Methods for producing strigolactones using cyanobacteria and yeast

Abstract

To provide highly productive methods for producing strigolactone by efficiently converting carlactone produced by cyanobacterium to strigolactone using yeast.SOLUTION: Provided is a method for producing strigolactone comprising the steps of: culturing a cyanobacterium having carlactone productivity to prepare a cyanobacterium culture fluid; culturing a yeast capable of expressing MAX1 enzyme to prepare a yeast culture fluid; mixing the yeast culture fluid and the cyanobacterium culture fluid to prepare a cyanobacterium-yeast coculture fluid; and culturing the cyanobacterium-yeast coculture fluid.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to methods for producing strigolactones using cyanobacteria and yeast. [Background technology]

[0002] Strigolactones are a group of carotenoid derivatives with lactone structures that are obtained from plants and are known to inhibit branching in plants. Strigolactones have important physiological functions in agricultural production, such as inhibiting branching in host plants themselves, inducing hyphal branching in arbuscular mycorrhizal fungi that supply plants with phosphorus and nitrogen, and stimulating seed germination in root parasitic plants. However, the amount of strigolactones that can be extracted from plants is limited.

[0003] It is known that the biosynthesis of strigolactones in plants involves two carotenoid oxidative cleavage enzymes, β-carotene-9-isomerase and 4-deoxyorobanchol synthase, which use carotenoids as substrates.

[0004] Non-Patent Document 1 analyzed the functions of the carotenoid oxidative cleavage enzymes CCD7 and CCD8, and the β-carotene-9-isomerase D27, and reported that the sequential action of these three enzymes on carotenoid substrates produces a compound named carlactone. Furthermore, carlactone has a butenolide ring, which is unique to the chemical structure of strigolactone, and has been shown to have similar physiological activity to strigolactone, suggesting that it is a biosynthetic intermediate of strigolactone.

[0005] Non-patent document 2 states that caractone actually exists as a metabolite in plants, and 13 We have reported that administering 1C-labeled curlactone to strigolactone biosynthetic mutants of rice resulted in the conversion of curlactone to strigolactone in the plant.

[0006] Non-patent document 3 reports that by mixing carlactone with rice 4-deoxyorobanchol synthase (CYP711A2), carlactone is converted to 4-deoxyorobanchol, a strigolactone.

[0007] Non-Patent Document 4 reports research on strigolactone production using Escherichia coli capable of producing caractone and yeast (Saccharomyces cerevisiae ECL-3 strain) into which AtMAX1 (CYP711A1) and ATR1 have been introduced.

[0008] As mentioned above, carlactone is a plant hormone called strigolactone and its precursor, and carlactone and strigolactone are effective in eliminating root parasitic plants. However, there are currently no reports of a method for mass-producing carlactone and strigolactone using cyanobacteria and yeast. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Adrian Alder et al., “The Path from β-Carotene to Carlactone, a Strigolactone-Like Plant Hormone”, Science 16 Mar 2012:Vol.335, Issue 6074, pp.1348-1351, DOI:10.1126 / science.1218094 [Non-patent document 2] Yoshiya Seto et al., “Carlactone is an endogenous biosynthetic precursor for strigolactones”, PNAS January 28, 2014 111 (4) 1640-1645; https: / / doi.org / 10.1073 / pnas.1314805111 [Non-patent document 3] Zhang et al., “Rice cytochrome P450 MAX1 homologs catalyze distinct steps in strigolactone biosynthesis”, Nat Chem Biol.2014 Dec;10(12):1028-33.doi:10.1038 / nchembio.1660. [Non-patent document 4] Wu et al., “Establishment of Strigolactone-Producing Bacterium-Yeast Consortium”, Sci Adv 2021 Sep 17;7(38):eabh4048.doi: 10.1126 / sciadv.abh4048. Summary of the Invention [Problem to be solved by the invention]

[0010] One approach to mass-producing strigolactone is to establish a system for producing carlactone, a substrate for the strigolactone biosynthetic pathway, in an industrial-scale production host and efficiently converting it to strigolactone. Therefore, an objective of the present invention is to establish a highly productive method for producing strigolactone by efficiently converting carlactone produced using cyanobacteria to strigolactone by co-cultivating it with yeast. [Means for solving the problem]

[0011] To solve the above problems, the inventors focused on the fact that strigolactones are produced in the chloroplasts and endoplasmic reticulum within plant cells and constructed a strigolactone production system using cyanobacteria, which have an intracellular environment similar to that of chloroplasts, as the production host. Furthermore, the inventors combined yeast with endoplasmic reticulum-containing bacteria capable of activating strigolactone-metabolizing enzymes to construct a strigolactone production pathway through co-cultivation of cyanobacteria and yeast. Using yeast expressing the rice 4-deoxyorobanchol synthase (CYP711A2), they succeeded in producing 4-deoxyorobanchol, a strigolactone.

