Yeast and food additive

Abstract

To provide a technique for efficiently producing lipoxygenase.SOLUTION: The present invention provides a yeast having a Poaceae lipoxygenase expressed on the cell surface.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to yeast and food additives.

Background Art

[0002] In the processing of cereals (such as wheat flour) derived from gramineous plants, such as bread making and noodle making, adjustment of viscoelasticity and the like can affect the quality of the product. Examples of components in cereals related to such viscoelasticity include gluten, glutenin, and the like.

[0003] Glutenin is a polymer having polypeptide chains crosslinked via disulfide bonds and has the property of being rich in elasticity. In recent years, it has been reported that structural changes due to crosslinking between glutenins can have a great influence on the viscoelasticity of gluten.

[0004] Lipoxygenase has been identified as an oxidase that promotes crosslinking between glutenins. There are three types of isozymes of lipoxygenase in wheat. Each lipoxygenase is involved in the peroxidation reaction of linoleic acid. For example, among the isozymes of lipoxygenase, "lipoxygenase 3" has particularly high activity and has a favorable influence on volume increase, elasticity improvement, whitening effect, etc. in bread making and the like (for example, Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] On the other hand, in the mass production of lipoxygenase, since lipoxygenase has a large molecular weight, it has been difficult to correctly maintain the folded structure in conventional heterologous protein expression systems (systems for expressing proteins inside Escherichia coli or yeast). In particular, in conventional protein purification processes, disruption of microorganisms and the like are required, and it may have been difficult to stably produce lipoxygenase that maintains the correct structure. Therefore, conventionally, a practical lipoxygenase expression technology has not been sufficiently established.

[0007] The present invention has been made in view of the above circumstances, and an object thereof is to provide an efficient production technology for lipoxygenase.

Means for Solving the Problems

[0008] As a result of investigations by the present inventors, it has been found that the above problems can be solved by expressing a predetermined lipoxygenase on the cell surface in yeast, and the present invention has been completed. More specifically, the present invention provides the following.

[0009] (1) Yeast in which a gramineous plant lipoxygenase is expressed on the cell surface.

[0010] (2) The yeast according to (1), wherein the gramineous plant lipoxygenase is wheat lipoxygenase.

[0011] (3) The yeast according to (2), wherein the wheat lipoxygenase is wheat lipoxygenase 3.

[0012] (4) The yeast according to (1), wherein the lipoxygenase is expressed on the cell surface via a linker protein.

[0013] (5) A food additive containing the yeast according to any one of (1) to (4).

[0014] (6) The food additive according to (5), which is used for bread making or noodle making.

Advantages of the Invention

[0015] According to the present invention, an efficient production technique for lipoxygenase is provided.

Brief Description of the Drawings

[0016]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0017] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited thereto.

[0018] <Yeast of the Present Invention> In one aspect, the yeast of the present invention expresses the Poaceae plant lipoxygenase on the cell surface.

[0019] Various techniques for producing target proteins using yeast have been attempted. On the other hand, as a result of the studies by the present inventors, it has been found that by expressing the Poaceae plant lipoxygenase not inside the yeast but outside, the protein can be expressed while correctly maintaining the folded structure. By adopting such an expression system, for example, the Poaceae plant lipoxygenase having a correct structure can be efficiently produced without performing crushing of microorganisms required in the conventional protein purification process.

[0020] Yeast expressing the Poaceae plant lipoxygenase on the cell surface can be directly used for bread making, noodle making, etc., and high-quality bread and noodles can be obtained more easily than before.

[0021] Hereinafter, the configuration of the inventive yeast will be described in detail.

[0022] (1) Poaceae plant lipoxygenase The Poaceae plant lipoxygenase corresponds to the target protein to be expressed on the cell surface of yeast. The type of Poaceae plant lipoxygenase expressed on the cell surface can be one or more than two kinds.

[0023] "Cell surface expression" includes that the target protein (such as Poaceae plant lipoxygenase) is expressed on any part of the outer surface (extracellular surface) of the cell surface of yeast. In such a mode, a part or the whole of the target protein is distributed on the extracellular surface of yeast from the cell surface toward the outside of the cell.

[0024] The Poaceae plant lipoxygenase may be directly expressed on the extracellular surface of yeast, or may be expressed via a linker protein (described later) expressed on the extracellular surface of yeast.

[0025] "Poaceae" includes any plant belonging to the Poaceae family, preferably an edible plant. Examples of Poaceae plants include wheat, barley, rye, corn, rice, etc. Among these, wheat is preferred from the viewpoint that the effects of the present invention are particularly easily obtained.

[0026] "Lipoxygenase" (LOX) is a dioxygenase belonging to EC 1.13.11 and is an enzyme that catalyzes the oxidation of polyunsaturated fatty acids. Lipoxygenase is known to mediate the oxidation of thiol groups in wheat flour dough constituent proteins in bread making and noodle making, cause structural changes in the proteins, and improve the elasticity of the dough through interaction with lipids in the dough.

