Genetically recombinant yeast, method for producing target protein, kit, and method for producing genetically recombinant yeast

Abstract

The present invention addresses the problem of providing: a substrate; provided are: a genetically recombinant yeast capable of highly expressing a target protein in a strictly controlled manner without using methanol; a method for producing a target protein using the genetically recombinant yeast; a kit for producing the genetically recombinant yeast; and a method for producing the genetically recombinant yeast. According to the present invention, provided is a genetically recombinant yeast comprising a first exogenous sucrose-induced promoter for expressing a protein of interest.

Description

TECHNICAL FIELD

[0001] The present application relates to a genetically recombinant yeast for expressing a target protein. The present application also relates to a method for producing a target protein using the genetically recombinant yeast, a kit for producing the genetically recombinant yeast, and a method for producing the genetically recombinant yeast. BACKGROUND

[0002] Recombinant proteins are used as pharmaceuticals or industrial enzymes, and in most cases, are produced by genetically recombinant microorganisms. Methanol-assimilating yeasts are used as efficient protein expression systems. As methanol-assimilating yeasts capable of high expression and high-density culture of proteins, Ogataea polymorpha, Candida boidinii, Ogataea methanolica, and Pichia pastoris (Komagataella phaffii) are known. In particular, Pichia pastoris has been widely used for commercial use.

[0003] In Pichia pastoris, the AOX1 promoter has been used as a promoter for expressing proteins. The AOX1 promoter is a methanol-inducible promoter that is strictly controlled and can perform strong inducible expression using methanol as a carbon source.

[0004] Furthermore, in recent years, as an alternative promoter that does not use methanol, an AOX1 promoter variant (Patent Literature 1), a G1 promoter variant (Patent Literature 2), an FMD / MOX promoter (Patent Literature 3), and the like have been developed. PRIOR ART DOCUMENTS PATENT LITERATURE

[0005] Patent Literature 1: International Publication No. WO2006 / 089329 Patent Literature 2: International Publication No. WO2017 / 021541 Patent Literature 3: International Publication No. WO2017 / 109082 SUMMARY TECHNICAL PROBLEM TO BE SOLVED BY THE INVENTION

[0006] In the case of using the AOX1 promoter, methanol is used. However, in the case of using methanol, which is flammable and toxic, countermeasures against explosion and health risks are required, and thus in the case of particularly large-scale industrial use, there is a problem in that expensive equipment is required. Furthermore, by using methanol as a carbon source, the survival rate of cells is reduced compared to carbon sources such as glucose, and it is possible that the yield of the product is affected. Therefore, even if it is desired to increase protein expression, the amount of addition of the carbon source is limited. Furthermore, oxygen consumption and heat generation due to metabolism are large, and an increase in cooling costs is also a problem.

[0007] On the other hand, the promoters described in Patent Literatures 1 to 3 have problems that their expression ability is inferior to that of the AOX1 promoter or has only the same degree of expression ability, or expression is performed depending on the release of suppression, and it is difficult to achieve strict expression control. Also, in order to improve the productivity of a protein, in the case of expressing a plurality of foreign genes (for example, the case of expressing a protein A so as to function as a chaperone for a target protein B, etc.), a plurality of alternative promoters are required, but the number of existing promoters is limited.

[0008] Therefore, a novel promoter which does not use methanol and has high expression amount with strict control is expected.

[0009] The present application is to provide a genetically modified yeast which can highly express a target protein with strict control without using methanol. The present application is also to provide a method for producing a target protein using the genetically modified yeast, a kit for producing the genetically modified yeast, and a method for producing the genetically modified yeast. Means for solving the technical problem

[0010] The present inventors have conducted intensive studies in order to solve the above problem, and as a result, have found that by using an exogenous sucrose-inducible promoter as a promoter for expressing a target protein in yeast, the target protein can be highly expressed with strict control without using methanol. The present application has been accomplished based on the above insight.

[0011] According to the present application, the following inventions are provided. <1> A genetically modified yeast comprising a first exogenous sucrose-inducible promoter for expressing a target protein. <2> The genetically modified yeast according to <1>, further comprising an exogenous gene encoding a target protein. <3> The genetically modified yeast according to <1> or <2>, further comprising an exogenous gene conferring sucrose assimilation. <4> The genetically modified yeast according to <3>, wherein the exogenous gene conferring sucrose assimilation is linked to a second exogenous sucrose-inducible promoter. <5> The genetically modified yeast according to <3> or <4>, wherein the exogenous gene conferring sucrose assimilation is a maltase gene. <6> The genetically modified yeast according to any one of <3> to <5>, wherein the first exogenous sucrose-inducible promoter and the exogenous gene conferring sucrose assimilation are derived from a yeast having an a-glucosidase capable of hydrolyzing sucrose. The genetically recombined yeast according to <6>, wherein the yeast having an alpha-glucosidase capable of hydrolyzing sucrose belongs to any one of the genera of Ogataea, Candida, Cyberlindnera, Wickerhamomyces, Clavispora, Debaromyces, Meyerozyma, Scheffersomyces, Metschnikowia, Spathaspora, Babjeviella, Hypopichia, or Yamadazyma. The genetically recombined yeast according to <7>, wherein the yeast having an alpha-glucosidase capable of hydrolyzing sucrose is Ogataea Parapolymorpha. The genetically recombined yeast according to any one of <1> to <8>, which is of the genus Komagataella. A method for producing a target protein, comprising the step of culturing the genetically recombined yeast according to any one of <1> to <9> in a medium containing sucrose as a carbon source. The method for producing a target protein according to <10>, wherein the target protein is an antibody, a biologically active protein, a biologically active polypeptide, an extracellular matrix, an artificial protein, or an enzyme. The method for producing a target protein according to <10> or <11>, wherein the concentration of sucrose in the medium during the culturing is 0.5 to 2 g / mL. The method for producing a target protein according to any one of <10> to <12>, wherein the rate of addition of sucrose is 2 to 100 g / hour per 0.9 L of the medium at the start of the culturing. A kit for producing the genetically recombined yeast according to any one of <1> to <9>, the kit comprising: a yeast; and an expression construct containing a sucrose-inducible promoter. The kit according to <14>, wherein the expression construct contains a foreign gene encoding a target protein, and the sucrose-inducible promoter induces the expression of the foreign gene encoding the target protein. A method for producing the genetically recombined yeast according to any one of <1> to <9>, the method comprising the step of introducing an expression construct containing a sucrose-inducible promoter into a yeast. The method for producing the genetically recombined yeast according to <16>, wherein the expression construct containing a sucrose-inducible promoter is introduced into the genome of the yeast. The method for producing a genetically recombined yeast according to <16> or <17>, wherein the expression construct comprising a sucrose-inducible promoter comprises a sucrose-inducible promoter and a foreign gene encoding a target protein, and the sucrose-inducible promoter is an expression construct that induces expression of the foreign gene encoding the target protein. The method for producing a genetically recombined yeast according to <16>, wherein the expression construct comprising a sucrose-inducible promoter is introduced into the genome of a yeast having a foreign gene that confers sucrose assimilation. Effects of the Invention