[0012] The present invention was completed based on the above findings, and provides a method for producing strigolactone, comprising the steps of: preparing a cyanobacterial culture solution by culturing cyanobacteria capable of producing cyanolactone; preparing a yeast culture solution by culturing yeast; mixing the cyanobacterial culture solution with the yeast culture solution to prepare a cyanobacterial-yeast co-culture solution; and co-cultivating the cyanobacterial-yeast co-culture solution.

[0013] The present invention also provides a culture containing strigolactone or a purified product thereof obtained by the method for producing strigolactone. [Effects of the Invention]

[0014] According to the method for producing strigolactone of the present invention, approximately 1,200 pg of strigolactone (4-deoxyorobanchol) can be produced per mL of culture medium using cyanobacteria and yeast, which is 10,000 times the amount of 4-deoxyorobanchol extracted from rice roots. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a diagram illustrating an outline of the method for producing strigolactone of the present embodiment. [Figure 2] FIG. 1 is a process diagram of a method for producing strigolactone of the present embodiment. [Figure 3] FIG. 1 shows the results of an analysis of strigolactones produced by cyanobacteria-yeast co-culture. DETAILED DESCRIPTION OF THE INVENTION

[0016] The method for producing strigolactone according to an embodiment of the present invention will now be described. Figure 1 is a diagram illustrating an overview of the method for producing strigolactone according to the present embodiment.

[0017] 1. Preparation of cyanobacterial culture medium First, the step of preparing a cyanobacterial culture solution by culturing cyanobacteria capable of producing curlactone will be described.

[0018] The type of cyanobacteria that can be used in this embodiment is not particularly limited, and includes all types. Preferred examples include, but are not limited to, cyanobacteria belonging to the genera Synechocystis, Synechococcus, Thermosynechococcus, Trichodesmium, Acaryochloris, Crocosphaera, and Anabaena, and more preferred examples include cyanobacteria belonging to the genera Synechocystis, Synechococcus, Thermosynechococcus, and Anabaena. More preferred are Synechocystis sp. PCC6803, Synechocystis sp. PCC7509, Synechocystis sp. PCC6714, Synechococcus elongatus PCC7942, Synechococcus elongatus UTEX 2973, Synechococcus sp. PCC7002, Thermosynechococcus elongatus BP-1, Trichodesmium erythraeum IMS101, Acaryochloris mariana MBIC11017, Crocosphaera watsonii WH8501, and Anabaena sp. PCC7120. Particularly preferred is Synechococcus elongatus PCC7942.

[0019] β-Carotene-9-isomerase is an enzyme that catalyzes the conversion of all-trans-β-carotene to 9-cis-β-carotene. The recombinant cyanobacterium of the present invention has a β-carotene-9-isomerase gene that expresses this enzyme. The β-carotene-9-isomerase gene is not limited as long as it expresses an enzyme that can convert trans-β-carotene to 9-cis-β-carotene in the cyanobacterium. Examples of such genes include those from the following plants or algae: rice (Oryza sativa, Oryza brachyantha); sorghum (Sorghum bicolor); foxtail millet (Setaria italica); wheatgrass (Brachypodium distachyon); wheatgrass (Aegilops tauschii); corn (Zea mays); asparagus (Asparagus officinalis); wild bamboo (Musa acuminata); oil palm (Elaeis guineensis); date palm (Phoenix dactylifera); tobacco (Nicotiana tabacum); Arabidopsis thaliana; Arabidopsis lyrata; rapeseed (Brassica napus); shepherd's purse (Camelina sativa); alfalfa (Medicago truncatula); Physcomitrella patens Examples of the D27 genes include those derived from Chlamydomonas patens; Chlamydomonas reinhardtii; Dunaliella Bardawil; Monoraphidium neglectum; Volvox carteri; and Coccomyxa subellipsoidea.