[0027] The wheat-derived lipoxygenase present in wheat germ has three isozymes, namely, lipoxygenase 1, lipoxygenase 2, and lipoxygenase 3. Among these, lipoxygenase 3 has the function of introducing an oxygen molecule to the 9th carbon of linoleic acid, and it has been shown to have a stronger impact on bread-making properties and the like than other isozymes. In particular, lipoxygenase 3 is rare and has been difficult to isolate stably in the past. However, according to the present invention, lipoxygenase 3 can be produced stably. Therefore, in a preferred embodiment of the present invention, the Poaceae plant lipoxygenase is wheat lipoxygenase 3.

[0028] The enzyme activity of lipoxygenase can be evaluated, for example, by the peroxidation reaction of linoleic acid shown in the examples. According to lipoxygenase having sufficient enzyme activity (particularly, wheat lipoxygenase 3), for example, in bread-making, an increase in the baked volume of bread and the like can be brought about due to an increase in protein by cross-linking between glutenins.

[0029] In a preferred embodiment of the present invention, wheat lipoxygenase 3 is encoded by the gene sequence of SEQ ID NO: 1 or 3.

[0030] In a preferred embodiment of the present invention, wheat lipoxygenase 3 has the amino acid sequence of SEQ ID NO: 2 or 4.

[0031] (2) Yeast The type of yeast and the like are not particularly limited as long as the cell surface expression of the Poaceae plant lipoxygenase is possible. As the yeast, usually, any yeast having a cell wall-related gene is used as the host yeast.

[0032] Examples of the cell wall-related genes expressed by yeast include the CCW12 gene, CCW14 gene, DAN1 gene, TIP1 gene, CWP1 gene, and the like. With the cell surface expression of the Poaceae plant lipoxygenase in yeast, usually, some or all of the functions of the above cell wall-related genes are deficient.

[0033] Examples of the yeast include yeast belonging to the genus Saccharomyces, Candida, Pichia, Zygosaccharomyces, Hansenula, Kluyveromyces, Schwanniomyces, Komagataella, Yarrowia, and the like.

[0034] (3) Linker protein Poaceae lipoxygenase may be expressed on the cell surface of yeast via a linker protein.

[0035] The linker protein is not particularly limited as long as it can immobilize Poaceae lipoxygenase on the cell surface of yeast.

[0036] In a preferred embodiment of the present invention, the linker protein is a cell surface-localized protein of yeast (described later).

[0037] Preferred examples of the cell surface-localized protein include GPI-anchored proteins. "GPI" (glycosylphosphatidylinositol) is a glycolipid having a basic structure of ethanolamine phosphate-6 mannose α-1,2 mannose α-1,6 mannose α-1,4 glucosamine α-1,6 inositol lipid.

[0038] In a preferred embodiment of the present invention, the linker protein is encoded by the gene sequences of SEQ ID NOs: 5 and 7.

[0039] In a preferred embodiment of the present invention, the linker protein has the amino acid sequences of SEQ ID NOs: 6 and 8.

[0040] (4) Other components of yeast Yeast may or may not have any other components as long as it does not inhibit the cell surface expression of Poaceae lipoxygenase.

[0041] In a preferred embodiment of the present invention, the yeast of the present invention includes those composed of yeast, Poaceae lipoxygenase, and a linker protein.

[0042] The yeast may be cultured and propagated under conditions suitable for yeast growth as needed.

[0043] (5) Use of the yeast The yeast of the present invention can be applied to any use that utilizes the Poaceae plant lipoxygenase.

[0044] Examples of the use of the yeast of the present invention include food additives.

[0045] The form of the food additive is not particularly limited, and examples include powders and dispersions.

[0046] Examples of food additives include those used in bread making, noodle making, etc.

[0047] An example of using the yeast of the present invention in bread making will be described below. For example, by adding the yeast of the present invention to the dough and fermenting it as it is, the addition of conventional purified lipoxygenase becomes unnecessary, so that higher-quality bread can be produced more efficiently (however, in the present invention, the mode of adding conventional purified lipoxygenase during bread making is not excluded).

[0048] <Method for producing yeast> The yeast of the present invention can be produced by any method capable of expressing the target protein on the cell surface in yeast.

[0049] For example, the yeast of the present invention can be produced according to the method of Inokuma et al. (Inokuma K, Kitada Y, Bamba T, Kobayashi Y, Yukawa T, den Haan R, van Zyl WH, Kondo A, Hasunuma T. “Improving the functionality of surface-engineered yeast cells by altering the cell wall morphology of the host strain.” Appl Microbiol Biotechnol. 2021 Aug;105(14-15):5895-5904. doi: 10.1007 / s00253-021-11440-6.) or according to JP-A-2020-156336.

[0050] As a preferred method for producing the yeast of the present invention, the method shown in the examples can be mentioned.

