Methods for Analyzing Microbial Metabolites

Abstract

Provided is a method by which the time required for analysis can be shortened and various substances produced by microorganisms can be analyzed. A method for analyzing metabolites of microorganisms according to the present invention comprises: a step for supplying a mobile phase containing carbon dioxide in a liquid state, a subcritical state, or a supercritical state to a container in which microorganisms cultured in a medium are housed together with the medium, and thereby moving, to the mobile phase, metabolite components of the microorganisms present in the microorganisms and the medium; a step for introducing, into a column, the mobile phase to which the metabolite components have been moved; and a step for mass-analyzing the metabolite components contained in the mobile phase which has passed through the column.

Description

[Technical field]

[0001] The present invention relates to a method for analyzing microbial metabolites. [Background technology]

[0002] In recent years, attempts have been made to obtain useful substances from the metabolites of microorganisms (recombinants, hereinafter referred to as "recombinant microorganisms") by genetically modifying the microorganisms to give them the ability to produce useful substances or to increase their production ability. For example, Patent Document 1 describes the obtaining of isoprene or terpenes from bacteria such as Clostridium bacteria and Moorella bacteria that have been genetically modified. Isoprene is a hydrocarbon represented by the structural formula CH2=C(CH3)CH=CH2. Terpene is a hydrocarbon whose structural unit is isoprene, and is a general term for various substances with different molecular structures. These isoprenes and terpenes have a variety of uses, such as raw materials for resins, raw materials for fragrances, food additives, detergents, electronic materials, and raw materials for pharmaceuticals and agrochemicals.

[0003] One method for investigating whether a microorganism has acquired an expected ability through genetic recombination is to analyze the metabolites of the microorganism using a chromatograph mass spectrometer. Patent Document 1 describes a method in which a recombinant microorganism (bacterial strain) grown by culturing on an agar medium is inoculated into a liquid medium, and the liquid medium is placed in a vial together with a mixed gas of carbon monoxide, carbon dioxide, and hydrogen, and then the liquid medium is sealed and cultivated, and the gas phase (gas in the head space above the vial) is analyzed with a gas chromatograph mass spectrometer. Since isoprene contained in the metabolites of the recombinant microorganism volatilizes from the liquid medium during cultivation in the vial, the isoprene production ability of the recombinant microorganism can be examined by analyzing the gas phase. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. WO2018 / 155272 [Patent Document 2] JP 2019-184502 A [Patent Document 3] International Publication No. WO2016 / 084963 Summary of the Invention [Problem to be solved by the invention]

[0005] In the method described in Patent Document 1, it is necessary to culture the recombinant microorganism in a liquid medium and sufficiently volatilize the desired substance (isoprene) contained in the metabolites of the recombinant microorganism, which takes a long time for analysis. Furthermore, if the substance produced by the microorganism is not volatile, it cannot be analyzed.

[0006] An object of the present invention is to provide a method capable of shortening the time required for analysis and analyzing various substances produced by microorganisms. [Means for solving the problem]

[0007] The method for analyzing microbial metabolites according to the present invention, which has been achieved to solve the above problems, comprises the steps of: A step of supplying a mobile phase containing carbon dioxide in a liquid state, a subcritical state, or a supercritical state to a container in which a microorganism cultured in a medium is accommodated together with the medium, thereby transferring components of the metabolites of the microorganism present in the microorganism and the medium to the mobile phase; introducing the mobile phase into a column in which the metabolite components have been transferred; subjecting the metabolite components contained in the mobile phase passed through the column to mass spectrometry; has. Effect of the Invention

[0008] In the method according to the present invention, carbon dioxide is pumped at high pressure (7.38 MPa or more) to supply liquid, subcritical, or supercritical carbon dioxide to the container while the microorganisms cultured in the culture medium are housed in the container. The mobile phase containing carbon dioxide in a liquid, subcritical, or supercritical state under high pressure conditions comes into contact with the microorganisms and the culture medium in the container, and the components of the substances to be analyzed (metabolites) contained in the microorganisms and the culture medium are transferred to the mobile phase. The mobile phase to which the metabolite components have been transferred in this way is introduced into a column, and the components of the substances contained in the mobile phase that has passed through the column are subjected to mass spectrometry.