[0020] In this embodiment, the carotenoid oxidative cleavage enzyme is (i) an enzyme that catalyzes the conversion of 9-cis-β-carotene produced by β-carotene-9-isomerase to 9-cis-β-apo-10'-carotenol, and / or (ii) an enzyme that catalyzes the conversion of 9-cis-β-carotene and 9-cis-β-apo-10'-carotenol to carlactone ((Z)-(R)-carlactone). The recombinant cyanobacterium used in this embodiment has a carotenoid oxidative cleavage enzyme gene so as to express the enzyme. The carotenoid oxidative cleavage enzyme gene is not limited, as long as it expresses an enzyme that can convert 9-cis-β-carotene in the cyanobacterium to carlactone.Examples of such genes include those from the following plants or algae: rice (Oryza sativa, Oryza brachyantha); sorghum (Sorghum bicolor); foxtail millet (Setaria italica); wheatgrass (Brachypodium distachyon); wheatgrass (Aegilops tauschii); corn (Zea mays); asparagus (Asparagus officinalis); wild bamboo (Musa acuminata); oil palm (Elaeis guineensis); date palm (Phoenix dactylifera); tobacco (Nicotiana tabacum); Arabidopsis thaliana; Arabidopsis lyrata; rapeseed (Brassica napus); shepherd's purse (Camelina sativa); alfalfa (Medicago truncatula); Physcomitrella patens Examples of CCD7 and CCD8 genes derived from Chlamydomonas reinhardtii, Dunaliella bardawil, Monoraphidium neglectum, Volvox carteri, and Coccomyxa subellipsoidea include the CCD7 gene and its orthologous genes, such as the MAX3 gene, D17 gene, RMS5 gene, and DAD3 gene, and orthologous genes including these can also be used in the present invention as carotenoid oxidative cleavage enzyme genes. Furthermore, the CCD8 gene and its orthologous genes, such as the MAX4 gene, D10 gene, RMS1 gene, and DAD1 gene, can also be used in the present invention as carotenoid oxidative cleavage enzyme genes.

[0021] In a preferred embodiment, the recombinant cyanobacterium used in this embodiment has both of the following carotenoid oxidative cleavage enzymes: (i) a gene encoding an enzyme that catalyzes the conversion of 9-cis-β-carotene to 9-cis-β-apo-10'-carotenol, and (ii) a gene encoding an enzyme that catalyzes the conversion of 9-cis-β-carotene to 9-cis-β-apo-10'-carotenol into (Z)-(R)-carlactone.

[0022] Furthermore, the enzymes encoded by the β-carotene-9-isomerase gene and the carotenoid oxidative cleavage enzyme gene may have one or more amino acids deleted, substituted, or added in their amino acid sequences, as long as they have the catalytic reaction described above.

[0023] Vectors such as plasmid vectors can be used to introduce genes into cyanobacteria. Expression vectors are preferred. For example, an expression vector is constructed containing DNA fragments of heterologous carotenoid oxidative cleavage enzyme genes and β-carotene-9-isomerase genes and a promoter for their expression. Examples of promoters that can be used include the lac, tac, or trc promoter, promoters related to derivatives inducible by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG), and promoters isolated from cyanobacteria, such as the Rubisco operon (rbc), PSI reaction center protein (psaAB), and PSII D1 protein (psbA). Various promoters that function in cyanobacteria can be used, but are not limited to these. Furthermore, the expression vector may further incorporate a marker gene (e.g., a resistance gene for drugs such as kanamycin, chloramphenicol, spectinomycin, or erythromycin) for selecting a host into which the vector has been appropriately introduced. The expression vector is then introduced into the parent cyanobacterium or the modified cyanobacterium of the present invention using known methods for transformation. Vectors can be introduced into cyanobacteria by common methods such as natural transformation, electroporation, conjugation, etc. After transformation, the cyanobacteria are cultured in a selective medium, such as a medium containing an antibiotic, to select transformants having the desired traits.

[0024] The recombinant cyanobacteria used in this embodiment have the ability to produce curlactone. Furthermore, the recombinant cyanobacteria of the present invention secrete curlactone extracellularly. Therefore, by culturing the recombinant cyanobacteria of the present invention under appropriate conditions and then recovering the secreted curlactone, efficient curlactone production can be achieved.

[0025] Cyanobacteria can generally be cultured using liquid culture or a modified version thereof using BG-11 medium (J Gen Microbiol., 1979, 111:1-61). For caractone production, it is preferable to culture until the cells' metabolism is activated and sufficient carotenoids accumulate in the cells; for example, aeration and agitation culture or shaking culture for 1 to 7 days is suitable. When expression of the introduced gene is induced by adding IPTG or the like, the induction period is not limited to the following, but it is preferable to add it 1 to 3 days after the start of culture.