[0051] Hereinafter, an example of the method for producing the yeast of the present invention will be described. In the gene sequence design of yeast that expresses the target protein on the cell surface layer, usually, from upstream to downstream, the following regions are linked in the following order. Between each region and at the ends of each region, an arbitrary region may or may not be included as long as the expression of each region is not inhibited. · Promoter region · Secretion signal region · Region of target protein (Poaceae lipoxygenase) · Linker protein region (anchor domain) · Terminator region

[0052] (1) Promoter region The promoter region is a region having promoter activity, and transcriptional activity occurs when a transcription factor binds to this region.

[0053] The type, origin, etc. of the promoter constituting the promoter region are not particularly limited.

[0054] From the perspective of facilitating cell surface expression in yeast, the promoter is preferably a promoter used as the linker protein (anchor domain) region described below. Examples of such promoters include the SED1 promoter, TDH3 promoter, PGK1 promoter, and the like.

[0055] In a preferred embodiment of the present invention, the promoter is the SED1 promoter. Note that SED1 is a cell surface-localized protein during the stationary phase of the yeast Saccharomyces cerevisiae.

[0056] In a preferred embodiment of the present invention, the promoter has the gene sequence of SEQ ID NO: 9.

[0057] (2) Secretion signal region The secretion signal region is a region having the function of secreting the target protein (Poaceae lipoxygenase) outside the yeast cell.

[0058] The type, origin, etc. of the secretion signal constituting the secretion signal region are not particularly limited.

[0059] Examples of the secretion signal include the SED1 secretion signal peptide, α-factor signal peptide, MFα signal peptide, killer toxin signal peptide, and the like from the perspective of facilitating cell surface expression in yeast.

[0060] In a preferred embodiment of the present invention, the secretion signal is the SED1 secretion signal peptide. The secretion signal sequence of SED1 can be used in combination with the SED1 promoter sequence and the SED1 anchor domain (described below).

[0061] (3) Target protein region In the present invention, the target protein region is a region that expresses the above-described Poaceae lipoxygenase.

[0062] The DNA encoding the target protein may be a sequence encoding the full length of the target protein, or may be a partial sequence thereof as long as it has the activity of the target protein.

[0063] In a preferred embodiment of the present invention, the target protein (wheat lipoxygenase 3) is encoded by the gene sequence of SEQ ID NO: 1 or 3.

[0064] In a preferred embodiment of the present invention, the target protein (wheat lipoxygenase 3) has the amino acid sequence of SEQ ID NO: 2 or 4.

[0065] (4) Linker protein region The linker protein region corresponds to the anchor domain.

[0066] The "anchor domain" includes a region having an activity (anchor activity) capable of immobilizing the target protein on the cell surface of yeast.

[0067] As the linker protein region (anchor domain), any protein known as a cell surface-localized protein can be employed. In the present invention, the "cell surface-localized protein" includes a protein that is fixed to the cell surface and is localized on the surface of the fixed cell membrane.

[0068] The type, origin, etc. of the linker protein are not particularly limited as long as it functions in yeast.

[0069] Examples of the linker protein include GPI (glycosylphosphatidylinositol) anchor type proteins, transmembrane proteins, prenylated proteins, etc. from the viewpoint of facilitating cell surface expression in yeast.

[0070] In a preferred embodiment of the present invention, the linker protein is a GPI anchor type protein. GPI-anchored proteins have glycolipids (GPI) at their C-terminals, and by covalently binding GPI to phosphatidylinositol (PI) in the cell membrane, they bind to the cell membrane surface.

[0071] Examples of GPI-anchored proteins include SED1, GPI1, PGA1, etc.

[0072] In a preferred embodiment of the present invention, the GPI-anchored protein is SED1.

[0073] In cell surface expression in yeast, generally, the GPI-anchoring region is adopted. In the present invention, the "GPI-anchoring region" includes the whole cell surface-localized protein (GPI-anchored protein) and its cell wall-binding region (GPI-anchor attachment signal region). In this embodiment, as the "anchor domain", a region including the whole GPI-anchored protein or its cell wall-binding region (GPI-anchor attachment signal region) is used. The "cell wall-binding region" is usually a region on the C-terminal side of the cell surface-localized protein.

[0074] In a preferred embodiment of the present invention, the linker protein is encoded by the gene sequences of SEQ ID NO: 5 and 7.

[0075] In a preferred embodiment of the present invention, the linker protein has the amino acid sequences of SEQ ID NO: 6 and 8.

[0076] (5) Terminator region The terminator region is a region having terminator activity (activity to terminate transcription).

[0077] The type and origin of the terminator constituting the terminator region are not particularly limited.

[0078] Examples of the terminator include DIT1 terminator, TDH3 terminator, PGK1 terminator, ADH1 terminator, etc.

[0079] In a preferred embodiment of the present invention, the terminator has the gene sequence of SEQ ID NO: 10.

