Method for producing β-carotene and β-carotene derivative, and genetically modified microorganism

Abstract

The purpose of the present disclosure is to provide: a method for producing β-carotene and a β-carotene derivative; and a genetically modified microorganism. The present disclosure provides: a method for producing β-carotene and / or a β-carotene derivative using a genetically modified microorganism that belongs to the genus Lipomyces; and a genetically modified microorganism that belongs to the genus Lipomyces, and that has a β-carotene biosynthesis pathway and produces β-carotene and / or a β-carotene derivative through fermentation.

Description

Method for producing β-carotene and β-carotene derivatives and genetically modified microorganisms

[0001] The present invention relates to a method for producing β-carotene and β-carotene derivatives, and to a genetically modified microorganism.

[0002] Carotenoids are known to have various physiological activities, but because animals cannot synthesize carotenoids on their own, they must be ingested from food, etc. In recent years, carotenoids have been used for a variety of purposes, including food flavorings, coloring agents, nutritional supplements, cosmetics, animal feed additives, and supplements. In particular, β-carotene not only has antioxidant properties, but is also known to be a precursor to vitamin A, which has effects such as maintaining the function of the skin and mucous membranes and maintaining eyesight.

[0003] Carotenoids can be produced by extraction from natural sources, chemical synthesis, or microbial fermentation, but currently most are produced by chemical synthesis. Because the raw materials for chemical synthesis are derived from fossil fuels and the production process requires a large amount of heat, a resource-recycling process using microbial fermentation is anticipated. However, carotenoid production by microbial fermentation has a low yield, and to date, with the exception of astaxanthin, no carotenoids have been commercialized.

[0004] For example, there have been reports of the production of β-carotene and retinol by modifying metabolic pathways and studying culture methods in the budding yeast Saccharomyces cerevisiae (hereinafter referred to as S. cerevisiae) (Patent Document 1, Non-Patent Document 1, Non-Patent Document 2). There have also been reports of the production of β-carotene and retinol in the oleaginous yeast Yarrowia lipolytica (hereinafter referred to as Y. lipolytica) (Patent Document 2, Non-Patent Document 3). There has also been a report on the production of lycopene using the oleaginous yeast Lipomyces starkeyi (hereinafter referred to as L. starkeyi) (Non-Patent Document 4).

[0005] U.S. Patent No. 9,297,031 International Publication No. 2023,044,937

[0006] Yijin Zhao et al. , Biotechnology and Bioengineering, 2021, 118, 2043-2052 Qiongyue Hu et al. , Bioresources and Bioprocessing, 2022, 9:22 Yongshuo Ma et al. , Nature Communications, 2022, 13:572 Hirosuke Kanamoto et al. , Carotenoids: Biosynthetic and Biofunctional Approaches, Springer Nature Singapore, 2021, 153-163

[0007] As described above, attempts have been made to produce β-carotene and retinol by microbial fermentation using the oleaginous yeast Y. lipolytica, but problems have arisen, such as the long culture time and the difficulty of culturing due to mycelium formation, which are factors hindering commercialization. In particular, Y. lipolytica has a tendency to form mycelium, and this mycelium formation is a factor that causes a decrease in culture efficiency.

[0008] Although there have been reports on lycopene production using the oleaginous yeast L. starkeyi, the productivity is low, and no strain applicable to industrial production has been identified. Furthermore, there have been no reports on the production of β-carotene and retinol using L. starkeyi, and their potential has not yet been fully explored.

[0009] Yeast cells take two forms: a round "yeast form" and a filamentous "hyphal form," with some species taking one or both forms. While S. cerevisiae and L. starkeyi basically take the "yeast form," Y. lipolytica can switch from the "yeast form" to the "hyphal form" due to changes in the external environment. When Y. lipolytica transitions to the "hyphal form," problems such as adhesion of the hyphae to the walls of the culture tank arise, making efficient fermentation difficult. Given this background, there is a demand for a method for producing β-carotene and retinol using a host that takes the "yeast form" and does not form hyphae. However, reports on the "yeast form" budding yeast S. cerevisiae have shown that the production efficiency of β-carotene and retinol is still insufficient, and its productivity has not yet reached the level of commercial production.

[0010] Therefore, an object of the present disclosure is to provide a new method for producing β-carotene and β-carotene derivatives, and a genetically modified microorganism.

[0011] The present inventors have discovered that by using a genetically modified microorganism of the genus Lipomyces, which is a type of oleaginous yeast, β-carotene and β-carotene derivatives can be produced more efficiently than by conventional methods, and have completed the present invention.

[0012] The present disclosure includes the following: 1. A method for producing at least one of β-carotene and a β-carotene derivative using a genetically modified microorganism of the genus Lipomyces. 2. The method according to 1 above, wherein the β-carotene derivative is at least one of retinol, β-cryptoxanthin, zeaxanthin, canthaxanthin, astaxanthin, fucoxanthin, capsanthin, abscisic acid, β-ionone, and retinal. 3. The method according to 1 above, wherein the genetically modified microorganism is Lipomyces starchii. 4. The method according to 1 above, comprising culturing the genetically modified microorganism to produce at least one of β-carotene and a β-carotene derivative in a culture, wherein the genetically modified microorganism has a β-carotene biosynthetic pathway. 5. 5. The method according to 4, wherein the β-carotene biosynthetic pathway comprises at least one selected from farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, phytoene synthase, phytoene desaturase, and lycopene cyclase. 6. The method according to 5, wherein the β-carotene biosynthetic pathway comprises at least one selected from Xanthophyllomyces dendrous-derived geranylgeranyl diphosphate synthase, Mucor circinelloides-derived phytoene synthase, Mucor circinelloides-derived lycopene synthase, and Mucor circinelloides-derived lycopene cyclase. 7. The method according to 6, wherein the β-carotene biosynthetic pathway comprises at least one protein selected from the following [1] to [4]:[1] At least one protein selected from the following (1a) to (1c): (1a) a protein comprising the amino acid sequence represented by SEQ ID NO: 36; (1b) a mutant protein comprising an amino acid sequence in which 1 to 37 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 36, and having geranylgeranyl diphosphate synthase activity; (1c) a homologous protein comprising an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 36, and having geranylgeranyl diphosphate synthase activity; [2] At least one protein selected from the following (2a) to (2c): (2a) a protein comprising the amino acid sequence represented by SEQ ID NO: 37; (2b) a mutant protein comprising an amino acid sequence in which 1 to 60 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 37, and having phytoene synthase activity; (2c) a homologous protein comprising an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 37, and having phytoene synthase activity. (3) At least one protein selected from the following (3a) to (3c): (3a) a protein comprising the amino acid sequence represented by SEQ ID NO: 37; (3b) a mutant protein comprising an amino acid sequence represented by SEQ ID NO: 37 in which 1 to 60 amino acids have been deleted, substituted, inserted or added, and having lycopene cyclase activity; (3c) a homologous protein comprising an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 37, and having lycopene cyclase activity; [4] At least one protein selected from the following (4a) to (4c): (4a) a protein comprising the amino acid sequence represented by SEQ ID NO: 38; (4b) a mutant protein comprising an amino acid sequence represented by SEQ ID NO: 38 in which 1 to 58 amino acids have been deleted, substituted, inserted or added, and having phytoene desaturase activity; (4c) a homologous protein comprising an amino acid sequence represented by SEQ ID NO: 38 in which 50% or more identity has been added, and having phytoene desaturase activity. 8. The method according to 1, wherein the genetically modified microorganism is a microorganism in which the activity of a lycopene cyclase is not inhibited by lycopene produced in the genetically modified microorganism. 9. The method according to 1, comprising any one of the following (α1) to (α7):(α1) The genetically modified microorganism has at least one of β-carotene dioxygenase and retinal reductase, and by culturing the genetically modified microorganism, retinol is produced in the culture. (α2) The genetically modified microorganism has β-carotene dioxygenase, and by culturing the genetically modified microorganism, retinal is produced in the culture. (α3) The genetically modified microorganism has acyltransferase, and by culturing the genetically modified microorganism, retinol palmitate is produced in the culture. (α4) The genetically modified microorganism has β-carotene hydroxylase, and by culturing the genetically modified microorganism, zeaxanthin is produced in the culture. (α5) The genetically modified microorganism has β-carotene hydroxylase, and by culturing the genetically modified microorganism, β-cryptoxanthin is produced in the culture. (α6) The genetically modified microorganism has β-carotene ketolase, and by culturing the genetically modified microorganism, canthaxanthin is produced in the culture. (α7) The genetically modified microorganism has β-carotene hydroxylase and β-carotene ketolase, and astaxanthin is produced in a culture by culturing the genetically modified microorganism. 10. The method according to 9 above, which is any one of the following (β1), (β2), and (β4) to (β7):(β1) A protein comprising the (α1), wherein the β-carotene dioxygenase in the (α1) is a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 39 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 39. (β2) A protein comprising the (α2), wherein the β-carotene dioxygenase in the (α2) is a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 39 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 39. (β4) A protein comprising the (α4), wherein the β-carotene hydroxylase in the (α4) is a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 52 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 52, or a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 54 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 54. (β5) The protein comprises the (α5), wherein the β-carotene hydroxylase in the (α5) is a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 56 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 56. (β6) The protein comprises the (α6), wherein the β-carotene ketolase in the (α6) is at least one of a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 58 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 58, or a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 60 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 60. (β7) The protein comprises the (α7), wherein the β-carotene hydroxylase in the (α7) is a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 62 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 62, and the β-carotene ketolase is a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 58 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 58. 11. The method according to 1 above, wherein the efficiency of conversion of lycopene to β-carotene by the genetically modified microorganism is 30% or more. 12. The method according to 11 above, wherein the efficiency of β-carotene production is 1.2 μg / wet cell weight / h or more.13. The method according to 12 above, wherein the β-carotene derivative is retinol and the retinol production efficiency is 0.26 mg / L / h or more. 14. A genetically modified microorganism of the genus Lipomyces, which has a β-carotene biosynthetic pathway and produces at least one of β-carotene and a β-carotene derivative by fermentation. 15. The genetically modified microorganism according to 14 above, which further has at least one of β-carotene dioxygenase and retinal reductase and produces retinol by fermentation. 16. A method for producing a β-carotene derivative, comprising culturing the genetically modified microorganism according to 14 or 15 above to produce a β-carotene derivative in a culture and recovering the β-carotene derivative from the culture. 17. A method for producing at least one of β-carotene and a β-carotene derivative using a microorganism in which lycopene produced intracellularly does not inhibit the activity of lycopene cyclase. 18. The method according to 17 above, wherein the microorganism is a microorganism capable of producing farnesyl diphosphate intracellularly using an endogenous enzyme.

[0013] According to the production method of the present disclosure, at least one of β-carotene and a β-carotene derivative can be efficiently produced by using a genetically modified microorganism of the genus Lipomyces.

[0014] Figure 1 shows the results of analyzing the amounts of β-carotene and lycopene produced per gram of wet cell weight (g). Figure 2 shows the results of analyzing the amount of retinol produced. Figure 3 shows an embodiment of the retinol biosynthetic pathway, including the β-carotene biosynthetic pathway. Figure 4 shows an embodiment of the astaxanthin biosynthetic pathway, including the β-carotene biosynthetic pathway. Figure 5A shows chromatograms of a retinal preparation and the dodecane layer of the culture medium, detected with UV at a wavelength of 350 nm. Figure 5B shows the results of analyzing the amount of retinal produced. Figure 6 shows chromatograms of a retinol palmitate preparation and the cell lysate, detected with UV at a wavelength of 325 nm. Figure 7 shows chromatograms of a retinol palmitate preparation and the cell lysate, detected with m / z = 269. Figure 8 shows chromatograms of a zeaxanthin preparation and the cell lysate, detected with visible light at a wavelength of 450 nm. Figure 9 shows chromatograms of a β-cryptoxanthin preparation and cell lysate detected with visible light at a wavelength of 450 nm. Figure 10 shows chromatograms of a canthaxanthin preparation and cell lysate detected with visible light at a wavelength of 450 nm. Figure 11 shows chromatograms of an astaxanthin preparation and cell lysate detected with visible light at a wavelength of 450 nm. Figure 12 shows the results of a comparison of retinol productivity for different sugar sources by retinol-producing L. starkeyi strains.

[0015] 1. Method for Producing β-Carotene and β-Carotene Derivatives The method of this embodiment (hereinafter also referred to as the present production method) is characterized by producing at least one of β-carotene and β-carotene derivatives using a genetically modified microorganism of the genus Lipomyces.

