Cells and method for producing ceramide using the same

Abstract

To provide cells that can produce α-hydroxyphytoceramide at a higher rate compared to wild-type cells. [Solution] Cells in which the ORM1 and ORM2 genes are deficient and the function of the KEI1 gene is reduced. This invention aims to promote a manufacturing process that reduces the burden on the global environment compared to conventional chemical synthesis of long-chain ceramides, by utilizing genome editing technology to realize a long-chain ceramide production process modeled after the natural production process. Because it is possible to construct a process that is more efficient and stable than conventional microbial production of long-chain ceramides, the burden on the environment can be further reduced and it has industrial application value. It will serve as a strong foundation for developing long-chain ceramide products that are useful in the pursuit of beauty and health.

Description

Technical Field

[0001] The present invention relates to cells and a method for producing ceramides using the same.

Background Art

[0002] Ceramide is one of the sphingolipids and is a general term for lipid molecules having a structure in which a sphingoid base and a long-chain fatty acid are amide-bonded. Ceramide is contained in the stratum corneum, which is the outermost layer of mammalian skin, and is a major component constituting the intercellular lipids of keratinocytes present in the stratum corneum. Ceramide is considered to play an important role, for example, in skin moisturization and prevention of allergen invasion. It is also known that ceramide decreases due to aging, ultraviolet rays, etc., and it has been clarified that this is one of the causes of dry skin and rough skin. For these reasons, the development of cosmetics or food and drink products for supplementing ceramide has been promoted. The cosmetics or food and drink products contain not only ceramide but also sphingolipids other than ceramide, or precursors of ceramide (such as sphingoid bases), and there are also those for the purpose of supporting the biosynthesis of ceramide.

[0003] For the development of the above-mentioned cosmetics or food and drink products, some technical means for producing sphingoid bases or sphingolipids have been proposed. For example, Patent Document 1 discloses a method for producing a target substance, which includes culturing yeast having the ability to produce a target substance in a medium containing an additive capable of associating with, binding to, solubilizing, and / or capturing the target substance, and recovering the target substance from the cells of the yeast and / or the medium, wherein the target substance is selected from the group consisting of sphingoid bases and sphingolipids. It is described that the production of phytosphingosine, which is one of the sphingoid bases, increased in the yeast mutant strain EYS4423 (Δcha1 Δlcb4 Δorm2 Δcka2[ScLCB1 ScLCB2][ScTSC10][PcSUR2]) due to the presence of α-cyclodextrin.

[0004] Furthermore, regarding sphingolipids, fungal sphingolipids possess an inositol-phosphate head group essential for cell survival, and this head group is added by inositol phosphorylceramide (IPC) synthase. Studies using Saccharomyces cerevisiae have revealed that the essential protein encoded by the KEI1 gene is a subunit of IPC synthase. It is known that temperature-responsive mutations in the Kei1 gene cause growth inhibition at 37°C (Non-Patent Literature 1). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Patent Application No. 2018-510904 [Non-patent literature]

[0006] [Non-Patent Document 1] Sato K, et al., “Kei1: a novel subunit of inositolphosphorylceramidesynthase, essential for its enzyme activity and Golgi localization.”, Mol BiolCell. 2009 Oct;20(20):4444-57. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The present invention aims to provide cells that can produce α-hydroxyphytoceramide at a higher rate compared to wild-type cells. The present invention also aims to provide a method for producing ceramide using said cells. [Means for solving the problem]

[0008] The inventors of the present invention have discovered that Saccharomyces cerevisiae lacking the ORM1 and ORM2 genes, and having a known temperature-responsive mutation in the KEI1 gene, maintains its proliferative capacity and has a high α-hydroxyphytoceramide production capacity compared to wild-type Saccharomyces cerevisiae, leading to the completion of the present invention.

[0009] The present invention includes, for example, the following: [1] Cells in which the ORM1 and ORM2 genes are deficient, and the function of the KEI1 gene is reduced. [2] The cells described in [1], in which the function of the KEI1 gene is reduced, and in which the KEI1 gene has a temperature-responsive mutation. [3] Cells of microbial origin, as described in [1] or [2]. [4] The cells described in [1] to [3], which are yeast cells, Aspergillus oryzae cells, or Acetylobacteria cells. [5] A yeast cell belonging to the genus Saccharomyces, as described in any of [1] to [4]. [6] Saccharomyces cerevisiae cells, as described in any of [1] to [5]. [7] A method for producing ceramide, comprising the step of culturing cells as described in any of [1] to [6]. [8] The method for producing the above-mentioned ceramide, comprising α-hydroxyphytoceramide, as described in [7]. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide cells that can produce α-hydroxyphytoceramide at a higher rate compared to wild-type cells. Furthermore, according to the present invention, it is possible to provide a method for producing ceramide using these cells. [Brief explanation of the drawing]

[0011] [Figure 1] It is a graph showing the amount of α-hydroxy phytoceramide per volume of the culture broth and the dry cell weight when yeast is cultured at 26.5°C. [Figure 2] It is a graph showing the amount of α-hydroxy phytoceramide per volume of the culture broth and the dry cell weight when yeast is cultured at 28°C. [Figure 3] It is a graph showing the amount of α-hydroxy phytoceramide per volume of the culture broth and the dry cell weight when yeast is cultured at 30°C.

Mode for Carrying Out the Invention

[0012] Hereinafter, the modes for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[0013] 〔Cell〕 The cell according to this embodiment has a deletion of the ORM1 gene and the ORM2 gene, and the function of the KEI1 gene is reduced. As a result, compared with wild-type cells, α-hydroxy phytoceramide can be highly produced. Since α-hydroxy phytoceramide is a kind of ceramide, it is considered that other ceramides can also be highly produced according to the cell according to this embodiment.

