Production of proteins in fungal cells in the absence of inducing substrates

Abstract

To provide methods for cost-effective commercial scale production of enzymes / polypeptides by filamentous fungi without the need to provide inducing substrates.SOLUTION: A method for producing a heterologous protein of interest in a Trichoderma sp. fungal cell in the absence of an inducing substrate comprises: (i) introducing into the fungal cell a polynucleotide construct comprising, in the 5' to 3' direction, (a) a first nucleic acid sequence comprising a constitutive promoter and (b) a second nucleic acid sequence operably linked to the first nucleic acid sequence, where the second nucleic acid sequence encodes an Ace3 protein comprising about 90% sequence identity to a specific sequence and comprises "Lys-Ala-Ser-Asp" as the last four C-terminal amino acids, and (ii) fermenting the cells of the step (i) under conditions suitable for fungal cell growth and protein production, where such suitable growth conditions do not include an inducing substrate.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a joint venture of U.S. Provisional Patent Application No. 62 / 403,787, filed October 4, 2016. Priority is claimed to the specification, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing The sequence listing text file submitted via EFS contains the following date: October 2, 2017 The file "NB41159WOPCT_SEQ This sequence listing is in accordance with 37 C.FR § 1.52 (e) and is incorporated herein by reference in its entirety.

[0003] Field This disclosure relates generally to the fields of molecular biology, biochemistry, protein production and filamentous fungi. Certain embodiments of the present disclosure provide methods for increasing the production of one or more proteins of interest, including: The present invention relates to mutant filamentous fungal cells, compositions thereof, and methods thereof. More particularly, certain embodiments The present disclosure relates to mutant filamentous fungal (host) cells derived from a parent filamentous fungal cell, the mutant host The cells are capable of expressing / producing one or more proteins of interest (POI) in the absence of an inducing substrate. This includes genetic modifications that enable [Background technology]

[0004] Cellulose, a component of lignocellulosic plant material, is the most abundant and diverse material found in nature. Similarly, filamentous fungi are known to be efficient decomposers of plant biomass. In fact, the number of industrially relevant lignocellulolytic enzymes (collectively referred to as For example, filamentous fungi are the primary source of cellulose β-( 1,4) Extracellular cellulase enzymes that hydrolyze linked glycosidic bonds to produce glucose (e.g., cellobiohydrolase, endoglucanase, β-glucosidase) It is known that these fungi can grow in the cells by (Granting ability to utilize loin).

[0005] In particular, the filamentous fungus Trichoderma reesei (T .reesei; fungus Hypocrea jecorina The anamorph of Azotobacter jecorina is known to be an efficient producer of cellulase enzymes. (For example, International Publication No. 1998 / 15619 and International Publication No. 2005 / Brochure No. 028636, Brochure No. 2006 / 074005, Brochure No. 199 Brochure No. 2 / 06221, Brochure No. 1992 / 06209, Brochure No. 199 Please refer to the pamphlet No. 2 / 06183, the pamphlet No. 2002 / 12465, etc. Therefore, filamentous fungi such as T. reesei can Derived ethanol, fabrics and clothing, detergents, textiles, food and feed additives, and other industrial uses Because it has the ability to produce enzymes that are useful for the manufacture of products for industrial use, It has come.

[0006] The expression of these industrially relevant enzymes in Trichoderma ( It is known that the production of β-lactams (β-lactams) depends on the carbon source available for growth. The production of cellulase enzymes by filamentous fungi is an energy consuming process and therefore To ensure efficient production of these enzymes, both induction and repression mechanisms exist in filamentous fungi. For example, the enzymes required for the degradation of plant cell wall material (i.e., cellulase / Various genes encoding hemicellulases are "activated" in the presence of an "inducing" substrate. and a readily metabolizable carbon source (e.g., D-glucose) that is preferred over plant biomass. In the presence of α-aminobutyric acid, a mechanism known as "carbon catabolite repression" (hereafter "CCR") occurs. Thus, cellulase genes are strongly repressed by glucose. Cellulose and certain disaccharides (e.g., sophorose, lactose, gentiobiotics) For example, the major cellobiohydrolase 1 (cbh1) is induced several thousand fold by Expression levels were significantly higher in the presence of cellulose or sophorose-inducing media compared to glucose-containing media. It is "upregulated" several thousand fold in media containing a carbon source (Ilmen et al. ., 1997). Furthermore, in the "induced" T. reesei cultures, Addition of a carbon source (cellulose or sophorose) overcomes the induction and increases cellulase activity. It has been shown to result in downregulation of gene expression (El-Gogary et al., 1989; Ilmen et al., 1997). Genes that comprise the enzyme system (the gene members of this system act synergistically to The expression of β-lactamase (β-lactamase), which is required for efficient hydrolysis of cellulose to soluble oligosaccharides, was increased at the transcriptional level. At a minimum, the temperature is adjusted and regulated by the

[0007] More specifically, genome-wide analysis revealed that T. reesei In total, 10 cellulolytic enzymes and 16 xylanolytic enzymes were encoded by the genes. The presence of α-amylase was revealed (Martinez et al., 2008). In particular, the most abundant The secreted enzymes are two major cellobiohydrolases (EC.3.2.1.91), bh1 (cellobiohydrolase 1) and cbh2 (cellobiohydrolase 2), and There are two major specific endo-β-1,4-xylanases (EC 3.3.1.8), xy n1 (endo-1,4-β-xylanase 1) and xyn2 (endo-1,4-β-xylanase 2) 2) and are referred to herein as “major industrially relevant hemicellulases and cellulases.” These MIHCs are known as cellulose and xylan decomposition enzymes. The enzymes act together with additional enzymes that produce soluble oligosaccharides and monosaccharides, e.g., cellobiose. It also produces sophoroin, D-glucose, xylobiose and D-xylose. The glycosylation product is the product of the transglycosylation activity of some of these enzymes (Vaher et al., 2003). More specifically, these soluble oligosaccharides and monosaccharides (i.e. That is, cellobiose, D-glucose, xylobiose, D-xylose and sophorone. This study demonstrated that β-lactamase (C-lactamase) affects the expression of MICH in T. reesei. For example, the presence of D-glucose induces CCR, which is thought to be due to the Sophorose results in the secretion of large amounts of MIHC. D-xylose is considered to be the most potent inducer of β-lactamase. Regulates the expression of xyn1 and xyn2.

[0008] Generally, enzymes / polypeptides produced by filamentous fungi such as Trichoderma Commercial-scale production of peptides is generally carried out using methods such as batch, fed-batch, and continuous-flow processes. , by solid or submerged culture. For example, Trichoderma One of the most problematic and expensive aspects of industrial cellulase production in 2. Inducer (i.e., induction substrate) appropriate for Trichoderma host cells For example, the present invention provides a method for producing cellulase enzymes on a commercial scale as well as in laboratory scale experiments. The production of the enzyme is achieved by growing fungal cells on solid cellulose (i.e., the inducing substrate). or by a disaccharide inducer (i.e., an inducing substrate) such as "lactose". The protein is "induced" by culturing cells in its presence.

[0009] Unfortunately, on an industrial scale, both "induction" methods suffer from the high costs associated with cellulase production. For example, as described above, cellulase synthesis has the disadvantage of resulting in Thus, cells under the control of an inducible promoter The key factor affecting the yield of enzymes is the relationship between the cellulose substrate and the glucose concentration. The key to successful commercialization of regulated gene products is maintaining a proper balance between Cellulose is an effective and inexpensive inducer, but When filamentous fungal cells are grown on solid cellulose, controlling glucose concentration can be problematic At low concentrations of cellulose, glucose production is too slow to support vigorous cell growth and function. On the other hand, if glucose production is faster than its consumption, the cells cannot meet metabolic demands. Glucose synthesis can be stopped by glucose repression. Therefore, substrate addition should be slowed down. And expensive process control schemes are required to monitor the glucose concentration. (Ju and Afolabi, 1999). Furthermore, the solid properties of cellulosic materials Because of this, it can be difficult to achieve slow, continuous delivery of the substrate.

[0010] Some of the problems with using cellulose as an "inducing substrate" are: This can be overcome by using soluble "inducing substrates" such as "sophorose" or "gentiobiose". For example, when lactose is used as the "inducing substrate", lactose must be provided in high concentrations to act as an inducer and carbon source. (See, for example, Seiboth, et al., 2002.) Sophorose is a Although sophorose is a more potent inducer than sophorose, it is expensive and difficult to manufacture. For example, a mixture of glucose, sophorose and other disaccharides (i.e., glucose (produced by the enzymatic conversion of cocos) can be used for the efficient production of cellulase. However, this incurs higher production costs than using glucose alone. Although it is easier to handle and control than solid cellulose, it is difficult to use as an inducing substrate. The use of sophorose can make the cost of producing cellulase prohibitively expensive.

[0011] Based on the above, inducing substrates that are expensive to produce (e.g., sophorose, lactose) are Cost-effective production of enzymes / polypeptides from filamentous fungi without the need or requirement for the provision of enzymes / polypeptides (e.g., enzyme preparations, enzyme chemistry, etc.) There remains an ongoing and unmet need in the art for high commercial scale production. More specifically, it is clear that one or more endogenous ligands are expressed by a filamentous fungal host cell. To produce cellulolytic enzymes on a commercial scale (such filamentous fungal cells can be grown in the absence of an inducing substrate), There remains a need in the art for an improved method for expressing one or more of these genes under Additionally, one or more heterologous protein products expressed and produced in such filamentous fungal host cells. (Heterologous genes encoding such proteins are introduced into fungal host cells, and the cells To produce cost-effective vectors capable of expressing such heterologous genes in the absence of an inducing substrate. There is a further unmet need in the art. Summary of the Invention [Means for solving the problem]

[0012] Certain embodiments of the present disclosure are directed to the use of inducing substrates that are expensive to produce (e.g., sophorose, lactose, etc.). The enzyme / polypeptide synthesis by filamentous fungi does not require or require the provision of a specific enzyme / polypeptide (e.g., taurate ... Accordingly, certain other embodiments relate to commercial scale production of one or more target tanks. Mutant filamentous fungal cells, compositions thereof, and methods for increasing protein production - Patents.com For example, certain embodiments of the present disclosure relate to mutant filamentous fungal cells derived from a parent filamentous fungal cell. This mutant cell has about 90% sequence identity to the Ace3 protein of SEQ ID NO:6. The introduced polynucleotide construct encodes the Ace3 protein, which The mutant cells showed a significantly higher phenotype than the parent cells when the mutant and parent cells were cultured under similar conditions. This allows the production of higher amounts of the protein of interest (POI) in the absence of an inducing substrate. In certain embodiments, the mutant cells will not differentiate from the parent cells when the mutant cells and the parent cells are cultured under similar conditions. In comparison to normal cells, they produce greater amounts of POI in the presence of an inducing substrate.

[0013] In another embodiment of the mutant cell, the mutant cell comprises a polypeptide having about 90% sequence identity to SEQ ID NO:6. The loaded Ace3 protein has “Lys-Ala” as the last four C-terminal amino acids. In another embodiment, the amino acid sequence of the present invention comprises about 90% sequence identity to SEQ ID NO:6. The encoded Ace3 protein is operably linked to SEQ ID NO:6, The sequence includes the N-terminal amino acid fragment of SEQ ID NO: 98 preceding the sequence. In certain embodiments, the Ace-3 protein has about 90% sequence identity to SEQ ID NO:12. In another embodiment, the introduced polynucleotide encoding the Ace3 protein has a homology to the The nucleic acid sequence of the ... Contains the ORF sequence.

[0014] In yet other embodiments of the mutant cell, the POI is an endogenous POI or a heterologous POI. Thus, in certain embodiments, the mutant cells contain an introduced poI encoding a heterologous POI. In another embodiment, the heterologous POI is encoded by a polynucleotide construct. The nucleotide construct is expressed under the control of a cellulose-inducible gene promoter. In other particular embodiments, the endogenous POI is a lignocellulolytic enzyme. Thus, in other embodiments, the lignocellulolytic enzyme is a cellulase enzyme, a hemicellulase enzyme, or a lignocellulolytic enzyme. In certain other embodiments, the enzyme is selected from the group consisting of a glycerol-binding protein (GPCR), ... The lignocellulolytic enzymes are cbh1, cbh2, egl1, egl2, and egl3. , egl4, egl5, egl6, bgl1, bgl2, xyn1, xyn2, xyn3 ,bxl1,abf1,abf2,abf3,axe1,axe2,axe3,man1 , agl1, agl2, agl3, glr1, swo1, cip1 and cip2 In yet another embodiment, the heterologous POI is selected from the group consisting of α-amylase, alkaloid enzyme, α-amylase, β-amylase, cellulase, dextranase, α-glucosidase enzymes, α-galactosidase, glucoamylase, hemicellulase, pentosanase, xylase Lactase, invertase, lactase, naringanase, pectinase, pullulanase , acid protease, alkaline protease, bromelain, neutral protease, papain , pepsin, peptidase, rennet, renin, chymosin, subtilisin, thermolysin Aspartic acid proteinase, trypsin, lipase, esterase, phospholipase enzymes, phosphatases, phytases, amidases, iminoacylases, glutaminase, Lysozyme, penicillin acylase, isomerase, oxidoreductase, catalase , chloroperoxidase, glucose oxidase, hydroxysteroid dehydrogenase Enzymes, peroxidases, lyases, aspartate β-decarboxylases, humara enzymes, histidases, transferases, ligases, aminopeptidases, carboxylases Peptidase, chitinase, cutinase, deoxyribonuclease, α-galactosidase β-galactosidase, β-glucosidase, laccase, mannosidase, muta Enzymes, polyphenol oxidases, ribonucleases and transglutaminases is selected from the group consisting of:

[0015] In another embodiment of the mutant cell, the polynucleotide construct has the sequence A polynucleotide sequence encoding the Ace3 protein having approximately 90% sequence identity with In certain embodiments, the polynucleotide comprises a promoter sequence operably linked to the 5' end of the polynucleotide. The peptide construct contains Ace3, which has about 90% sequence identity to SEQ ID NO:6. A naturally occurring ac operably linked to the 3' end of a polynucleotide sequence encoding a protein. In certain other embodiments, the polynucleotide comprises a polynucleotide sequence comprising an e3 terminator sequence. The construct is integrated into the genome of the fungal cell. The construct is integrated into the telomeric site of the fungal cell genome. In this embodiment, the polynucleotide construct is a glucoamylase (gl) gene of the fungal cell genome. a1) is integrated into a gene locus. The construct has about 90% sequence identity to SEQ ID NO:4, SEQ ID NO:11 or SEQ ID NO:13. In another embodiment, the encoded Ace3 protein comprises a nucleotide sequence having , SEQ ID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 14. Thus, in another embodiment, the encoded Ace3 protein comprises the amino acid sequence , SEQ ID NO:6, SEQ ID NO:12 or SEQ ID NO:14.

[0016] In other embodiments, the mutant cell contains the wild-type xylanase regulator 1 of SEQ ID NO:48. (Xyr1) protein, or the mutant xylanase regulator 1 (X yr1) Genetic modification that expresses a polynucleotide encoding a protein. In certain embodiments, the mutant cell expresses endogenous carbon catabolite repressor 1 (Cre1). A gene that reduces or suppresses the expression of a gene encoding an Ace1 protein or an Ace1 protein. In yet another embodiment, the mutant cell comprises expression of the Ace2 protein. In another embodiment, the filamentous fungal cell is selected from the group consisting of Ascomycota. a) subphylum Pezizomycotina cells. In one embodiment, the filamentous fungal cell is a Trichoderma spp. cell. be.

[0017] In other embodiments, the disclosure provides a sequence similar to SEQ ID NO:6, SEQ ID NO:12, or SEQ ID NO:14. Polynucleotide ORF encoding Ace3 protein with 90% sequence identity Regarding.

[0018] In other embodiments, the disclosure provides a method for producing a lignocellulosic fraction produced by a mutant cell of the disclosure. In another embodiment, the disclosure relates to a heterologous enzyme produced by the mutant cells of the disclosure. Regarding POIs.

[0019] In another embodiment, the disclosure provides a method for growing Trichoderma in the absence of an inducing substrate. The present invention relates to a method for producing an endogenous protein of interest in a fungal cell of the genus rma, the method comprising the steps of: i) a first nucleic acid sequence comprising, in a 5' to 3' direction: (a) a promoter; and (b) a first a second nucleic acid sequence operably linked to the nucleic acid sequence of SEQ ID NO:6, The Ace3 protein, which shares approximately 90% sequence identity with the last four C-terminal ends, is encoded by Polynucleotide conjugates containing the amino acids "Lys-Ala-Ser-Asp" (ii) introducing the construct into a fungal cell; and (iii) inhibiting the growth of the fungal cell and the production of the protein. The cells of step (i) are grown under conditions suitable for growth (such suitable growth conditions being free of an inducing substrate). In certain embodiments of the method, the polynucleotide construct is The tract comprises a third nucleic acid sequence operably linked to the 3' end of the second nucleic acid, the third nucleic acid sequence being The nucleic acid sequence comprises the native ace3 terminator sequence. The polynucleotide construct is integrated into the genome of the fungal cell. The nucleotide construct is integrated into the telomeric site of the fungal cell genome. In an embodiment, the polynucleotide construct is a glycosyltransferase that is a glycosyltransferase of a fungal cell genome. In another embodiment of the method, the polynucleotide is integrated into the gla1 locus. The constructs are approximately 90% identical to SEQ ID NO:4, SEQ ID NO:11 or SEQ ID NO:13. In another embodiment, the encoded Ace3 tag comprises a nucleotide sequence having sequence identity to the Ace3 tag. The protein has 95% sequence identity to SEQ ID NO:6, SEQ ID NO:12 or SEQ ID NO:14. In another embodiment, the encoded Ace3 protein comprises an amino acid sequence having the following structure: It comprises the amino acid sequence of SEQ ID NO:6, SEQ ID NO:12 or SEQ ID NO:14.

[0020] In a particular embodiment of the method, the promoter in step (i) is the rev3 promoter ( SEQ ID NO: 15), bxl promoter (SEQ ID NO: 16), tkl1 promoter (SEQ ID NO: No. 17), PID104295 promoter (SEQ ID NO: 18), dld1 promoter ( SEQ ID NO: 19), xyn4 promoter (SEQ ID NO: 20), PID72526 promoter - (SEQ ID NO: 21), axe1 promoter (SEQ ID NO: 22), hxk1 promoter ( SEQ ID NO: 23), dic1 promoter (SEQ ID NO: 24), opt promoter (SEQ ID NO: No. 25), gut1 promoter (SEQ ID NO: 26), and pki1 promoter (SEQ ID NO: No. 27).

[0021] In a related embodiment of the method, the endogenous POI is a lignocellulolytic enzyme. In certain embodiments, the lignocellulolytic enzyme is a cellulase enzyme, a hemicellulase enzyme, or a combination thereof. The enzymes are cbh1, cbh2, egl1, egl2, egl3, egl4, egl5, Selected from the group consisting of egl6, bgl1, bgl2, swo1, cip1 and cip2 In another embodiment, the hemicellulase enzymes are selected from the group consisting of xyn1, xyn2, xyn3, xyn4, xyn5, xyn6, xyn7, xyn8, xyn9, xyn10, xyn11, xyn12, xyn13, xyn14, xyn15, xyn16, xyn17, xyn18, xyn19, xyn110, xyn111, xyn12, xyn13, yn4, bxl1, abf1, abf2, abf3, axe1, axe2, axe3, m Selected from the group consisting of an1, agl1, agl2, agl3 and glr1.

[0022] In another embodiment of the method, step (i) comprises the step of: In certain embodiments, the nucleic acid sequence further comprises a nucleotide construct encoding a heterologous POI. The polynucleotide construct is under the control of a cellulose-inducible gene promoter. In certain embodiments, the cellulose-inducible gene promoter is cbh1, cb Selected from h2, egl1, egl2, xyn2 or stp1.

[0023] In another embodiment, the method further comprises administering to the subject a wild-type xylanase regulator 1 ( Xyr1) protein, or a mutant xylanase regulator 1 (Xy r1) further includes a genetic modification that expresses a polynucleotide encoding a protein. In another embodiment, the method further comprises activating an endogenous carbon catabolite repressor 1 (Cre1) protein. A gene that reduces or suppresses the expression of the gene encoding the protein or Ace1 protein In yet another embodiment, the method further comprises expressing an Ace2 protein. It further includes genetic modifications such as

[0024] In other embodiments, the present disclosure provides a method for culturing Trichoderma in the absence of an inducing substrate. erma species fungal cells, comprising: (i) a first nucleic acid sequence comprising, in a 5' to 3' direction, (a) a promoter; and (b) a a second nucleic acid sequence operably linked to the nucleic acid sequence of SEQ ID NO:1, 2) encoding the Ace3 protein, which has approximately 90% sequence identity to (ii) introducing the nucleotide construct into a fungal cell; and (iii) inhibiting the growth and proliferation of the fungal cell. and under conditions favorable for the production of the protein (such favorable growth conditions do not include the inducing substrate). In another embodiment, the polynucleotide conjugate is a polynucleotide conjugate. The construct comprises a third nucleic acid sequence operably linked to the 3' end of the second nucleic acid, In another embodiment of the method, the nucleic acid sequence of 3 comprises the naturally occurring ace3 terminator sequence. The polynucleotide construct is integrated into the genome of the fungal cell. In this study, the polynucleotide construct was integrated into the telomeric site of the genome of fungal cells. In other particular embodiments, the polynucleotide construct is In another embodiment of the method, the gene is integrated into the glucoamylase (gla1) locus. The oligonucleotide construct is SEQ ID NO: 4, SEQ ID NO: 11 or SEQ ID NO: 13. In certain embodiments, the coding sequence comprises a nucleotide sequence having about 90% sequence identity to the coding sequence. The Ace3 protein was 9 to SEQ ID NO:6, SEQ ID NO:12 or SEQ ID NO:14. In another particular embodiment, the encoded amino acid sequence has 5% sequence identity to the The Ace3 protein has the amino acid sequence of SEQ ID NO:6, SEQ ID NO:12 or SEQ ID NO:14. include.

[0025] Thus, in another embodiment of method 8, the endogenous POI is a lignocellulolytic enzyme. In certain embodiments, the lignocellulolytic enzyme is a cellulase enzyme, a hemicellulase enzyme, or a In another embodiment, the enzyme is selected from the group consisting of a selenium enzyme, ... The lulase enzymes are cbh1, cbh2, egl1, egl2, egl3, egl4, egl5, From the group consisting of l5, egl6, bgl1, bgl2, swo1, cip1 and cip2 In another embodiment, the hemicellulase enzyme is selected from the group consisting of xyn1, xyn2, xyn 3, xyn4, bxl1, abf1, abf2, abf3, axe1, axe2, axe 3, man1, agl1, agl2, agl3 and glr1 .