[0016] <β-Carotene and β-Carotene Derivatives> In this specification, "β-carotene" is a type of terpenoid. It is a fat-soluble substance biosynthesized from eight isoprene units, and is a precursor of the "β-carotene derivatives" described below. In this specification, the "β-carotene derivatives" are those having the basic structure of β-carotene (C ４０ The term refers to a group of compounds that have a tetraterpene skeleton, the skeleton of which has been partially modified by oxidation, reduction, esterification, or other chemical reactions. This includes redox products derived from the β-carotene molecule and derivatives with ester bonds.

[0017] Examples of β-carotene derivatives include retinol (vitamin A1), β-cryptoxanthin, zeaxanthin, canthaxanthin, astaxanthin, fucoxanthin, capsanthin, abscisic acid, β-ionone, retinal (vitamin A2), lycopene, lutein, retinoic acid (vitamin A acid), retinol acetate, retinol palmitate (retinyl palmitate), Examples of the β-carotene derivatives include retinol, retinal, β-cryptoxanthin, zeaxanthin, canthaxanthin, astaxanthin, and retinol palmitate, and retinol linoleate, retinol linolenate, retinol oleate, retinol linolenate, retinol oleate, retinol linolenate, retinol oleate, retinol linolenate, retinol oleate, retinol linoleate, retinol linoleate ester, retinol linolenate glyceride, retinol linolenate triglyceride, retinol linolenate ethyl ester, retinol linolenate methyl ester, retinol linolenate amide, retinol linolenate phenylamide, retinol linolenate hexylamide, retinol linolenate stearylamide, retinol linolenate oleylamide, retinol linolenate linoleylamide, and retinol linolenate linolenylamide. Among these, preferred β-carotene derivatives are retinol, retinal, β-cryptoxanthin, zeaxanthin, canthaxanthin, astaxanthin, and retinol palmitate, with retinol being most preferred.

[0018] As used herein, the term "β-carotene biosynthetic pathway" refers to a series of steps leading to the synthesis of β-carotene, including the linking of isoprene units, synthesis of diterpenes, oxidation and dehydration of diterpenes, and synthesis of β-carotene.

[0019] In this embodiment, "having a β-carotene biosynthetic pathway" may mean that the microorganism originally contains an enzyme involved in the β-carotene biosynthetic pathway, or that a foreign gene encoding the enzyme has been introduced. Alternatively, a foreign gene encoding the enzyme may be further introduced into a genetically modified microorganism that originally contains an enzyme involved in the β-carotene biosynthetic pathway. Furthermore, expressing an enzyme contained in one biological species in another biological species that does not contain the enzyme is referred to as heterologous expression.

[0020] Whether a genetically modified microorganism has a β-carotene biosynthetic pathway can be confirmed by culturing the microorganism, diluting the culture appropriately, centrifuging it, and detecting the amount of β-carotene contained in at least one of the supernatant and the bacterial cells using HPLC or the like.

[0021] A diagram showing one aspect of the retinol synthesis pathway, including the β-carotene biosynthesis pathway, is shown in Figure 3. As shown in Figure 3, retinol is synthesized from farnesyl diphosphate (FPP) to obtain geranylgeranyl diphosphate (GGPP), phytoene, lycopene, β-carotene, and retinal.

[0022] A diagram showing one embodiment of the astaxanthin biosynthetic pathway, including the β-carotene biosynthetic pathway, is shown in Figure 4. As shown in Figure 4, there are two pathways in which astaxanthin is synthesized from β-carotene via β-cryptoxanthin and zeaxanthin, and another pathway in which astaxanthin is synthesized via canthaxanthin.

[0023] <Genetically modified microorganisms of the genus Lipomyces> Examples of microorganisms of the genus Lipomyces include Lipomyces starkeyi, Lipomyces arxii, Lipomyces doornjongii, Lipomyces japonicus, Lipomyces kockii, Lipomyces kononenkooae, Lipomyces lipofer, and Lipomyces mesembrius. mesembrius, Lipomyces oligophaga, Lipomyces orientalis, Lipomyces smithiae, Lipomyces spencer-martinsiae, Lipomyces suomiensis, Lipomyces tetrasporus, Lipomyces yamadae, Lipomyces yarrowii, Lipomyces yarrowii), Lipomyces starkeyoides, Lipomyces starkeyoides var. barbatus, Lipomyces starkeyoides var. minimus, Lipomyces starkeyoides var. macrosporus, Lipomyces starkeyoides var. microsporus), Lipomyces starkeyoides var. minoris,Lipomyces starkeyoides var. major, Lipomyces starkeyoides var. minor, Lipomyces starkeyoides var. macro, Lipomyces starkeyoides var. micro, Lipomyces starkeyoides var. macros, Lipomyces starkeyoides micros Lipomyces starkeyoides var. micros, Lipomyces starkeyoides var. macro, Lipomyces starkeyoides var. mino, Lipomyces starkeyoides var. macrospor, Lipomyces starkeyoides var. microspor, Lipomyces starkeyoides majorus Lipomyces starkeyoides var. majors, Lipomyces starkeyoides var. minors, Lipomyces starkeyoides var. macroris, and Lipomyces starkeyoides var. microris. Among these, Lipomyces starkeyi (e.g., L. starkeyi CBS1807) is preferred.

[0024] It has been reported that lycopene can inhibit the activity of lycopene cyclase (Yongshuo Ma et al., Nature Communications, 2022, 13:572). It has also been reported that lycopene produced in microorganisms similarly inhibits the activity of lycopene cyclase (Yongshuo Ma et al., Nature Communications, 2022, 13:572).

[0025] On the other hand, the present inventors have found that in the case of the genus Lipomyces, there is almost no inhibition of the activity of lycopene cyclase by lycopene produced in the microorganism, and that β-carotene and β-carotene derivatives may be produced efficiently. Furthermore, because microorganisms of the genus Lipomyces usually have a yeast-like morphology, they are less likely to form mycelia and adhere to the walls of a culture tank. Therefore, the present inventors have found that the present production method, by using a genetically modified microorganism of the genus Lipomyces, enables the production of at least one of β-carotene and β-carotene derivatives with higher efficiency than conventional methods.

[0026] The genetically modified microorganism of this embodiment is a Lipomyces microorganism. Because Lipomyces microorganisms typically do not have an endogenous β-carotene biosynthetic pathway, it is preferable to introduce foreign genes encoding some or all of the enzymes involved in the β-carotene biosynthetic pathway. Alternatively, the β-carotene biosynthetic pathway may be enhanced by enhancing the expression of endogenous genes possessed by Lipomyces microorganisms.

[0027] When a foreign gene encoding an enzyme involved in the β-carotene biosynthesis pathway is introduced into the genus Lipomyces, the nucleotide sequence of the foreign gene and the amino acid sequence of the enzyme can be obtained from the literature and the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and can be codon-optimized for L. starkeyi.

[0028] The genetically modified microorganism in this embodiment has a β-carotene biosynthetic pathway, and the β-carotene biosynthetic pathway preferably includes at least one enzyme selected from farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, phytoene synthase, phytoene desaturase, and lycopene cyclase.

[0029] Farnesyl diphosphate synthase is an enzyme that synthesizes farnesyl diphosphate (FPP), an intermediate required for carotenoid synthesis. Examples of sources of farnesyl diphosphate synthase include L. starkeyi, S. cerevisiae, and Y. lipolytica.

[0030] Geranylgeranyl diphosphate synthase (GGPPS) is an enzyme that synthesizes geranylgeranyl diphosphate (GGPP), a precursor of an isoprenoid unit. Examples of geranylgeranyl diphosphate synthase include those derived from Xanthophyllomyces dendrorhous, Haematococcus pluvialis (Accession No. QHF16627.1), Pantoea agglomerans (Accession No. BAB79600.1), Arabidopsis thaliana (Accession No. CAB16803.1), and Mucor circinelloides (Accession No. CI8001.1). Specific examples of geranylgeranyl diphosphate synthase include geranylgeranyl diphosphate synthase derived from Xanthophyllomyces dendrorhous (amino acid sequence: SEQ ID NO: 36, nucleotide sequence: SEQ ID NO: 1).

[0031] Phytoene synthase (PSY) is an enzyme involved in the synthesis of phytoene in the carotenoid biosynthetic pathway. Examples of phytoene synthase include those derived from Mucor circinelloides, Xanthophyllomyces dendrorhous (Accession No. Q7Z859.1), Pantoea agglomerans (Accession No. BAB79604.1), Arthrospira platensis (Accession No. BAA20384.1), and Blakeslea trispora (Accession No. BAA20384.1). AAO46893.1) and Aspergillus oryzae (Accession No. BAE63386.1). Specific examples of phytoene synthase include phytoene synthase derived from Mucor circinelloides (amino acid sequence: SEQ ID NO: 37, nucleotide sequence: SEQ ID NO: 2).

[0032] Phytoene desaturase (PDS) is an enzyme that desaturates phytoene and is also called lycopene synthase. Examples of phytoene desaturases include those derived from Mucor circinelloides, Xanthophyllomyces dendrothrous (Accession No. AAO53257.1), Arabidopsis thaliana (Accession No. CAB10200.1), and Rhodobacter capsulatus (Accession No. CAA36533.1). Specific examples of phytoene desaturases include phytoene desaturase derived from Mucor circinelloides (amino acid sequence: SEQ ID NO: 38, nucleotide sequence: SEQ ID NO: 3).

[0033] Lycopene cyclase (LC) is an enzyme that cyclizes lycopene molecules during carotenoid synthesis, and is also called β-carotene synthase. Examples of lycopene cyclase include those derived from Mucor circinelloides, Xanthophyllomyces dendrorhous (Accession No. Q7Z859.1), Nonlabens dokdonensis (Accession No. AGC77519.1), and Flavobacterium sp. (strain P99-3) (Accession No. BAC77673.1), and Algoriphagus marincola (Accession No. KPQ14994.1). Specific examples of lycopene cyclases include lycopene cyclase derived from Mucor circinelloides (amino acid sequence: SEQ ID NO: 37, nucleotide sequence: SEQ ID NO: 2).

[0034] There are reports of heterologous expression of phytoene synthase / lycopene cyclase and phytoene desaturase derived from Mucor circinelloides in S. cerevisiae and Y. lipolytica (Xiang Jia et al., The Royal Society of Chemistry, 2018, 8, 34967-34972, Yongshuo Ma et al., Nature Communications, 2022, 13:572).

[0035] The genetically modified microorganism in this embodiment has a β-carotene biosynthetic pathway, and the β-carotene biosynthetic pathway preferably includes at least one selected from geranylgeranyl diphosphate synthase derived from Xanthophyllomyces dendrorhous, phytoene synthase derived from Mucor circinelloides, lycopene synthase derived from Mucor circinelloides, and lycopene cyclase derived from Mucor circinelloides.

[0036] The genetically modified microorganism in this embodiment preferably has a β-carotene biosynthetic pathway, and the β-carotene biosynthetic pathway preferably contains at least one protein selected from the following [1] to [4]: [1] At least one protein selected from the following (1a) to (1c): (1a) a protein comprising the amino acid sequence represented by SEQ ID NO: 36; (1b) a mutant protein comprising an amino acid sequence in which 1 to 37 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 36, and having geranylgeranyl diphosphate synthase activity; (1c) a homologous protein comprising an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 36, and having geranylgeranyl diphosphate synthase activity; [2] At least one protein selected from the following (2a) to (2c): (2a) a protein comprising the amino acid sequence represented by SEQ ID NO: 37; (2b) a mutant protein comprising an amino acid sequence in which 1 to 60 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 37, and having phytoene synthase activity; (2c) a homologous protein comprising an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 37, and having phytoene synthase activity. [3] At least one protein selected from the following (3a) to (3c): (3a) a protein comprising the amino acid sequence represented by SEQ ID NO: 37; (3b) a mutant protein comprising an amino acid sequence in which 1 to 60 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 37, and having lycopene cyclase activity; (3c) a homologous protein comprising an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 37, and having lycopene cyclase activity; [4] At least one protein selected from the following (4a) to (4c): (4a) a protein comprising the amino acid sequence represented by SEQ ID NO: 38; (4b) a mutant protein comprising an amino acid sequence in which 1 to 58 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 38, and having phytoene desaturase activity; (4c) a homologous protein comprising an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 38, and having phytoene desaturase activity.