[0012] According to the present application, a target protein can be highly expressed in a strictly controlled manner without using methanol. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Indicates a MAL cluster region for converting the AOX2 CDS of Pichia pastoris. Contains AG1, AGT1, MAL-activator in the cluster region, and has sucrose assimilation. Figure 2 Indicates the relationship of product A and product B. Figure 3 Indicates that a MAL cluster-introduced yeast strain is selected by YNB-sucrose addition medium. Left: confirms colony formation of the MAL cluster-introduced yeast strain, right: no colony formation in a yeast strain into which the MAL cluster is not introduced. Figure 4 Indicates the structure of a plasmid vector manufactured to have a sucrose-inducible promoter. Figure 5 Is a schematic diagram indicating that a linearized plasmid is inserted into the MAL1 site on the MAL cluster by homologous recombination. Figure 6 Indicates the structure of a plasmid vector manufactured to have a methanol-inducible promoter. DETAILED DESCRIPTION

[0014] Hereinafter, one example of an embodiment of the present application will be described. However, the present application is not limited to the following embodiment, and can be appropriately modified and implemented within the scope of the object of the present application. In the present specification, a numerical range indicated by "~" indicates a range including the values before and after the numerical range as the minimum value and the maximum value, respectively.

[0015] <Genetically recombined yeast> The genetically recombined yeast of the present application comprises a first foreign sucrose-inducible promoter for expressing a target protein.

[0016] The first exogenous sucrose-inducible promoter in the present application is a promoter for expressing a target protein. The first exogenous sucrose-inducible promoter is located upstream of a gene encoding a target protein to induce expression of the gene encoding the target protein. By using a sucrose-inducible promoter, the target protein can be expressed in a strictly controlled manner.

[0017] The gene encoding a target protein can be a gene originally possessed by yeast or an exogenous gene, but is preferably an exogenous gene. That is, the genetically recombined yeast of the present application preferably further comprises an exogenous gene encoding a target protein.

[0018] As the first exogenous sucrose-inducible promoter, there is no particular limitation, and for example, the promoter of an alpha-glucosidase gene, an alpha-glucoside transporter gene, or a MAL-activator gene included in a MAL cluster described in Katrin Viigand et al., Genome Mining of Non-Conventional Yeasts: Search and Analysis of MALClusters and Proteins, Genes 2018, 9, 354; doi:10.3390 / genes9070354 can be used. Details of the alpha-glucosidase gene, the alpha-glucoside transporter gene, or the MAL-activator gene will be described later.

[0019] As the first exogenous sucrose-inducible promoter, the promoter of an alpha-glucosidase gene is more preferable in the above, and the promoter of an AG1 gene is further preferable. Specifically, the promoter having the base sequence described in SEQ ID NO: 16 can be mentioned.

[0020] As the promoter of an AG1 gene, as long as it has a promoter activity, it can be a promoter having a base sequence having a sequence homology of 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and particularly preferably 98% or more, to the base sequence described in SEQ ID NO: 16.

[0021] The sequence homology refers to a value calculated by the following formula. % Sequence homology = [(number of identical bases) / (alignment length)] x 100 The sequence homology in two base sequences can be determined by any method known to those skilled in the art, and can be determined using a BLAST (Basic Local Alignment Search Tool) program (J. Mol. Biol. 215:403-410, 1990) or the like.

[0022] The genetically recombined yeast of the present application preferably further comprises an exogenous gene that confers sucrose assimilation. As the exogenous gene that confers sucrose assimilation, a gene of an enzyme related to sucrose assimilation, a gene of a transporter protein related to sucrose assimilation, a gene of a transcriptional activator of an enzyme gene related to sucrose assimilation, and the like can be used.

[0023] As the exogenous gene that confers sucrose assimilation, specifically, an α-glucosidase gene, an α-glucoside transporter gene, or a MAL-activator gene (a gene described in Genes 2018, 9, 354; doi:10.3390 / genes9070354 and the like) and the like can be mentioned.

