt catctgggtt cgttgcgctg agctctagct aggccagaag 900 acacagccga gacatgccta ttcgacgtgc ctacggggcg gaggcagagg gcagaggaca 960 ggagggcagg aggacatccc aacaaaggct ccccatgccg gaaacgcaaa gagaagcatc 1020 ggagagctgc gaagcacccg ccgaaacagc cactacctta cccagcaaga gcagcagctc 1080 tcttgaatgg cctgactggc gacaccgcgg gtccctagcg ccggggcctt gcctggatgg 1140 caccggtccc tggagcggga tcctcgtcgc tggtgctcga cgggctgtgg tgctgctgtg 1200 cgtcttgcgc tgccggctgg gctgggctgg gctgggctgg atctgagctg cttgcagcgc 1260 cgttccctca ggcttcggaa tcgcagccgt cgcggcgttg cccgaacagc cacccggagc 1320 ttggcctgcc tctgcgaggt gctaatgctg aaactggtcg accctgtata ttacccgtga 1380 tgctctggca ccgcagctcc agacccagac ccgggtctag gttcccaata tcgatcctgt 1440 agcattgata tcgaacggta cttgctgggt actacctccc actcttgctg ctcggtaccc 1500 tgccctgtct cacctgtgca tgcacagaag caccatcact atccattgac ttccaatttt 1560 gcatctgcat ccgtcggcct atcttgccgg catctacttc tacttctact tctacgtctc 1620 cgcctgcact cgcattgacc tctttttgca tctacatctg cggcagagct gt gcgctatc taccttacct 1680 cgtcttgaac ccccctgatc cgagcatcgt atccctccat aaccacctcg 1740 gctcgctacc tcacctcctt cgtgataggt attcaatcgt ggtgtctctt gctcatctgc 1800 atctggaatt cttccctttc tcctcttgac atcttctacc tattatcgcg ctcttcctcc 1860 tccgtctgca accccccaga cgccgccgaa cccgatctgt ggctgccatt agcctttcgg 1920 cgcgcattcc tctacgccac ccggtttggg tcccccttca cccccagcac atattccgac 1980 aaccctgtgt ctgcagccgc atggcacaag ccctcgacat ttcctcgtcg tcaggacccg 2040 gacccctcgt cgccctgccc gagcgaactc tcggcccccc agcctcggcc atcacgcctc 2100 cctcgaccgt ccagcgctcg gcctggacgt cgtcgcctcg cgcgcccgcc atggacatgt 2160 tcaacttcac aaccgctgcc tcggcatccg cagtcgcctc gtatgccccc gccggcggca 2220 gcaatagcaa tagcaatagc aatagcggcg gcaacagcgg cggcctgggc gacatgaact 2280 tctctcagat gccgcagtac tctggccgca atcccctgca gtcgatgctc tcccagacca 2340 aggagggccc cgatgccaag cggataaagg tcgagtcgcc gcactcggcg ctggattcga 2400 tcgattcatg gcttcagttt gacgaggatg ccgacaagtt tggcagcttc gagattgact 2460 tttccaaaca gtacaacacg gccaaccagc caaggtgagc tgcgctcctg aaccgcca at 2520 ctggtgtttc ccgctctcta acacattgcg acattaagag cttccacaaa catgacgccg 2580 ggactgggca gcggtctcta cgccaatccc accgccccct tcagagaaga agactttctc 2640 gacgacagcg cctttgacca ctctctgtcc gaagacgacg agatgttcga caacatcaac 2700 atcaatgacc agctgtccaa gcttggcacg ctgccctcga ccgaagctca gtcatccagg 2760 agtctcttct ccacgccgtc gatgggctgg gagaagcctc agcaaggcgt ccagcccagc 2820 gccgtcatgg gcgacgagat cccgaggcgc atgactagcg attcctggga cccgattccg 2880 cagggcacca attacaccct cacgccggac gagcgccgcc gactgctcga aatcgccatg 2940 ggccccggca gattggcgtc ctacgtcaac cccagccggt tcaacatggg cttcggatct 3000 acgatgcagc ccagcataag ccaggagttt ggcttcggct tcggcgctcc gaaacagggg 3060 ctgagccata tgggcatgta ccagaggaac aactcggccg acacgagcgc atcccccctc 3120 tcgcagagtc gggaattgaa acacaagcag cccaaggtcg agacgccgcc gaagacggtg 3180 cccacaaagc ggcccagcac gctttcggag acgagcagca tcggcaaaga aaaggtcaag 3240 tccgccgacc gcatggcgca caacgacgtt gagcgcaagt atcgaaccaa tctcaaggac 3300 aagattaccg agctgcgaaa tgcggtgccg gcgctgcagc aggtggctga aggggagccg 336 0 gacgctgcgc agggcattgc caaagtgagc aaggtgagtt ggaaaacagc atgccccatc 3420 ttgtcagtga ctcgtgaaga cggcatcact gactctgatc ctatagggca ccgtattgac 3480 aaaagcgact gaatacattc atcagctcga gcaacgcaac aaggatatca tgacggagca 3540 taagcagctt atgcgacgac tgcaagcctt cgaagcactt ctcaacaact cgatgagagt 3600 cgacatcatg cccagccata gcatgacgct cttcgacccg agaggattct gttga 3655 <210> 3 <211> 17

