WO2011161063A1 - Leaa from trichoderma reesei - Google Patents

Leaa from trichoderma reesei Download PDF

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WO2011161063A1
WO2011161063A1 PCT/EP2011/060254 EP2011060254W WO2011161063A1 WO 2011161063 A1 WO2011161063 A1 WO 2011161063A1 EP 2011060254 W EP2011060254 W EP 2011060254W WO 2011161063 A1 WO2011161063 A1 WO 2011161063A1
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protein
nucleic acid
trichoderma reesei
promoter
glucanase
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Christian Kubicek
Bernhard Seiboth
Rita Linke
Razieh Karimi
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Technische Universitaet Wien
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Technische Universitaet Wien
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a transcription promoting protein methyltransferase of Trichoderma reesei.
  • filamentous fungi such as
  • Trichoderma reesei can be used to produce valuable compounds. Due to their glycosylation and secretion capacities, filamentous fungi are preferred hosts for producing secreting proteins.
  • Trichoderma reesei shows a relatively high expression of cellu- lases and hemicellulases which indicates that the expression of these enzymes is somehow up-regulated.
  • the present invention relates to an isolated poly ⁇ peptide with protein methyltransferase activity having at least 80% identity with amino acid sequence SEQ ID No. 1.
  • the polypeptide of the pre ⁇ sent invention which may be encoded by nucleotide sequence SEQ ID No. 2 from Trichoderma reesei, is able to regulate the ex ⁇ pression rate of a specific group of proteins which are all re ⁇ lated to biomass degradation (carbohydrate-active enzymes, CA- Zymes) . It could be shown herein that if the concentration of the polypeptide of the present invention is reduced within the cell the amount of the aforementioned proteins is also reduced. On the other hand an increase of the concentration of the poly ⁇ peptide of the present invention within the cell leads to an in ⁇ creased production of CAZymes. This data clearly demonstrate that the regulation of the expression of the protein methyl- transferase having at least 80% identity with amino acid se ⁇ quence SEQ ID No. 1 allows the regulation of the expression rate of specific proteins within a cell.
  • bio- mass degrading proteins are usually clustered within the genome of fungi like the enzymes involved in the biosynthesis of secon ⁇ dary metabolites.
  • the latter are known to occur in clusters, frequently near the telomere end of the chromosomes.
  • the Aspergilli such clusters of secondary metabo ⁇ lite genes have been demonstrated to be epigenetically regulated at an upper hierarchic level by the protein methyltransferase LaeA, by reversing the repressing heterochromatin structure resulting from methylation of K9 on histone 3A and binding of the heterochromatin protein HepA to histone 3A.
  • cellulase forma ⁇ tion is regulated by an LaeA orthologue which exhibits at least 80% identity with amino acid sequence SEQ ID No. 1.
  • the findings of the present invention can be used to provide host cells, in particular genetically modified fungi like
  • Trichoderma reesei which show an increased or reduced CAZyme expression activity.
  • Trichoderma reesei cells which do not express biomass degrading enzymes (by inactivating (e.g. gene deletion or disruption) of the gene encoding for the protein methyltransferase of the pre ⁇ sent invention) .
  • biomass degrading enzymes by inactivating (e.g. gene deletion or disruption) of the gene encoding for the protein methyltransferase of the pre ⁇ sent invention
  • cells which show a high activity of said enzymes can be achieved by increasing the expression rate of the protein methyltransferase according to the present invention. This can be achieved by introducing some more copies of a nucleic acid molecule harboring a nucleic acid stretch encoding for the pro ⁇ tein methyltransferase of the present invention.
  • the promoter region of the native gene may be modified to com ⁇ prise homologous or heterologous promoter which are much strong ⁇ er than the native promoter.
  • one of the above identified enzymes, which are regulated by said pro ⁇ tein methyltransferase may be exchanged by genetic manipulation by a heterologous nucleic acid molecule encoding for a product of interest. Since the genome sequence of Trichoderma reesei, for instance, is known in the art such manipulations can be eas ⁇ ily performed.
  • the polypeptide showing transcription promoting activity may be at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, in particular 100%, identical with amino acid se ⁇ quence SEQ ID No . 1.
  • SEQ ID No . 1 SEQ ID No . 1 :
  • a further aspect of the present invention relates to an iso ⁇ lated nucleic acid molecule encoding an isolated polypeptide having at least 80% identity with amino acid sequence SEQ ID No. 1.
  • the nucleic acid molecule according to the present invention exhibits preferably at least 80% identity with nucleic acid se ⁇ quence SEQ ID No . 2.
  • Nucleic acid sequence SEQ ID No. 2 is directly derived from Trichoderma reesei. However, according to the present invention this sequence may of course vary provided that the encoded pro ⁇ tein still exhibits the transcription promoting protein methyl- transferase of the polypeptide disclosed herein.
  • the nucleic acid molecule exhibits at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, in particular 100%, identity with nucleic acid sequence SEQ ID No. 2.
  • a further aspect of the present invention relates to a vec ⁇ tor comprising an isolated nucleic acid molecule according to the present invention.
  • the nucleic acid molecule of the present invention is provided in a vector.
  • the vector may be an expression vector capable to express the polypeptide of the present invention.
  • the vector of the present invention may also be a recombination vector which allows to transfer a nucleic acid molecule encoding the polypep ⁇ tide of the present invention into the genome of a host cell.
  • the vector of the present invention may comprise further ele ⁇ ments such as additional coding sequences within the same tran ⁇ scription unit, controlling elements such as promoters, ribosome binding sites, transcription terminators, polyadenylation sites, additional transcription units under control of the same or dif ⁇ ferent promoters, sequences that permit cloning, expression, ho ⁇ mologous recombination, and transformation of a host cell.
  • controlling elements such as promoters, ribosome binding sites, transcription terminators, polyadenylation sites, additional transcription units under control of the same or dif ⁇ ferent promoters, sequences that permit cloning, expression, ho ⁇ mologous recombination, and transformation of a host cell.
  • the vector of the present invention preferably comprises further at least one promoter operably linked to said nucleic acid molecule.
  • At least one promoter is provided within the vector.
  • "Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter "operably linked" to a coding sequence is pre ⁇ sent in the cell in such a way that expression of the coding se ⁇ quence can be directly influenced by the promoter.
  • the at least one promoter is selected from the group consisting of tefl promoter (transcription elongation factor 1), gpgl promoter, pkil promoter, enol promoter and pgkl promoter.
  • promoters which can be used to regulate gene expression in host organisms such as Trichoderma reesei.
  • Particularly preferred promoters are those regulating the protein expression of the following pro- teins in Trichoderma reesei: GH5 glycoside hydrolase (protein ID 81087), Bradorhizobium bleomycin resistance (protein ID 103009), unknown hypothetical protein (protein ID 106270), HHE domain protein, conserved (protein ID 70608), hypothetical conserved protein (protein ID 109925), PTHll-type GPCRs (protein ID
  • protein ID 79202 protein ID 79202
  • unknown protein protein ID 60370
  • hypothetical secreted protein protein ID 122889
  • Flavonol reduc ⁇ tase/cinnamoyl-CoA reductase protein ID 111716
  • Zinc-binding oxidoreductase protein ID 23292
  • hypothetical protein protein ID 124198
  • unknown protein protein ID 109523
  • Kynurenine aminotransferase glutamine transaminase K
  • GPRl/FUN34/yaaH-like protein protein ID 60810
  • hypothetical protein protein ID 54352
  • Preferred promoters are selected from the group of promoters selected from the group consisting of cbhl, cbh2, xynl, xyn2, xyn3, egll, gna3, envl, cDNAl, bxll, pkil, gpdA, gpdl or hexl promoters.
  • Preferred pro ⁇ moters are also promoters disclosed, for instance, in Nakari- Setala et al . (Appl . Env. Microbiol. 61 (1995), 3650-3655; cDNAl promotor) , Rahman et al . (Biosci. Biotechnol. Biochem. 73
  • the promoters may be of heterologous or homologous origin.
  • Suitable promoters can also be provided by using the method disclosed in US 5,989,870.
  • Another aspect of the present invention relates to a recom ⁇ binant host cell comprising a nucleic acid molecule or a vector according to the present invention.
  • more than one copy of the nucleic acid molecule or vector according to the present inven ⁇ tion are preferably provided in said cells.
  • the host cell is a fungus, preferably a fungus of the class of Sordariomycetes , more preferably a fungus of the family of Hy- pocreaceae, even more preferably a fungus of the genus of
  • Trichoderma in particular Trichoderma reesei.
  • a further aspect of the present invention relates to a ge ⁇ netically modified Trichoderma reesei cell overexpressing a pol ⁇ ypeptide with transcription promoting protein methyltransferase having at least 80% identity with amino acid sequence SEQ ID No. 1 compared to the genetically unmodified wild-type Trichoderma reesei cell.
  • telomere ⁇ telomere ⁇ fers to the property of the genetically Trichoderma reesei cell to express (i.e. to produce or to synthesize) said polypeptide to a higher extent than the genetically unmodified wild-type Trichoderma reesei cell from which the genetically modified
  • Trichoderma reesei cell is derived and which is used to obtain said genetically modified Trichoderma reesei cell.
  • the genetically modified Trichoderma reesei cell expresses at least 20 %, pref ⁇ erably at least 30 %, more preferably at least 40 %, particu ⁇ larly at least 50 %, more polypeptide of the present invention than the genetically unmodified wild-type Trichoderma reesei cell. This amount can be determined by methods known in the art such as ELISA or other methods involving antibodies specifically binding to the polypeptide of the present invention.
  • the methods for obtaining a genetically modified Trichoderma reesei cell ac ⁇ cording to the present invention are well known in the art.
  • the Trichoderma reesei cell according to the present inven ⁇ tion comprises preferably a vector according as defined above.
  • said cell comprises at least one mutation within the 5 ' -region of the genome location comprising nucleic acid sequence SEQ ID No. 2 or a variant thereof having at least 80% identity to SEQ ID No . 2.
  • the Trichoderma reesei genome location comprising the 5' and 3' region of nucleic acid SEQ ID No. 2 comprises the following nucleotide sequence (SEQ ID No. 3; italic: intron, underlined: coding region) :
  • the 5' region of SEQ ID No. 2 as evidenced above may be mutated by incorporating additional or alternative regulatory sequences.
  • These regulatory sequences include promot ⁇ ers, such as those listed above in connection with the vectors of the present invention.
  • the at least one mutation is a deletion, an insertion or a point mutation .
  • said Trichoderma reesei cell comprises within the 5'- region of the genome location comprising nucleic acid sequence SEQ ID No. 2 a promoter, a transcription factor binding site or a functional fragment thereof is inserted resulting in an in ⁇ creased expression of the polypeptide encoded by the nucleic ac ⁇ id sequence SEQ ID No. 2 or a variant thereof having at least 80% identity to SEQ ID No. 2 compared to the wild-type Tricho ⁇ derma reesei.
  • the Trichoderma reesei cell of the present invention pref ⁇ erably comprises further a recombinant nucleic acid coding re ⁇ gion operatively linked to a Trichoderma reesei promoter
  • nucleic acid coding region operatively linked to a Trichoderma reesei promoter sequence, wherein the nucleic acid coding region is under the
  • the nucleic acid coding region encodes preferably for a pep ⁇ tide, a polypeptide, a protein or a functional DNA or RNA.