[0014] The type of the cell according to this embodiment is not particularly limited as long as it is a cell that originally has the ORM1 gene, the ORM2 gene, and the KEI1 gene, that is, a wild-type cell having the above three genes. Whether a wild-type cell has the above three genes or not can be analyzed by, for example, a known method. Examples of such analysis means include BLAST search using the known nucleotide sequences of the ORM1 gene, the ORM2 gene, and the KEI1 gene as queries, respectively.

[0015] The cells according to this embodiment may be, for example, cells derived from animals, plants, or microorganisms, and are preferably cells derived from microorganisms. Examples of animal-derived cells include cells from cows, pigs, chickens, sheep, fish, horses, etc. Examples of plant-derived cells include cells from rice, corn, pineapple, peach, yuzu, beet, taro, etc. As the cells derived from microorganisms, it is preferably a yeast cell, an Aspergillus oryzae cell, or an acetic acid bacterium cell, and more preferably a yeast cell.

[0016] Examples of yeast cells include yeast cells belonging to the genus Saccharomyces, yeast cells belonging to the genus Schizosaccharomyces, yeast cells belonging to the genus Pichia, yeast cells belonging to the genus Zygosaccharomyces, yeast cells belonging to the genus Kluyveromyces, yeast cells belonging to the genus Candida, yeast cells belonging to the genus Rhodotorula, yeast cells belonging to the genus Debaryomyces, yeast cells belonging to the genus Cryptococcus, yeast cells belonging to the genus Hansenula, yeast cells belonging to the genus Bullera, yeast cells belonging to the genus Sporobolomyces, yeast cells belonging to the genus Brettanomyces, and yeast cells belonging to the genus Yarrowia, etc. Yeast cells belonging to the genus Saccharomyces are preferred.

[0017] Examples of yeast cells belonging to the genus Saccharomyces include Saccharomyces cerevisiae cells, Saccharomyces pastorianus cells, Saccharomyces bayanus cells, Saccharomyces uvarum cells, Saccharomyces paradoxus cells, Saccharomyces kudriavzevii cells, and Saccharomyces eubayanus cells, with Saccharomyces cerevisiae cells being more preferred.

[0018] Examples of koji mold cells may include koji mold cells belonging to the genus Aspergillus, Rhizopus, Mucor, Monascus, and Neurospora.

[0019] Examples of koji mold cells belonging to the genus Aspergillus include Aspergillus oryzae cells, Aspergillus sojae cells, Aspergillus tamarii cells, Aspergillus kawachii cells, Aspergillus usamii mutant shirousamii cells, Aspergillus niger cells, and Aspergillus awamori cells, Aspergillus chevalieri cells, Aspergillus clavatus cells, Aspergillus fumigatus cells, and Aspergillus flavus cells. Examples include Aspergillus flavus cells, Aspergillus luchuensis cells, Aspergillus nidulans cells, Aspergillus puulaauensis cells, and Aspergillus fischeri cells.

[0020] Examples of koji mold cells belonging to the genus Rhizopus include Rhizopus oryzae cells and Rhizopus oligosporus cells.

[0021] Examples of Aspergillus cells belonging to the genus Mucor include Mucor indicus cells, Mucor circinelloides cells, and Mucor Miehei cells.

[0022] Examples of Aspergillus cells belonging to the genus Monascus include Monascus purpureus cells.

[0023] Examples of Aspergillus cells belonging to the genus Neurospora include Neurospora intermedia cells.

[0024] Examples of acetic acid bacteria cells may include acetic acid bacteria cells belonging to the genus Acetobacter, acetic acid bacteria cells belonging to the genus Gluconobacter, acetic acid bacteria cells belonging to the genus Komagataeibacter, and acetic acid bacteria cells belonging to the genus Gluconacetobacter.

[0025] Examples of acetic acid bacteria cells belonging to the genus Acetobacter include Acetobacter aceti cells and Acetobacter pasteurianus cells.

[0026] Examples of acetic acid bacteria cells belonging to the genus Gluconobacter include Gluconobacter oxydans cells.

[0027] Examples of acetic acid bacteria cells belonging to the genus Komagataeibacter include Komagataeibacter xylinus cells and Komagataeibacter europaeus cells.

[0028] Examples of acetic acid bacteria cells belonging to the genus Gluconacetobacter include Gluconacetobacter diazotrophicus cells and Gluconacetobacter hansenii cells.

[0029] The ORM1 and ORM2 genes (hereinafter collectively referred to as "ORM genes") each encode proteins that negatively regulate the activity of serine palmitoyltransferase (SPT), and are present in many eukaryotes, including yeast. SPT is an enzyme that functions in the early stages of sphingolipid biosynthesis, a component of the cell membrane, and catalyzes the following reactions. Palmitoyl-CoA + Serine → 3-Ketosphinganine → Sphingosine → Ceramide

[0030] Typical ORM proteins and the polynucleotides encoding them are shown in Table 1 below. Table 1 below shows the motifs and entries of typical ORM proteins and the polynucleotides encoding them that are registered in GenomeNet. The specific sequences corresponding to each registration name are disclosed at https: / / www.genome.jp / entry / K17308.

