Erythritol-inducible promoter and method for producing target substance using same

Abstract

To provide the production of substances of interest in cells of the genus Trichoderma without promoting cellulase production.SOLUTION: Disclosed is an erythritol-inducible promoter comprising DNA selected from (a) DNA comprising a specific nucleotide sequence; (b) DNA comprising a nucleotide sequence having at least 90% identity to the specific nucleotide sequence; (c) DNA comprising the specific nucleotide sequence with deletion, substitution, addition or insertion of one or a few nucleotides therein. In the genus Trichoderma bacteria, the erythritol-inducible promoter can induce the expression of a gene of substance of interest without promoting intrinsic cellulase induction-expression process. Thus, the invention allows for production of a substance of interest of high purity in the genus Trichoderma bacteria.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an erythritol-inducible promoter and a method for producing a target substance using the promoter. [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). Non-Patent Document 2 describes the use of Trichoderma reesei as a microorganism for the production of heterologous proteins derived from humans and other microorganisms, taking advantage of its high protein production capacity.

[0003] To date, constitutive expression promoters such as the promoter of the enolase gene eno1 and the promoter of the translation elongation factor tef1 have been reported as promoters for inducing the expression of target protein genes in Trichoderma (Non-Patent Document 3). However, these promoters do not have a high ability to induce gene expression, and are not sufficient as promoters for expressing target substances.

[0004] Trichoderma promoters with higher gene expression induction capabilities include the promoters of the cellulase genes cbh1 and cbh2 and the promoter of the xylanase gene xyn3 (Non-Patent Document 3). However, because these promoters are activated under cellulase production conditions, activation of these promoters simultaneously expresses multiple cellulase genes within the cells of Trichoderma fungi, resulting in high cellulase production, making it impossible to produce a single target protein. Cellulase production can be suppressed by deleting the cellulase gene in Trichoderma fungi, but it is extremely difficult to delete all of the multiple cellulase genes on the genome. Furthermore, deleting a major cellulase gene significantly impairs Trichoderma fungi's growth under cellulase production conditions. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Chemistry and Biology, 2012, 50(8):592-599, 2012 [Non-patent document 2] Appl Biochem Biotechnol, 2011, 165(5-6):1169-77 [Non-patent document 3] Front Bioeng Biotechnol, 2018, 11(6):Article 135, doi:10.3389 / fbioe.2018.00135 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides an erythritol-inducible promoter, an expression vector containing the promoter, a gene expression cassette and a transformed Trichoderma fungal cell, as well as a method for producing a target substance using the transformed Trichoderma fungal cell. [Means for solving the problem]

[0007] In one aspect, the present invention provides an erythritol-inducible promoter comprising a DNA selected from the following (a) to (c): (a) DNA consisting of any one of the nucleotide sequences of SEQ ID NOs: 1 to 4; (b) DNA consisting of a nucleotide sequence having at least 90% identity to any of the nucleotide sequences of SEQ ID NOs: 1 to 4; and (c) DNA consisting of a nucleotide sequence in which one or several nucleotides have been deleted, substituted, added, or inserted relative to any of the nucleotide sequences of SEQ ID NOs: 1 to 4. In another aspect, the present invention provides an expression vector comprising the erythritol-inducible promoter. In another aspect, the present invention provides an erythritol-inducible gene expression cassette comprising a gene encoding a target substance or an enzyme involved in the synthesis of the target substance and the erythritol-inducible promoter. In another aspect, the present invention provides a transformed Trichoderma fungal cell comprising the expression vector or the gene expression cassette. In another aspect, the present invention provides a method for producing a The present invention also provides a method for producing a target substance, which comprises culturing the transformed Trichoderma cells in a medium containing erythritol. [Effects of the Invention]

[0008] The erythritol-inducible promoter provided by the present invention enables the induction of gene expression of a target substance in Trichoderma fungi without promoting the cellulase expression induction process inherent to the cells, thereby enabling the production of a target substance with higher purity in Trichoderma fungi. [Brief explanation of the drawings]