  • a nucleic acid molecule encoding for a protein, polypeptide, peptide or functional DNA of interest may be introduced into the genome of Trichoderma reesei by gene replacement at the genome location comprising one or more of the genes encoding the proteins listed in table 1 of the example section.
  • Methods for per ⁇ forming a gene replacement are well known in the art (see e.g. Guangtao Z et al . , J. Biotechnol. 139 (2009): 146-151).
  • the ge ⁇ nomic sequence of Trichoderma reesei is known in the art (Marti ⁇ nez D et al., Nat. Biotechnol.
  • genes are examples that are abundantly expressed on lactose, and present in LAEl-regulated genomic clusters: alcohol oxidase AOX1 (Trire2: 80659), a hexose transporter (Trire2: 105260), the lactate/pyruvate transporter (Trire2: 121441), a major facilitator superfamily protein (Tri- re2:70972), and GPR1 (Trire2 : 60810) .
  • Cel6A Trire2 : 72567)
  • Cel7A Trire2 : 123989
  • Cel61B Trire2: 120961
  • Cel5A Trire2 : 120312
  • Yet another aspect of the present invention relates to a method for the recombinant production of a peptide, a polypep ⁇ tide, a protein or a functional DNA or RNA comprising the step of cultivating a genetically modified Trichoderma reesei cell according to the present invention.
  • Trichoderma reesei As described above and shown in the examples the expression of specific proteins in Trichoderma reesei is controlled by the protein methyltransferase of the present invention.
  • This control mechanism allows to generate recombinant Trichoderma reesei cells which harbor heterologous nucleic acid molecules within the genomic loci of the nucleic acid molecules naturally regu ⁇ lated by the polypeptide of the present invention within the cell.
  • biomass degrading enzymes naturally occurring in Trichoderma reesei cells and to isolate them.
  • genetically modified Trichoderma reesei cells as de ⁇ scribed above have to be used which overexpress the polypeptide of the present invention in comparison to wild-type Trichoderma reesei cells.
  • Fig. 1 shows the effect of loss-of-function of lael on bio ⁇ mass formation and cellulase/hemicellulase enzyme formation by T. reesei.
  • Biomass on cellulose is quantified as the fungal protein that can be extracted from the cellulose-fungus debris by 0.1 M NaOH (1 h, 30 °C) and refered to 1 L of culture, whereas that on glycerol is given by the biomass dry weight per L.
  • Fig. 2 shows the expression of the two cellulase genes cbhl, encoding CEL7A (a) and cbh2, encoding CEL6A (b) in T. reesei QM 9414 and the Alael mutant during growth or incubation, respec ⁇ tively, on glycerol, lactose and sophorose.
  • Expression in QM 9414 is given with full bars and set to 1.0 for every condition. The respective expression levels in relation to the wild-type are shown with open bars. Data are means of triplicate determi ⁇ nations from two biological replica.
  • Fig. 3 shows the biomass formation (A), cellulase production (B) and extracellular protein (C) during growth of T. reesei QM 9414 (QM) and several mutant strains bearing an additional copy of the lael gene (DO, Dl, D2, D3, D7) on lactose.
  • the three bars represent (from left to right) values for 48, 72 and 96 hrs of cultivation .
  • Fig. 4 shows the biomass formation (A), cellulase production (B) and extracellular protein (C) during growth of T. reesei QM 9414 (QM) and several mutant strains bearing an additional copy of the tefl:lael gene construct (Wl , P8, 01, Ml-2, M2-3, El, Nl) on lactose.
  • the three bars represent (from left to right) values for 48, 72 and 96 hrs of cultivation.
  • T. reesei QM9414 (ATCC 26921), an early cellulase producing mutant and H. jecorina KU70, a derivative of the QM 9414 uridine auxotrophic pyr4 negative strain TU-6 (ATCC MYA-256) , and which bears a deletion in the ku70 gene and is thus deficient in non ⁇ homologous end joining, were used in this example.
  • Escherichia coli JM109 Promega, USA was used for plasmid construction and amplification .
  • T. reesei was grown in Mandels-Andreotti medium [26], using Avicel cellu ⁇ lose, lactose, oat spelts xylan or glycerol as a carbon source (1 %, w/v) as stated at the respective results.
  • Induction of cellulases by sophorose (0.5 mM) in pregrown, washed mycelia was performed as described (Sternberg et al . , J Bacteriol 139
  • T. reesei a 1.2 kb lael coding region was replaced by the T. reesei pyr4 (orotidine 5'- phosphate decarboxylase-encoding) gene. This was performed by amplifying around 1 kb of the up- and downstream non-coding region of lael from genomic DNA of T. reesei QM9414 using the primer pairs given in the following table: Table A: Oligonucleotide primers used for construction of vec ⁇ tors for lael deletion and amplification
  • Resulting PCR fragments were ligated by T/A cloning into pGEM-T Easy (Promega, USA) .
  • the upstream non-coding region was excised by digestion with Xhol/Hindlll and the downstream region by Xhol/Apal from the pGEM-T Easy backbone, and then both frag ⁇ ments were ligated into a Apal/Hindlll restricted vector
  • PCR fragments were cloned into pGEM-T Easy, lael was then ex ⁇ cised with Spel/Pstl and ptrA by PstI /Hindi I I , respectively, lael was subsequently ligated into pBluescript SK(+), previously cut with Spel/Pstl, followed by the cloning of ptrA into the re ⁇ sulting plasmid plaelptrA.
  • RNA was reverse transcribed with the RevertAidTM First Strand cDNA Kit
  • RNAs were extracted using TRIzol® reagent (In- vitrogen Life Technologies, USA) , according to the manufac ⁇ turer's instructions, and then purified using the RNeasy MinE- lute Cleanup Kit (Qiagen, Germany) . The RNA quality and quantity were determined using a Nanodrop spectrophotometer. High quality purified RNAs were submitted to Roche-NimbleGene (40 ⁇ g per 3- microarray set) where cDNAs were synthesized, amplified and la ⁇ beled and then used for subsequent hybridization.
  • T. reesei strains in which lael was deleted and strains, which expressed lael under the strong constitutive expression signals of the tefl (translation elongation factor 1-alpha encoding) promoter region .
  • T. reesei pyr4 orotidine 5'- phosphate decarboxylase-encoding
  • Oligonucleotide primers used for construction of vectors for lael deletion and overexpression
  • TrLaelATGCla GCTATCGATGTCTCGAAACGCTCCCAAC
  • the two resulting PCR fragments were digested with Hin- dlll/XhoI (upstream region) and Apal/Xhol (downstream region) and ligated into a Apal /Hindlll restricted vector pBluescript SK(+) (Stratagene, La Jolla, California), followed by the inser ⁇ tion of the 2.7 kb Sail fragment of T. reesei pyr4 in the Xhol site resulting in pRKBSl.
  • a 1,820-bp lael PCR fragment including the coding and terminator region with the oligonucleotides TrLaelATGCla and TrLaelTermHind was amplified and the fragment was inserted downstream of the tefl promoter region (Genbank accession number Z23012.1) into the Clal /Hindlll sites of pLHlhphtefl resulting in vector
  • pRKBS3 which contains the E. coll hygromycin B phosphotrans ⁇ ferase (hph) under T. reesei expression signals as selection marker (Akel et al . , Eukaryot Cell 8 (2009): 1837-1844) .
  • RNAs were ground in liquid nitrogen using a mortar and pestle.
  • Total RNAs were extracted using TRIzol® reagent (Invi- trogen Life Technologies, USA), according to the manufacturer's instructions, and then purified using the RNeasy MinElute Clean ⁇ up Kit (Qiagen, Germany) .
  • the RNA quality and quantity were de ⁇ termined using a Nanodrop spectrophotometer. High quality purified RNAs were submitted to Roche-NimbleGen (40 ⁇ g per 3- microarray set) where cDNAs were synthesized, amplified and la ⁇ belled and then used for subsequent hybridization.
  • T. reesei high density oligonucleotide (HDO) microarray (Roche-NimbleGen, Inc., USA) was constructed, using 60-mer probes (7 probes per gene, 10 transcripts with less than 7 probes; a total of 63836 probes) representing the 9.143 genes of T. reesei.
  • HDO high density oligonucleotide
  • Microarray scanning, data acquisition and identification of probe sets showing a significant difference (p ⁇ 0.05) in ex ⁇ pression level between the different strains were performed by Roche-NimbleGen (www . nimblegen . com) .
  • Transcripts showing sig ⁇ nificantly down-regulated expression in the lael strain (at least 2-fold changes) were annotated manually.
  • the dataset was also manually screened for the downregulation of genes encoding carbohydrate active enzymes to at least 2-fold changes.
  • the mi ⁇ croarray data and the related protocols are available at the GEO web site (www . ncbi . nlm. nih . gov/geo/ ) under accession number:
  • GSE22687 platform GPL10642
  • T. reesei genes have not yet been mapped to chromosomes, but their appearance on genomic scaffolds is known.
  • we aligned them onto an ordered list of genes on the individual scaffolds. Distances ( numbers of genes) between positive hits were re ⁇ corded. Clustering of transcripts was considered to appear if the distance between them was at least 3-fold smaller than the average distribution of the 769 significantly regulated
  • RNA DNase treated (DNase I, RNase free; Fermentas) RNA (5yg) was reverse transcribed with the RevertAidTM First Strand cDNA Kit (Fermentas) according to the manufacturer's protocol with a com ⁇ bination of oligo-dT and random hexamer primers of the following table :
  • the protein contained the expected SAM domain, and four S and three T resi ⁇ dues were detected which fulfill the consensus of phosphoryla ⁇ tion by respective protein kinases (analyzed by NetPhos v 2.0; Blom et al . J. Mol. Biol. 294: 1351-1362 (1999)). Consistent with data from Aspergillus LaeA (Bok and Keller, Eukaryot. Cell 3, 527 (2004)), a conventional nuclear localization signal was not found.
  • Lael null mutants were created by replacing the lael coding region with the orotidine-5-decarboxylase gene pyr410 in T. reesei KU70 (a ku70 delta strain) .
  • Growth of the Alael- deleted strain on simple carbon sources such as glycerol was similar to that of the parent strain, but growth on cellulose was severely impaired ( Figure 1 a) , indicating that the loss of lael function indeed leads to a defect in growth on cellulose.
  • microarrays representing all 9130 unique al ⁇ leles in the genome of T. reesei were used to examine their transcript levels when grown on lactose as a carbon source. 765 genes exhibited an at least twofold decrease in their hybridiza ⁇ tion intensity in the Alael strain compared to QM 9414.
  • carbohydrate-active enzyme encoding genes were de ⁇ tected, which in majority comprised glycosyl hydrolases involved in cellulose and hemicellulose degradation (GHs; Table 1) : they included all 10 cellulases (CEL5A, CEL5B, CEL6A, CEL7A, CEL7B, CEL12, CEL45, CEL61A, CEL61B and CEL74), both known swollenins (SWOl, SW02 ; proteins carrying an expansin-like domain and that disrupt the crystalline cellulase structure) and CIPs (CIPl, CIP2; proteins that contain a signal peptide and a cellulose- binding domain) , 5 of the 7 known ⁇ -glucosidases (CEL1A, CEL1B, CEL3C and CEL3D) , and all 4 xylanases (XYN1-XYN4) .