[0031] [Table 1] TIFF2026056127000002.tif215149 TIFF2026056127000003.tif221149 TIFF2026056127000004.tif221149 TIFF2026056127000005.tif221149 TIFF2026056127000006.tif221149 TIFF2026056127000007.tif222149 TIFF2026056127000008.tif226149 TIFF2026056127000009.tif221149 TIFF2026056127000010.tif226149 TIFF2026056127000011.tif221149 TIFF2026056127000012.tif226149 TIFF2026056127000013.tif221149 TIFF2026056127000014.tif226149 TIFF2026056127000015.tif226149 TIFF2026056127000016.tif221149 TIFF2026056127000017.tif221149 TIFF2026056127000018.tif226149 TIFF2026056127000019.tif226149 TIFF2026056127000020.tif221149 TIFF2026056127000021.tif226149 TIFF2026056127000022.tif226149 TIFF2026056127000023.tif226149 TIFF2026056127000024.tif209149

[0032] Examples of ORM1 genes include genes encoding proteins that have 50% or more sequence identity with the amino acid sequences shown in SEQ ID NOs. 1 and 44-78, and that have the function of negatively regulating SPT activity. This sequence identity may be, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or 100%.

[0033] The amino acid sequence shown in SEQ ID NO: 1 is the ORM1 protein of Saccharomyces cerevisiae. The amino acid sequence shown in SEQ ID NO: 44 is the amino acid sequence of Entry 497200 in Table 1. The amino acid sequence shown in SEQ ID NO: 45 is the amino acid sequence of Entry 109563382 in Table 1. The amino acid sequence shown in SEQ ID NO: 46 is the amino acid sequence of Entry 102282650 in Table 1. The amino acid sequence shown in SEQ ID NO: 47 is the amino acid sequence of Entry 396901 in Table 1. The amino acid sequence shown in SEQ ID NO: 48 is the amino acid sequence of Entry 107052201 in Table 1. The amino acid sequence shown in SEQ ID NO: 49 is the amino acid sequence of Entry 395220 in Table 1. The amino acid sequence shown in SEQ ID NO: 50 is the amino acid sequence of Entry 101103947 in Table 1. The amino acid sequence shown in SEQ ID NO: 51 is the amino acid sequence of Entry 129101258 in Table 1. The amino acid sequence shown in SEQ ID NO: 52 is the amino acid sequence of Entry 106517519 in Table 1. The amino acid sequence shown in SEQ ID NO: 53 is the amino acid sequence of Entry 113058128 in Table 1. The amino acid sequence shown in SEQ ID NO: 54 is the amino acid sequence of Entry 113114021 in Table 1. The amino acid sequence shown in SEQ ID NO: 55 is the amino acid sequence of Entry 113114022 in Table 1. The amino acid sequence shown in SEQ ID NO: 56 is the amino acid sequence of Entry 121583636 in Table 1. The amino acid sequence shown in SEQ ID NO: 57 is the amino acid sequence of Entry 121584963 in Table 1. The amino acid sequence shown in SEQ ID NO: 58 is the amino acid sequence of Entry 795897 in Table 1. The amino acid sequence shown in SEQ ID NO: 59 is the amino acid sequence of Entry 122827835 in Table 1. The amino acid sequence shown in SEQ ID NO: 60 is the amino acid sequence of Entry 128611931 in Table 1. The amino acid sequence shown in SEQ ID NO: 61 is the amino acid sequence of Entry 129422407 in Table 1. The amino acid sequence shown in SEQ ID NO: 62 is the amino acid sequence of Entry 107393894 in Table 1.The amino acid sequence shown in SEQ ID NO: 63 is the amino acid sequence of Entry 121322612 in Table 1. The amino acid sequence shown in SEQ ID NO: 64 is the amino acid sequence of Entry 130196183 in Table 1. The amino acid sequence shown in SEQ ID NO: 65 is the amino acid sequence of Entry 124385630 in Table 1. The amino acid sequence shown in SEQ ID NO: 66 is the amino acid sequence of Entry GSTEN00028692G001 in Table 1. The amino acid sequence shown in SEQ ID NO: 67 is the amino acid sequence of Entry 106836350 in Table 1. The amino acid sequence shown in SEQ ID NO: 68 is the amino acid sequence of Entry 106836351 in Table 1. The amino acid sequence shown in SEQ ID NO: 69 is the amino acid sequence of Entry 123275594 in Table 1. The amino acid sequence shown in SEQ ID NO: 70 is the amino acid sequence of Entry 100050034 in Table 1. The amino acid sequence shown in SEQ ID NO: 71 is the amino acid sequence of Entry 100050100 in Table 1. The amino acid sequence shown in SEQ ID NO: 72 is the amino acid sequence of Entry 100051420 in Table 1. The amino acid sequence shown in SEQ ID NO: 73 is the amino acid sequence of Entry 100051562 in Table 1. The amino acid sequence shown in SEQ ID NO: 74 is the amino acid sequence of Entry 103544122 in Table 1. The amino acid sequence shown in SEQ ID NO: 75 is the amino acid sequence of Entry 103544123 in Table 1. The amino acid sequence shown in SEQ ID NO: 76 is the amino acid sequence of Entry 103544905 in Table 1. The amino acid sequence shown in SEQ ID NO: 77 is the amino acid sequence of Entry 103545562 in Table 1. The amino acid sequence shown in SEQ ID NO: 78 is the amino acid sequence of Entry 103566635 in Table 1.

[0034] In this specification, sequence identity refers to the percentage (%) of matching bases or residues when the base sequences or amino acid sequences being compared are multiple-aligned. Multiple alignment refers to aligning base sequences or amino acid sequences by inserting appropriate gaps so that corresponding base sequences or amino acid sequence portions are aligned, in order to make them comparable to each other. For multiple alignment, known multiple alignment programs can be used. For example, Clustal W, Clustal X, and the BLAST program can be suitably used.

[0035] Examples of ORM2 genes include genes encoding proteins that have 50% or more sequence identity with the amino acid sequences shown in SEQ ID NOs. 2 and 44-78, and that have the function of negatively regulating SPT activity. This sequence identity may be, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or 100%.