[0080] (6) Cell surface expression of the target protein As a method for cell surface expression of the target protein, rice lipoxygenase, any method known in the art can be employed. Typical examples of such methods include the method using an expression cassette and a cassette vector.

[0081] The "expression cassette" includes a construct in which genes corresponding to each region are ligated so that each region described above is expressed in a host microorganism or the like.

[0082] The "cassette vector" includes a vector into which an expression cassette is inserted for expressing the target protein. Examples of the form of the cassette vector include plasmid vectors.

[0083] For example, by introducing the above cassette vector into a host yeast by any method, the target protein can be cell surface expressed in the yeast. As the introduction method, any transformation method can be employed, for example, the lithium acetate method, the electroporation method, etc.

[0084] The yeast transformed with the cassette vector may be screened using, as an index, the activity of the rice lipoxygenase, which is the target protein. For example, by the method shown in the examples, the activity of the rice lipoxygenase is evaluated, and yeast showing the desired enzyme activity can be obtained as yeast satisfying the requirements of the present invention.

Examples

[0085] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0086] (Test 1) Sequence design of yeast expressing lipoxygenase 3 protein on the cell surface As lipoxygenase 3 (LOX3), sequence design of yeast expressing "rLOX3 isozyme" or "mini rLOX3 isozyme" on the cell surface was performed.

[0087] In this example, as shown in FIGS. 1 and 2, a promoter sequence (SED1 promoter), upstream of the linker sequence (SED1 secretion signal (N-terminal region of the linker protein)), wheat lipoxygenase 3 sequence (rLOX3, or mini rLOX3), downstream of the linker sequence (SED1 anchoring domain (C-terminal region of the linker protein)), and a terminator sequence (D1T1 terminator) were ligated in this order. Note that "MCS" is a ligation site.

[0088] The yeast designed in this example has a structure in which the lipoxygenase 3 protein is bound via a linker protein (GPI-anchor type protein (based on an artificial DNA sequence)) on the outer surface of the cell wall.

[0089] "rLOX3 isozyme" and "mini rLOX3 isozyme" are wheat lipoxygenase 3s in which the inventors performed codon optimization, respectively, and have the following gene sequences.

[0090] [Table 1]

[0091] [Table 2]

[0092] [Table 3]

[0093]

Table 4

[0094]

Table 5

[0095]

Table 6

[0096]

Table 7

[0097]

Table 8

[0098]

Table 9

[0099]

Table 10

[0100] The design of yeast that expresses the target protein on the cell surface was based on the method of Inokuma et al. (Inokuma K, Kitada Y, Bamba T, Kobayashi Y, Yukawa T, den Haan R, van Zyl WH, Kondo A, Hasunuma T. “Improving the functionality of surface-engineered yeast cells by altering the cell wall morphology of the host strain.” Appl Microbiol Biotechnol. 2021 Aug;105(14-15):5895-5904. doi: 10.1007 / s00253-021-11440-6.).

[0101] Based on the above method of Inokuma et al., yeast that expresses lipoxygenase 3 protein on the cell surface was designed. In this example, since the active site is contained in the C-terminal region of the lipoxygenase protein and the N-terminal region of the lipoxygenase protein is susceptible to cleavage, an anchor protein was attached to the C-terminal side of the lipoxygenase protein based on the orientation of the lipoxygenase protein and designed to be localized on the cell surface. Furthermore, in the lipoxygenase protein, since it is oriented outermost in the N-terminal region, it was assumed that it might affect the uptake of linoleic acid substrate by the lipoxygenase protein. Therefore, not only the full-length lipoxygenase protein (rLOX3 isozyme) but also the lipoxygenase protein with the N-terminal region truncated (mini rLOX3 isozyme) was used.

[0102] In this example, "pAUR101 DNA" (TaKaRa) was used as the chromosomal integration type yeast shuttle vector.

[0103] (Test 2) Acquisition of yeast that expresses lipoxygenase 3 protein on the cell surface Based on the design of the above "Test 1", yeast was actually prepared by the following method.

[0104] (1) Preparation of linker protein A cassette vector for expressing the linker protein required for cell surface expression in yeast was prepared by the following method.

[0105] (1-1) Cloning of cassette vector (linker protein) A cassette vector for expressing the linker protein was cloned by the following method.

[0106] The designed linker protein gene was amplified by PCR using "Prime STAR Max DNA polymerase" (TaKaRa) with primers ("CellDisplay Gibson Fw" (SEQ ID NO: 11) and "CellDisplay Gibson Rv" (SEQ ID NO: 12)).

[0107]

Table 11

[0108] The obtained PCR reaction product was purified using "FavorPrep GEL / PCR Purification Mini Kit" (FAVORGEN).