[0037] The above [1] to [4] may be the following [1'] to [4'], respectively. [1'] At least one protein selected from the following (1a') to (1c'): (1a') a protein consisting of the amino acid sequence represented by SEQ ID NO: 36 (1b') a mutant protein consisting of an amino acid sequence represented by SEQ ID NO: 36 in which 1 to 37 amino acids are deleted, substituted, inserted or added, and having geranylgeranyl diphosphate synthase activity (1c') a homologous protein consisting of an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 36 and having geranylgeranyl diphosphate synthase activity [2'] At least one protein selected from the following (2a') to (2c'): (2a') a protein consisting of the amino acid sequence represented by SEQ ID NO: 37 (2b') a mutant protein consisting of an amino acid sequence represented by SEQ ID NO: 37 in which 1 to 60 amino acids are deleted, substituted, inserted or added, and having phytoene synthase activity (2c') A homologous protein having phytoene synthase activity, which comprises an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 37. [3'] At least one protein selected from the following (3a') to (3c'): (3a') A protein consisting of the amino acid sequence represented by SEQ ID NO: 37. (3b') A mutant protein having lycopene cyclase activity, which comprises an amino acid sequence represented by SEQ ID NO: 37 in which 1 to 60 amino acids are deleted, substituted, inserted or added. (3c') A homologous protein having lycopene cyclase activity, which comprises an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 37. [4'] At least one protein selected from the following (4a') to (4c'): (4a') A protein consisting of the amino acid sequence represented by SEQ ID NO: 38. (4b') A mutant protein having phytoene desaturase activity, which comprises an amino acid sequence represented by SEQ ID NO: 38 in which 1 to 58 amino acids are deleted, substituted, inserted or added. (4c') A homologous protein having an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 38 and having phytoene desaturase activity.

[0038] In the mutant proteins of [1] to [4] above, "amino acids deleted, substituted, inserted or added" may mean that 1 to 30 amino acids are deleted, substituted, inserted or added at any position in the same sequence. In [1] to [4] above, the number of amino acids deleted, substituted, inserted or added is 1 to 25, preferably 1 to 20, 1 to 15, 1 to 10, more preferably 1 to 5, and most preferably 1 to 3.

[0039] The amino acids to be deleted, substituted, inserted, or added may be naturally occurring or non-naturally occurring. Naturally occurring amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine.

[0040] Examples of amino acids that can be substituted for each other are shown below. Amino acids in the same group can be substituted for each other. Group A: Hydrophobic amino acids Group B: Acidic amino acids Group C: Polar amino acids Group D: Basic amino acids Group E: Secondary amino acids Group F: Amino acids with a hydroxyl group Group G: Aromatic amino acids Group H: Sulfur-containing amino acids

[0041] More specifically, they are as follows: Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: asparagine, glutamine Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid Group E: proline, 3-hydroxyproline, 4-hydroxyproline Group F: serine, threonine, homoserine Group G: phenylalanine, tyrosine

[0042] The homologous proteins in [1] to [4], (β1), (β2), and (β4) to (β7) preferably have at least 50% identity, more preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more identity, compared to the amino acid sequence on which they are based. When even higher identity is required, the homologous proteins preferably have 85% or more, even more preferably 90% or more, 95% or more, particularly preferably 97% or more, and most preferably 99% or more identity.

[0043] In this embodiment, the percentage of sequence identity between two amino acid sequences or two nucleotide sequences is calculated as the ratio of matching residues when the two sequences are aligned so that the residues contained in the two sequences are most identical. For example, the percentage of sequence identity can be determined using a mathematical algorithm.

[0044] Examples of such mathematical algorithms include the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482, the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, the similarity search method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448, and the similarity search method of Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877, and an improved version of the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, although mathematical algorithms are not limited to these examples.

[0045] Alignment for determining the percentage of sequence identity can be performed using programs based on these mathematical algorithms, and the programs can be executed by a computer, if appropriate. Examples of such programs include, but are not limited to, the PC / Gene program CLUSTAL (available from Intelligenetics, Mountain View, Calif.), MAFFT (Katoh, K., Misawa, K., Kuma, K., & Miyata, T. (2002), 30(14), 3059-3066., http: / / mafft.cbrc.jp / alignment / server / ), and MUSCLE (Edgar R. C. (2004). Nucleic acids research, 32(5), 1792-1797., http: / / www.ebi.ac.uk / Tools / msa / muscle / ), BLAST, FASTA, and TFASTA.

[0046] Alignment using these programs can be performed, for example, using default parameters. The CLUSTAL program is described in Higgins et al. (1988) Gene 73:237-244, Higgins et al. (1989) CABIOS 5:151-153, Corpet et al. (1988) Nucleic Acids Res. 16:10881-90, Huang et al. (1992) CABIOS 8:155-65, and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.

[0047] BLAST is described in Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). 215(3), 403-410., Mount D. W. (2007). CSH protocols, 2007, pdb. top17., etc. Programs called BLASTP and BLASTN have been developed based on BLAST, and the percentage of sequence identity can be calculated using these programs with default settings.

[0048] Specific examples of the DNA encoding the protein described in [1] above include DNA selected from the group consisting of (D1) to (D4) below: (D1) DNA encoding the protein described in [1] above (D2) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 (D3) DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 and encodes the homologous protein described in [1] (1c) above (D4) DNA consisting of a nucleotide sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 and encodes the homologous protein described in [1] (1c) above

[0049] Specific examples of DNA encoding the protein described in [2] or [3] above include DNA selected from the group consisting of (D5) to (D8) below: (D5) DNA encoding the protein described in [2] or [3] above (D6) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2 (D7) DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 2 and encodes the homologous protein described in [2] (2c) or [3] (3c) above (D8) DNA consisting of a nucleotide sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the nucleotide sequence represented by SEQ ID NO: 2 and encodes the homologous protein described in [2] (2c) or [3] (3c) above

[0050] Specific examples of the DNA encoding the protein described in [4] above include DNA selected from the group consisting of (D9) to (D12) below: (D9) DNA encoding the protein described in [4] above (D10) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 3 (D11) DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 and encodes the homologous protein described in [4] (4c) above (D12) DNA consisting of a nucleotide sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the nucleotide sequence represented by SEQ ID NO: 3 and encodes the homologous protein described in [4] (4c) above

[0051] Examples of recombinant DNAs containing DNA encoding the protein according to any one of [1] to [4] above include recombinant DNAs containing DNA according to any one of (D1) to (D12) above. The recombinant DNA containing DNA according to any one of (D1) to (D12) above refers to recombinant DNA in which the DNA is incorporated into an expression vector that is capable of autonomous replication in a parent strain or integration into a chromosome and contains a promoter at a position where the DNA can be transcribed.

[0052] In the above (D3) to (D5), "hybridizing" means that DNA hybridizes to DNA having a specific base sequence or a part of the DNA. Therefore, the DNA having the specific base sequence or a part of the DNA can be used as a probe in Northern or Southern blot analysis, and can also be used as an oligonucleotide primer in PCR analysis.

[0053] The DNA used as a probe may be at least 100 bases long, preferably at least 200 bases long, more preferably at least 500 bases long. The DNA used as a primer may be at least 10 bases long, preferably at least 15 bases long.

[0054] Methods for DNA hybridization experiments are well known, and those skilled in the art can determine hybridization conditions according to the present specification. The hybridization conditions can be determined according to the methods described in Molecular Cloning, 4th Edition (2012), Methods for General and Molecular Bacteriology, ASM Press (1994), Immunology Methods Manual, Academic Press (1996), and many other standard textbooks.

[0055] Alternatively, DNA that hybridizes under stringent conditions can be obtained by following the instructions provided with a commercially available hybridization kit, such as the Random Primed DNA Labeling Kit (manufactured by Roche Diagnostics), which prepares a probe by the random prime method and hybridizes under stringent conditions.

[0056] The above-mentioned stringent conditions include incubating the DNA-immobilized filter and the probe DNA overnight at 42°C in a solution containing 50% formamide, 5xSSC (750 mmol / L sodium chloride, 75 mmol / L sodium citrate), 50 mmol / L sodium phosphate (pH 7.6), 5xDenhardt's solution, 10% dextran sulfate, and 20 μg / L denatured salmon sperm DNA, followed by washing the filter in a 0.2xSSC solution at approximately 65°C.

[0057] Examples of DNA that can hybridize under the above-mentioned stringent conditions include DNA that has at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity to DNA consisting of at least one base sequence selected from the group consisting of SEQ ID NOs: 11 to 14, 23 to 25, and 41 to 46, when calculated based on the above-mentioned parameters using, for example, BLAST or FASTA.

[0058] The DNA encoding the "mutant protein" in [1] to [4] above can be obtained, for example, by subjecting a DNA consisting of the base sequence represented by any one of SEQ ID NOs: 1 to 4 as a template to error-prone PCR or the like.

[0059] Alternatively, DNA encoding the mutant protein of the above [2] can also be obtained by PCR using a pair of PCR primers each having a nucleotide sequence at its 5' end designed to introduce a desired mutation (deletion, substitution, insertion, or addition) [Gene, 77, 51 (1989)].

[0060] Alternatively, the DNA can be obtained by following the instructions provided with a commercially available site-directed mutagenesis kit, such as the PrimeSTAR® Mutagenesis Basal Kit (manufactured by Takara Bio Inc.), which can introduce a mutation (deletion, substitution, insertion, or addition) at the desired site.

[0061] That is, first, a pair of mutagenesis primers is designed with a template of a plasmid having a base sequence designed to introduce the desired mutation (deletion, substitution, insertion, or addition), with a 15-base overlap on the 5' side. The overlapping portion contains the desired mutation. Next, PCR is performed using the mutagenesis primers and a template of a plasmid having the base sequence into which the desired mutation is to be introduced. The amplified fragment obtained is transformed into Escherichia coli, yielding a plasmid having the base sequence into which the desired mutation has been introduced.

[0062] Mutations may also be introduced by, for example, mutagenesis. Examples of mutagenesis include X-ray irradiation, ultraviolet irradiation, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS). Random mutations may also be induced in vitro by directly treating DNA with hydroxylamine.

[0063] As a method for obtaining DNA encoding the "homologous protein" in [1] to [4] above, for example, the DNA of (D4) can be obtained as follows: various gene sequence databases are searched for base sequences that have an identity of 50% or more, preferably 65% ​​or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more, to the base sequence represented by SEQ ID NO: 1, or various protein sequence databases are searched for amino acid sequences that have an identity of 50% or more, preferably 65% ​​or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more, to the amino acid sequence represented by SEQ ID NO: 36, and probe DNA or primer DNA that can be designed based on the base sequence or amino acid sequence obtained by the search, and a microorganism containing the DNA, and the DNA can be obtained by a method similar to the method for obtaining DNA described above.

[0064] When the genetically modified microorganism of this embodiment produces retinol, it preferably has at least one of β-carotene dioxygenase and retinal reductase in addition to the β-carotene biosynthetic pathway described above. β-Carotene dioxygenase is an enzyme that converts β-carotene to retinal. Examples of β-carotene dioxygenase include those derived from Uncultured marine bacterium 66A03, Donghaeana dokdonensis (Accession No. WP_041566834.1), Homo sapiens (Accession No. NP_059125.2), Gallus gallus (Accession No. NP_001351831.1), and Halobacterium salinarum (Accession No. AAG20274.1). Specific examples of β-carotene dioxygenase include β-carotene dioxygenase derived from Uncultivated marine bacterium 66A03 (amino acid sequence: SEQ ID NO: 39, nucleotide sequence: SEQ ID NO: 16, SEQ ID NO: 30).

[0065] Heterologous expression of β-carotene dioxygenase derived from Uncultivated marine bacterium 66A03 in S. cerevisiae and Y. lipolytica has been reported (Qiongyue Hu et al., Bioresources and Bioprocessing, 2022, 9:22; Park, Hyemin, et al., Metabolic Engineering, 2022, 73: 26-37).

[0066] Retinal reductase is an enzyme that converts retinal to retinol. Examples of retinol reductase include alcohol dehydrogenases (ADHs), retinol dehydrogenase (RDHs), and aldo-keto reductase (AKR). Examples of sources of alcohol dehydrogenase include S. cerevisiae, L. starkeyi, and Y. lipolytica. Examples of retinol dehydrogenase include those derived from L. starkeyi, Homo sapiens, Mus musculus, Bos taurus, and Rattus norvegicus. Examples of sources of aldo-keto reductases include S. cerevisiae and Escherichia coli. Specific examples of retinal reductases include alcohol dehydrogenase derived from L. starkeyi (amino acid sequence: SEQ ID NO: 40, nucleotide sequence: SEQ ID NO: 41) and S. cerevisiae (amino acid sequence: SEQ ID NO: 65, nucleotide sequence: SEQ ID NO: 33).

[0067] When the genetically modified microorganism of this embodiment produces a β-carotene derivative, a heterologous β-carotene hydroxylase gene or β-carotene ketolase gene may be expressed. Specifically, for the production of zeaxanthin and β-cryptoxanthin, β-carotene hydroxylase (β-carotene hydroxylase, CrtZ) is preferred, for the production of canthaxanthin, β-carotene ketolase (β-carotene ketolase, CrtW) is preferred, and for the production of astaxanthin, both CrtZ and CrtW are preferred, and enhanced expression is even more preferred. Furthermore, when the genetically modified microorganism of this embodiment produces retinol palmitate among the β-carotene derivatives, it preferably contains an acyltransferase. The genetically modified microorganism of this embodiment may inherently contain an acyltransferase, or an exogenous gene encoding the enzyme may be introduced into the microorganism. Furthermore, an exogenous gene encoding the acyltransferase may be further introduced into a genetically modified microorganism that inherently contains the acyltransferase.