<212> PRT <213> Artificial Sequence <22 0> <223> Artificial Peptides <22 0> <221> MISC_FEATURE <222> (5) .. (5)

<223> Xaa is V or I <22 0> <221> MISC_FEATURE <222> (15) .. (15)

<223> Xaa is D or V <4 0 0> 3

Ala His Asn Asp Xaa Glu Arg Lys Tyr Arg Thr Asn Leu Lys Xaa Lys 1. 5 10 15 lie <210> 4 <211> 15

<212> PRT <213> Artificial Sequence <22 0> <223> Artificial Peptides <22 0> <221> MISC_FEATURE <222> (2) .. (2)

<223> Xaa is V or I <22 0> <221> MISC_FEATURE <222> (6) .. (6)

<223> Xaa is T or A <22 0> <221> MISC_FEATURE <222> (7) .. (7)

<223> Xaa is V or I <22 0> <221> MISC_FEATURE <222> (9) .. (9) <223> Xaa can be any amino acid residue <4 0 0> 4

Lys Xaa Ser Lys Gly Xaa Xaa Leu Xaa Lys Ala Thr Glu Tyr lie 1. 5 10 15 <210> 5

<211> 6 <212> PRT <213> Artificial Sequence <22 0> <223> Artificial Peptides <22 0> <221> MISC_FEATURE <222> (2) .. (2)

<223> Xaa is R or K <4 0 0> 5

Arg Xaa Leu Gin Ala Phe 1. 5

<210> 6 <211> 8 <212> PRT <213> Artificial Sequence <22 0> <223> Artificial Peptides <4 0 0> 6

Leu Phe Asp Pro Arg Gly Phe Cys 1. 5 <210> 7 <211> 9

<212> PRT <213> Artificial Sequence <22 0> <223> Artificial Peptides <22 0> <221> MISC_FEATURE <222> (3) .. (3)

<223> Xaa is F or Y <22 0> <221> MISC_FEATURE <222> (4) .. (4)

<223> Xaa is D or E <22 0> <221> MISC_FEATURE <222> (7) .. (7)

<223> Xaa is F or Y <22 0> <221> MISC_FEATURE <222> (9) .. (9) <223> Xaa is R or K <4 0 0> 7

Gly Ser Xaa Xaa was given by Asp Xaa Ser Xaa 1. 5

<210> 8 <211> 8 <212> PRT <213> Artificial Sequence <22 0> <223> Artificial Peptides <22 0> <221> MISC_FEATURE <222> (5) .. (5)

<223> Xaa is S or T <22 0> <221> MISC_FEATURE <222> (7) .. (7)

<223> Xaa is V, I or L <400> 8

Pro Gly Leu Gly Xaa Gly Xaa Tyr 1. 5 <210> 9

<211> 21 <212> PRT <213> Artificial Sequence <22 0> <223> Artificial Peptides <22 0> <221> MISC_FEATURE <222> (13). , (13)