[0109] The chromosomal integration type yeast shuttle vector (pAUR101 DNA) was digested with restriction enzymes SalI and KpnI. Then, the restriction enzyme product was purified using "FavorPrep GEL / PCR Purification Mini Kit" (FAVORGEN). The purified restriction enzyme product was mixed with the digested shuttle vector to prepare a mixed DNA solution.

[0110] An equal volume of "Gibson Assembly Master Mix" (NEB) was added to the mixed DNA solution, and the mixture was incubated at 50 °C for 1 hour using a thermal cycler (BioRad). After incubation, transformation was performed using "E. coli JM109 competent cells" (TaKaRa), and it was spread on an LB agar medium (Bacto™ Tryptone, Bacto™ Yeast Extract, Bacto™ Agar) containing 150 μg / mL of ampicillin (Nacalai Tesque), and cultured overnight at 37°C. After culturing, the formation of E. coli colonies was confirmed.

[0111] (1-2) DNA sequencing analysis of the cassette vector (linker protein) Using a Shikazinia DNA extraction reagent (Kanto Chemical), a DNA extract was prepared from the E. coli colonies by the following method.

[0112] Using the DNA extract from the E. coli colonies as a template for the PCR reaction, the target DNA sequence was amplified with primers ("pAUR101 Fw" (SEQ ID NO: 13) and "pAUR101 Rv" (SEQ ID NO: 14)) using "TaKaRa Ex-Taq Hot Start" (TaKaRa). The temperature conditions for the PCR reaction were as follows: After a reaction at 95°C for 2 minutes, the following temperature cycle was performed 30 times: 95°C for 20 seconds → 55°C for 30 seconds → 72°C for 2 minutes.

[0113] The obtained PCR reaction product was purified using the "FavorPrep GEL / PCR Purification Mini Kit" (FAVORGEN).

[0114] For the purified PCR reaction product, nucleotide sequencing was performed by commissioned analysis (GeneWiz) using sequencing primers ("pAUR101 Fw" (SEQ ID NO: 13), "pAUR101 Rv" (SEQ ID NO: 14), "CellDisplay SEQ No.1" (SEQ ID NO: 15), "CellDisplay SEQ No.2" (SEQ ID NO: 16)). As a result, it was confirmed that the cassette vector as designed was obtained. Note that the cassette vector (linker protein) prepared in this example is also referred to as the "pAUR101-CellDisplay vector" hereinafter.

[0115]

Table 12

[0116] (1-3) Plasmid extraction of the cassette vector (linker protein) The transformant (E. coli) of the "pAUR101-CellDisplay vector" was cultured overnight at 37 °C in an LB liquid medium containing 150 μg / mL of ampicillin. After culturing, the bacterial cell culture solution was collected by centrifugation, and the "pAUR101-CellDisplay vector" was extracted and purified using the "FavorPrep Plasmid Extraction Mini Kit" (FAVORGEN).

[0117] (2) Preparation of lipoxygenase 3 (rLOX3 isozyme) A cassette vector expressing the lipoxygenase 3 (rLOX3 isozyme) protein was prepared by the following method.

[0118] (2-1) Molecular cloning of the cassette vector (rLOX3 isozyme) A cassette vector expressing "rLOX3 isozyme" was cloned by the following method.

[0119] "rLOX3 isozyme" was amplified by PCR using "Prime STAR Max DNA polymerase" (TaKaRa) and primers ("Full-LOX3 Gibson Fw" (SEQ ID NO: 17) and "Full-LOX3 Gibson Rv" (SEQ ID NO: 18)).

[0120]

Table 13

[0121] The obtained PCR reaction product was purified using the "FavorPrep GEL / PCR Purification Mini Kit" (FAVORGEN).

[0122] The "pAUR101-CellDisplay vector" was digested with the restriction enzyme SrfI. Subsequently, the restriction enzyme product was purified using the "FavorPrep GEL / PCR Purification Mini Kit" (FAVORGEN). The purified restriction enzyme product was mixed with the digested "pAUR101-CellDisplay vector" to prepare a mixed DNA solution.

[0123] An equal volume of "Gibson Assembly Master Mix" (NEB) was added to the mixed DNA solution, and the mixture was incubated at 50 °C for 1 hour using a thermal cycler (BioRad). After incubation, transformation was performed using "E. coli JM109 competent cells" (TaKaRa), and the cells were spread on an LB agar medium (Bacto™ Tryptone, Bacto™ Yeast Extract, Bacto™ Agar) containing 150 μg / mL of ampicillin (Nacalai Tesque), and cultured overnight at 37 °C. After culturing, the formation of E. coli colonies was confirmed.

[0124] (2-2) DNA sequencing analysis of the cassette vector (rLOX3 isozymes) Using a Schizochytrium DNA extraction reagent (Kanto Chemical), a DNA extract was prepared from E. coli colonies by the following method.