[0068] When the genetically modified microorganism of this embodiment produces β-cryptoxanthin, it preferably has CrtZ in addition to the above-mentioned β-carotene biosynthetic pathway, and more preferably has it enhanced. When the genetically modified microorganism of this embodiment produces zeaxanthin, it similarly preferably has CrtZ in addition to the above-mentioned β-carotene biosynthetic pathway, and more preferably has it enhanced. When the genetically modified microorganism of this embodiment produces astaxanthin, it similarly preferably has CrtZ in addition to the above-mentioned β-carotene biosynthetic pathway, and more preferably has it enhanced. CrtZ is an enzyme that converts β-carotene to β-cryptoxanthin, β-cryptoxanthin to zeaxanthin, and canthaxanthin to astaxanthin.

[0069] Examples of β-carotene hydroxylase (CrtZ) include those derived from Brevundimonas vesicularis (Accession No. ABC50108.1), Erwinia uredovora (Pantoea ananatis) (Accession No. D90087.2), Agrobacterium aurantiacum (Accession No. GM621472.1), Alcaligenes sp. PC-1 (Accession No. D58422.1), and Brevundimonas sp. SD212 (Accession No. AB181388.1), Paracoccus marcussii (Accession No. MT175370.1), Paracoccus sp. N81106 (Accession No. AB206672), Pantoea agglomerans (Accession No. M87280.1), Capsicum annuum (Accession No. GU122940.1), and Haematococcus pluvialis (Accession No. KP866868).

[0070] Among these, when producing β-cryptoxanthin, CrtZ derived from Pantoea agglomerans (Accession No. M87280.1) and CrtZ derived from Haematococcus pluvialis (Accession No. KP866868, amino acid sequence: SEQ ID NO: 56, nucleotide sequence: SEQ ID NO: 55) are preferred, and CrtZ derived from Haematococcus pluvialis (Accession No. KP866868, amino acid sequence: SEQ ID NO: 56, nucleotide sequence: SEQ ID NO: 55) is more preferred.

[0071] When producing zeaxanthin, Brevundimonas sp. SD212-derived CrtZ (Accession No. AB181388.1), Haematococcus pluvialis-derived CrtZ (Accession No. KP866868, amino acid sequence: SEQ ID NO: 56, nucleotide sequence: SEQ ID NO: 55), Erwinia uredovora-derived CrtZ (Accession No. D90087.2, amino acid sequence: SEQ ID NO: 52, nucleotide sequence: SEQ ID NO: 51), or Pantoea agglomerans-derived CrtZ (Accession No. M87280.1, amino acid sequence: SEQ ID NO: 54, nucleotide sequence: SEQ ID NO: 53) is preferred, and Erwinia uredovora-derived CrtZ (Accession No. D90087.2, amino acid sequence: SEQ ID NO: 52, nucleotide sequence: SEQ ID NO: 51) or CrtZ derived from Pantoea agglomerans (Accession No. M87280.1, amino acid sequence: SEQ ID NO: 54, nucleotide sequence: SEQ ID NO: 53) is more preferred.

[0072] When producing astaxanthin, a strain derived from Brevundimonas sp. SD212 (Accession No. AB181388.1, amino acid sequence: SEQ ID NO: 62, nucleotide sequence: SEQ ID NO: 61) is more preferred.

[0073] When the genetically modified microorganism of this embodiment produces canthaxanthin, it preferably has CrtW in addition to the above-mentioned β-carotene biosynthetic pathway, and more preferably has it enhanced. Similarly, when the genetically modified microorganism of this embodiment produces astaxanthin, it preferably has CrtW in addition to the above-mentioned β-carotene biosynthetic pathway, and more preferably has it enhanced. CrtW is an enzyme that converts β-carotene to canthaxanthin and zeaxanthin to astaxanthin.

[0074] Examples of β-carotene ketolase (CrtW) include those derived from Brevundimonas vesicularis (Accession No. ABC50116.1), Bradyrhizobium sp. ORS278 (Accession No. AF218415.1), and Alcaligenes sp. PC-1 (Accession No. D58422.1), Agrobacterium aurantiacum (Accession No. AB206672.1), Haematococcus pluvialis (Accession No. D45881.1), Paracoccus sp. N81106 (Accession No. P54972), or Chlamydomonas reinhardtii (Accession No. AY860820).

[0075] Among these, when producing canthaxanthin, CrtW derived from Brevundimonas vesicularis (Accession No. ABC50116.1), CrtW derived from Paracoccus sp. N81106 (Accession No. P54972, amino acid sequence: SEQ ID NO: 58, nucleotide sequence: SEQ ID NO: 57), or CrtW derived from Chlamydomonas reinhardtii (Accession No. AY860820, amino acid sequence: SEQ ID NO: 60, nucleotide sequence: SEQ ID NO: 59) is preferred. More preferred is CrtW derived from N81106 (Accession No. P54972, amino acid sequence: SEQ ID NO: 58, nucleotide sequence: SEQ ID NO: 57) or CrtW derived from Chlamydomonas reinhardtii (Accession No. AY860820, amino acid sequence: SEQ ID NO: 60, nucleotide sequence: SEQ ID NO: 59).

[0076] When producing astaxanthin, the genetically modified microorganism in this embodiment has CrtZ and CrtW, and can convert β-carotene to canthaxanthin, canthaxanthin to astaxanthin, or β-carotene to astaxanthin via β-cryptoxanthin and zeaxanthin.

[0077] When producing astaxanthin, CrtW derived from Brevundimonas vesicularis (Accession No. ABC50116.1) and CrtW derived from Paracoccus sp. N81106 are preferred, and CrtW derived from Paracoccus sp. N81106 (Accession No. P54972, amino acid sequence: SEQ ID NO: 58, nucleotide sequence: SEQ ID NO: 57) is more preferred.

[0078] When the genetically modified microorganism of this embodiment produces retinol palmitate, it preferably has an acyltransferase in addition to the retinol biosynthetic pathway described above. Acyltransferase is an enzyme that catalyzes the reaction of producing retinol palmitate by condensing retinol with palmitic acid. Introduction of this enzyme can improve the efficiency of retinol palmitate production.

[0079] Examples of acyltransferases include those derived from Homo sapiens (Accession No. AF071510.1), Saccharomyces cerevisiae (Accession No. Z75153.1), and Lipomyces starkeyi (Accession No. KAK9317287.1, amino acid sequence: SEQ ID NO: 66, nucleotide sequence: SEQ ID NO: 67). Of these, those derived from Lipomyces starkeyi (Accession No. KAK9317287.1, amino acid sequence: SEQ ID NO: 66, nucleotide sequence: SEQ ID NO: 67) are preferred when producing retinol palmitate.

[0080] One aspect of the present production method includes, for example, any one of the following (α1) to (α7). These configurations enable the production of a target product (retinol, retinal, retinol palmitate, zeaxanthin, β-cryptoxanthin, canthaxanthin, or astaxanthin) in a culture in addition to the β-carotene biosynthesis pathway based on the various enzymes possessed by the genetically modified microorganism in the above embodiment. (α1) The genetically modified microorganism has at least one of β-carotene dioxygenase and retinal reductase, and by culturing the genetically modified microorganism, retinol is produced in the culture. (α2) The genetically modified microorganism has β-carotene dioxygenase, and by culturing the genetically modified microorganism, retinal is produced in the culture. (α3) The genetically modified microorganism has acyltransferase, and by culturing the genetically modified microorganism, retinol palmitate is produced in the culture. (α4) The genetically modified microorganism has β-carotene hydroxylase, and by culturing the genetically modified microorganism, zeaxanthin is produced in the culture. (α5) The genetically modified microorganism has β-carotene hydroxylase, and by culturing the genetically modified microorganism, β-cryptoxanthin is produced in the culture. (α6) The genetically modified microorganism has β-carotene ketolase, and by culturing the genetically modified microorganism, canthaxanthin is produced in the culture. (α7) The genetically modified microorganism has β-carotene hydroxylase and β-carotene ketolase, and astaxanthin is produced in the culture by culturing the genetically modified microorganism.

[0081] Furthermore, the method including any one of (α1) to (α7) preferably corresponds to any one of the following (β1) to (β7): (β1) including (α1), wherein the β-carotene dioxygenase in (α1) is derived from Uncultured marine bacterium 66A03; (β2) including (α2), wherein the β-carotene dioxygenase in (α2) is derived from Uncultured marine bacterium 66A03; or (β3) including (α3), wherein the acyltransferase in (α3) is derived from Lipomyces starkeyi. One embodiment is a protein containing the amino acid sequence shown in SEQ ID NO: 66, but is not limited to this protein and also includes other acyltransferases having the same reaction activity.(β4) The β-carotene hydroxylase in (α4) is at least one of β-carotene hydroxylase derived from Erwinia uredovora (Pantoea ananatis) (Accession No. D90087.2) and β-carotene hydroxylase derived from Pantoea agglomerans (Accession No. M87280.1). (β5) The β-carotene hydroxylase in (α5) is β-carotene hydroxylase derived from Haematococcus pluvialis (Accession No. KP866868). (β6) A method comprising the (α6) above, wherein the β-carotene ketolase in the (α6) is at least one of a β-carotene ketolase derived from Paracoccus sp. N81106 (Accession No. P54972) or a β-carotene ketolase derived from Chlamydomonas reinhardtii (Accession No. AY860820); (β7) A method comprising the (α7) above, wherein the β-carotene hydroxylase in the (α7) is a β-carotene hydroxylase derived from Brevundimonas sp. SD212 (Accession No. AB181388.1), and the β-carotene ketolase is derived from Paracoccus sp. The β-carotene ketolase is at least one of β-carotene ketolase derived from Chlamydomonas reinhardtii (Accession No. AY860820) and β-carotene ketolase derived from Chlamydomonas reinhardtii (Accession No. P54972).

[0082] <In vivo method and in vitro method> Specific aspects of the method of this embodiment include an in vivo method and an in vitro method, with the in vivo method being preferred from the viewpoint of production efficiency. These are described below.

[0083] (In vivo method) In the method of this embodiment, the in vivo method is a method in which a target product is produced within a microorganism using a microorganism in a growing or growing state. The microorganism used in the in vivo method is preferably the genetically modified microorganism of this embodiment described above. An example of an in vivo method is a fermentation method. The fermentation method produces a target substance by utilizing the functions of the growth process of a microorganism, and usually involves bacterial cell growth.

[0084] In this embodiment, the in vivo method includes culturing a genetically modified microorganism of the genus Lipomyces to produce at least one of β-carotene and a β-carotene derivative in a culture, and the genetically modified microorganism preferably has a β-carotene biosynthetic pathway. The culture includes cells of the genetically modified microorganism and a medium. When the medium is liquid, the culture is also referred to as a culture solution.

[0085] Specifically, one embodiment of the in vivo method is preferably a method comprising the following steps (x1) and (x2): (x1) a step of preparing a genetically modified microorganism belonging to the genus Lipomyces and having a β-carotene biosynthetic pathway; and (x2) a step of producing at least one of β-carotene and a β-carotene derivative in at least one of a culture supernatant and intracellular cells using the genetically modified microorganism prepared in (x1).

[0086] In this embodiment, the microorganism can be cultured according to a conventional method used for culturing microorganisms. The medium for culturing the microorganism may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the microorganism and allows efficient cultivation of the microorganism. The medium may be either liquid or solid, with liquid being preferred.

[0087] The carbon source may be any that can be assimilated by the microorganism, and examples thereof include sugars such as glucose, fructose, sucrose, galactose, cellobiose, maltose, molasses containing these, starch (e.g., soluble starch, corn starch) or starch hydrolysates, organic acids such as acetic acid or propionic acid, and alcohols such as glycerol, ethanol or propanol, etc. Among these, glucose, galactose or cellobiose is preferred.

[0088] Examples of nitrogen sources include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal, soybean meal hydrolysate, various fermentation bacteria and digested products thereof, and the like.

[0089] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0090] The microorganism of this embodiment used in the method of this embodiment may be a microorganism capable of producing an acceptor carbohydrate such as glucose, lactose, lactose monohydrate, etc. In this production method, glucose, lactose, lactose monohydrate, etc. may be added to the medium during cultivation.

[0091] In the method of this embodiment, instead of adding glucose, lactose, lactose monohydrate, or the like to the medium during cultivation, glucose, lactose, lactose monohydrate, or the like may be supplied to the microorganism of this embodiment by simultaneously culturing a microorganism capable of producing glucose, lactose, lactose monohydrate, or the like from sugar with the microorganism of this embodiment.