[0009] According to the method of the present invention, the components of the target substance produced by the microorganism are transferred to a mobile phase containing carbon dioxide in a liquid state, subcritical state, or supercritical state, and therefore there is no need to culture the recombinant microorganism in a liquid medium to volatilize the components of the target substance, thereby shortening the time required for analysis. In addition, since the components of the target substance do not need to be volatile, various substances can be analyzed. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram showing an example of a supercritical fluid extraction-supercritical fluid chromatography mass spectrometry apparatus (SFE-SFC-MS) used when carrying out an embodiment of the method for analyzing microbial metabolites according to the present invention. [Diagram 2] 2 is a flowchart showing a method for analyzing microbial metabolites according to the present embodiment. [Diagram 3] FIG. 2 is a diagram showing the procedure for cutting out a microbial colony cultured on an agar medium together with a part of the agar medium and placing it in a container in the method of the present embodiment. [Figure 4] 4 is a graph showing an example of the change over time in the mixing ratio of a modifier in a mobile phase used in the method of the present embodiment. [Diagram 5] Chromatograms obtained by transferring metabolite components to the mobile phase from (a) a terpene non-producing strain, (b) a terpene producing strain, and (c) a high terpene producing strain using the method of this embodiment. [Figure 6]Graph showing peak areas of chromatograms obtained using the method of the present embodiment and a conventional method for a terpene non-producing strain, a terpene producing strain, and a high terpene producing strain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] An embodiment of the method for analyzing microbial metabolites according to the present invention will be described with reference to FIGS.

[0012] (1) Apparatus for carrying out the method for analyzing microbial metabolites according to the present embodiment FIG. 1 is a schematic diagram showing the configuration of a chromatograph mass spectrometer used in carrying out the method for analyzing microbial metabolites according to the present embodiment. This chromatograph mass spectrometer not only delivers carbon dioxide but also has a pressurizing function for bringing the carbon dioxide into a supercritical state, and is therefore called a supercritical fluid extraction-supercritical fluid chromatograph mass spectrometer (see Patent Document 2), and is hereinafter referred to as "SFE-SFC-MS." Note that "SFE" is an abbreviation for "Supercritical Fluid Extraction," "SFC" is an abbreviation for "Supercritical Fluid Chromatograph," and "MS" is an abbreviation for "Mass Spectrometer." However, as described later, in this embodiment, carbon dioxide in not only a supercritical state but also a subcritical state or liquid state is used as an extraction liquid or a mobile phase.

[0013] The SFE-SFC-MS 10 includes a liquefied carbon dioxide delivery section 11, a supercritical fluid extraction (SFE) section 12, a supercritical fluid chromatography (SFC) section 13, and a mass spectrometry (MS) section 14.

[0014] The liquefied carbon dioxide liquid delivery unit 11 includes a gas cylinder 111, a gas flow path 112, a pressure pump 113, a modifier container 114, a modifier flow path 115, and a liquid delivery pump .

[0015] The gas cylinder 111 is a carbon dioxide cylinder, and supplies carbon dioxide to the gas flow passage 112 . Pressurization The pump 113 is provided in the gas flow passage 112 and pressurizes the carbon dioxide to a pressure of 7.38 MPa or more, thereby maintaining the carbon dioxide in a liquid state, a subcritical state, or a supercritical state.

[0016] The modifier container 114 is a container that contains a modifier. The modifier is also called an entrainer. The modifier is mainly an auxiliary solvent having polar molecules, and is added to carbon dioxide to facilitate the movement of components contained in the metabolite. The modifier used can be appropriately changed by replacing the modifier container 114 according to the metabolite to be analyzed. The modifier flow path 115 connects the modifier container 114 and the gas flow path 112, and merges with the gas flow path 112 at a first junction 1121. The liquid delivery pump 116 is provided in the modifier flow path 115. The modifier is introduced from the modifier container 114 through the modifier flow path 115 by the action of the liquid delivery pump 116 into the gas flow path 112, and is mixed with the carbon dioxide flowing through the gas flow path 112. Hereinafter, the flow path downstream of the first junction 1121 as viewed from the gas flow path 112 is referred to as the mobile phase flow path 15.