[0009] [Figure 1] Relative expression levels of 122079 genes after the addition of erythritol, glucose, or sorbitol. 0h: immediately after addition, 2h: 2 hours after addition. [Figure 2] Relative expression levels of 68466 genes after the addition of erythritol, glucose, or sorbitol. 0h: immediately after addition, 2h: 2 hours after addition. [Figure 3] Relative expression level of 68606 genes after addition of erythritol, glucose, or sorbitol. 0h: immediately after addition, 2h: 2 hours after addition. [Figure 4] Relative expression levels of 68585 genes after the addition of erythritol, glucose, or sorbitol. 0h: immediately after addition, 2h: 2 hours after addition. DETAILED DESCRIPTION OF THE INVENTION

[0010] All patents, non-patent documents, and other publications cited herein are hereby incorporated by reference in their entirety.

[0011] Herein, 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 analysis using the homology analysis program of the genetic information processing software GENETYX Ver. 12, with the unit size to compare (ktup) set to 2.

[0012] 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.

[0013] 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 1 to 10, more preferably 1 to 6, even more preferably 1 to 3, and even more preferably 1 or 2 nucleotides have been deleted, substituted, added, or inserted. As used herein, "addition" of a nucleotide includes addition of nucleotides to one or both ends of a sequence.

[0014] As used herein, "upstream" and "downstream" in relation to a gene refer to the upstream and downstream in 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, respectively, on the DNA sense strand. For example, a "promoter linked upstream of a gene" means that a promoter is present on the 5' side of the gene on the DNA sense strand.

[0015] As used herein, the term "operably linked" between a gene and a regulatory region such as a promoter means that the gene and regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. Procedures for "operably linking" a gene with a regulatory region are well known to those skilled in the art.

[0016] As used herein, the term "native" when used with respect to a cellular function, property, or trait 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 that the function, property, or trait is not inherently present in the cell but is introduced from outside. For example, "exogenous" nucleotides or DNA are nucleotides or DNA introduced into a cell from outside. The 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).

[0017] As used herein, "promoter activity" refers to the activity of promoting 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 reporter gene product produced. Examples of reporter genes include genes encoding enzymes that act on chromogenic substrates, such as the β-galactosidase (LacZ) gene, the β-glucuronidase (GUS) gene, the luciferase gene, the β-lactamase gene, and the EtbC (2,3-dihydroxyethylbenzene 1,2-dioxygenase) gene, as well as genes encoding fluorescent proteins, such as the GFP (Green Fluorescent Protein) gene. Alternatively, promoter activity can be confirmed by measuring the expression level of mRNA transcribed from the reporter gene using sequencing, quantitative RT-PCR, or other methods.

[0018] 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 the expression of mRNA of a target gene (i.e., a gene to which the promoter is operably linked) in the presence of erythritol at a level at least 40-fold, preferably at least 50-fold, and more preferably at least 100-fold higher than in the absence of erythritol or in the presence of cellulose, glucose, or sorbitol. More specifically, the term refers to a promoter that can induce the expression of mRNA of a target gene to the above level after 8 hours in the presence of 0.2 w / v% erythritol.

[0019] It is desirable to produce a target substance in Trichoderma fungi without promoting cellulase production inherent to the cells. The present inventors have identified a gene from Trichoderma fungi that is barely expressed in the absence of erythritol but whose expression is significantly enhanced with the addition of erythritol. Erythritol is a sugar alcohol found in fruits, mushrooms, and the like. In conventional cultures of Trichoderma fungi for cellulase production, cellulose and glucose are primarily used as carbon sources, with erythritol being substantially unused. Furthermore, as shown in the Examples below, erythritol does not induce the expression of cellulosic biomass degrading enzymes inherent to the cells (see Table 4). Therefore, by using erythritol as an inducer and expressing the target substance gene using the promoter of these genes, it is possible to produce a target substance in Trichoderma fungi without promoting the cellulase expression induction process inherent to the cells. The present invention enables the production of a target substance with higher purity in Trichoderma fungi.