<223> Xaa is R or K <22 0> <221> MISC_FEATURE <222> (18) .. (18)

<223> Xaa is I or L <4 0 0> 9

Arg Glu Gly Leu Tyr Ser Thr Pro Leu Ser Trp Glu Xaa Pro Gin Pro 1. 5 10 15

Gly Xaa Arg Met Asp 20 <210> 10 <211> 17

<212> PRT <213> Artificial Sequence <22 0> <223> Artificial Peptides <22 0> <221> misc_feature <222> (11) 7. (11) <223> Xaa can any naturally occurring amino acid <4 0 0> 10

Gly Arg Ala Pro Gin Leu Ser Gin Gin Xaa Gin Gin Gin Gin Gin 1. 5 10 15

Gin <210> 11 <211> 19

<212> PRT <213> Artificial Sequence <22 0> <223> Artificial Peptides <22 0> <221> MISC_FEATURE <222> (8) .. (8)

<223> Xaa is R or K <22 0> <221> MISC_FEATURE <222> (17). , (17)

<223> Xaa is S or T <4 0 0> 11

Pro Pro Glu Val Pro Pro Xaa Glu Gly Leu Tyr Ser Thr Pro Leu 1. 5 10 15

Xaa Trp Glu <210> 12

<211> 20 <212> PRT <213> Artificial Sequence <22 0> <223> Artificial Peptides <22 0> <221> MISC_FEATURE <222> (5) .. (5)

<223> Xaa is A or P <22 0> <221> MISC_FEATURE <222> (17). , (17)

<223> Xaa is L or M <4 0 0> 12

Pro Ser Thr Glu Xaa Gin Ser Ser Arg Ser Leu Tyr Ser Thr Pro Ser 1. 5 10 15

Xaa Gly Trp Glu 20 <210> 13 <211> 23

<212> PRT <213> Artificial Sequence <22 0> <223> Artificial Peptides <4 0 0> 13

Thr Lys Ala Lys Pro Gly Glu Lys Met Ala Gin Leu Glu Ser Ile Ser 1. 5 10 15

Thr Ser Gin Gin Pro Leu Asp 20 <210> 14 <211> 15

<212> PRT <213> Artificial Sequence <22 0> <223> Artificial Peptides <22 0> <221> misc_feature <222> (9) .7 (9) <223> Xaa may be any naturally occurring amino acid <4 0 0> 14

Ala Leu Glu Asn Ser Ala Gin Gly Xaa Asp Lys Phe Gin Pro Gly 1. 5 10 15 <210> 15 <211> 49

<212> PRT <213> Artificial Sequence <22 0> <223> Artificial Peptides <22 0> <221> MISC_FEATURE <222> (5) .. (5)

<223> Xaa is V or I <22 0> <221> MISC_FEATURE <222> (15) .. (15)

<223> Xaa is D or V <22 0> <221> MISC_FEATURE <222> (18) .. (18) <223> Xaa is a peptide having 20 to 40 amino acid residues <22 0>

<221> MISC_FEATURE <222> (20) .. (20)

<223> Xaa is V or I <22 0> <221> MISC_FEATURE <222> (24) .. (24)

<223> Xaa is T or A <22 0> <221> MISC_FEATURE <222> (25). , (25)

<223> Xaa is V or I <22 0> <221> misc_feature <222> (27) 7. (27) <223> Xaa can be any naturally occurring amino acid <22 0> <221> MISC_FEATURE <222> ( 34) .. (34) <223> Xaa is a peptide having 15 o 20 amino acid residues <22 0> <221> misc_feature <222> (36) .. (36) <223> Xaa can any naturally occurring amino acid <22 0> <221> MISC_FEATURE <222> (41) .. (41) <223> Xaa is a peptide having 15 to 25 amino acid residues <4 0 0> 15