[0092] Cultivation is preferably carried out under aerobic conditions, such as shaking culture or submerged aeration and stirring culture. The culture temperature is usually 30 to 37°C, and the culture time is usually 24 hours to 3 days. The pH of the culture solution during cultivation is usually maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0093] By culturing as described above, at least one of β-carotene and a β-carotene derivative can be produced in the culture. Furthermore, by analyzing the culture using a high performance liquid chromatography (HPLC) LC-20AD (Shimadzu Corporation), it is possible to confirm and quantify the production of at least one of β-carotene and a β-carotene derivative.

[0094] By the above-mentioned cultivation, at least one of β-carotene and a β-carotene derivative is produced and accumulated in the culture, and at least one of β-carotene and a β-carotene derivative can be produced by collecting at least one of β-carotene and a β-carotene derivative from the culture.

[0095] Typically, after centrifugation of the culture, at least one of β-carotene and a β-carotene derivative can be collected from the supernatant. When at least one of β-carotene and a β-carotene derivative accumulates in the cells, the cells can be disrupted by ultrasonication or the like, and then centrifuged to remove the cells. From the resulting supernatant, at least one of β-carotene and a β-carotene derivative can be collected by an ion exchange resin method or the like.

[0096] (In vitro method) In this embodiment, the in vitro method is a method in which a culture obtained by culturing a microorganism or a processed product of the culture is used as an enzyme source, the enzyme source and a substrate for the enzyme are present in an aqueous medium, and at least one of β-carotene and a β-carotene derivative is produced by an enzymatic reaction. Among these methods, a reaction system in which microbial cells in a dormant or stationary state without proliferation are used as an enzyme source is called a bacterial cell reaction method. However, the form of the reaction between the microorganism and the acceptor substrate is not limited to the specific method described below.

[0097] The in vitro method is preferably a method comprising the following steps (y1) and (y2): (y1) a step of preparing a culture obtained by culturing a microorganism of the genus Lipomyces having a β-carotene biosynthetic pathway or a processed product of the culture to form an enzyme source constituting the β-carotene biosynthetic pathway, and (y2) a step of causing the enzyme source prepared in (y1) and a substrate for the enzyme to be present in an aqueous medium and producing at least one of β-carotene and a β-carotene derivative by enzymatic reaction.

[0098] The enzyme source in step (y1) is a culture obtained by culturing the genetically modified microorganism of this embodiment, or a processed product of the culture. Examples of the culture or the processed product of the culture include a concentrate of the culture, a dried product of the culture, bacterial cells obtained by centrifuging the culture, a dried product of the bacterial cells, a freeze-dried product of the bacterial cells, a surfactant-treated product of the bacterial cells, an ultrasonically treated product of the bacterial cells, a mechanically ground product of the bacterial cells, a solvent-treated product of the bacterial cells, an enzyme-treated product of the bacterial cells, a protein fraction of the bacterial cells, an immobilized product of the bacterial cells, and an isolated and purified purified enzyme obtained by extraction from the bacterial cells. Among these, a solvent-treated product of the bacterial cells and an isolated and purified purified enzyme obtained by extraction from the bacterial cells are preferred.

[0099] Examples of aqueous media include water, buffer solutions such as phosphates, carbonates, acetates, borates, citrates, Tris, etc., alcohols such as methanol and ethanol, esters such as ethyl acetate, ketones such as acetone, amides such as acetamide, etc. Another example of an aqueous medium is the culture medium of the microorganism used as the enzyme source.

[0100] In this embodiment, the microorganisms can be cultured according to a conventional method used for culturing microorganisms, similar to the "in vivo method" described above.

[0101] When extracting the enzyme sources constituting the β-carotene biosynthetic pathway from a culture of a microorganism, for example, the microorganism can be separated from the medium by centrifugation, and the microorganism can be disrupted to extract the enzyme sources.

[0102] The isolated and purified enzyme used in the "in vitro method" can be obtained using the genetically modified microorganism of the present embodiment described above. The solvent-treated bacterial cells used in the "in vitro method" can be obtained using the genetically modified microorganism of the present embodiment described above, but it is more preferable to use, for example, xylene as the solvent.

[0103] Using the enzyme source, a substrate for the enzyme is placed in an aqueous medium and reacted, and then the reaction product is analyzed using a liquid chromatograph mass spectrometer (e.g., LCMS-8040 manufactured by Shimadzu Corporation), whereby it is possible to confirm and quantify the production of at least one of β-carotene and a β-carotene derivative.

[0104] At least one of β-carotene and a β-carotene derivative is obtained by isolating and purifying at least one of β-carotene and a β-carotene derivative by an ion exchange resin method, HPLC, or the like.

[0105] In the present production method, the β-carotene production efficiency per wet cell weight (g) is preferably 1.2 μg / wet cell weight / h or more, more preferably 2.0 μg / wet cell weight / h or more, even more preferably 3.0 μg / wet cell weight / h or more, 4.0 μg / wet cell weight / h or more, 5.0 μg / wet cell weight / h or more, and particularly preferably 5.3 μg / wet cell weight / h or more.

[0106] In this specification, the "production efficiency of β-carotene" is defined by the following formula: β-carotene production efficiency (μg / wet cell weight / h) = [mass of β-carotene in the culture (μg) / wet cell weight (g)] / culture time (h)

[0107] When the β-carotene derivative is retinol, the retinol production efficiency is preferably 0.26 mg / L / h or more, more preferably 0.3 mg / L / h or more, even more preferably 0.4 mg / L / h or more, 0.5 mg / L / h or more, 0.6 mg / L / h or more, 0.7 mg / L / h or more, 0.8 mg / L / h or more, 0.9 mg / L / h or more, and particularly preferably 1.0 mg / L / h or more.

[0108] When the β-carotene derivative is retinal, the production efficiency of retinal is preferably 0.01 mg / L / h or more, more preferably 0.05 mg / L / h or more, even more preferably 0.1 mg / L / h or more, 0.2 mg / L / h or more, 0.3 mg / L / h or more, 0.4 mg / L / h or more, and particularly preferably 0.53 mg / L / h or more.

[0109] As used herein, the "retinol production efficiency" is defined by the following formula: Retinol production efficiency (mg / L / h) = [mass of retinol in the culture (mg) / volume of culture medium (L)] / culture time (h).

[0110] In this specification, the "conversion efficiency of lycopene to β-carotene" can be calculated using the following formula based on the mass of β-carotene and the mass of lycopene per wet weight of cells in the culture after culturing the genetically modified microorganism for a predetermined time: Conversion efficiency of lycopene to β-carotene = (mass of β-carotene per wet weight of cells) / [(mass of β-carotene per wet weight of cells) + (mass of lycopene per wet weight of cells)] × 100 (%)

[0111] The masses of lycopene and β-carotene can be analyzed by the method described in the "Analysis Examples" below. When a genetically modified microorganism has an exogenous gene that produces a β-carotene derivative from β-carotene, the "conversion efficiency from lycopene to β-carotene" can be calculated using the strain before the exogenous gene was introduced, i.e., a strain that produces β-carotene but does not produce a β-carotene derivative.

[0112] The genetically modified microorganism in this embodiment preferably has a conversion efficiency from lycopene to β-carotene of 30% or more, more preferably 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, even more preferably 90% or more, particularly preferably 95% or more, and most preferably 97% or more.

[0113] In the genetically modified microorganism of this embodiment, it is preferable that the activity of lycopene cyclase is not inhibited by lycopene produced by the genetically modified microorganism.

[0114] The absence of inhibition of the activity of lycopene cyclase by lycopene produced in a microorganism can be confirmed by the fact that when a lycopene cyclase having a known mutation that suppresses the activity inhibition by lycopene (Yongshuo Ma et al., Nature Communications, 2022, 13:572) is expressed in the microorganism, no improvement in β-carotene production efficiency is observed compared to when a lycopene cyclase without the mutation is expressed in the microorganism.

[0115] Specifically, the absence of inhibition of the activity of lycopene cyclase by lycopene produced in a microorganism means that when the lycopene cyclase having the mutation is expressed in a microorganism, the "degree of improvement in β-carotene production efficiency" represented by the following formula is preferably 350% or less, more preferably 250% or less, even more preferably 150% or less, 100% or less, 75% or less, 50% or less, 30% or less, 20% or less, 10% or less, 7% or less, 5% or less, and particularly preferably 3% or less, compared to when a lycopene cyclase not having the mutation is expressed in a microorganism.

[0116] Improvement in β-carotene production efficiency=[β-carotene production amount when lycopene cyclase having the mutation is expressed in a microorganism / β-carotene production amount when lycopene cyclase not having the mutation is expressed in a microorganism]×100(%)

[0117] The conditions for β-carotene production by each microorganism used to calculate the improvement in β-carotene production efficiency are the same, except for whether the lycopene cyclase possessed by the microorganism has the above mutation. Furthermore, when the microorganism has a foreign gene that produces a β-carotene derivative from β-carotene, the improvement in β-carotene production efficiency can be calculated using the strain before the foreign gene was introduced, i.e., a strain that produces β-carotene but does not produce a β-carotene derivative.

[0118] One aspect of this embodiment includes a method for producing at least one of β-carotene and a β-carotene derivative using a microorganism in which lycopene produced intracellularly does not inhibit the activity of lycopene cyclase. In this aspect, the microorganism is preferably a microorganism capable of producing farnesyl diphosphate intracellularly using an endogenous enzyme. In this aspect, the microorganism is not particularly limited as long as it has the characteristic that lycopene produced intracellularly does not inhibit the activity of lycopene cyclase, but is preferably an oleaginous yeast, and more preferably a microorganism of the genus Lipomyces.

[0119] The fact that the activity of lycopene cyclase is not inhibited by lycopene produced in the bacterial cells can be confirmed by the fact that when a lycopene cyclase having a known mutation that suppresses the activity inhibition by lycopene (Yongshuo Ma et al., Nature Communications, 2022, 13:572) is expressed in the microorganism, no improvement in β-carotene production efficiency is observed compared to when a lycopene cyclase without the mutation is expressed in the microorganism.

[0120] Specifically, the absence of inhibition of the activity of lycopene cyclase by lycopene produced within the bacterial cells means that when the lycopene cyclase having the mutation is expressed in a microorganism, the "degree of improvement in β-carotene production efficiency," represented by the following formula, is preferably 350% or less, more preferably 250% or less, even more preferably 150% or less, 100% or less, 75% or less, 50% or less, 30% or less, 20% or less, 10% or less, 7% or less, 5% or less, and particularly preferably 3% or less, compared to when a lycopene cyclase not having the mutation is expressed in a microorganism.

[0121] Improvement in β-carotene production efficiency=[β-carotene production amount when lycopene cyclase having the mutation is expressed in a microorganism / β-carotene production amount when lycopene cyclase not having the mutation is expressed in a microorganism]×100(%)

[0122] The conditions for β-carotene production by each microorganism used to calculate the improvement in β-carotene production efficiency are the same, except for whether the lycopene cyclase possessed by the microorganism has the above mutation. Furthermore, when the microorganism has a foreign gene that produces a β-carotene derivative from β-carotene, the improvement in β-carotene production efficiency can be calculated using the strain before the foreign gene was introduced, i.e., a strain that produces β-carotene but does not produce a β-carotene derivative.

[0123] 2. Genetically Modified Microorganism The genetically modified microorganism of this embodiment is a microorganism of the genus Lipomyces, has a β-carotene biosynthetic pathway, and produces at least one of β-carotene and a β-carotene derivative by fermentation.

[0124] As used herein, "fermentation" refers to the transformation of organic matter by the action of microorganisms. The genetically modified microorganism of this embodiment produces at least one of β-carotene and a β-carotene derivative through fermentation. Examples of genetically modified microorganisms of the genus Lipomyces of this embodiment include the genetically modified microorganisms used in the present production method described in Section 1 above.

[0125] A method for producing a genetically modified microorganism of this embodiment includes transforming a parent strain of the genus Lipomyces with recombinant DNA containing DNA encoding an enzyme constituting the β-carotene biosynthetic pathway. Examples of the enzyme constituting the β-carotene biosynthetic pathway include a protein represented by at least one selected from the above [1] to [4].

[0126] Specifically, for example, a DNA fragment of an appropriate length containing a portion encoding the protein described in any one of [1] to [4] above, preferably a DNA selected from the group consisting of (D1) to (D12) above, is prepared as needed. Furthermore, by substituting bases in the nucleotide sequence of the portion encoding the protein so that it contains codons optimal for expression in host cells, a transformant with improved production efficiency can be obtained. Information on codon usage frequencies in parent strains used in this production method is available from public databases.