[0024] As the α-glucosidase gene, a maltase gene, an isomaltase gene, a sucrase gene, and the like can be mentioned, and specifically, as the maltase gene (also including a gene having a function of a part of the isomaltase gene), MAL32 (S. Cerevisiae), MAL1 (O. polymorpha), AG1 (O. parapolymorpha), AG1 (L. elongisporus), AG2 (M. guiliermondii), AG1.2 (C. fabianii), AGL1 (S. stipitis), MAL6 (S. stipitis), MAL7 (S. stipitis), MAL8 (S. stipitis), MalT (A. oryzae), AG1 (L. starkeyi), AG2 (L. starkeyi), AG4 (L. starkeyi), AG5 (L. starkeyi), AG6 (L. starkeyi) can be mentioned, and as the isomaltase gene, AG1 (T. delbrueckii), AG1 (M. guiliermondii), AG1.1 (C. fabianii), AG3 (L. starkeyi), AG7 (L. starkeyi), AG8 (L. starkeyi), and the like can be mentioned.

[0025] As the α-glucoside transporter gene, there can be mentioned MALl (S. stipitis), MAL2 (O. polymorpha), MAL2 (S. stipitis), MAL3 (S. stipitis), MAL4 (S. stipitis), MAL5 (S. stipitis), MAL31 (S. cerevisiae), MalP (A. oryzae), AGTl (O. parapolymorpha), AGTl. l (M. guiliermondii), AGTl.2 (M. guiliermondii), AGTl.3 (M. guiliermondii), AGT2. l (L. starkeyi), AGT2.2 (L. starkeyi), AGT3 (L. starkeyi), AGT4 (L. starkeyi), AGT5 (L. starkeyi), AGT6 (L. starkeyi), and the like.

[0026] As the MAL-activator gene, there can be mentioned MAL33 (S. cerevisiae), MAL-ACT (O. parapolymorpha), MAL-ACTl (O. polymorpha), MAL-ACT2 (O. polymorpha), MalR (A. oryzae), SUCl. l (S. stipitis), SUCl.2 (S. stipitis), SUCl.4 (S. stipitis), and the like.

[0027] The exogenous gene conferring sucrose assimilation is preferably linked to the 2ndexogenous sucrose-inducible promoter.

[0028] The 2ndexogenous sucrose-inducible promoter can be the same promoter as the 1stexogenous sucrose-inducible promoter, or a different promoter from the 1stexogenous sucrose-inducible promoter, but is preferably the same promoter as the 1stexogenous sucrose-inducible promoter. As a specific example of the 2ndexogenous sucrose-inducible promoter, the same promoters as described for the 1stexogenous sucrose-inducible promoter can be mentioned.

[0029] The 1st sucrose-inducible promoter and the exogenous gene conferring sucrose assimilation are preferably derived from a yeast having an α-glucosidase capable of hydrolyzing sucrose.

[0030] As the yeast having an α-glucosidase capable of hydrolyzing sucrose, a yeast belonging to any of the genera of Ogataea, Candida, Cyberlindnera, Wickerhamomyces, Clavispora, Debaromyces, Meyerozyma, Scheffersomyces, Metschnikowia, Spathaspora, Babjeviella, Hypopichia, or Yamadazyma can be cited, but is not particularly limited. The yeast having an α-glucosidase capable of hydrolyzing sucrose is further preferably a genus of Ogataea, and is particularly preferably Ogataea Parapolymorpha.

[0031] In the case where the genetically recombined yeast of the present application has an exogenous gene conferring sucrose assimilation, the positional relationship between the gene encoding the target protein and the exogenous gene conferring sucrose assimilation is not particularly limited. The gene encoding the target protein can be inserted into the genome of the genetically recombined yeast, or can exist extrachromosomally. The exogenous gene conferring sucrose assimilation can also be inserted into the genome of the genetically recombined yeast, or can exist extrachromosomally. In the case where the gene encoding the target protein and the exogenous gene conferring sucrose assimilation are inserted into the genome of the genetically recombined yeast, the insertion positions of the respective genes on the genome are not particularly limited. The gene encoding the target protein and the exogenous gene conferring sucrose assimilation can be inserted into separate positions on the genome, or can exist at positions adjacent to each other. In the case where the gene encoding the target protein and the exogenous gene conferring sucrose assimilation exist at adjacent positions, the gene encoding the target protein can exist upstream of the exogenous gene conferring sucrose assimilation, or can exist downstream.

[0032] The genetically recombined yeast of the present application can cite a yeast belonging to the genera of Komagataella, Pichia, Ogataea, Candida, Saccharomyces, Torulopsis, Zygosaccharomyces, Schizosaccharomyces, Yarrowia, Kluyveromyces, Debaryomyces, Geotrichum, Wicherhamia, Fellomyces, Sporobolomyces, and the like.

[0033] Among the genus Komagataella, Komagataella pastoris, Komagataella phaffii, and the like are preferred. Both Komagataella pastoris and Komagataella phaffii have the alternative name of Pichia pastoris.

[0034] Among the genus Pichia, Pichia methanolica and the like are preferred. Among the genus Ogataea, Ogataea angusta, Ogataea polymorpha, Ogataea parapolymorpha, Ogataea minuta, and the like are preferred. Among the genus Candida, Candida boidinii and the like are preferred.

[0035] Among the above, the genetically recombinant yeast of the present application is particularly preferably the genus Komagataella. As an example of a strain that can be specifically used, Komagataella pastoris ATCC 76273 (Y-11430, Pichia pastoris CBS 7435) can be given. This strain can be obtained from the American Type Culture Collection or Thermo Fisher Scientific Inc., and the like.

[0036] <Kit for producing genetically recombinant yeast> The kit for producing genetically recombinant yeast of the present application contains a yeast and an expression construct containing a sucrose-inducible promoter. As the yeast, with respect to the genetically recombinant yeast, a yeast belonging to the above genus can be used, but is not particularly limited. The yeast is particularly preferably the genus Komagataella.

