Erythritol utilization-deficient mutant trichoderma spp. and method for producing target substance using same

Abstract

To provide erythritol-utilization deficient mutant Trichoderma spp. and a method for producing a target substance using the same.SOLUTION: Disclosed is a mutant Trichoderma spp. with reduced or disappeared ability to utilize erythritol, where the expression of at least one polypeptide selected from the group consisting of following (A)-(D) has been decreased or eliminated, and a gene encoding a target substance or an enzyme involved in the synthesis thereof has been introduced and an erythritol-inducible promoter is ligated to the upstream of the gene: (A) a polypeptide comprising an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto; (B) a polypeptide comprising an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 90% identity thereto; (C) a polypeptide comprising an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 90% identity thereto; (D) a polypeptide comprising an amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 90% identity thereto.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a mutant Trichoderma fungus defective in erythritol assimilation, and a method for producing a target substance using the same. [Background technology]

[0002] Trichoderma fungi are capable of producing large amounts of enzymes such as cellulase and xylanase, and have long been attracting attention as microorganisms for producing cellulolytic enzymes (Non-Patent Document 1). For example, Trichoderma reesei has a high protein production capacity and is expected to be used for the production of heterologous proteins derived from humans and other microorganisms (see Non-Patent Document 2).

[0003] When a target protein is industrially produced using a microorganism, a large amount of carbon sources such as glucose and cellulose are used. On the other hand, in the presence of a high concentration of carbon source, the fungus is subjected to osmotic stress, which results in a decrease in protein productivity (Non-Patent Document 3). In order to prevent a decrease in the productivity of the target substance due to osmotic stress, it is desirable to reduce the osmotic stress of the fungus during culture. It has been reported that when yeast and the filamentous fungus Aspergillus nidulans are subjected to osmotic stress, sugar alcohols such as glycerol and erythritol are produced intracellularly to increase the osmotic resistance of the fungus cell and avoid osmotic stress (Non-Patent Documents 4 and 5).

[0004] Studies have been conducted on the production of erythritol from cellulosic raw materials using Trichoderma reesei (Non-Patent Document 6). On the other hand, it has been reported that Trichoderma reesei assimilates erythritol and that the assimilation rate is equal to or higher than that of glucose (Non-Patent Document 7). Although the erythritol assimilation pathway of Trichoderma has not been elucidated, the present inventors have discovered an erythritol-inducible promoter that works in Trichoderma (Patent Document 1). It is considered that the produced erythritol is re-assimilated in Trichoderma and erythritol is not retained in the cells for a long time. In fact, even in the prior art, Trichoderma reesei has only been able to produce erythritol at a level of about several mg / L (Non-Patent Document 6). Patent Document 2 describes that it is preferable to inactivate genes encoding erythrulose kinase (SEQ ID NO: 19), erythritol isomerase 1 and 2 (SEQ ID NO: 23 and 25) of Trichoderma reesei in modified microorganisms for erythritol synthesis. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent application 2021-178778 [Patent Document 2] European Patent Application Publication No. 3929299 [Non-patent literature]

[0006] [Non-Patent Document 1] Chemistry and Biology, 2012, 50(8):592-599, 2012 [Non-Patent Document 2] Appl Biochem Biotechnol, 2011, 165(5-6):1169-1177 [Non-Patent Document 3] Fungal Biol, 2010, 114(10):855-862 [Non-Patent Document 4] Journal of the Brewing Society of Japan, 2010, 105(10):618-627 [Non-Patent Document 5] J Bacteriol, 1986, 168(3):1358-1365 [Non-Patent Document 6] AMB Express, 2014, 4:34 [Non-Patent Document 7] Appl Environ Microbiol, 2006, 72(3):2126-2133 Summary of the Invention [Problem to be solved by the invention]

[0007] If erythritol can be efficiently accumulated in Trichoderma cells, as in yeast and Aspergillus described in Non-Patent Documents 4 and 5, it is expected that osmotic stress on the bacterial cells caused by the carbon source in the medium can be reduced, and the decrease in productivity of the target substance caused by osmotic stress can be improved. Furthermore, the present inventors have previously discovered an erythritol-inducible promoter that works in Trichoderma bacteria, and have found that this promoter can induce expression of a gene for the target substance without promoting the cellulase expression process of Trichoderma bacteria, making it possible to produce the target substance with a higher purity (Patent Document 1). In the production of the target substance using this erythritol-inducible promoter, it is also desirable to cause the host Trichoderma fungus to accumulate erythritol in order to efficiently induce expression of the target substance.

[0008] The present invention provides a mutant Trichoderma fungus defective in erythritol assimilation ability, and a method for producing a target substance using the mutant Trichoderma fungus. [Means for solving the problem]

[0009] In one aspect, the present invention relates to a mutant Trichoderma fungus having reduced or eliminated erythritol assimilation ability, wherein expression of at least one polypeptide selected from the group consisting of the following (A) to (D) is reduced or eliminated: (A) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto; (B) a polypeptide consisting of the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 90% identity thereto; (C) a polypeptide consisting of the amino acid sequence of SEQ ID NO:6 or an amino acid sequence having at least 90% identity thereto; and (D) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 90% identity thereto; and a gene encoding a substance of interest or an enzyme involved in the synthesis of the substance of interest and an erythritol-inducible promoter linked upstream of the gene are introduced into the vector. Mutant Trichoderma spp., to provide. In another aspect, the present invention provides a method for producing a target substance using the mutant Trichoderma fungus. Effect of the Invention

[0010] The erythritol assimilation-deficient mutant Trichoderma fungus provided by the present invention has improved resistance to osmotic stress caused by the carbon source in the culture medium and is capable of accumulating higher concentrations of erythritol intracellularly, making it advantageous for producing target substances using erythritol-inducible promoters. [Brief description of the drawings]

[0011] [Figure 1] Relative expression levels of genes after addition of erythritol, glucose or sorbitol. (A) 122079 genes, (B) 68466 genes, (C) 68606 genes, (D) 68585 genes. 0h: immediately after addition, 2h: 2 hours after addition. [Diagram 2] Erythritol concentration in culture supernatant after addition of erythritol to cultures of Trichoderma mutant strains lacking the 122079 gene, the 68466 gene, the 68606 gene, and the 68585 gene. [Diagram 3]Effect of intracellular erythritol accumulation on the expression level of the 122079 gene in Trichoderma mutants lacking the 122079 gene, the 68466 gene, the 68606 gene, and the 68585 gene. [Figure 4] Effect of intracellular erythritol accumulation on the expression level of the 68466 gene in Trichoderma mutants lacking the 122079 gene, the 68466 gene, the 68606 gene, and the 68585 gene. [Diagram 5] Effect of intracellular erythritol accumulation on the expression level of the 68606 gene in Trichoderma mutants lacking the 122079 gene, the 68466 gene, the 68606 gene, and the 68585 gene. [Figure 6] Effect of intracellular erythritol accumulation on the expression level of the 68585 gene in Trichoderma mutants lacking the 122079 gene, the 68466 gene, the 68606 gene, and the 68585 gene. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] All patents, non-patent publications, and other publications cited herein are hereby incorporated by reference in their entirety.

[0013] In the present specification, the identity of an amino acid sequence or a nucleotide sequence is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing an analysis using a homology analysis program of the genetic information processing software GENETY Ver. 12 with a unit size to compare (ktup) of 2.

[0014] As used herein, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence means identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% or more.

[0015] As used herein, "an amino acid sequence in which one or several amino acids have been deleted, substituted, added, or inserted" refers to an amino acid sequence in which preferably one to three, more preferably one or two, amino acid residues have been deleted, substituted, added, or inserted. As used herein, "addition" of an amino acid residue includes addition of an amino acid residue to one or both ends of a sequence. As used herein, "a nucleotide sequence in which one or several nucleotides have been deleted, substituted, added, or inserted" refers to a nucleotide sequence in which preferably one to ten, more preferably one to five, even more preferably one to three, nucleotides have been deleted, substituted, added, or inserted. As used herein, "addition" of a nucleotide includes addition of a nucleotide to one or both ends of a sequence.