Ala His Asn Asp Xaa Glu Arg Lys Tyr Arg Thr Asn Leu Lys Xaa Lys 1. 5 10 15

Ile Xaa Lys Xaa Ser Lys Gly Xaa Xaa Leu Xaa Lys Ala Thr Glu Tyr 20 25 30

Ile Xaa Arg Xaa Leu Gin Ala Phe Xaa Leu Phe Asp Pro Arg Gly Phe 35 40 45

Cys <210> 16 <211> 59

<212> PRT <213> Artificial Sequence <22 0> <223> Artificial Peptides <22 0> <221> MISC_FEATURE <222> (3) .. (3)

<223> Xaa is F or Y <22 0> <221> MISC_FEATURE <222> (4) .. (4)

<223> Xaa is D or E <220> <221> MISC_FEATURE <222> (7) .. (7)

<223> Xaa is F or Y <220> <221> MISC_FEATURE <222> (9) .. (9)

<223> Xaa is R or K <220>

<221> MISC_FEATURE <222> (10) .. (10) <223> Xaa is a peptide having 200 to 300 amino acid residues <22 0> <221> MISC_FEATURE <222> (15) .. (15)

<223> Xaa is V or I <22 0> <221> MISC_FEATURE <222> (25). , (25)

<223> Xaa is D or V <22 0> <221> MISC_FEATURE <222> (28) .. (28) <223> Xaa is a peptide having 20 to 40 amino acid residues <22 0> <221> MISC_FEATURE < 222> (30). , (30)

<223> Xaa is V or I <22 0> <221> MISC_FEATURE <222> (34). , (34)

<223> Xaa is T or A <22 0> <221> MISC_FEATURE <222> (35). , (35)

<223> Xaa is V or I <22 0> <221> misc feature <222> (37) 7. (37) <223> Xaa can any natural occurring amino acid <22 0> <221> MISC_FEATURE <222> (44) (44) <223> Xaa is a peptide having 15 o 20 amino acid residues <22 0> <221> misc_feature <222> (46) 7. (46) <223> Xaa can be any naturally occurring amino acid <22 0> <221> MISC_FEATURE <222> (51) .. (51) <223> Xaa is a peptide having 15 to 25 amino acid residues <4 0 0> 16

Gly Ser Xaa Xaa, Asp Xaa Ser Xaa Xaa Ala His Asn Asp Xaa Glu 1. 5 10 15

Arg Lys Tyr Arg Thr Asn Leu Lys Xaa Lys Ile Xaa Lys Xaa Ser Lys 20 25 30

Gly Xaa Xaa Leu Xaa Lys Ala Thr Glu Tyr Ile Xaa Arg Xaa Leu Gin 35 40 45

Ala Phe Xaa Leu Phe Asp Pro Arg Gly Phe Cys 50 55 <210> 17

<211> 20 <212> DNA <213> Artificial Sequence <22 0> <223> Primer <4 0 0> 17 tgagagcggt ggtatccacg 20 <210> 18 <211> 25

<212> DNA <213> Artificial Sequence <22 0> <223> Primer <4 0 0> 18 ggtaccacca gacatgacaa tgttg 25 <210> 19 <211> 23

<212> DNA <213> Artificial Sequence <22 0> <223> Primer <4 0 0> 19 tggatcgtca actggttcta cga 23 <210> 20 <211> 23

<212> DNA <213> Artificial Sequence <22 0> <223> Primer <4 0 0> 20 gcatgtgtag caacgtggtc ttt 23

<210> 21 <211> 21 <212> DNA <213> Artificial Sequence <22 0> <223> Primer <400> 21 agctactcaa caacgtgacg c 21

<210> 22 <211> 21 <212> DNA <213> Artificial Sequence <22 0> <223> Primer <4 0 0> 22 atcccactgc tgctcaggta c 21 <210> 23

<211> 22 <212> DNA <213> Artificial Sequence <22 0> <223> Primer <4 0 0> 23 ggcctttgtt cttcatgact cc 22 <210> 24 <211> 19

<212> DNA <213> Artificial Sequence <22 0> <223> Primer <4 0 0> 24 gtgagccaag gcatcttcg 19 <210> 25

<211> 22 <212> DNA <213> Artificial Sequence <22 0> <223> Primer <4 0 0> 25 cggtatgata tcagaggttg gc 22 <210> 26 <211> 17