[0125] Using the DNA extract from E. coli colonies as a template for the PCR reaction, the target DNA sequence was amplified with primers ("pAUR101 Fw" (SEQ ID NO: 13) and "pAUR101 Rv" (SEQ ID NO: 14)) using "TaKaRa Ex-Taq Hot Start" (TaKaRa). Note that for the temperature conditions of the PCR reaction, after reacting at 95°C for 2 minutes, the following temperature cycle was performed 30 times: 95°C for 20 seconds → 55°C for 30 seconds → 72°C for 4 minutes.

[0126] The obtained PCR reaction product was purified using the "FavorPrep GEL / PCR Purification Mini Kit" (manufactured by FAVORGEN).

[0127] For the purified PCR reaction product, sequencing primers ("pAUR101 Fw" (SEQ ID NO: 13), "pAUR101 Rv" (SEQ ID NO: 14), "CellDisplay SEQ No.1" (SEQ ID NO: 15), "CellDisplay SEQ No.2" (SEQ ID NO: 16), "CellDisplay SEQ No.3" (SEQ ID NO: 19), "LOX3-SEQ No.1" (SEQ ID NO: 20), "LOX3-SEQ No.2" (SEQ ID NO: 21), "LOX3-SEQ No.3" (SEQ ID NO: 22), "LOX3-SEQ No.4" (SEQ ID NO: 23)) were used to determine the nucleotide sequence by entrusted analysis (GeneWiz). As a result, it was confirmed that the cassette vector was obtained as designed. Note that the cassette vector (rLOX3 isozymes) prepared in this example is also hereinafter referred to as the "pAUR101-LOX3-CellDisplay vector".

[0128]

Table 14

[0129] (2-3) Plasmid extraction of the cassette vector (rLOX3 isozymes) The transformant (E. coli) of the "pAUR101-LOX3-CellDisplay vector" was cultured overnight at 37°C in an LB liquid medium containing 150 μg / mL of ampicillin. After cultivation, the cells in the culture broth were collected by centrifugation, and the "pAUR101-rLOX3-CellDisplay vector" was extracted and purified using the "FavorPrep Plasmid Extraction Mini Kit" (FAVORGEN).

[0130] (3) Preparation of Lipoxygenase 3 (mini rLOX3 Isozyme) A cassette vector expressing the lipoxygenase 3 (mini rLOX3 isozyme) protein was prepared by the following method.

[0131] (3-1) Molecular Cloning of Cassette Vector (mini rLOX3 Isozyme) A cassette vector expressing the "mini rLOX3 isozyme" was cloned by the following method.

[0132] The "mini rLOX3 isozyme" was amplified by PCR using "Prime STAR Max DNA polymerase" (TaKaRa) and primers ("Mini-LOX3 Gibson Fw" (SEQ ID NO: 24), "Mini-LOX3 Gibson Rv" (SEQ ID NO: 25)).

[0133]

Table 15

[0134] The obtained PCR reaction product was purified using the "FavorPrep GEL / PCR Purification Mini Kit" (FAVORGEN).

[0135] The "pAUR101-CellDisplay vector" was digested with the restriction enzyme SrfI. Subsequently, the restriction enzyme product was purified using the "FavorPrep GEL / PCR Purification Mini Kit" (FAVORGEN). The purified restriction enzyme product was mixed with the cleaved "pAUR101-CellDisplay vector" to prepare a mixed DNA solution.

[0136] An equal volume of "Gibson Assembly Master Mix" (New England Biolabs) was added to the mixed DNA solution, and the mixture was incubated at 50 °C for 1 hour using a thermal cycler (Bio-Rad). After incubation, transformation was carried out using "E. coli JM109 competent cells" (Takara) and spread on Bacto™ Tryptone, Bacto™ Yeast Extract, Bacto™ Agar, and cultured overnight at 37 °C. After culturing, the formation of E. coli colonies was confirmed.

[0137] (3-2) DNA sequencing analysis of the cassette vector (mini rLOX3 isozymes) Using Shigecinia DNA extraction reagent (Kanto Chemical Co., Inc.), a DNA extract was prepared from E. coli colonies by the following method.

[0138] The DNA extract from E. coli colonies was used as a template for PCR reaction, and the target DNA sequence was amplified using "TaKaRa Ex-Taq Hot Start" (Takara) with primers ("pAUR101 Fw" (SEQ ID NO: 13), "pAUR101 Rv" (SEQ ID NO: 14)). The temperature conditions for the PCR reaction were as follows: after a reaction at 95 °C for 2 minutes, the following temperature cycle was performed 30 times: 95 °C for 20 seconds → 55 °C for 30 seconds → 72 °C for 4 minutes.

[0139] The obtained PCR reaction product was purified using "FavorPrep GEL / PCR Purification Mini Kit" (FAVORGEN).