[0017] The SFE unit 12 is provided in the mobile phase flow path 15, and has a branch flow path 121, a sample container (corresponding to the "container" in the present invention) 122, and first to third on-off valves 1231 to 1233. The branch flow path 121 branches off from the mobile phase flow path 15 at a branch point 151 in the mobile phase flow path 15, and merges with the mobile phase flow path 15 at a second junction 152 downstream of the branch point 151 in the mobile phase flow path 15. The sample container 122 is provided in the branch flow path 121, and is a container for accommodating a sample 20 that is a combination of a microbial colony 22 and a part of the agar medium 21 surrounding the microbial colony 22, as described below. The first on-off valve 1231 is an on-off valve provided in the branch flow path 121 between the branch point 151 and the sample container 122, the second on-off valve 1232 is an on-off valve provided in the branch flow path 121 between the sample container 122 and the second junction 152, and the third on-off valve 1233 is an on-off valve provided in the mobile phase flow path 15 between the branch point 151 and the second junction 152.

[0018] The SFC section 13 is provided downstream of the SFE section 12 in the mobile phase flow path 15, and includes a trap column 131 and an SFC analytical column 132 arranged in series from the upstream side. The trap column 131 and the SFC analytical column 132 are filled with different packing materials. The packing material filled in the trap column 131 is one that is more likely to capture the substance to be analyzed than the packing material filled in the SFC analytical column 132. Note that the trap column 131 may not be used, and the SFC analytical column 132 may serve as the trap column 131.

[0019] In this embodiment, an MS / MS type mass spectrometer is used for the MS section 14, but other mass spectrometers may also be used.

[0020] A back pressure control valve 16 is provided in the mobile phase flow path 15 between the SFC section 13 and the MS section 14. The back pressure control valve 16 is provided upstream of the mobile phase to maintain the pressure of the mobile phase so that the state of the mobile phase can be maintained.

[0021] A makeup flow path 172 is provided in the mobile phase flow path 15 between the SFC section 13 and the back pressure control valve 16, and a makeup container 171 for storing a makeup solution is connected to the makeup flow path 172. The makeup liquid is a liquid for promoting detection of the analysis target components by the MS section 14, and methanol, ethanol, or the like can be used, and a salt such as ammonium formate can be added to methanol, etc., as necessary.

[0022] (2) Method for Analyzing Metabolites of Microorganisms According to the Present Invention The method for analyzing microbial metabolites according to this embodiment will be described in detail below with reference to Figures 2 and 3. Microorganisms are cultured in advance on an agar medium 21 by a conventional method to form colonies 22 of the microorganisms (Step 1 in Figure 2; see Figure 3(a)).

[0023] Here, the metabolites of microorganisms refer to organic compounds that accumulate in cells during the culture process or are excreted into the medium, including fermentation products. Examples of organic compounds that are metabolites of microorganisms include hydrocarbons, amino acids, organic acids, polysaccharides, proteins, antibiotics, and alcohols.

[0024] Examples of the hydrocarbon include aliphatic hydrocarbons, aromatic hydrocarbons, and alicyclic hydrocarbons. Examples of the aliphatic hydrocarbon include alkanes such as ethyl, propane, butane, pentane, hexane, heptane, octane, nonane, decane, undecane, dodecane, heptadecane, and octadecane; alkenes such as ethylene, propylene, butene, pentene, hexene, octene, nonene, decene, butadiene, and isoprene; and alkynes such as ethyne, propyne, butyne, and pentyne. Examples of the aromatic hydrocarbon include benzene, toluene, xylene, and ethylbenzene. Examples of the alicyclic hydrocarbon include cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclobutene, cyclopentene, cyclohexene, cycloheptene, and cyclooctene.

[0025] Among aliphatic hydrocarbons, aromatic hydrocarbons, and alicyclic hydrocarbons, substances that are insoluble in water are also called fat-soluble components. In particular, isoprenoids are examples of fat-soluble components. Isoprenoids are hydrocarbons whose constituent units are isoprene, and are substances that are biosynthesized through the isoprenoid pathway. Examples of isoprenoids include terpenes, terpenoids, steroids, and carotenoids. Examples of terpenes include monoterpenes, sesquiterpenes, diterpenes, sesterterpenes, triterpenes, and tetraterpenes. Examples of terpenoids include monoterpenoids, sesquiterpenoids, diterpenoids, sesterterpenoids, triterpenoids, and tetraterpenoids. Steroids are a general term for compounds that have a cyclopentanohydrophenanthrene ring skeleton, and examples of these include β-estradiol and progesterone. Carotenoids are C 10-methyl-1,10-dimethyl-1,10-triphenylphosphine oxide (C10-methyl-1,10-triphenylphosphine oxide) composed of eight isoprene units. 40 H 56 Examples of these include carotene, lycopene, lutein, zeaxanthin, and astaxanthin.