[0016] As used herein, "upstream" and "downstream" in relation to a gene refer to the upstream and downstream of the transcription direction of the gene. For example, the "upstream sequence" and "downstream sequence" of a gene refer to the sequences located on the 5' and 3' sides of the gene in the DNA sense strand, respectively. For example, "a promoter linked upstream of a gene" means that a promoter is present on the 5' side of the gene in the DNA sense strand.

[0017] As used herein, the term "operably linked" between a gene and a control region such as a promoter means that the gene and the control region are linked in such a way that the gene can be expressed under the control of the control region. Procedures for "operably linked" between a gene and a control region are well known to those skilled in the art.

[0018] As used herein, the term "native" in reference to a function, property, or trait of a cell is used to indicate that the function, property, or trait is inherently present in the cell. In contrast, the term "exogenous" is used to indicate a function, property, or trait that is not inherently present in the cell, but is introduced from the outside. For example, "exogenous" nucleotides or DNA are nucleotides or DNA that are introduced from the outside into a cell. Exogenous nucleotides or DNA may be derived from the same organism as the cell into which it is introduced, or from a different organism (i.e., heterologous nucleotides or heterologous DNA).

[0019] In this specification, an "erythritol assimilation-related gene" is defined as a gene whose expression is reduced or eliminated, thereby reducing or eliminating the erythritol assimilation ability of Trichoderma cells. The erythritol assimilation ability of Trichoderma can be measured, for example, by the following methods: culturing Trichoderma in a medium containing erythritol as the sole carbon source and measuring the growth rate of the fungus; measuring the erythritol concentration in the medium containing erythritol at appropriate times while culturing Trichoderma in the medium containing erythritol to determine the uptake rate of erythritol into the fungus; culturing Trichoderma for a predetermined time in the medium containing erythritol, and then measuring the amount of erythritol in the fungus to determine the amount of erythritol that is not metabolized to another substance and that accumulates as erythritol.

[0020] In this specification, "promoter activity" means an activity that promotes the transcription of DNA (gene) into mRNA. Promoter activity can be confirmed by using an appropriate reporter gene. For example, promoter activity can be confirmed by linking DNA encoding a detectable protein, i.e., a reporter gene, downstream of a promoter and measuring the amount of the gene product produced by the reporter gene. Examples of reporter genes include genes of enzymes that act on color-developing substrates, such as the β-galactosidase (LacZ) gene, the β-glucuronidase (GUS) gene, the luciferase gene, the β-lactamase gene, and the gene encoding EtbC (2,3-dihydroxy-ethylbenzene 1,2-dioxygenase), and genes of fluorescent proteins, such as the gene encoding GFP (Green Fluorescent Protein). Alternatively, promoter activity can also be confirmed by measuring the expression amount of mRNA transcribed from the reporter gene by sequencing, quantitative RT-PCR, or the like.

[0021] As used herein, the term "erythritol-inducible promoter" refers to a promoter that has promoter activity in the presence of erythritol, and preferably refers to a promoter that can induce expression of mRNA of a target gene (i.e., a gene to which the promoter is operably linked) in the presence of erythritol at least 40-fold, preferably at least 50-fold, and more preferably at least 100-fold, as compared to the absence of erythritol or the presence of cellulose, glucose, or sorbitol.

[0022] The present inventors have identified genes from Trichoderma that are hardly expressed under conditions in which erythritol is not added, but whose expression is significantly improved under conditions in which erythritol is added. In strains in which these genes have been deleted, erythritol accumulated intracellularly. These genes are presumed to be genes involved in erythritol assimilation in Trichoderma. In the production of a target substance using Trichoderma, if a mutant Trichoderma in which the above-mentioned erythritol assimilation-related gene has been deleted is cultured in an erythritol-containing medium to accumulate erythritol intracellularly, it is possible to reduce osmotic stress caused by the carbon source in the medium, and improve the decrease in productivity of the target substance in Trichoderma due to osmotic stress.

[0023] In addition, in the production of a target substance using a filamentous fungus such as Trichoderma, it is desired to produce the target substance without promoting the cellulase production process inherent to the cell. The present inventors previously discovered an erythritol-inducible promoter that works in Trichoderma (Patent Document 1). Erythritol does not induce the expression of cellulosic biomass decomposition enzymes inherent to Trichoderma cells (see the Examples in Patent Document 1). If a target gene operably linked to the erythritol-inducible promoter is introduced into a mutant Trichoderma in which the above-mentioned erythritol assimilation-related genes have been deleted, and the mutant is cultured in an erythritol-containing medium, the erythritol accumulated in the cell can selectively induce the expression of the target gene via the erythritol-inducible promoter without promoting the cellulase expression induction process inherent to the cell. Therefore, the present invention makes it possible to produce a target substance in Trichoderma with higher purity and higher yield.

[0024] The present invention provides a mutant Trichoderma bacterium deficient in erythritol assimilation. The erythritol assimilation-deficient mutant Trichoderma bacterium of the present invention (hereinafter also simply referred to as "mutant Trichoderma bacterium of the present invention") is a Trichoderma bacterium modified so as to reduce or eliminate erythritol assimilation ability. The erythritol assimilation ability of the mutant Trichoderma bacterium of the present invention (erythritol reduction rate calculated based on the amount of erythritol remaining in a medium for culturing Trichoderma bacterium 2 hours after adding erythritol to the medium, as measured by HPLC) is preferably reduced to 50% or less, more preferably 70% or less, and even more preferably 90% or less, compared to that of the parent Trichoderma bacterium (the Trichoderma bacterium before being modified to be deficient in erythritol assimilation ability).

[0025] In a preferred embodiment, the mutant Trichoderma fungus of the present invention is a mutant Trichoderma fungus modified so that expression of a polypeptide associated with erythritol assimilation is reduced or eliminated. In another preferred embodiment, the mutant Trichoderma fungus of the present invention is a mutant Trichoderma fungus modified so that expression of a gene associated with erythritol assimilation is reduced or eliminated. As shown in Example 1 below, the following genes have been identified as erythritol assimilation-related genes in Trichoderma fungi. Furthermore, the polypeptides encoded by the following genes are polypeptides associated with erythritol assimilation in Trichoderma fungi. The names of genes in Trichoderma fungi in this specification are identified according to the Ensembl Fungi database (fungi.ensembl.org / Trichoderma_reesei / Info / Index / ). GENE ID SEQ ID:No Estimation function GENE PEPTIDE TRIREDRAFT_122079 1 2 predicted_protein TRIREDRAFT_68466 3 4 glycerone_kinase TRIREDRAFT_68606 5 6 triose-phosphate_isomerase TRIREDRAFT_68585 7 8 galactose-6-phosphate_isomerase

[0026] Therefore, examples of the polypeptides involved in erythritol assimilation whose expression is reduced or eliminated in the mutant Trichoderma of the present invention include the following (A) to (D): (A) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto; (B) a polypeptide consisting of the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 90% identity thereto; (C) a polypeptide consisting of the amino acid sequence of SEQ ID NO:6 or an amino acid sequence having at least 90% identity thereto; and (D) A polypeptide consisting of the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having at least 90% identity thereto. Examples of polypeptides consisting of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 include polypeptides consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, added or inserted relative to the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, respectively.