[0036] The amino acid sequence shown in SEQ ID NO: 2 is the ORM2 protein of Saccharomyces cerevisiae. The amino acid sequence shown in SEQ ID NO: 44 is the amino acid sequence of Entry 497200 in Table 1. The amino acid sequence shown in SEQ ID NO: 45 is the amino acid sequence of Entry 109563382 in Table 1. The amino acid sequence shown in SEQ ID NO: 46 is the amino acid sequence of Entry 102282650 in Table 1. The amino acid sequence shown in SEQ ID NO: 47 is the amino acid sequence of Entry 396901 in Table 1. The amino acid sequence shown in SEQ ID NO: 48 is the amino acid sequence of Entry 107052201 in Table 1. The amino acid sequence shown in SEQ ID NO: 49 is the amino acid sequence of Entry 395220 in Table 1. The amino acid sequence shown in SEQ ID NO: 50 is the amino acid sequence of Entry 101103947 in Table 1. The amino acid sequence shown in SEQ ID NO: 51 is the amino acid sequence of Entry 129101258 in Table 1. The amino acid sequence shown in SEQ ID NO: 52 is the amino acid sequence of Entry 106517519 in Table 1. The amino acid sequence shown in SEQ ID NO: 53 is the amino acid sequence of Entry 113058128 in Table 1. The amino acid sequence shown in SEQ ID NO: 54 is the amino acid sequence of Entry 113114021 in Table 1. The amino acid sequence shown in SEQ ID NO: 55 is the amino acid sequence of Entry 113114022 in Table 1. The amino acid sequence shown in SEQ ID NO: 56 is the amino acid sequence of Entry 121583636 in Table 1. The amino acid sequence shown in SEQ ID NO: 57 is the amino acid sequence of Entry 121584963 in Table 1. The amino acid sequence shown in SEQ ID NO: 58 is the amino acid sequence of Entry 795897 in Table 1. The amino acid sequence shown in SEQ ID NO: 59 is the amino acid sequence of Entry 122827835 in Table 1. The amino acid sequence shown in SEQ ID NO: 60 is the amino acid sequence of Entry 128611931 in Table 1. The amino acid sequence shown in SEQ ID NO: 61 is the amino acid sequence of Entry 129422407 in Table 1. The amino acid sequence shown in SEQ ID NO: 62 is the amino acid sequence of Entry 107393894 in Table 1.The amino acid sequence shown in SEQ ID NO: 63 is the amino acid sequence of Entry 121322612 in Table 1. The amino acid sequence shown in SEQ ID NO: 64 is the amino acid sequence of Entry 130196183 in Table 1. The amino acid sequence shown in SEQ ID NO: 65 is the amino acid sequence of Entry 124385630 in Table 1. The amino acid sequence shown in SEQ ID NO: 66 is the amino acid sequence of Entry GSTEN00028692G001 in Table 1. The amino acid sequence shown in SEQ ID NO: 67 is the amino acid sequence of Entry 106836350 in Table 1. The amino acid sequence shown in SEQ ID NO: 68 is the amino acid sequence of Entry 106836351 in Table 1. The amino acid sequence shown in SEQ ID NO: 69 is the amino acid sequence of Entry 123275594 in Table 1. The amino acid sequence shown in SEQ ID NO: 70 is the amino acid sequence of Entry 100050034 in Table 1. The amino acid sequence shown in SEQ ID NO: 71 is the amino acid sequence of Entry 100050100 in Table 1. The amino acid sequence shown in SEQ ID NO: 72 is the amino acid sequence of Entry 100051420 in Table 1. The amino acid sequence shown in SEQ ID NO: 73 is the amino acid sequence of Entry 100051562 in Table 1. The amino acid sequence shown in SEQ ID NO: 74 is the amino acid sequence of Entry 103544122 in Table 1. The amino acid sequence shown in SEQ ID NO: 75 is the amino acid sequence of Entry 103544123 in Table 1. The amino acid sequence shown in SEQ ID NO: 76 is the amino acid sequence of Entry 103544905 in Table 1. The amino acid sequence shown in SEQ ID NO: 77 is the amino acid sequence of Entry 103545562 in Table 1. The amino acid sequence shown in SEQ ID NO: 78 is the amino acid sequence of Entry 103566635 in Table 1.

[0037] In this specification, "a gene is missing" means that a gene lacks its function within a cell. Specific embodiments of "a missing ORM1 gene and ORM2 gene" include, for example, a state in which the ORM1 gene and ORM2 gene are deleted from the cell's genomic DNA, a state in which there is a mutation in the ORM1 gene and ORM2 gene that prevents translation into a normal protein, and a state in which the cell has the base sequence encoding the ORM1 gene and ORM2 gene, but the transcription of the gene is suppressed and no protein is produced. Preferably, the ORM1 gene and ORM2 gene are deleted from the cell's genomic DNA, or there is a mutation in the ORM1 gene and ORM2 gene that prevents translation into a normal protein.

[0038] More specific examples of mutations in the ORM1 gene that prevent translation into a normal protein include cases where the gene has nonsense mutations, frameshift mutations, splicing site mutations, etc. Mutations in the ORM1 gene may include, for example, a substitution of positions 122 to 129 of the amino acid sequence shown in SEQ ID NO: 1 from RSYVMFHL (SEQ ID NO: 3) to GLCHVPSD (SEQ ID NO: 4), and a deletion of amino acid residues from positions 130 to 222. In this case, the amino acid sequence of the ORM1 protein is the amino acid sequence shown in SEQ ID NO: 5.