[0026] Examples of amino acids include glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, cystine, methionine, phenylalanine, tyrosine, tryptophan, proline, hydroxyproline, asparagine, glutamine, aspartic acid, glutamic acid, lysine, histidine, and arginine. Individual amino acids can be classified into acidic amino acids, basic amino acids, or neutral amino acids based on differences in side chains. Specifically, glutamic acid and aspartic acid can be classified into acidic amino acids, lysine, arginine, and histidine can be classified into basic amino acids, and glycine, alanine, threonine, methionine, cysteine, serine, valine, leucine, isoleucine, tryptophan, phenylalanine, tyrosine, proline, glutamine, and asparagine can be classified into neutral amino acids.

[0027] Examples of organic acids include acetic acid, lactic acid, pyruvic acid, succinic acid, malic acid, itaconic acid, citric acid, acrylic acid, propionic acid, and fumaric acid. Examples of polysaccharides include xanthan, dextran, alginate, hyaluronic acid, curdlan, gellan, sucrose, and pullulan. Examples of proteins include hormones, lymphokines, interferons, and enzymes (amylase, glucoamylase, invertase, lactase, protease, lipase, etc.). Examples of antibiotics include antibacterial agents (β-lactams, macrolides, ansamycins, tetracyclines, chloramphenicol, peptide antibiotics, aminoglycosides, etc.), antifungal agents (polyoxin B, griseofulvin, polyene macrolides, etc.), anticancer agents (daunomycin, adriamycin, dactinomycin, mithramycin, bleomycin, etc.), protease / peptidase inhibitors (leupeptin, antipain, pepstatin, etc.), and cholesterol biosynthesis inhibitors (compactin, lovastatin, pravastatin, etc.).

[0028] Examples of alcohols include ethanol, isopropanol, glycerin, propylene glycol, trimethylene glycol, 1-butanol, and sorbitol. Other organic compounds produced by fermentation include acrylamide, diene compounds (such as isoprene), and pentanediamine.

[0029] Techniques for producing the above organic compounds by culturing microorganisms capable of producing the organic compounds are widely known. This embodiment can be widely applied to such techniques. In microbial fermentation, microorganisms grow by themselves using a carbon source, a nitrogen source, and the like. In this sense, in this embodiment, the metabolites also include microbial cells. Examples of microbial cells include any microorganism capable of producing an organic compound.

[0030] Microorganisms capable of producing organic compounds include both microorganisms that inherently have the ability to produce organic compounds and microorganisms that do not inherently have or do not substantially have the ability to produce organic compounds but have acquired the ability to produce organic compounds by introducing an organic compound-producing gene by genetic recombination. Regarding microorganisms capable of producing organic compounds, various microorganisms are known depending on the type of organic compound, and in this embodiment, these known microorganisms can be widely used.

[0031] Next, the colony 22 of the microorganisms cultured on the agar medium 21 is placed in the sample container 122 together with a part of the agar medium 21 (referred to as the "agar medium piece 211") by the method described below (step 2). First, the tip of the pipette 23 is inserted into a part of the agar medium 21 including the position where the colony 22 is formed (FIGS. 3(a) and (b)), and the pipette 23 is pulled out from the agar medium 21 (FIG. 3(c)). As a result, the sample 20, which is a combination of the colony 22 and the agar medium piece 211, is excised into the pipette 23. Next, the tip of the pipette 23 is inserted into the sample container 122 (FIG. 3(d)), and the sample 20 in the pipette 23 is pushed out from the pipette 23 into the sample container 122 on whose bottom a first filter 1224 described below is placed (FIG. 3(e)). Thereafter, the tip of the pipette 23 is removed from the sample container 122 (ibid. (f)), a cylindrical body 1225 and a second filter 1226 described below are placed inside the sample container 122, and the lid 1221 is then attached to the sample container 122 (ibid. (g)).

[0032] A bottom opening 1222 is provided at the bottom of the sample container 122, and a lid opening 1223 is provided at the lid 1221. A first filter 1224 is provided to cover the bottom opening 1222, and a cylindrical body 1225 is placed on the first filter 1224. The sample 20 is accommodated inside the cylindrical body 1225. A second filter 1226 is placed on the cylindrical body 1225. With the above configuration, a flow path of the mobile phase is formed in the sample container 122 from the lid opening 1223 through the second filter 1226, the cylindrical body 1225 (the sample 20 in the cylindrical body 1225), and the first filter 1224 toward the bottom opening 1222 (or in the opposite direction).