[0026] In another embodiment of the method, step (i) comprises the step of: In certain embodiments, the nucleic acid sequence further comprises a nucleotide construct encoding a heterologous POI. The polynucleotide construct is under the control of a cellulose-inducible gene promoter. In another particular embodiment of the method, the promoter in step (i) is rev3 promoter (SEQ ID NO: 15), bxl promoter (SEQ ID NO: 16), tkl1 promoter PID104295 promoter (SEQ ID NO: 18), dld1 promoter promoter (SEQ ID NO: 19), xyn4 promoter (SEQ ID NO: 20), PID7252 6 promoter (SEQ ID NO: 21), axe1 promoter (SEQ ID NO: 22), hxk1 promoter promoter (SEQ ID NO: 23), dic1 promoter (SEQ ID NO: 24), opt promoter 25), gut1 promoter (SEQ ID NO:26), and pki1 promoter In another embodiment, the method further comprises the step of: The wild-type xylanase regulator 1 (Xyr1) protein, which is designated as SEQ ID NO: 48, or SEQ ID NO: Polypeptide 46 encoding the mutant xylanase regulator 1 (Xyr1) protein In another particular embodiment, the method further comprises a genetic modification that expresses the nucleotide. Endogenous carbon catabolite repressor 1 (Cre1) protein or Ace1 protein Further included are genetic modifications that reduce or suppress the expression of genes encoding genes. In one embodiment, the method further comprises modifying a gene that expresses the Ace2 protein.

[0027] In other specific embodiments, the present disclosure provides methods for culturing Trichoderma in the absence of an inducing substrate. The present invention relates to a method for producing a heterologous protein of interest in a fungal cell of the species O. oderma, comprising the steps of: (i) a first nucleic acid sequence comprising, in the 5' to 3' direction, (a) a constitutive promoter; and b) a second nucleic acid sequence operably linked to the first nucleic acid sequence, the second nucleic acid sequence having the sequence The last four encode the Ace3 protein, which has approximately 90% sequence identity to number 6. Polynucleotides containing "Lys-Ala-Ser-Asp" as the C-terminal amino acids (ii) introducing the nucleotide construct into a fungal cell; and (iii) inhibiting the growth and proliferation of the fungal cell. The enzyme is grown under conditions favorable for the production of protein (such favorable growth conditions do not include an inducing substrate). and fermenting the cells of step (i). Thus, in a particular embodiment of the method, The fungal cell contains an introduced polynucleotide construct encoding a heterologous POI. and the construct is added before, during, or after step (i). In another embodiment, the polynucleotide construct is introduced into a fungal cell. a third nucleic acid sequence operably linked to the 3' end of the nucleic acid of In another embodiment of the method, the polynucleotide comprises the ace3 terminator sequence of The construct is integrated into the genome of the fungal cell. The tide construct is integrated into the telomeric site of the genome of the fungal cell. In the present invention, the polynucleotide construct is a nucleotide sequence encoding the glucoamylase (gla) gene of the fungal cell genome. 1) It is integrated into a gene locus.

[0028] In another particular embodiment of the method, the polynucleotide construct comprises SEQ ID NO:4, A nucleotide sequence having about 90% sequence identity to SEQ ID NO:11 or SEQ ID NO:13. In another embodiment, the encoded Ace3 protein comprises SEQ ID NO:6, SEQ ID NO: SEQ ID NO: 12 or SEQ ID NO: 14. In one embodiment, the encoded Ace3 protein is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:12, or The polynucleotide encoding the heterologous POI comprises the amino acid sequence of SEQ ID NO: 14. The nucleotide construct is expressed under the control of a cellulose-inducible gene promoter. In other specific embodiments, the heterologous POI is α-amylase, alkaline α-amylase, β-amylase, cellulase, dextranase, α-glucosidase, α-galactosidase idase, glucoamylase, hemicellulase, pentosanase, xylanase, invertase Tase, lactase, naringanase, pectinase, pullulanase, acid protease, Alkaline protease, bromelain, neutral protease, papain, pepsin, peptidase dase, rennet, renin, chymosin, subtilisin, thermolysin, aspartic acid Proteinases, trypsins, lipases, esterases, phospholipases, phosphatases enzymes, phytases, amidases, iminoacylases, glutaminase, lysozymes, penicillins Phosphoryl acylase, isomerase, oxidoreductase, catalase, chloroperoxylase idase, glucose oxidase, hydroxysteroid dehydrogenase, peroxidase lyase, aspartate β-decarboxylase, fumarase, histidase , transferases, ligases, aminopeptidases, carboxypeptidases, chitinases Enzymes, cutinases, deoxyribonucleases, α-galactosidases, β-galactosidases sidase, β-glucosidase, laccase, mannosidase, mutanase, polyphenolase selected from the group consisting of ribonuclease, ribonuclease and transglutaminase will be done.

[0029] In another embodiment of the method, the promoter in step (i) is the rev3 promoter (sequence No. 15), bxl promoter (SEQ ID NO: 16), tkl1 promoter (SEQ ID NO: 1 7. Selected from the group consisting of the PID104295 promoter (SEQ ID NO: 18), the dld1 promoter (SEQ ID NO: 19), the xyn4 promoter (SEQ ID NO: 20), the PID72526 promoter (SEQ ID NO: 21), the axe1 promoter (SEQ ID NO: 22), the hxk1 promoter (SEQ ID NO: 23), the dic1 promoter (SEQ ID NO: 24), the opt promoter (SEQ ID NO: 25), the gut1 promoter (SEQ ID NO: 26), and the pki1 promoter (SEQ ID NO: 27). In yet another embodiment, the method further comprises a polynucleotide encoding the wild-type xylanase regulator 1 (Xyr1) protein of SEQ ID NO: 48 or the mutant xylanase regulator 1 (Xyr1) protein of SEQ ID NO: 46. expressing a gene modification. In another embodiment, the method further comprises a gene modification that reduces or suppresses the expression of the endogenous carbon catabolite repressor 1 (Cre1) protein or the Ace1 protein. In yet another embodiment, the method further comprises a gene modification that includes the expression of the Ace2 protein. In another embodiment, the present disclosure relates to a method for producing a target heterologous protein in fungal cells of the genus Trichoderma in the absence of an inducer substrate, the method comprising (i) introducing into the fungal cells a polynucleotide construct comprising, in the 5' to 3' direction, (a) a first nucleic acid sequence comprising a constitutive promoter and (b) a second nucleic acid sequence operably linked to the first nucleic acid sequence, the second nucleic acid sequence encoding an Ace3 protein having about 90% sequence identity to SEQ ID NO: 12, and (ii) growing the fungal cells and In yet another embodiment, the method further comprises a polynucleotide encoding the wild-type xylanase regulator 1 (Xyr1) protein of SEQ ID NO: 48 or the mutant xylanase regulator 1 (Xyr1) protein of SEQ ID NO: 46. expressing a gene modification. In another embodiment, the method further comprises a gene modification that reduces or suppresses the expression of the endogenous carbon catabolite repressor 1 (Cre1) protein or the Ace1 protein. In yet another embodiment, the method further comprises a gene modification that includes the expression of the Ace2 protein. In another embodiment, the present disclosure relates to a method for producing a target heterologous protein in fungal cells of the genus Trichoderma in the absence of an inducer substrate, the method comprising (i) introducing into the fungal cells a polynucleotide construct comprising, in the 5' to 3' direction, (a) a first nucleic acid sequence comprising a constitutive promoter and (b) a second nucleic acid sequence operably linked to the first nucleic acid sequence, the second nucleic acid sequence encoding an Ace3 protein having about 90% sequence identity to SEQ ID NO: 12, and (ii) growing the fungal cells and

[0030] In another embodiment, the present disclosure relates to a method for producing a target heterologous protein in fungal cells of the genus Trichoderma in the absence of an inducer substrate, the method comprising (i) introducing into the fungal cells a polynucleotide construct comprising, in the 5' to 3' direction, (a) a first nucleic acid sequence comprising a constitutive promoter and (b) a second nucleic acid sequence operably linked to the first nucleic acid sequence, the second nucleic acid sequence encoding an Ace3 protein having about 90% sequence identity to SEQ ID NO: 12, and (ii) growing the fungal cells and (ii) growing the fungal cells and In yet another embodiment, the method further comprises a polynucleotide encoding the wild-type xylanase regulator 1 (Xyr1) protein of SEQ ID NO: 48 or the mutant xylanase regulator 1 (Xyr1) protein of SEQ ID NO: 46. expressing a gene modification. In another embodiment, the method further comprises a gene modification that reduces or suppresses the expression of the endogenous carbon catabolite and under conditions favorable for protein production (such favorable growth conditions do not include an inducing substrate). and b) fermenting the cells of step (i). In certain embodiments, the fungal cells are heterologous. The introduced polynucleotide construct encoding the POI (this construct The lactate is introduced into the fungal cell before, during, or after step (i). In another embodiment, the polynucleotide construct is made 3' to the second nucleic acid. a third nucleic acid sequence operably linked thereto, the third nucleic acid sequence being a naturally occurring ace3 terminus In another embodiment of the method, the polynucleotide construct comprises a In certain embodiments, the polynucleotide construct is integrated into the genome of the fungal cell. In another embodiment, the polynucleotide is integrated into the genome of the fungal cell at a telomeric site. The optide construct was integrated into the glucoamylase (gla1) locus of the fungal genome. In yet another embodiment, the polynucleotide construct comprises the sequence set forth in SEQ ID NO:4. , a nucleotide having about 90% sequence identity to SEQ ID NO:11 or SEQ ID NO:13 In certain embodiments, the encoded Ace3 protein comprises the sequence SEQ ID NO:6, Contains an amino acid sequence having 95% sequence identity to sequence number 12 or SEQ ID NO:14 In another embodiment, the encoded Ace3 protein is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:12, and SEQ ID NO:4. or the amino acid sequence of SEQ ID NO: 14. In another embodiment of the method, the heterologous POI is encoded The polynucleotide construct encoding the cellulose-inducible gene promoter In certain embodiments, the heterologous POI is expressed under conditions of α-amylase, alkaline α-amylase, enzymes, β-amylase, cellulase, dextranase, α-glucosidase, α-galactosidase, Lactosidase, glucoamylase, hemicellulase, pentosanase, xylanase, Invertase, lactase, naringanase, pectinase, pullulanase, acid protease ase, alkaline protease, bromelain, neutral protease, papain, pepsin , peptidase, rennet, renin, chymosin, subtilisin, thermolysin, aspartate Lagic acid proteinase, trypsin, lipase, esterase, phospholipase, phosphatase Phytase, amidase, iminoacylase, glutaminase, lysozyme , penicillin acylase, isomerase, oxidoreductase, catalase, chlorope peroxidase, glucose oxidase, hydroxysteroid dehydrogenase, peroxidase, lyase, aspartate β-decarboxylase, fumarase, histidine Glutamate, transferase, ligase, aminopeptidase, carboxypeptidase enzyme, chitinase, cutinase, deoxyribonuclease, α-galactosidase, β- Galactosidase, β-glucosidase, laccase, mannosidase, mutanase, poly The group consisting of ribonuclease, ribonuclease and transglutaminase is selected from.

[0031] In another embodiment of the method, the promoter in step (i) is the rev3 promoter (sequence No. 15), bxl promoter (SEQ ID NO: 16), tkl1 promoter (SEQ ID NO: 1 7, PID104295 promoter (SEQ ID NO: 18), dld1 promoter (SEQ ID NO: No. 19), xyn4 promoter (SEQ ID NO: 20), PID72526 promoter (SEQ ID NO: Sequence number 21), axe1 promoter (sequence number 22), hxk1 promoter (sequence number No. 23), dic1 promoter (SEQ ID NO: 24), opt promoter (SEQ ID NO: 25 ), gut1 promoter (SEQ ID NO: 26), and pki1 promoter (SEQ ID NO: 2 7). In yet another embodiment, the method comprises the step of: Live xylanase regulator 1 (Xyr1) protein, or a mutation of SEQ ID NO: 46 Polynucleotides encoding the xylanase regulator 1 (Xyr1) protein In another embodiment, the method further comprises modifying a gene expressing an endogenous carbon catabolite. The gene encoding the light repressor 1 (Cre1) protein or the Ace1 protein In yet another embodiment, the method further comprises the steps of: The method further comprises modifying a gene that expresses the Ace2 protein.

[0032] In other specific embodiments, the present disclosure provides a method for the production of an endogenous protein in the absence of an inducing substrate. Trichoderma reesei strains for increasing The present invention relates to a method for genetically modifying a gene comprising: (i) an Ace3-S protein having SEQ ID NO: 3; Ace3-SC protein of SEQ ID NO: 8 or Ace3-LC protein of SEQ ID NO: 10 T. reesei, which contains a genomic copy of the ace3 gene encoding a protein Screening and identifying T. reesei strains (identified T. reesei strains) i) The strain contains the Ace3-L protein of SEQ ID NO: 6, the Ace3-LN protein of SEQ ID NO: 14, The genome of the ace3 gene encoding the Ace3 protein or the Ace3-EL protein of SEQ ID NO: 12 (ii) T. reesei identified in step (i) ei) strain comprising, in a 5' to 3' direction, (a) a first nucleic acid sequence comprising a promoter; and (b) a polynucleotide sequence comprising a second nucleic acid sequence operably linked to the first nucleic acid sequence; A step of constructing a second nucleic acid sequence having about 90% sequence identity to SEQ ID NO:6. and the last four C-terminal amino acids are “Lys- Ala-Ser-Asp" or the second nucleic acid sequence is about 9 relative to SEQ ID NO:12. (iii) fungal cells, which encode an Ace-3 protein having 0.0% sequence identity to the Ace-3 protein; The growth of the cells and the production of the protein are carried out under favorable conditions (such favorable growth conditions are achieved by providing an inducing substrate). and fermenting the cells of step (ii) with a culture medium (not including the culture medium).

[0033] In other specific embodiments, the present disclosure relates to a mutant filamentous fungal cell derived from a parent filamentous fungal cell, The mutant cells contain the native ace3 gene promoter replaced with an alternative promoter. The mutant cells showed a non-inducing effect on the inducing substrate when the mutant and parent cells were cultured under similar conditions. In the presence of a specific In an embodiment, the alternative promoter is reesei promoter. In another embodiment, the alternative promoter is the rev3 promoter (SEQ ID NO: 15), bxl promoter (SEQ ID NO: 16), tkl1 promoter promoter (SEQ ID NO: 17), PID104295 promoter (SEQ ID NO: 18), dld1 Promoter (SEQ ID NO: 19), xyn4 promoter (SEQ ID NO: 20), PID725 26 promoter (SEQ ID NO: 21), axe1 promoter (SEQ ID NO: 22), hxk1 promoter (SEQ ID NO: 23), dic1 promoter (SEQ ID NO: 24), opt promoter promoter (SEQ ID NO:25), gut1 promoter (SEQ ID NO:26), and pki1 promoter The promoter is selected from the group consisting of: [Brief description of the drawings]

[0034] [Figure 1] Figure 1 shows a schematic diagram of the Ace3 protein coding region. More specifically, Figure 1A shows a schematic diagram of the Ace3 protein coding region based on the annotation of T. reesei strains QM6a and RUT-C30 aligned to the same DNA sequence in the genome (Figure 1A). Predicted exons and introns are indicated by arrows and dashed lines, respectively. Dashed vertical lines indicate nonsense mutations in the RUT-C30 genome. The cloned ace3-S (short) open reading frame of SEQ ID NO: 2 and the cloned ace3-L (long) open reading frame of SEQ ID NO: 5 were screened and tested as described in this application. Figure 1B shows an amino acid alignment of the Ace3-L protein (sequence number 6) from T. reesei strain RUT-C30, the Ace3-S protein (sequence number 3), the Ace3-SC protein (sequence number 8), the Ace3-LN protein (sequence number 14), the Ace3-LC protein (sequence number 10) and the Ace3-EL protein (sequence number 12) from T. reesei strain QM6a. [Diagram 2] FIG. 2 shows a schematic diagram of the Ace3 expression vectors pYL1 (FIG. 2A), pYL2 (FIG. 2B), pYL3 (FIG. 2C) and pYL4 (FIG. 2D). [Diagram 3]Figure 3 shows polyacrylamide gel electrophoresis (SDS-PAGE) of T. reesei parental and mutant cell supernatants when parental and mutant cells were grown in defined medium in srMTPs containing 20% ​​lactose (lac) or 20% glucose (glu). Equal volumes of culture supernatant were used in each lane. M is a molecular weight marker, and the parental T. reesei strain served as the control strain. [Figure 4] Figure 4 shows SDS-PAGE of T. reesei parental and mutant cell supernatants when grown in shake flasks in defined medium supplemented with 1.5% glucose / sophorose (sop) or 1.5% glucose (glu). Equal volumes of culture supernatant were used in each lane. Total protein concentrations of culture supernatants are shown below each corresponding lane. M is a molecular weight marker and KD is kilodaltons. [Diagram 5] Figure 5 shows an SDS-PAGE of T. reesei parental and mutant cells grown in 2 L fermentors in defined medium supplemented with glucose / sophorose (sop) or glucose (glu) as the carbon source, where M is a molecular weight marker and zero (0) is the seed culture supernatant. [Figure 6] FIG. 6 shows a schematic diagram of a promoter replacement construct made by fusing a DNA fragment containing the 5' region upstream of the native promoter of the ace3 locus, a loxP-flanked hygromycin B resistance (selection marker) cassette, and a DNA fragment containing a promoter of interest operably fused (linked) to the 5' end of the ace3 ORF. [Figure 7] FIG. 7 shows a schematic diagram of the Ace3 expression vector pYL8 containing the dic1 promoter. [Figure 8]Figure 8 shows SDS-PAGE of T. reesei parental and its modified (daughter) cell supernatants when the parental and modified strains were grown in shake flasks in defined medium supplemented with 2.5% glucose / sophorose ("Sop", inducing conditions) or 2.5% glucose ("Glu", non-inducing conditions). Equal volumes of culture supernatant were used in each lane. M is a molecular weight marker and KD is kilodaltons. [Figure 9] Figure 9 shows protein production in small-scale (2L) fermentations. T. reesei parent and daughter strains "LT83" were grown in defined medium with glucose / sophorose (Sop, inducing conditions) or glucose (Glu, non-inducing conditions) as the carbon source. The total protein produced by the parent strain on glucose / sophorose at the end of the fermentation was arbitrarily set to 100, and the relative amount of protein produced by each strain at each time point was plotted. [Figure 10] Figure 10 shows SDS-PAGE of T. reesei parental and its mutant (daughter) cell supernatants when the parental and modified strains were grown in shake flasks in defined medium supplemented with 2.5% glucose / sophorose ("Sop", inducing conditions) or 2.5% glucose ("Glu", non-inducing conditions). Equal volumes of culture supernatant were used in each lane. M is a molecular weight marker and KD is kilodaltons. [Figure 11] Figure 11 shows a schematic diagram of the ace3 locus. The arrows at the 5' end (N-terminus) of the ace3 locus indicate the different transcription start sites in the form suggested by the cDNA sequence (arrow 1), the RutC-30 annotation form (arrow 2) and the QM6a annotation form (arrow 3). The arrows at the 3' end (C-terminus) of the ace3 locus indicate the different stop codons in the RutC-30 annotation form (arrow 4) and the QM6a annotation form (arrow 5). [Figure 12]Figure 12 shows a schematic diagram of the different ace3 forms that were cloned. Thus, as shown in Figure 12 and described in Example 6, the following ace3 forms were cloned and tested: "ace3-S" of SEQ ID NO: 1, containing 1,713 bp of exon 3, 148 bp of intron 3 and 177 bp of exon 4; "ace3-SC" of SEQ ID NO: 7, containing 1,713 bp of exon 3, 148 bp of intron 3 and 144 bp of exon 4; "ace3-L" of SEQ ID NO: 4, containing 258 bp of exon 2, 423 bp of intron 2, 1,635 bp of exon 3, 148 bp of intron 3 and 144 bp of exon 4; "ace3-LC" of SEQ ID NO:9, which contains exon 2, intron 2 of 423 bp, exon 3 of 1,635 bp, intron 3 of 148 bp, and exon 4 of 177 bp; "ace3-EL" of SEQ ID NO:11, which contains exon 1 of 61 bp, intron 1 of 142 bp, exon 2 of 332 bp, intron 2 of 423 bp, exon 3 of 1,635 bp, intron 3 of 148 bp, and exon 4 of 144 bp; and "ace3-LN" of SEQ ID NO:13, which contains exon 2 of 258 bp, exon 3 of 1,635 bp, intron 3 of 148 bp, and exon 4 of 144 bp. [Figure 13] Figure 13 shows the nucleic acid sequence of the ace3-SC gene form (SEQ ID NO:7; includes 1,713 bp of exon 3, 148 bp of intron 3, and 144 bp of exon 4) and the encoded Ace3-SC protein sequence (SEQ ID NO:8). As shown in Figure 13 for the ace3-SC gene form, nucleotides shown in bold black text represent intron sequences. [Figure 14] Figure 14 shows the nucleic acid sequence of the ace3-S gene form (SEQ ID NO:1; includes 1,713 bp of exon 3, 148 bp of intron 3, and 177 bp of exon 4) and the encoded Ace3-S protein sequence (SEQ ID NO:3). As shown in Figure 14 for the ace3-S gene form, nucleotides shown in bold black text represent intron sequences. [Figure 15]Figure 15 shows the nucleic acid sequence of the ace3-L gene form (SEQ ID NO: 4; containing 258 bp exon 2, 423 bp intron 2, 1,635 bp exon 3, 148 bp intron 3 and 144 bp exon 4) and the encoded Ace3-L protein sequence (SEQ ID NO: 6). As shown in Figure 15 for the ace3-L gene form, nucleotides shown in bold black text represent intron sequences. [Figure 16] Figure 16 shows the nucleic acid sequence of the ace3-LC gene form (SEQ ID NO:9, including 258 bp exon 2, 423 bp intron 2, 1,635 bp exon 3, 148 bp intron 3 and 177 bp exon 4) and the encoded Ace3-LC protein sequence (SEQ ID NO:10). As shown in Figure 16 for the ace3-LC gene form, nucleotides shown in bold black text represent intron sequences. [Figure 17] Figure 17 shows the nucleic acid sequence of the ace3-EL gene form (SEQ ID NO:11; containing 61 bp exon 1, 142 bp intron 1, 332 bp exon 2, 423 bp intron 2, 1,635 bp exon 3, 148 bp intron 3 and 144 bp exon 4) and the encoded Ace3-EL protein sequence (SEQ ID NO:12). As shown in Figure 17 for the ace3-EL gene form, nucleotides shown in bold black text represent intron sequences. [Figure 18] Figure 18 shows the nucleic acid sequence of the ace3-LN gene form (SEQ ID NO: 13; includes 258 bp exon 2, 1,635 bp exon 3, 148 bp intron 3 and 144 bp exon 4) and the encoded Ace3-LN protein sequence (SEQ ID NO: 14). As shown in Figure 18 for the ace3-LN gene form, nucleotides shown in bold black text represent intron sequences. [Figure 19] FIG. 19 shows a schematic diagram of the DNA fragment arrangement designed for integration of the ace3 form into the gla1 locus. [Figure 20] FIG. 20 shows a schematic diagram of the vector pMCM3282. [Figure 21] FIG. 21 shows a schematic diagram of the vector pCHL760. [Figure 22] FIG. 22 shows a schematic diagram of the vector pCHL761. [Diagram 23] Figure 23 shows SDS-PAGE of secreted proteins produced by submerged cultures (i.e., shake flasks) of T. reesei parent cells (Figure 23, cell identification number 1275.8.1) and mutant T. reesei (daughter) cells (Figure 23, cell identification numbers 2218, 2219, 2220, 2222 and 2223) under induced ("Glu / Sop") and non-induced ("Glu") culture conditions.