[0039] More specific examples of mutations in the ORM2 gene that prevent translation into a normal protein include cases where the gene has nonsense mutations, frameshift mutations, splicing site mutations, etc. A mutation in the ORM2 gene may, for example, be a deletion of amino acid residues at positions 142 to 216 of the amino acid sequence shown in SEQ ID NO: 2. In this case, the amino acid sequence of the ORM2 protein is the one shown in SEQ ID NO: 6.

[0040] The KEI1 gene is known to be an essential gene in yeast, and its deficiency is lethal. As mentioned above, the KEI1 protein is a subunit of IPC synthase (Aur1) and is thought to be necessary for both Aur1 activity (the activity of converting ceramide to IPC) and the localization of Aur1 to the Golgi apparatus.

[0041] Typical KEI1 proteins and the polynucleotides encoding them are shown in Table 2 below. Table 2 below shows the motifs and entries of typical KEI1 proteins and the polynucleotides encoding them that are registered on GenomeNet. The specific sequences corresponding to each registration name are disclosed at https: / / www.genome.jp / entry / K22724.

[0042] [Table 2] TIFF2026056127000026.tif223149 TIFF2026056127000027.tif222149 TIFF2026056127000028.tif223149 TIFF2026056127000029.tif223149 TIFF2026056127000030.tif113149

[0043] Examples of KEI1 genes include genes that have 50% or more sequence identity with the amino acid sequences shown in SEQ ID NOs. 7 and 79-87, and that encode a protein having the subunit activity of the above-mentioned IPC synthase (Aur1). The above sequence identity may be, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or it may be 100%.

[0044] The protein consisting of the amino acid sequence shown in SEQ ID NO: 7 is the KEI1 protein of Saccharomyces cerevisiae. The protein consisting of the amino acid sequence shown in SEQ ID NO: 79 is the amino acid sequence with Entry ACHE_51240S in Table 2. The protein consisting of the amino acid sequence shown in SEQ ID NO: 80 is the amino acid sequence with Entry ACLA_074520 in Table 2. The protein consisting of the amino acid sequence shown in SEQ ID NO: 81 is the amino acid sequence with Entry AFUA_2G15580 in Table 2. The protein consisting of the amino acid sequence shown in SEQ ID NO: 82 is the amino acid sequence with Entry AFLA_001987 in Table 2. The protein consisting of the amino acid sequence shown in SEQ ID NO: 83 is the amino acid sequence with Entry AKAW2_60196A in Table 2. The protein consisting of the amino acid sequence shown in SEQ ID NO: 84 is the amino acid sequence with Entry ANIA_07601 in Table 2. The protein consisting of the amino acid sequence shown in SEQ ID NO: 85 is the amino acid sequence with Entry AO090012000272 in Table 2. The protein consisting of the amino acid sequence shown in SEQ ID NO: 86 is the amino acid sequence with Entry APUU_80326A in Table 2. The protein consisting of the amino acid sequence shown in SEQ ID NO: 87 is the amino acid sequence with Entry NFIA_090810 in Table 2.

[0045] In this specification, "reduced gene function" means, for example, a decrease in function compared to the wild-type gene function, either constitutively or only under specific conditions, and may be a decrease of, for example, 10% to less than 100%, 20% to less than 100%, 30% to less than 100%, 40% to less than 100%, 50% to less than 100%, 60% to less than 100%, 70% to less than 100%, 80% to less than 100%, or 90% to less than 100%. Furthermore, "gene function" includes not only the physiological activity function of the protein encoded by the gene, but also the function of the gene in expressing (transcribe or translating) said protein.

[0046] Specific examples of "reduced function of the KEI1 gene" include, for example, a state in which a mutation in the KEI1 gene leads to the translation of a protein with reduced function, and a state in which a cell has a base sequence encoding the KEI1 gene, but the transcription of the gene is suppressed and protein production is reduced (however, the gene is not deleted). Preferably, the state in which a mutation in the KEI1 gene leads to the translation of a protein with reduced function is preferred, and more preferably, the KEI1 gene has a temperature-responsive mutation.

[0047] In this specification, "temperature-responsive mutation" means a mutation in which the function of a gene decreases in response to ambient temperature. A temperature-responsive mutation may be, for example, a mutation in which the function of a gene decreases compared to the wild-type gene under conditions of 26.5°C or higher, or between 26.5°C and 33°C.

[0048] KEI1 genes with reduced function can be obtained by methods known to those skilled in the art. For example, they can be easily obtained by randomly introducing mutations into wild-type KEI1 genes and then screening them for reduced function compared to wild-type genes. In particular, KEI1 genes with temperature-sensitive mutations can be easily obtained by randomly introducing mutations into wild-type KEI1 genes and then culturing them under conditions of 26.5°C or higher, and then screening them for reduced function compared to wild-type genes.

[0049] Temperature-responsive mutations in the KEI1 gene may include, for example, a substitution of the phenylalanine residue at position 103 with an isoleucine residue in the amino acid sequence shown in SEQ ID NO: 7, a substitution of QN to KT between positions 193 and 194, and a deletion of amino acid residues between positions 195 and 221. When cultured at 37°C, the KEI1 gene with these temperature-responsive mutations exhibits such reduced gene function that yeast growth is inhibited. The amino acid sequence of the KEI1 protein in this case is the amino acid sequence shown in SEQ ID NO: 8.

[0050] The cells according to this embodiment may have mutations in genes other than the gene described above. Such genes may be, for example, genes involved in the synthesis of sphingoid bases or sphingolipids, or other genes.

[0051] The cells according to this embodiment can be produced, for example, by the method described in detail in the examples below.