[0033] Next, the sample container 122 is attached to the SFE unit 12 of the SFE-SFC-MS 10, and the bottom opening of the sample container 122 is 1222 and lid opening 1223 are connected to branch channel 121, thereby communicating the inside of sample container 122 with branch channel 121 in the same manner as used in the conventional SFE-SFC-MS10.

[0034] Next, with the first on-off valve 1231 closed and the third on-off valve 1233 open, carbon dioxide gas is supplied from the gas cylinder 111 to the gas flow path 112, and a predetermined pressure is applied to the carbon dioxide gas by the liquid feed pump 113. If necessary, a modifier is supplied from the modifier container 114 to the modifier flow path 115 by the liquid feed pump 116, and the modifier is mixed with the carbon dioxide at the first junction 1121. This results in a mobile phase containing carbon dioxide in a liquid state, subcritical state, or supercritical state. Note that, as described later, the supply of the modifier may not be performed during a part of the analysis time, and when the substance (metabolite) to be analyzed is composed of non-polar molecules, the supply of the modifier may not be performed throughout the entire analysis time.

[0035] Next, by supplying the mobile phase to the sample container 122 using the method described below, metabolite components metabolized by the microorganisms and present in the colonies 22 and the agar medium pieces 211 are transferred to the mobile phase (Step 3: SFE). There are two types of SFE methods: static extraction and dynamic extraction.

[0036] In the static extraction, first, the first on-off valve 1231 is closed, the third on-off valve 1233 is opened, and the second on-off valve 1232 is opened to supply the mobile phase into the sample container 122. Then, the second on-off valve 1232 is closed when a predetermined amount of the mobile phase is supplied into the sample container 122. As a result, the components of the metabolites are transferred to the mobile phase stored in the sample container 122 (without the mobile phase flowing). After a predetermined time has elapsed, the first on-off valve 1231 and the second on-off valve 1232 are opened and the third on-off valve 1233 is closed. As a result, the components transferred to the mobile phase are discharged from the sample container 122 together with the mobile phase. Note that, in the stage of supplying the mobile phase into the sample container 122, instead of opening the second on-off valve 1232 and closing the first on-off valve 1231, the first on-off valve 1231 may be opened and the second on-off valve 1232 may be closed.

[0037] In dynamic extraction, the first on-off valve 1231 and the second on-off valve 1232 are opened, and the third on-off valve 1233 is closed. This causes the metabolite components to move into the mobile phase flowing through the sample container 122, and the components are discharged from the sample container 122 together with the mobile phase.

[0038] In this embodiment, either static extraction or dynamic extraction may be performed, or both may be performed.

[0039] The metabolite components that have migrated to the mobile phase are introduced into the trap column 131 of the SFC section 13 together with the mobile phase, and are captured by the trap column 131 .

[0040] After step 3 (SFE) is completed by the above operations, the first on-off valve 1231 and the second on-off valve 1232 are closed, and then the third on-off valve 1233 is opened to supply the mobile phase (not including metabolite components) into the trap column 131. As a result, the metabolite components captured in the trap column 131 are discharged from the trap column 131 and introduced into the SFC analysis column 132, where they are separated in time by component (step 4: SFC).

[0041] In this case, by adjusting the mixing ratio of the modifier contained in the mobile phase, it is possible to control the discharge rate of the metabolite components from the trap column 131 and the retention time of the components in the SFC analytical column 132. For example, by increasing the mixing ratio of the modifier, the discharge of the metabolite components from the trap column 131 is promoted. In addition, by increasing the mixing ratio of the modifier at the start of discharge from the trap column 131 and then decreasing the mixing ratio, it is possible to increase the retention time in the SFC analytical column 132 while promoting discharge from the trap column 131, thereby improving the accuracy of time separation of each component. Specific examples of the types and mixing ratios of modifiers will be described later.

[0042] Each component separated by time in the SFC analytical column 132 is introduced into the MS section 14, where mass analysis is performed (step 5). This makes it possible to quantify the amount of useful substances of interest contained in the metabolites.