[0026] By the above-mentioned cultivation, the cyanobacteria produces curlactone and secretes the curlactone into the culture medium.

[0027] 1. Creating yeast strains The type of yeast that can be used in this embodiment is not particularly limited, and any type can be used. Preferably, a yeast belonging to the genus Saccharomyces, Debaryomyces, Schizosaccharomyces, Candida, Yarrowia, Rhodotorula, Lipomyces, Kluyveromyces, Rhodosporidium, Trichoderma, Torulopsis, Pichia, or the like is used as a parent strain, but is not limited thereto. For example, Saccharomyces cerevisiae, Saccharomyces bayanus, Debaryomyces nilssonii, Debaryomyces hansenii, Schizosaccharomyces pombe, Candida glabrata, Candida tropicalis, Candida utilis, Candida boidinii, Yarrowia lipolytica, Rhodotorula glutinis, Lipomyces lipoferus, Examples of suitable strains include Saccharomyces lipoferus, Kluyveromyces lactis, Rhodosporidium toruloides, Trichoderma reesei, Torulopsis colliculosa, and Pichia farinosa. Among these, the genus Saccharomyces is the most preferred because it is easy to use genetic engineering techniques.

[0028] In this embodiment, the 4-deoxyorobanchol synthase expressed in yeast cells is an enzyme that catalyzes the conversion of carlactone ((Z)-(R)-carlactone) to 4-deoxyorobanchol. The recombinant yeast used in this embodiment has a 4-deoxyorobanchol synthase gene so as to express this enzyme. The 4-deoxyorobanchol synthase gene is not limited as long as it expresses an enzyme that can convert carlactone to 4-deoxyorobanchol. Examples of such genes include those from the following plants or algae: rice (Oryza sativa, Oryza brachyantha); sorghum (Sorghum bicolor); foxtail millet (Setaria italica); wheatgrass (Brachypodium distachyon); wheatgrass (Aegilops tauschii); corn (Zea mays); asparagus (Asparagus officinalis); wild bamboo (Musa acuminata); oil palm (Elaeis guineensis); date palm (Phoenix dactylifera); tobacco (Nicotiana tabacum); Arabidopsis thaliana; Arabidopsis lyrata; rapeseed (Brassica napus); camelina sativa; alfalfa (Medicago truncatula); and peanut (Arachis Examples of MAX1 genes include those derived from Helianthus annuus; Marchantia polymorpha; Physcomitrella patens; Chlamydomonas reinhardtii; Dunaliella bardawil; Monoraphidium neglectum; Volvox carteri; and Coccomyxa subellipsoidea.

[0029] In this embodiment, the MAX1 enzyme and NADPH-P450 reductase (ATR1), which is required for its activation, must be expressed in yeast. An expression vector for the rice MAX1 enzyme is introduced into the WAT11 yeast strain, which expresses ATR1, for co-expression. The vector is preferably an expression vector. For example, an expression vector is constructed containing a DNA fragment of the 4-deoxyorobanchol synthase gene and a promoter for its expression. Promoters that can be induced by the addition of galactose can be used, but are not limited to these. Various promoters that function in yeast can be used. Furthermore, the expression vector may further incorporate a marker gene required for selection during cloning in E. coli (e.g., resistance genes for drugs such as ampicillin, kanamycin, chloramphenicol, spectinomycin, and erythromycin) or a marker gene for selecting a host yeast into which the vector has been appropriately introduced (e.g., metabolic genes such as leucine, tryptophan, histidine, uracil, and adenine). The expression vector is then introduced into the parent yeast strain WAT11 for transformation by known methods. Vectors can be introduced into yeast by common methods such as transformation using polyethylene glycol, conjugation, etc. After transformation, yeast is cultured in a selective medium, for example, a medium lacking amino acids or nucleic acids, allowing the selection of transformants having desired traits.

[0030] 2.Preparing yeast culture Next, the step of preparing a yeast culture solution by culturing yeast will be described. Yeast culture can generally be performed by liquid culture using SGI medium or a modified method thereof. For expression of MAX1 and ATR1, it is preferable to culture the cells until their metabolism is sufficiently activated; for example, shaking culture for 2 to 4 days is suitable.

[0031] 3. Preparing Cyanobacteria-Yeast Co-cultures Next, we will explain the process of preparing a cyanobacteria-yeast co-culture by mixing the cyanobacteria culture solution and the yeast culture solution. After mixing the recombinant cyanobacteria culture solution cultured for 3 days with the recombinant yeast culture solution cultured for 2 days, we further added YPGE medium containing galactose, an inducer of yeast MAX1 gene expression, to prepare a co-culture solution.