[0052] [Method for producing ceramide] The method for producing ceramide according to this embodiment includes a step of culturing the cells according to this embodiment (culture step). As described above, the cells according to this embodiment can produce α-hydroxyphytoceramide in high quantities compared to wild-type yeast. Furthermore, it is thought that they can also produce other ceramides in high quantities. Therefore, the cells according to this embodiment are useful for the production of ceramide.

[0053] In the culture process, ceramide is produced within eukaryotic cells by culturing cells in a culture medium, and then accumulates within the yeast cells or in the culture medium.

[0054] The culture process can be carried out under general conditions for culturing cells and can be set as appropriate by those skilled in the art. For example, the culture temperature may be 20°C to 35°C. The culture in the culture process may be carried out at a constant temperature or at a temperature that is varied as appropriate. When culturing at a temperature that is varied, it may include culturing at 26.5°C to 30°C. The pH of the culture medium may be 6 to 8 or 7.0 to 7.5. The culture time may be 1 to 7 days or 2 to 3 days. The culture medium is not particularly limited as long as it allows cells to proliferate, but examples include Eagle's MEM medium, DMEM, Modified Eagle medium (MEM), SD medium, SG medium, SDTE medium, YPD medium, etc.

[0055] The method for producing ceramide according to this embodiment may further include a step of purifying the ceramide produced in the culture step from the culture (purification step). The culture in the purification step may be the culture medium after the culture step, or it may include the cells and culture medium after the culture step.

[0056] In the purification process, various purification methods commonly used in this field, such as size exclusion chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography (HPLC), dialysis, salting out, ammonium sulfate precipitation, precipitation, and crystallization, can be used in appropriate combinations. If the culture contains the cells and culture medium after the above-mentioned culture process, the supernatant obtained by removing cell lysates, for example by disrupting the cells and centrifuging them, can be applied to the above purification method. [Examples]

[0057] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0058] (1) Microorganisms used and culture method Escherichia coli DH5α (TOYOBO) was used for all plasmid amplification. DH5α was cultured in Luria-Bertani medium containing 100 μg / mL ampicillin (5.0 g / L Yeast Extract Dried (Nacalai Tesque), 10.0 g / L Tryptone, and 5.0 g / L NaCl). Saccharomyces cerevisiae (S. cerevisiae) YPH499 [MATa ura3-52 lys2-801 ade2-101 trp1- 63 his3-Δ200 leu2-Δ1 (Stratagene)] was used as the host for the ceramide metabolic pathway transfection strains. For culturing yeast strains, SC medium [6.7 g / L BD Difco® Yeast Nitrogen Base without Amino Acids (Difco Laboratories), 20 g / LD(+)-Glucose] with amino acids and nucleic acids added according to the strain's nutritional requirements, or YPD medium [10.0 g / L Yeast Extract Dried (Nacalai Tesque), 20.0 g / L Bacto Peptone (Thermo Fisher Scientific), 20.0 g / LD(+)-Glucose] were used. For yeast transformation, SC medium was used with amino acids and nucleic acids appropriately removed according to the strain's nutritional requirements. Pre-culture was performed at 30°C and 200 rpm.

[0059] (2) Plasmid creation All plasmids created in this example were created using the In-Fusion (registered trademark) HD Cloning Kit (Takara). Briefly, a homologous sequence of 15 bases at the ends of the restriction enzyme-treated vector was added to both ends of the base sequence to be cloned by PCR, and the vector, the cloning sequence, and the In-Fusion enzyme were mixed and incubated at 50 °C for 15 minutes to create the plasmid. Table 3 shows the amino acid sequences of the proteins expressed from the wild-type ORM1 gene, ORM2 gene, and KEI gene of yeast used in this example, and the amino acid sequences of the proteins expressed from the ORM1 gene, ORM2 gene, and KEI gene into which mutations were introduced in this example. Table 4 shows the plasmids created in this example. Table 5 shows the primers used for the plasmids. Furthermore, Table 6 shows the DNA fragments created when creating the plasmids created in this example.

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Table 3

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Table 4

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Table 5

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Table 6

[0064] <Construction of p2gRNA-ORM1, p2gRNA-ORM2, and p2gRNA-KEI1> The gRNA expression vectors p2gRNA-ORM1 (SEQ ID NO: 12), p2gRNA-ORM2 (SEQ ID NO: 13), or p2gRNA-KEI1 (SEQ ID NO: 14) were constructed according to the following procedure. First, DNA fragment 1 (linear plasmid, SEQ ID NO: 40) containing a gRNA scaffold linked to the SNR52 promoter (SNR52p) and the SUP4 terminator (SUP4t) was prepared. DNA fragment 1 was prepared by extension via PCR using the Sc-gRNA F primer (SEQ ID NO: 18) and the Sc-gRNA R primer (SEQ ID NO: 19) with the p2gRNA-CCW12 plasmid (SEQ ID NO: 11) as a template. Next, as the DNA fragment to be incorporated into DNA fragment 1, the annealed product of the ORM1 gRNA F primer (SEQ ID NO: 22) and the ORM1 gRNA R primer (SEQ ID NO: 23), the ORM2 gRNA F primer (SEQ ID NO: 24) and the ORM2 gRNA R primer (SEQ ID NO: 25), or the KEI1 gRNA F primer (SEQ ID NO: 26) and the KEI1 gRNA R primer (SEQ ID NO: 27) was used. By incorporating the annealed DNA fragment into DNA fragment 1 (p2gRNA vector), p2gRNA-ORM1 (SEQ ID NO: 12), p2gRNA-ORM2 (SEQ ID NO: 13), or p2gRNA-KEI1 (SEQ ID NO: 15) was obtained.