[0043] According to the method of the present embodiment, since the components of the analyte produced by the microorganism are transferred to a mobile phase containing carbon dioxide, there is no need to culture the recombinant microorganism in a liquid medium to volatilize the components of the analyte. Therefore, the time required for analysis can be shortened. In addition, since the components of the analyte do not need to be volatile, various substances can be analyzed.

[0044] In this embodiment, the microbial colonies 22 formed on the agar medium 21 are cut out together with a part of the agar medium (agar medium piece 211) and stored in a container as is. Raco Alternatively, the colonies 22 may be scraped off and stored in the sample container 122, but this requires time and effort, and it is difficult to reliably scrape off the entire colony of the microorganisms formed on the agar medium. In contrast, the method of this embodiment allows the entire colony 22 to be easily collected in a short time. Note that the method of storing the microorganisms together with the medium in the container is not limited to the method used in this embodiment.

[0045] (3) Examples of quantitative analysis of metabolites The results of an experiment in which a quantitative analysis of metabolites was performed will be described below with reference to Figs. 4 to 6. In this experiment, (a) a wild-type Escherichia coli strain not capable of producing terpenes (Escherichia coli (hereinafter referred to as "E. coli") MG1655 strain, accession number NITE BP-1686, hereinafter referred to as "non-terpene producing strain"), (b) a strain obtained by genetically modifying the non-terpene producing strain to impart the ability to produce terpenes (terpene producing strain), and (c) a strain obtained by genetically modifying the non-terpene producing strain to impart a higher terpene production ability than the terpene producing strain by performing a genetic modification operation different from that in (b) (high terpene producing strain) were used as microorganisms, and limonene (C 10 H 16 ) were the subject of analysis.

[0046] Terpene (isoprene) is a hydrocarbon used as a material for isoprene-based rubber, and limonene, a type of terpene, is a fat-soluble compound known as one of the aromatic components contained in citrus fruits.

[0047] (b) The terpene-producing strain was obtained by preparing competent cells of the (a) non-terpene-producing strain and introducing the plasmid pACYC177-Ptac-opt_LMS-ispA* expressing the limonene synthase gene and the farnesyl diphosphate synthase gene by electroporation. (c) The terpene-high-producing strain was obtained by constructing the MG1655 poxB::Ptac-dxs strain in which the dxs gene was enhanced at the poxB locus on the chromosome of the non-terpene-producing strain, preparing competent cells of this strain, and introducing the plasmid pACYC177-Ptac-opt_LMS-ispA* expressing the limonene synthase gene and the farnesyl diphosphate synthase gene by electroporation.

[0048] The plasmids used for constructing (b) the terpene-producing strain and (c) the high terpene-producing strain were constructed as follows, in accordance with the procedure described in Patent Document 3. Note that the following procedure is as described in Patent Document 3, and therefore a detailed description thereof will be omitted here.

[0049] (1) Obtaining plant-derived limonene synthase genes In order to efficiently express the plant-derived limonene synthase gene, the secondary structure was eliminated, the codons were optimized to be identical to those of P. ananatis, and the chloroplast targeting signal was further cut off. The gene obtained in this manner was named opt_LMS. Plants that produce plant-derived limonene synthase include Alaska spruce (Picea sitchensis), big fir (Abies grandis), spearmint (Mentha spicata), Citrus unshiu, and lemon (Citrus limon). In this example, spearmint-derived limonene synthase was used.

[0050] (2) Obtaining a mutant farnesyl diphosphate synthase gene from E. coli In order to efficiently express the ispA mutant (S80F) gene with high geranyl diphosphate production activity, which is a mutant strain of the ispA gene encoding E. coli farnesyl diphosphate synthase, we obtained a mutant farnesyl diphosphate synthase gene derived from E. coli with codons optimized to eliminate secondary structures. This gene was named ispA*.

[0051] (3) Construction of plasmid for simultaneous expression of opt_LMS and ispA* genes opt_LMS and ispA* were each chemically synthesized and then cloned into a standard vector pUC57 (GenScript) to obtain plasmids, which were named pUC57-opt_LMS and pUC57-ispA*, respectively.