[0127] A recombinant DNA can be prepared by inserting the DNA encoding the proteins constituting the β-carotene biosynthetic pathway downstream of a promoter in an appropriate expression vector. A parent strain (Lipomyces) can be transformed with the recombinant DNA to obtain the genetically modified microorganism of this embodiment.

[0128] Examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.

[0129] When the expression vector is used, any promoter may be used as long as it functions in the cells of a yeast strain, and examples thereof include promoters such as the THD3 promoter (SEQ ID NO: 4), 70486 promoter (SEQ ID NO: 11), ACT1 promoter (SEQ ID NO: 7), TEF1 promoter (SEQ ID NO: 24), PHO5 promoter, PGK promoter (SEQ ID NO: 28), GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MFα1 promoter, and CUP1 promoter.

[0130] By inserting the DNA fragment described in any one of (D1) to (D12) above downstream of the promoter of an appropriate expression vector, a recombinant DNA that can be used to produce the genetically modified microorganism of this embodiment can be produced.

[0131] When introducing two or more genes into the genetically modified microorganism of this embodiment, it is sufficient that each gene is retained in a usable state in the host. For example, all of the genes may be retained on a single expression vector, or all may be retained on a chromosome. Alternatively, each gene may be retained separately on multiple expression vectors, or may be retained separately on a single or multiple expression vectors and on a chromosome. Two or more genes may be introduced as an operon. "Introducing two or more genes" may include, for example, introducing genes encoding two or more proteins (e.g., enzymes), introducing genes encoding two or more subunits that constitute a single protein complex (e.g., an enzyme complex), and combinations thereof.

[0132] As explained above, the present specification discloses the following: 1. A method for producing at least one of β-carotene and a β-carotene derivative using a genetically modified microorganism of the genus Lipomyces. 2. The method according to 1 above, in which the β-carotene derivative is at least one of retinol, β-cryptoxanthin, zeaxanthin, canthaxanthin, astaxanthin, retinol palmitate, fucoxanthin, capsanthin, abscisic acid, β-ionone, and retinal. 3. The method according to 1 or 2 above, in which the genetically modified microorganism is Lipomyces starchii. 4. The method according to any one of 1 to 3 above, which comprises culturing the genetically modified microorganism to produce at least one of β-carotene and a β-carotene derivative in a culture, wherein the genetically modified microorganism has a β-carotene biosynthetic pathway. 5. 5. The method according to 4, wherein the β-carotene biosynthetic pathway comprises at least one selected from farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, phytoene synthase, phytoene desaturase, and lycopene cyclase. 6. The method according to 5, wherein the β-carotene biosynthetic pathway comprises at least one selected from Xanthophyllomyces dendrous-derived geranylgeranyl diphosphate synthase, Mucor circinelloides-derived phytoene synthase, Mucor circinelloides-derived lycopene synthase, and Mucor circinelloides-derived lycopene cyclase. 7. The method according to any one of 4 to 6, wherein the β-carotene biosynthetic pathway comprises at least one protein selected from the following [1] to [4]:[1] At least one protein selected from the following (1a) to (1c): (1a) a protein comprising the amino acid sequence represented by SEQ ID NO: 36; (1b) a mutant protein comprising an amino acid sequence in which 1 to 37 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 36, and having geranylgeranyl diphosphate synthase activity; (1c) a homologous protein comprising an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 36, and having geranylgeranyl diphosphate synthase activity; [2] At least one protein selected from the following (2a) to (2c): (2a) a protein comprising the amino acid sequence represented by SEQ ID NO: 37; (2b) a mutant protein comprising an amino acid sequence in which 1 to 60 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 37, and having phytoene synthase activity; (2c) a homologous protein comprising an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 37, and having phytoene synthase activity. (3) At least one protein selected from the following (3a) to (3c): (3a) a protein comprising the amino acid sequence represented by SEQ ID NO: 37; (3b) a mutant protein comprising an amino acid sequence represented by SEQ ID NO: 37 in which 1 to 60 amino acids have been deleted, substituted, inserted or added, and having lycopene cyclase activity; (3c) a homologous protein comprising an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 37, and having lycopene cyclase activity; [4] At least one protein selected from the following (4a) to (4c): (4a) a protein comprising the amino acid sequence represented by SEQ ID NO: 38; (4b) a mutant protein comprising an amino acid sequence represented by SEQ ID NO: 38 in which 1 to 58 amino acids have been deleted, substituted, inserted or added, and having phytoene desaturase activity; (4c) a homologous protein comprising an amino acid sequence represented by SEQ ID NO: 38 in which 50% or more identity has been added, and having phytoene desaturase activity. 8. The method according to any one of 1 to 7 above, wherein the genetically modified microorganism is a microorganism in which the activity of lycopene cyclase is not inhibited by lycopene produced in the genetically modified microorganism. 9. The method according to any one of 1 to 8 above, comprising any one of the following (α1) to (α7):(α1) The genetically modified microorganism has at least one of β-carotene dioxygenase and retinal reductase, and by culturing the genetically modified microorganism, retinol is produced in the culture. (α2) The genetically modified microorganism has β-carotene dioxygenase, and by culturing the genetically modified microorganism, retinal is produced in the culture. (α3) The genetically modified microorganism has acyltransferase, and by culturing the genetically modified microorganism, retinol palmitate is produced in the culture. (α4) The genetically modified microorganism has β-carotene hydroxylase, and by culturing the genetically modified microorganism, zeaxanthin is produced in the culture. (α5) The genetically modified microorganism has β-carotene hydroxylase, and by culturing the genetically modified microorganism, β-cryptoxanthin is produced in the culture. (α6) The genetically modified microorganism has β-carotene ketolase, and by culturing the genetically modified microorganism, canthaxanthin is produced in the culture. (α7) The genetically modified microorganism has β-carotene hydroxylase and β-carotene ketolase, and astaxanthin is produced in a culture by culturing the genetically modified microorganism. 10. The method according to 9 above, which is any one of the following (β1), (β2), and (β4) to (β7):(β1) A protein comprising the (α1), wherein the β-carotene dioxygenase in the (α1) is a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 39 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 39. (β2) A protein comprising the (α2), wherein the β-carotene dioxygenase in the (α2) is a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 39 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 39. (β4) A protein comprising the (α4), wherein the β-carotene hydroxylase in the (α4) is a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 52 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 52, or a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 54 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 54. (β5) The protein comprises the (α5), wherein the β-carotene hydroxylase in the (α5) is a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 56 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 56. (β6) The protein comprises the (α6), wherein the β-carotene ketolase in the (α6) is at least one of a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 58 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 58, or a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 60 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 60. (β7) The protein comprises the (α7), wherein the β-carotene hydroxylase in the (α7) is a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 62 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 62, and the β-carotene ketolase is a homologous protein comprising the amino acid sequence represented by SEQ ID NO: 58 or an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 58. 11. The method according to any one of 1 to 10, wherein the efficiency of conversion of lycopene to β-carotene by the genetically modified microorganism is 30% or more.12. The method according to 11 above, wherein the β-carotene production efficiency is 1.2 μg / wet cell weight / h or more. 13. The method according to 12 above, wherein the β-carotene derivative is retinol and the retinol production efficiency is 0.26 mg / L / h or more. 14. A genetically modified microorganism of the genus Lipomyces, which has a β-carotene biosynthetic pathway and produces at least one of β-carotene and a β-carotene derivative by fermentation. 15. The genetically modified microorganism according to 14 above, which further has at least one of β-carotene dioxygenase and retinal reductase and produces retinol by fermentation. 16. A method for producing a β-carotene derivative, comprising culturing the genetically modified microorganism according to 14 or 15 above to produce a β-carotene derivative in a culture, and recovering the β-carotene derivative from the culture. 17. 17. A method for producing at least one of β-carotene and a β-carotene derivative using a microorganism in which the activity of lycopene cyclase is not inhibited by lycopene produced within the microorganism's cells. 18. The method according to 16, wherein the microorganism is a microorganism capable of producing farnesyl diphosphate within the microorganism's cells using an endogenous enzyme.

[0133] [Analysis Examples] In the Examples, lycopene, β-carotene, zeaxanthin, β-cryptoxanthin, canthaxanthin, astaxanthin, retinol, retinal, and retinol palmitate were analyzed according to the following procedures.

[0134] For the β-carotene-producing strain, zeaxanthin-producing strain, β-cryptoxanthin-producing strain, canthaxanthin-producing strain, and astaxanthin-producing strain in Examples 2 and 7, 1 ml of the culture solution containing the cultured microorganisms was centrifuged, and 200 μl of 0.5 mm glass beads and 500 μl of ethyl acetate were added to the precipitated cells, which were then disrupted and extracted using a Multi-Beads Shocker (Yasui Kikai Co., Ltd.) at 2,500 rpm for 15 minutes. The supernatant obtained by centrifuging the disrupted cell solution was used as the analysis sample.

[0135] For the retinol-producing strains in Examples 4 and 5, the culture medium containing the microorganisms after cultivation was centrifuged, and the upper dodecane layer was collected and used as an analytical sample. Lycopene, β-carotene, zeaxanthin, β-cryptoxanthin, canthaxanthin, astaxanthin, retinol, and retinal were analyzed using a high-performance liquid chromatography (HPLC) LC-20AD (Shimadzu Corporation) under the conditions listed in Table 1A, and retinol palmitate was analyzed using an LC / MS (Agilent 1260 infinity LC, Agilent 610) under the conditions listed in Table 1B.

[0136]

[0137]

[0138] Example 1: Construction of a β-carotene-producing L. starkeyi strain. Microorganisms of the genus Lipomyces can synthesize geranylgeranyl diphosphate (GGPP), an intermediate in the β-carotene biosynthetic pathway, using endogenous enzymes. However, because the endogenous geranylgeranyl diphosphate synthase (GGS1) of L. starkeyi used in this example was expected to have low enzymatic activity, geranylgeranyl diphosphate synthase (CrtE) derived from Xanthophyllomyces dendrorhous was introduced. In addition, phytoene synthase / lycopene cyclase and phytoene desaturase derived from Mucor circinelloides were selected as transgenes.

[0139] The construction of a β-carotene-producing L. starkeyi strain is described in detail below. All nucleic acid ligations were performed using the InFusion cloning method. An expression cassette for CrtE was constructed by ligating artificially synthesized X. dendrorhous-derived CrtE (SEQ ID NO: 1) with the TDH3 promoter (SEQ ID NO: 4) and TDH3 terminator (SEQ ID NO: 5).

[0140] A selection marker was constructed by linking the hygromycin resistance gene hph (SEQ ID NO: 6) to the ACT1 promoter (SEQ ID NO: 7) and the ACT1 terminator (SEQ ID NO: 8). The expression cassette, selection marker, and homologous sequence were linked so that the sequences of the TGL3 gene (SEQ ID NOs: 9 and 10) were located at both ends as homologous sequences for introducing them into the genome.

[0141] The ligated expression construct was subjected to plasmid extraction using a QIAprep Spin Miniprep Kit (QIAGEN) and treated with NotI restriction enzyme (Takara Bio). Approximately 5 μg / L of the restriction enzyme-treated DNA was transformed into the L. starkeyi CBS1807Δlslig4 strain by electroporation and cultured in a selective medium to obtain a transformed strain (hereinafter referred to as the CrtE-introduced L. starkeyi strain). The transformation method was according to Takaku et al., Journal of Microbiological Methods, 2020, 169, 105816. The CBS1807Δlslig4 strain was prepared by genetically modifying the parent strain CBS1807 obtained from the Central Office for Mold Cultures (Utrecht, The Netherlands) using the method described in Oguro et al., 2017.

[0142] Similarly, an expression cassette for phytoene synthase / lycopene cyclase was constructed by linking an artificially synthesized phytoene synthase / lycopene cyclase (SEQ ID NO: 2) derived from M. circinelloides to the 70486 promoter (SEQ ID NO: 11) and the 70486 terminator (SEQ ID NO: 12). Also, an expression cassette for phytoene desaturase was constructed by linking an M. circinelloides-derived phytoene desaturase (SEQ ID NO: 3) to the TDH3 promoter (SEQ ID NO: 4) and the TDH3 terminator (SEQ ID NO: 5).

[0143] In addition, a selection marker was constructed by linking the G418 resistance gene KanR (SEQ ID NO: 13) to the ACT1 promoter (SEQ ID NO: 7) and ACT1 terminator (SEQ ID NO: 8). The two expression cassettes, selection marker, and homologous sequences were linked so that the LIG4 gene sequences (SEQ ID NOs: 14 and 15) were located at both ends as homologous sequences for introducing them into the genome. The ligated expression construct was extracted as a plasmid using a QIAprep Spin Miniprep Kit (QIAGEN) and treated with NotI restriction enzyme (Takara Bio).

[0144] The CrtE-introduced L. starkeyi strain obtained above was transformed with 10 μg / L of restriction enzyme-treated DNA by electroporation, and the resulting transformant was cultured in a selective medium to obtain a β-carotene-producing L. starkeyi strain.