[0020] The erythritol-inducible promoter provided by the present invention (hereinafter also simply referred to as "the promoter of the present invention") is a DNA selected from the following (a) to (c), and includes a DNA that has promoter activity in the presence of erythritol: (a) DNA consisting of any one of the nucleotide sequences of SEQ ID NOs: 1 to 4; (b) DNA consisting of a nucleotide sequence having at least 90% identity to any of the nucleotide sequences of SEQ ID NOs: 1 to 4; and (c) DNA consisting of a nucleotide sequence in which one or several nucleotides have been deleted, substituted, added, or inserted relative to any of the nucleotide sequences of SEQ ID NOs: 1 to 4.

[0021] The promoters represented by SEQ ID NOs: 1 to 4 are promoters derived from Trichoderma reesei. As mentioned above, erythritol has not been used in conventional cultivation of Trichoderma fungi, and therefore, it was not previously known that the promoters represented by SEQ ID NOs: 1 to 4 were erythritol-inducible promoters. The promoters represented by SEQ ID NOs: 1 to 4 are erythritol-inducible promoters that have been discovered for the first time in Trichoderma fungi.

[0022] The promoter of the present invention can be obtained by any method, including conventional chemical synthesis or genetic engineering. For example, the promoter DNA of the present invention can be artificially synthesized based on the nucleotide sequences of SEQ ID NOS: 1 to 4. For artificial DNA synthesis, commercially available DNA synthesis services provided by, for example, GenScript, Inc. can be used. Alternatively, the promoter of the present invention can be cloned from Trichoderma species, such as Trichoderma reesei.

[0023] The promoter of the present invention can also be produced by introducing a mutation into the DNA of the nucleotide sequence of SEQ ID NO: 1 to 4. Examples of the method for introducing the mutation include ultraviolet irradiation and site-directed mutagenesis. Examples of the method for site-directed mutagenesis include a method using splicing overlap extension (SOE) PCR (Gene, 1989, 77:61-68), the ODA method (Gene, 1995, 152:271-276), and the Kunkel method (Proc. Natl. Acad. Sci. USA, 1985, 82(2):488-492). Alternatively, the promoter can be produced using the Site-Directed Mutagenesis System Mutan-SuperExpress Km Kit (Takara Bio Inc.), Transformer TM Commercially available site-directed mutagenesis kits, such as the Site-Directed Mutagenesis Kit (Clonetech) and the KOD-Plus-Mutagenesis Kit (Toyobo), can also be used. The promoter of the present invention can be obtained by selecting from the mutated DNAs those that have promoter activity in the presence of erythritol. For example, a reporter gene can be operably linked downstream of the mutated DNA, and the expression level of the reporter gene can be analyzed in the presence of erythritol to select DNA of an erythritol-inducible promoter.

[0024] Alternatively, methods for deleting, substituting, adding, or inserting nucleotides into a nucleotide sequence are described, for example, in Dieffenbach et al. (Cold Spring Harbar Laboratory Press, New York, 581-621, 1995).

[0025] By using the above-mentioned techniques, it is possible to obtain an erythritol-inducible promoter consisting of the nucleotide sequence of SEQ ID NOs: 1 to 4, 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 NOs: 1 to 4.

[0026] The promoter of the present invention has the function of controlling the expression of genes located downstream thereof. By using the promoter of the present invention, a DNA fragment having an expression control region with excellent transcriptional activity can be obtained. For example, a DNA fragment containing a gene of interest and the promoter of the present invention operably linked upstream of the gene of interest can be constructed. In addition to the promoter of the present invention and the gene of interest, the DNA fragment may also contain a cis-element or terminator that improves the transcriptional activity of the promoter. Furthermore, the DNA fragment may also contain a selectable marker gene such as a drug resistance gene or an auxotrophic marker gene. Preferably, the DNA fragment containing the gene of interest and the promoter of the present invention is an erythritol-inducible gene expression cassette for expressing the gene of interest.