  • the majority of the other affected GHs (21 of 28) comprised
  • the 25 carbohydrate-active enzyme clusters in the T. reesei genome contain an average five-fold increase in carbohydrate- active enzyme gene density compared to the expected density for randomly distributed genes. 765 of the total 9130 genes in the T. reesei genome to be at least 2-fold downregulated in the Alael strain were identified, thus implying that at a random distribution one at every twelfth gene should be found. If the genes would however be clustered as calculated above, the aver ⁇ age gene density of LAEl-affected genes should be around 2.5. To investigate this, the 765 identified genes on the T. reesei scaffolds were mapped and searched for potential clusters. In ⁇ deed, 28 areas on 21 scaffolds were found that exhibited a four ⁇ fold increase of gene density over the random distribution.
  • gene density is defined as the number of genes present in a proposed cluster, divided by the number of genes whose expres ⁇ sion was affected at least 2-fold by the lael deletion 32 of the 67 downregulated CAZyme genes were located within these clusters. Interestingly, 13 of these 29 clusters were found in areas not previously predicted.
  • cel7A and cel6A were used as cellulase model genes and their ex ⁇ pression in the parent strain and in the Alael strain was spe ⁇ cifically tested by Real Time PCR. These cells were cultivated on lactose, sophorose (a disaccharide confering high cellulase induction in resting cells) and on the non-inducing carbon source glycerol ( Figure 2) . The data confirmed the finding of the microarray experiments as gene expression was absent in the Alael strains on all three carbon sources. The results also dem ⁇ onstrate that the nature of the inducer does not influence the epigenetic regulation of cellulase formation.
  • LAE1 as a regulator of cellulase and hemi- cellulase biosynthesis in T. reesei
  • two approaches were used: in one, a second copy of T. reesei lael was introduced into its genome.
  • coli hygromycin B phos ⁇ photransferase (hph) expression cassette as fungal selection marker.
  • a 1,820-bp lael PCR fragment including the coding and terminator region was amplified with the oligonucleotides
  • TrLaelATGCla and TrLaelTermHind (Table A, above) and inserted downstream of the tefl promoter region at a Clal/Hindlll cleavage site resulting in vector Ptefllaelhph .

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Abstract

The present invention relates to isolated polypeptide with transcription promoting protein methyltransferase activity having at least 80% identity with amino acid sequence SEQ ID No. 1 and uses thereof.

Description

LEAA FROM TRICHODERMA REESEI
The present invention relates to a transcription promoting protein methyltransferase of Trichoderma reesei.
It is known in the art that filamentous fungi such as
Trichoderma reesei can be used to produce valuable compounds. Due to their glycosylation and secretion capacities, filamentous fungi are preferred hosts for producing secreting proteins.
Most of the industrial production of enzymes for plant bio- mass hydrolysis is performed with mutants of the fungus Tricho¬ derma reesei (the anamorph of the tropical ascomycete Hypocrea jecorina) . It is known that the genome contains a lower number of cellulase and hemicellulase genes than the genomes of other fungi. Although the number of said genes is relatively low
Trichoderma reesei shows a relatively high expression of cellu- lases and hemicellulases which indicates that the expression of these enzymes is somehow up-regulated.
It is an object of the present invention to provide means to improve the expression of homologous and heterologous proteins, in particular enzymes, in fungi, in particular in Trichoderma reesei. This means should be able to up- as well as down- regulate protein expression in fungi. Furthermore this means may also be used in recombinant protein expression.
Therefore the present invention relates to an isolated poly¬ peptide with protein methyltransferase activity having at least 80% identity with amino acid sequence SEQ ID No. 1.
It surprisingly turned out that the polypeptide of the pre¬ sent invention, which may be encoded by nucleotide sequence SEQ ID No. 2 from Trichoderma reesei, is able to regulate the ex¬ pression rate of a specific group of proteins which are all re¬ lated to biomass degradation (carbohydrate-active enzymes, CA- Zymes) . It could be shown herein that if the concentration of the polypeptide of the present invention is reduced within the cell the amount of the aforementioned proteins is also reduced. On the other hand an increase of the concentration of the poly¬ peptide of the present invention within the cell leads to an in¬ creased production of CAZymes. This data clearly demonstrate that the regulation of the expression of the protein methyl- transferase having at least 80% identity with amino acid se¬ quence SEQ ID No. 1 allows the regulation of the expression rate of specific proteins within a cell.
The above described effect is a result of the fact that bio- mass degrading proteins are usually clustered within the genome of fungi like the enzymes involved in the biosynthesis of secon¬ dary metabolites. The latter are known to occur in clusters, frequently near the telomere end of the chromosomes. In another fungal genus, the Aspergilli, such clusters of secondary metabo¬ lite genes have been demonstrated to be epigenetically regulated at an upper hierarchic level by the protein methyltransferase LaeA, by reversing the repressing heterochromatin structure resulting from methylation of K9 on histone 3A and binding of the heterochromatin protein HepA to histone 3A. Because of the clustered co-occurrence of cellulase and secondary metabolite syn¬ thesis genes in the Trichderma reesei genome, cellulase forma¬ tion is regulated by an LaeA orthologue which exhibits at least 80% identity with amino acid sequence SEQ ID No. 1.
As indicated in Table 1 of the examples section the regula¬ tion of the expression of the polypeptide having amino acid se¬ quence SEQ ID No. 1 results in the regulation of the protein ex¬ pression of the following proteins (classification in accordance with Henrissat B and Bairoch A, Biochem J. 316(1996) : 695-696; www.cazy.org/Glycoside-Hydrolases.html): GH 5 endo-B-1,4- glucanase Cel5A, GH5 endo-β-Ι , 4-glucanase CEL5B, GH6 Cellobiohy- drolase 2 CEL6A, GH7 endo-β, 4-glucanase EGL1, GH7 cellobiohy- drolase 1 CEL7A, GH12 endo-β-Ι , 4-glucanase 12a, GH45 endo-B-1,4- glucanase EG5, GH61 endo-β-Ι , 4-glucanase CEL61A, GH61endo-B-l , 4- glucanase CEL61B, GH1 β-glucosidase CEL1B, GH1 β-glucosidase CEL1A, GH3 β-glycosidase of uncertain specificity, GH3 β- glucosidase CEL3D, GH3 β-glucosidase CEL3C, CIP2, CIPl, CBM13 protein, swollenin, swollenin-like, 84 % ID to 123992, GH10 xy- lanase XYN3, GHllxylanase XYN1, GH11 xylanase XYN2, GH30 xy- lanase XYN4, GH3 β-xylosidase BXL1, GH43 β-xylosidase/ - arabinofuranosidase, GH74 xyloglucananase CEL74a, hemicellulose side chain cleaving enzymes, CE5 acetyl xylan esterase AXE1, GH67 -glucuronidase AGU1, GH62, -L-arabinofuranosidase ABF2, GH54, L- -arabinofuranosidase ABF1, GH95 -fucosidase, GH95 - fucosidase, GH92 -1 , 2-mannosidase, GH92 -1 , 2-mannosidase, GH47 oi-1 , 2-mannosidase, GH2 β-mannosidase, GH27 -galactosidase AGL1, GH27 a-galactosidase AGL3 and GH28 polygalacturonase.
The findings of the present invention can be used to provide host cells, in particular genetically modified fungi like
Trichoderma reesei, which show an increased or reduced CAZyme expression activity. For instance, it is possible to provide Trichoderma reesei cells which do not express biomass degrading enzymes (by inactivating (e.g. gene deletion or disruption) of the gene encoding for the protein methyltransferase of the pre¬ sent invention) . However, it is also possible to provide cells which show a high activity of said enzymes. The latter effect can be achieved by increasing the expression rate of the protein methyltransferase according to the present invention. This can be achieved by introducing some more copies of a nucleic acid molecule harboring a nucleic acid stretch encoding for the pro¬ tein methyltransferase of the present invention. Alternatively the promoter region of the native gene may be modified to com¬ prise homologous or heterologous promoter which are much strong¬ er than the native promoter. In order to produce heterologous proteins in a fungus like Trichoderma reesei under the control of the protein methyltransferase of the present invention one of the above identified enzymes, which are regulated by said pro¬ tein methyltransferase, may be exchanged by genetic manipulation by a heterologous nucleic acid molecule encoding for a product of interest. Since the genome sequence of Trichoderma reesei, for instance, is known in the art such manipulations can be eas¬ ily performed.
Methods for the production of such genetically modified fun¬ gi, in particular Trichoderma reesei, are well known in the art. For instance, in WO 2006/060126 methods for transforming and cultivating Trichoderma reesei cells are disclosed.
It is emphasized that also a functional fragment of the pol¬ ypeptide showing protein methyltransferase activity and having at least 80% identity with amino acid sequence SEQ ID No. 1 is subject of the present invention. "Functional fragments" of the protein methyltransferase of the present invention refer to pos¬ sible fragments which still retain the activity of the full length polypeptide.
According to the present invention the polypeptide showing transcription promoting activity may be at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, in particular 100%, identical with amino acid se¬ quence SEQ ID No . 1. SEQ ID No . 1 :
MSRNAPNGCVPPSQATAPPSPATSLRLTVGEPVSEPATESGERVLQDGFWEHGRFYGSWKPGKY LFPIDKEELNRLDVFHKYFLVARDEKVTSTPLRKDGRPKIMDLGTGTGIWAYNVVEEYAKDAEI MAVDLNQIQPALI PRGV KQFDIEEPSWDPLLRDCELIHMRLLYGS IRDDKWPHVYRKAFEHL APGIGYIEQLEIDWMPRWENEDLPRHSALQEWAQLFQRAMHRYHRSVTVSGEATRRRMEAAGFT DFSETTIRCYVNPWSPDRHQRECARWFNLAFSLGLEAMSMMPMIDKLGMTKDDIVDLCSRAKKE MCILRYRAYCTL
A further aspect of the present invention relates to an iso¬ lated nucleic acid molecule encoding an isolated polypeptide having at least 80% identity with amino acid sequence SEQ ID No. 1.
The nucleic acid molecule according to the present invention exhibits preferably at least 80% identity with nucleic acid se¬ quence SEQ ID No . 2.