[0065] <Construction of p2gRNA-ORM1-ORM2> PCR was performed using p2gRNA-ORM2 (SEQ ID NO: 13) as a template with the primers ORM1-ORM2 gRNA F (SEQ ID NO: 20) and ORM1-ORM2 gRNA R (SEQ ID NO: 21) to create an ORM2 gRNA expression cassette. The resulting DNA fragment was cloned into p2gRNA-ORM1 digested with SmaI (New England Biolabs) to create p2gRNA-ORM1-ORM2 (SEQ ID NO: 14).

[0066] <Construction of pUC19-kei1ts_donor> A plasmid carrying the gene encoding kei1ts, a temperature-responsive mutant of Kei1, was constructed by the following method. Using the genomic DNA of the YPH499 strain as a template, fragments (KEI1 donor F1R1 (SEQ ID NO: 41), KEI1 donor F2R2 (SEQ ID NO: 42), KEI1 donor F3R3 (SEQ ID NO: 43)) obtained by PCR using primers KEI1 donor F1 (SEQ ID NO: 32) and KEI1 donor R1 (SEQ ID NO: 33), KEI1 donor F2 (SEQ ID NO: 34) and KEI1 donor R2 (SEQ ID NO: 35), KEI1 donor F3 (SEQ ID NO: 36) and KEI1 donor R3 (SEQ ID NO: 37) were ligated with pUC19 (NIPPON GENE, SEQ ID NO: 16) cut with BamHI-HF (New England Biolabs) using In-Fusion to construct pUC19-kei1ts_donor (SEQ ID NO: 17).

[0067] (3) Preparation of donor DNA <Preparation of donor DNA for ORM1 and ORM2 disruption> 100 pM of ORM1 donor F (SEQ ID NO: 28) and ORM1 donor R (SEQ ID NO: 29), and ORM2 donor F (SEQ ID NO: 30) and ORM2 donor R (SEQ ID NO: 31) were mixed in equal amounts and heated at 100°C for 5 minutes. Then, it was cooled to 25°C and annealed to prepare donor DNA for ORM1 and ORM2 disruption.

[0068] <Preparation of donor DNA for introducing temperature-responsive mutation of KEI1> Using pUC19-kei1ts_donor as a template, the fragment obtained by PCR using KEI1 donor F4 (SEQ ID NO: 38) and KEI1 donor R4 (SEQ ID NO: 39) was used as donor DNA for introducing the temperature-responsive mutation of KEI1.

[0069] (4) Preparation of yeast strains Yeast transformation was carried out according to the One-step transformation method using lithium acetate (Chen DC, et al., Curr Genet 1992, 21:83-84.). The yeast strains used in this example are shown in Table 7.

[0070] <Creation of YPH499(+ADE2) strain> In the YPH499 strain, to repair the mutation in the ADE2 gene encoding AIR carboxylase that catalyzes the sixth step of the purine biosynthesis pathway, plasmid pGK402 (Ishii et al., 2009, SEQ ID NO: 9) was digested with EcoRV-HF (New England Biolabs) and transformed into the YPH499 strain to create the YPH499(+ADE2) strain.

[0071] <Creation of YPH499 orm1Δ strain> First, plasmid pCL-TEF1p-Cas9-CYC1t (Inokuma et al., 2021, SEQ ID NO: 10) was introduced into the YPH499(+ADE2) strain (hereinafter referred to as "YPH499(+ADE2) / pCL-TEF1p-Cas9-CYC1t strain"). Subsequently, p2gRNA-ORM1 and ORM1 disruption donor DNA were introduced into the YPH499(+ADE2) / pCL-TEF1p-Cas9-CYC1t strain, and the YPH499 orm1Δ strain was created by homologous recombination via cleavage of the target region by CRISPR-Cas9.

[0072] <Creation of YPH499 orm2Δ strain> First, plasmid pCL-TEF1p-Cas9-CYC1t (Inokuma et al., 2021, SEQ ID NO: 10) was introduced into the YPH499(+ADE2) strain. Subsequently, p2gRNA-ORM2 and ORM2 disruption donor DNA were introduced into the YPH499(+ADE2) / pCL-TEF1p-Cas9-CYC1t strain, and the YPH499 orm2Δ strain was created by homologous recombination via cleavage of the target region by CRISPR-Cas9.

[0073] <Generation of YPH499 orm1 / 2Δ strain> First, the plasmid pCL-TEF1p-Cas9-CYC1t (Inokuma et al., 2021, SEQ ID NO: 10) was introduced into the YPH499(+ADE2) strain. Subsequently, p2gRNA-ORM1-ORM2 and donor DNA for ORM1 disruption and donor DNA for ORM2 disruption were introduced into the YPH499(+ADE2) / pCL-TEF1p-Cas9-CYC1t strain, and the YPH499 orm1 / 2Δ strain was generated by homologous recombination via cleavage of the target region by CRISPR-Cas9.

[0074] <Generation of YPH499 orm1 / 2Δkei1ts strain> First, the plasmid pCL-TEF1p-Cas9-CYC1t (Inokuma et al., 2021, SEQ ID NO: 10) was introduced into YPH499 orm1 / 2Δ (hereinafter, this strain is also referred to as "YPH499 orm1 / 2Δ / pCL-TEF1p-Cas9-CYC1t strain"). Subsequently, p2gRNA-kEI1 and donor DNA for introducing the temperature-responsive mutation of KEI1 were introduced into the YPH499 orm1 / 2Δ / pCL-TEF1p-Cas9-CYC1t strain, and the YPH499 orm1 / 2Δkei1ts strain was generated by homologous recombination via cleavage of the target region by CRISPR-Cas9.