[0027] The DNA fragment containing the promoter of the present invention can be constructed so as to have restriction enzyme recognition sequences at both ends. The promoter of the present invention can be introduced into a vector using the restriction enzyme recognition sequences. For example, the promoter of the present invention can be introduced into a vector by cleaving the vector with a restriction enzyme and adding a DNA fragment containing the promoter of the present invention and having a restriction enzyme cleavage sequence at its end (restriction enzyme method).

[0028] A DNA fragment containing the promoter of the present invention may be directly introduced into the genome of a host cell. For example, a DNA fragment containing the promoter of the present invention may be introduced upstream of a gene of interest in the genome of a host cell. Alternatively, for example, a gene expression cassette containing the aforementioned gene of interest and the promoter of the present invention may be introduced into the genome of a host cell.

[0029] Alternatively, by incorporating the promoter of the present invention into an expression vector that enables expression of a target gene, an expression vector that can improve expression of the target gene at the transcription level can be obtained. In the expression vector, the promoter of the present invention can be operably linked upstream of DNA encoding the target gene. The expression vector having the promoter of the present invention may be a vector for introduction into the genome of a host cell, or a vector that is maintained outside the genome. Those that can replicate within the host cell are preferred.

[0030] 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), and 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 vectors pNAN8142 (Biosci Biotechnol Biochem, 1996, 60:383-389), pMA91 (Biosci Biotechnol Biochem, 1998, 62:1615-1618), and the like.

[0031] In the expression vector or DNA fragment, the target gene placed downstream of the promoter of the present invention is not particularly limited. For example, the target gene is a gene encoding a target substance or an enzyme involved in the synthesis of the target substance. The target gene may be a heterologous gene encoding a heterologous expression product, a homologous gene introduced from the outside, a gene encoding an expression product inherent to the host cell, or a gene encoding any other protein, peptide, nucleic acid, etc. Examples of target substances include enzymes, hormones, cytokines, other physiologically active peptides, transporters, non-coding RNA, etc. Examples of enzymes include oxidoreductases, transferases, hydrolases, lyases, isomerases, and synthetases (ligases or synthetases). Preferred examples include cellulosic biomass decomposition enzymes such as cellulase and hemicellulase, exoglucanases, endoglucanases, β-glucosidases, proteases, lipases, mannases, arabinases, galactases, and amylases, with cellulases and hemicellulases being more preferred. Examples of hemicellulases include xylanases, β-xylosidases, and α-arabinofuranosidases, with xylanases being preferred.

[0032] The transformant of the present invention can be obtained by introducing an expression vector or DNA fragment containing the promoter of the present invention into a host cell using a common transformation method, such as electroporation, transformation, transfection, conjugation, protoplast method, particle gun method, Agrobacterium method, etc.

[0033] The host cells into which the vector or DNA fragment is introduced are not particularly limited as long as the promoter of the present invention can function as a promoter in the cells, but are preferably cells of the genus Trichoderma. Other examples of host cells include cells of filamentous fungi such as those of the genera Aspergillus, Penicillium, Neurospora, Fusarium, Chrysosporium, Humicola, Emericella, Hypocrea, Acremonium, Chrysosporium, Myceliophthora, Piromyces, Talaromyces, Thermoascus, and Thielavia.

[0034] Examples of the Trichoderma fungus include Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma harzianum, Trichoderma koningii, and Trichoderma viride, and preferably Trichoderma reesei and its mutants. For example, Trichoderma reesei QM9414 and its mutants, preferably Trichoderma reesei PC-3-7 (ATCC66589), Trichoderma reesei PCD-10 (FERM P-8172), Trichoderma reesei E1AB1 (sometimes referred to as JN13), or their mutants, can be used as host cells. 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).

[0035] The transformant of the present invention can be used to produce a target substance. For example, a transformant containing an expression vector or a DNA fragment having a gene encoding a target substance or an enzyme involved in the synthesis of the target substance and a promoter of the present invention operably linked upstream of the gene is cultured in the presence of erythritol to express the gene under the control of the promoter of the present invention, thereby producing the target substance. Examples of target substances are as described above.