[0140] For the purified PCR reaction product, nucleotide sequencing was performed by a consigned analysis (GeneWiz) using sequence primers (「pAUR101 Fw」(SEQ ID NO: 13), 「pAUR101 Rv」(SEQ ID NO: 14), 「CellDisplay SEQ No.1」(SEQ ID NO: 15), 「CellDisplay SEQ No.2」(SEQ ID NO: 16), 「CellDisplay SEQ No.3」(SEQ ID NO: 19), 「LOX3-SEQ No.1」(SEQ ID NO: 20), 「LOX3-SEQ No.2」(SEQ ID NO: 21), 「LOX3-SEQ No.3」(SEQ ID NO: 22), 「LOX3-SEQ No.4」(SEQ ID NO: 23)). As a result, it was confirmed that the cassette vector as designed was obtained. The cassette vector (mini rLOX3 isozyme) prepared in this example is also referred to as the 「pAUR101-Mini-LOX3-CellDisplay vector」 below.

[0141] (3-3) Plasmid Extraction of Cassette Vector (Mini rLOX3 Isozyme) The transformant (E. coli) of the 「pAUR101-Mini-LOX3-CellDisplay vector」 was cultured overnight at 37 °C in an LB liquid medium containing 150 μg / mL of ampicillin. After culturing, the bacterial cell culture solution was collected by centrifugation, and the 「pAUR101-Mini-LOX3-CellDisplay vector」 was extracted and purified using the 「FavorPrep Plasmid Extraction Mini Kit」 (FAVORGEN).

[0142] (4) Expression of Yeast Expressing Lipoxygenase 3 Protein on the Cell Surface Yeast expressing lipoxygenase 3 protein on the cell surface was obtained using each cassette vector by the following method.

[0143] The two types of cassette vectors obtained above (the "pAUR101-LOX3-CellDisplay vector" and the "pAUR101-Mini-LOX3-CellDisplay vector") were integrated into the genome of budding yeast (Saccharomyces cerevisiae BY4741) according to the attached instructions using the lithium acetate method of "Yeastmarker (trademark) Transformation System 2" (manufactured by TaKaRa) in the Aureobasidin A (aureobasidin A) resistant yeast transformation system (TaKaRa) to obtain yeast that expresses lipoxygenase 3 protein on the cell surface layer. The obtained yeast was spread on YPD agar medium containing 0.5 μg / ml of aureobasidin A and cultured overnight at 30°C.

[0144] DNA was extracted from the obtained yeast (colonies) using the Shikazinia DNA extraction reagent (Kanto Chemical Co., Inc.).

[0145] Using the DNA extract as a template for the PCR reaction, the target DNA sequence was amplified with primers ("pAUR101 Fw" (SEQ ID NO: 13) and "pAUR101 Rv" (SEQ ID NO: 14)) using "TaKaRa Ex-Taq Hot Start" (TaKaRa). The temperature conditions for the PCR reaction were as follows: after 95°C for 2 minutes, the following temperature cycle was performed 30 times: 95°C for 20 seconds → 55°C for 30 seconds → 72°C for 4 minutes.

[0146] The obtained PCR reaction product was electrophoresed on a 1% agarose gel (Nacalai Tesque, Inc.), and it was confirmed that a genomic insertion strain of the gene that expresses lipoxygenase 3 on the surface layer was constructed as designed.

[0147] The obtained yeast was subjected to the following activity evaluation. Hereinafter, yeast that expresses "rLOX3 isozyme" on the cell surface layer is also referred to as "rLOX3-displaying yeast". Yeast that expresses "mini rLOX3 isozyme" on the cell surface layer is also referred to as "mini rLOX3-displaying yeast".

[0148] (Test 3) Activity evaluation of "rLOX3-displaying yeast" The enzyme activity was evaluated using "rLOX3-displaying yeast" by the following method.

[0149] (1) Preparation of substrate Linoleic acid emulsion was prepared as a substrate for lipoxygenase. The linoleic acid emulsion was prepared based on the method of Surrey (1964). Specifically, linoleic acid (100 μl) was dissolved in a mixture of Tween20 (0.12 mL), 50 mM acetate buffer (pH 5.5, 2.5 mL), and 1.0 mM sodium hydroxide (0.32 mL). After the linoleic acid was dissolved, the mixture was diluted to 50 mL with 50 mM acetate buffer. The dilution was used as the substrate. The substrate was sealed under nitrogen and stored in the dark at 4°C until use.

[0150] (2) Cultivation of yeast "rLOX3-displaying yeast" was inoculated into YPD liquid medium containing 0.5 μg / ml aureobasidin A and pre-cultured at 30°C for about 1 day. Then, the pre-culture solution was added to YPD liquid medium (100 - 300 mL) containing 0.5 μg / ml aureobasidin A and cultured at 16°C for 1 - 3 days to obtain a cell culture solution.

[0151] In addition, non-recombinant budding yeast (wild-type yeast) was inoculated into YPD liquid medium and pre-cultured at 30°C for about 1 day. Then, the pre-culture solution was added to YPD liquid medium (100 - 300 mL) and cultured at 16°C for 1 - 3 days to obtain a cell culture solution.