[0027] Furthermore, examples of erythritol assimilation-related genes whose expression is reduced or eliminated in the mutant Trichoderma of the present invention include the following (a) to (d): (a) a gene consisting of the nucleotide sequence of SEQ ID NO:1 or a nucleotide sequence having at least 90% identity thereto; (b) a gene consisting of the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence having at least 90% identity thereto; (c) a gene consisting of the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least 90% identity thereto; and (d) A gene consisting of the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence having at least 90% identity thereto. Examples of genes consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7 include genes consisting of a nucleotide sequence in which one or several nucleotides have been deleted, substituted, added or inserted relative to the nucleotide sequence of SEQ ID NO: 1, 3, 5 or 7, respectively.

[0028] In one embodiment, the mutant Trichoderma fungus of the present invention may have reduced or eliminated expression of at least one polypeptide selected from the group consisting of (A) to (D). In another embodiment, the mutant Trichoderma fungus of the present invention may have reduced or eliminated expression of at least one gene selected from the group consisting of (a) to (d). In one embodiment, the mutant Trichoderma fungus of the present invention has reduced expression of any one of the genes (a) to (d) to preferably 50% or less, more preferably 70% or less, and even more preferably 90% or less, compared to that of the parent Trichoderma fungus. The gene expression levels can be quantified by real-time PCR.

[0029] Examples of parent Trichoderma fungi of the mutant Trichoderma fungus of the present invention include Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma harzianum, Trichoderma koningii, Trichoderma viride, etc., and preferably Trichoderma reesei and its mutants. For example, Trichoderma reesei QM9414 strain and its mutants, preferably Trichoderma reesei PC-3-7 strain (ATCC66589), Trichoderma reesei PCD-10 strain (FERM P-8172), Trichoderma reesei E1AB1 strain (sometimes called JN13 strain), or their mutants, can be preferably used as the parent Trichoderma fungus. The E1AB1 strain is a Trichoderma reesei PC-3-7 strain in which β-glucosidase (BGL) derived from Aspergillus aculeatus is expressed using the egl1 promoter (see Enzyme Microb Technol, 2016, 82:89-95, and Examples 1 to 3 of WO2013 / 115305).

[0030] Methods for reducing or eliminating the expression of a gene or polypeptide associated with erythritol assimilation in Trichoderma include, for example, deleting or inactivating the erythritol assimilation-associated gene in the Trichoderma cells, inactivating the mRNA transcribed from the gene, or inhibiting translation of the mRNA of the gene into protein, etc. Methods for deleting or inactivating a target gene in Trichoderma cells include removing a part or all of the nucleotide sequence of the target gene from the genome or replacing it with another nucleotide sequence, inserting another polynucleotide fragment into the sequence of the target gene, mutating the transcription initiation region or translation initiation region of the target gene, etc.

[0031] Preferably, a part or the whole of the nucleotide sequence of the target gene is deleted. More specific examples include a method of site-specifically deleting or inactivating an erythritol assimilation-related gene on the genome of a Trichoderma fungal cell, or its transcription initiation region or translation initiation region, and a method of randomly deleting or inactivating a gene in a Trichoderma fungal cell and then selecting a cell having the desired mutation. Specific methods for introducing a mutation into a target region on the genome or removing, substituting or inserting a nucleotide sequence include site-specific mutagenesis, homologous recombination, and SOE (splicing by overlap extension)-PCR (Gene, 1989, 77:61-68). Examples of methods for randomly deleting or inactivating a gene in a cell include a method of randomly cloning an inactivated gene and introducing a DNA fragment into the cell to cause homologous recombination between the inactivated gene and the gene on the genome of the cell, and a method of inducing a mutation by irradiating the cell with ultraviolet light, gamma rays or the like. If necessary, the mutated Trichoderma bacteria are subjected to evaluation of their erythritol assimilation ability or genetic analysis to select those having the desired mutation. The procedure for evaluating erythritol assimilation ability is as described above.

[0032] Methods for inactivating the mRNA of a target gene in a cell include target-specific mRNA inhibition using siRNA. The basic procedure of siRNA is well known to those skilled in the art (see, for example, JP-A-2007-512808), and reagents or kits for siRNA are commercially available. Those skilled in the art can prepare a desired target-specific siRNA and inhibit the target mRNA based on known literature or the manual of a commercially available product.

[0033] The mutant Trichoderma fungus of the present invention can be used for producing a target substance. Typically, the target substance can be produced by using the mutant Trichoderma fungus of the present invention as a host and introducing a gene encoding the target substance or an enzyme involved in the synthesis of the target substance into the host and expressing the gene. In order to promote expression of the introduced gene, the gene is preferably operably linked to a promoter that functions in Trichoderma fungus.

[0034] In a preferred embodiment, the mutant Trichoderma of the present invention is a recombinant Trichoderma in which expression of a polypeptide associated with erythritol assimilation is reduced or eliminated, and a gene encoding a target substance or an enzyme involved in the synthesis thereof and an erythritol-inducible promoter operably linked upstream of the gene have been introduced. The erythritol-inducible promoter is a DNA selected from the following (i) to (ii), and includes a DNA having promoter activity in the presence of erythritol: (i) DNA consisting of any one of the nucleotide sequences of SEQ ID NOs: 13 to 16; and (ii) DNA consisting of a nucleotide sequence having at least 90% identity to any of the nucleotide sequences of SEQ ID NOs: 13 to 16; Examples of DNAs consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequences of SEQ ID NOs: 13 to 16 include DNAs consisting of a nucleotide sequence in which one or several nucleotides have been deleted, substituted, added, or inserted relative to the nucleotide sequences of SEQ ID NOs: 13 to 16, respectively.

[0035] The promoters of SEQ ID NOs: 13 to 16 are promoters derived from Trichoderma reesei discovered by the present inventors, and are promoters of the erythritol assimilation-related genes of SEQ ID NOs: 1 to 4 described above, respectively (Patent Document 1). The promoters of SEQ ID NOs: 13 to 16 are erythritol-inducible promoters discovered for the first time in a fungus belonging to the genus Trichoderma.

[0036] The method for obtaining the erythritol-inducible promoter is not particularly limited, and the promoter can be obtained by a conventional chemical synthesis method or a genetic engineering method. For example, the promoter DNA can be artificially synthesized based on the nucleotide sequences of SEQ ID NOs: 13 to 16. For artificial DNA synthesis, a commercially available DNA synthesis service provided by, for example, GenScript, Inc. can be used. Alternatively, the promoter can be cloned from a Trichoderma fungus such as Trichoderma reesei.

[0037] The erythritol-inducible promoter can also be produced by introducing a mutation into the DNA of the nucleotide sequence of SEQ ID NO: 13 to 16. Examples of the method for introducing the mutation include ultraviolet irradiation and site-directed mutagenesis. An erythritol-inducible promoter can be obtained by selecting from the mutated DNAs those having promoter activity in the presence of erythritol. For example, a reporter gene is operably linked downstream of the mutated DNA, and the expression level of the reporter gene is analyzed in the presence of erythritol, thereby selecting the DNA of the erythritol-inducible promoter.

[0038] By using the above-mentioned technique, it is possible to obtain an erythritol-inducible promoter consisting of the nucleotide sequence of SEQ ID NO:13 to 16 or a nucleotide sequence having at least 90% identity thereto, or an erythritol-inducible promoter consisting of a nucleotide sequence in which one or several nucleotides have been deleted, substituted, added, or inserted relative to the sequence of SEQ ID NO:13 to 16.

[0039] A gene encoding a target substance or an enzyme involved in the synthesis thereof (hereinafter referred to as a target gene) is operably linked to the erythritol-inducible promoter. For example, a DNA fragment containing a target gene and an erythritol-inducible promoter linked upstream of the target gene can be constructed. The DNA fragment may contain a cis-element or a terminator that improves the transcription activity of the promoter, in addition to the promoter and the target gene. Furthermore, the DNA fragment may contain a selection marker gene such as a drug resistance gene or an auxotrophic marker gene. Preferably, the DNA fragment containing the target gene and the erythritol-inducible promoter is an erythritol-inducible gene expression cassette for expressing the target gene. The DNA fragment can be constructed to have restriction enzyme recognition sequences at both ends. The DNA fragment can be introduced into a vector using the restriction enzyme recognition sequences. For example, the vector can be cleaved with a restriction enzyme, and the DNA fragment having a restriction enzyme cleavage sequence at the end can be added thereto, thereby introducing the target gene and the erythritol-inducible promoter into the vector (restriction enzyme method).