[0036] The culture conditions for the transformant are not particularly limited as long as they allow the growth of the transformant cells and the production of the target substance. The medium used for the culture may be either a synthetic medium or a natural medium, as long as it contains components necessary for normal cell growth and the production of the target substance, such as a carbon source, a nitrogen source, inorganic salts, and vitamins. The initial concentration of erythritol in the culture medium is preferably 0.1 to 10% (w / v).

[0037] It is desirable to use a non-cellulase-inducing carbon source as the carbon source added to the medium to avoid stimulating the cellulase expression induction process inherent in the cells. For example, non-cellulase-inducing carbohydrates such as glucose and fructose, sugar alcohols such as sorbitol, alcohols such as ethanol and glycerol, and organic acids such as acetic acid can be used as carbon sources. Alternatively, erythritol may be used as a carbon source. On the other hand, the medium preferably does not contain a cellulase-inducing carbon source such as cellulose. To produce a target substance while further reducing catabolite repression of the 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, such as glucose, in a nitrogen source such as aqueous ammonia or an aqueous solution containing an ammonium salt, and culture the solution by feeding the solution, from the viewpoints of culture efficiency and suppressing foaming during culture.

[0038] Examples of nitrogen sources 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, and potassium carbonate. Examples of vitamins include biotin and thiamine. Furthermore, if necessary, substances required for the growth of the transformant can be added.

[0039] Cultivation 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. The temperature is also preferably 10 to 50°C, more preferably 20 to 42°C, more preferably 25 to 35°C. The pH during cultivation is 3 to 9, preferably 4 to 5. The cultivation time is 10 hours to 10 days, preferably 2 to 7 days.

[0040] After culturing, 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, the cells may be disrupted by ultrasound, pressure, or the like, followed by removal of cellular components by decantation, filtration, centrifugation, or the like, and the remaining fraction containing the target substance may be recovered. Alternatively, the target substance can be produced and secreted outside the cells by operably linking a secretory signal peptide that functions in the transformant to a gene encoding the target substance.

[0041] If necessary, the target substance can be purified by subjecting the collected fraction to dialysis, salting out, ion exchange, distillation, solvent extraction, or a combination of these. In the method for producing a target substance according to the present invention, the culture of the transformant 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.

[0042] As exemplary embodiments of the present invention, the following substances, manufacturing methods, uses, methods, etc. are further disclosed herein, but the present invention is not limited to these embodiments.

[0043] [1] An erythritol-inducible promoter consisting of a DNA selected from the following (a) to (c): (a) DNA consisting of any one of the nucleotide sequences of SEQ ID NOs: 1 to 4; (b) DNA consisting of a nucleotide sequence having at least 90% identity to any of the nucleotide sequences of SEQ ID NOs: 1 to 4; and (c) DNA consisting of a nucleotide sequence in which one or several nucleotides have been deleted, substituted, added, or inserted relative to any of the nucleotide sequences of SEQ ID NOs: 1 to 4. [2] Preferably, in the presence of erythritol, the expression of mRNA of a target gene is 40-fold or more, preferably 50-fold or more, and more preferably 100-fold or more, compared to the expression in the absence of erythritol or in the presence of cellulose, glucose, or sorbitol. More preferably, after 8 hours in the presence of 0.2 w / v% erythritol, the expression of mRNA of the target gene is induced at an amount 40 times or more, preferably 50 times or more, more preferably 100 times or more, compared to the amount in the absence of erythritol or in the presence of cellulose, glucose or sorbitol. [1] The erythritol-inducible promoter described in [1]. [3] An expression vector comprising the erythritol-inducible promoter according to [1] or [2]. [4] The expression vector according to [3], preferably comprising a gene encoding a target substance or an enzyme involved in the synthesis thereof, and the erythritol-inducible promoter linked upstream of the gene. [5] An erythritol-inducible gene expression cassette comprising a gene encoding a target substance or an enzyme involved in the synthesis thereof and the erythritol-inducible promoter according to [1] or [2] linked upstream of the gene. [6] The target substance is Preferably it is an enzyme, More preferably, it is a cellulase, hemicellulase, exoglucanase, endoglucanase, β-glucosidase, protease, lipase, mannase, arabinase, galactase, or amylase; More preferably, it is cellulase, xylanase, β-xylosidase, or α-arabinofuranosidase; More preferably, it is cellulase or xylanase. The expression vector according to [3] or [4], or the gene expression cassette according to [5]. [7] A transformed Trichoderma fungus cell containing the expression vector according to [3] or [4], or the gene expression cassette according to [5]. [8] The transformed Trichoderma fungus cell according to [7], wherein the Trichoderma fungus is preferably Trichoderma reesei or a mutant thereof. [9] A method for producing a target substance, comprising culturing the transformed Trichoderma cell according to [7] or [8] in a medium containing erythritol.