[0035] A brief description of biological sequences The following sequence complies with 37 C.FR §§1.821-1.825 ("Requirements"). nts for Patent Applications Containing N ucleotide Sequence and / or Amino Acid Seq Disclosures—the Sequence Rules” It is compatible with WIPO Standard ST.25 (2009), the European Patent Convention (EPC) and the PCT Rules 5.2 and 49.5(a-bis) and Article 2 of the Administrative Instructions Consistent with the sequence listing requirements of Chapter 08 and Annex C. Nucleotide and amino acid sequence data The symbols and format used for the data are specified in 37 C.FR §1.822. They follow the rules.

[0036] SEQ ID NO:1 is a gene encoding the Ace3-S protein of SEQ ID NO:3. Trichoderma reesei wild-type strain QM6a nucleic acid sequence It is a column.

[0037] SEQ ID NO:2 is an open reading frame of the nucleic acid sequence encoding the Ace3-S protein of SEQ ID NO:3. Open reading frame (ORF).

[0038] SEQ ID NO:3 is the sequence of Trichoderma reesei ( This is the amino acid sequence of the Ace3 protein (QM6a strain), hereinafter referred to as "Ace3-S" .

[0039] SEQ ID NO:4 is a gene encoding the Ace3-L protein of SEQ ID NO:6. Nucleic acid of Trichoderma reesei strain Rut-C30 It is an array.

[0040] SEQ ID NO:5 is the ORF of the nucleic acid sequence encoding the Ace3-L protein of SEQ ID NO:6. be.

[0041] SEQ ID NO:6 is the sequence of Trichoderma reesei ( This is the amino acid sequence of the Ace3 protein (Rut-C30 strain), hereafter referred to as "Ace3-L". It is called.

[0042] SEQ ID NO: 7 is a transcription factor that contains a gene encoding the Ace3-SC protein of SEQ ID NO: 8. The nucleic acid sequence of Trichoderma reesei.

[0043] SEQ ID NO:8 is Trichoderma reesei A This is the amino acid sequence of the ce3 protein, hereinafter referred to as "Ace3-SC."

[0044] SEQ ID NO:9 contains a gene encoding the Ace3-LC protein of SEQ ID NO:10; The nucleic acid sequence of Trichoderma reesei.

[0045] SEQ ID NO:10 is Trichoderma reesei This is the amino acid sequence of the Ace3 protein, hereinafter referred to as "Ace3-LC."

[0046] SEQ ID NO:11 contains the gene encoding the Ace3-EL protein of SEQ ID NO:12 , which is the nucleic acid sequence of Trichoderma reesei .

[0047] SEQ ID NO:12 is Trichoderma reesei This is the amino acid sequence of the Ace3 protein, hereinafter referred to as "Ace3-EL."

[0048] SEQ ID NO:13 contains the gene encoding the Ace3-LN protein of SEQ ID NO:14 , which is the nucleic acid sequence of Trichoderma reesei .

[0049] SEQ ID NO:14 is the sequence of Trichoderma reesei This is the amino acid sequence of the Ace3 protein, hereinafter referred to as "Ace3-LN."

[0050] SEQ ID NO:15 is a nucleic acid sequence comprising the rev3 promoter sequence.

[0051] SEQ ID NO: 16 is a nucleic acid sequence comprising the β-xyl promoter sequence.

[0052] SEQ ID NO:17 is a nucleic acid sequence comprising the tki1 promoter sequence.

[0053] SEQ ID NO: 18 is a nucleic acid sequence comprising the PID104295 promoter sequence.

[0054] SEQ ID NO:19 is a nucleic acid sequence comprising the dld1 promoter sequence.

[0055] SEQ ID NO:20 is a nucleic acid sequence comprising the xyn4 promoter sequence.

[0056] SEQ ID NO:21 is a nucleic acid sequence comprising the PID72526 promoter sequence.

[0057] SEQ ID NO:22 is a nucleic acid sequence comprising the axe3 promoter sequence.

[0058] SEQ ID NO:23 is a nucleic acid sequence comprising the hxk1 promoter sequence.

[0059] SEQ ID NO:24 is a nucleic acid sequence comprising the dic1 promoter sequence.

[0060] SEQ ID NO:25 is a nucleic acid sequence comprising the opt promoter sequence.

[0061] SEQ ID NO:26 is a nucleic acid sequence comprising the gut1 promoter sequence.

[0062] SEQ ID NO:27 is a nucleic acid sequence comprising the pki1 promoter sequence.

[0063] SEQ ID NO:28 is the nucleic acid sequence of primer TP13.

[0064] SEQ ID NO:29 is the nucleic acid sequence of primer TP14.

[0065] SEQ ID NO:30 is the nucleic acid sequence of primer TP15.

[0066] SEQ ID NO:31 is the nucleic acid sequence of primer TP16.

[0067] SEQ ID NO:32 is the nucleic acid sequence of primer TP17.

[0068] SEQ ID NO:33 is the nucleic acid sequence of primer TP18.

[0069] SEQ ID NO:34 is the nucleic acid sequence of primer TP19.

[0070] SEQ ID NO:35 is the nucleic acid sequence of primer TP20.

[0071] SEQ ID NO:36 is the nucleic acid sequence of primer TP21.

[0072] SEQ ID NO:37 is the nucleic acid sequence of primer TP22.

[0073] SEQ ID NO:38 is the nucleic acid sequence of primer TP23.

[0074] SEQ ID NO:39 is the nucleic acid sequence of primer TP24.

[0075] SEQ ID NO:40 is the nucleic acid sequence of primer TP25.

[0076] SEQ ID NO:41 is the nucleic acid sequence of primer TP26.

[0077] SEQ ID NO:42 is the nucleic acid sequence of plasmid pYL1.

[0078] SEQ ID NO:43 is the nucleic acid sequence of plasmid pYL2.

[0079] SEQ ID NO:44 is the nucleic acid sequence of plasmid pYL3.

[0080] SEQ ID NO:45 is the nucleic acid sequence of plasmid pYL4.

[0081] SEQ ID NO: 46 is the T. reesei xyr1 (A824V) mutant tandem This is the amino acid sequence of protein.

[0082] SEQ ID NO: 47 is the amino acid sequence of the T. reesei Ace2 protein. It is a column.

[0083] SEQ ID NO: 48 is the amino acid sequence of the T. reesei wild-type xyr1 protein. It is an acid sequence.

[0084] SEQ ID NO:49 is the nucleic acid sequence of the primer.

[0085] SEQ ID NO:50 is the nucleic acid sequence of the primer.

[0086] SEQ ID NO:51 is the nucleic acid sequence of the primer.

[0087] SEQ ID NO:52 is the nucleic acid sequence of the primer.

[0088] SEQ ID NO:53 is the nucleic acid sequence of the primer.

[0089] SEQ ID NO:54 is the nucleic acid sequence of the primer.

[0090] SEQ ID NO:55 is the nucleic acid sequence of the primer.

[0091] SEQ ID NO:56 is the nucleic acid sequence of the primer.

[0092] SEQ ID NO:57 is the nucleic acid sequence of the primer.

[0093] SEQ ID NO:58 is the nucleic acid sequence of the primer.

[0094] SEQ ID NO:59 is the nucleic acid sequence of the primer.

[0095] SEQ ID NO:60 is the nucleic acid sequence of the primer.

[0096] SEQ ID NO:61 is the nucleic acid sequence of the primer.

[0097] SEQ ID NO:62 is the nucleic acid sequence of the primer.

[0098] SEQ ID NO:63 is the nucleic acid sequence of the primer.

[0099] SEQ ID NO:64 is the nucleic acid sequence of the primer.

[0100] SEQ ID NO:65 is the nucleic acid sequence of the primer.

[0101] SEQ ID NO:66 is the nucleic acid sequence of the primer.

[0102] SEQ ID NO:67 is the nucleic acid sequence of the primer.

[0103] SEQ ID NO:68 is the nucleic acid sequence of the primer.

[0104] SEQ ID NO:69 is the nucleic acid sequence of the primer.

[0105] SEQ ID NO:70 is the nucleic acid sequence of the primer.

[0106] SEQ ID NO:71 is the nucleic acid sequence of the primer.

[0107] SEQ ID NO:72 is the nucleic acid sequence of the primer.

[0108] SEQ ID NO:73 is the nucleic acid sequence of the primer.

[0109] SEQ ID NO:74 is the nucleic acid sequence of the primer.

[0110] SEQ ID NO:75 is the nucleic acid sequence of the primer.

[0111] SEQ ID NO:76 is the nucleic acid sequence of the primer.

[0112] SEQ ID NO:77 is the nucleic acid sequence of the primer.

[0113] SEQ ID NO:78 is the nucleic acid sequence of the primer.

[0114] SEQ ID NO:79 is the nucleic acid sequence of the primer.

[0115] SEQ ID NO:80 is the nucleic acid sequence of the primer.

[0116] SEQ ID NO:81 is the nucleic acid sequence of the primer.

[0117] SEQ ID NO: 82 is an artificial nucleic acid sequence.

[0118] SEQ ID NO: 83 is an artificial nucleic acid sequence.

[0119] SEQ ID NO: 84 is an artificial nucleic acid sequence.

[0120] SEQ ID NO: 85 is an artificial nucleic acid sequence.

[0121] SEQ ID NO: 86 is an artificial nucleic acid sequence.

[0122] SEQ ID NO: 87 is an artificial nucleic acid sequence.

[0123] SEQ ID NO: 88 is an artificial nucleic acid sequence.

[0124] SEQ ID NO: 89 is an artificial nucleic acid sequence.

[0125] SEQ ID NO: 90 is an artificial nucleic acid sequence.

[0126] SEQ ID NO: 91 is an artificial nucleic acid sequence.

[0127] SEQ ID NO:92 is the nucleic acid sequence of the primer.

[0128] SEQ ID NO:93 is the nucleic acid sequence of the primer.

[0129] SEQ ID NO:94 is the nucleic acid sequence of the primer.

[0130] SEQ ID NO:95 is the nucleic acid sequence of the primer.

[0131] SEQ ID NO:96 is the nucleic acid sequence of the primer.

[0132] SEQ ID NO:97 is the nucleic acid sequence of the primer.

[0133] SEQ ID NO:98 is the 45 (45%) amino acid sequence of the N-terminal sequence of the Ace3-EL protein of SEQ ID NO:12. ) amino acid sequence comprising the amino acid fragment at position

[0134] SEQ ID NO: 99 is the ORF of the nucleic acid sequence encoding the Ace3-SC protein.

[0135] SEQ ID NO:100 is the ORF of the nucleic acid sequence encoding the Ace3-LC protein.

[0136] SEQ ID NO:101 is the ORF of the nucleic acid sequence encoding the Ace3-EL protein.

[0137] SEQ ID NO: 102 is the ORF of the nucleic acid sequence encoding the Ace3-LN protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0138] I. Overview Certain embodiments of the present disclosure provide methods for the preparation of mutants for increasing the production of one or more proteins of interest. More particularly, certain embodiments of the present disclosure relate to heterophyllous fungal cells, compositions thereof, and methods thereof. The embodiment is a feed containing an induction agent (i.e., lactose, sophorose, gentiobiose, cellulose, etc. A mutant that can produce one or more proteins of interest in the absence of an inducing substrate (such as a sucrose substrate). Thus, certain embodiments of the present disclosure relate to heterophyllic fungal cells that are variants derived from a parent filamentous fungal cell. For heterophyllic fungal (host) cells, the mutant host cells grow in the absence of an inducing substrate (target tan). This involves genetic modification that allows expression of a gene of interest (encoding a protein of interest). The gene of interest (encoding a protein) is an endogenous fungal gene (e.g., cbh1, ch b2, xyn1, xyn2, xyn3, egl1, egl2, egl3, bgl1, bg l2, etc.) or a gene heterologous to the filamentous fungal cell.

[0139] Thus, in other specific embodiments, the mutant fungal host cell of the present disclosure comprises an Ace3-L TA Ace3-EL protein (SEQ ID NO: 12) and Ace3- LN protein (SEQ ID NO: 14). A gene that increases the expression of the "activator of cellulase expression 3" (ace3) gene, which activates cellulase expression In other embodiments, the mutant fungal host cell of the present disclosure comprises an Ace3-L protein. (SEQ ID NO: 6), Ace3-EL protein (SEQ ID NO: 12) and Ace3-LN protein A polypeptide encoding an Ace3 protein selected from the group consisting of: Genetic modification to increase expression of oligonucleotide open reading frames (ORFs) Thus, in certain embodiments, the mutant fungal host cell of the present disclosure comprises Ace3-L Protein (SEQ ID NO: 6), Ace3-EL protein (SEQ ID NO: 12) and Ace3 -LN protein (SEQ ID NO: 14), and Ace3, which encodes an Ace3 protein having about 90% to about 99% sequence identity. 3. Includes genetic modifications that increase expression / production of the gene or its ORF.

[0140] In certain embodiments, Ace3 proteins (i.e., Ace3-L, Ace3-EL or Ace3-LN protein) in filamentous fungal host cells. In another embodiment, the filamentous chromosome is an ace3 expression cassette integrated into the genome (chromosome). Genetic modification to increase expression of the Ace3 protein in bacterial cells was performed by inserting an ace3 expression cassette. (i.e., encoding the Ace3-L, Ace3-EL, or Ace3-LN protein) In another embodiment, the present invention is an episomally maintained plasmid construct comprising: In this study, we investigated whether Ace3-L, Ace3-EL or Ace3-LN proteins were expressed in fungal cells. A genetic modification that increases expression of the ace3 gene is integrated into the telomere site. In certain embodiments, such an expression cassette is a telomeric vector / plasmid. Alternatively, the plasmid is present in two or more copies. The ORF of the ace3 gene or ace3 is operably linked to a heterologous promoter. In embodiments, the Ace3-L, Ace3-EL or Ace3-LN protein is expressed in a filamentous fungal cell. Genetic modification to increase expression of the ace3 gene (or its ORF) encoding a protein ace3-L, Ace3-EL or Ace3-LN protein coding The native ace3 promoter (i.e., the ace3 gene is naturally The modification is related to the naturally occurring ace3 promoter region.

[0141] II. Definition Before describing the present compositions and methods in further detail, the following terms and phrases are defined. Terms not defined should be accorded their ordinary meaning as used and known to those of ordinary skill in the art. do.

[0142] All publications and patents cited herein are hereby incorporated by reference. can be.

[0143] When a range of values ​​is given, the upper and lower limits of the range are used unless the context clearly dictates otherwise. Each intervening value between the lower and upper limits, to one-tenth of the unit of the lower limit, and any value within that range It is understood that other stated or intervening values ​​are encompassed within the scope of the present compositions and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges. and subject to any specifically excluded limits in the stated ranges. All of the above are encompassed by the compositions and methods of the invention. If a range includes any of the inclusive limits, then ranges excluding either or both of those inclusive limits also include those limits that are within the scope of the present invention. and methods.

[0144] Some ranges are provided herein with numerical values ​​preceded by the term "about." The term " " is used herein to refer to the exact number that it precedes, as well as the number that it precedes. Literal support for near or approximate numbers ) to indicate whether a number is close or approximate to a specifically recited number. In determining the amount, the unlisted near or approximate number shall be considered in the context in which it is presented. For example, a number may be used that provides a substantially equivalent number to a specifically recited number. In this context, the term "about" is used unless that term is clearly defined otherwise in the context. , refers to a range of -10% to +10% of that number. Another example is the phrase "a pH value of about 6." refers to a pH value between 5.4 and 6.6, unless the pH value is specifically defined otherwise.

[0145] The headings provided herein are incorporated by reference in their entirety. It is not intended to limit the various aspects or embodiments of the compositions and methods. The terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0146] In accordance with this Detailed Description, the following abbreviations and definitions apply: "A," "an," and "the" are used unless the context clearly indicates otherwise. Please note that unless otherwise specified, multiple referents are included. Reference to an "enzyme" includes a plurality of such enzymes and reference to "a dose" includes one or more doses. and equivalents thereof known to those skilled in the art.

[0147] It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement should not be construed as limiting the scope of the invention in any manner that would render the invention “sole” in any manner whatsoever in connection with the recitation of claim elements. "lely", "only", "excluding", Use of exclusionary terminology, such as "not including" or " "The term 'negative' limitation is intended to serve as a predicate to the use of the term."

[0148] Additionally, the term "comprising" as used herein When the term "comprising" is used, the component following the term "comprising" is changed to "including, but not limited to, "including, but not limited to" Please note that the following phrase after the term "comprising" An ingredient is necessary or essential, but a composition that contains that ingredient may contain other non-essential or optional ingredients. It may further include a part.

[0149] The term "consisting of" is used herein. When a term is used, the component following the term "consisting of" is "Including and limited to Please note that "consisting of" means "consisting of" The ingredient following the term f) is therefore necessary or essential and the composition No other ingredients are included.

[0150] As will be apparent to those of skill in the art upon reading this disclosure, the particular implementations described and illustrated herein may Each of the forms does not depart from the scope or spirit of the compositions and methods described herein. The present invention can be easily separated or combined with any of the features of the other several embodiments without any need for modification. Any of the methods described have distinct components and features that can be used to The steps may be carried out in the order of events shown, or in any other order which is logically possible.

[0151] As used herein, the term "ascomycota fungal cell" refers to a fungal cell of the phylum Ascomycota in the kingdom Fungi. Ascomycota refers to any organism in the family Ascomycota. Examples of ascomycota fungal cells include Trichoderma Trichoderma species, Aspergillus species, Myceliophthora species and Penicillium species Filamentous fungi within the subphylum Pezizomycotina, such as species of Including, but not limited to, bacteria.

[0152] As used herein, the term "filamentous fungi" refers to fungi from the subfamily Eumycota. It refers to all filamentous forms of fungi in the phylum Oomycota and subphylum Oomycota. For example, These include, but are not limited to, Acremonium, Aspergillus, (Aspergillus), Emericella, Fusarium Fusarium), Humicola, Mucor, Myceliophthora, Neurospora pora, Penicillium, Scytharia alidium, Thielavia, Tolipocladium ypocladium, or Trichoderma species. In some embodiments, the filamentous fungus is Aspergillus aculeatus. gillus aculeatus, Aspergillus awamori us awamori), Aspergillus foetidus etidus, Aspergillus japonicus icus, Aspergillus nidulans s), Aspergillus niger, or Aspergillus It may be Aspergillus oryzae.

[0153] In some embodiments, the filamentous fungus is Fusarium bactridioides. ium bactridioides, Fusarium cerealis cerealis), Fusarium crook wellense), Fusarium culmorum , Fusarium graminearum, Fusari Fusarium graminum, Fusarium heterosporum Fusarium heterosporum, Fusarium negundii arium negundi, Fusarium oxysporum sporum, Fusarium reticulatum m), Fusarium roseum, Fusarium sambu Fusarium sambucinum, Fusarium sarcochromium (F usarium sarcochroum, Fusarium sporotrichioides (Fu sarium sporotrichioides, Fusarium sulfureum sarium sulphureum, Fusarium torulosum torulosum, Fusarium trichothechioides othecioides or Fusarium venenatum In some embodiments, the filamentous fungus is Humicola insolens. Humicola insolens), Humicola lanuginosa (Humicola lanuginosa), Mucor miehei, Miceli Myceliophthora thermophila, Neurospora crassa, Cytalidium te Scytalidium thermophilum, or Chieravi The species is Thielavia terrestris.

[0154] In some embodiments, the filamentous fungus is Trichoderma harzianum. rma harzianum), Trichoderma koningii (Trichoderma k oningii, Trichoderma longibrachiatum (Trichoderma longibrachiatum) ngibrachiatum, Trichoderma reesei eesei) or Trichoderma viride In some embodiments, the filamentous fungus is Trichoderma reesei. Rma reesei) American Type Culture Collection, No. 56765 T. reesei, available from the Culture Collection Trichoderma reesei (Trichoderma reesei) strain “Rut-C30” In some embodiments, the filamentous fungus is a U.S. Department of ent of Agriculture, Northern Regional Res. NRRL number 15709 and Trichoderma from T. reesei strain "RL-P37" available at The cells are Trichoderma reesei cells.

[0155] As used herein, "mutated filamentous fungal cells," "mutated fungal cells," "mutated cells," etc. The term refers to the parent (control) of the subphylum Pezizomycotina. ) refers to a filamentous fungal cell derived from (i.e., obtained from) a filamentous fungal cell. A "mutated" fungal cell as defined in the document is derived from a "parent" fungal cell, but this "mutated" cell The mutant strains of the present disclosure contain at least one genetic modification that is not present in the "parent" cell. When comparing a "filamentous fungal cell" to a "parental filamentous fungal cell," the "parental" cell has at least one gene The “mutated” cells containing the modification were compared to the genetically unmodified (parent) “control” cells. It fulfills its role.

[0156] As used herein, the term "genetic modification" refers to a modification / change in a nucleic acid sequence. Modifications include, but are not limited to, substitutions, deletions, or deletions of at least one nucleotide in a nucleic acid sequence. These may include deletions, insertions or chemical modifications.

[0157] As defined herein, "mutated cells containing a genetic modification" and "Ace3-L protein" , the gene encoding the Ace3-EL protein and / or the Ace3-LN protein The phrase "mutated cells containing a genetic modification that increases expression of the Ace3-L protein" refers to The gene encoding the Ace3-EL protein and / or the Ace3-LN protein The method includes introducing at least one copy of a gene or an ORF into a filamentous fungal cell, Therefore, the exogenously introduced Ace3-L protein, Ace3 -EL protein, and / or Ace3-LN protein-encoding gene, or Filamentous fungal cells that contain at least one copy of the ORF are referred to as parental fungal cells (unmodified). As compared to a mutant fungal cell that contains a genetic modification.