[0040] The DNA fragment containing the gene of interest and the erythritol-inducible promoter may be directly introduced into the genome of the host cell. For example, the DNA fragment may be introduced upstream of the gene of interest in the genome of the host cell. Also, for example, a gene expression cassette containing the gene of interest and the erythritol-inducible promoter described above may be introduced into the genome of the host cell.

[0041] Alternatively, by incorporating the erythritol-inducible promoter into an expression vector that enables expression of a target gene, an expression vector that can improve the expression of the target gene at the transcription level can be obtained. In the expression vector, the erythritol-inducible promoter can be operably linked upstream of the target gene. The expression vector may be a vector for introduction into the genome of a host cell, or a vector that is retained outside the genome. It is preferable that the expression vector is replicable in the host cell.

[0042] Examples of vectors include pBluescript II SK(-) (Stratagene), pUC vectors such as pUC18 / 19 and pUC118 / 119 (Takara Bio), pET vectors (Takara Bio), pGEX vectors (GE Healthcare), pCold vectors (Takara Bio), pHY300PLK (Takara Bio), pUB110 (Plasmid, 1986, 15(2):93-103), pBR322 (Takara Bio), pRS403 (Stratagene), pMW218 / 219 (Nippon Gene), pRI vectors such as pRI909 / 910 (Takara Bio), pBI vectors (Clontech), IN3 vectors (Implanta Innovations), pPTR1 / 2 (Takara Bio), pDJB2 (Gene, 1985, 36:321-331), pAB4-1 (Mol Gen Genet, 1987, 206: 71-75), pLeu4 (Gene, 1989, 84: 335-343), pPyr225 (Mol Genet Genomics, 2002, 268: 397-406), pFG1 (Curr Genet, 1990, 18: 447-451), yeast expression vector pNAN8142 (Biosci Biotechnol Biochem, 1996, 60: 383-389), pMA91 (Biosci Biotechnol Biochem, 1998, 62: 1615-1618), and the like.

[0043] The gene of interest introduced into the mutant Trichoderma fungus of the present invention may be a gene encoding a substance of interest to be produced using the mutant Trichoderma fungus of the present invention, or an enzyme involved in the synthesis of the substance. The gene of interest may be a heterologous gene encoding a heterologous expression product, a gene derived from the same species and introduced from outside, a gene encoding an expression product inherent to the mutant Trichoderma fungus that is the host, or a gene encoding any other protein, peptide, nucleic acid, etc. Examples of the substance of interest include enzymes, hormones, cytokines, other physiologically active peptides, transporters, non-coding RNA, etc. Examples of the enzyme include oxidoreductase, transferase, hydrolase, lyase, isomerase, synthetase (ligase or synthetase), etc., and preferred examples include cellulosic biomass decomposition enzymes such as cellulase and hemicellulase, exoglucanase, endoglucanase, β-glucosidase, protease, lipase, mannase, arabinase, galactase, amylase, etc., and more preferably cellulase or hemicellulase. Examples of the hemicellulase include xylanase, β-xylosidase, α-arabinofuranosidase, etc., and among these, xylanase is preferred.

[0044] The recombinant Trichoderma fungus of the present invention can be obtained by introducing an expression vector or DNA fragment containing the target gene and an erythritol-inducible promoter into the host mutant Trichoderma fungus of the present invention using a common transformation method, such as electroporation, transformation, transfection, conjugation, protoplast, particle gun, or Agrobacterium.

[0045] The resulting recombinant Trichoderma fungus is cultured in the presence of erythritol to express the target gene under the control of the erythritol-inducible promoter, thereby producing a target substance, examples of which are as described above.

[0046] The culture conditions for the recombinant Trichoderma fungus are not particularly limited as long as the recombinant Trichoderma fungus can grow and produce a target substance. The medium used for the culture may be either a synthetic medium or a natural medium, so long as it contains components normally required for the growth of Trichoderma fungus and the production of a target substance, such as a carbon source, a nitrogen source, inorganic salts, and vitamins. The concentration of erythritol in the culture is preferably 0.001 to 10% (w / v), more preferably 0.1 to 10% (w / v), as the initial concentration in the medium.

[0047] As the carbon source to be added to the medium, it is desirable to use a cellulase-non-inducing carbon source in order to avoid stimulating the cellulase expression induction process inherent to Trichoderma cells. For example, non-cellulase-inducing carbohydrates such as glucose and fructose, sugar alcohols such as sorbitol, alcohols such as ethanol and glycerol, organic acids such as acetic acid, etc. can be used as carbon sources. In order to produce a target substance while further reducing catabolite repression of cells, cells may be cultured while feeding a non-cellulase-inducing carbon source such as glucose. In this case, it is preferable to dissolve the non-cellulase-inducing carbon source, for example, glucose, in an aqueous solution containing ammonia water or an ammonium salt, which is a nitrogen source, and feed the solution to the medium for culture, from the viewpoint of culture efficiency and suppression of foaming during culture.

[0048] Examples of the nitrogen source include ammonia, ammonium salts such as ammonium sulfate, nitrogen compounds such as amines, and natural nitrogen sources such as peptone and soybean hydrolysate. Examples of inorganic salts include potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, potassium carbonate, etc. Examples of vitamins include biotin and thiamine. Furthermore, if necessary, a substance required for the growth of the recombinant Trichoderma fungus can be added.

[0049] The culture is preferably carried out under aerobic conditions such as shaking culture or aeration and agitation culture. The culture temperature is preferably 10° C. or higher, more preferably 20° C. or higher, more preferably 25° C. or higher, and preferably 50° C. or lower, more preferably 42° C. or lower, more preferably 35° C. or lower. Also, the temperature is preferably 10 to 50° C., more preferably 20 to 42° C., more preferably 25 to 35° C. The pH during culture is 3 to 9, preferably 4 to 5. The culture time is 10 hours to 10 days, preferably 2 to 7 days.

[0050] After the culture, the target substance can be obtained by recovering the target substance from the culture. If necessary, the recovered target substance may be further purified. The method for recovering or purifying the target substance from the culture is not particularly limited, and may be performed according to a known recovery or purification method. For example, the culture may be recovered, and if necessary, a bacterial cell disruption treatment may be performed using ultrasonic waves or pressure, and then the cell components may be removed by decantation, filtration, centrifugation, or the like, and the remaining fraction containing the target substance may be recovered. Alternatively, the target substance can be secreted outside the cells by operably linking a secretion signal peptide that functions in Trichoderma to a gene encoding the target substance.

[0051] If necessary, the collected fraction can be subjected to dialysis, salting out, ion exchange, distillation, solvent extraction, or a combination of these to purify the target substance. In the method for producing a target substance according to the present invention, the cultivation of the recombinant Trichoderma fungus and the collection of the target substance may be carried out by any of a batch system, a semi-batch system, and a continuous system.

[0052] As exemplary embodiments of the present invention, the following substances, manufacturing methods, uses, methods, etc. are further disclosed in this specification, but the present invention is not limited to these embodiments.