[10] The method of producing according to [9], further comprising recovering the target substance from the culture obtained by the culturing.

[11] The target substance is Preferably it is an enzyme, More preferably, it is a cellulase, hemicellulase, exoglucanase, endoglucanase, β-glucosidase, protease, lipase, mannase, arabinase, galactase, or amylase; More preferably, it is cellulase, xylanase, β-xylosidase, or α-arabinofuranosidase; More preferably, it is cellulase or xylanase. The manufacturing method according to [9] or

[10] .

[0044]

[12] Use of a DNA selected from the following (a) to (c) as an erythritol-inducible promoter: (a) DNA consisting of any one of the nucleotide sequences of SEQ ID NOs: 1 to 4; (b) DNA consisting of a nucleotide sequence having at least 90% identity to any of the nucleotide sequences of SEQ ID NOs: 1 to 4; and (c) DNA consisting of a nucleotide sequence in which one or several nucleotides have been deleted, substituted, added, or inserted relative to any of the nucleotide sequences of SEQ ID NOs: 1 to 4.

[13] The erythritol-inducible promoter is Preferably, in the presence of erythritol, the expression of mRNA of the target gene is 40 times or more, preferably 50 times or more, more preferably 100 times or more, compared to the expression in the absence of erythritol or in the presence of cellulose, glucose or sorbitol. More preferably, after 8 hours in the presence of 0.2 w / v% erythritol, the expression of mRNA of the target gene is induced at an amount 40 times or more, preferably 50 times or more, more preferably 100 times or more, compared to the amount in the absence of erythritol or in the presence of cellulose, glucose or sorbitol.

[12] The use described in

[12] .

[14] Use of the expression vector according to [4] or the erythritol-inducible gene expression cassette according to [5] for expressing a gene encoding a target substance or an enzyme involved in the synthesis of the target substance.

[15] Use of the transformed Trichoderma fungus cell according to [7] or [8] for producing a target substance. [Example]

[0045] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0046] Example 1 Expression analysis by RNAseq analysis Trichoderma reesei (T. reesei) strain PC-3-7 was used to culture 1 x 10 spores. 5 The cells 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 composition was as follows: 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% Tween 80, 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 follows: 6 mg H3BO3, 26 mg (NH4)6Mo7O 24 4H2O, 100mg FeCl3·6H2O, 40mg CuSO4·5H2O, 8mg MnCl2·4H2O, and 200mg ZnCl2 were diluted to 100mL with distilled water. 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 an additional 8 hours, the cells were harvested. As a control, cells cultured under the above conditions for 80 hours in the absence of erythritol were used.

[0047] Total RNA was extracted from the collected fungal cells using the QIAGEN RNeasy Plant Mini Kit. A next-generation sequencing library (cDNA library) was constructed using the TruSeq RNA Sample Prep v3 Kit (Illumina). The resulting cDNA library was sequenced using the Miseq System with the Miseq Reagent Kit (300 cycles) (Illumina). The resulting sequence information was mapped to the T. reesei QM6a CDS sequence 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) was calculated, which is the number of mapped reads corrected for gene length and total number of reads. The expression level of each gene was analyzed based on the RPKM obtained. As a result, four genes were found to have RPKM values ​​more than 50 times higher under erythritol-supplemented conditions than under the control, and were hardly expressed under the control conditions: 122079 (TRIREDRAFT_122079), 68466 (TRIREDRAFT_68466), 68606 (TRIREDRAFT_68606), and 68585 (TRIREDRAFT_68585) (Table 1).