[0032] 4. Co-culture process Next, we will explain the co-cultivation step of culturing the cyanobacteria-yeast co-culture solution. For strigolactone production, it is preferable to culture the cells until their metabolism is activated and strigolactone is sufficiently accumulated in the cells. For example, shaking culture is suitable for 1 to 3 days.

[0033] 5. Strigolactone extraction process Ethyl acetate is used to extract strigolactones from cyanobacteria-yeast. Ethyl acetate is added to the cyanobacteria-yeast culture medium and mixed thoroughly. The separated ethyl acetate layer is transferred to a new centrifuge tube. This process is repeated three times. Physiological saline is added to the ethyl acetate layer and mixed thoroughly, after which the separated ethyl acetate layer is transferred to a new tube. Solid sodium sulfate is added to the ethyl acetate layer to dehydrate it, and after separating it into appropriate sample volumes, the solvent is removed using an evaporator. This sample is redissolved in 50% acetonitrile and subjected to LC-MS / MS.

[0034] The present invention will be described in more detail below using specific examples, but the present invention is not limited to the following embodiments. [Example]

[0035] 1. Construction of the curlactone-producing plasmid and preparation of transformants The following three genes (DbD27, OsCCD7, and AtCCD8) were amplified by PCR using PrimeStar DNA polymerase (TaKaRa) according to the instructions for PrimeStar DNA polymerase (TaKaRa).

[0036] The DbD27 gene fragment was amplified using the DbD27-f1 and SD-DbD27-r2 primer sets from the Dunaliella D27 gene (Davidi and Pick, Plant Cell Rep 2017), which had been codon-optimized for Synechococcus elongatus PCC7942. The OsCCD7 gene fragment was amplified using the rice CCD7 gene (NCBI-Protein ID: XP_015637185) cDNA clone as template with the SD-OsCCD7-f3 and SD-OsCCD7-r4 primer sets. The AtCCD8 gene fragment was amplified using the Arabidopsis CCD8 gene (NCBI-Protein ID: NP_195007) cDNA clone as template with the SD-AtCCD8-f5 and AtCCD8-r6 primer sets. Primer information for each primer set is listed in the table below.

[0037] [Table 1]

[0038] The three DNA fragments were recombined using PrimeStar GXL polymerase (TaKaRa) and the primer set DbD27-f1 and AtCCD8-r6. The resulting DNA fragment was cloned into the pHRA-lacI-Plac-Ptrc vector using the In-Fusion cloning kit (TaKaRa) and designated pHRA-SL3. A DNA fragment containing the lacI-trc promoter-DbD27-OsCCD7-AtCCD8 was amplified using the primer set p24-B0015ter-f and B0011-Cm-r and cloned into the p24EX plasmid vector using the In-Fusion cloning kit. The resulting plasmid was designated p24EX-SL3. The primer information is shown in the table below.

[0039] [Table 2]

[0040] p24EX-SL3 was used to transform Synechococcus elongatus PCC7942. 20 mL of logarithmic-phase culture was concentrated to 0.5 mL, and 1 μg of p24EX-SL3 was added. The culture was shaken by end-over-end rotation overnight in the dark, followed by one hour in the light. Transformants were selected by plating on BG11 agar medium containing 10 μg / μl chloramphenicol. The resulting transformants were used in the following cyanobacterial culture preparation process.

[0041] 2. Cultivation of Curlactone-producing Cyanobacteria Figure 2 shows a process diagram for the strigolactone production method. The cyanobacterial CL-producing strain was inoculated into 40 mL of BG11 medium containing 7.5 μg / mL chloramphenicol at a concentration of OD750 = 0.05. After 48 hours of cultivation, IPTG (final concentration 1 mM) was added. Bacterial cells were harvested 24 hours after IPTG addition.

[0042] 3. Creating yeast strains For MAX1 and ATR1, a budding yeast strain (Saccharomyces cerevisiae WAT11, pYeDP60-OsCYP711A2) expressing the rice MAX1 enzyme and Arabidopsis ATR1 was used, as in Non-Patent Document 3.

[0043] 4.MAX1 yeast culture solution preparation process The MAX1 yeast was inoculated into 10 mL of SGI medium (20 g / LD (+)-glucose, 1 g / L bactocasamino acids, 6.7 g / L yeast nitrogen base without amino acid, 40 mg / L L-tryptophan) and cultured with shaking at 30°C for 48 hours.