<212> DNA <213> Artificial Sequence <22 0> <223> Primer <4 0 0> 26 ctgggccatg agcttgc 17 <210> 27 <211> 19

<212> DNA <213> Artificial Sequence <22 0> <223> Primer <4 0 0> 27 tagccaggcg aattatgcg 19

<210> 28 <211> 20 <212> DNA <213> Artificial Sequence <22 0> <223> Primer <4 0 0> 28 ggtcctgggt atcgcctatc 20 <210> 29

<211> 20 <212> DNA <213> Artificial Sequence <22 0> <223> Primer <4 0 0> 29 agatttctga cgcggacctc 20 <210> 30

<211> 20 <212> DNA <213> Artificial Sequence <22 0> <223> Primer <4 0 0> 30 agggcaatag acttgacggg 20 <210> 31

<211> 21 <212> DNA <213> Artificial Sequence <22 0> <223> Primer <4 0 0> 31 taccaactct tcgcaaacgt g 21 <210> 32 <211> 23

<212> DNA <213> Artificial Sequence <22 0> <223> Primer <4 0 0> 32 ccaggagtga tccagaagaa gtc 23

Claims (15)

Claims: A method for producing at least one secondary metabolite in a genetically modified fungal cell, characterized in that the method comprises cultivating a genetically modified fungal cell capable of producing the at least one secondary metabolite, wherein the unmodified fungal cell comprises at least one gene encoding at least one polypeptide comprising at least two motifs selected from the group consisting of the amino acid sequences AHNDX1ERKYRTNLKX2KI (SEQ ID NO: 3), KX3SKGX4X5LX5KATEYI (SEQ ID NO: 4), RX7LQAF (SEQ ID NO: 5), LFDPRGFC (SEQ ID No. 6) and variants thereof having an identity of at least 60%, wherein Xi is V or I, X2 is D or V, X3 is V or I, X4 is T or A, X5 is V or 1. Xg is any amino acid and X7 is R or K, and the expression of said polypeptide in the genetically modified fungal cell is reduced compared to the unmodified fungal cell and / or the polypeptide in the genetically modified fungal cell comprises at least one mutation compared to the unmodified fungal cell whereby the formation of the at least one secondary metabolite in the genetically modified fungal cell is increased compared to the unmodified fungal cell. 2. The method according to claim 1, characterized in that the at least one polypeptide at least one further motif selected from the group consisting of the amino acid sequences GSX8X9IDXi0SXii (SEQ ID NO. 7), PGLGX12GX13Y (SEQ ID NO. 8), REGLY-STPLSWEX14PQPGX15RMD (SEQ ID No. 9), GRAPQLSQQQXQQQQQQ (SEQ ID NO: 10), PPPEVPPXi7EGLYSTPLXi8WE (SEQ ID NO: 11), PSTEX19QSSRSLYSTPSX20GWE (SEQ ID NO: 12), TKAKPGEKMAQLESISTSQQPLD (SEQ ID NO: 13), ALENSAQGX21DKFQPG (SEQ ID NO: 11). 14) and variants thereof having an identity of at least 60%, wherein X8 is F or Y, X9 D or E, Χ40 F or Y, X41 R or K, X42 S or T, Xi3 V, I or L, X14 R or K, X15 I or L, X46 is any amino acid, X17 R or K, X18 S or T, X19 A or P, X2o L or M and X2i is any amino acid. 3. The method according to claim 1 or 2, characterized in that the polypeptide has a range of at least 60% ident, preferably at least 70% ident, even more preferably at least 80% ident, with amino acid residues 118 to 157 and / or 394 to 494 is the amino acid sequence SEQ ID NO: 1. 4. The method according to any one of claims 1 to 3, characterized in that the polypeptide is at least 60% ident, preferably at least 70% ident, even more preferably at least 80% ident, with the amino acid sequence SEQ ID NO. 5. The method according to any one of claims 1 to 4, characterized in that the at least one mutation is a deletion, substitution or insertion of at least one amino acid residue. 