[0048] [Table 1]

[0049] Example 2 Expression analysis of erythritol-inducible expression genes The erythritol specificity of the induction of expression of the four genes found in Example 1 was verified by real-time PCR. For 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 induce high expression of AaBGL1 using the egl1 promoter, was used. The E1AB1 strain was cultured at a spore count of 1 × 10 5 The 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 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, 1.28% diammonium hydrogen citrate, and 50 mM tartaric acid buffer (pH 4.0) (all percentages are w / v%). The composition of Trace element 2 was as described in Example 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 cells were harvested immediately (0 h) and 2 hours after the addition of erythritol. As a control, cells cultured for a total of 50 hours without adding any carbon source after 48 hours of culture were used.

[0050] cDNA was synthesized using RNA extracted from the collected cells (TaKaRa PrimeScript TMThe synthesized cDNA was analyzed using the Agilent Technologies Brilliant III Ultra-Fast SYBR Green QPCR Master Mixes. The primers used for real-time PCR are listed in Table 2. The expression level of each gene was determined by relative quantification (ΔΔCt method). The normalization gene was the phosphoglycerate kinase gene pgk1 (TRIREDRAFT_21406), a glycolytic enzyme. The relative expression levels of each gene relative to the expression level of the pgk1 gene are shown in Figures 1–4. Compared to immediately after the addition of erythritol (0 h), the expression levels of the four genes significantly increased 2 hours after the addition (2 h). In contrast, no increase in the expression levels of the four genes was observed in the control, glucose, or sorbitol-added conditions. This indicates that the expression of the four genes, 122079, 68466, 68606, and 68585, is specifically induced by erythritol.

[0051] [Table 2]

[0052] Example 3 Construction of plasmid DNA for gene transfer We verified that the promoter regions of the four genes whose erythritol-specific expression induction was confirmed in Example 2 function as erythritol-inducible promoters. The following six DNA fragments were prepared by PCR using T. reesei genomic DNA as a template. Fragment 1: Approximately 1.0 kbp of the promoter region upstream of the 122079 gene (SEQ ID NO: 1) Fragment 2: Approximately 0.8 kbp of the promoter region upstream of the 68466 gene (SEQ ID NO: 2) Fragment 3: Approximately 0.8 kbp of the promoter region upstream of the 68606 gene (SEQ ID NO: 3) Fragment 4: Approximately 0.8 kbp of the promoter region upstream of the 68585 gene (SEQ ID NO: 4) Fragment 5: The region from the coding region of the xylanase gene xyn3 (TRIREDRAFT_120229) to 0.5 kbp downstream Fragment 6: The region from approximately 1.0 kbp upstream to the ORF and approximately 0.3 kbp downstream of the pyr4 gene (TRIREDRAFT_74020) (approximately 2.4 kbp) The primers used to amplify the fragments are shown in Table 3.

[0053] [Table 3]

[0054] Fragments 5 and 6 were ligated to construct XYN3-pyr4. XYN3-pyr4 was then ligated to each of fragments 1 to 4. The resulting fragments were inserted into the HincII restriction enzyme 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. Ligation of DNA fragments was performed according to the protocol of the In-Fusion HD Cloning kit (Takara Bio).

[0055] The constructed plasmid was transformed into competent E. coli DH5α Competent Cells (Takara Bio), and from the resulting ampicillin-resistant transformants, strains harboring the plasmid containing the target gene 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 cells using NucleoSpin Plasmid EasyPure (Machley-Nagel).