[0053] [1] A mutant Trichoderma bacterium having reduced or eliminated erythritol assimilation ability, in which expression of at least one polypeptide selected from the group consisting of the following (A) to (D) is reduced or eliminated: (A) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto; (B) a polypeptide consisting of the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 90% identity thereto; (C) a polypeptide consisting of the amino acid sequence of SEQ ID NO:6 or an amino acid sequence having at least 90% identity thereto; and (D) A polypeptide consisting of the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having at least 90% identity thereto. [2] The mutant Trichoderma sp. according to [1], wherein the expression of at least one gene selected from the group consisting of the following (a) to (d) is reduced or eliminated: (a) a gene consisting of the nucleotide sequence of SEQ ID NO:1 or a nucleotide sequence having at least 90% identity thereto; (b) a gene consisting of the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence having at least 90% identity thereto; (c) a gene consisting of the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least 90% identity thereto; and (d) A gene consisting of the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence having at least 90% identity thereto. [3] Preferably, the mutant Trichoderma bacterium according to [1] or [2] has an erythritol assimilation ability reduced to 50% or less compared to that of the parent Trichoderma bacterium. [4] The mutant Trichoderma bacterium according to [3], wherein the erythritol assimilation ability is defined as an erythritol reduction rate calculated based on the amount of erythritol remaining in a medium for culturing the Trichoderma bacterium, measured by HPLC, 2 hours after adding erythritol to the medium. [5] The mutant Trichoderma fungus according to [2], wherein the expression level of any one of the genes (a) to (d) is reduced to 50% or less compared to that of the parent Trichoderma fungus. [6] The mutant Trichoderma fungus according to any one of [1] to [5], wherein the Trichoderma fungus is Trichoderma reesei or a mutant strain thereof. [7] The mutant Trichoderma fungus according to any one of [1] to [6], preferably having a gene encoding a target substance or an enzyme involved in the synthesis of the target substance and an erythritol-inducible promoter linked upstream of the gene introduced therein. [8] The mutant Trichoderma sp. according to [7], wherein the erythritol-inducible promoter is composed of a DNA selected from the following (i) and (ii): (i) a DNA consisting of any one of the nucleotide sequences of SEQ ID NOs: 13 to 16; and (ii) A DNA consisting of a nucleotide sequence having at least 90% identity to any of the nucleotide sequences of SEQ ID NOs: 13 to 16. [9] The mutant Trichoderma fungus according to [7] or [8], wherein the target substance is preferably an enzyme, a hormone, a cytokine, a physiologically active peptide, a transporter, or a non-coding RNA.

[10] The enzyme, Preferably, it is an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, or a synthetase; More preferably, it is a cellulase, a hemicellulase, an exoglucanase, an endoglucanase, a β-glucosidase, a protease, a lipase, a mannase, an arabinase, a galactase, or an amylase; More preferably, it is a cellulase or a hemicellulase. and The hemicellulase is preferably a xylanase, a β-xylosidase, or an α-arabinofuranosidase. [9] A mutant Trichoderma fungus described in [9].

[11] Cultivating the mutant Trichoderma fungus according to any one of [7] to

[10] above in a medium containing erythritol; and recovering the target substance from the culture obtained by the culture; A method for producing a target substance, comprising:

[12] The method according to

[11] , wherein the medium preferably contains erythritol at an initial concentration of 0.001 to 10% (w / v).

[13] The method according to

[11] or

[12] , wherein the carbon source contained in the medium is a non-cellulase-inducing carbon source.

[14] The method described in

[13] , wherein the cellulase-non-inducing carbon source is preferably glucose, fructose, sugar alcohol, alcohols, or organic acids.

[15] The method according to

[11] or

[12] , wherein a solution of glucose dissolved in an aqueous solution containing ammonia water or an ammonium salt is added to the medium during the culture. EXAMPLES

[0054] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0055] Example 1 Identification of genes related to erythritol assimilation (1) Expression analysis by RNA sequencing Trichoderma reesei (T. reesei) strain PC-3-7 was used to culture spores at a concentration of 1 × 10 5 The bacteria were inoculated into the medium at a concentration of 100 cells / mL and cultured at 28°C with shaking at 220 rpm (PRIS PRXYg-98R). The medium had the following composition: 3% cellulose, 0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 0.1% Bacto Peptone, 0.05% Bacto Yeast extract, 0.1% Tween80, 0.1% Trace element 2, 1.28% diammonium hydrogen citrate, 50 mM tartrate buffer (pH 4.0) (all percentages are w / v%). Trace element 2 was prepared as follows: 6 mg H3BO3, 26 mg (NH4)6Mo7O 24100mg FeCl3·6H2O, 40mg CuSO4·5H2O, 8mg MnCl2·4H2O, and 200mg ZnCl2 were diluted with distilled water to a total volume of 100mL. After culturing for 72 hours under the above conditions, erythritol was added to the medium to a final concentration of 0.2w / v%. After culturing for another 8 hours, the bacteria were harvested. As a control, bacteria were used that had been cultivated under the above conditions for 80 hours in the absence of erythritol.

[0056] Total RNA was extracted from the collected fungi (using the RNeasy Plant Mini Kit from QIAGEN), and the extracted total RNA was subjected to the TruSeq RNA sample Prep v3 kit (Illumina) to construct a next-generation sequencer library (cDNA library). The obtained cDNA library was sequenced by the Miseq system using the Miseq Reagent Kit (300 cycles) (Illumina). The obtained sequence information was mapped to the CDS sequence of T. reesei QM6a obtained from the Ensembl Fungi database (fungi.ensembl.org / Trichoderma_reesei / Info / Index / ) using CLC Genomics Workbench. For each gene, the RPKM (Reads Per Kilobase of exon per Million mapped reads), which is the value obtained by correcting the number of mapped reads by the length of the gene and the total number of reads, was calculated. The expression level of each gene was analyzed based on the obtained RPKM. As a result, four genes were found to have RPKM values ​​that were more than 50 times higher under erythritol-supplemented conditions compared to the control, and were hardly expressed under control conditions: 122079 (TRIREDRAFT_122079), 68466 (TRIREDRAFT_68466), 68606 (TRIREDRAFT_68606), and 68585 (TRIREDRAFT_68585) (Table 1).

[0057] [Table 1]

[0058] (2) Expression analysis by real-time PCR The erythritol specificity of the expression induction of the four genes found in (1) was verified by real-time PCR. For the verification, the T. reesei E1AB1 strain (Enzyme Microb Technol, 2016, 82: 89-95), which was obtained by modifying the T. reesei PC-3-7 strain to highly express AaBGL1 using the egl1 promoter, was used. The E1AB1 strain was cultured at 1 × 10 spores. 5 The bacteria were inoculated at a concentration of 100 cells / mL and cultured at 28°C with shaking at 220 rpm (PRIS PRXYg-98R). The medium had the following composition: 1% glucose, 0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 0.1% Bacto Peptone, 0.05% Bacto Yeast extract, 0.1% Tween80, 0.1% Trace element 2, 1.28% diammonium hydrogen citrate, 50 mM tartrate buffer (pH 4.0) (all percentages are w / v%). The composition of Trace element 2 was as described above in (1). After 48 hours of culture under the above conditions, erythritol, glucose, or sorbitol was added to the medium to a final concentration of 0.2 w / v%. The bacteria were harvested immediately (0 h) and 2 hours (2 h) after the addition of erythritol. As a control, bacteria were cultured for a total of 50 hours without adding any carbon source after 48 hours of culture.

[0059] cDNA was synthesized using RNA extracted from the collected bacteria (TaKaRa PrimeScript TMII 1st strand cDNA Synthesis Kit was used). Real-time PCR analysis was performed using the synthesized cDNA (Agilent Technologies, Brilliant III Ultra-Fast SYBR Green QPCR Master Mixes was used). The primers in Table 2 were used for real-time PCR. The expression level of each gene was determined by relative quantification (ΔΔCt method). The phosphoglycerate kinase gene pgk1 (TRIREDRAFT_21406), a glycolytic enzyme, was used as the normalization gene.