[0158] In other embodiments, the parent fungal cell of the present disclosure comprises an Ace3 protein as disclosed herein. The endogenous ace3 gene encoding either the Ace3-S protein or the Ace3-S protein (A ce3-SC protein, Ace3-L protein, Ace3-LC protein, Ace The presence of the ace3 gene, which encodes the Ace3-EL and Ace3-LN proteins For example, the parent fungal cell is screened for the presence of the Ace3-L protein, Ace3-L protein, The ace3 gene encodes the ce3-EL or Ace3-LN protein. If it is determined that the modified fungal cell contains an endogenous copy of the For example, by replacing the endogenous promoter of the ace3 gene with a heterologous promoter. Similarly, if the parent fungal cell is capable of producing the Ace3-S protein, the Ace3-SC protein, or contain an endogenous copy of the ace3 gene encoding the Ace3-LC protein. If so determined, the mutant fungal cell may be genetically modified to, for example, express the Ace3 -L protein, Ace3-EL protein, and / or Ace3-LN protein A polynucleotide construct encoding the This can be achieved by using the Ace3-S, Ace3-SC, or Ace3-LC proteins. The method may further comprise genetic modification of the endogenous ace3 gene which encodes a gene for a target gene.

[0159] In other embodiments, the mutant filamentous fungal cells of the present disclosure will contain additional genetic modifications. For example, in certain embodiments, such mutant filamentous fungal cells (i.e., Ace3 of the present disclosure) can be used. -L protein, encoding the Ace3-EL and / or Ace3-LN proteins The exogenously introduced copy of the gene or ORF is a carbon catabolite repressor. A gene encoding the sarcoprotein "Cre1" or Ace1 repressor protein The present invention further includes genetic modifications that reduce the expression and / or activity of.

[0160] In other embodiments, such mutant filamentous fungal cells (i.e., the Ace3-L protein of the present disclosure) can be used. The genes encoding the proteins Ace3-EL and / or Ace3-LN or an exogenously introduced copy of the ORF) is set forth in SEQ ID NO:25 or SEQ ID NO:27. By introducing at least one copy of the xylanase regulator 1 (Xyr1) shown It further includes genetic modifications.

[0161] As used herein, the "Ace3-L" protein form (SEQ ID NO:6) and " The "Ace3-LN" protein form (SEQ ID NO: 14; see FIG. 1B) contains the same Ace3-LN However, the headings "Ace3-L" and "Ace3-LN" For comparison with the specific genes encoding such protein forms, see the Although used in a specific embodiment, this is in no way a limitation of the present disclosure.

[0162] As used herein, the term "host cell" refers to a cell that is capable of expressing a specific For an incoming sequence (i.e., a polynucleotide sequence to be introduced into a cell), It refers to a filamentous fungal cell that has the capacity to act as a host and an expression vehicle.

[0163] A "heterologous" nucleic acid construct or sequence is not native to the cell in which it is expressed. or have a portion of the sequence that is not present in the natural form. Regulatory sequences that do not naturally regulate the expression of the same gene ( Generally, heterologous nucleic acid sequences refer to those is not endogenous to the cell or part of the genome in which it resides, but is a Into cells by gene transfer, transformation, microinjection, electroporation, etc. A "heterologous" nucleic acid construct is one that contains regulatory sequences that are not found in the native cell. / DNA coding sequence combinations with identical or different regulatory sequences / DNA coding Similarly, heterologous proteins may contain a combination of sequences that are not related to each other in the same relationship as they are in nature. The term often refers to two or more subsequences not found in a sequence (e.g., a fusion protein).

[0164] As used herein, "DNA construct" or "expression construct" The term refers to a nucleic acid sequence that comprises at least two DNA polynucleotide fragments. The term refers to the introduction of nucleic acid sequences into fungal host cells using DNA or expression constructs. This DNA can be synthesized in vitro (e.g., by PCR) or by any other suitable procedure. In some preferred embodiments, the DNA construct may be generated by a method In certain embodiments, the target gene includes a sequence (e.g., encoding an Ace3-L protein). In some embodiments, the target polynucleotide sequence is operably linked to a promoter. In some embodiments, the DNA construct further comprises at least one selectable marker. In an embodiment, the DNA construct comprises sequences that are homologous to a host cell chromosome. In this embodiment, the DNA construct contains sequences that are heterologous to the host cell chromosome.

[0165] As used herein, the term "cellulase," "cellulolytic enzyme," or "cellulase" refers to a The term "exoglucanase enzyme" includes exoglucanases, exo-cellobiohydrolases, endoglucanases, means bacterial or fungal enzymes such as lucanase and / or β-glucosidase These different types of cellulase enzymes convert cellulose and its derivatives into glucose. For example, many microorganisms, such as the wood-rotting fungus Trichoderma The genus Trichoderma, the composting bacteria Thermomonospora nospora) (now Thermobifida), Bacillus subtilis Bacillus, and Cellulomonas; Genus Streptomyces; and fungus genus Humicola ola, Aspergillus, and Fusarium arium produces enzymes that hydrolyze cellulose. Enzymes are mixtures of proteins that have three different functions that help convert cellulose to glucose. Compounds, namely endoglucanases (EG), cellobiohydrolases (CBH), and It is a mixture of β-glucosidases (BGs). EG, cellobiohydrolase (CBH) and β-glucosidase (β The terms "EG", "CBH" and "BG" are interchangeable with their abbreviations, respectively. It is used for the purpose.

[0166] As used herein, the term "carbon limitation" refers to the inability of a microorganism to produce a desired protein. There is enough carbon to produce just enough food, but not enough to fully satisfy the needs of living things. For example, there is not enough carbon to sustain growth. , the greatest amount of carbon is used for the production of proteins.

[0167] As used herein, the term "promoter" refers to a promoter for a downstream gene or Designates a nucleic acid sequence that functions to direct the transcription of an open reading frame (ORF). The promoter is generally appropriate for the host cell in which the target gene is being expressed. The target, together with other transcriptional and translational regulatory nucleic acid sequences (also called "control sequences"), regulates the function of a given gene. In general, transcriptional and translational regulatory sequences include promoters, which are required for the expression of a gene. -sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences sequences, as well as enhancer or activator sequences. In certain embodiments, the promoter is an inducible promoter. The promoter is a constitutive promoter.

[0168] As used herein, a "promoter sequence" refers to a promoter sequence that is specific to a particular filamentous fungus for expression purposes. A "promoter" is a nucleic acid that directs the transcription of a nucleic acid. As used herein, a promoter is defined as an array of regulatory sequences that regulates transcription. The desired nucleic acid sequence near the start site is, for example, T A "constitutive" promoter is one that can express the promoter under most environmental and developmental conditions. An "inducible promoter" is a promoter that is active under environmental or developmental regulation. It is a promoter active in The term "operably linked" refers to a nucleic acid expression control sequence (e.g., a promoter, The term refers to the functional linkage of a nucleic acid sequence (or an array of transcription factor binding sites) to a second nucleic acid sequence, and is used to control expression. The sequence directs the transcription of a nucleic acid corresponding to a second sequence. Thus, the nucleic acid is transfected into another nucleus. "Operably linked" means that a polypeptide is placed into a functional relationship with a nucleic acid sequence. For example, a secretory linker The DNA encoding the signal peptide is involved in the secretion of the polypeptide. Operable with DNA encoding a polypeptide when expressed as a preprotein a promoter or enhancer is a cognate to a promoter sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to the nucleic acid sequence to facilitate translation. Generally, an "operably linked" gene is referred to as a coding sequence when it is positioned in such a way that it is operably linked to the coding sequence. By "linked to" it is meant that the DNA sequences being linked are contiguous, and In the case of , it means that it is adjacent and in the leading face. The sensors do not have to be contiguous. Linking can be accomplished by ligation at appropriate restriction sites. If no such site exists, a synthetic oligonucleotide may be prepared according to conventional techniques. In another embodiment, the linkage is a DNA adapter or linker. A was combined with the nucleotide sequence in a sequence-independent, scarless manner to create a seamless cloning method. Seamless cloning is usually performed using Gibson Assembly (NEB) , NEBuilder HiFi DNA Assembly (NEB), Golden Gate Assembly (NEB), and GeneArt Seamless cloning and assembly system(ThermoFisher This is performed using commercially available systems such as those from MIT Scientific. The present invention is not limited to these.

[0169] As used herein, the term "coding sequence" refers to a sequence that encodes a protein. A coding sequence refers to a nucleotide sequence that directly specifies the amino acid sequence of a protein product. It is generally determined by an open reading frame, which usually begins with an ATG start codon. Coding sequences typically include DNA, cDNA, and recombinant nucleotide sequences.

[0170] As defined herein, an "open reading frame" (hereinafter "ORF") is a (ii) a series of two or more codons representing amino acids; and (iii) a stop codon. A nucleic acid or nucleic acid sequence (naturally occurring) comprising a continuous reading frame consisting of a donor. ORF means any sequence that begins at the 5' It is read (or translated) in the 3' direction from

[0171] As used herein, the term "gene" refers to any gene involved in the production of a polypeptide chain. By "coding" we mean the segment of DNA preceding or following the coding region (e.g., the 5' untranslated (5'UTR) sequence or "leader" sequence, and 3'UTR sequence or "trailer" sequence. " sequences, as well as the intervening sequences (introns) between the individual coding segments (exons). The gene may or may not contain an enzyme (e.g., a protease, enzymes, xylanases, amylases, glucoamylases, cellulases, oxidases, lipases The gene may code for a commercially important industrial protein or peptide, such as a ribozyme (RI), a ribozyme (RI), or a ribozyme (RI). The gene may be a naturally occurring gene, a mutated gene or a synthetic gene.

[0172] The term "recombinant" when used in reference to a cell, nucleic acid, protein, or vector The term as used herein refers to a cell, nucleic acid, protein, or vector that is a heterologous nucleic acid. by the introduction of a heterologous nucleic acid or protein, or by modification of a naturally occurring nucleic acid or protein. that the cell has been modified by a method known as gene therapy, or that the cell is derived from a cell so modified Thus, for example, a recombinant cell contains a structure not found within the native (non-recombinant) form of the cell. Expressing genes that are otherwise abnormally expressed, under-expressed, or not at all Expressing naturally occurring genes that are not expressed.

[0173] The term "vector" as used herein refers to a vector carrying a nucleic acid sequence for introduction into one or more cell types. A vector is defined as a polynucleotide designed to contain a cloning vector, expression vector, shuttle vector, plasmid, phage or virus particle Expression vectors include vectors, DNA constructs, and cassettes. It may include regulatory sequences such as signal sequences, coding sequences and transcription terminators.

[0174] As used herein, an "expression vector" refers to a vector capable of expressing a protein in a suitable host. A DNA construct comprising a coding sequence operably linked to a suitable control sequence capable of Such control sequences include promoters that cause transcription, sequences that control transcription, and an optional operator sequence encoding a suitable ribosome binding site on the mRNA These may include sequences, enhancers, and sequences which control the termination of transcription and translation.

[0175] As used herein, the term "secretory signal sequence" refers to a signal sequence that is secreted by a larger polypeptide. As a component of a peptide, it passes through the secretory pathway of the cell in which it is synthesized into a larger polypeptide. means a DNA sequence that codes for a polypeptide that is to be secreted (i.e., a "secretory peptide"). The larger polypeptide is usually cleaved during passage through the secretory pathway to produce the secretory peptide. is removed.

[0176] As used herein, the term "induction" refers to an "inducer" (i.e. In response to the presence of an inducing substrate, the target protein (hereafter referred to as the target protein) is synthesized in filamentous fungal cells at an extremely high rate. It refers to an increase in the transcription of genes that result in the synthesis of a protein called a POI (promoter's interest). To measure the “induction” of a gene of interest (GOI) or ORF of interest, Filamentous fungal (host) cells are treated with a candidate inducer, and the inducer The results are compared to parental filamentous fungal (control, unmodified) cells that have not been treated with the If we assign a relative protein activity value of 100% to the parent (control) cells, the mutant host cells Induction of the GOI encoding the POI in the is greater than 100%, greater than 110%, more preferably greater than 150%, Preferably, the antibody is 200-500% (i.e., 2-5 times higher than the control), or more preferably This is usually achieved when the temperature is 1000-3000% higher.

[0177] As used herein, "inducer" or "inducer "inducers" or "inducing substrate" or "inducing substrates" are used interchangeably and are used interchangeably to describe Induces bacterial cells to produce "increased amounts" of polypeptides (e.g., enzymes, receptors, antibodies, etc.) or to produce a compound / substance other than that which is produced in the absence of an inducing substrate. Inducing substrates are any compound that induces the synthesis of glycerol. Examples of inducing substrates include sophorose, lactose, and gentibiose. and cellulose, but are not limited to.

[0178] As used herein, the term "inducing feed" includes at least an "inducing substrate." The term "inducing feed" refers to a composition that is fed to filamentous fungal cells, such as a fungal feed, to induce the production of POI. .

[0179] As used herein, the terms "isolated" or "purified" is a nucleic acid or amino acid that is removed from at least one component to which it is naturally attached Refers to...

[0180] As defined herein, the term "protein of interest" or "POI" refers to a protein derived from a filamentous fungal cell. The term "protein" refers to a polypeptide that is desired to be expressed in a cell. They can be enzymes, substrate-binding proteins, surface active proteins, structural proteins, etc. The protein of interest can be expressed in a mammalian cell line and can be intended for commercialization. by genes or heterologous genes (i.e., to the mutant cell and / or to the parent cell) The protein of interest can be encoded intracellularly or as a secreted (extracellular) protein. It can be expressed as a quality.

[0181] As used herein, "polypeptide" and "protein" (and / or The terms are used interchangeably and are separated by peptide bonds. It refers to a polymer of any length that contains linked amino acid residues. Conventional one-letter or three-letter codes are used to indicate the molecular weight of the polymer. The polymer may be linear or branched. It may be of any shape, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also refer to groups that are naturally occurring or that have been modified by intervention (e.g., disulfide bond formation, glycosylation, etc.). or any other manipulation, such as silencing, lipidation, acetylation, phosphorylation, or conjugation with a labeling moiety. Also included are amino acid polymers that have been modified by, for example, amino one or more analogs of an acid (including, for example, unnatural amino acids, etc.), and Polypeptides containing other known modifications are also included within the definition.

[0182] As used herein, functionally and / or structurally similar proteins are defined as " Such proteins are considered to be "genus and / or species related proteins." They may be derived from different organisms or different classes of organisms (e.g., bacteria and fungi). Proteins may also be characterized by their sequence, as determined by primary sequence analysis, or by secondary or tertiary structure analysis. The term encompasses homologs as determined by immunological cross-reactivity or as determined by immunological cross-reactivity.

[0183] As used herein, the phrase "substantially no activity" or similar terms The phrase states that the specific activity cannot be detected in the mixture or does not interfere with the intended purpose of the mixture. This means that it is present in small amounts.

[0184] As used herein, the term "derived polypeptide" refers to an N-terminally and C-terminally Addition of one or more amino acids to either or both of the amino acid sequences, substitution of one or more amino acids at different sites in the or deletion of one or more amino acids at one or more sites in the amino acid sequence, and / or Alternatively, the protein may be modified by the insertion of one or more amino acids at one or more sites in the amino acid sequence. This refers to a protein that is obtained or can be obtained from a protein. The method comprises modifying a DNA sequence encoding a naturally occurring protein and injecting the DNA sequence into a suitable host. and expressing the modified DNA sequence to form an induced protein. can be achieved.

[0185] Related (and derived) proteins include "mutated proteins" is a sequence similar to that of the reference / It is different from the parent protein (e.g., the wild-type protein). Mutant protein and parent protein The number of different amino acid residues between the It may be 8, 9, 10, 15, 20, 30, 40, 50 or more amino acid residues. The mutant protein has a similar sequence to the reference protein by at least about 70%, at least about 75%, At least about 80%, at least about 85%, at least about 90%, at least about 91%, At least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 9 The mutant proteins may share 9% or more amino acid sequence identity. The sequence of the target protein differs from that of the reference protein in terms of the motifs, domains, epitopes, conserved regions, etc. It may be possible.

[0186] As used herein, the term "homologous protein" refers to a protein that is similar to a reference protein. Homologs are proteins that have the activity and / or structure of a homologue. Homologs are not necessarily evolutionarily related. Therefore, the term does not refer to the same or similar species obtained from different organisms. It is intended to encompass similar or corresponding enzymes (i.e., with respect to structure and function). In some embodiments, quaternary, tertiary and / or nucleotide sequences similar to the reference protein are used. In some embodiments, it is desirable to identify homologs having a primary structure. In some embodiments, the protein induces a similar immune response as the reference protein. The protein is genetically engineered to produce an enzyme with a desired activity.

[0187] The degree of homology between sequences can be determined using any suitable method known in the art. (e.g., Smith and Waterman, 1981; Needleman and Wunsch,1970;Pearson and Lipman,1988; Wisconsin Genetics Software Package(Gene GAP, BEST from tics Computer Group, Madison, WI programs such as FIT, FASTA and TFASTA; and Devereux et al., 1984).

[0188] As used herein, the terms "substantially similar" and "substantially identical" are used interchangeably. The phrase "polynucleotide" in the context of at least two nucleic acids or polypeptides generally refers to a polynucleotide. The peptide or polypeptide has at least about 70% repeat sequence identity compared to a control (i.e., wild-type) sequence. % identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, At least about 98% identity, or at least about 99% identity, or more Sequence identity is determined using the methods of BLAST, ALIGN, and CL. It can be determined using standard parameters by known programs such as USTAL. can.

[0189] As used herein, the term "gene" refers to a gene that expresses a protein or RNA. When referring to a nucleic acid that encodes and indicates a gene, it is synonymous with the term "allele." The vegetative form of a fungus is generally haploid, so the expression of a specific gene is required to confer a particular phenotype. One copy (ie, a single allele) is sufficient.

[0190] As used herein, the terms "wild-type" and "native" are used interchangeably. "Genes" refers to genes, proteins, fungal cells or strains as they are synthesized and found in nature.

[0191] As used herein, "gene deletion" refers to the removal of a gene from the genome of a host cell. A gene is a regulatory element that is not located immediately adjacent to the coding sequence of the gene (e.g. For example, an enhancer element is included in the gene deletion, the deletion of the coding sequence, In addition, in some cases, for example, a promoter sequence and / or a terminator sequence (these "Deletion of flanking enhancer elements" refers to a deletion of flanking enhancer elements, including but not limited to:

[0192] As used herein, "disruption of a gene" refers to the disruption of a cell from producing a functional gene within a host cell. Any genetic or chemical manipulation that substantially prevents a plant from producing a substance, e.g., a protein. Exemplary disruption methods include disrupting a polypeptide coding sequence, a promoter sequence, or a promoter mutation. Complete sequence of any part of a gene, including a motor, enhancer, or other regulatory element. This includes total or partial deletions or mutations, and mutations can be substitutions, insertions, deletions, inversions, etc. The present invention includes all of the above-mentioned mutations, and combinations and variations thereof, all of which are functional. The gene can also be expressed by RNAi, antisense, CR, or other methods that substantially prevent the production of a functional gene product. Using ISPR / Cas9 or any other method that abolishes, reduces or decreases gene expression It can be used to destroy it.

[0193] As used herein, "the Ace3-L protein, Ace3-EL and and / or "genetic modification" that increases the expression of the gene encoding the Ace3-LN protein. The phrase "mutated [host] cell" includes such Ace3-L protein, Ac The ace3 gene forms encoding the e3-EL and / or Ace3-LN proteins ( or its ORF) into a host cell. In other particular embodiments, the parent fungal cell is transformed (e.g., by transformation). The cells express Ace3-L protein, Ace3-EL and / or Ace3-LN protein. When the gene naturally contains an endogenous gene form encoding Ace3, it is referred to as "Ace3-L protein, Ace3 -Increase the expression of genes encoding the EL and / or Ace3-LN proteins The phrase "mutated [host] cell," including "genetically modified," refers to a cell that is This involves replacing the promoter with a heterologous promoter.

[0194] As used herein, "aerobic fermentation" refers to growth in the presence of oxygen.

[0195] As used herein, the term "cell broth" refers to a cell culture in liquid / submerged culture. The term "culture medium" collectively refers to the medium and cells in culture.

[0196] As used herein, the term "cell mass" refers to the amount of cells present in a liquid / submerged culture. The amount of cells refers to the amount of cellular components present in a sample, including intact and lysed cells. Cell mass can be expressed as dry weight or wet weight. It can be expressed as wet weight.

[0197] As used herein, a "functional polypeptide / protein" refers to a polypeptide or protein that has an enzymatic activity, binding and the like, and in order to eliminate or reduce the activity, Proteins that have not been mutagenized, truncated, or otherwise modified. A functional polypeptide may be thermally stable or unstable, as described herein. could be.

[0198] As used herein, a "functional gene" refers to an active gene product, i.e., a gene that is capable of producing an active gene product. Typically, a functional gene is a gene that can be used by cellular components to produce a protein. is that cellular components cannot be used to produce an active gene product or that the cell A disrupted gene that has been modified to reduce its ability to be used by the component to produce an active gene product. This is the exact opposite of the genes that were

[0199] As used herein, a "protein of interest" refers to a protein produced in a submerged culture of filamentous fungal cells. Proteins of interest are proteins for which production is desired. Generally, proteins of interest are used for industrial, pharmaceutical, animal health, and food and beverage applications, and are commercially important and can be produced in large quantities. The protein of interest is expressed by a number of fungal cells. They should be distinguished from other proteins, which are generally not products but are the main These are likely background protein contaminants.

[0200] As used herein, a "mutated fungal host cell" refers to a mutant fungal host cell that is capable of expressing a mutated gene. The amount of protein is increased by at least 5%, at least 10%, or at least If both have a 15% or greater increase, the "biomass" of the fungal cells will be greater than that of the "parent fungal cells." "Substantially more protein per unit of protein" (where The amount of protein produced was normalized to the total biomass of the cells in which protein production was measured. where biomass can be expressed as wet weight (e.g., cell pellet) or dry weight. (can be done).

[0201] III.Activator of Cellulase Expression 3( ace3) Recently, cellulase / hemicellulase production has been "induced" (i.e., with different induction compositions). (e.g., by adding sophorose, lactose) Trichoderma reesei (Tri choderma reesei) culture (Hakkinen et al., 2014 Transcriptional profiling data from Putative "regulators" of gene expression were identified, and candidate genes encoding regulatory proteins were identified. Hakkinen et al. (2014) identified gene ID number 77 513 (gene ID number is the same as in T. reesei Database 2.0) We identified this candidate gene as a marker for the schizophrenia syndrome (Hakkinen et al., Fig. 2 and (See Table 2.) This candidate gene was named "Activator of Cellula The name of the coded expression is "ace3" (hereafter "ace3"). The protein (i.e., the candidate transcription factor) was named "Ace3". In a study by Inen et al. (2014), T. reesei Q M6a strain (genome.jgi.doe.gov / Trire2 / Trire2.ho The predicted a based on the publicly available genome sequence of The ce3 ORF (SEQ ID NO: 2) was used, but the predicted annotation of this QM6a The gene (gene ID 77513) consists of two exons and one intron ( See, for example, FIG. 1).