[0152] Cells were collected by centrifugation from each of the obtained cell culture solutions.

[0153] (3) Enzyme reaction As samples, "rLOX3-displaying yeast" and non-recombinant budding yeast (wild-type yeast) were prepared. 8 ml of acetic acid buffer was added to a 50 ml tube and incubated at 30 °C. To a 1.5 ml tube, each sample (0.1 g) and 200 μl of the above acetic acid buffer were added and stirred to prepare a sample solution, which was stored in ice. Next, 3.8 ml of substrate was added to 8 ml of acetic acid buffer and stirred, then 200 μl of the sample solution was added and immediately stirred to conduct an enzymatic reaction. After the reaction, 1 ml of the reaction solution was taken each time and sterilization filtered through a 0.45 μm filter. Further, the reaction solution was incubated at 30 °C for 10, 20, 30, 40, 50, or 60 seconds, stirred, and then sterilization filtered through a 0.45 μm filter.

[0154] For each filtered sample, the absorbance at 234 nm was measured. In this test, "1 U of enzyme (lipoxygenase 3) activity" was defined as "the amount of enzyme of 1 M peroxide generated per minute" based on the absorbance at 234 nm.

[0155] The enzyme activities of lipoxygenase 3 protein in each sample are shown in Table 16. As shown in Table 16, the activity of "rLOX3-displaying yeast" was significantly higher compared to that of "wild-type yeast".

[0156]

Table 16

[0157] (Test 4) Evaluation of bread making using "rLOX3-displaying yeast" Using the following method, a bread making test based on the straight dough method was conducted using "rLOX3-displaying yeast".

[0158] As samples, "rLOX3-displaying yeast" and non-recombinant budding yeast (wild-type yeast) were prepared. To 50 g of wheat flour, 30 mL of water and each sample (1 g of yeast) were added, and the mixture was kneaded with a mixer (“Micro-Mixer”, manufactured by National MFC) for 5 minutes to obtain dough. Subsequently, the dough was rounded and fermented in an oven at 30°C for 90 minutes. After fermentation, the dough was baked in an oven at 200°C for 20 minutes. After baking, the volume of the bread was measured using the rapeseed displacement method.

[0159] The volume of each bread is shown in Table 17. As shown in Table 17, the volume (bulk) of the bread increased by using the “rLOX3-displaying yeast”. From this, it was shown that the “rLOX3-displaying yeast” has an effect equal to or better than that of conventional yeast in bread making.

[0160]

Table 17

[0161] In addition, the “rLOX3-displaying yeast” does not require operations (such as yeast disruption) for the isolation of lipoxygenase 3 protein, and by simply adding it to food materials as it is, the above-mentioned good effects can be obtained. Therefore, the yeast of the present invention can be easily introduced in the production of various foods (bread, noodles, etc.) and can contribute to the improvement of product quality.

[0162] (Test 5) Activity evaluation of “mini rLOX3-displaying yeast” Enzyme activity evaluation was performed in the same manner as in Test 3, except that “mini rLOX3-displaying yeast” was used instead of “rLOX3-displaying yeast”.

[0163] The enzyme activity of lipoxygenase 3 protein in each sample is shown in Table 18. As shown in Table 18, the activity of the “mini rLOX3-displaying yeast” was significantly higher compared to that of the “wild-type yeast”.

[0164]

Table 18

[0165] (Test 6) Evaluation of bread making using "mini rLOX3-displaying yeast" Bread making evaluation was carried out in the same manner as in Test 4, except that "mini rLOX3-displaying yeast" was used instead of "rLOX3-displaying yeast".

[0166] The volume of each loaf of bread is shown in Table 19. As shown in Table 19, the use of "mini rLOX3-displaying yeast" increased the volume of the bread. From this, it was shown that "mini rLOX3-displaying yeast" has an effect equal to or better than that of conventional yeast in bread making.

[0167] [Table 19]

[0168] In addition, "mini rLOX3-displaying yeast" does not require operations (such as yeast disruption) for the isolation of lipoxygenase 3 protein, and by simply adding it to food materials as it is, the above-mentioned good effects can be obtained. Therefore, the yeast of the present invention can be easily introduced in the production of various foods (such as bread and noodles) and can contribute to the improvement of product quality.

Claims

1. Yeast in which a gramineous plant lipoxygenase is expressed on the cell surface.

2. The yeast according to Claim 1, wherein the gramineous plant lipoxygenase is wheat lipoxygenase.

3. The yeast according to Claim 2, wherein the wheat lipoxygenase is wheat lipoxygenase 3.

4. The yeast according to Claim 1, wherein the lipoxygenase is expressed on the cell surface via a linker protein.

5. A food additive comprising the yeast according to any one of Claims 1 to 4.

6. The food additive according to Claim 5, which is used for bread making or noodle making.

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