[0037] The expression construct preferably contains a foreign gene encoding a target protein, and the above sucrose-inducible promoter induces the expression of the foreign gene encoding the target protein.

[0038] The expression construct containing a sucrose-inducible promoter can be obtained by inserting the sucrose-inducible promoter into an appropriate vector. The vector for inserting the sucrose-inducible promoter is not particularly limited as long as it can replicate in a host, and for example, a plasmid, a bacteriophage DNA, or a viral vector, and the like can be given. Also, in the case where the vector itself does not have the ability to replicate, it can also be a DNA fragment that can replicate by being inserted into a chromosome or the like of a host.

[0039] As the plasmid, plasmids derived from Escherichia coli (e.g., pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBlueScript, etc.), plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, etc.), plasmids derived from yeast (e.g., YEp13 and the like YEp series, YRp series, Yip series, YCp50 and the like YCp series, etc., pPIC9, pPICZ, pPICZα, and the like Pichia vector series), and the like can be mentioned. As the phage DNA, lambda phage (Charon 4A, Charon 21A, EMBL3, EMBL4, lambda gt10, lambda gt11, lambda ZAP, etc.) can be mentioned. Furthermore, animal viruses such as retroviruses or vaccinia viruses, or insect virus vectors such as baculoviruses can also be used.

[0040] In order to insert the sucrose-inducible promoter into the vector, first, the purified DNA is cleaved with an appropriate restriction enzyme, and inserted into an appropriate restriction enzyme site or a multiple cloning site of the vector DNA, whereby the vector is ligated. Alternatively, the vector and the sucrose-inducible promoter can be ligated by making a part of each of them have the same region, and using an in vitro method using PCR or the like, or an in vivo method using yeast or the like.

[0041] The expression construct containing the sucrose-inducible promoter preferably further contains a foreign gene encoding a target protein downstream of the sucrose-inducible promoter. The method of inserting the foreign gene is the same as the method of inserting the sucrose-inducible promoter into the vector.

[0042] <Method for producing genetically recombined yeast> The method for producing genetically recombined yeast of the present application includes a step of introducing an expression construct containing a sucrose-inducible promoter into yeast. As the expression construct containing the sucrose-inducible promoter and the yeast, the above-described expression construct and yeast in the present specification can be used, respectively. As the expression construct containing the sucrose-inducible promoter, it is preferable to contain a sucrose-inducible promoter and a foreign gene encoding a target protein, the above-described sucrose-inducible promoter being an expression construct that induces the expression of the foreign gene encoding the target protein.

[0043] As the method of introducing the expression construct containing the sucrose-inducible promoter into yeast, any method of introducing DNA into yeast can be used, and is not particularly limited, and for example, electroporation, the spheroplast method, the lithium acetate method, and the like can be mentioned.

[0044] The expression construct containing the sucrose-inducible promoter can be introduced into the genome of the yeast, or the expression construct containing the sucrose-inducible promoter can be introduced into the yeast as extrachromosomal DNA, but is preferably introduced into the genome of the yeast. The introduction into the genome of the yeast can be by homologous recombination, or can be by means other than homologous recombination.

[0045] In a preferred mode of the present application, the expression construct containing the sucrose-inducible promoter is introduced into the yeast having the exogenous gene conferring sucrose assimilation. The expression construct containing the sucrose-inducible promoter is preferably introduced into the genome of the yeast having the exogenous gene conferring sucrose assimilation.

[0046] As to the confirmation of whether the sucrose-inducible promoter is integrated into the yeast, it can be performed by the PCR (polymerase chain reaction) method, the Southern hybridization method, or the like. For example, DNA is prepared from the transformant, DNA-specific primers are designed and PCR is performed. Thereafter, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis, or the like, and is stained with ethidium bromide or the like, and then the amplified product is detected as a single band, whereby it is possible to confirm the transformation.

[0047] The genetically recombinant yeast of the present application can be selected by previously introducing an antibiotic resistance gene serving as a selection marker, culturing using a medium containing the antibiotic, and obtaining a proliferated colony. As the antibiotic, Zeocin, Kanamycin, Hygromycin, Puromycin, Blasticidin, or the like can be used.

[0048] The genetically recombinant yeast into which the expression construct containing the sucrose-inducible promoter manufactured as described above is introduced can be used in the production method of the target protein after being cultured and proliferated as appropriate by a conventional method.

[0049] <Production method of target protein> The production method of the target protein of the present application includes a step of culturing the genetically recombinant yeast of the present application in a medium containing sucrose as a carbon source.

[0050] As the sucrose source, a biomass or a chemical synthetic product, or the like can be used, but is not particularly limited. The sucrose concentration in the medium in the culture is preferably 0.5 to 2 g / mL, more preferably 0.5 to 0.7 g / mL, and further preferably 0.55 to 0.65 g / mL.

[0051] The addition rate of sucrose is preferably 2 to 100 g / hour, more preferably 2 to 20 g / hour, and further preferably 3 to 15 g / hour, per 0.9 L of the medium amount at the start of the culture.

[0052] As the target protein, an antibody, a biologically active protein, a biologically active polypeptide, an extracellular matrix, an artificial protein, an enzyme, or the like can be mentioned, but is not particularly limited.