[0052] Next, PCR was performed using a specific primer and pUC57-opt_LMS as a template, and the PCR fragment obtained was used to perform further PCR to obtain a pUC57-opt_LMS fragment. PCR was also performed using a specific primer and pUC57-ispA* as a template to obtain a pUC57-ispA* fragment. The pUC57-opt_LMS fragment and the pUC57-ispA* fragment were ligated to pACYC177 (manufactured by Nippon Gene Co., Ltd.) digested with restriction enzymes Pstl and Pcal using In-Fusion HD cloning kit (manufactured by Clontech Co., Ltd.) to obtain a plasmid for simultaneous expression of the opt_LMS and ispA* genes. This was named pACYC177-Ptac-opt_LMS-ispA*.

[0053] These three strains were analyzed by a conventional quantitative analysis method. Specifically, the above three strains were cultured on an agar medium for growth, and then the fungal cells on the surface of the medium were scraped off and inoculated into a liquid medium for limonene fermentation contained in a sealed container, followed by further culture. After the culture was completed, the gas components in the sealed container were measured using a gas chromatograph quantitative analyzer. When limonene was not detected in the terpene non-producing strain, but was detected in the terpene producing strain and the high terpene producing strain, the amount of limonene detected in the high terpene producing strain was about five times that detected in the terpene producing strain.

[0054] Detailed conditions for analysis using the method of this embodiment will be described below.

[0055] The samples were prepared as follows: Each strain was cultured on agar medium 21 in the presence of oxygen, a growth promoter, until the colony 22 reached a diameter of about 3 mm, and then the amount of oxygen supplied was reduced and the culture was continued. Then, the colony 22 and the surrounding agar medium 21 within a diameter of 5 mm were cut out by the method described above, and the colony 22 and the agar medium piece 211 were placed in a container.

[0056] In SFE (step 3), ammonium formate methanol with an ammonium formate concentration of 15 mmol / L (millimoles per liter) was used as a modifier, and metabolites were extracted by combining static extraction and dynamic extraction as follows (see FIG. 4). First, a mobile phase with a modifier mixing ratio (volume ratio) of 10% was supplied to the sample container 122 at a pressure of 15 MPa and a supply rate of 1 mL / min for 0.6 minutes without flowing out of the sample container 122, to perform static extraction ("static SFE1" in FIG. 4). Next, while the mobile phase in the sample container 122 was being flowed out, a mobile phase not containing a modifier (mixture ratio of 0%) was supplied to the sample container 122 at a pressure of 15 MPa and a supply rate of 1 mL / min for 0.2 minutes to perform dynamic extraction ("dynamic SFE1" in FIG. 4). As a result, the metabolites that had migrated to the mobile phase in the sample container 122 were discharged to the SFC unit 13 by the mobile phase supplied to the sample container 122. Next, a mobile phase not containing a modifier was supplied to the sample container 122 at a pressure of 15 MPa and a supply rate of 1 mL / min for 1.2 minutes without allowing the mobile phase to flow out of the sample container 122, to perform static extraction ("static SFE2" in the same publication). Immediately thereafter, while allowing the mobile phase to flow out of the sample container 122, the mixture ratio of the modifier in the mobile phase was continuously increased from 0% to 12% in 1 minute while being supplied to the sample container 122 at a pressure of 15 MPa and a supply rate of 3 mL / min, to perform dynamic extraction ("dynamic SFE2" in the same publication).

[0057] In SFC (step 4), the mixture ratio of modifier in the mobile phase was continuously increased from 12% to 30% in 1.5 min, while the mobile phase was supplied to the SFC section 13 at a pressure of 15 MPa and a supply rate of 3 mL / min (Figure 4). The temperature of the SFC analytical column 132 was maintained at 40°C.

[0058] The eluate eluted from the SFC analytical column 132 was mixed with a makeup solution consisting of methanol supplied from a makeup container 171 at a supply rate of 0.1 mL / min, and introduced into the MS section 14 .

[0059] An example of the chromatogram obtained for each of (a) the non-terpene producing strain, (b) the terpene producing strain, and (c) the high terpene producing strain is shown in Figure 5. At retention times where no peaks appear in the chromatogram of the non-terpene producing strain, peaks derived from limonene appear in the chromatograms of the terpene producing strain and the high terpene producing strain. The peak area is larger in the high terpene producing strain than in the terpene producing strain.

[0060] When the inside of the sample container 122 was checked after the analysis was completed, the agar medium pieces 211 were found to remain with almost the same shape and size as before the analysis. Therefore, it is considered that the agar medium pieces 211 were hardly washed away during the extraction of metabolites and did not adversely affect the analysis.