[0044] 5. Preparing Cyanobacteria-Yeast Co-cultures A co-culture solution was prepared by mixing 100 μL of a recombinant cyanobacterial culture that had been producing carlactone with 100 μL of a recombinant yeast culture that had been cultured for two days, and then adding 500 μL of YPGE medium (5 g / LD (+)-glucose, 10 g / L yeast extract, 10 g / L bactopeptone, 3% (vol / vol) ethanol) containing galactose (final concentration 20 g / L), an inducer of yeast MAX1 gene expression. The recombinant cyanobacterial culture was added in amounts of 500 μL, 250 μL, or 100 μL, and the total culture volume was adjusted using BG11 medium.

[0045] 6. Co-culture process A culture solution having the composition shown in Table 3 (total 1100 μL) was prepared and cultured in a test tube with shaking at 28° C. for 19 hours.

[0046] [Table 3]

[0047] 7.Strigolactone extraction process After the shaking culture was completed, 500 μL of ethyl acetate was added to the tube containing the culture medium. 40 μL (400 pmol) of d6-5-deoxystrigol (d6-5DS) was also added as an internal standard. The mixture was then vigorously mixed, and the separated ethyl acetate layer was transferred to a new tube. This procedure was repeated three times for each sample. 1 mL of saturated NaCl was added, and the mixture was shaken vigorously and allowed to stand. After standing, the ethyl acetate layer from the tube, which had separated into two layers, was collected in a new tube. Two teaspoons of anhydrous sodium sulfate were added to the ethyl acetate layer, which was then filtered through a cotton plug. The solvent was then evaporated to dryness under reduced pressure using an evaporator.

[0048] The bacterial cell samples and supernatant samples obtained above were subjected to liquid chromatography mass spectrometry (LC / MS / MS) as follows: The dried samples were dissolved in 40 μL of 50% acetonitrile and subjected to LC / MS / MS. LC / MS / MS was performed using an Acquity UPLC BEH-C18 column (2.1 × 50 mm, 1.7 μm; Waters) combined with a Nexera UPLC system (Shimadzu) and a TripleTOF® 5600 MS system (Sciex) under the following conditions: flow rate 0.3 mL / min, mobile phase A 0.5% formic acid water, B acetonitrile, column open 40°C, gradient: 20% B at 0 min, 40% B at 4 min, 70% B at 7 min, 99% B at 9 min, and 99% B at 11 min. 4-deoxyorobanchol was detected in positive ion mode at m / z = 331.1 > 216.1. The internal standard d6-5-deoxystrigol was detected in positive ion mode at m / z = 337.2 > 222.1.

[0049] Figure 3 shows the analysis results of strigolactone (4-deoxyorobanchol: 4DO) produced by co-culture of cyanobacteria and yeast. As shown in Figure 3, when 500 μL of cyanobacteria culture solution and 100 μL of yeast culture solution were used, approximately 1200 pg of strigolactone (4-deoxyorobanchol) was produced per mL of culture solution (1320 pg per 1100 μL culture system). The amount of strigolactone produced increased in proportion to the volume of cyanobacteria culture solution used in the co-culture. According to Non-Patent Document 2, the amount of strigolactone extracted from rice roots is approximately 10 pg per gram. Approximately 9 mg of cells can be obtained from 1 mL of cyanobacteria-yeast culture solution, so the amount of strigolactone obtained from this embodiment is more than 10,000 times the amount extracted from rice roots.

Claims

1. A step of preparing a cyanobacterial culture solution by culturing a cyanobacterium capable of producing curlactone; A step of preparing a yeast culture solution by culturing yeast; Mixing the cyanobacteria culture solution and the yeast culture solution to prepare a cyanobacteria-yeast co-culture solution; A co-cultivation step of culturing the cyanobacteria-yeast co-culture solution; 1. A method for producing strigolactone, comprising: the cyanobacterium capable of producing carlactone has a CCD7 gene and a CCD8 gene as carotenoid oxidative cleavage enzyme genes and a D27 gene as a β-carotene-9-isomerase gene; The yeast has MAX1 and ATR1. Methods for producing strigolactones.

2. 2. The method for producing strigolactone according to claim 1, wherein the strigolactone is 4-deoxyorobanchol.

3. 3. The method for producing strigolactone according to claim 1, further comprising the step of adding ethyl acetate after the co-cultivation step to extract strigolactone.

4. A method for producing a culture or a purified product thereof containing strigolactone by the method for producing strigolactone according to any one of claims 1 to 3.

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