[0060] [Table 2]

[0061] The relative expression levels of each gene to the expression level of the pgk1 gene are shown in Figures 1(A) to (D). Compared to immediately after the addition of erythritol (0 h), the expression levels of the four genes increased significantly 2 hours after the addition (2 h). On the other hand, no increase in the expression levels of the four genes was observed in the control or under glucose or sorbitol addition conditions. This indicated that the expression of the four genes, 122079, 68466, 68606, and 68585, was specifically induced in an erythritol-specific manner. These four genes were presumed to be erythritol assimilation-related genes.

[0062] Example 2 Construction of a plasmid for deleting genes related to erythritol assimilation The following four DNA fragments were prepared by PCR using the genomic DNA of Trichoderma reesei as a template. Fragment 1: DNA containing the 122079 gene (SEQ ID NO: 9), fragment 2: DNA containing the 68466 gene (SEQ ID NO: 10), fragment 3: DNA containing the 68606 gene (SEQ ID NO: 11), and fragment 4: DNA containing the 68585 gene (SEQ ID NO: 12). These DNA fragments were inserted into the HincII restriction enzyme cleavage site of pUC118 (Takara Bio) to construct plasmids pUC-122079, pUC-68466, pUC-68606, and pUC-68585. The following four DNA fragments were prepared using each of the constructed plasmids as a template. Fragment 5: a fragment of about 5.2 kbp amplified using pUC-122079 as a template, fragment 6: a fragment of about 5.0 kbp amplified using pUC-68466 as a template, fragment 7: a fragment of about 4.8 kbp amplified using pUC-68606 as a template, fragment 8: a fragment of about 4.8 kbp amplified using pUC-68585 as a template. As a selection marker for transformation, fragment 9: a region (about 2.7 kbp) from about 1.0 kbp upstream to the ORF and about 0.3 kbp downstream of the pyr4 gene (TRIREDRAFT_74020) was prepared using the genomic DNA of T. reesei as a template. Furthermore, a selection marker cassette for transformation was prepared by binding fragment 10 of about 0.7 kbp and fragment 11 of about 1.0 kbp upstream and downstream of fragment 9, respectively, to remove the selection marker. The gene deletion plasmids pUC-Δ122079-pyr4, pUC-Δ68466-pyr4, pUC-Δ68606-pyr4, and pUC-Δ68585-pyr4 were constructed by combining the prepared transformation selection marker cassette with fragments 5 to 8. The DNA fragments were combined according to the protocol of the In-Fusion HD Cloning kit (Takara Bio), and the primers used are shown in Table 3.

[0063] [Table 3]

[0064] The constructed plasmid was transformed into competent cells E. coli DH5α Competent Cells (Takara Bio), and among the ampicillin-resistant transformants obtained, strains carrying the plasmid with the target gene inserted were selected by colony PCR. The selected transformants were cultured in LB medium supplemented with ampicillin (37°C, 1 day), and the plasmid was recovered and purified from the resulting bacteria using NucleoSpin Plasmid EasyPure (Machley-Nagel).

[0065] Example 3: Preparation of mutant Trichoderma strains lacking erythritol assimilation-related genes The Δpyr4 strain of T. reesei E1AB1 (Enzyme and Microbial Technology, 2016, 82: 89-95) was transformed with the plasmid constructed in Example 2. The introduction was performed by the protoplast PEG method (Biotechnol Bioeng. 2012, 109(1): 92-99). Transformants were selected using the pyr4 gene as a selection marker on selective medium (2% glucose, 1.1 M sorbitol, 2% agar, 0.5% (NH4)2SO4, 0.2% KH2PO4 (pH 5.5), 0.06% CaCl2·2H2O, 0.06% CsCl2, 0.06% MgSO4·7H2O, and 0.1% Trace element 1 (all percentages are w / v%). Trace element 1 was prepared as follows: 0.5 g FeSO4·7H2O, 0.2 g CoCl2, 0.16 g MnSO4·H2O, 0.14 g ZnSO4·7H2O was diluted to 100 mL with distilled water. The selected transformants were stabilized by subculture, and then strains that stably retained the target gene were selected by colony PCR. The selected mutant strains were named 122079 deletion strain (Δ122079), 68466 deletion strain (Δ68466), 68606 deletion strain (Δ68606), and 68585 deletion strain (Δ68585), respectively.

[0066] Example 4 Verification of erythritol assimilation ability of mutant Trichoderma strains (1) Cultivation of mutant strains The mutant Trichoderma strain prepared in Example 3 was cultured to verify its erythritol assimilation. As a preculture, 50 mL of medium was placed in a 500 mL flask, and 1 × 10 spores of the strain prepared in Example 2 were added. 5 The bacteria were inoculated at a concentration of 100 cells / mL and cultured at 28°C with shaking at 220 rpm (Pris Co., Ltd., PRXYg-98R). The medium composition was as follows: 1% glucose, 0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 0.1% Bacto Peptone, 0.05% Bacto Yeast extract, 0.1% Tween 80, 0.1% Trace element 2, 50 mM tartrate buffer (pH 4.0) (all percentages are w / v%). Trace element 2 was prepared as follows: 6 mg H3BO3, 26 mg (NH4)6Mo7O 24 100mg FeCl3·4H2O, 100mg FeCl3·6H2O, 40mg CuSO4·5H2O, 8mg MnCl2·4H2O, and 200mg ZnCl2 were diluted with distilled water to make 100mL.

[0067] After 48 hours of pre-culture, the main culture was carried out using a BTR-25NA1S-8M (Biot) jar fermenter. The above pre-culture solution was inoculated at 1% (v / v%) and cultured for 24 hours. Glucose was used as the carbon source at a concentration of 2%, and the other medium components were as follows: 0.42% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 0.1% Bacto Peptone, 0.05% Bacto Yeast extract, 0.1% Tween 80, 0.1% Trace element 2, 0.2% Antifoam PE-L. The jar fermenter settings were as follows: temperature: 28°C, aeration: 0.5vvm, pH: 4.5 (adjusted with 5% ammonia water), stirring speed: 700 rpm. After 24 hours of culture, erythritol was added to a final concentration of 0.2%, and the culture supernatant and bacteria were collected 0, 2, 4, and 8 hours after the addition.

[0068] (2) Quantitative determination of erythritol in culture supernatant 1 mL of the culture medium collected in (1) was centrifuged at 15,000 rpm at 4°C for 5 minutes, and the supernatant was passed through a filter DISMIC-13CP (0.2 μm cellulose acetate membrane, ADVANTEC) to remove the bacterial cells. The culture supernatant to be subjected to HPLC was appropriately diluted with 37 mM sulfuric acid, and insoluble matter was removed using an AcroPrep 96 filter plate (0.2 μm GHP membrane, Nippon Pole). The analytical conditions for HPLC are shown below. Analyzer: Prominence-I LC-2030C 3D plus (Shimadzu) Analytical column: ICSep ICE ION 300 7.8mmI.D.×300mm(CONCISE SEPARATIONS) Eluent: 0.01N sulfuric acid Flow rate: 0.5mL / min Column temperature: 50℃ A calibration curve was prepared by similarly analyzing erythritol solutions of known concentrations, and the erythritol concentration in the culture supernatant was calculated based on the prepared calibration curve.

[0069] In the case of the E1AB1 strain, erythritol completely disappeared from the culture supernatant 4 hours after the addition of erythritol, whereas in all of the mutants, the 122079 deletion strain (Δ122079), the 68466 deletion strain (Δ68466), the 68606 deletion strain (Δ68606), and the 68585 deletion strain (Δ68585), the erythritol assimilation rate was lower than that of the E1AB1 strain from 2 hours after the addition, and erythritol remained in the culture supernatant even 8 hours after the addition in the Δ122079, Δ68606, and Δ68585 strains (Figure 2). This indicates that the erythritol assimilation ability of filamentous fungi is reduced by deleting the 122079 gene, the 68466 gene, the 68606 gene, or the 68585 gene.