[0202] As described herein and further described in the Examples section below, the applicants of the present disclosure Based on the T. reesei "RUT-C30" annotation, For the ace3 ORF, the clone described in Hakkinen et al., 2014 was used. The cloned ace3 ORF (i.e., Ace3 of T. reesei “QM When comparing and evaluating the 6a strain annotations, surprising and unexpected results were obtained. For example, as described in Example 1 below, the longer Ace3 transcript of SEQ ID NO:6 was found to be A T. reesei protein of SEQ ID NO:4 (referred to herein as "Ace3-L") T. reesei "RUT-C30 strain" ace3 gene (or ORF of SEQ ID NO: 5) F) T. reesei "strain QM6a" ace3 of SEQ ID NO: 1 The gene (and the ORF of SEQ ID NO:2) is encoded by the shorter Ace3 protein of SEQ ID NO:3 ( Herein, this gene is referred to as "Ace3-S."

[0203] In contrast, the publicly available genome of T. reesei strain Rut-C30 Genome sequence (genome.jgi.doe.gov / TrireRUTC30_1 / Tr ireRUTC30_1.home.html) The ORF (gene ID 98455) contains three exons and two introns. Longer protein sequences (i.e., A from T. reesei QM6a) ce3-S) (Figure 1A). More specifically, the "RUT-C30" model Therefore, the predicted initiation codon is located upstream of that of the “QM6a” model, and the C-terminus is Sense mutations (Poggi-Parodi et al., 2014) and longer This results in an N-terminal sequence and a shorter C-terminal sequence (see, for example, FIG. 1B).

[0204] Similarly, as described in Example 6 below, the position of the 5' end of the ace3 gene coding region Therefore, applicants have identified the 5′ endogenous ... The ends were further evaluated. As briefly mentioned above, the Joint Genome Ins The annotation of DNA sequences in the Japanese Genomic Identification (JGI) is based on the same DNA sequence. However, the results were different between the mutant strain Rut-C30 and the wild-type strain QM6a. The 5' end of the chromosome region is upstream of exon 3 and is suggested to be within intron 2. In the case of Rut-C30, the 5' end of the coding region is exon 2. It is located within (Figure 11).

[0205] Further analysis of the genomic DNA sequence and additional cDNA sequences identified "exon 1" and It was suggested that there may be an "intron 1" (as shown in Figure 11). Furthermore, the 3' end of the ace3 coding region of Rut-C30 was identical to that of the wild-type isolate QM6a. Compared with the sequence, it contained a mutation that created a premature stop codon (Figure 11). As described in Example 6, Applicants have demonstrated that these differences in the ace3 gene are The effect of overexpression of different possible forms was examined.

[0206] Furthermore, as described in the Examples below, Applicant has developed "pYL1", "pYL2", "pY These were named "pYL3" and "pYL4" (see plasmid maps in Figures 2A-2D). T. reesei was transformed with one of four different ace3 expression vectors. ) cells to produce the Ace3-S protein (SEQ ID NO: 3) and Ac A gene encoding the e3-L protein (SEQ ID NO:6) was constructed (Example 1) and tested. (Examples 2 to 4). More specifically (Example 1), these expression vectors were transformed into E. coli The bacterial ColE1 ori and AmpR genes for replication and selection in E. coli In addition, the expression vector (see Figures 2A-2D) ) is the T. reesei pyr2 selection marker and its natural terminator The ace3 ORF coding sequence (ace3-L or ace3-S) operably linked heterologous T. reesei promoter sequences (i.e., xk1 or pki1 promoter).

[0207] Subsequently, stable T. reesei transformants (i.e. Mutant host cells (A4-7, B2-1, C2-28 and D3-1) were treated as “induced” and “non-induced” Under both "induction" and "reaction" conditions, the sustained release microtiter plate (srMTP) (Example 2), Tested / screened in shake flasks (Example 3) and small-scale fermentations (Example 4) The culture supernatants were collected and analyzed by polyacrylamide gel electrophoresis (Figure 3-Figure 4). 5). As shown in these Examples, all of the host cells tested (i.e. , parent, mutant A4-7, mutant B2-1, mutant C2-28 and mutant D3-1) were induced by (i.e., sophorose or lactose) were used to secrete large amounts of protein. In contrast, surprisingly, the absence of inducing substrates (i.e., sophorose or lactose) Below, the mutant host expressing ace3-L ORF when glucose is the only carbon source. Only chief cells (i.e., mutants A4-7 and C2-28) produce secreted proteins. The parental (control) cells and mutant host cells expressing the ace3-S ORF (i.e. It was observed that the mutants B2-1 and D3-1 did not produce detectable secreted protein. It was done.

[0208] In another embodiment, the present disclosure provides a method for the expression of an ace3-L (Example 5) expression construct. The protein was produced under "non-induced" conditions using 13 different promoters driven by For example, the 13 promoters tested included: (i) holm amidase gene (rev3; protein ID 103041) promoter (SEQ ID NO: 15), (ii) β-xylosidase gene (bxl; protein ID 121127) (iii) a promoter (SEQ ID NO: 16), (iv) a transketolase gene (tkl1; (protein ID 2211) promoter (SEQ ID NO: 17); (iv) a promoter of unknown function Gene (protein ID 104295) promoter (SEQ ID NO: 18), (v) oxy The promoter of the dld1 gene (protein ID 5345) (SEQ ID NO: 19), (vi) xylanase IV gene (xyn4; protein ID 111849) (vii) promoter (SEQ ID NO: 20), (vii) α-glucuronidase gene (protein I D 72526) promoter (SEQ ID NO: 21), (viii) acetyl xylan ester axelase gene 1 (axel; protein ID 73632) promoter (SEQ ID NO: 22 ), (ix) hexose kinase gene (hxk1; protein ID 73665) pro motor (SEQ ID NO: 23), (x) mitochondrial carrier protein gene (dic1 (protein ID 47930) promoter (SEQ ID NO: 24), (xi) oligopeptide The promoter of the opt transporter gene (opt; protein ID 44278) (sequence No. 25), (xii) glycerol kinase gene (gut1; protein ID 58 356) promoter (SEQ ID NO: 26) and (xiii) the pyruvate kinase gene ( The pki1 (protein ID 78439) promoter (SEQ ID NO: 27) was included. As shown in 3, parent T. reesei cells were cultured with sophorose inducer In contrast, the expression of the 13 different promoters produced secreted proteins only in the presence of the Mutant (daughter) T. reesei strains containing and expressing Ace3-L driven by either one of the T. reesei cells secreted similar amounts of TA under both induced and uninduced conditions. As described in Example 5, T. reesei ) The parent strain and its transformants were further tested in shake flask experiments and small-scale fermentations. As shown in FIG. 8, parental (control) T. reesei cells express sophorose ( The daughter strain LT83 produced secreted proteins only in the presence of the "Sop" inducer. secreted similar amounts of proteins under both induced ("Sop") and uninduced ("Glu") conditions. Similarly, as shown in FIG. ) strain produces secreted proteins only in the presence of a sophorose inducer ("Sop"). However, the daughter line LT83 was expressed under both induced ("Sop") and uninduced ("Glu") conditions. Similar amounts of secreted protein were produced.

[0209] Example 6 of the present disclosure describes the effect of overexpressing different possible forms of the ace3 gene. Experimental studies of the mutant strain Rut-C30 / wild-type strain QM6a genome have been described (e.g., (See the above discussion of sequence annotation, Figures 11 and 12). The different forms of ace3 shown in Figure 12 are from the T. reesei ace3 gene. A gene overexpression vector was inserted into the glucoamylase gene of T. reesei. When designed to allow targeted integration of ace3 into the gla1 locus, These were overexpressed in T. reesei. The constructs shown differ by having different forms of the ace3 gene. Similarly, 2% lactose or 2% The strains in Table 7 were grown in liquid medium containing glucose, and the amount of total secreted protein was measured from the culture supernatant. Overexpression of ace3-L, ace3-EL and ace3-LN forms (i.e. RutC-30 C-terminal mutation) was found to be effective in both media (i.e., 2% lactose or 2% glucose) improved total protein production (Table 8). In medium containing ace3, overexpression of all forms of the ace3 gene resulted in the production of the entire protein. Although the level of improvement was limited to the ace3 genes ace3-L and ace3-E, The results were highest in the strains overexpressing the L and ace3-LN forms (Table 8). Overexpressing the ace3-L, ace3-EL and ace3-LN forms of the ce3 gene In the case of 'non-inducing conditions' (i.e., when glucose was used as the carbon source), It is clear that secreted proteins are observed.

[0210] Example 7 of the present disclosure includes a 5' region upstream of the native promoter of the ace3 locus. DNA fragment, a loxP-flanked hygromycin B resistance selection marker cassette, and and a promoter of interest operably fused to the 5' end of the ace3 open reading frame. A promoter replacement construct was created by fusing a fragment containing the promoter For example, in a particular embodiment, The promoter replacement construct was expressed in Trichoderma reesei. Replacing the endogenous ace3 gene promoter in T. reesei cells with an alternative promoter It is used to

[0211] In Example 8 of the present disclosure, an endogenous non-lignocellulosic gene promoter of interest is It has been reported that the promoter of a cellulose gene is replaced with that of a cellulose gene. The T. reesei glucoamylase expression construct was synthesized using DNA polynucleotides. The T. reesei glucoside fragments were assembled from The ORF sequence encoding amylase is 5' (upstream) T. reesei c operably linked to the bh1 promoter and 3' (downstream) T. reesei ) operably linked to the cbh1 terminator, this construct serves as a selectable marker. The T. reesei pyr2 gene was also included in the culture. Transformants capable of secreting T. reesei glucoamylase enzyme. To identify the mutant (daughter) T. reesei cells (i.e., Ac (including genetic modifications that increase expression of the gene encoding the e3-L protein) Transform the yeast with the coamylase expression construct, select for transformants, and grow the yeast on glucose as a carbon source. in liquid medium containing coagulant (i.e., without an inducing substrate such as sophorose or lactose) As shown in Figure 10, parent T. reesei cells were cultured at 100°C for 1 h at 20°C. 1,029 μg / mL glucoamylase in a defined medium containing glucose / sophorose (inducing conditions) In a defined medium containing glucose (non-inducing conditions), the enzyme was produced at only 38 μg / mL. It only produced glucoamylase, but not ace driven by the dic1 promoter. The engineered (daughter) strain “LT88” containing 3-L expressed 3-fold higher glutamate under “induction” (Sop) conditions. coamylase (i.e., compared to the parent (control) strain) and ) conditions (i.e., compared to the parent (control) strain). Thus, these results suggest that mutant (daughter) cells containing the Ace3-L ORF These mutant cells not only produced extracellular proteins in the absence of inducers, but also The cells also grew better than the parental (control) T. reesei cells under such inductive conditions. These results indicate that the β-lactamase produced more total protein than the β-lactamase.

[0212] Example 9 describes a method for incorporating a naturally associated heterologous gene of interest promoter into a lignocellulosic target gene. It has been reported that the promoter of a gene of Buttiauxella spp. is replaced with that of a gene of Buttiauxella spp. OR encoding phytase (i.e., heterologous GOI) from Buttiauxella species F is the T. reesei cbh1 promoter at the 5' end and operably linked at its terminus to the T. reesei cbh1 terminator The DNA construct further comprises a selection marker. To identify transformants capable of secreting phytase enzyme, mutant T. reesei was used. T. reesei cells (i.e., Transforming a strain of yeast (containing a genetic modification that increases expression) with a phytase expression construct; Transformants were selected and cultured in liquid medium containing glucose as a carbon source (i.e., sophorotropic The culture is incubated in the absence of an inducing substrate such as glucose or lactose.

[0213] Example 10 of the present disclosure includes the T. reesei pki1 promoter Streptococcus pyogenes expressed under enes) containing the cas9 gene and a guide RNA expressed under the U6 promoter. The construction of native ace3 promoter replacement vectors has been described. For example, cas9 The mediated ace3 promoter replacement vectors (pCHL760 and pCHL761) were transformed into T . Function of the ace3 promoter-replaced strain transformed into T. reesei parent cells To test the activity of the inducer substrate (Sophorosporin) in a 50 ml submerged culture in a shake flask, Cells were grown in the presence and absence of glycerol (glycerol). In the parental cells (Fig. 23, ID 1275.8.1), glucose / sophorose (induction) In comparison with the case of 0.1% sucrose, the production of secreted proteins was significantly lower in defined medium containing glucose (non-inducing). In contrast, transformants 2218, 2219, 2220, 2222 and 2223 produced similar amounts of secreted proteins under induced and uninduced conditions, and Mutant cells carrying the xk1 or dic1 promoter (i.e., endothelial cells expressing endothelial cells at the ace3 locus) The gene (which replaces the natural ace3 promoter) produces extracellular proteins in the absence of an inducer. It showed that he was alive.

[0214] Thus, as discussed and described herein, certain embodiments of the present disclosure include one or more Endogenous fungal lignocellulolytic enzymes (i.e., cellulolytic enzymes, e.g., cellulolytic enzymes For the production of enzymes such as glyceryl phosphate, ... In particular embodiments of the present disclosure, the mutant host cells of the present disclosure are grown in the complete absence of an inducing substrate. (i.e., Ace3-L protein, Ace3-EL and / or Ace3-LN protein) In a mutant host cell containing a genetic modification that increases expression of the endogenous enzyme, The mutant host cells, compositions and methods of the present disclosure relate, inter alia, to the production of the compounds of the present disclosure. Such mutant host cells of the present invention can be grown on an inducing substrate to produce such cellulolytic enzymes. Due to the fact that such cells do not require induction (i.e., only in the presence of an inducing substrate) In contrast to the parent cells, which produce cellulose-degrading enzymes, It is particularly useful for significantly reducing costs / expenses.

[0215] For example, in certain embodiments, the present disclosure provides a method for the production of a medicament comprising the steps of: and / or one or more heterologous proteins of interest in the absence of an inducing substrate. In particular embodiments, the present invention relates to a mutant fungal host cell capable of expressing / producing The mutant fungal host cells of the present disclosure (i.e., Ace3-L protein, Ace3-EL and and / or genetic modification to increase the expression of Ace3-LN) are endogenous target genes. Further, the method further comprises the steps of: expressing a lignocellulosic protein and / or a heterologous protein of interest; For example, in certain embodiments, endogenous non-lignocellulosic proteins of interest are modified to The genes encoding the specific enzymes are altered in the mutant fungal host cell. In an embodiment, a gene encoding an endogenous non-lignocellulosic protein of interest (or O The promoters naturally associated with lignocellulosic proteins encode filamentous lignocellulosic proteins. A promoter from a fungal gene (e.g., encoding a non-lignocellulosic protein of interest) operably linked to an endogenous gene encoding a 5'-lignocellulosic gene promoter Similarly, in certain other embodiments, the mutant fungal host cell of the present disclosure (i.e., Increase the expression of Ace3-L protein, Ace3-EL and / or Ace3-LN The gene modification (including the genetic modification to express a heterologous protein of interest) is modified to express a heterologous protein of interest. In other particular embodiments, the gene encoding the heterologous protein of interest is naturally associated with the gene. The promoters selected were derived from fungal genes encoding lignocellulosic proteins. A motor (e.g., operably linked to a heterologous gene encoding a heterologous protein of interest) 5'-lignocellulosic gene promoter).

[0216] Thus, in certain embodiments, the present disclosure provides a mutant filamentous fungal cell derived from a parent filamentous fungal cell. Regarding the Ace-L protein, the mutant cells showed a higher expression level of the Ace-L protein-encoding gene than the parent cells. The encoded Ace3-L protein contains a genetic modification that increases its expression and is represented by SEQ ID NO: No. 6. The Ace3-L protein has about 90% sequence identity to the Ace3-L protein of In embodiments, the encoded Ace has about 90% sequence identity to SEQ ID NO:6. The 3 protein contains the sequence "Lys-Ala-Ser-Asp" as the last four C-terminal amino acids. In another embodiment, the nucleic acid sequence of the present invention is a nucleic acid sequence having about 90% sequence identity to SEQ ID NO:6. The Ace3 protein is operably linked to and precedes SEQ ID NO:6. In another embodiment, the Ace- The three proteins have approximately 90% sequence identity to SEQ ID NO:12.

[0217] In other specific embodiments, the polynucleotide of the disclosure is SEQ ID NO:4, SEQ ID NO:11 or or a nucleotide sequence having about 90% sequence identity to SEQ ID NO: 13. In embodiments of the present disclosure, the polynucleotide is SEQ ID NO:5, SEQ ID NO:101 or SEQ ID NO: Contains a nucleotide sequence having about 90% sequence identity to number 102.

[0218] IV. Filamentous Fungal Host Cells In certain embodiments of the present disclosure, a gene encoding an Ace3-L polypeptide or Mutant fungal cells (i.e., parent fungal cells) containing genetic modifications that increase expression of RF. More specifically, in certain embodiments, mutant filamentous fungal cells (i.e., derived from (i.e., relative to the parent (control) cell) is a gene encoding the Ace3-L protein of SEQ ID NO:6. In a preferred embodiment, the gene includes a genetic modification that increases expression of a gene (or ORF). Increase the expression of the gene (or ORF) encoding the Ace3-L protein in column 6. Such mutant fungal cells, which contain genetic modifications that allow them to express endogenous targets in the absence of an inducing substrate, In another embodiment, the method can produce at least one of the target proteins of SEQ ID NO:6. A gene that increases the expression of the gene (or ORF) that encodes the Ace3-L protein Such mutant fungal cells, containing a genetic modification, are capable of expressing a heterologous protein of interest in the absence of an inducing substrate. It is possible to produce at least one of the qualities.

[0219] Thus, in certain embodiments, filamentous fungal cells for the manipulations and uses of the present disclosure include: Phylum Ascomycota, subphylum Pezizomycota tina), especially filamentous fungi derived from fungi having a vegetative hyphal state. include, but are not limited to, Trichoderma spp., Aspergillus spp. Aspergillus species, Fusarium species, Scedosporium Scedosporium species, Penicillium species, Chrysosporium species, Cephalosporium species halosporium) species, Talaromyces species, Geos Geosmithia species, Myceliophthora ora species, and Neurospora species of commercial importance These include filamentous fungal cells used for the production of various industrial and pharmaceutical proteins.

[0220] Specific filamentous fungi include, but are not limited to, Trichoderma reesei. a reesei (formerly Trichoderma longibrachiatum) a longibrachiatum and Hypocrea jecorina (Hypocre a jecorina), Aspergillus niger s niger), Aspergillus fumigatus atus, Aspergillus itaconi cus), Aspergillus oryzae, Aspergillus Aspergillus nidulans, Aspergillus Aspergillus terreus, Aspergillus sojae Aspergillus sojae, Aspergillus japonicus rgillus japonicus), Scedosporium prolificans (Sce dosporium prolificans, Neurospora crassa (Neur ospora crassa, Penicillium funiculosum funiculosum, Penicillium chrysogenum chrysogenum), Talaromyces (Geosmithia) emersonii (Tala romyces(Geosmithia)emersonii), Fusarium venena Fusarium venenatum, Myceliophthora thermophila eliophthora thermophila) and Chrysosporium lucnowi Chrysosporium lucknowense is one of them.

[0221] V. Recombinant Nucleic Acids and Molecular Biology In certain embodiments, the present disclosure provides Ace3-L, Ace3-EL or Ace3-LN Mutant yarn containing genetic modifications that increase expression of a gene or ORF that codes for a protein As described above, such mutant host cells can be transformed in the absence of an inducing substrate. and capable of producing one or more proteins of interest (i.e., in contrast to the unmodified parent (control) cells). (specifically).

[0222] Thus, in certain embodiments, the present disclosure provides Ace3-L, Ace3-EL or Ace3-L. The present invention relates to a recombinant nucleic acid comprising a gene or ORF encoding the ce3-LN protein. In this embodiment, the recombinant nucleic acid expresses Ace3-L, Ace3-EL or contains a polynucleotide expression cassette for producing the Ace3-LN protein. In certain embodiments, the polynucleotide expression cassette is contained within an expression vector. In embodiments, the expression vector is a plasmid. The nucleotide expression cassette or its expression vector may be selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: At least 85% relative to No. 11, SEQ ID NO: 101, SEQ ID NO: 13 or SEQ ID NO: 102 In another embodiment, the recombinant nucleic acid comprises a nucleotide sequence having sequence identity to the The nucleotide expression cassette or its expression vector has a sequence identity of about 90% to SEQ ID NO:6. It includes a nucleotide sequence encoding an Ace3-L protein having identity.

[0223] In other particular embodiments, the recombinant nucleic acid (or a polynucleotide expression cassette thereof) For use in filamentous fungi, the vector (or expression vector thereof) may further comprise one or more selectable markers. Selectable markers for this purpose include, but are not limited to, alsl, amdS, hygR, pyr2, p yr4, pyrG, sucA, bleomycin resistance marker, blasticidin resistance marker , pyrithiamine resistance marker, chlorimuron ethyl resistance marker, neomycin resistance marker markers, adenine pathway genes, tryptophan pathway genes, thymidine kinase markers, etc. In a particular embodiment, the selectable marker is pyr2, and its components and uses The method of use is generally described in WO 2011 / 153449. Thus, in certain embodiments, a polynucleotide encoding the Ace3 protein of the present disclosure is The nucleic acid construct comprises a nucleic acid sequence encoding a selectable marker operably linked thereto. Contains the acid sequence.

[0224] In another embodiment, the recombinant nucleic acid, polynucleotide construct, polynucleotide The expression cassette or its expression vector is Ace3-L, Ace3-EL or Ace3 - a heterologous promoter driving expression of the gene (or ORF) encoding the LN protein More particularly, in certain embodiments, the heterologous promoter is a constitutive promoter. or an inducible promoter. In certain embodiments, the heterologous promoter is rev3 Promoter, bxl promoter, tkl1 promoter, PID104295 Promo promoter, dld1 promoter, xyn4 promoter, PID72526 promoter , axe1 promoter, hxk1 promoter, dic1 promoter, opt promoter The promoter is selected from the group consisting of the gut1 promoter and the pki1 promoter. Without wishing to be bound by any particular theory or mechanism of action, the present invention Conferring higher expression levels under course-limiting conditions (i.e., relative to excess glucose concentration) eru, rev3, bxl, tkl, PID104295, dld1, xyn4, PID7 Pros such as 2526, axe1, hxk1, dic1, opt, gut1 and pki1 Motors are believed to be particularly useful in the present disclosure. Nucleic acid (or polynucleotide construct, polynucleotide expression cassette or the expression vector) is operably located 5' of the nucleic acid sequence encoding the Ace3 protein It contains a linked promoter.