[0053] The antibody is a hetero-tetramer protein of two polypeptide chains of L chain and H chain, and is not particularly limited as long as it has the ability to bind to a specific antigen. The antibody can be a partial antibody (fragmented antibody). As the partial antibody, a Fab antibody, a (Fab)2 antibody, a scFv antibody, a diabody, or the like can be mentioned, but is not particularly limited. As the antibody, a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, or a bispecific antibody, or the like is preferred. The type of the antibody is also not particularly limited, and can be any one of IgG1, IgG2, IgG3, IgG4, or the like, IgA, IgD, IgE, IgM, or the like, but when used as a medicine, IgG and IgM are preferred.

[0054] As the biologically active protein and the biologically active polypeptide, for example, a hormone, a growth factor, a coagulation factor, a neural protein, an apolipoprotein, a tumor suppressor, an antigen protein and peptide including an endogenous or exogenous tumor, a bacterial surface protein and peptide, a viral surface protein and peptide, a protozoan cell surface protein and peptide, a fungal surface protein and peptide, and a parasite including a viral reverse transcriptase and a related viral specific enzyme can be mentioned. As other proteins, a receptor such as an insulin receptor, a hormone receptor, a growth factor receptor, or the like can be mentioned. In the examples described later, a gene recombined gelatin was used as the target protein.

[0055] As the enzyme, an enzyme derived from a microorganism, an enzyme produced by an animal or a plant can be mentioned. For example, a phytase, an amylase, a glucosidase, a cellulase, a lipase, a protease, a glutaminease, a peptidase, a nuclease, an oxidase, a lactase, a xylanase, a trypsin, a pectinase, an isomerase, or the like can be mentioned, but is not limited to these.

[0056] As for the culture of the gene recombined yeast, a usual yeast culture method can be appropriately used, and the culture conditions can be appropriately set under conditions suitable for the growth of the yeast.

[0057] As the culture medium for the culture, any one of a natural culture medium or a synthetic culture medium can be used as long as it contains a carbon source of sucrose, a nitrogen source, an inorganic substance, and a trace nutrient required by the strain used, as needed.

[0058] As the carbon source of the culture medium, sucrose can be contained, or a carbon source other than sucrose can be contained. As the carbon source other than sucrose, for example, saccharides such as glucose, maltose, fructose, mannose, trehalose, mannitol, sorbitol, starch, dextrin, molasses, or organic acids such as citric acid, succinic acid, or glycerol can be used.

[0059] As the nitrogen source of the culture medium, for example, an organic nitrogen source such as corn steep liquor, soybean meal, or peptone, or an inorganic nitrogen source such as ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, ammonium phosphate, or ammonia can be used. Furthermore, nitrogen-containing natural substances such as peptone, polypeptone, Bacto peptone, meat extract, fish meat extract, yeast extract, corn steep liquor, soybean powder, soybean meal, dried yeast, casein amino acid, and soluble plant protein can also be used as the nitrogen source.

[0060] As the inorganic substance of the culture medium, for example, calcium salts, magnesium salts, potassium salts, sodium salts, phosphate salts, manganese salts, zinc salts, iron salts, copper salts, molybdenum salts, cobalt salts, or the like can be appropriately used. Specifically, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, zinc sulfate, sodium chloride, potassium chloride, calcium chloride, or the like can be used. Furthermore, phosphoric acid, sulfuric acid, potassium hydroxide, TES (Trace Element Solution), or the like can be appropriately used.

[0061] As the culture method, a liquid culture method is preferable, and can be any one of batch culture, fed-batch culture, continuous culture, or perfusion culture.

[0062] The culture temperature and pH are only required to be conditions suitable for the propagation of the genetically recombinant yeast. The temperature is preferably 20 to 40°C, and more preferably 25 to 35°C. The pH is preferably 2 to 9, and more preferably 5 to 8. The culture time is preferably 1 hour to 14 days, more preferably 6 hours to 10 days, and further preferably 12 hours to 7 days. The culture is preferably performed by shaking or aeration stirring.

[0063] In the case where the non-secretory target protein is secreted outside the yeast cell, a base sequence encoding a signal sequence can be introduced into the 5' end of the target protein gene. The base sequence encoding a signal sequence is not particularly limited as long as it is a base sequence encoding a signal sequence that the yeast can secrete and express. As specific examples of the base sequence encoding a signal sequence, base sequences encoding signal sequences of Saccharomyces cerevisiae Mating Factor a (MFa), acid phosphatase (PHOl) of Saccharomyces acidifaciens, acid phosphatase (PHOl) of Komagataella pastoris, invertase (SUC2) of Saccharomyces cerevisiae, PLB1 of Saccharomyces cerevisiae, bovine serum albumin (BSA), human serum albumin (HSA), and immunoglobulin can be given.

[0064] By culturing the genetically recombined yeast of the present application, the target protein can be accumulated in the host or in the culture solution and recovered. As to the recovery method of the target protein, a publicly known purification method can be appropriately combined and used. For example, the genetically recombined yeast is cultured with a culture medium, and the bacterial bodies are removed from the culture supernatant by centrifugal separation or filtration treatment of the culture solution. By using the methods of salting-out (ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (protein fraction precipitation method based on acetone or ethanol, etc.), dialysis, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, ultrafiltration, etc. alone or in combination, the target protein can be recovered from the culture supernatant.

[0065] The present application is more specifically described by the following examples, but the present application is not limited by the examples. Example

[0066] Example 1: Cloning of MAL cluster The genome of Ogataea Parapolymorpha containing the MAL cluster having SEQ ID NO: 1 and the genome of Pichia pastoris CBS7435 having AOX2 were used as templates.