[0070] Example 5 Measurement of intracellular erythritol accumulation in deletion mutant strains The amount of erythritol accumulated in the cells of the mutant strains prepared in Example 3 was measured using a gas chromatography-mass spectrometer (GC-MS). 7 mL of cold ethanol was added to 5 mL of the culture solution collected 4 hours and 8 hours after the addition of erythritol in Example 4 (1), and the mixture was quickly cooled to -30°C. The cooled culture solution was centrifuged at 5,000g and 4°C for 5 minutes, the supernatant was removed, and 5 mL of cold 60% methanol was added and suspended to wash the precipitated bacteria. After washing the bacteria twice, the bacteria were frozen at -80°C. The frozen bacteria were freeze-dried. The freeze-dried bacteria were disrupted using a multi-beads shocker (Yasui Kikai), and 5 mg of the disrupted bacteria was added with 0.5 mL of methanol containing 37 μM adipic acid as an internal standard and thoroughly suspended, and 0.5 mL of ice-cold chloroform and 0.2 mL of water were added and thoroughly stirred. The mixture was centrifuged at 14,000×g at 4° C. for 5 minutes, and 0.5 mL of the supernatant was collected. The resulting supernatant was dried using a centrifugal evaporator.

[0071] 20μL of pyridine containing 40mg / mL methoxyamine was added to the dried sample and reacted at 30℃ and 800rpm for 90 minutes, then 80μL of MSTFA+1% TMCS was added and reacted at 37℃ and 800rpm for 30 minutes. After the reaction, the sample was cooled to room temperature in the dark and then subjected to GC-MS analysis. The GC-MS analysis conditions are shown below. Analyzer: Agilent 7890A GC system (Agilent Technologies) Column: DB-5MS+DG 30m×0.25mm×0.25μm (Agilent Technologies) Vaporization chamber temperature: 250℃ Injection method: Split injection (split ratio 50:1) Oven temperature: 60°C for 3.5 minutes, then increase temperature at 10°C / min, and hold at 325°C for 10 minutes Column flow rate: Helium gas 1.14mL / min Interface: 250℃ Ion source: 200℃ A calibration curve was prepared by adding 100 μL of 100 μM adipic acid solution to an erythritol solution of known concentration and analyzing it in the same manner, and the amount of erythritol accumulated in the bacterial cells was calculated based on the calibration curve.

[0072] The intracellular erythritol concentration (nmol / mg-dry cell) per mg of dry cells of the E1AB1 strain and the deletion mutant strain was determined. In the E1AB1 strain, erythritol was hardly detected intracellularly 4 hours after the addition of erythritol, and the value was below the detection limit at 8 hours, indicating that the erythritol taken up into the cells was rapidly assimilated (Table 4). On the other hand, in the deletion mutant strain, intracellular accumulation of erythritol was observed 4 hours after the addition of erythritol, and even at 8 hours, erythritol was detected in the cells at about 100 times more than that of the E1AB1 strain, indicating that the erythritol taken up into the cells was not assimilated and accumulated within the cells (Table 4). These results demonstrate that the amount of erythritol accumulated within the cells can be increased by deleting the 122079 gene, 68466 gene, 68606 gene, or 68585 gene.

[0073] [Table 4]

[0074] Example 6 Expression analysis of erythritol assimilation-related genes in deletion mutants For the mutant Trichoderma strains prepared in Example 3, the effect of intracellular erythritol accumulation on the expression levels of erythritol assimilation-related genes was examined by real-time PCR. cDNA was synthesized using RNA extracted from the fungus recovered in Example 4(1) (TaKaRa Co., Ltd. PrimeScript TMII 1st strand cDNA Synthesis Kit was used). Real-time PCR analysis was performed using the synthesized cDNA (Agilent Technologies, Brilliant III Ultra-Fast SYBR Green QPCR Master Mixes was used). The primers used for real-time PCR were listed in Table 2. The expression level of each gene was determined by relative quantification (ΔΔCt method). The phosphoglycerate kinase gene pgk1 (TRIREDRAFT_21406), a glycolytic enzyme, was used as the normalization gene.

[0075] The relative expression levels of each gene with respect to the expression level of the pgk1 gene are shown in Figures 3 to 6. In the parent strain E1AB1, an increase in the expression levels of the four erythritol assimilation-related genes was observed 2 hours after the addition of erythritol compared to immediately after the addition (0 h), but the expression levels of all genes were significantly reduced from 4 hours after the addition compared to 2 hours after the addition. On the other hand, in the deletion mutants Δ122079, Δ68466, Δ68606, and Δ68585, an increase in the expression of the four genes was confirmed 2 hours after the addition, and furthermore, unlike the E1AB1 strain, they showed very high expression levels even 4 and 8 hours after the addition. This indicates that the expression level and expression persistence of the erythritol assimilation-related genes are improved by deleting the 122079 gene, 68466 gene, 68606 gene, or 68585 gene.

[0076] Example 7: Construction of recombinant strains carrying erythritol-inducible promoter-linked genes The mutant Trichoderma strain prepared in Example 3 was introduced with the xylanase XYN3 gene linked to an erythritol-inducible promoter to prepare a recombinant strain expressing XYN3 in an erythritol-inducible manner.

[0077] (1) Construction of plasmid for gene transfer The following six DNA fragments were prepared by PCR using the genomic DNA of T. reesei as a template. Fragment 12: Approximately 1.0 kbp of the promoter region upstream of the 122079 gene (SEQ ID NO: 13) Fragment 13: Approximately 0.8 kbp of the promoter region upstream of the 68466 gene (SEQ ID NO: 14) Fragment 14: Approximately 0.8 kbp of the promoter region upstream of the 68606 gene (SEQ ID NO: 15) Fragment 15: Approximately 0.8 kbp of the promoter region upstream of the 68585 gene (SEQ ID NO: 16) Fragment 16: The region from the coding region of the xylanase gene xyn3 (TRIREDRAFT_120229) to 0.5 kbp downstream Fragment 17: The region from about 1.0 kbp upstream to the ORF and about 0.3 kbp downstream of the pyr4 gene (TRIREDRAFT_74020) (about 2.4 kbp) The primers used to amplify the fragments are shown in Table 5.

[0078] [Table 5]

[0079] Fragments 16 and 17 were ligated to prepare XYN3-pyr4. XYN3-pyr4 was then ligated to each of fragments 1 to 4. The resulting fragments were inserted into the HincII restriction enzyme cleavage site of pUC118 (Takara Bio) to construct four vectors: pUC-P122079-XYN3-pyr4, pUC-P68466-XYN3-pyr4, pUC-P68606-XYN3-pyr4, and pUC-P68585-XYN3-pyr4. The DNA fragments were ligated according to the protocol of the In-Fusion HD Cloning kit (Takara Bio). The constructed plasmid was transformed into competent cells E. coli DH5α Competent Cells (Takara Bio), and among the transformants obtained as ampicillin-resistant strains, strains carrying the plasmid into which the target gene had been inserted were selected by colony PCR. The selected transformants were cultured in LB medium supplemented with ampicillin (37°C, 1 day), and plasmids were recovered and purified from the resulting bacteria using NucleoSpin Plasmid EasyPure (Machrey-Nagel).

[0080] (2) Construction of recombinant Trichoderma strains expressing XYN3 In order to perform transformation again on the mutant Trichoderma strains prepared in Example 3, strains that acquired 5-fluoroorotic acid (5-FOA) resistance and grew were selected again using PDA medium containing 0.2% 5-FOA monohydrate. Δ122079, Δ68466, Δ68606, and Δ68585 were each plated on a 5-FOA-containing medium, and the grown strains were obtained as Δ122079Δpyr4, Δ68466Δpyr4, Δ68606Δpyr4, and Δ68585Δpyr4. The plasmid prepared in (1) was introduced into the obtained strains and E1AB1Δpyr4 by the protoplast PEG method. Transformants were selected using the pyr4 gene as a selection marker on selective medium (2% glucose, 1.1 M sorbitol, 2% agar, 0.5% (NH4)2SO4, 0.2% KH2PO4 (pH 5.5), 0.06% CaCl2·2H2O, 0.06% CsCl2, 0.06% MgSO4·7H2O, 0.1% trace element 1 (all percentages are w / v%)). The selected transformants were stabilized by subculture, and then strains that stably retained the target gene were selected by colony PCR.