SEQ ID No . 2 :
ATGTCTCGAAACGCTCCCAACGGGTGTGTTCCACCCTCCCAAGCTACTGCTCCGCCTTCGCCAG CCACAAGTCTGCGACTAACAGTTGGGGAACCGGTCAGCGAGCCGGCCACTGAATCCGGGGAGAG AGTTCTCCAGGATGGGTTCTGGGAGCACGGTCGCTTTTATGGTTCTTGGAAGCCTGGGAAATAC CTTTTCCCCATAGACAAGGAGGAGCTCAATAGGTTAGATGTCTTTCACAAGTATTTCCTCGTTG CAAGAGACGAGAAAGTCACTTCAACTCCCCTGAGGAAAGATGGACGGCCGAAAATCATGGATCT CGGCACAGGCACGGGCATCTGGGCGTATAATGTTGTGGAAGAGTATGCCAAGGATGCCGAAATC ATGGCCGTGGATCTCAATCAAATTCAACCAGCTCTGCACTTGGCCCCTGGCATTGGCTATATCG AGCAACTGGAGATTGACTGGATGCCGCGATGGGAGAATGAGGATCTCCCCAGACATTCGGCTCT TCAAGAATGGGCTCAGCTATTCCAACGTGCCATGCATCGCTACCACCGCAGCGTCACGGTATCA GGCGAGGCTACCAGACGCAGAATGGAAGCGGCTGGCTTTACAGATTTCTCCGAAACAACGATCC GGTGCTACGTAAACCCGTGGTCTCCCGATCGCCATCAGCGGGAGTGTGCCCGTTGGTTCAACCT CGCCTTCAGCCTCGGCCTTGAGGCCATGAGCATGATGCCAATGATTGACAAACTCGGCATGACC AAGGACGATATC
Nucleic acid sequence SEQ ID No. 2 is directly derived from Trichoderma reesei. However, according to the present invention this sequence may of course vary provided that the encoded pro¬ tein still exhibits the transcription promoting protein methyl- transferase of the polypeptide disclosed herein.
According to the present invention the nucleic acid molecule exhibits at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, in particular 100%, identity with nucleic acid sequence SEQ ID No. 2.
A further aspect of the present invention relates to a vec¬ tor comprising an isolated nucleic acid molecule according to the present invention.
In order to transfer a nucleic acid molecule encoding the polypeptide of the present invention to a host cell the nucleic acid molecule of the present invention is provided in a vector. The vector may be an expression vector capable to express the polypeptide of the present invention. However, the vector of the present invention may also be a recombination vector which allows to transfer a nucleic acid molecule encoding the polypep¬ tide of the present invention into the genome of a host cell. The vector of the present invention may comprise further ele¬ ments such as additional coding sequences within the same tran¬ scription unit, controlling elements such as promoters, ribosome binding sites, transcription terminators, polyadenylation sites, additional transcription units under control of the same or dif¬ ferent promoters, sequences that permit cloning, expression, ho¬ mologous recombination, and transformation of a host cell.
The vector of the present invention preferably comprises further at least one promoter operably linked to said nucleic acid molecule.
In order to control the transcription of the nucleic acid molecule of the present invention within the cell at least one promoter is provided within the vector. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A promoter "operably linked" to a coding sequence is pre¬ sent in the cell in such a way that expression of the coding se¬ quence can be directly influenced by the promoter.
According to a preferred embodiment of the present invention the at least one promoter is selected from the group consisting of tefl promoter (transcription elongation factor 1), gpgl promoter, pkil promoter, enol promoter and pgkl promoter.
Of course it is also possible to use any other promoter which can be used to regulate gene expression in host organisms such as Trichoderma reesei. Particularly preferred promoters are those regulating the protein expression of the following pro- teins in Trichoderma reesei: GH5 glycoside hydrolase (protein ID 81087), Bradorhizobium bleomycin resistance (protein ID 103009), unknown hypothetical protein (protein ID 106270), HHE domain protein, conserved (protein ID 70608), hypothetical conserved protein (protein ID 109925), PTHll-type GPCRs (protein ID
109146), MSF permease (protein ID 78585), glutathione-S- transferase (protein ID 112022), Catalase (protein ID 58472), mannose- 6-phosphate isomerase (protein ID 60445) , unknown pro¬ tein (protein ID 105287), translation initiation regulator Gnc20
(protein ID 22839) , imidazole proprionase-related amidohydrolase
(protein ID 110757), MSF transporter (protein ID 3405), short chain dehydrogenase/reductase (protein ID 106164), MSF permease
(protein ID 79202), unknown protein (protein ID 60370), hypothetical secreted protein (protein ID 122889), Flavonol reduc¬ tase/cinnamoyl-CoA reductase (protein ID 111716), Zinc-binding oxidoreductase (protein ID 23292), hypothetical protein (protein ID 124198), unknown protein (protein ID 109523), Kynurenine aminotransferase, glutamine transaminase K (protein ID 122820), GPRl/FUN34/yaaH-like protein (protein ID 60810), hypothetical protein (protein ID 54352), Predicted Zn-dependent hydrolase
(beta-lactamase superfamily) (protein ID 70197), sulfite transporter ssul (protein ID 2076), unknown protein (protein ID
4851), unknown secreted protein (protein ID 110830), unknown esterase/lipase (protein ID 70491) and MSF permease (protein ID 105260). These genes are regularly upregulated under cellulose inducing conditions (e.g. cultivation of Trichoderma reesei on lactose) .
The vector according to the present invention, which can be transformed into a Trichoderma reesei cell comprises in the 5' region of the nucleic acid molecule exhibiting at least 80% identity with nucleic acid sequence SEQ ID No. 2 or of the nu¬ cleic acid molecule encoding for a polypeptide having at least 80% identity with amino acid sequence SEQ ID No. l a promoter which allows to control the expression rate of the polypeptide of the present invention within a cell. Preferred promoters are selected from the group of promoters selected from the group consisting of cbhl, cbh2, xynl, xyn2, xyn3, egll, gna3, envl, cDNAl, bxll, pkil, gpdA, gpdl or hexl promoters. Preferred pro¬ moters are also promoters disclosed, for instance, in Nakari- Setala et al . (Appl . Env. Microbiol. 61 (1995), 3650-3655; cDNAl promotor) , Rahman et al . (Biosci. Biotechnol. Biochem. 73
(2009), 1083-1089; egl3 and xyn3 promoter), WO 98/23764 Al
(short cbhl promoter) , EP 0 952 223 Al (cbhl-promoter of Trichoderma viridae) , US 7,393,664, US 7,517,685 (hexl promoter) and Mach et al . (Curr. Genetics 25 (1994): 567-570; pkil). In a pre¬ ferred embodiment of the present invention the promoters may be of heterologous or homologous origin.
Suitable promoters can also be provided by using the method disclosed in US 5,989,870.
Another aspect of the present invention relates to a recom¬ binant host cell comprising a nucleic acid molecule or a vector according to the present invention.
In order to increase the amount of the polypeptide of the present invention within the host cell more than one copy of the nucleic acid molecule or vector according to the present inven¬ tion are preferably provided in said cells.
According to a preferred embodiment of the present invention the host cell is a fungus, preferably a fungus of the class of Sordariomycetes , more preferably a fungus of the family of Hy- pocreaceae, even more preferably a fungus of the genus of
Trichoderma, in particular Trichoderma reesei.
A further aspect of the present invention relates to a ge¬ netically modified Trichoderma reesei cell overexpressing a pol¬ ypeptide with transcription promoting protein methyltransferase having at least 80% identity with amino acid sequence SEQ ID No. 1 compared to the genetically unmodified wild-type Trichoderma reesei cell.
The term "overexpressing a polypeptide", as used herein, re¬ fers to the property of the genetically Trichoderma reesei cell to express (i.e. to produce or to synthesize) said polypeptide to a higher extent than the genetically unmodified wild-type Trichoderma reesei cell from which the genetically modified
Trichoderma reesei cell is derived and which is used to obtain said genetically modified Trichoderma reesei cell. According to a preferred embodiment of the present invention the genetically modified Trichoderma reesei cell expresses at least 20 %, pref¬ erably at least 30 %, more preferably at least 40 %, particu¬ larly at least 50 %, more polypeptide of the present invention than the genetically unmodified wild-type Trichoderma reesei cell. This amount can be determined by methods known in the art such as ELISA or other methods involving antibodies specifically binding to the polypeptide of the present invention. The methods for obtaining a genetically modified Trichoderma reesei cell ac¬ cording to the present invention are well known in the art.
The Trichoderma reesei cell according to the present inven¬ tion comprises preferably a vector according as defined above.
According to a preferred embodiment of the present invention said cell comprises at least one mutation within the 5 ' -region of the genome location comprising nucleic acid sequence SEQ ID No. 2 or a variant thereof having at least 80% identity to SEQ ID No . 2.