[0225] In another embodiment, a recombinant nucleic acid (or polynucleotide construct, polynucleotide The octide expression cassette, or its expression vector, contains the native ace3 terminator sequence. Thus, in certain embodiments, the recombinant nucleic acid (also The polynucleotide construct, polynucleotide expression cassette, or expression vector thereof The promoter is operably linked to the 5' end of the nucleic acid sequence encoding the Ace3 protein. a nucleic acid sequence encoding the Ace3 protein, Contains the native ace3 terminator sequence (e.g., 5'-Pro-ORF-Term-3 ', where 'Pro' is a constitutive promoter, 'ORF' codes for Ace3, "Term" is the native ace3 terminator sequence).

[0226] Thus, in certain embodiments, filamentous fungal host cells of the present disclosure can be transformed using Standard techniques for fungal transformation and fungal cultivation, which are well known to those skilled in the art, are used. Therefore, the DNA construct or vector is introduced into a fungal host cell. Methods for introducing genes include transformation, electroporation, nuclear microinjection, and transformation. Introduction, transfection (e.g., lipofection-mediated and DEAE-dextrose Phosphate-mediated transfection), incubation with calcium phosphate DNA precipitate , high-velocity guns using DNA-coated microparticles, gene guns or biolistic transformation, General transformation techniques include those known in the art and include techniques such as rotoplast fusion. (e.g., Ausubel et al., 1987; Sambrook et al. al., 2001 and 2012, and Campbell et al., 1989 (See, for example, Expression can be achieved by, for example, the methods described in U.S. Pat. Nos. 6,022,725 and 6,268,328. Details: Harkki et al., 1991 and Harkki et al., 19 For the transformation of Aspergillus strains, See also Cao et al. (2000).

[0227] In general, transformation of Trichoderma species usually requires 10 Permeabilized protoplasts at a density of 107 / mL, especially 2 x 106 / mL or Use cells in an appropriate solution (e.g., 1.2 M sorbitol and 50 mM CaC l2) Mix these protoplasts or cells in a volume of 100 μL with the desired DNA. Generally, a high concentration of polyethylene glycol (PEG) is added to the uptake solution. Additives such as dimethyl sulfoxide, heparin, spermidine, and potassium chloride also A similar procedure can be used with other fungal host cells. For example, U.S. Pat. Nos. 6,022,725 and 6,022,725 are also available. See US Pat. No. 6,268,328, both of which are incorporated by reference. ).

[0228] In certain embodiments, the present disclosure provides a method for the preparation of a fungal host cell comprising the steps of: Regarding protein expression and production (i.e., genes that increase Ace3-L expression) The endogenous protein is produced by a mutant fungal host cell of the present disclosure that contains the mutation. In other embodiments, the present disclosure provides a method for the production of a fungal host cell comprising the step of: Thus, the present disclosure relates generally to the field of recombinant genetics. The basic theory that sets out the general methodology used in this disclosure is For the text, see Sambrook et al., (2nd Edition, 1989) Kriegler (1990) and Ausubel et al. (1994) Included.

[0229] Thus, in certain embodiments, a heterologous gene or OR encoding a protein of interest is F is introduced into a filamentous fungal (host) cell. In certain embodiments, the heterologous gene or ORF is , typically before being transformed into a filamentous fungal (host) cell for replication and / or expression. These intermediate vectors can be, for example, plasmids. or a prokaryotic vector, such as a shuttle vector. Expression of a gene or ORF is under the control of its native promoter. The expression of a heterologous gene or ORF is driven by a heterologous constitutive promoter or a heterologous inducible promoter. The gene is placed under the control of a heterologous promoter which may be an inhibitor.

[0230] Those skilled in the art will appreciate that a native promoter can be modified with one or more This document is known to be modified by the substitution, replacement, addition or deletion of nucleotides. The practice of the invention encompasses, but does not impose, such modifications to the promoter. isn't it.

[0231] The expression vector / construct usually contains all the additional elements required for the expression of the heterologous sequence. A transcription unit or expression cassette containing the construct is included. For example, a typical expression cassette The vector comprises a 5' promoter operably linked to a heterologous nucleic acid sequence encoding a protein of interest. Contains and is required for efficient polyadenylation of the transcript, a ribosome binding site, and translation termination Additional elements of the cassette may include enhancers and If genomic DNA is used as the structural gene, functional splice donor sites and The nucleic acid may include an intron having an acceptor site and an intron having an acceptor site.

[0232] In addition to a promoter sequence, the expression cassette should also contain a promoter sequence to provide for efficient termination. The gene may contain a transcription termination region downstream of the promoter sequence. In the present invention, any fungal type may be used. Although the terminator is likely to be functional, the preferred terminator is trichoderma. Terminator from Trichoderma cbhI gene, Aspergillus Aspergillus nidulans trpC gene (Ye lton et al., 1984; Mullaney et al., 1985), Aspergillus awamori or Aspergillus Aspergillus niger glucoamylase gene (Nu nberg et al., 1984; Boel et al., 1984), and / or Mucor miehei carboxyl protease gene This includes European Patent Specification No. 0215594.

[0233] The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells can be used. Standard bacterial expression vectors include bacteriophages λ and M13, as well as pBR322-based plasmids, pSKF, pET23D and other plasmids, MBP, GST, and LacZ fusion expression systems. Additionally, epitope tags, such as c-myc, can be added to the recombinant protein.

[0234] Elements that can be included in an expression vector also include replicons, recombination vectors, and the like. Genes encoding antibiotic resistance that allow for the selection of bacteria carrying the mide, or There may be unique restriction sites in non-essential regions of the plasmid to allow for insertion of the sequence Any of the many resistance genes known in the art may be suitable, and selection may be performed. The specific antibiotic resistance genes identified are not definitive. DNA replication or integration in Trichoderma reesei It is preferable to select the ion exchange coefficient so as not to interfere with the

[0235] The transformation method of the present invention involves stabilizing all or a part of a transformation vector into the genome of a filamentous fungus. However, the formation of autonomously replicating extrachromosomal transformation vectors can result in stable integration. Transformation that results in maintenance is also contemplated.

[0236] Many standard transfection methods can be used to express large amounts of heterologous proteins. To generate a Trichoderma reesei cell line The cellulase-producing strains of Trichoderma were found to be DNA- Published methods for introducing constructs include Lorito, Hayes, Di Pietro and Harman,1993,Curr.Genet.24:349 -356;Goldman, VanMontagu and Herrera-Estr. ella,1990,Curr.Genet.17:169-174;Penttila ,Nevalainen,Ratto,Salminen and Knowles,1 987, Gene 6:155-164, Aspergillus ) see Yelton, Hamer and Timberlake, 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474 For Fusarium, see Bajar, Podila and Kola ttukudy,1991,Proc.Natl.Acad.Sci.USA 88:8 202-8212 for Streptomyces genus, opwood et al.,1985,The John Innes Founda tion, Norwich, UK, and for Bacillus , Brigidi, DeRossi, Bertarini, Riccardi and Matteuzzi,1990,FEMS Microbiol.Lett.55:13 5-138).

[0237] Any of the known procedures for introducing foreign nucleotide sequences into host cells may be used. These include calcium phosphate transfection, polybrene transfection, and protoplast fusion. , electroporation method, biolistics method, liposome method, microinjection vector, plasma vector, viral vector, and cloned genome in host cells. known methods for introducing murine DNA, cDNA, synthetic DNA or other foreign genetic material This includes the use of any other method that can be used to (See U.S. Pat. No. 6,255,115). Agrobacterium-mediated transfection methods are also useful. The particular genetic engineering procedure used must at least partially modify the host cell to be capable of expressing the heterologous gene. It is only necessary to be able to successfully introduce one more gene.

[0238] After the expression vector is introduced into the cells, the transfected cells express the cellulase gene. The transformed cells are cultured under conditions favoring expression of the gene under the control of the promoter sequence. Large batches of can be cultured as described herein. The product is then recovered from the culture using a suitable technique.

[0239] Thus, the invention herein relates to cellulase genes, the expression of which occurs naturally, Cellulase gene promoters, including fusion DNA sequences, and various heterologous constructs The present invention provides for the expression and enhanced secretion of a desired polypeptide under the control of a desired sequence. The invention also provides methods for the high level expression and secretion of such desired products.

[0240] VI. Protein of interest As noted above, certain embodiments of the present disclosure include mutant filamentous fungal cells (derived from a parent filamentous fungal cell). ), the mutant cells express Ace3-L, Ace3-EL or Ace3-LN protein. a genetic modification that increases (compared to the parent cell) expression of a gene encoding a protein; The encoded Ace3-L, Ace3-EL or Ace3-LN protein is A mutant cell line having about 90% sequence identity to the Ace3-L protein of sequence number 6. expresses at least one protein of interest (POI) in the absence of an inducing substrate.

[0241] Certain embodiments of the present disclosure are directed to the production of a protein (i.e., a target protein) in the absence of an inducing substrate. The present invention is particularly useful for enhancing the intracellular and / or extracellular production of a target protein. The protein of interest may be an endogenous protein (i.e., endogenous to the host cell) or a heterologous protein. It may be a seed protein (i.e., not native to the host cell). Proteins that can be produced include, but are not limited to, hormones, enzymes, growth factors, These include cytokines and antibodies.

[0242] For example, the target protein may be, but is not limited to, hemicellulase, peroxidase, Proteases, cellulases, xylanases, lipases, phospholipases, esterases, Cutinase, pectinase, keratinase, reductase, oxidase, phenolase Oxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosana enzymes, mannanases, β-glucanases, hyaluronidases, chondroitinase, Casein, amylase, glucoamylase, acetyl esterase, aminopeptidase, Amylases, arabinases, arabinosidases, arabinofuranosidases, carboxylases Peptidase, catalase, deoxyribonuclease, epimerase, α-galactosidase β-galactosidase, α-glucanase, glucanylase, endo-β- Glucanase, glucose oxidase, glucuronidase, invertase, isomerase These may include enzymes.

[0243] In certain embodiments, the protein of interest may include, but is not limited to, the polypeptides described in WO 03 / 027 Brochure No. 306, Brochure No. 200352118, Brochure No. 20035205 Brochure No. 4, Brochure No. 200352057, Brochure No. 200352055 Brochure, No. 200352056 Brochure, No. 200416760 Bread Fret, Brochure No. 9210581, Brochure No. 200448592 , Brochure No. 200443980, Brochure No. 200528636, Brochure No. 200501065, Brochure No. 2005 / 001036, Brochure No. 2005 / 093050, Brochure No. 200593073, Brochure No. 200674005, Brochure No. 2009 / 149202, Brochure No. 2011 / 038019, Brochure No. 2010 / 141779 , Brochure No. 2011 / 063308, Brochure No. 2012 / 125951 , Brochure No. 2012 / 125925, Brochure No. 2012125937 Brochure No. 2011 / 153276, Brochure No. 2014 / 093275 Brochure, No. 2014 / 070837 Brochure, No. 2014 / 07084 Brochure No. 1, Brochure No. 2014 / 070844, Brochure No. 2014 / 093 Brochure No. 281, 2014 / 093 Brochure No. 282, 2014 / 0 Brochure No. 93287, Brochure No. 2014 / 093294, Brochure No. 2015 Disclosed in Brochure No. 2016 / 084596 and Brochure No. 2016 / 069541 These include enzymes that have been

[0244] The optimal conditions for protein production depend on the choice of host cell and the choice of protein to be expressed. Such conditions may vary depending on the particular application, through routine experimentation and / or optimization. This can be easily verified by the dealer.

[0245] The protein of interest can be purified or isolated after expression. Depending on whether other components such as tetracycline, ... Standard purification methods include electrophoretic, molecular, immunological, and cloning techniques. Chromatographic techniques, such as ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. For example, the target protein can be detected by a method such as chromatofocusing. The protein can be purified using a standard anti-target protein antibody column. Ultrafiltration and diafiltration techniques are also useful. The time required to purify the protein depends on the intended use of the protein. In some cases, purification of the protein is not necessary. be.

[0246] In other specific embodiments, the genetically modified fungal cells of the present disclosure (i.e., ace Mutant fungal host cells containing genetic modifications that increase expression of 3-L) were used to generate increased levels of To confirm that the target protein is produced, various screening methods are used. The expression vector can be used to encode a polypeptide for a target protein that functions as a detectable label. The target protein may itself encode a selectable or screenable peptide fusion. The labeled proteins can serve as markers that can be detected by Western blotting. , dot blotting (Cold Spring Harbor Protocols Methods available on the website), ELISA, or whole cells if the label is GFP Detection can be by fluorescence or FACS. For example, a 6-histidine tag can be used to identify the target protein. The tag was included as a fusion to the protein and was detected by Western blotting. If the target protein is expressed at a high enough level, it can be combined with Coomassie / silver staining to The increased expression in mutant host cells over parental (control) cells was measured using SDS-PAGE. In addition, the method can be performed to detect cell 1 Protein activity or amount per cell, protein activity or amount per milliliter of medium Improved levels of the protein of interest can be detected using other methods, such as detecting increased amounts of This, or a combination of these methods, can be used to This allows the culture or fermentation to continue efficiently for a long period of time.

[0247] Specific productivity determination is another way to evaluate protein production. Specific productivity (Qp) is , can be calculated by the following formula: Qp = gP / gDCW·hr (where "gP" is the number of grams of protein produced in the tank and "gDCW" is the The dry cell weight (DCW) in the tank in grams, and "hr" is the generation time and growth time. (The fermentation time (hours) from the time of inoculation, including the time of inoculation.)

[0248] In other embodiments, the mutant fungal host cell exhibits reduced expression of the mutated fungal host cell compared to the (unmodified) parent cell. At least about 0.5%, for example, at least about 0.5%, at least about 0.7%, at least about 1%, at least about 1.5%, at least about 2.0%, at least about 2.5%, or can produce at least about 3%, or more, of the protein of interest.

[0249] VII. Fermentation In certain embodiments, the present disclosure provides a method for producing a protein of interest, comprising fermentation of a mutant fungal cell. In general, the present invention provides a method for producing a mutant fungal cell that secretes a protein of interest. Fermentation methods well known in the art can be used to ferment the mutant fungal cells. In some embodiments, the fungal cells are grown under batch or continuous fermentation conditions. A typical batch fermentation is a closed system in which the composition of the medium is set at the beginning of the fermentation and the At the start of the fermentation, the medium is inoculated with the desired organism. Fermentation can be carried out without adding any ingredients to the fermentation mixture. Considered a "batch" with respect to source addition, controlling factors such as pH and oxygen concentration The metabolic and biomass composition of batch systems is often determined by the In batch cultures, cells undergo a static lag phase and then a high logarithmic growth phase. Left untreated, the cells progress through the stationary phase, where proliferation slows or stops. Stationary phase cells eventually die. Generally, exponential phase cells produce most of the product. is responsible for the production of

[0250] A suitable variation of the standard batch system is the "fed-batch fermentation" system. In this variation of the typical batch system, the substrate is gradually absorbed as the fermentation progresses. The fed-batch system is likely to inhibit cellular metabolism due to catabolite repression. Fed-batch is useful when it is desired to have a limited amount of substrate in the medium. In systems where it is difficult to measure the actual substrate concentration, pH, dissolved oxygen, and It is estimated based on the change in measurable factors such as the partial pressure of waste gases such as CO2 and the batch Fermentation and fed-batch fermentation are common and well known in the art.

[0251] Continuous fermentation involves the continuous addition of a defined fermentation medium to a bioreactor and the addition of an equal volume of medium for processing. Continuous fermentation is an open system in which the culture is grown at a constant temperature and the conditioned medium is removed simultaneously. Continuous fermentation maintains a high density of 0.1–1.5 μg / ml, with cells primarily in logarithmic growth phase. or allows for the adjustment of one or more factors that affect the concentration of the product. For example, in one embodiment In this method, the limiting nutrients, such as carbon or nitrogen sources, are maintained at a constant ratio and all other parameters are In other systems, the cell concentration is measured by the turbidity of the medium. Many of the factors that affect growth can be varied continuously while keeping the temperature constant. The continuous system attempts to maintain steady-state growth conditions. Cell loss due to fermentation must be balanced with the cell growth rate during fermentation. Methods for regulating nutrients and growth factors in processes and maximizing the rate of product formation Techniques for this purpose are well known in the field of industrial microbiology.

[0252] Certain embodiments of the present disclosure relate to fermentation procedures for culturing fungi. Fermentation procedures for the production of cellulase enzymes are known in the art. For example, cellulase enzymes include It can be produced by solid-state or submerged culture, including batch, fed-batch and continuous-flow processes. Cultures are generally made up of an aqueous mineral salts medium, organic growth factors, carbon and energy sources, and a growth medium containing nutrients, molecular oxygen, and, of course, a starting inoculum of the filamentous fungal host being used. It takes place inside.

[0253] Ensure adequate microbial growth and provide carbon and energy to the cells during the microbial conversion process. To maximize the uptake of nutrients and achieve maximum cell yield at maximum cell density in the fermentation medium, In addition to the nutrients and energy sources, oxygen, assimilable nitrogen, and microbial inoculum, It is necessary to provide adequate amounts of inorganic nutrients in the right proportions.

[0254] The composition of the aqueous mineral medium depends on the microorganism and substrate used, as is known in the art. The nutrients can vary widely depending in part on the quality of the medium. In addition, suitable amounts of phosphorus, magnesium, calcium, potassium, sulfur, and sodium In a suitable soluble, absorbable ionic complex form, copper, malic acid, and the like are preferably included. Several trace elements, such as calcium, molybdenum, zinc, iron, boron, and iodine, are also present in this These may also be included in suitable soluble, absorbable forms, all of which are known in the art. This is what is being done.

[0255] Fermentation reactions occur at favorable oxygen pressures that are effective in supporting vigorous growth of microbial species. Air, oxygen-enriched air, or substantially pure oxygen provided to maintain the contents of the tank. Aerobic processes in which the necessary molecular oxygen is supplied by a molecular oxygen-containing gas, such as molecular oxygen. It is.

[0256] Fermentation temperature can be varied somewhat, but Trichoderma reesei For filamentous fungi such as Erma reesei, the temperature is generally in the range of about 20°C to 40°C. Generally, the temperature is preferably within the range of about 25°C to 34°C.

[0257] Microorganisms also require an assimilable source of nitrogen. The assimilable source of nitrogen can be any nitrogen-containing any compound capable of releasing nitrogen in a form suitable for metabolic utilization by microorganisms. Various organic nitrogen source compounds, such as protein hydrolysates, can be used. However, it is usually made up of inexpensive nitrogen-containing compounds such as ammonia, ammonium hydroxide, and urea. and ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride Various ammonium salts, such as ammonium ions, or various other ammonium compounds, can be used. Ammonia gas itself is convenient for large-scale operations and can be used in aqueous fermentation in suitable quantities. (fermentation medium) can be used by bubbling ammonia. , may also be used to aid in pH control.

[0258] The pH range of the aqueous microbial fermentation (fermentation mixture) is illustratively in the range of about 2.0 to 8.0. For filamentous fungi, the pH should usually be in the range of about 2.5 to 8.0; for Trichoderma In Trichoderma reesei, the pH is usually around 3.0. The preferred pH range for a microorganism depends on the medium and the particular microorganism used. The amount of the medium used will depend to some extent on the type of medium used and therefore can be easily determined by one of skill in the art. It changes somewhat with changes in

[0259] It is possible to carry out the fermentation in such a way that the carbon-containing substrate can be controlled as the limiting factor. It is preferred, because it provides good conversion of carbon-containing substrates to the cells and does not provide a significant amount of unconverted Contamination of cells by the substrate is avoided. The latter is because traces of water-soluble substrates can be easily washed away. However, this can be a problem with non-water-soluble substrates. This can result in the need for additional product processing steps, such as suitable washing steps.

[0260] As noted above, the time to reach this level is not critical and depends on the specific microorganism and procedure performed. However, the method for determining the carbon source concentration in the fermentation medium is Methods for determining whether a desired carbon source level has been achieved are well known in the art. It is well known.

[0261] Fermentation can be carried out as a batch or continuous operation, with fed-batch operation being preferred due to its ease of control, uniformity and This is highly favorable in terms of production of the most efficient amounts of product and the most economical use of all equipment.

[0262] If necessary, one of the carbon and energy source substances may be added before the aqueous mineral medium is fed to the fermenter. Part or all of the absorbable nitrogen source, such as ammonia, is added to the aqueous mineral medium. can be added.

[0263] Each of the streams introduced into the reactor is controlled at a predetermined rate or is fed by a carbon and energy group feed stream. The concentration of fermentation products, pH, dissolved oxygen, oxygen or carbon dioxide in the fermenter exhaust gas, and dry cell weight Depending on the need, it can be determined by monitoring cell density, translucency, etc. It is preferable to control as much as possible, consistent with efficient use of carbon and energy sources. to obtain a more rapid cell growth rate and the highest possible yield of microbial cells per substrate supply. Therefore, the feed rates of the various materials can be varied.

[0264] In batch or, preferably, fed-batch operation, any apparatus, reactor, or fermentation means, tank, or the vessel, piping, associated circulation or cooling systems, etc., are first The mixture is then sterilized, for example at about 121° C. for at least about 15 minutes. The mixture is then sterilized with the necessary nutrients, including oxygen. Cultivation of the selected microorganism in a sterile reactor in the presence of all nutrients and a carbon-containing substrate. The type of fermenter used is not important.

[0265] Recovery and purification of enzymes (e.g., cellulases) from the fermentation broth are also known to those skilled in the art. The fermentation broth is generally free of cell debris, e.g., cells, The cellulase enzyme product is then added to the biomass containing various suspended solids and other biomass contaminants. These are preferably removed from the fermentation broth by means known in the art. It is.

[0266] Suitable processes for such removal include, for example, centrifugation to produce a cell-free filtrate. Conventional solidification processes such as separation, filtration, dialysis, microfiltration, rotary vacuum filtration, or other known processes may be used. These include separation techniques for the liquid phase, such as ultrafiltration, evaporation, or precipitation prior to crystallization. It is preferred to further concentrate the fermentation broth or cell-free filtrate.

[0267] Precipitation of the protein components of the supernatant or filtrate is carried out with a salt, e.g., ammonium sulfate. This is followed by various chromatographic techniques, such as ion exchange chromatography, Purify by affinity chromatography or similar art-recognized methods. can be carried out. EXAMPLES

[0268] The following examples illustrate embodiments of the present disclosure, but are provided by way of illustration only. It should be understood that from the above discussion and these examples, those skilled in the art will be able to understand various uses and Various modifications and adaptations of the present disclosure may be made to suit various applications and conditions. Such modifications are also intended to be within the scope of the invention as defined in the appended claims.