[0081] Example 8. Production of target protein using recombinant strains (1) Production of XYN3 by culturing recombinant strains The recombinant strains derived from Δ122079 and E1AB1 prepared in Example 7 were cultured to produce proteins by erythritol induction. For the culture, 50 mL of medium was placed in a 500 mL flask, and 1 × 10 spores of the recombinant strains prepared in Example 7 were added. 5The cells were inoculated at a concentration of 100 cells / mL and cultured at 28°C with shaking at 220 rpm (PRIS PRXYg-98R). The medium had the following composition: 1% glucose, 0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 0.1% Bacto Peptone, 0.05% Bacto Yeast extract, 0.1% Tween80, and 0.1% Trace element 1. After 48 hours of culture, erythritol was added to the medium to a final concentration of 0.2 w / v%, and the culture medium was collected. After further culture for 4 and 24 hours, the culture medium was collected again.

[0082] (2) XYN3 activity measurement The xylanase (XYN3) activity in the culture medium recovered in (1) was measured by the pNP (p-Nitrophenol) method. The culture supernatant was diluted to prepare an enzyme solution. 1 mM pNP-β-Xylobioside solution (50 mM Na-acetate buffer, pH 5.0) was used as the substrate solution. 80 μL of the substrate solution was added to 20 μL of the enzyme solution, and the reaction was stopped by adding 100 μL of 1 M Na2Co3 solution. The absorbance at 420 nm was then measured. A calibration curve was prepared by performing the same procedure using p-nitrophenol. The amount of enzyme that liberates 1 μmol of pNP per minute was defined as 1 U, and the activity of XYN3 in each culture medium was calculated. As a result, in the 122079 promoter-inducible XYN3 expression strain (E1AB1P122079), 68466 promoter-inducible XYN3 expression strain (E1AB1P68466), and 68585 promoter-inducible XYN3 expression strain (E1AB1P68585) using the E1AB1 strain as a host, the XYN3 activity was improved 4 hours after the addition of erythritol compared to 0 hours after the addition, but the activity at 24 hours was equivalent to or slightly higher than that at 4 hours (Table 6).

[0083] On the other hand, in the 122079 promoter-inducible XYN3 expression strain (Δ122079P122079), 68466 promoter-inducible XYN3 expression strain (Δ122079P68466), and 68585 promoter-inducible XYN3 expression strain (Δ122079P68585) using Δ122079 as a host, the XYN3 activity after 4 hours of erythritol addition was higher than that of the promoter-inducible XYN3 expression strain using E1AB1 as a host, and furthermore, the activity after 24 hours was about 2.5 times higher than that after 4 hours (Table 6). The 68606 promoter-inducible XYN3 expression strain (Δ122079P68606) also showed an increase in XYN3 activity by the addition of erythritol, although the increase rate was small. Furthermore, 68466 promoter-inducible XYN3 expression strains (Δ68466P68466, Δ68606P68466, and Δ68585P68466, respectively) using Δ68466, Δ68606, and Δ68585 as hosts were cultured by the method of Example 9, and the XYN3 activity in the culture supernatant was measured. As a result, the XYN3 activity was higher than that of the XYN3 expression strain (E1AB1P68466) using E1AB1 as a host (Table 7). Furthermore, in all of the deletion mutants Δ122079, Δ68466, Δ68606, and Δ68585, it was confirmed that the transcription of genes linked to each of the four erythritol-inducible promoters P122079, P68466, P68606, and P68585 was induced in response to the addition of erythritol, and the expression was maintained for a long period of time. Therefore, it was considered that by linking a gene of interest downstream of the erythritol-inducible promoter in the deletion mutant, it would be possible to induce transcription of the gene of interest for a long period of time in the presence of erythritol.The above results demonstrated that the erythritol assimilation-related gene deletion mutant exhibited high protein productivity when protein expression was induced using an erythritol-inducible promoter.

[0084] [Table 6]

[0085] [Table 7]

Claims

1. A mutant Trichoderma fungus having reduced or eliminated erythritol assimilation ability, The expression of at least one polypeptide selected from the group consisting of the following (A) to (D) is reduced or eliminated: (A) a polypeptide consisting of the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 90% identity thereto; (B) a polypeptide consisting of the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 90% identity thereto; (C) a polypeptide consisting of the amino acid sequence of SEQ ID NO:6 or an amino acid sequence having at least 90% identity thereto; and (D) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 90% identity thereto; and a gene encoding a substance of interest or an enzyme involved in the synthesis of the substance of interest and an erythritol-inducible promoter linked upstream of the gene are introduced into the vector. Mutant Trichoderma spp.

2. The mutant Trichoderma fungus according to claim 1, wherein expression of at least one gene selected from the group consisting of the following (a) to (d) is reduced or eliminated: (a) a gene consisting of the nucleotide sequence of SEQ ID NO:1 or a nucleotide sequence having at least 90% identity thereto; (b) a gene consisting of the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence having at least 90% identity thereto; (c) a nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having at least 90% identity thereto; A gene consisting of a nucleic acid sequence; and (d) A gene consisting of the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence having at least 90% identity thereto.

3. The mutant Trichoderma bacterium according to claim 1, wherein the erythritol assimilation ability is reduced to 50% or less compared to that of the parent Trichoderma bacterium.

4. A mutant Trichoderma fungus as described in claim 2, having an erythritol assimilation ability reduced to 50% or less compared to that of the parent Trichoderma fungus.

5. The mutant Trichoderma fungus according to claim 1 , wherein the Trichoderma fungus is Trichoderma reesei or a mutant thereof.

6. The mutant Trichoderma fungus described in claim 2, wherein the Trichoderma fungus is Trichoderma reesei or a mutant strain thereof.

7. The mutant Trichoderma fungus according to claim 1 , wherein the erythritol-inducible promoter is a DNA selected from the following (i) and (ii): (i) DNA consisting of any of the nucleotide sequences of SEQ ID NOs: 13 to 16; and (ii) A DNA consisting of a nucleotide sequence having at least 90% identity to any one of the nucleotide sequences of SEQ ID NOs: 13 to 16.

8. The mutant Trichoderma fungus according to claim 2, wherein the erythritol-inducible promoter is composed of a DNA selected from the following (i) and (ii): (i) DNA consisting of any of the nucleotide sequences of SEQ ID NOs: 13 to 16; and (ii) A DNA consisting of a nucleotide sequence having at least 90% identity to any one of the nucleotide sequences of SEQ ID NOs: 13 to 16.

9. The mutant Trichoderma fungus according to claim 4, wherein the erythritol-inducible promoter is composed of a DNA selected from the following (i) and (ii): (i) DNA consisting of any of the nucleotide sequences of SEQ ID NOs: 13 to 16; and (ii) A DNA consisting of a nucleotide sequence having at least 90% identity to any one of the nucleotide sequences of SEQ ID NOs: 13 to 16.

10. The mutant Trichoderma fungus described in claim 9, wherein the Trichoderma fungus is Trichoderma reesei or a mutant strain thereof.

11. Cultivating the mutant Trichoderma fungus according to any one of claims 1 to 10 in a medium containing erythritol; and recovering the target substance from the culture obtained by the culture; A method for producing a target substance, comprising:

12. The method according to claim 11, wherein the medium contains erythritol at an initial concentration of 0.001 to 10% (w / v).

13. The method of claim 11 , wherein the carbon source contained in the medium is a non-cellulase-inducing carbon source.

Images