6. The method according to any one of claims 1 to 5, characterized in that the polypeptide comprises at least one mutation in at least one motif selected from the group consisting of the amino acid sequences AHNDX1ERKYRTNLKX2KI (SEQ ID NO: 3), KX3SKGX4X5LX6KATEYI (SEQ ID NO: 4), RX7LQAF (SEQ ID NO: 5), LFD-PRGFC (SEQ ID NO: 6) and variants thereof having an identity of at least 60%. 7. The method according to any one of claims 2 to 6, characterized in that the polypeptide comprises at least one mutation in at least one motif selected from the group consisting of the amino acid sequences GSXsXglDXxoSXn (SEQ ID No. 7), PGLGX12GX13Y (SEQ ID No. 8), REGLYSTPLSWEX14PQPGX15RMD (SEQ ID NO: 9), GRAPQLSQQQXi6QQQQQQ (SEQ ID NO: 10), PPPEVPPXi7EGLYSTPLXi8WE (SEQ ID NO: 11), PSTEX19QSSRSLYSTPSX20GWE (SEQ ID NO: 12), TKAKPGEK-MAQLESISTSQQPLD (SEQ ID NO: 13), ALENSAQGX21DKFQPG (SEQ ID No. 14) and variants thereof having an identity of at least 60%. 8. The method according to any one of claims 1 to 7, characterized in that the reduction of the expression of the polypeptide in the genetically modified fungal cell by modification of the gene or parts thereof is achieved. 9. The method according to claim 8, characterized in that the promoter of the gene or the polypeptide coding region of the gene is mutated. 10. The method according to claim 9, characterized in that a mutation of the promoter is a deletion, insertion or substitution of at least one nucleotide. 11. The method according to any one of claims 1 to 10, characterized in that the fungal cell is a fungal cell of the class of Sordariomycetes, in particular the family Hypocreaceae, Clavici-pitaceae, Glomerellaceae or Nectriaceae. 12. The method according to any one of claims 1 to 11, characterized in that the fungal cell is a fungal cell of the genus Trichoderma, Claviceps, Colletotrichum or Fusarium. 13. The method according to any one of claims 1 to 12, characterized in that the fungal cell is selected from the group consisting of Trichoderma reesei, Trichoderma harzianum, Trichoderma virens, Trichoderma atroviride, Tolypocladium ophio-glossoides, Ophiocordyceps unilateralis, Hirsutella minnesoten-sis , Metarhizium rohertsii, Metarhizium anisopliae, Metarhizium majus, Neonectria ditissima, Nectria haematococca, Villosiclava virens, Colletotrichum suhlineola, Colletotrichum fioriniae, Colletotrichum graminicola, Claviceps purpurea, Stachybotrys chartarum, Stachybotrys chlorohalonata, Fusarium avenaceum, Colletotrichum gloeosporioides, Fusarium langsethiae, Fusarium pseudograminearum, Fusarium graminearum, Fusarium fujikuroi, Fusarium oxysporum, Verticillium longisporum, Fusarium verticil-lioides, Colletotrichum orbiculare, Thielaviopsis punctulata, Madurella mycetomatis, Verticillium dahlia, Phaeoacremonium minimum, Thielavia terrestris, Magnaporthe oryzae, Neurospora tet-raspe rma, Myceliophthora thermophila, Neurospora crassa, Metarhicon acridum, Magnaporthiopsis poae, Sordaria macrospora, Po-dospora anserine, Metarhizium guizhouense, Chaetomium globosum, Metarhizium brunneum, Gaeumannomyces graminis, Sporothrix schenckii, Sporothrix brasiliensis, Ophiostoma piceae, Colle- totrichum higginsianum, Metarhizium album and Diaporthe ampeli-na. 14. The method according to any one of claims 1 to 13, characterized in that the at least one secondary metabolite is selected from the group consisting of polyketides, alkaloids, terpenes, melanins, pteridines, phenylpropanoids, flavonoids, and non-ribosomal peptides. 15. The method according to any one of claims 1 to 14, characterized in that the genetically modified fungal cell comprises at least one nucleic acid molecule coding for at least one heterologous protein.