[0269] Example 1 Generation of ace3 overexpression in filamentous fungal cells 1A. Overview In this example, a mutant Trichoderma reesei expressing the ace3 gene was Derma reesei cells (i.e., an exemplary filamentous fungus) were transformed into protoplasts. Using transformation, a nucleic acid containing the pyr2 gene, a heterologous promoter, and the ace3 gene was and was produced by transforming parent T. reesei cells. As shown generally in Figures 1 and 2, two different promoters and two different versions The ace3 ORF of the ace3 gene (Fig. 1; ace3-SC and ace3-L) was synthesized in four different combinations. These were used in combination to construct four different ace3-expressing vectors (Figures 2A-2D). k1 (the gene encoding hexokinase) and pki1 (the gene encoding pyruvate kinase) The promoter of the gene that controls ace3 was chosen to drive constitutive expression of ace3. However, other promoters may also be used and may be selected by one of skill in the art.

[0270] 1B. Trichoderma reesei host cells The T. reesei parent host cell shown in the following examples is Sheir-N It is generally described by Eiss and Montenecourt, 1984. Thus, T. reesei pyr2 gene deleted T. reesei reesei strain RL-P37 (NRRL accession no. 15709).

[0271] 1C. Construction of Ace3 Expression Vector As described in the above detailed description of the present disclosure, Ace3 is an induced For the production of cellulases and hemicellulases under inductive conditions (i.e., in the presence of lactose), Recently shown to be essential for T. reesei transcription factors (Ha For more details, see Hakkinen et al. (2014) reported that T. reesei strain QM6a (genome.j Referred to gi.doe.gov / Trire2 / Trire2.home.html We used the predicted ace3 ORF based on the publicly available genome sequence of The predicted annotation of QM6a (protein ID 77513) is two exons and one It consists of two introns (see, for example, FIG. 1).

[0272] In addition, the publicly available genome of T. reesei strain Rut-C30 Genome sequence (genome.jgi.doe.gov / TrireRUTC30_1 / Tri (see reRUTC30_1.home.html) The ORF (protein ID 98455) contains three exons and two introns. Longer protein sequences (i.e., from T. reesei QM6a) The short ace3-S (Figure 1) contains The initiation codon predicted by the model is located upstream of that of the “QM6a” model, and is C-terminal There is a nonsense mutation in (Poggi-Parodi et al., 2014), This results in a long N-terminal sequence and a shorter C-terminal protein sequence (Figure 1).

[0273] In this example, the short ace3 ORF (based on the QM6a annotation, but Ace3-S) containing a RUT-C30 nonsense mutation that truncates the C-terminus of the protein and a long Both ace3 ORFs (based on the RUT-C30 annotation (Ace3-L)) As shown in Figure 1, short ace3 (Ace3-S) and long ace3 (Ace3-S) were cloned. Both e3(Ace3-L) ORFs contain the C-terminal nonsense nucleotide sequence found in RUT-C30. The heterologous hexose kinase was used to drive expression of the ace3 ORF (Figure 1). The heterologous pyruvate kinase (hxk1) promoter and the heterologous pyruvate kinase (pki1) promoter were Tested.

[0274] Therefore, four Ace3 expression vectors, pYL1 pYL2, pYL3 and pYL4 (FIGS. 2A to 2D) were constructed. The marker contains the bacterial ColE1 ori for replication and selection in E. coli and Contains the vector backbone carrying the AmpR gene. In addition to the two selection markers, the T. reesei promoter sequence (i.e. hxk1 or pki1 promoter) and its natural terminator e3 ORF (ace3-L or ace3-SC) also exists. Q5 High-fidelity delity DNA polymerase (New England Biolab s) and the primers shown in Table 1 below, From genomic DNA, the T. reesei promoter and ace3 O RF was PCR amplified.

[0275] The specific primers used to PCR amplify the fragments of each vector were as follows: To construct the vector pYL1, the hxk1 promoter was Ace3-L was amplified using pairs TP13 (SEQ ID NO: 7) and TP14 (SEQ ID NO: 8). The ORF was amplified using primers TP15 (SEQ ID NO: 9) and TP16 (SEQ ID NO: 10). The vector backbone was amplified using primer pair TP17 (SEQ ID NO: 11) and TP18 (SEQ ID NO: The complete sequence of plasmid pYL1 is provided as SEQ ID NO:21. do.

[0276] To construct the vector pYL2, the hxk1 promoter was amplified using primer pair TP13 ( Ace3-SC ORF was amplified using TP19 (SEQ ID NO: 13) and TP20 (SEQ ID NO: 14). was amplified using primers TP20 (SEQ ID NO: 14) and TP16 (SEQ ID NO: 10). The vector backbone was generated using primer pair TP17 (SEQ ID NO: 11) and TP18 (SEQ ID NO: 12) ) The complete sequence of plasmid pYL2 is provided as SEQ ID NO:22.

[0277] To construct the vector pYL3, the pki1 promoter was amplified by the primer pair TP21 ( Ace3-L ORF was amplified using TP22 (SEQ ID NO: 16) and TP23 (SEQ ID NO: 17). was amplified using primers TP23 (SEQ ID NO: 17) and TP16 (SEQ ID NO: 10). The vector backbone was generated using primer pair TP17 (SEQ ID NO: 11) and TP24 (SEQ ID NO: 18) ) The complete sequence of plasmid pYL3 is provided as SEQ ID NO:23.

[0278] To construct the vector pYL4, the pki1 promoter was amplified using the primer pair TP21 ( Ace3-SC OR F is amplified using primers TP26 (SEQ ID NO: 20) and TP16 (SEQ ID NO: 10). The vector backbone was prepared using primer pair TP17 (SEQ ID NO: 11) and TP24 (SEQ ID NO: 1 8) The complete sequence of plasmid pYL4 is provided as SEQ ID NO:24.

[0279] For each vector, the three PCR fragments were assembled and analyzed using the Gibson method. assembly cloning kit (New England Biolabs; The NEB DH5 ELISA kit was used according to the manufacturer's protocol using the ELISA kit (catalog number E5510S). The resulting vector was sequenced using Sanger sequencing. The maps are shown in FIGS. 2A to 2D.

[0280] [Table 1]

[0281] 1. Transformation of T. reesei The expression vectors pYL1, pY2, pY3 and pYL4 were transformed with PacI enzyme (Ne and linearized using England Biolabs and polyethylene glycol (P T. reesei parent host cells were transformed by EG)-mediated protoplast transformation. The cells were transformed with The transformants were grown on Vogel's minimal medium agar plates. The strain was selected for uridine prototrophy, acquired by the pyr2 marker. Stable transformants were obtained by two successive transfers onto a top plate. After that, single colonies were obtained by dilution of the spore suspension. The mutant (i.e., engineered) host cells carrying pYL4 and pYL5 were designated as mutants A4-7, They were named Mutation B2-1, Mutation C2-28 and Mutation D3-1.

[0282] Example 2 Protein production in sustained release microtiter plates (srMTPs) This example shows the production of a transformant (see Example 1) that secretes the enzyme under non-inducing conditions. The screening method used to identify the markers is described below. For example, the markers obtained from Example 1 are Stable transformants were tested in slow release microtiter plates (srMTPs). The srMTP used was 20% glucose (wt / wt) or 20% lactose (wt / wt). (weight) and is a 24-well PDMS elastomer plate, International Publication No. 2014 It was prepared as described in brochure no. 047520.

[0283] In both the "non-induced" and "induced" conditions, parental and mutant T. reesei ( T. reesei host cells were tested. In the "non-inducing condition", 20% glucose (wt in srMTP containing 2.5% glucose (weight / volume) supplemented with Cells were grown in 1.25 ml liquid broth of medium. In the "inducing condition", 20% lactate was added. 2.5% glucose / sophorose (wt / wt) in srMTP containing / volume) supplemented with defined medium (sophorose and lactose were found to potently inhibit cellulase enzyme expression). Cells were grown in 1.25 ml liquid broth containing 1000 ng / ml of ribosomal RNA (which acts as a potent inducer).

[0284] The preparation of glucose / sophorose was carried out as described in U.S. Pat. No. 7,713,725. The defined medium is generally described in WO 2013 / 096056. Prepared as described, 9 g / L casamino acids, 5 g / L (NH4)2SO4, 4.5g / L KH2PO4, 1g / L MgSO4 7H2O, 1g / L CaCl2 2H2O, 33 g / L PIPPS buffer (pH 5.5), 0.25 ml / L T. repens Contains trace amounts of T. reesei. The components are 191.41g / L citric acid·H2O, 200g / L FeSO4·7H2O , 16g / L ZnSO4·7H2O, 0.56g / L CuSO4·5H2O, 1.2 g / L MnSO4·H2O and 0.8 g / L H3BO3. All srM The TP was incubated at 28°C for approximately 120 hours with continuous shaking at 280 rpm. Ta.

[0285] After incubation, supernatants from all cultures were harvested and subjected to polyacrylamide gel electrophoresis. Equal volumes of culture supernatants were incubated in a reducing environment at 90°C for 15 min and analyzed using PAGE. After that, the cells were washed with 4-12% NuPage (Inv Loading dye for polyacrylamide gels (Iseritron, Carlsbad CA) The gel was then dissolved in SimplyBlue™ (Invitrogen). were stained and imaged (see FIG. 3).

[0286] As shown in Figure 3, all host cells tested showed no significant difference in the expression of the inducer (i.e., The enzyme secreted large amounts of protein in the presence of sophorose and lactose in srMTP. However, when glucose was the only carbon source and the inducer (i.e., sophoro In the absence of glucose or lactose, mutant host cells expressing the Ace3-L ORF Only the mutants A4-7 and C2-28 produced secreted proteins. and parent host cells and mutant cells expressing the Ace3-SC ORF (i.e., mutant Mutants B1-1 and D3-1 produced extracellular proteins that exceeded the detection limit of PAGE. This result indicates that Ace3-L (i.e., in contrast to Ace3-S) Inducer-free protein production in T. reesei This clearly shows that.

[0287] The relative concentrations of secreted proteins were determined using Zorbax C3 reverse phase chromatography ( For example, the secreted protein profile of the above host cells was determined by The method was used to analyze whether all host cells (i.e., parental and mutant cells) were expressing the phenotype under induction conditions. Below, a similar cellulase protein profile (cellulase is approximately 40% CBH1, It was observed that the CBH2-containing EG1 / EG2 complex was produced by the EG1 / EG2 complex. Under non-inducing conditions, cellulase enzymes were not produced in the parental cells and in the Ace3-S-expressing mutant host cells. In contrast, surprisingly, no significant changes were detected in the Ace3-L-expressing mutant host cells ( That is, mutants A4-7 and C2-28) produced similar ratios of cellulase enzymes as under inducing conditions. It was found to produce

[0288] Briefly, the assay was performed as follows: the supernatant samples were diluted with 50 mM sodium acetate. Dilute with thorium buffer (pH 5.0) and add 20 ppm EndoH to deglycosylate. The mixture was then incubated at 37°C for 3 hours. 10 μl of 90% acetonitrile was added to 100 μl of the mixture. 1 of the EndoH-treated sample, passed through a 0.22 μm filter, and then injected. . Agilent Zorbax300 SB C3 RRHD 1.8um(2.1× A DAD detector (Agilent Technologies) equipped with a 100 mm column An Agilent 1290 with HPLC was used. The column was heated at 60°C and a flow rate of 1.0 Operated at 1 mL / min with 0.1% trifluoroacetate in MiliQ water as running buffer A. Trifluoroacetic acid (TFA) and 0.07% in acetonitrile as running buffer B The DAD detector was operated at 220 nm and 280 nm with a 4 nm window. The injection volume was 10 μL.

[0289] Moreover, it is noteworthy that Ace3-L-expressing mutant host cells were able to uptake glucose-containing sr Approximately 20-30% of the total extracellular protein was produced in MTP (i.e., lactose-containing (compared to the total extracellular protein produced with srMTP). This relatively low expression was This may be due to the high glucose supply rate in the srMTP containing the course. The fact that the production rates of cellulase and hemicellulase are higher at low growth rates is sufficient. It has been established (Arvas et al., 2011). Nevertheless, srM TP growth assay to screen stable colonies for protein production It was a relatively high-throughput assay.

[0290] Taken together, mutant T. reesei expressing the Ace3-L ORF The host cells are able to produce the protein in the absence of an inducer, albeit at a relatively low rate. More specifically, this low production rate of cellulase and hemicellulase was As shown in Examples 3 and 4 below, it is believed that the s Related to the rMTP proliferation method.

[0291] Example 3 Protein production in shake flasks To further explore and validate the srMTP results presented above, we investigated the inducer substrate In the presence and absence of 50 mL of submerged culture in shake flasks, parent host cells and A The ce3-L expressing mutant host cells were grown. More specifically, the inducing conditions (i.e., carbon source glucose / sophorose as carbon source) and non-inducing conditions (i.e., glucose / sophorose as carbon source). In submerged (liquid) culture under both conditions, the parent T. reesei host cell , mutant A4-7 cells and mutant C2-28 cells were grown and their respective extracellular ( The protein production levels (secreted protein) of the 25-well plate with a bottom baffle were compared. Add 50 mL of YEG broth in a 100 mL Erlenmeyer flask to each host cell (i.e., T. reesei T. reesei parent host cells, mutant A4-7 host cells and mutant C2-28 host cells YEG broth was supplemented with 5 g / L yeast extract and 22 g / L The cell cultures were grown for 48 h followed by an additional 24 h in fresh These seed cultures were then subcultured in YEG. In a 1 L shake flask, 50 mL of defined medium supplemented with 1.5% glucose (non-inducing condition ), or in 50 mL of defined medium containing 1.5% glucose / sophorose (inducing conditions). I was inoculated.

[0292] All shake flasks were incubated at 28°C with continuous shaking at 200 rpm. After 3 days of incubation, the supernatants from all cell cultures were harvested and analyzed as described above. Analysis was performed using PAGE as described in Example 2. Bio-Rad reagents (Thermo S Scientific®; Catalog No. 23236), and bovine serum as a standard. A 5-fold dilution of albumin (BSA) was used to measure the Bradford dye-binding activity at 595 nm. Total protein in the supernatant was measured by high performance liquid chromatography (HPLC). Glucose concentration was measured by LC analysis and was found to be 0.01% in the cultures after 3 days of incubation. No glucose was detected.

[0293] As shown in Figure 4, parental (control) T. reesei cells were able to tolerate glucose / Produces 464μg / mL of total secreted protein in defined medium containing sophorose (inducible) However, in defined medium containing glucose (non-inducing), total secretion was only 140 μg / L. In contrast, mutant A4-7 and C2-28 cells only produced the protein. produced similar amounts of secreted proteins under induced and uninduced conditions, and both were The secreted protein produced by induction was greater than that produced by parental (control) cells. Therefore, these results suggest that the mutant cells carrying the Ace3-L ORF (i.e., mutant A4 C2-7 and C2-28 cells) produce extracellular proteins in the absence of inducers. Not only that, but these mutant cells also outgrow the parental (control) T. reesei under such inducing conditions. (T. reesei) cells.

[0294] Example 4 Protein production in small-scale fed-batch fermentation This example shows that mutant Ace3-L expressing cells (i.e., mutant A4-7 and C2-28 cells) (cells) produced similar amounts of cellulase in small-scale fermentations in the presence and absence of inducer substrate. and hemicellulase enzymes. Fermentation of Saccharomyces eesei was carried out in a 2 L flask, generally as described in U.S. Pat. No. 7,713,725. The experiment was carried out in a bioreactor using a seed culture in citrate minimal medium. In the present study, the supernatants from all cultures were harvested at different time points during the fermentation, and equal volumes of the culture supernatants were added to the P The parent (control) T. reesei was subjected to AGE analysis. As shown in FIG. Cells produced secreted proteins only in the presence of the sophorose inducer. In addition, mutant A4-7 and C2-28 cells (Figure 5) showed increased expression of IFN-γ under both induced and uninduced conditions. , produced similar amounts of protein.

[0295] Example 5 Heterologous promoter for Ace3 expression This example uses 13 different promoters to drive the expression of ace3-L, It is shown that protein production is enhanced under "induction" conditions. Using mouse transformation, a plasmid containing the pyr2 gene, a heterologous promoter, and the ace3-L gene was By transforming parent T. reesei cells with a telomere vector Using this technique, we generated T. reesei cells expressing the ace3-L gene.

[0296] Therefore, 13 T. reesei ( T. ree The 13 promoters tested included, but were not limited to, (i) formamidase gene (rev3; protein ID 103041) promoter (ii) β-xylosidase gene (bxl; protein ID No. 15); 121127) promoter (SEQ ID NO: 16), (iii) transketolase gene ( tkl1; Protein ID 2211) promoter (SEQ ID NO: 17); (iv) functional Unknown gene (Protein ID 104295) promoter (SEQ ID NO: 18) , (v) oxidoreductase gene (dld1; protein ID 5345) promoter (vi) xylanase IV gene (xyn4; protein ID 111849) promoter (SEQ ID NO: 20), (vii) α-glucuronidase gene (Protein ID 72526) promoter (SEQ ID NO: 21), (viii) acetyl Xylan esterase gene 1 (axe1; protein ID 73632) promoter (SEQ ID NO: 22), (ix) hexose kinase gene (hxk1; protein ID 73665) promoter (SEQ ID NO: 23), (x) mitochondrial carrier protein Gene (dic1; protein ID 47930) promoter (SEQ ID NO: 24), (x i) Oligopeptide transporter gene (opt; protein ID 44278) (xii) glycerol kinase gene (gut1; (protein ID 58356) promoter (SEQ ID NO: 26), and (xiii) pyruvate Kinase gene (pki1; protein ID 78439) promoter (SEQ ID NO: 27 Protein ID (PID) numbers are available at genome.jgi.doe.go. v / Trire2 / Trire2.home.html. Therefore, The 13 promoters mentioned above are those that promote proliferation of the genes that generally respond to high glucose concentrations. It is expressed at moderate to low levels, at low glucose concentrations or under sophorose-inducing conditions. It was chosen to drive expression since it is expressed at higher levels.

[0297] Table 2 below lists 13 promoters and their corresponding sequences constructed using standard molecular biology procedures. The expression vectors tested in this example (Table 2) are summarized below. , bacterial ColE1 ori and A for replication and selection in E. coli The mpR gene and Saccharomyces cerevisiae 2μ ori and Ura3 genes for replication and selection in S. cerevisiae Additionally, the vector backbone contains the T. reesei telomere sequence ( "TrTEL"), T. reesei pyr2 selection marker, T. reesei A T. reesei promoter sequence and its native terminator sequence. The ace3-L ORF, which is involved in the dic1 promoter, is a representative vector map. The vector pYL8 containing the chromosome is shown in FIG. 7. Therefore, other vectors (e.g., pYL 9, pYL12, etc.) have the same sequence as shown in Figure 7, except for different promoter sequences. It has columns.

[0298] [Table 2]

[0299] The expression vector was transformed into 100 ng / ml of ... by T. reesei parent host cells (containing a non-functional pyr2 gene) ) (Ouedraogo et al., 2015; Pentt The transformants were plated on Vogel's minimal medium agar plates. The cells were grown and selected for uridine prototrophy conferred by the pyr2 marker. Stable transformants were obtained by two successive transfers on agar plates, It was then grown twice in succession on nonselective PDA plates and on Vogel agar plates. Single colonies were obtained by one round of growth followed by plate dilution of the spore suspension.

[0300] The parent and transformed (daughter) T. reesei were cultured under both "non-induced" and "induced" conditions. (T. reesei) host cells were tested. For example, in "non-inducing conditions", standard 24-well plates were in a 100-well microtiter plate (MTP) supplemented with 2.5% glucose (weight / volume). Cells were grown in 1.25 ml liquid broth of defined medium. medium, 1.25 ml of defined medium supplemented with 2.5% glucose / sophorose (wt / vol) In liquid broth (sophorose acts as a potent inducer of cellulase enzyme expression) Following incubation, supernatants from all cultures were harvested and analyzed by Biol. -Rad Reagent (Thermo Scientific®; Catalog No. 232 36) and a 5-fold dilution of bovine serum albumin (BSA) as a standard were used at 595 nm. Total secreted protein was measured by Bradford dye-binding assay.

[0301] As shown in Table 3, the parent T. reesei cells express sophorose inducible In contrast, high levels of secreted protein were produced only in the presence of the enzyme. Mutant (daughter) T. reesei containing and expressing promoter-driven Ace3-L (T. reesei) cells secrete similar amounts of β-lactamase under both induced and uninduced conditions. As shown in Table 3, the protein levels of each modified (daughter) strain were Produced by the parent strain (LT4) under Glu / Sop-inducing conditions Shown as ratio to protein (concentration).

[0302] [Table 3]

[0303] Furthermore, shake flask experiments as generally described in Example 3 demonstrated that T. reesei The T. reesei (T sei) parent strain and its transformants described above were further tested. .reesei) daughter line “LT83” (wherein the daughter line LT83 is a dic1 promoter ( Representative results for a plasmid containing the ace3-L ORF driven by SEQ ID NO:28 are shown in FIG. As shown in Figure 8, parental (control) T. reesei cells express sophorose ("Sop") inducer, but produced secreted protein only in the presence of the inducer. 3 produced similar amounts of secreted proteins under both induced ("Sop") and uninduced ("Glu") conditions. produced protein.

[0304] Similarly, small-scale fermentations were carried out generally as described in Example 4. As shown in FIG. 9, the parent (control) T. reesei strain was The daughter strain LT83 produced secreted proteins only in the presence of the promoter ("Sop"). The Glu expression level was 1.37, and the Glu expression level was 1.37. produced proteins.

[0305] Example 6 Cloning of T. reesei ACE3 overexpression constructs The genome sequence of T. reesei is available from the Joint Genome Inst. Officially from the Institute (https: / / genome.jgi.doe.gov / ) However, the location of the 5' end of the ace3 coding region is unclear. The annotation of DNA sequences at the Point Genome Institute is Although the DNA sequence is the same, the mutant strain Rut-C30 (genome.jgi.doe. gov / TrireRUTC30_1 / TrireRUTC30_1.home.htm l) and the wild-type strain QM6a (genome.jgi.doe.gov / Trire2 / Tr In the case of QM6a, the ace3 code region The 5' end of the In the case of Rut-C30, the 5' end of the ace3 coding region is Further analysis of the genomic DNA sequence and additional cDNA sequences. Further analysis revealed the possible presence of exon 1 and intron 1 as shown in Figure 11. Furthermore, the 3' end of the ace3 coding region of Rut-C30 is Compared with the sequence of the live isolate QM6a (Fig. 11, arrow 5), a premature stop codon (Fig. 11, The ace3 gene contains a mutation that creates the 4-terminal endothelial cell line (arrow 4). The effect of overexpressing these different possible forms was experimentally examined as described herein. (See, e.g., Figures 11, 12, and 13-18).