The Trichoderma reesei genome location comprising the 5' and 3' region of nucleic acid SEQ ID No. 2 comprises the following nucleotide sequence (SEQ ID No. 3; italic: intron, underlined: coding region) :
CCTTTTACCAACTTGGCAGCCCTTGCCTCTTCGTTGCTGGCTAGTAGGGGAGGCAGGCCATTGATCCCGG GCTCGCGTCAATCCACCAAGCCCCAAAGAGCCCTAGAAGCTCGCGACACTTGTCATTGAACCAACACGAC TCTCAACCGCCGTCTGTCGATTCTCACTTCGGCATTCGTCGACCTCCTCCCTCCAGCCGCTGGTCCACTC CGGACCCGAGCTTCGCGCCAGTCTTAAAGGGCTAGCCGTCCTCGCCCCCCCTTCTCCAGTCCGCCGAACG ACAGTCTCAACCTCAACCCTGGGAGTCGTGATAATTTTCTATACCGCCCTTCTCCGCTCTCTCAGCCGTC ACTCGCTTGTGTCTTCCAACCGGAGCTGTCCACGCTGCCGCCTGGGACGATTACGCCCTTGCCTGTCGCT GCATTCGTGCTGCAAATTGGTTGCTCCGGTGGCTCAGGTGACCCTGGTCGAGGACTTCTACCTCTTTCAA GGAGCCTCGTGAGAGTTGATACAGCAGCTGCCCTCGACAGGTCACACAAACACATGGGATCGTCGCTTAT TCTGACCTACGGCAGCTGCGCGGCAACCCACCCGTACCACAGGCATCGGTATCTGTGTCACCTCCTTGAC ATCTCGGCCAGTTGAATTTCTGATGTAAGTTGCCATCTGCCTTGTTGTTGTTCGTCTGGCTGCCCTACAC CATCTTGCCCGCCTGCTCGGCTCCCATCTACTCCTTGATTTATCTTTCCATCTTGTCTTACTGCCATGAT GCGCGTTGAAAGGCGCCATGCATGCCATCGTGTCCGGCCAGGATATGAATGCTGTGCTCAGTCAGCCGTC TTCCTTTTTGGAAGCCAATCAGCAAGGTTGAACTGTCTTTCTCTTTCCTTGCCATCATCAACCCTTGCGG CAGATGTTTATGTCTCTCGCCCCTTGCCTTGAAGTGGGCACCTCCCCGCGATTTCAAGCTGTTACACTCT TCCCCCCCTTCGGCCTCTTCTGTCGCCTTCTACCGCCCTTGGACTTCTATTTCGGTAGAAGTGCCACTTG TTCCTTGCCCAAGACATCCTCACGCGTTATACTGTACTTAACAAGCAGGGTCTCGCTCCACTCCTTGTTA TAACACCATCACATGCGCTTCTTCTTTTCAAAAAAACGGCTTGGGATTAACAAACTTGTTGTAATTAGCT ATCTAACATCTCCTTGACCGGCTCAAAATCACCGGCTATAACTACTGACCCTTCCTCTCCCCTTCCCCCT TCCTGGACCCCTTGGCACTGGACTCTGGAACATCCGCCTGGAGCCCGCCACCTGCATTCCCAAGGATTGA CCCGCCCCTCCCGTTGGCCCTCATAACCTTCGCCATTACTCACTATAAACGCCATGTCTCGAAACGCTCC CAACGGGTGTGTTCCACCCTCCCAAGCTACTGCTCCGCCTTCGCCAGCCACAAGTCTGCGACTAACAGTT GGGGAACCGGTCAGCGAGCCGGCCACTGAATCCGGGGAGAGAGTTCTCCAGGATGGGTTCTGGGAGCACG GTCGCTTTTATGGTTCTTGGAAGCCTGGGAAATACCTTTTCCCCATAGACAAGGrrrGrcrcrrrGrAGC GTCAATACCCTCCGCGCCTGTTCGTACAACTAACAACATCACCAGGAGGAGCTCRATAGGTTAGATGTCT TTCACAAGTATTTCCTCGTTGCAAGAGACGAGAAAGTCACTTCAACTCCCCTGAGGAAAGATGGACGGCC GAAAATCATGGATCTCGGCACAGGCACGGGCATCTGGGCGTATAATGTTGTGGAAGAGTAAGTTATTATA
GAGGTAGTTTCACACTACGCTGTGCCAGTTGCTCACATTTTTCAGGTATGCCAAGGATGCCGAAATCATG GCCGTGGATCTCAATCAAATTCAACCAGCTCTGTAAGTTGTGAGCTTTCAATCGCTTGACCTTTTTTTTT TTTTTTTTTTTCAAACGCTAATGCATTTGCTGAATAGCATTCCTCGAGGTGTAACAACCAAGCAGTTTGA CATTGAAGAGCCCTCGTGGGATCCACTGCTTCGGGACTGCGAATTGATCCATATGCGATTGCTATACGGC AGCATAAGAGATGACAAGTGGCCCCATGTCTACCGCAAGGCCTTTGAGTGCGTAACTCGTGTAACCAACA ACCrGCrCCGrrrcrGACGrrCGrrrACAGGCACTTGGCCCCTGGCATTGGCTATATCGAGCAACTGGAG ATTGACTGGATGCCGCGATGGGAGAATGAGGATCTCCCCAGACATTCGGCTCTTCAAGAATGGGCTCAGC TATTCCAACGTGCCATGCATCGCTACCACCGCAGCGTCACGGTATCAGGCGAGGCTACCAGACGCAGAAT GGAAGCGGCTGGCTTTACAGATTTCTCCGAAACAACGATCCGGTGCTACGTAAACCCGTGGTCTCCCGAT CGCCATCAGCGGGAGTGTGCCCGTTGGTTCAACCTCGCCTTCAGCCTCGGCCTTGAGGCCATGAGCATGA TGCCAATGATTGACAAACTCGGCATGACCAAGGACGATATCGTCGACCTCTGTAGCAGAGCCAAGAAGGA GATGTGCATTCTGCGGTACCGCGCCTATTGCACTCTGTAAGCCGCCCGCCCCCCTGACAAACACAATTTG CCGGAGCCACAACTAATCACGCTTGCAGACACATTTGGACAGCCAGAAAACCGAACGAAGATGAGTCTCA AACTTTCAAAGAAAGAGACTCCGATACGCAGCCATCTAGAAGAGAGGAATCCTCTGCTTAAGACGCCATG GTCGCCAATAGATCGGATAGAGAAGAGAAACAGCATCCTTGCGTCCACATAATACAAACGGCGACCGGCA GGGATGCGAGAGCCAGCGTTCTGCGGCTTGTCCTGTTTTCGAAGCTACAGAAGCCCACCGTGCTATGTGA GCTACCGCTTCATATATGATCGTCGCCGACGCTGAACAATGTCATGCAACCATCACCAAGATTCTCATAC AGGTATAGACGAAGCCTATTCAAGAACGAGAAGTATGCGAGGTGTGGATACTTTGGCTCTGTCTCATTCG AATGAAGCACTGCTAGTGGGTTCATGGCCGGATATATGATCTCTCGTCTTCGTTCTTTACGCTCTTGACG ATATGATGCCCGACAAAGGCCAGCCCGTTTGGTTACAATGAAAGATGACCTTGGGCTTAGACCTACTCCT CTTCTCTCCCTCTTCCTTCATCCTTTCTCTTCCCCTTGATCCCGAGGACTTATCACATGACGACGAAGAA CGGGGAAACTCTGATGGCTAGAAGCATTGTAGGGACTTAGACGAAGGTGGGGAACAGGTGTACAGAAACA TGTCGGCCAGAGTGTTATGGGACTCGGCCGTTGCAAACACGAAGAGATGGCGATAAGAGCCTGAATGGGG TTGGCGTTACAGGGGCATTGTGTATGGCTTTACTCTCTGCTGTCGATTGGATAGATCGTGTCTTTCGAAC TTGAGGATGTTGACATCACTCGTGCTGGTTTTTCTGATTATGTTGTTGGTTAATCGCTTCTGCTAGCAGG GGCATCTCGGCAAGGGGTGGGCATGACCAAGAGATGCCGGAATCACCCCATTACGAAACACTACCCAAGC TGCTAAAACATCCCCATGTGGCCAGATGCAAAGGGAACGAAAAAAAGAAGAAGAGTAAAGAAAAAGCAAA CAACACTTTGAAGATATACTATTAGGGCCCTTTGTATGATACACTTGAGACTGCCTCCTGCATGGTTTCA TCTGGGACGCTGATGCATGGATACCAGCGCCATGTAACCCGGGACAAGGTCCCCTTAGGTTTGGGTAGTC TAGGTGGTAACCTAGGCCAGTAGACAGGGGAAGGGTATGGGGGCAGACCGGGCAAATCATTTCAGGGACG GGGCAGCAAACTACGAGTGAAAGATTGAGAGGCCGAGAGGAAACTTGATACGGGTGGAAAGAGTTTGCTT CTGTTCAAAGGGGGATGTTGTTGGAGAATGGAAAGCGTGAGTCTTTTGGTGAGAATGATGTTGTGATGTT G
The 5' region of SEQ ID No. 2 as evidenced above (SEQ ID No. 3) may be mutated by incorporating additional or alternative regulatory sequences. These regulatory sequences include promot¬ ers, such as those listed above in connection with the vectors of the present invention. According to a preferred embodiment of the present invention the at least one mutation is a deletion, an insertion or a point mutation .
According to a further preferred embodiment of the present invention said Trichoderma reesei cell comprises within the 5'- region of the genome location comprising nucleic acid sequence SEQ ID No. 2 a promoter, a transcription factor binding site or a functional fragment thereof is inserted resulting in an in¬ creased expression of the polypeptide encoded by the nucleic ac¬ id sequence SEQ ID No. 2 or a variant thereof having at least 80% identity to SEQ ID No. 2 compared to the wild-type Tricho¬ derma reesei.
The Trichoderma reesei cell of the present invention pref¬ erably comprises further a recombinant nucleic acid coding re¬ gion operatively linked to a Trichoderma reesei promoter
sequence and/or a vector comprising a nucleic acid coding region operatively linked to a Trichoderma reesei promoter sequence, wherein the nucleic acid coding region is under the
transcriptional control of said promoter sequence.
The nucleic acid coding region encodes preferably for a pep¬ tide, a polypeptide, a protein or a functional DNA or RNA.
According to a preferred embodiment of the present invention a nucleic acid molecule encoding for a protein, polypeptide, peptide or functional DNA of interest may be introduced into the genome of Trichoderma reesei by gene replacement at the genome location comprising one or more of the genes encoding the proteins listed in table 1 of the example section. Methods for per¬ forming a gene replacement are well known in the art (see e.g. Guangtao Z et al . , J. Biotechnol. 139 (2009): 146-151). The ge¬ nomic sequence of Trichoderma reesei is known in the art (Marti¬ nez D et al., Nat. Biotechnol. 26 (2008): 553-560), therefore the gene replacement can easily be performed using the methods known in the art. The following genes are examples that are abundantly expressed on lactose, and present in LAEl-regulated genomic clusters: alcohol oxidase AOX1 (Trire2: 80659), a hexose transporter (Trire2: 105260), the lactate/pyruvate transporter (Trire2: 121441), a major facilitator superfamily protein (Tri- re2:70972), and GPR1 (Trire2 : 60810) .
Alternatively, if the purpose is heterologous overexpression of proteins, the following cellulase genes could be replaced: Cel6A (Trire2 : 72567) , Cel7A (Trire2 : 123989) , Cel61B (Trire2: 120961), and Cel5A (Trire2 : 120312)
Yet another aspect of the present invention relates to a method for the recombinant production of a peptide, a polypep¬ tide, a protein or a functional DNA or RNA comprising the step of cultivating a genetically modified Trichoderma reesei cell according to the present invention.
As described above and shown in the examples the expression of specific proteins in Trichoderma reesei is controlled by the protein methyltransferase of the present invention. This control mechanism allows to generate recombinant Trichoderma reesei cells which harbor heterologous nucleic acid molecules within the genomic loci of the nucleic acid molecules naturally regu¬ lated by the polypeptide of the present invention within the cell. Of course, it is also possible to exploit this effect to overexpress biomass degrading enzymes naturally occurring in Trichoderma reesei cells and to isolate them. To achieve this object genetically modified Trichoderma reesei cells as de¬ scribed above have to be used which overexpress the polypeptide of the present invention in comparison to wild-type Trichoderma reesei cells.
The present invention is further illustrated by the follow¬ ing figures and examples, however, without being restricted thereto .
Fig. 1 shows the effect of loss-of-function of lael on bio¬ mass formation and cellulase/hemicellulase enzyme formation by T. reesei. Growth of T. reesei QM 9414 and the corresponding Alael strain on 1 % (w/v) cellulose (a) and 1 % (w/v) glycerol (b) . Biomass on cellulose is quantified as the fungal protein that can be extracted from the cellulose-fungus debris by 0.1 M NaOH (1 h, 30 °C) and refered to 1 L of culture, whereas that on glycerol is given by the biomass dry weight per L. Cellulase (c) and hemicellulase (d) formation by T. reesei QM 9414 and the corresponding Alael strain on 1 % (w/v) lactose and 1 % (w/v) xylan, respectively. Experiments are means of 3-5 biological replicas .
Fig. 2 shows the expression of the two cellulase genes cbhl, encoding CEL7A (a) and cbh2, encoding CEL6A (b) in T. reesei QM 9414 and the Alael mutant during growth or incubation, respec¬ tively, on glycerol, lactose and sophorose. Expression in QM 9414 is given with full bars and set to 1.0 for every condition. The respective expression levels in relation to the wild-type are shown with open bars. Data are means of triplicate determi¬ nations from two biological replica.
Fig. 3 shows the biomass formation (A), cellulase production (B) and extracellular protein (C) during growth of T. reesei QM 9414 (QM) and several mutant strains bearing an additional copy of the lael gene (DO, Dl, D2, D3, D7) on lactose. The three bars represent (from left to right) values for 48, 72 and 96 hrs of cultivation .