[0056] Example 4: Preparation of transformed strains The T. reesei E1AB1Δpyr4 strain was transformed with the plasmid constructed in Example 3. Introduction was performed using 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% (NH)SO, 0.2% KHPO (pH 5.5), 0.06% CaCl·2H0, 0.06% CsCl, 0.06% MgSO·7H0, 0.1% Trace element 1; all percentages are w / v%). The composition of Trace element 1 was as follows: 0.5g FeSO4·7H2O, 0.2g CoCl2, 0.16g MnSO4·H2O, and 0.14g ZnSO4·7H2O were diluted to 100mL with distilled water. The selected transformants were stabilized by subculture, and then strains that stably retained the target gene were further selected by colony PCR.

[0057] Example 5 Cultivation of transformed strains The transformed strain prepared in Example 4 was cultured to carry out protein production by erythritol induction. For the culture, 50 mL of medium was placed in a 500 mL flask, and 1 × 10 spores of the strain prepared in Example 4 were added. 5 The 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 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, and 50 mM tartrate buffer (pH 4.0) (all percentages are w / v%). The composition of Trace element 2 was as described in Example 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 recovered. After an additional 4 hours of culture, the culture medium was again recovered.

[0058] Example 6 XYN3 activity measurement The xylanase (XYN3) activity in the culture medium recovered in Example 5 was measured using 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 mixture was incubated at 50°C for 10 minutes. 100 μL of 1 M Na2Co3 solution was then added to terminate the reaction. The absorbance at 420 nm was then measured. A calibration curve was prepared using p-nitrophenol, with the amount of enzyme liberating 1 μmol of pNP per minute defined as 1 U, and the XYN3 activity in each culture medium was calculated.

[0059] As shown in Table 4, XYN3 activity was barely observed in the parent strain E1AB1 4 hours after erythritol addition, whereas in the 122079 promoter-expressing strain (P122079), the 68466 promoter-expressing strain (P68466), and the 68585 promoter-expressing strain (P68585), XYN3 activity significantly increased 4 hours after erythritol addition compared to 0 hours after addition. These results demonstrated that these promoters function as erythritol-inducible promoters, enabling the induced production of target substances in the presence of erythritol. Furthermore, since the parent strain E1AB1 did not exhibit XYN3 activity under erythritol-added conditions, it was demonstrated that erythritol does not induce the expression of cellulosic biomass degrading enzymes inherent in the cells. The erythritol-induced expression of the 68606 gene was confirmed in Examples 1 and 2, demonstrating that the 68606 promoter functions as an erythritol-inducible promoter. On the other hand, the 68606 promoter-expressing strain (P68606) did not exhibit XYN3 activity in the presence of erythritol, which was thought to be because this experimental system was not suitable for the P68606 strain.

[0060] [Table 4]

Claims

1. An erythritol-inducible promoter consisting of DNA selected from the following (a) to (c): (a) DNA consisting of any one of the nucleotide sequences of SEQ ID NOs: 1, 2 and 4; (b) DNA consisting of a nucleotide sequence having at least 90% identity to any of the nucleotide sequences of SEQ ID NOs: 1, 2, and 4; and (c) DNA consisting of a nucleotide sequence in which 1 to 10 nucleotides have been deleted, substituted, added, or inserted relative to any of the nucleotide sequences of SEQ ID NOs: 1, 2, and 4.

2. An erythritol-inducible expression vector comprising the erythritol-inducible promoter of claim 1.

3. 3. The erythritol-inducible expression vector according to claim 2, comprising a gene encoding a target substance or an enzyme involved in the synthesis thereof, and the erythritol-inducible promoter linked upstream of the gene.

4. An erythritol-inducible gene expression cassette comprising a gene encoding a target substance or an enzyme involved in the synthesis thereof and the erythritol-inducible promoter according to claim 1 linked upstream of said gene.

5. A transformed Trichoderma fungal cell comprising the expression vector according to claim 2 or 3 or the gene expression cassette according to claim 4.

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

7. A method for producing a target substance, comprising culturing the transformed Trichoderma cell according to claim 5 in a medium containing erythritol.

8. The method according to claim 7 , further comprising recovering the target substance from the culture obtained by the culturing.

Images