[0306] Therefore, the different forms of ace3 shown in FIG. 12 are consistent with those of T. reesei ) where the T. reesei ace3 gene was overexpressed. The overexpression vector for this purpose was the T. reesei glucoamylase gene. It was designed to allow targeted integration of ace3 into the gla1 locus. All plasmid (vector) constructs were transformed into T. reesei The ace3 gene is under the control of the T. reesei dic1 promoter. , and expressed with the native ace3 terminator. These vectors were transformed into T. reesei ( For the selection of T. reesei transformants, the pyr4 marker was inserted into its natural promoter. After integration into the gla1 locus, the pyr Repeats of the pyr4 promoter were included to allow excision of the 4 genes. The vector backbone, which allows replication and amplification in E. coli, is EcoRI-XhoI. digested pRS426 (Colot et al., 2006). Required for targeted integration 5' and 3' terminal fragments of the T. reesei gla1 locus The dic1 promoter, the different forms of the ace3 coding region, and the terminator The flanking fragments were generated by PCR using the primers shown in Table 4. The type was wild-type T. reesei QM6a (ATCC accession no. 13631 ) genomic DNA.

[0307] [Table 4]

[0308] For the dic1 promoter, ace3 gene and terminator, the template was The previous plasmid pYL8 (see Example 5) harbors these fragments. The selection marker (pyr4) was obtained from the previous plasmid with NotI digestion. The PCR primers used to generate the desired DNA fragment are shown in Table 4. The R products and digestion fragments were separated using agarose gel electrophoresis. The correct fragment was extracted from the gel using a gel extraction kit (Qiagen) according to the protocol of The fragment was isolated. The plasmid was prepared as described in PCT / EP2013 / 050126 ( The yeast phase described in WO 2013 / 102674 (published as a pamphlet) The same recombination method was used to construct the above fragments. The plasmid was rescued from yeast. A small number of clones were selected and the plasmid p53 was transformed into E. coli. DNA was isolated and sequenced, and an overview of the plasmids is shown in Table 5.

[0309] [Table 5]

[0310] Thus, the constructs shown in Table 5 contain different forms of the ace3 gene. The SC morphology differs by the presence of a gene that contains exons 3 and 4 and intron 3. It is the short form of the gene (see Figures 12 and 13, SEQ ID NO: 7). The SC form of SEQ ID NO:7 has 1,713 bp of exon 3 and 148 bp of intron 3. and 144 bp of exon 4. Son 4) has the same mutations as T. reesei RutC-30 strain.

[0311] The S form is a short form of the gene that contains exons 3 and 4 and intron 3. but does not have a mutation at the (3') C-terminus of exon 4 (Figures 12 and 14, SEQ ID NO: 1). More specifically, the S form is a 1,713 bp exon 3, 148b p intron 3 and 177 bp of exon 4. In the "SC" and "S"), the translation initiation codon is As per the annotation of the QM6a strain, a long intron 2 (see Figure 12) Both the "SC" and "S" forms contain part of the coding region for the putative DNA binding domain. is lacking.

[0312] The L form contains exons 2, 3, and 4, as well as intron 2 (a long intron) and The long form of the gene including intron 3 (see, for example, FIG. 12 and FIG. 15, SEQ ID NO: 4 More specifically, the L form has a 258-bp exon 2, a 423-bp intron 3, and a 18-bp intron 4. intron 2, 1,635 bp exon 3, 148 bp intron 3 and 144 bp The (3') C-terminus of the L form is similar to that of T. reesei ) It has the same mutations as the RutC-30 strain.

[0313] The LC form contains exons 2, 3, and 4, as well as intron 2 (long intron) and and intron 3 (see, for example, Figures 12 and 16, SEQ ID NO: 9). More specifically, the LC form has a 258-bp exon 2, a 423-bp intron 2 of 1,635 bp, exon 3 of 148 bp, and intron 3 of 177 bp. The LC form contains exon 4 of 100 bp. The LC form has no mutations in the C-terminus. In terms of morphology, the translation initiation codon is annotated by JGI for the Rut-C30 strain. It is in exon 2.

[0314] The EL form contains exons 1, 2, 3, and 4, as well as introns 1 and 2 (long A particularly long version of the ace3 gene that contains intron 3 ( See, e.g., Figures 12 and 17, SEQ ID NO: 11). More specifically, the EL form It consists of 61 bp in exon 1, 142 bp in intron 1, 332 bp in exon 2, and 42 3 bp intron 2, 1,635 bp exon 3, 148 bp intron 3 and It contains 144 bp of exon 4. The (3') C-terminus of the EL form is identical to that of T. reesei (T. reesei RutC-30 strain.

[0315] The LN form contains exons 2, 3, and 4, and intron 3 but not intron 2. 12 and 18, SEQ ID NO: 13. The (3') C-terminus of the LN form is T. reesei RutC- 30. Therefore, as mentioned above, L, LC, LN and The EL and EL forms encode the complete putative DNA binding domain.

[0316] Transformation into T. reesei strain RL-P37 All plasmids shown in Table 5 were digested with MssI to generate fragments for targeted integration. The proteins were released and separated by agarose gel electrophoresis. Alignment of DNA sequences within representative fragments used for transformation of T. reesei A gel extraction kit (Qiagen) was used according to the manufacturer's protocol. Therefore, the correct fragment was isolated from the gel. Approximately 10 μg of the purified fragment was used. Using this method, the pyr4-mutant of T. reesei RL-P37 was The preparation and transformation of protoplasts was carried out according to the method described in WO 2013 / 023361. The pyr4 selection was performed as described in the / 102674 brochure. .

[0317] The transformants were streaked onto selective media plates. The grown clones were selected from the primates listed in Table 6. Correct integration was screened by PCR using the expected sigma. The resulting clones were purified to single cell clones and subjected to PCR amplification using the primers listed in Table 6. Correct integration and clonal purity were confirmed by CR and also by Southern blotting. The results were then rescreened for phenotype (data not shown).

[0318] [Table 6]

[0319] Cultivation of different ace3 transformants In a 24-well microtiter plate, 2% lactose or 2 The strains shown in Table 7 were grown in liquid medium containing 0.4% glucose. The other components of the medium were 5%KH2PO4, 0.5%(NH4)2SO4, 0.1%MgSO4, 0.1%CaC l2, 0.9% casamino acids, 0.048% citric acid x H2O, 0.05% FeSO4 x 7 H2O, 0.0003%MnSO4×H2O, 0.004%ZnSO4×7H2O, 0. 0.0002% H3BO3 and 0.00014% CuSO4 × 5H2O. 100 mM PIPPS (Calbiochem) was included to maintain the pH at .5.

[0320] [Table 7]

[0321] Infors HT microton shaker at 80% humidity, 28°C, 800R Cultures were cultured in PM. Culture samples were taken on days 3-7. BioRad P Protein Assay was performed according to the manufacturer's protocol. The amount of secreted protein was measured. Overexpression of the ce3L, EL and LN forms improved production of the total protein. In media with lactose as a source, overexpression of all forms of the ace3 gene resulted in the production of all Protein production was improved to some extent, but the level of improvement was limited by the L and EL mutations of the ace3 gene. and LN morphology. Glucose (i.e., non-inducing conditions) was used with transformants overexpressing morphogenetic It is clear that high levels of secreted protein (Table 8) were observed when

[0322] [Table 8]

[0323] Example 7 Knock-in of endogenous Ace3 heterologous promoter The promoter replacement construct (see FIG. 6) was inserted into the native promoter of the ace3 locus. A DNA fragment containing the 5' region upstream of the promoter, flanked by loxP, and hygromycin B resistance selection marker cassette, and The promoter was generated by fusing a fragment containing a promoter of interest operably fused to the (For more information on gene / promoter replacement for use in filamentous fungi, see (See PCT application no. PCT / US2016 / 017113).

[0324] Therefore, the above promoter replacement constructs were used to express Trichoderma reesei (Tr ichoderma reesei cells, transformants were isolated, and diagnostic PC Genomic DNA was extracted for R and the promoter replacement code at the native ace3 locus was This method is used to confirm the homologous recombination of the construct. The native ace3 promoter is replaced by the set and any desired promoter. Transformants can be identified. The hygromycin B resistance cassette is then inserted into the cre recombinase. It is removed by the action of enzymes (Nagy, 2000).

[0325] The efficiency of homologous integration at the ace3 locus was determined using the method described in WO 2016 / 100272. No. 2016 / 100571, and International Publication No. As illustrated in the brochure 2016 / 100568, a suitably designed guide The gene was enhanced by the action of cas9, which was directed by RNA to the native ace3 promoter. It is possible.

[0326] The conditional promoter replacement (CPR) strategy was developed based on the publication by Hu et al. (2007). Aspergillus fumigatus It has been described in A. fumigatus NiiA nitrogen regulator. A tunable promoter (pNiiA) was used to express the endogenous promoter of the selected gene. Deletion and replacement strategies are generally described. A similar method was used to culture Trichoderma reesei ) to replace the endogenous promoter of the ace3 gene with an alternative promoter. can.

[0327] Example 8 Lignocellulosic target gene promoters of endogenous non-lignocellulosic target gene promoters Motor replacement Trichoderma reesei glucoamylase Expression constructs are assembled from DNA polynucleotide fragments (e.g., (See U.S. Patent No. 7,413,879). Here, T. Reesey (T. The ORF sequence encoding T. reesei glucoamylase is 5' (upstream) operably linked to the T. reesei cbh1 promoter and 3' (downstream) This DNA was operably linked to the T. reesei cbh1 terminator. The A construct contains the T. reesei pyr2 gene as a selectable marker. It further contained genes.

[0328] Thus, the mutant (daughter) T. reesei cells of the present disclosure (i.e. (including a genetic modification that increases the expression of a gene encoding the Ace3-L protein), The glucoamylase expression construct was transformed into T. reesei. To identify those transformants capable of secreting the glucoamylase enzyme. To achieve this, transformants were selected and grown in liquid medium containing glucose as a carbon source (i.e., The strain was cultured in a culture medium containing no inducing substrates such as sophorose or lactose.

[0329] Therefore, the T. reesei glucoamylase-expressing (parent) strain (control ), and a modified T. reesei (daughter) glucoamylase expressing strain (i.e. That is, the Ace3-L ORF (containing and expressing the Ace3-L ORF) was grown in shake flasks and The supernatants of the cell cultures were harvested and cultured as described generally in Examples 2 and 3 above. Analysis was performed using GE.

[0330] More specifically, as shown in FIG. 10, the parent T. reesei cells were In a defined medium containing glucose / sophorose (inducing condition), the glucose concentration was 1,029 μg / mL. In the defined medium containing glucose (non-inducing condition), the enzyme produced only 38 μg / ml. In contrast, the dic1 promoter only produced 1000 g / L of glucoamylase. The engineered (daughter) strain “LT88” containing ace3-L driven by the “inducible” (Sop) Produces 3-fold more glucoamylase under inducing conditions (i.e., compared to the parent (control) strain under inducing conditions) (with "Glu") and produced 2.5-fold more glucoamylase under "non-inducing" ("Glu") conditions. (i.e., compared to the parental (control) strain under inducing conditions) or the parental (control) strain under non-inducing conditions. ) strain produced 67-fold more glucoamylase under “non-induced” (“Glu”) conditions compared to the Therefore, these results suggest that the modified (daughter) cells containing the Ace3-L ORF are Not only do these mutant cells produce extracellular proteins in the absence of inducers, Under such induction conditions, more than parental (control) T. reesei cells , indicating that the entire protein is produced.

[0331] Example 9 Lignocellulosic target gene promoters using naturally associated heterologous target gene promoters Replacement by tar Phytase expression constructs were synthesized by cloning the DNA polynucleotide fragments (See, for example, U.S. Patent No. 8,143,046). The ORF encoding the phytase from Buttiauxella sp. operably linked at its 5' end to the T. reesei cbh1 promoter and operable at the 3' end with the T. reesei cbh1 terminator The DNA construct contains a selectable marker, Aspergillus nidulans. Aspergillus nidulans amdS gene. T. reesei cells (i.e., encoding the Ace3-L protein) (including genetic modifications to increase expression of the gene) were transformed with a phytase expression construct. During cultivation, Buttiauxella phytase enzyme is produced. To identify transformants capable of secreting ribosomal DNA, transformants were selected and treated with ribosomal DNA as a carbon source. In a liquid medium containing glucose (i.e., an inducing substrate such as sophorose or lactose) The culture was performed using a 100% ethanol-free medium.

[0332] Example 10 Construction of native ace3 promoter replacement vector In this example, two ace3 promoter replacement vectors, pCHL760 and pCH L761 was constructed using standard molecular biology procedures. Vector backbone pMCM328 2 (Figure 20) was transformed into pMB1 or pMB2 for replication and selection in E. coli. In addition, N. crassa cpc 1 promoter and the A. nidulans trpC terminator -expressed in Trichoderma reesei The vector contained the hph hygromycin selection marker for promoter replacement. The pki1 promoter of T. reesei is used to express the toxin. Streptococcus pyogenes optimized for sorghum s pyogenes) cas9 codon, and guide R expressed under the U6 promoter NA (e.g., WO 2016 / 100568 and WO 2016 / 100568). (See Brochure No. 2016 / 100272).

[0333] Therefore, pMCM3282 was digested with EcoRV and the ace3 native promoter was placed The promoter region of T. reesei hxk1 or dic1 is adjacent to adjacent 5' end of approximately 1 kb from the T. reesei ace3 locus and three fragments with 3'-terminal homologous sequences were synthesized using the Gibson Assembl y into pMCM3282 / EcoRV, and pCHL760 (Figure 21) and and pCHL761 (Figure 22) was obtained.

[0334] Q5 High-fidelity DNA polymerase (New England Biolabs and the primers listed in Table 9 below were used to amplify T. reesei (T reesei) genomic DNA, 5'ace3 homologues were identified together with hxk1 or dic1. The sequence and the 3'ace3 homology sequence were PCR amplified.

[0335] [Table 9]

[0336] More specifically, the specific primers used to PCR amplify the fragments of each vector are described. To construct pCHL760, the primer pair CL1840 and CL1791 were used to amplify the 5' upstream homologous region, and primer pairs CL1794 and The 3' downstream homologous region was amplified using primer pair CL1792 and CL1831. CL1793 was used to amplify the dic1 promoter.

[0337] To construct pCHL761, the primer pair CL1840 and CL1800 were used. The 5' upstream homologous region was amplified using primer pair CL1803 and CL1831. , amplify the 3' downstream homologous region using primer pair CL1801 and CL1802. The ic1 promoter was amplified.

[0338] Vector pMCM3282 (Figure 20) contains, in the 5' to 3' direction, the eesei U6 promoter, E. coli ccdB cassette, and C It contains the structural region of the single-stranded guide RNA (sgRNA) involved in as9 binding, and is expressed in the U6 gene. The ccdB cassette contains an intron derived from Trichoderma sp. a) Five different target sites in the ace3 gene were replaced with specific sequences. Final ace3 To construct the promoter replacement vector, pCHL760 (Figure 21) and pCHL Insertion of a guide RNA sequence into 761 (Figure 22).

[0339] Therefore, for the production of different sgRNA sequences, the following primers with an AarI restriction site were used: The oligonucleotides shown in Table 10 were designed.

[0340] [Table 10]

[0341] More specifically, CL1821 and CL1822, CL1823 and CL1824, CL1825 and CL1826, CL1827 and CL1828, CL1829 and CL1830 and CL1830 were annealed to generate double-stranded DNA, which were then subjected to type IIS seam synthesis. into pCHL760 and pCHL761 at the AarI site using the less cloning method. The final plasmid with the correctly inserted guide RNA sequence was , which had lost the virulent ccdB gene.

[0342] Transformation of T. reesei Cas9-mediated ace3 promoter replacement vectors pCHL760 and pCHL761 was transformed into T. leuconostigmine by polyethylene glycol (PEG)-mediated protoplast transformation. The transformants were transformed into T. reesei parent cells and hygromycin-resistant transformants were selected. To select for the transformants, the transformants were grown on Vogel's minimal medium agar containing hygromycin. Some of these transformants had incorporated the plasmid but did not contain the genomic DNA. The transformants were unstable, with no stable integration into A. culture medium to eliminate the plasmid and the hygromycin resistance marker.

[0343] To screen for dic1 promoter replacement transformants, genomic DNA was Extract A and use the primer pair CL1858 and CL1848 (predicted for the desired integration). The expected size of the product was 2,412 bp) and CL1853 and CL18 18 (the expected size product for the desired integration was 2,431 bp) (See, for example, Table 11.) The PCR products were then amplified using the DNA sequence The plasmid was analyzed by determination to confirm integration of the desired promoter.

[0344] To screen for hxk1 promoter replacement transformants, primers Vs. CL1858 and CL1898 (the expected size product for the desired integration is 1,784 bp) and CL1853 and CL1850 (which were the desired integration The expected size of the product for this was 2,178 bp) and then PCR was performed using Correct integration was confirmed by DNA sequencing of the CR products (see, e.g., Table 11). I want to.

[0345] [Table 11]

[0346] Protein production in shake flasks To test the functionality of the ace3 promoter replacement strain, we used inducer substrate (Sophoro Cells were grown in 50 ml submerged cultures in shake flasks in the presence and absence of 5-HT15 (pH 7.0) and 5-HT15 (pH 7.0) cultures. The results were analyzed under induction conditions (glucose / sophorose as carbon source) and non-induction conditions (glucose / sophorose as carbon source). In both liquid cultures, parent T. reesei host cells were cultured on 100% glucose. Cell (ID number 1275.8.1), mutant cell ID numbers 2218, 2219, 2220, 2 221, 2222 and 2223 were grown and their respective extracellular secreted proteins were The production levels were compared. Briefly, 250 mL Erlenmeyer flasks with bottom baffles were used. Add 50 mL of YEG broth to each host cell (i.e., T. reesei ei) Parent host cells and its mutant cells) mycelia were added separately. YEG broth was 5 g / The medium contained 20 g / L yeast extract and 22 g / L glucose. The cell culture was grown for 48 hours. These seed cultures were then subcultured in fresh YEG for an additional 24 hours. was placed in a 250 mL shake flask with a bottom baffle and supplemented with 2.5% glucose. 50 mL of defined medium (non-inducing conditions) or 2.5% glucose / sophorose 50 mL of defined medium (inducing conditions) was inoculated. All shake flasks were incubated at 200 rpm for 1 h. The plates were incubated at 28°C with continuous shaking. After 4 days of incubation, all plates were The supernatant from the cell culture was collected and analyzed using SDS-PAGE as shown in FIG. and analyzed.

[0347] As seen in the SDS-PAGE above, in parental cells (Figure 23, ID 1275.8.1) The ratio of glucose (uninduced) to sophorose (induced) was 1:1. In the constant medium, the production of secreted proteins was very low. In contrast, transformant 2218 2219, 2220, 2222 and 2223 show similar amounts of cleavage under induced and uninduced conditions. These results therefore support the conclusion that the ace3 gene is a naturally occurring Mutant cells with the hxk1 or dic1 promoter replacing the ace3 promoter produced extracellular protein in the absence of inducer.

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Claims

1. 1. A method for producing a heterologous protein of interest (POI) in a fungal cell of the genus Trichoderma in the absence of an inducing substrate, comprising: (i) introducing into the fungal cell a polynucleotide construct comprising, in a 5' to 3' direction, (a) a first nucleic acid sequence comprising a constitutive promoter, and (b) a second nucleic acid sequence operably linked to the first nucleic acid sequence, wherein the second nucleic acid sequence encodes an Ace3 protein having 90% sequence identity to SEQ ID NO:6 and comprising "Lys-Ala-Ser-Asp" as the last four C-terminal amino acids; (ii) fermenting the cells of step (i) under conditions suitable for fungal cell growth and protein production, said conditions being free of an inducing substrate; The method includes:

2. 2. The method of claim 1, wherein the fungal cell comprises an introduced polynucleotide construct encoding the heterologous protein of interest, the construct being introduced into the fungal cell before, during, or after step (i).

3. The method of claim 1, wherein the polynucleotide construct comprises a third nucleic acid sequence operably linked 3' to the second nucleic acid, the third nucleic acid sequence comprising a native ace3 terminator sequence.

4. The method of claim 1 , wherein the polynucleotide construct is integrated into the genome of the fungal cell.

5. The method of claim 1 , wherein the polynucleotide construct is integrated into a telomeric site of the genome of the fungal cell.

6. The method of claim 1 , wherein the polynucleotide construct is integrated into the glucoamylase (gla1) locus of the genome of the fungal cell.

7. 2. The method of claim 1, wherein the polynucleotide construct comprises a nucleotide sequence having 90% sequence identity to SEQ ID NO:4, SEQ ID NO:11 or SEQ ID NO:

13.

8. The method of claim 1, wherein the encoded Ace3 protein comprises an amino acid sequence having 95% sequence identity to SEQ ID NO:6, SEQ ID NO:12 or SEQ ID NO:

14.

9. The heterologous target protein may be α-amylase, alkaline α-amylase, β-amylase, cellulase, dextranase, α-glucosidase, α-galactosidase, glucoamylase, hemicellulase, pentosanase, xylanase, invertase, lactase, naringanase, pectinase, pullulanase, acid protease, alkaline protease, bromelain, neutral protease, papain, pepsin, peptidase, rennet, renin, chymosin, subtilisin, thermolysin, aspartic acid proteinase, trypsin, lipase, esterase, phospholipase, phosphatase, phytase, amidase, iminoacylase, glutaminase , lysozyme, penicillin acylase; isomerase, oxidoreductase, catalase, chloroperoxidase, glucose oxidase, hydroxysteroid dehydrogenase, peroxidase, lyase, aspartate β-decarboxylase, fumarase, histidase, transferase, ligase, aminopeptidase, carboxypeptidase, chitinase, cutinase, deoxyribonuclease, α-galactosidase, β-galactosidase, β-glucosidase, laccase, mannosidase, mutanase, polyphenol oxidase, ribonuclease and transglutaminase.

10. 2. The method of claim 1, wherein the promoter is selected from the group consisting of rev3 promoter (SEQ ID NO: 15), bxl promoter (SEQ ID NO: 16), tkl1 promoter (SEQ ID NO: 17), PID104295 promoter (SEQ ID NO: 18), dld1 promoter (SEQ ID NO: 19), xyn4 promoter (SEQ ID NO: 20), PID72526 promoter (SEQ ID NO: 21), axe1 promoter (SEQ ID NO: 22), hxk1 promoter (SEQ ID NO: 23), dic1 promoter (SEQ ID NO: 24), opt promoter (SEQ ID NO: 25), gut1 promoter (SEQ ID NO: 26) and pki1 promoter (SEQ ID NO: 27).

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