[0067] As shown in Figure 1 , the 5' region of the MAL cluster was amplified using KP-MAL3 and KP-MAL4 with the genome of Ogataea Parapolymorpha as a template. The base sequence of the amplified fragment is shown in SEQ ID NO: 2. A part of the region of AOX2 was amplified using KP-MAL1 and KP-MAL2 with the genome of Pichia pastoris CBS7435 having AOX2 as a template. The base sequence of the amplified fragment is shown in SEQ ID NO: 3. And, using the two amplificates obtained in the above, a sequence amplified product (product A) connected by PCR reaction was obtained.

[0068] Likewise, the 3' region of the MAL cluster was amplified using KP-MAL5 and KP-MAL6 with the genome of Ogataea Parapolymorpha as a template. The base sequence of the amplified fragment is shown in SEQ ID NO: 4. A part of the region of AOX2 was amplified using KP-MAL7 and KP-MAL8 with the genome of Pichia pastoris CBS7435 having AOX2 as a template. The base sequence of the amplified fragment is shown in SEQ ID NO: 5. Using the two amplificates obtained in the above, a sequence amplified product (product B) connected by PCR reaction was obtained.

[0069] The relationship of product A to product B is shown in Figure 2 .

[0070] Primer for MAL cluster amplification KP-MAL1 (Forward): 5'-TTGTCAGCTTAAAGGACTCC-3' (SEQ ID NO: 6)

[0071] KP-MAL2 (Reverse): 5'-TTCCGGGAATACCATTTTTCTCAGTTGATTTGTTTGTGGG-3' (SEQ ID NO: 7)

[0072] KP-MAL3 (Forward): 5'-CAAATCAACTGAGAAAAATGGTATTCCCGGAAAGAACTCG-3' (SEQ ID NO: 8)

[0073] KP-MAL4 (Reverse): 5'-CCTGGTCTTCTGTGAGTATC-3' (SEQ ID NO: 9)

[0074] KP-MAL5 (Forward): 5'-TGGGCAGGCTTGTCTGTTTG-3' (SEQ ID NO: 10)

[0075] KP-MAL6 (Reverse): 5'-CATAGATACAACATAAACTAGGAATGTTTCCAGCGGTATG-3' (SEQ ID NO: 11)

[0076] KP-MAL7 (Forward): 5'-CATACCGCTGGAAACATTCCTAGTTTATGTTGTATCTATG-3' (SEQ ID NO: 12)

[0077] KP-MAL8 (Reverse): 5'-CTGACTGGACTTAGCGAAG-3' (SEQ ID NO: 13)

[0078] The product A and product B obtained in the above and Pichia pastoris CBS7435 (OD600: 200-300, 60 μL) were simultaneously introduced into Pichia pastoris CBS7435 by an electroporation method (Micropulser electroporator (Bio-Rad)) to thereby perform transformation of Pichia pastoris CBS7435. As described above, the product A and product B were inserted into the AOX2 site of the Pichia yeast genome by homologous recombination. To confirm that the MAL cluster composed of the product A and product B has been introduced into the CDS at the AOX2 site by homologous recombination, a colony in which the MAL cluster was integrated was obtained by performing a screening by a YNB-sucrose addition medium. Figure 3 ).

[0079] Example 2: Production of a plasmid vector in which a gene of a target protein is linked downstream of a sucrose-inducible MAL promoter As a model protein, recombinant gelatin CBE3 described below was used. (WO2008 / 103041 Al). Molecular weight: 51.6 kD Structure: GAP [(GXY) 63 ]3G Number of amino acids: 571 RGD sequence: 12 Imino acid content: 33% Approximately 100% of the amino acids are a repeating structure of GXY. CBE3 has an ERGD sequence. The amino acid sequence of CBE3 does not include a serine residue, a threonine residue, an asparagine residue, a tyrosine residue, and a cysteine residue. Isoelectric point: 9.34 Hydrophilic repeating unit ratio in a polymer: 26.1% The amino acid sequence is shown in SEQ ID NO: 14. (SEQ ID NO: 14) GAP (GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPP)3G

[0080] A plasmid vector was manufactured in which a gene encoding a target protein and a Zeocin resistance gene were linked downstream of a sucrose-inducible MAL promoter. The base sequence of the manufactured plasmid vector is shown in SEQ ID NO: 15. The structure of the manufactured plasmid vector is shown in Figure 4 .

[0081] The base sequence of P-mal is shown in SEQ ID NO: 16. MF-a: The signal sequence of a target protein is shown in SEQ ID NO: 17. NRC3: The base sequence encoding a target protein (CBE3) is shown in SEQ ID NO: 18.

[0082] <Example 3> Transformation of Pichia pastoris The plasmid vector (plasmid vector having a sucrose-inducible promoter) manufactured in Example 2 was introduced into the Pichia pastoris CBS7435 strain manufactured in Example 1 by homologous recombination. Specifically, the plasmid vector of SEQ ID NO: 15 (plasmid vector manufactured in Example 2) in which a gene of a target protein was linked downstream of a sucrose-inducible MAL promoter was linearized with a restriction enzyme EcoRI. The linearized plasmid (2 to 10 μg of linearized plasmid) was introduced into the Pichia pastoris CBS7435 strain (OD600: 200 to 300, 60 μL) manufactured in Example 1 by an electroporation method. The electroporation method was performed using an electroporation device (Micropulser electroporator (Bio-Rad)).

[0083] The Pichia pastoris CBS7435 strain transformed by the electroporation method was inoculated on a YPD agar medium plate containing an antibiotic Zeocin (500 μg / mL) as a selection marker and screening was performed, and a proliferated colony was obtained. Thereby, as shown in Figure 5As shown, a transformed yeast in which an expression unit including a MAL promoter (also expressed as P-mal) and an AG1 gene is integrated in the sequence of the MAL promoter in the expression unit on the MAL cluster including a MAL promoter and an NRC3 (CBE3) gene was obtained.