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Table 7

[0076] (5) Evaluation of ceramide production The four yeast strains prepared in (4) were cultured in YPD medium for 72 hours under temperature conditions of 26.5 °C, 28 °C, or 30 °C. Thereafter, the amount of α-hydroxy phytoceramide per volume of the culture solution was measured by the following procedure. Also, the dry cell weight per volume of the culture solution was measured. The results are shown in Figures 1 to 3.

[0077] 500 μL of culture medium was collected and centrifuged at 12,000 rpm for 30 seconds. The supernatant was then removed, 500 μL of 150 mM NH4HCO3 (pH 8) was added, and the mixture was centrifuged again at 12,000 rpm for 30 seconds at room temperature, and the supernatant was removed again. Subsequently, 400 μL of 150 mM NH4HCO3 (pH 8) was added to suspend the mixture, and this suspension was transferred to a 1.5 ml screw-cap microtube (self-standing type, Biomedical Science) containing one 0.2 mL single PCR tube's worth of 0.6 mm zirconia beads (Biomedical Science). The mixture was then disrupted at 1,500 rpm for 5 minutes using a benchtop bead disruptor, ShakeMaster Neo (Biomedical Science). 20 μL of the disrupted sample was diluted 10-fold by adding 180 μL of 150 mM NH4HCO3 (pH 8). To the solution, 10 μL of 5 ng / μL C19 ceramide (d18:1 / 19:0) (Cayman Chemical) and 990 μL of CHCl3 / MeOH=17 / 1 were added as internal standard solutions. Ceramide was extracted by shaking at 1,500 rpm for 120 minutes at 4°C using a well plate compatible constant temperature shaking incubator Maximizer (Taitec). The extract was centrifuged at 12,000 rpm for 30 seconds at room temperature, and the lower organic layer was collected in a microcentrifuge tube. The solution was then evaporated using a vacuum freeze-dryer (Labconco) and a centrifugal concentrator (Labconco).

[0078] To the sample dried by evaporation, 500 μL of a chloroform:methanol = 1:2 mixed solvent was added and vortexed for 5 minutes. Then, sonication was performed for 10 minutes, followed by centrifugation at 10000 × g for 1 minute. 5 μL of the supernatant after centrifugation was collected and analyzed by liquid chromatography-mass spectrometry (LC-MS / MS). An ExionLC system (SCIEX) and a Unison UK-C8 (2.1 mm × 150 mm, particle size 3 μm) (Imtakt) column were used for liquid chromatography. Eluent A was water containing 0.2% formic acid and 0.0442% ammonium formate, and eluent B was methanol containing 0.2% formic acid and 0.0316% ammonium formate. The eluent gradient was set to 10% (2 min) → 100% (15 min) → 10% (3 min) for eluent B. Mass spectrometry was performed using a Triple Quad 6500 + LC-MS / MS (SCIEX), with ionization performed by electrospray ionization. The analysis was conducted in positive ion mode, and the MS parameters for specific analytes were set as follows: For the C26-α-hydroxyphytoceramide ion, the mass at the first quadrupole (Q1) was set to 712.600 Da and the mass at the second quadrupole (Q2) was set to 694.800 Da. For the C19 ceramide ion, the mass at Q1 was set to 580.400 Da and the mass at Q2 was set to 264.100 Da. After standardizing the signal value with an internal standard, quantification was performed by comparing it with the area value of the standard sample.

[0079] As shown in Figures 1-3, under all temperature conditions, the dry cell weight per unit area of ​​culture medium of the YPH499 orm1 / 2Δ kei1ts strain was higher than that of the YPH499 orm1 / 2Δ strain and the YPH499 kei1ts strain, and was comparable to or greater than that of the YPH499 strain. Furthermore, under all temperature conditions, the YPH499 orm1 / 2Δ kei1ts strain had the highest amount of α-hydroxyphytoceramide per unit area of ​​culture medium.

[0080] These results indicate that the YPH499 orm1 / 2Δ kei1ts strain maintains or improves its proliferative capacity and also has a higher α-hydroxyphytoceramide production capacity compared to the YPH499 strain (wild-type).

[0081] From the above, it was revealed that Saccharomyces cerevisiae lacking the ORM1 and ORM2 genes, and possessing a known temperature-responsive mutation in the KEI1 gene, maintains its proliferative capacity and has a high α-hydroxyphytoceramide production capacity compared to wild-type Saccharomyces cerevisiae. Furthermore, this invention aims to promote a manufacturing process that reduces the burden on the global environment compared to conventional chemical synthesis of long-chain ceramides, by utilizing genome editing technology to realize a long-chain ceramide production process modeled after the natural production process. Because it is possible to construct a process that is more efficient and stable than conventional microbial production of long-chain ceramides, the burden on the environment can be further reduced, and it has industrial application value. It will serve as a strong foundation for developing long-chain ceramide products useful in the pursuit of beauty and health.

Claims

1. Cells in which the ORM1 and ORM2 genes are deficient, and the function of the KEI1 gene is reduced.

2. The cell according to claim 1, wherein the reduced function of the KEI1 gene is due to the KEI1 gene having a temperature-responsive mutation.

3. The cell according to claim 1, which is a cell derived from a microorganism.

4. The cell according to claim 1, which is a yeast cell, an Aspergillus oryzae cell, or an Acetylobacter cell.

5. The cell according to claim 1, which is a yeast cell belonging to the genus Saccharomyces.

6. The cell according to claim 1, which is a Saccharomyces cerevisiae cell.

7. A method for producing ceramide, comprising the step of culturing cells according to any one of claims 1 to 6.

8. The manufacturing method according to claim 7, wherein the ceramide comprises α-hydroxyphytoceramide.

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