Fig. 4 shows the biomass formation (A), cellulase production (B) and extracellular protein (C) during growth of T. reesei QM 9414 (QM) and several mutant strains bearing an additional copy of the tefl:lael gene construct (Wl , P8, 01, Ml-2, M2-3, El, Nl) on lactose. The three bars represent (from left to right) values for 48, 72 and 96 hrs of cultivation.
EXAMPLE :
Materials and Methods
Strains
T. reesei QM9414 (ATCC 26921), an early cellulase producing mutant and H. jecorina KU70, a derivative of the QM 9414 uridine auxotrophic pyr4 negative strain TU-6 (ATCC MYA-256) , and which bears a deletion in the ku70 gene and is thus deficient in non¬ homologous end joining, were used in this example. Escherichia coli JM109 (Promega, USA) was used for plasmid construction and amplification .
For cellulase respectively xylanase production, T. reesei was grown in Mandels-Andreotti medium [26], using Avicel cellu¬ lose, lactose, oat spelts xylan or glycerol as a carbon source (1 %, w/v) as stated at the respective results. Induction of cellulases by sophorose (0.5 mM) in pregrown, washed mycelia was performed as described (Sternberg et al . , J Bacteriol 139
(1979) : 761-769) .
Construction of a Alael strain of T. reesei
To delete the lael gene of T. reesei, a 1.2 kb lael coding region was replaced by the T. reesei pyr4 (orotidine 5'- phosphate decarboxylase-encoding) gene. This was performed by amplifying around 1 kb of the up- and downstream non-coding region of lael from genomic DNA of T. reesei QM9414 using the primer pairs given in the following table: Table A: Oligonucleotide primers used for construction of vec¬ tors for lael deletion and amplification
Figure imgf000014_0001
*Respective restriction sites are underlined
Resulting PCR fragments were ligated by T/A cloning into pGEM-T Easy (Promega, USA) . The upstream non-coding region was excised by digestion with Xhol/Hindlll and the downstream region by Xhol/Apal from the pGEM-T Easy backbone, and then both frag¬ ments were ligated into a Apal/Hindlll restricted vector
pBluescript SK(+) (Stratagene, USA). The resulting plasmid was cleaved with Xhol, dephosphorylated and the 2.7 kb Sail fragment of T. reesei pyr4 inserted resulting in pAlael.
lael gene amplification in T. reesei
To introduce a second copy of lael into the genome of T. reesei QM 9414, 900 bp of the upstream and 500 bp of the down¬ stream non-coding region of lael were amplified from genomic DNA of T. reesei QM9414 using the primer pairs given in Table A. As a selection marker, 2 kb of the A. oryzae ptrA (pyrithiamine re¬ sistance conferring) gene was amplified from plasmid pME2892 (Kubodera et al . 2000) using the primer pair given in Table A. PCR fragments were cloned into pGEM-T Easy, lael was then ex¬ cised with Spel/Pstl and ptrA by PstI /Hindi I I , respectively, lael was subsequently ligated into pBluescript SK(+), previously cut with Spel/Pstl, followed by the cloning of ptrA into the re¬ sulting plasmid plaelptrA.
mRNA extraction and Real Time PCR
DNase treated (DNase I, RNase free; Fermentas) RNA (5yg) was reverse transcribed with the RevertAid™ First Strand cDNA Kit
(Fermentas) according to the manufacturer's protocol with a com¬ bination of the provided oligo-dT and random hexamer primers. All real-time RT-PCR experiments were performed on a Bio-Rad
(USA) iCycler IQ. For the reaction the IQ SYBR Green Supermix
(Bio-Rad, USA) was prepared for 25 μΐ assays with standard MgCl2 concentration (3 mM) and a final primer concentration of 100 nM each. All assays were carried out in 96-well plates which were covered with optical tape. The amplification protocol consisted of an initial denaturation step (3 min at 95°C) followed by 40 cycles of denaturation (15 sec at 95°C), annealing (20 sec at 57°C) and elongation (10 sec at 72°C) . Determination of the PCR efficiency was performed using triplicate reactions from a dilu¬ tion series of cDNA (l.OOE-00, l.OOE-01, 1.00E-02 and 1.00E-03). Amplification efficiency was then calculated from the given slopes in the IQ5 Optical system Software v2.0. Expression ra¬ tios were calculated using REST© Software (Pfaffl M.W. et al . Nucleic Acid Research 30(2009): e36) ) . All samples were analyzed in two independent experiments with three replicates in each run. Transcriptome analysis of lael loss-of function
Mycelia from both stages were freeze-dried and ground in liquid nitrogen using a mortar and pestle. For each of the two experimental conditions, five independent replicates of mycelium were mixed. Total RNAs were extracted using TRIzol® reagent (In- vitrogen Life Technologies, USA) , according to the manufac¬ turer's instructions, and then purified using the RNeasy MinE- lute Cleanup Kit (Qiagen, Germany) . The RNA quality and quantity were determined using a Nanodrop spectrophotometer. High quality purified RNAs were submitted to Roche-NimbleGene (40 μg per 3- microarray set) where cDNAs were synthesized, amplified and la¬ beled and then used for subsequent hybridization.
A T. reesei high density oligonucleotide (HDO) microarray
(Roche-NimbleGen, Inc., USA) was constructed, using 60-mer probes representing the 9.130 genes of T. reesei. Microarray scanning, data acquisition and identification of probe sets showing a significant difference (p=0.05) in expression level between the two culture conditions considered were performed by Roche-NimbleGen . Transcripts showing significantly up-regulated expression (2-fold and 5-fold changes) were annotated using the eukaryotic orthologous groups (KOG) classification. The microar- ray data and the related protocols are available at the GEO web site (www.ncbi.nlm.nih.gov/geo/) under accession number:
GSE20516.
Construction of T. reesei strains with altered lael alleles
To study the function of LAE1, we constructed T. reesei strains in which lael was deleted and strains, which expressed lael under the strong constitutive expression signals of the tefl (translation elongation factor 1-alpha encoding) promoter region .
To delete the lael gene of T. reesei, the 1.2 kb lael coding region was replaced by the T. reesei pyr4 (orotidine 5'- phosphate decarboxylase-encoding) gene. This was performed by amplifying around 1 kb of the up- and downstream non-coding region of lael from genomic DNA of T. reesei QM9414 using the pri¬ mer pairs given in the following table:
Oligonucleotide primers used for construction of vectors for lael deletion and overexpression
Name rSequence (5 '-3'
lael gene deletion
5TrlaelHind TAAGCTTCACTCGCTTGTGTCTTC
5TrlaelXho TCTCGAGCGTTTATAGTGAGTAATGGC
3TrlaelXho TCTCGAGCTATTGCACTCTGTAAGCC
3TrlaelApa TGGGCCCTGGGTAGTGTTTCGTAATG tefl-lael construction
teflXhofw GCCTCGAGGGACAGAATGTAC
ClaSalrv AGTCGACATCGATGACGGTTTGTGTGATGTAGCGTG
TrLaelATGCla GCTATCGATGTCTCGAAACGCTCCCAAC
TrLaelTermHind CGAAGCTTGCCCAAGGTCATCTTTCATTG
*Respective restriction sites are underlined
The two resulting PCR fragments were digested with Hin- dlll/XhoI (upstream region) and Apal/Xhol (downstream region) and ligated into a Apal /Hindlll restricted vector pBluescript SK(+) (Stratagene, La Jolla, California), followed by the inser¬ tion of the 2.7 kb Sail fragment of T. reesei pyr4 in the Xhol site resulting in pRKBSl.
For expression of lael under a strong constitutive promoter, a 1,820-bp lael PCR fragment including the coding and terminator region with the oligonucleotides TrLaelATGCla and TrLaelTermHind was amplified and the fragment was inserted downstream of the tefl promoter region (Genbank accession number Z23012.1) into the Clal /Hindlll sites of pLHlhphtefl resulting in vector
pRKBS3, which contains the E. coll hygromycin B phosphotrans¬ ferase (hph) under T. reesei expression signals as selection marker (Akel et al . , Eukaryot Cell 8 (2009): 1837-1844) .
Fungal transformation
All vectors constructed were verified by sequencing. The strains were purified twice for mitotic stability, and integra¬ tion of the expression cassettes was verified by PCR analysis. Gene copy numbers of the integrated constructs were determined by Southern analysis, using chromosomal DNA cleaved with BamRI . Protoplast preparation and DNA mediated transformation was described (Guangtao et al., J Biotechnol 139 (2009): 146-151) .
Biochemical assays
Cellulase enzyme activities were determined using carboxy- methylcellulose (1 %, w/v) as described (Vaheri et al . , Biotech¬ nol Letts 1 (1979) : 41-46) . Protein in the culture supernatant was determined by the method of Bradford.
Transcriptome analysis of lael loss-of function and lael overexpression
Mycelia were ground in liquid nitrogen using a mortar and pestle. Total RNAs were extracted using TRIzol® reagent (Invi- trogen Life Technologies, USA), according to the manufacturer's instructions, and then purified using the RNeasy MinElute Clean¬ up Kit (Qiagen, Germany) . The RNA quality and quantity were de¬ termined using a Nanodrop spectrophotometer. High quality purified RNAs were submitted to Roche-NimbleGen (40 μg per 3- microarray set) where cDNAs were synthesized, amplified and la¬ belled and then used for subsequent hybridization.
A T. reesei high density oligonucleotide (HDO) microarray (Roche-NimbleGen, Inc., USA) was constructed, using 60-mer probes (7 probes per gene, 10 transcripts with less than 7 probes; a total of 63836 probes) representing the 9.143 genes of T. reesei.
Microarray scanning, data acquisition and identification of probe sets showing a significant difference (p < 0.05) in ex¬ pression level between the different strains were performed by Roche-NimbleGen (www . nimblegen . com) . Transcripts showing sig¬ nificantly down-regulated expression in the lael strain (at least 2-fold changes) were annotated manually. The dataset was also manually screened for the downregulation of genes encoding carbohydrate active enzymes to at least 2-fold changes. The mi¬ croarray data and the related protocols are available at the GEO web site (www . ncbi . nlm. nih . gov/geo/ ) under accession number:
GSE22687 (platform GPL10642) .
Analysis of genomic clustering of transcripts
T. reesei genes have not yet been mapped to chromosomes, but their appearance on genomic scaffolds is known. In order to identify whether the significantly regulated transcripts would be clustered to particular areas on these scaffolds, we aligned them onto an ordered list of genes on the individual scaffolds. Distances (=numbers of genes) between positive hits were re¬ corded. Clustering of transcripts was considered to appear if the distance between them was at least 3-fold smaller than the average distribution of the 769 significantly regulated
transcripts among all genes (9143), i.e. a third of 11.9, = 3.9.