[0145] A β-carotene-producing S. cerevisiae strain was constructed as a comparative strain as follows: First, the HMG-CoA reductase gene HMG1 (SEQ ID NO: 20), from which 530 amino acids at the N-terminus had been deleted, was linked to the TDH3 promoter (SEQ ID NO: 21) and TDH3 terminator (SEQ ID NO: 22), and introduced into the HMG1 locus on the genome of the YPH499 strain (Stratagene).

[0146] In this study, the G418 resistance gene KanR (SEQ ID NO: 32) was used as a selection marker linked to the PGK1 promoter (SEQ ID NO: 28) and PGK1 terminator (SEQ ID NO: 29). After transformation, the marker was removed using the Cre-loxP system (U. Gueldener et al., Nucleic Acids Research, 2002, 30(6)). Next, the X. dendrorhous-derived (SEQ ID NO: 23) was linked to the TEF1 promoter (SEQ ID NO: 24) and TDH1 terminator (SEQ ID NO: 25) and introduced into the URA3 locus.

[0147] Furthermore, phytoene synthase / lycopene cyclase (SEQ ID NO: 26) derived from Mucor circinelloides was linked to the TEF1 promoter (SEQ ID NO: 24) and TDH1 terminator (SEQ ID NO: 25), and phytoene desaturase (SEQ ID NO: 27) was linked to the PGK promoter (SEQ ID NO: 28) and PGK1 terminator (SEQ ID NO: 29) and introduced into the TRP1 locus. The resulting strains were designated as β-carotene-producing S. cerevisiae strains.

[0148] Example 2: Cultivation and analysis of β-carotene-producing strains β-carotene-producing L. starkeyi strains and β-carotene-producing S. cerevisiae strains were cultivated according to the following procedure. Isolated colonies or glycerol stocks were inoculated into YPD medium, and precultured for 48 hours for L. starkeyi and 24 hours for S. cerevisiae. The composition of YPD medium is shown in Table 2. 4 ml of the preculture solution was inoculated into YPD medium at 2% (v / v) and cultured at 30°C for 84 hours with shaking.

[0149]

[0150] As described in the analytical examples, the amounts of β-carotene and lycopene produced per wet cell weight (g) were analyzed and the results are shown in Table 3 and FIG.

[0151]

[0152] As shown in Table 3 and Figure 1, both the β-carotene-producing L. starkeyi strain and the β-carotene-producing S. cerevisiae strain produced β-carotene, but the production amount per wet cell weight of the β-carotene-producing L. starkeyi strain was significantly greater than that of the β-carotene-producing S. cerevisiae strain, the difference being approximately 4.7 times. The β-carotene production efficiency, calculated by dividing the β-carotene production amount by the 84-hour culture time, was 5.36 μg / wet cell weight / h for the L. starkeyi strain and 1.14 μg / wet cell weight / h for the S. cerevisiae strain.

[0153] Furthermore, the amount of lycopene produced per wet cell weight in the β-carotene-producing S. cerevisiae strain was significantly greater than that in the β-carotene-producing L. starkeyi strain, the difference being approximately 28 times.

[0154] As mentioned above, it has been reported in a literature article (Yongshuo Ma et al., Nature Communications, 2022, 13:572) that lycopene produced in Y. lipolytica cells inhibits the activity of lycopene cyclase, making the conversion reaction of lycopene to β-carotene less likely to proceed. In this example, the β-carotene-producing S. cerevisiae strain produced a large amount of lycopene and a small amount of β-carotene, which is thought to be due to the inhibition of lycopene cyclase activity by lycopene produced in the cells. In the β-carotene-producing S. cerevisiae strain, the conversion efficiency of lycopene to β-carotene is only 20%. On the other hand, the β-carotene-producing L. In the L. starkeyi strain, the conversion efficiency of lycopene to β-carotene was extremely high at 97%, and it is believed that the activity of lycopene cyclase was hardly inhibited by lycopene produced within the bacterial cells. Therefore, this example demonstrated that L. starkeyi is a microorganism suitable for producing β-carotene and β-carotene derivatives.

[0155] Example 3: Construction of a retinol-producing L. starkeyi strain. Because L. starkeyi does not have endogenous retinol biosynthesis genes, expression of a heterologous β-carotene dioxygenase gene is necessary to produce retinol. Therefore, the β-carotene dioxygenase gene from Uncultivated marine bacterium 66A03 was selected as the transgene. The gene and protein sequences were obtained from the literature and NCBI and codon-optimized for L. starkeyi.

[0156] The construction of a retinol-producing L. starkeyi strain is described in detail below. All nucleic acid ligations were performed using the InFusion cloning method. An expression cassette was constructed by ligating an artificially synthesized β-carotene dioxygenase (SEQ ID NO: 16) derived from Uncultured marine bacterium 66A03 with the 70486 promoter (SEQ ID NO: 11) and 70486 terminator (SEQ ID NO: 12).

[0157] Furthermore, a selection marker was constructed by linking the nourseothricin resistance gene sNAT1 (SEQ ID NO: 17) to the ACT1 promoter (SEQ ID NO: 7) and the ACT1 terminator (SEQ ID NO: 8). The expression cassette, selection marker, and homologous sequence were linked so that the sequences of the 18S rDNA gene (SEQ ID NOs: 18 and 19) were located at both ends as homologous sequences for introducing these into the genome.

[0158] Plasmids were extracted from the ligated expression constructs using a QIAprep Spin Miniprep Kit (QIAGEN) and then treated with ApaI restriction enzyme (Takara Bio Inc.). The β-carotene-producing L. starkeyi strain prepared in Example 1 was transformed by electroporation with approximately 5 μg / L of the restriction enzyme-treated DNA, and the resulting transformant was cultured in a selective medium, giving a retinol-producing L. starkeyi strain.

[0159] A retinol-producing S. cerevisiae strain was constructed as a comparative strain as follows. β-carotene dioxygenase (SEQ ID NO: 30) derived from Uncultivated marine bacterium 66A03 was ligated to the TEF1 promoter (SEQ ID NO: 24) and TEF1 terminator (SEQ ID NO: 31), and the oxidoreductase Env9 (SEQ ID NO: 33) derived from S. cerevisiae was ligated to the TDH3 promoter (SEQ ID NO: 34) and TDH3 terminator (SEQ ID NO: 35), and these were introduced into the DPP1 locus of the β-carotene-producing S. cerevisiae strain constructed in Example 1. In this case, the G418 resistance gene KanR (SEQ ID NO: 32) was used as a selection marker ligated to the PGK1 promoter (SEQ ID NO: 28) and PGK1 terminator (SEQ ID NO: 29). The resulting strain was designated as a retinol-producing S. cerevisiae strain.

[0160] Example 4: Cultivation and Analysis of Retinol-Producing Strains Retinol-producing L. starkeyi and S. cerevisiae strains were cultured according to the following procedure. Isolated colonies or glycerol stocks were inoculated into YPD medium, and precultured for 48 hours for L. starkeyi and 24 hours for S. cerevisiae. The composition of the YPD medium was the same as in Example 2. 4 ml of the preculture solution was inoculated into YPD medium at 2% (v / v) and cultured at 30°C for 24 hours with shaking. Subsequently, 800 μl of dodecane + 1% (w / v) dibutylhydroxytoluene (BHT) was added, and the culture was further cultured at 30°C for 48 hours.

[0161] As described in the analytical examples, the results of analyzing the amount of retinol produced are shown in Table 4 and FIG.

[0162]

[0163] Both the retinol-producing L. starkeyi strain and the retinol-producing S. cerevisiae strain produced retinol, but the production amount per culture medium was significantly greater for the retinol-producing L. starkeyi strain than for the retinol-producing S. cerevisiae strain, the difference being approximately four times. The retinol production efficiency, calculated by dividing the retinol production amount by the 72-hour culture time, was 1.01 mg / L / h for the retinol-producing L. starkeyi strain and 0.25 mg / L / h for the retinol-producing S. cerevisiae strain. Therefore, this example demonstrated that L. starkeyi is a microorganism suitable for producing retinol, a β-carotene derivative.

[0164] Example 5 Retinal Production in Retinol-Producing L. starkeyi Strains The retinol-producing L. starkeyi strain was cultured as described in Example 4 and analyzed as described in the analytical examples, and the results are shown in Figure 5A. As shown in Figure 5A, the retinol-producing L. starkeyi strain was shown to produce retinal.

[0165] The results of analyzing the amount of retinal produced are shown in Table 5 and Figure 5B.

[0166]

[0167] As shown in Table 5 and FIG. 5B, both the retinol-producing L. starkeyi strain and the retinol-producing S. cerevisiae strain produced retinal, but the production amount per culture medium of the retinol-producing L. starkeyi strain was significantly greater than that of the retinol-producing S. cerevisiae strain, the difference being approximately 30-fold. The retinol production efficiency, calculated by dividing the retinol production amount by the 72-hour culture time, was 0.53 mg / L / h for the retinol-producing L. starkeyi strain and 0.02 mg / L / h for the retinol-producing S. cerevisiae strain. Therefore, this example demonstrated that L. starkeyi is a microorganism suitable for producing retinal, a β-carotene derivative.

[0168] Example 6: Retinol palmitate production in a retinol-producing L. starkeyi strain. Retinol palmitate is an ester formed by condensation of retinol and palmitic acid by acyltransferase. Since there have been no previous reports demonstrating that retinol palmitate is produced by endogenous acyltransferase in L. starkeyi, the following verification was performed. In this verification, a strain was used in which the expression of three genes in the mevalonate pathway of the retinol-producing strain described in Example 3, HMG1, ERG10 (acetyl-CoA acetyltransferase), and ERG13 (HMG-CoA synthase), was enhanced.

[0169] The creation of a retinol-producing strain with enhanced expression of the above three genes is described in detail below. All nucleic acid ligations were performed using the InFusion cloning method. An expression cassette for tHMG1 was constructed by ligating truncated HMG1 (tHMG1) (SEQ ID NO: 42) amplified from L. starkeyi cDNA, with the transmembrane domain (amino acids 1-529) deleted, to the TDH3 promoter (SEQ ID NO: 4) and TDH3 terminator (SEQ ID NO: 5).

[0170] Similarly, ERG13 (SEQ ID NO: 44) amplified from L. starkeyi cDNA was ligated to the TDH3 promoter (SEQ ID NO: 4) and TDH3 terminator (SEQ ID NO: 5) to construct an expression cassette for ERG13. Furthermore, ERG10 (SEQ ID NO: 43) amplified from L. starkeyi cDNA was ligated to the 70486 promoter (SEQ ID NO: 11) and 70486 terminator (SEQ ID NO: 12) to construct an expression cassette for ERG10.

[0171] A selection marker was constructed by linking the Zeocin resistance gene ble (SEQ ID NO: 45) to the ACT1 promoter (SEQ ID NO: 7) and the ACT1 terminator (SEQ ID NO: 8). The expression cassette, selection marker, and homologous sequence were linked so that the sequences of the KU80 gene (SEQ ID NOs: 46 and 47) were located at both ends as homologous sequences for introducing these into the genome.

[0172] Plasmids from the ligated expression constructs were extracted using a QIAprep Spin Miniprep Kit (QIAGEN) and treated with NotI restriction enzyme (Takara Bio). The retinol-producing strain prepared in Example 3 was transformed with approximately 5 μg / L of the restriction enzyme-treated DNA by electroporation, and the transformant was cultured in a selective medium to obtain a transformant.

[0173] To confirm the production of retinol palmitate, the retinol-producing strain was cultured as follows: 50 ml of YPD medium (Table 2) was inoculated from an isolated colony or glycerol stock and cultured for 48 hours. After culturing, 10% (v / v) of the culture was inoculated into 250 ml of YPD medium and cultured at 30°C for 48 hours with shaking. 10% (v / v) of this culture was then inoculated into 1 L of S medium (Table 6) and cultured in a jar fermenter for 94 hours.

[0174]

[0175] After the cultivation, 0.5 mm glass beads and ethyl acetate were added to the collected cells, and the cells were disrupted and extracted using a multi-beads shocker. The centrifuged supernatant of the cell disruption solution was used as a sample for analyzing the cell contents.

[0176] LC / MS analysis was performed using an Agilent 1260 infinity LC and an Agilent 6100 under the conditions described in Table 1B of [Analysis Example]. In addition to detection at 325 nm, the production of retinol palmitate was confirmed by detection of the m / z = 269 fragment, with reference to a previous report (Breemen et al., J. Chromatogr. A. 794(1-2):245-51, 1998). Figure 6 shows chromatograms of the retinol palmitate preparation and cell lysate detected by UV (wavelength 325 nm). Figure 7 shows chromatograms of the retinol palmitate preparation and cell lysate detected at m / z = 269.