[0084] A schematic diagram showing insertion of a linearized plasmid into the AG1 site on the MAL cluster by homologous recombination is shown in Figure 5 .

[0085] Example 4: Cultivation and Protein Expression To investigate protein expression, the transformed yeast prepared in Example 3 was cultivated using a 3L fermenter. Fermentation was started by adding 9 mL of the yeast culture solution obtained by 24 hours of subculture to 900 mL of Batch Medium (225 mL of Batch Medium + 675 mL of water) in the fermenter, together with 23 mL of Biotin and 5.1 mL of antifoam agent (struktol J673, Schill and Seilacher). 5.1 mL of antifoam agent (struktol J673, Schill and Seilacher) and an aqueous sucrose solution were added 24 hours after the start of fermentation. TES was added 3.6 mL each at 24, 48, and 72 hours after the start of fermentation. Ammonia (about 130 mL in total) was automatically added from a pH sensor as feedback, serving as both a nitrogen source and a pH adjuster. Sucrose was added in a total of 970 mL from 24 hours after the start of fermentation to the end of cultivation. Sucrose addition rate: L (mL / min) = 0.262 x e 0.01t (e is natural logarithm: 2.718) t is the time from the start of fermentation (hr).

[0086] pH was 5.5, and pO2 was maintained at 25% from 2 hours after the start. Fermentation was stopped 100 hours after the start of cultivation, and a culture solution containing the produced protein was recovered.

[0087] [Table 1]

[0088] As for the expressed protein, the expression amount was evaluated based on GPC analysis (using equipment: Waters e2695 HPLC alliance). The results are shown in the following table (sucrose-inducible promoter introduction into yeast column of the following table).

[0089] As a comparative example, the measurement results of the expression amount of the protein when a methanol-inducible promoter was introduced into yeast are also shown in the following table (methanol-inducible promoter introduction into yeast column of the following table).

[0090] Preparation of yeast into which methanol inducible promoter is introduced Using pPICZα plasmid vector (invitrogen), a plasmid vector into which a gene of a target protein (CBE3) and a Zeocin resistance gene were ligated downstream of a methanol inducible AOXl promoter was manufactured. The base sequence of the manufactured plasmid vector is shown in SEQ ID NO: 19. The structure of the manufactured plasmid vector is shown in Figure 6 . The Pichia pastoris strain (X-33) transformed by the electroporation method was inoculated on a YPD agar medium plate containing an antibiotic Zeocin (500 μg / mL) as a selection marker and screening was performed, and a transformed yeast strain was obtained from the proliferated colonies.

[0091] Culturing and protein expression In order to investigate protein expression of the yeast into which the methanol inducible promoter was introduced, the transformed yeast manufactured in Example 0090 was cultured using a 3L fermenter as in the case of the yeast into which the sucrose inducible promoter was introduced. 8 mL of the yeast culture solution obtained by 24 hours of the propagation culture was collected, and Biotin 23 mL, an antifoaming agent (struktol J673, Schill and Seilacher) 5.1 mL were added to the Batch Medium 900 mL (Batch Medium 225 mL + water 675 mL) in the tank at the same time to start the fermentation. 5.1 mL of the antifoaming agent (struktol J673, Schill and Seilacher), methanol were added 24 hours after the start of the fermentation. TES was added 3.6 mL each at 24, 48, 72 hours after the start of the fermentation. Ammonia (about 100 mL in total) was automatically added from the pH sensor feedback, and ammonia served as a nitrogen source and a pH adjuster. Methanol was added 850 mL in total from 24 hours after the start to the end of the culture. Methanol addition rate: L (mL / min) = 0.229 x e 0.01t (e is natural logarithm: 2.718) t is the time from the start of the fermentation (hr). In addition, the methanol addition rate was set to be the same as the carbon equivalent per unit time of the sucrose addition rate.

[0092] [Table 2]

[0093] In the yeast into which the sucrose-inducible promoter of the present application is introduced, the protein expression amount is obtained above that of the conventional methanol-inducible promoter. Also, in the yeast into which the methanol-inducible promoter is introduced, the viability is reduced by the addition of methanol, but in the yeast into which the sucrose-inducible promoter is introduced, the sucrose addition has no effect on the viability, indicating the sustained protein expression.

[0094] Sequence of the MAL cluster (SEQ ID NO: 1): ATATTCAGTTCGAACTTTTTCTTTTCAATTTCCTCCGACTCTGCAGTGGAAACATATGCAATCAGCTTTTCTGCTGCCTTGAAATACTCATCGTTTTCCACGTCAAGTATCCAGGCAAATTTATAGTTGTTGATGCTTGAATTCAAAAACATGCGCACGTAATCTGGATATTGAGGGATGTAATTGAGGATTAGCCGAATCTTTCTGGTTCTAGCATAGAATCCAAAAAGCACTTCTGCAAAACGGTATCCATACCGCTGGAAACATTCC

[0095] (SEQ ID NO: 2) (fragment amplified with KP-MAL3 + KP-MAL4)

[0096] (SEQ ID NO: 3) (fragment amplified with KP-MALl + KP-MAL2)

[0097] (SEQ ID NO: 4) (fragment amplified with KP-MAL5 + KP-MAL6)