Real Time PCR
DNase treated (DNase I, RNase free; Fermentas) RNA (5yg) was reverse transcribed with the RevertAid™ First Strand cDNA Kit (Fermentas) according to the manufacturer's protocol with a com¬ bination of oligo-dT and random hexamer primers of the following table :
Primers for cellulase transcript quantification
by Real Time PCR
Gene Forward Primer (5' to 3' ) Reverse Primer (5' to 3' ) tefl * CCACATTGCCTGCAAGTTCGC GTCGGTGAAAGCCTCAACGCAC cel7a (cbhl) Ccgagcttggtagttactctg Ggtagccttcttgaactgagt eel 6a (cbh2) ACTACAACGGGTGGAACATTAC CGTGGATGTACAGCTTCTCG lael ACTGGAGATTGACTGGATGC TTCTGCGTCTGGTAGCCTC
* tefl was used as a reference gene
All real-time RT-PCR experiments were performed on a Bio-Rad iCycler IQ. For the reaction the IQ SYBR Green Supermix (Bio- Rad) was prepared for 25 μΐ assays with standard MgCl2 concentra¬ tion (3 mM) and a final primer concentration of 100 nM each. All assays were carried out in 96-well plates. The amplification protocol consisted of an initial denaturation step (3 min at 95°C) followed by 40 cycles of denaturation (15 sec at 95°C), annealing (20 sec at 57°C) and elongation (10 sec at 72°C) . De¬ termination of the PCR efficiency was performed using triplicate reactions from a dilution series of cDNA (1; 0.1; 0.01; 0.001) . Amplification efficiency was then calculated from the given slopes in the IQ5 Optical system Software v2.0. Expression ra¬ tios were calculated using REST© Software (Pfaffl et al , , Nu¬ cleic Acid Res . 30 (2002) : e 36) . All samples were analyzed in at least two independent experiments with three replicates in each run.
Statistical Analysis^
Basic statistical methods such as multiple regression analy¬ sis and analysis of variance (Anova) as well as multivariate ex¬ ploratory techniques (cluster and factor analyses) were performed using Statistica 6.1 (StatSoft, Inc., USA) data analysis software system.
Resul ts
To identify lael, 92 S-methionyl-adenosine-dependent methyl- transferases present in the T. reesei genome database
(http : //genome .jgi-psf.org/Trire2/Trire2. home . html ) were
screened. When any of the functionally verified Aspergillus LaeA proteins was used as a query in BLASTP, several hits with nega¬ tive probabilities of <e-30 were obtained, but using these as a query in BLASTP of the respective Aspergillus genome databases
(http : //www . broadinstitute .org/annotation/genome/aspergillus_gro up/MultiHome . html ) always resulted in several hits of similar negative probability and thus identified none of them clearly as a LaeA orthologue. Since this approach was therefore prone to lead to false positives, an iterative phylogenetic strategy for its identification was used: briefly, BLASTP was used to detect LaeA orthologues in species more closer related to the Asper- gilli (such as Coccidioides immitis) and then used the identi¬ fied protein to look for LaeA orthologues in Dothidiomycetes , and used the latter one to the Sordariomycetes and finally the Hypocreaceae . By this means 27 putative LaeA orthologues from Eurotiomycetes , Dothidiomycetes and Sordariomycetes were identi¬ fied (Table below) . A phylogenetic analysis of these protein se¬ quences produced a tree whose branching was consistent with the established phylogenetic relationship within these fungi, thus proving the orthology of the identified protein sequences. The T. reesei protein Trire2 : 41617 was thus identified as the puta¬ tive LaeA orthologue, LAE1.
Figure imgf000020_0001
An examination of genomic and cDNA sequences revealed that the total coding region of Lael comprises 1061 nucleotides (SEQ ID No. 3) and is interrupted by three introns (62, 58 and 278 nts long, from 5' to 3' ) , giving rise to a 221 aa mature protein (SEQ ID No. 1) with a molecular weight of 25832 Da and an isoelectric point of 5.83 (data calculated by ProtPARAM; Gasteiger et al . 2005 (In) John M. Walker (ed) : The Proteomics Protocols Handbook, Humana Press pp. 571-607 (2005)). It exhibited an overall amino acid identity of 28 and 24 % to the known proteins from A. nidulans and A. fumigatus, respectively. The protein contained the expected SAM domain, and four S and three T resi¬ dues were detected which fulfill the consensus of phosphoryla¬ tion by respective protein kinases (analyzed by NetPhos v 2.0; Blom et al . J. Mol. Biol. 294: 1351-1362 (1999)). Consistent with data from Aspergillus LaeA (Bok and Keller, Eukaryot. Cell 3, 527 (2004)), a conventional nuclear localization signal was not found.
Lael null mutants (Alael) were created by replacing the lael coding region with the orotidine-5-decarboxylase gene pyr410 in T. reesei KU70 (a ku70 delta strain) . Growth of the Alael- deleted strain on simple carbon sources such as glycerol was similar to that of the parent strain, but growth on cellulose was severely impaired (Figure 1 a) , indicating that the loss of lael function indeed leads to a defect in growth on cellulose. To test whether this is due to a loss of cellulase formation, two deletion mutants and the parent strain were cultivated on lactose, a carbon source which induces cellulase formation, but whose utilization is independent of cellulase formation. As shown in Figure 1 b, growth of the parent strains and the mu¬ tants is indeed similar. However, significantly reduced cellu¬ lase activity was found in the mutant cultures (Figure 1 c) .
Consistent findings were obtained with xylan as a carbon source, on which the Alael-mutants exhibited a somewhat impaired growth, but xylanase activity was even more impaired (Figure 1 d) .
The above data provided a first hint towards an effect of LAE1 on cellulase and hemicellulase formation in T. reesei, but the individual effects on the various cellulolytic and hemicel- lulolytic enzymes present in its genome cannot be deduced. In order to test whether the lael deletion indeed acts at the ex¬ pression of its biomass degrading enzymes, a genome-wide ap¬ proach was used: in total, 126 of the approximately 320 carbohy¬ drate-active enzyme (CAZyme) genes of T. reesei are found in 25 discrete regions of the chromosome ranging from 14 kb to 275 kb in length. Thus, microarrays representing all 9130 unique al¬ leles in the genome of T. reesei were used to examine their transcript levels when grown on lactose as a carbon source. 765 genes exhibited an at least twofold decrease in their hybridiza¬ tion intensity in the Alael strain compared to QM 9414. Among these, 65 carbohydrate-active enzyme encoding genes were de¬ tected, which in majority comprised glycosyl hydrolases involved in cellulose and hemicellulose degradation (GHs; Table 1) : they included all 10 cellulases (CEL5A, CEL5B, CEL6A, CEL7A, CEL7B, CEL12, CEL45, CEL61A, CEL61B and CEL74), both known swollenins (SWOl, SW02 ; proteins carrying an expansin-like domain and that disrupt the crystalline cellulase structure) and CIPs (CIPl, CIP2; proteins that contain a signal peptide and a cellulose- binding domain) , 5 of the 7 known β-glucosidases (CEL1A, CEL1B, CEL3C and CEL3D) , and all 4 xylanases (XYN1-XYN4) . The majority of the other affected GHs (21 of 28) comprised glycosidases ac¬ tive against various side chains in hemicelluloses .
Table 1. Changes in carbohydrate-active enzyme gene expression in T. reesei by knocking out the function of lael * (sequences and protein ID number are obtainable at genome. jgi- psf.org/Trire2/Trire2.home.html; Martinez D et al . , Nat. Bio- technol 26 (2008) : 553-560)
Protein ID downregulated p-value
Cellulases
GH 5 endo-β-Ι , 4-glucanase Cel5A 120312 17.338 0.000896
GH5 endo-β-Ι , 4-glucanase CEL5B 82616 5.179 0.00219
GH6 Cellobiohydrolase 2 CEL6A 72567 15.565 0.00106
GH7 endo-β , 4-glucanase EGL1 122081 3.019 0.00126
GH7 cellobiohydrolase 1 CEL7A 123989 6.841 0.000994
GH12 endo-β-Ι , 4-glucanase 12a 123232 15.789 0.00132
GH45 endo-β-Ι, 4-glucanase EG5 49976 14.409 0.000892
GH61 endo-β-Ι, 4-glucanase CEL61A 73643 25.659 0.000851
GH61endo-B-l, 4-glucanase CEL61B 120961 40.524 0.000836 GH1 β-glucosidase CEL1B 22197 3.454 0.000918
GH1 β-glucosidase CEL1A 120749 2.428 0.00107
GH3 β-glycosidase of uncertain 108671 2.833 0.00142 specificity
GH3 β-glucosidase CEL3D 46816 2.115 0.00525
GH3 β-glucosidase CEL3C 82227 3.555 0.00153 nonenzymatic cellulose attacking
enzymes
CIP2 123940 3.232 0.00118
CIPl 73638 16.794 0.000855
CBM13 protein 111094 8.529 0.00407 s ollenin 123992 3.714 0.000847 s ollenin-like, 84 % ID to 123992 111874 6.17 0.00153 xylanases
GH10 xylanase XYN3 120229 4.392 0.00202
GHllxylanase XYN1 74223 2.038 0.00213
GH11 xylanase XYN2 123818 23.487 0.000931
GH30 xylanase XYN4 111849 2.121 0.000885
GH3 β-xylosidase BXL1 121127 17.003 0.000896
GH43 β-xylosidase/ - 3739 2.752 0.000911 arabinofuranosidase
GH74 xyloglucananase CEL74a 49081 3.051 0.0009 hemicellulose side chain cleaving
enzymes
CE5 acetyl xylan esterase AXE1 73632 6.821 0.000951
GH67 -glucuronidase AGU1 72526 13.365 0.00101
GH62, -L-arabinofuranosidase ABF2 76210 14.836 0.000893
GH54, L- -arabinofuranosidase ABF1 55319 2.201 0.00158
GH95 -fucosidase 58802 9.946 0.00544
GH95 a-fucosidase 5807 3.606 0.0018
GH92 oi-1 , 2-mannosidase 74198 6.097 0.000712 GH92 oi-1 , 2-mannosidase 60635 2.154 0.00372
GH47 -1 , 2-mannosidase 45717 4.496 0.00133
GH2 β-mannosidase 69245 5.937 0.00181
GH27 -galactosidase AGL1 72632 5.777 0.00134
GH27 a-galactosidase AGL3 27259 2.065 0.00641 pectlnases
GH28 polygalacturonase 103049 2.382 0.00831
* values are given as means of two biological replica; ^own- regulation" is given as -fold decrease compared to the parent strain .
The 25 carbohydrate-active enzyme clusters in the T. reesei genome contain an average five-fold increase in carbohydrate- active enzyme gene density compared to the expected density for randomly distributed genes. 765 of the total 9130 genes in the T. reesei genome to be at least 2-fold downregulated in the Alael strain were identified, thus implying that at a random distribution one at every twelfth gene should be found. If the genes would however be clustered as calculated above, the aver¬ age gene density of LAEl-affected genes should be around 2.5. To investigate this, the 765 identified genes on the T. reesei scaffolds were mapped and searched for potential clusters. In¬ deed, 28 areas on 21 scaffolds were found that exhibited a four¬ fold increase of gene density over the random distribution.