[0177] As shown in Figure 6, a peak was observed at the same retention time between the standard and the sample at 325 nm, the absorption maximum wavelength of retinol palmitate. Furthermore, as shown in Figure 7, when detected at m / z = 269, a peak was observed at the same retention time between the standard and the sample, confirming that the mass spectrum also showed a fragment pattern similar to that of the standard. These results demonstrated that the retinol-producing L. starkeyi strain produces retinol palmitate.

[0178] Example 7: Construction of L. starkeyi strain producing β-carotene derivatives The β-carotene hydroxylase gene (CrtZ) and β-carotene ketolase gene (CrtW) shown below were selected as transgenes.

[0179] To generate zeaxanthin-producing L. starkeyi strains, CrtZ from Erwinia uredovora (Pantoea ananatis) (Accession No. D90087.2) or CrtZ from Pantoea agglomerans (Accession No. M87280.1) was selected.

[0180] To create a β-cryptoxanthin-producing L. starkeyi strain, CrtZ (Accession No. KP866868) from Haematococcus pluvialis was selected. To create a canthaxanthin-producing L. starkeyi strain, CrtW (Accession No. P54972) from Paracoccus sp. N81106 or CrtW (Accession No. AY860820) from Chlamydomonas reinhardtii was selected.

[0181] To construct an astaxanthin-producing L. starkeyi strain, CrtZ (AB181388.1) from Brevundimonas sp. SD212 and CrtW from Paracoccus sp. N81106 were selected. Gene and protein sequences were obtained from the literature and NCBI and codon-optimized for expression in L. starkeyi.

[0182] The construction of a β-carotene derivative-producing L. starkeyi strain is described in detail below. All nucleic acid ligations were performed using the InFusion cloning method. To construct a zeaxanthin-producing strain, an expression cassette for CrtZ was constructed by ligating artificially synthesized CrtZ derived from Pantoea ananatis (EuCrtZ, SEQ ID NO: 51) or CrtZ derived from Pantoea agglomerans (PagCrtZ, SEQ ID NO: 53) to the 70486 promoter (SEQ ID NO: 11) and 70486 terminator (SEQ ID NO: 12).

[0183] To create a β-cryptoxanthin-producing strain, an expression cassette for CrtZ was constructed by linking artificially synthesized Haematococcus pluvialis-derived CrtZ (HpCrtZ, SEQ ID NO: 55) to the 70486 promoter (SEQ ID NO: 11) and 70486 terminator (SEQ ID NO: 12). To create a canthaxanthin-producing strain, an expression cassette for CrtW was constructed by linking artificially synthesized Paracoccus sp. N81106-derived CrtW (PspCrtW, SEQ ID NO: 57) or Chlamydomonas reinhardtii-derived CrtW (CrCrtW, SEQ ID NO: 59) to the 70486 promoter (SEQ ID NO: 11) and 70486 terminator (SEQ ID NO: 12), respectively.

[0184] To create the astaxanthin-producing strain, an artificially synthesized CrtZ (BrevCrtZ, SEQ ID NO: 61) derived from Brevundimonas sp. SD212 was linked to the 70486 promoter (SEQ ID NO: 11) and 70486 terminator (SEQ ID NO: 12), and PspCrtW (SEQ ID NO: 57) was linked to the 3900 promoter (SEQ ID NO: 63) and 3900 terminator (SEQ ID NO: 64) to construct an expression cassette for CrtZ-CrtW.

[0185] In all of the above expression constructs, a selection marker was constructed by linking the nourseothricin resistance gene sNAT1 (SEQ ID NO: 17) to the ACT1 promoter (SEQ ID NO: 7) and the ACT1 terminator (SEQ ID NO: 8). The expression cassette, selection marker, and homologous sequence were linked so that the 18S rDNA gene sequences (SEQ ID NOs: 18 and 19) were located at both ends as homologous sequences for introducing them into the genome. The ligated expression constructs were used to extract plasmids using a QIAprep Spin Miniprep Kit (QIAGEN) and treated with Apa I restriction enzyme (Takara Bio).

[0186] Approximately 5 μg / L of restriction enzyme-treated DNA was transformed by electroporation and cultured in selective medium to obtain transformants, which were designated zeaxanthin-producing L. starkeyi strains 1 and 2, β-cryptoxanthin-producing L. starkeyi strain, canthaxanthin-producing L. starkeyi strains 1 and 2, and astaxanthin-producing L. starkeyi strain, respectively.

[0187]

[0188] Example 8: Cultivation and analysis of zeaxanthin-producing strains, β-cryptoxanthin-producing strains, canthaxanthin-producing strains, and astaxanthin-producing strains. Zeaxanthin-producing L. starkeyi strains 1 and 2, β-cryptoxanthin-producing L. starkeyi strains, canthaxanthin-producing L. starkeyi strains 1 and 2, and astaxanthin-producing L. starkeyi strain were cultivated according to the following procedure. Isolated colonies or glycerol stocks were inoculated into S medium and precultured for 48 hours. The composition of S medium is shown in Table 4. The preculture solution was inoculated into 4 ml of S medium at 2% (v / v) and cultured at 30°C for 96 hours with shaking. The results of analysis performed as described in the analytical examples are shown in Figures 8 to 11.

[0189] Figure 8 shows that the zeaxanthin-producing L. starkeyi strains 1 and 2 produce zeaxanthin, and Figure 9 shows that the β-cryptoxanthin-producing L. starkeyi strain produces β-cryptoxanthin.

[0190] Figure 10 shows that the canthaxanthin-producing L. starkeyi strains 1 and 2 produce canthaxanthin. Figure 11 shows that the astaxanthin-producing L. starkeyi strain produces astaxanthin.

[0191] Example 9 Comparison of Retinol Productivity of Retinol-Producing L. starkeyi Strains Using Different Sugar Sources The retinol-producing L. starkeyi strain obtained in Example 4 was cultured as follows. Isolated colonies or glycerol stocks were inoculated into YPD medium and precultured for 48 hours. The composition of YPD medium is shown in Table 2. For the subsequent main culture, YPD medium or a medium in which the glucose in YPD medium was replaced with another sugar source as shown below was used. The sugar sources used were glucose, sucrose, galactose, cellobiose, maltose, fructose, soluble starch, and corn starch, and were adjusted to a final concentration of 2% w / v in each medium. To avoid gelatinization, soluble starch was heated at 60°C for 2 hours to prepare a 20% (w / v) solution, which was then blended into the medium to achieve a final concentration of 2% w / v. Corn starch is insoluble, so it was not dissolved in water and was used at a concentration of 2% w / v. A medium without added sugar source (Negative ctl.) was prepared as a negative control. The preculture solution was inoculated at 2% (v / v) into 4 ml of YPD medium containing different sugar sources and cultured with shaking for 24 hours. Then, 800 μl of dodecane + 1% (w / v) dibutylhydroxytoluene (BHT) was added, and the medium was further cultured at 30°C for 144 hours. The results of the analysis performed as described in the analytical examples are shown in Figure 12.

[0192] As shown in Figure 12, in media containing each sugar source, all of the retinol-producing L. starkeyi strains produced retinol.

[0193] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. This application is based on a Japanese patent application (Patent Application No. 2024-135372) filed on August 14, 2024, the entirety of which is incorporated by reference. All references cited herein are incorporated in their entirety.

[0194]

Claims

1. A method for producing at least one of β-carotene and a β-carotene derivative using a genetically modified microorganism of the genus Lipomyces.

2. The β-carotene derivative is retinol, β-cryptoxanthin, zeaxanthin, canthaxanthin, astaxanthin, fucoxanthin, capsanthin, abscisic acid, β-ionone, retinal, lycopene, lutein, retinoic acid, retinol acetate, retinol palmitate, retinol linoleate, retinol linolenate, retinol oleic acid, retinol linolenic acid, retinol linolenic acid ester, retinol linolenic acid glyceride 2. The method of claim 1, wherein the retinyl linoleate is at least one of retinyl linoleate triglyceride, retinyl linoleate ethyl ester, retinyl linoleate methyl ester, retinyl linoleate amide, retinyl linoleate phenylamide, retinyl linoleate hexylamide, retinyl linoleate stearylamide, retinyl linoleate oleylamide, retinyl linoleate linoleylamide, and retinyl linoleate linolenylamide.

3. The method of claim 1, wherein the genetically modified microorganism is Lipomyces starkeyi.

4. The method according to claim 1, comprising culturing the genetically modified microorganism to produce at least one of β-carotene and a β-carotene derivative in a culture, wherein the genetically modified microorganism has a β-carotene biosynthetic pathway.

5. The method according to claim 4, wherein the β-carotene biosynthetic pathway comprises at least one enzyme selected from farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, phytoene synthase, phytoene desaturase, and lycopene cyclase.

6. The method according to claim 5, wherein the β-carotene biosynthetic pathway comprises at least one enzyme selected from the group consisting of geranylgeranyl diphosphate synthase derived from Xanthophyllomyces dendrorhous, phytoene synthase derived from Mucor circinelloides, lycopene synthase derived from Mucor circinelloides, and lycopene cyclase derived from Mucor circinelloides.

7. The method according to claim 6, wherein the β-carotene biosynthetic pathway comprises at least one protein selected from the following [1] to [4]: [1] At least one protein selected from the following (1a) to (1c): (1a) a protein comprising the amino acid sequence represented by SEQ ID NO: 36; (1b) a mutant protein comprising an amino acid sequence in which 1 to 37 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 36, and having geranylgeranyl diphosphate synthase activity; (1c) a homologous protein comprising an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 36, and having geranylgeranyl diphosphate synthase activity; [2] At least one protein selected from the following (2a) to (2c): (2a) a protein comprising the amino acid sequence represented by SEQ ID NO: 37; (2b) a mutant protein comprising an amino acid sequence in which 1 to 60 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 37, and having phytoene synthase activity; (2c) a homologous protein comprising an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 37, and having phytoene synthase activity. [3] At least one protein selected from the following (3a) to (3c): (3a) a protein comprising the amino acid sequence represented by SEQ ID NO: 37; (3b) a mutant protein comprising an amino acid sequence in which 1 to 60 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 37, and having lycopene cyclase activity; (3c) a homologous protein comprising an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 37, and having lycopene cyclase activity; [4] At least one protein selected from the following (4a) to (4c): (4a) a protein comprising the amino acid sequence represented by SEQ ID NO: 38; (4b) a mutant protein comprising an amino acid sequence in which 1 to 58 amino acids have been deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 38, and having phytoene desaturase activity; (4c) a homologous protein comprising an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 38, and having phytoene desaturase activity.

8. The method according to claim 1, wherein the genetically modified microorganism is a microorganism in which the activity of lycopene cyclase is not inhibited by lycopene produced within the genetically modified microorganism.

9. The method according to claim 1, comprising any one of the following (α1) to (α7): (α1) The genetically modified microorganism has at least one of β-carotene dioxygenase and retinal reductase, and by culturing the genetically modified microorganism, retinol is produced in the culture. (α2) The genetically modified microorganism has β-carotene dioxygenase, and by culturing the genetically modified microorganism, retinal is produced in the culture. (α3) The genetically modified microorganism has acyltransferase, and by culturing the genetically modified microorganism, retinol palmitate is produced in the culture. (α4) The genetically modified microorganism has β-carotene hydroxylase, and by culturing the genetically modified microorganism, zeaxanthin is produced in the culture. (α5) The genetically modified microorganism has β-carotene hydroxylase, and by culturing the genetically modified microorganism, β-cryptoxanthin is produced in the culture. (α6) The genetically modified microorganism has β-carotene ketolase, and by culturing the genetically modified microorganism, canthaxanthin is produced in the culture. (α7) The genetically modified microorganism has β-carotene hydroxylase and β-carotene ketolase, and astaxanthin is produced in the culture by culturing the genetically modified microorganism.

10. The method of claim 1, wherein the efficiency of conversion of lycopene to β-carotene by the genetically modified microorganism is 30% or more.

11. The method according to claim 10, wherein the efficiency of β-carotene production is 1.2 μg / wet cell weight / h or more.

12. The method according to claim 11, wherein the β-carotene derivative is retinol and the retinol production efficiency is 0.26 mg / L / h or more.

13. A genetically modified microorganism of the genus Lipomyces, which has a β-carotene biosynthetic pathway and produces at least one of β-carotene and a β-carotene derivative by fermentation.

14. The genetically modified microorganism according to claim 13, further comprising at least one of β-carotene dioxygenase and retinal reductase, and producing retinol by fermentation.

15. A method for producing a β-carotene derivative, comprising culturing the genetically modified microorganism according to claim 13 or 14 to produce a β-carotene derivative in the culture, and recovering the β-carotene derivative from the culture.

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