[0098] (SEQ ID NO: 5) (fragment amplified with KP-MAL7 + KP-MAL8) CATACCGCTGGAAACATTCCTAGTTTATGTTGTATCTATGAATATTTTTTTACATGGTGGATTTGTTCCAATCTAGTTGTTAGTTATTTGTCGTTATAGTCACTAATACAGTTACCATCGGAATCTTTTCATTTCTTTTTATACGTACGTATATGTACTAGATGAAGAATGCGACAAGGCCGACCAACAGCAATGGTGCTTGGTACCAAAGTTTGGAAGGTGCTACCGAATTGGCCGATGATATTGAAGTGGAGTTGTCATTCTCCTCATTGGTTGCTCCAGTAGTGGTAAAATTAACGGGGATATCTTCGGTGGAAGTGTCAATATCACCGTTCCAACAATAATTCTCAAAACATCTTCTACATTGCTCTGTTTGTGTTGCATTGGATCTCTCCTGCTGTCTACGAATGATTGCACATCCAACACAAGTACGCCATTCTTCATCAATAGTCATGTTCAAACGAGACGACACTTCAAAACCGTTTTGGATCATTCCTCTCTTCTCGGATTCAGAGTAGTCTAACTTGAAAGTTGAAGTGTTACTCCAATACGAGAAAGGTCTATTGGCCAGATAAACAACCAGGGGAGGAATGTGATCCGTGCCTTCAACAATGTCTGTCAAATTTCTAGCATCACAACCAAAGAAGGTTGGCTTACTCGAAAGATTCAAGTTTAGGAAAGTGGTGGTATCAGGTACATAAGGGAACGTTGTTCCATTTGCTTGAGAAGAAAACTGTCTCATGTAGGTGTTGACTAATGATGAACCGTTTGGCCAAGAGAGGTCAGTGTCTGCACTGTTGTCAAACGCAAAGATGATATCAACGTCACGCTCCTTTTGAAGTAGAGGCTGCAGAGGGACGTTTTGGTTATCCTCTCCACCATCAACCAAGTAAAGGGTGTCATTTTCCGCAATGGCACCTACTCCTGCATACGTACTTTTGTAGAAAGGGTTGGGGGAATAGATAGCAATGTCGTCTTCGTCTTCGCTAAGTCCAGTCAG.

[0099] (Sequence No. 15): Sequence of the plasmid vector that cloned the MAL promoter

[0100] (Sequence No. 16) P-mal: Sucrose-inducible MAL promoter (included in the MAL cluster)

[0101] (Sequence No. 17) MF-a: Signal sequence of the target protein ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAG AAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCT

[0102] (Sequence number 18) NRC3: Target protein (CBE3)

[0103] (Serial No. 19) pPICZα-CBE3

Claims

1. A recombinant yeast comprising a first exogenous sucrose-inducible promoter for expressing a target protein.

2. The recombinant yeast according to claim 1, further comprising a foreign gene encoding the target protein.

3. The recombinant yeast according to claim 1, further comprising an exogenous gene that imparts sucrose assimilation.

4. The recombinant yeast according to claim 3, wherein, The exogenous gene that confers sucrose assimilation is linked to a second exogenous sucrose-inducible promoter.

5. The recombinant yeast according to claim 3, wherein, The exogenous gene that confers sucrose assimilation is the maltase gene.

6. The recombinant yeast according to claim 3, wherein, The first exogenous sucrose-inducible promoter and the exogenous gene that confers sucrose assimilation are derived from yeast that possesses an α-glucosidase capable of hydrolyzing sucrose.

7. The recombinant yeast according to claim 6, wherein, Yeasts possessing α-glucosidase capable of hydrolyzing sucrose belong to any of the genera Ogataea, Candida, Cyberlindnera, Wickerhamomyces, Clavispora, Debaromyces, Meyerozyma, Scheffersomyces, Metschnikowia, Spathaspora, Babjeviella, Hypopichia, or Yamadazyma.

8. The recombinant yeast according to claim 7, wherein, The yeast that possesses an α-glucosidase capable of hydrolyzing sucrose is Ogataea Parapolymorpha.

9. The recombinant yeast according to any one of claims 1 to 8, wherein it belongs to the genus Komagataella.

10. A method for manufacturing a target protein, comprising the following steps: The recombinant yeast of any one of claims 1 to 8 is cultured in a medium containing sucrose as a carbon source.

11. The method for manufacturing the target protein according to claim 10, wherein, The target protein is an antibody, a bioactive protein, a bioactive peptide, an extracellular matrix, an artificial protein, or an enzyme.

12. The method for manufacturing the target protein according to claim 10, wherein, The sucrose concentration in the culture medium during cultivation is 0.5–2 g / mL.

13. The method for manufacturing the target protein according to claim 10, wherein, The rate of sucrose addition is 2–100 g / hour, based on the initial culture medium volume of 0.9 L.

14. A kit for manufacturing recombinant yeast according to any one of claims 1 to 8, the kit comprising: Yeast; and Expression constructs containing a sucrose-inducible promoter.

15. The kit according to claim 14, wherein, The expression construct contains a foreign gene encoding the target protein, and the sucrose-inducible promoter induces the expression of the foreign gene encoding the target protein.

16. A method for manufacturing recombinant yeast according to any one of claims 1 to 8, the method comprising the following steps: An expression construct containing a sucrose-inducible promoter was introduced into yeast.

17. The method for manufacturing recombinant yeast according to claim 16, wherein, The expression construct containing the sucrose-inducible promoter was introduced into the yeast genome.

18. The method for manufacturing recombinant yeast according to claim 16, wherein, The expression construct containing a sucrose-inducible promoter comprises a sucrose-inducible promoter and a foreign gene encoding the target protein, wherein the sucrose-inducible promoter is an expression construct that induces the expression of the foreign gene encoding the target protein.

19. The method for manufacturing recombinant yeast according to claim 16, wherein, The expression construct containing the sucrose-inducible promoter was introduced into the genome of a yeast containing a foreign gene that confers sucrose assimilation.

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