Clusters of expressed genes affected by lael-loss of function in T. reesei
scaffold cluster found** genes in expressed gene denCAZys predicted* cluster genes sity***
1 yes 618-690 72 23 3.13 5
yes 112-135 23 9 2.55 3
2 yes 410-417 7 2 3.5 2
3 no 4-27 23 6 3.83 2
yes 530-547 17 6 2.83 2 4 no 285-293 8 5 1.6 1
5 no 3-18 15 5 3 1
yes 209-233 24 8 3 1
6 no 134-138 4 4 1 1
7 no 226-252 26 8 3.25 2
yes 394-419 25 9 2.77 2
8 yes 167-192 25 7 3.51 3
10 yes 183-201 18 10 1.8 2
13 yes 16-34 18 5 3.6 1
no 43-61 18 6 3 1
14 no 187-194 7 3 2.33 1
16 no 120-134 14 4 3.5 2
19 yes 62-97 35 10 3.5 1
yes 157-189 32 12 2.67 1
22 no 48-98 50 17 2.94 1
27 yes 40-59 19 5 3.8 1
28 no 68-80 22 7 3.15 1
no 111-117 6 4 1.5 3
29 yes 91-102 11 7 1.57 2
30 no 4-19 15 4 3.75 1
31 yes 43-53 10 4 2.5 1
33 yes 29-42 13 6 2.16 1
44 no 4-15 11 5 1.2 1
* specifies that the expressed genes were found within the area of clusters proposed by Martinez et al . (Nature Biotechnol. 26, 553 (2008) ) .
** „found" means that a clustering of expressed genes was found, using the method outlined
*** gene density is defined as the number of genes present in a proposed cluster, divided by the number of genes whose expres¬ sion was affected at least 2-fold by the lael deletion 32 of the 67 downregulated CAZyme genes were located within these clusters. Interestingly, 13 of these 29 clusters were found in areas not previously predicted.
In order to confirm the results from microarray analysis, cel7A and cel6A were used as cellulase model genes and their ex¬ pression in the parent strain and in the Alael strain was spe¬ cifically tested by Real Time PCR. These cells were cultivated on lactose, sophorose (a disaccharide confering high cellulase induction in resting cells) and on the non-inducing carbon source glycerol (Figure 2) . The data confirmed the finding of the microarray experiments as gene expression was absent in the Alael strains on all three carbon sources. The results also dem¬ onstrate that the nature of the inducer does not influence the epigenetic regulation of cellulase formation.
Having identified LAE1 as a regulator of cellulase and hemi- cellulase biosynthesis in T. reesei, it was investigated whether an enhanced activity of LAE1 would even stimulate cellulase for¬ mation. To test this, two approaches were used: in one, a second copy of T. reesei lael was introduced into its genome.
In another one, lael downstream of the regulatory signals of the tefl (elongation factor la-encoding) gene was fused which allow high constitutive expression. To this end, 740 bp of the promoter region of H. jecorina tefl (Genbank accession number Z23012.1) were amplified by PCR using the oligonucleotides teflXhofw and teflClaSalrv (Table A, above); the Xhol/Sall- restricted tefl fragment was then cloned in the corresponding sites of pLHlhph, which contains an E. coli hygromycin B phos¬ photransferase (hph) expression cassette as fungal selection marker. A 1,820-bp lael PCR fragment including the coding and terminator region was amplified with the oligonucleotides
TrLaelATGCla and TrLaelTermHind (Table A, above) and inserted downstream of the tefl promoter region at a Clal/Hindlll cleavage site resulting in vector Ptefllaelhph .
Five and three T. reesei strains that contained a single lael copy and the tefl: lael construct, respectively, integrated ectopically in its genome, were examined for their ability to form cellulases on lactose (Figure 4) . Particularly the
tefl: lael copies indeed exhibited an up to 10-fold increased cellulase formation and 10-40 fold enhanced cel7A and cel6A gene expression, and this increased expression correlated with increased expression of lael in these strains.
The data presented clearly show that epigenetic manipulation significantly influences cellulase gene transcription in T.
reesei, and thus represents a so far overlooked area for strain improvement by recombinant techniques. Since LAE1 acts by inac¬ tivating the heterochromatin protein HepA or the H3K9 methyl- transferase Clr49 these findings further show that a loss-of- function in these genes also leads to an increase in cellulase formation. Orthologues for these two genes have been detected in T. reesei.

Claims

Claims :
1. Isolated polypeptide with transcription promoting protein me- thyltransferase activity having at least 80% identity with amino acid sequence SEQ ID No. 1.
2. Isolated nucleic acid molecule encoding an isolated polypep¬ tide according to claim 1.
3. Isolated nucleic acid molecule according to claim 2, charac¬ terised in that the nucleic acid molecule exhibits at least 80% identity with nucleic acid sequence SEQ ID No. 2.
4. Vector comprising an isolated nucleic acid molecule according to claim 2 or 3.
5. Vector according to claim 4 further comprising at least one promoter operably linked to said nucleic acid molecule.
6. Vector according to claim 5, characterised in that the at least one promoter is selected from the group consisting of tefl promoter (transcription elongation factor 1), gpgl promoter, pkil promoter, enol promoter and pgkl promoter.
7. Recombinant host cell comprising a nucleic acid molecule ac¬ cording to claim 2 or 3 or a vector according to any one of claims 4 to 6.
8. Host cell according to claim 7, characterised in that said cell comprises more than one copy of the nucleic acid molecule according to claim 2 or 3 or the vector according to any one of claims 4 to 6.
9. Host cell according to claim 7 or 8, characterised in that said host cell is a fungus.
10. Genetically modified Trichoderma reesei cell overexpressing a polypeptide according to claim 1 compared to the genetically unmodified wild-type Trichoderma reesei cell.
11. Trichoderma reesei cell according to claim 10, characterised in that the Trichoderma reesei cell comprises a vector according any one of claims 4 to 6.
12. Trichoderma reesei cell according to claim 10 or 11, charac¬ terised in that said cell comprises at least one mutation within the 5 ' -region of the genome location comprising nucleic acid se¬ quence SEQ ID No. 2 or a variant thereof having at least 80% identity to SEQ ID No. 2.
13. Trichoderma reesei cell according to claim 12, characterised in that the at least one mutation is a deletion, an insertion or a point mutation.
14. Trichoderma reesei cell according to claim 12 or 13, charac¬ terised in that within the 5 ' -region of the genome location com¬ prising nucleic acid sequence SEQ ID No. 2 a promoter, a tran¬ scription factor binding site or a functional fragment thereof is inserted resulting in an increased expression of the polypep¬ tide encoded by the nucleic acid sequence SEQ ID No. 2 or a var¬ iant thereof having at least 80% identity to SEQ ID No. 2 com¬ pared to the wild-type Trichoderma reesei.
15. Trichoderma reesei cell according to any one of claims 10 to 14, characterised in that the Trichoderma reesei cell comprises further a recombinant nucleic acid coding region operatively linked to a Trichoderma reesei promoter sequence and/or a vector comprising a nucleic acid coding region operatively linked to a Trichoderma reesei promoter sequence, wherein the nucleic acid coding region is under the transcriptional control of said pro¬ moter sequence.
16. Trichoderma reesei cell according to claim 15, characterised in that the nucleic acid coding region encodes for a peptide, a polypeptide, a protein or a functional DNA or RNA.
17. Trichoderma reesei cell according to claim 15 or 16, charac¬ terised in that the nucleic acid coding region is introduced in¬ to the genome of said Trichoderma reesei cell by gene replace¬ ment at the genome location comprising the coding regions for GH 5 endo-β-Ι, 4-glucanase Cel5A (protein ID 120312) , GH5 endo-B-l,4- glucanase CEL5B (protein ID 82616) , GH6 Cellobiohydrolase 2 CEL6Ac
(protein ID 72567), GH7 endo-β , 4-glucanase EGL1 (protein ID 122081), GH7 cellobiohydrolase 1 CEL7A (protein ID 123989), GH12 endo-B-l,4- glucanase 12a (protein ID 123232), GH45 endo-β-Ι , 4-glucanase EG5 (pro¬ tein ID 49976), GH61 endo-β-Ι , 4-glucanase CEL61A (protein ID 73643), GH61endo-B-l, 4-glucanase CEL61B (protein ID 120961), GH1 β-glucosidase CEL1B (protein ID 22197), GH1 β-glucosidase CEL1A (protein ID 120749), GH3 β-glycosidase of uncertain specificity (protein ID 108671) , GH3 β- glucosidase CEL3D (protein ID 46816) , GH3 β-glucosidase CEL3C (protein ID 82227) , CIP2 (protein ID 123940), CIPl (protein ID 73638) , CBM13 protein (protein ID 111094) , s ollenin (protein ID 123992) , swollenin- like, 84 % ID to 123992 (protein ID 111874), GH10 xylanase XYN3 (protein ID 120229) , GHllxylanase XYN1 (protein ID 74223), GH11 xylanase XYN2 (protein ID 123818), GH30 xylanase XYN4 (protein ID 111849) , GH3 β-xylosidase BXL1 (protein ID 121127), GH43 β-xylosidase/ - arabinofuranosidase (protein ID 3739), GH74 xyloglucananase CEL74a
(protein ID 49081), CE5 acetyl xylan esterase AXE1 (protein ID 73632), GH67 -glucuronidase AGU1 (protein ID 72526) , GH62, -L- arabinofuranosidase ABF2 (protein ID 76210), GH54, L-oi- arabinofuranosidase ABF1 (protein ID 55319), GH95 -fucosidase (pro¬ tein ID 58802), GH95 -fucosidase (protein ID 5807) , GH92 a-1, 2- mannosidase (protein ID 74198), GH92 -1 , 2-mannosidase (protein ID 60635), GH47 a-1 , 2-mannosidase (protein ID 45717), GH2 β-mannosidase
(protein ID 69245), GH27 a-galactosidase AGL1 (protein ID 72632) , GH27 a-galactosidase AGL3 (protein ID 27259) and/or GH28 polygalacturonase
(protein ID 103049) .
18. Method for the recombinant production of a peptide, a poly¬ peptide, a protein or a functional DNA or RNA comprising the step of cultivating a recombinant host cell according to any one of claims 7-9 or a genetically modified Trichoderma reesei cell according to any one of claims 10 to 17.
PCT/EP2011/060254 2010-06-21 2011-06-20 Leaa from trichoderma reesei Ceased WO2011161063A1 (en)

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US10273465B2 (en) 2009-09-23 2019-04-30 Danisco Us Inc. Glycosyl hydrolase enzymes and uses thereof
US10138499B2 (en) 2009-12-23 2018-11-27 Danisco Us Inc. Methods for improving the efficiency of simultaneous saccharification and fermentation reactions
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CN104640875A (en) * 2012-09-18 2015-05-20 维也纳技术大学 Modified fungal cell
CN111918959A (en) * 2018-03-26 2020-11-10 东丽株式会社 Trichoderma reesei mutant strain and method for producing protein
CN113106114A (en) * 2020-01-13 2021-07-13 中国科学院分子植物科学卓越创新中心 Factor for regulating and controlling trichoderma reesei protein expression efficiency, regulating and controlling method and application
CN113106114B (en) * 2020-01-13 2024-04-12 中国科学院分子植物科学卓越创新中心 Factor for regulating expression efficiency of trichoderma reesei protein, regulating method and application

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