[0061] Next, for each of the terpene-producing strain and the high-terpene-producing strain, chromatograms were obtained for multiple samples to determine the peak areas, and a portion of each of the multiple samples was taken and the peak areas of the chromatograms obtained by the conventional method (cultivating in a liquid medium to volatilize the metabolite components and analyzing by gas chromatography) were obtained. FIG. 6 shows a graph in which the peak areas obtained by the method of this embodiment are plotted on the horizontal axis and the peak areas obtained by the conventional method for the same samples are plotted on the vertical axis. From this graph, it can be seen that the larger the peak area obtained by the conventional method, the larger the peak area obtained by the method of this embodiment, and there is a correlation between the two. In other words, since the results obtained are similar to those of the conventional method, which is an already established analytical method, it can be said that the analytical results obtained by the method of this embodiment are valid.

[0062] (4) Modifications In the above embodiment, the microbial colony 22 is collected together with the portion 211 of the agar medium 21 using the pipette 23, but microorganisms collected by other methods may be stored together with the medium in the sample container 122. Also, instead of culturing the microorganisms outside the sample container 122, the microorganisms may be cultured in a medium stored in the sample container 122. In other respects as well, the present invention is not limited to the above embodiment, and various modifications are possible in line with the gist of the invention.

[0063] [Aspects] It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0064] (Section 1) The method for analyzing the metabolites of microorganisms according to paragraph 1 is as follows: A step of supplying a mobile phase containing carbon dioxide in a liquid state, a subcritical state, or a supercritical state to a container in which a microorganism cultured in a medium is accommodated together with the medium, thereby transferring components of the metabolites of the microorganism present in the microorganism and the medium to the mobile phase; introducing the mobile phase into a column in which the metabolite components have been transferred; subjecting the metabolite components contained in the mobile phase passed through the column to mass spectrometry; has.

[0065] In the method for analyzing microbial metabolites according to paragraph 1, a solvent containing carbon dioxide in a liquid state, subcritical state, or supercritical state is used to extract the microbial metabolites while the microorganisms cultured on a medium are contained in a container together with the medium, thereby making it possible to extract a larger amount of metabolites in a shorter time than when the metabolites are extracted in the form of volatile substances. Therefore, the microbial metabolites can be quantified with high accuracy in a short time.

[0066] The microorganisms may be cultured outside the vessel and then placed in the vessel together with the medium, or may be cultured in the vessel from the beginning.

[0067] The mobile phase may be carbon dioxide alone or may be a mixture of carbon dioxide and a modifier. The modifier is added to carbon dioxide to facilitate the extraction of the substance and to elute it from the column at a predetermined time. The modifier can be appropriately selected from materials of the mobile phase in a normal liquid chromatograph according to the metabolites to be analyzed and the type of column to be used.

[0068] (Section 2) The method for quantifying a microbial metabolite according to the second aspect of the present invention is the method according to the first aspect of the present invention, further comprising the steps of: Cultivating the microorganism on an agar medium outside the container; The microorganism is excised together with a portion of the agar medium; The microorganism is placed in a container together with a portion of the agar medium.

[0069] When microorganisms are cultured on an agar medium outside a container, in addition to the method of collecting microorganisms used in the method of quantifying microbial metabolites according to paragraph 2, a method of scraping the microorganisms from the agar medium can also be used. However, this method requires time and effort for scraping, and it is difficult to reliably scrape off the entire microbial colony formed on the agar medium. In contrast, according to the method of quantifying microbial metabolites according to paragraph 2, the microorganisms formed on the agar medium are cut out together with a part of the agar medium and placed in a container as is, so that the entire microorganism (colony) can be easily collected in a short time.

[0070] (Section 3) The method for quantifying a microbial metabolite according to paragraph 3 comprises the steps of: The components of the metabolites of the microorganisms are fat-soluble components, The mobile phase is a mixture of carbon dioxide and ammonium formate in methanol.

[0071] According to the method for quantifying microbial metabolites relating to item 3, by using a mobile phase containing a mixture of carbon dioxide and ammonium formate / methanol, fat-soluble components such as terpenes can be efficiently transferred to the mobile phase. [Explanation of symbols]

[0072] 10…Supercritical fluid extraction-Supercritical fluid chromatography mass spectrometer (SFE-SFC-MS) 11...Liquefied carbon dioxide delivery section 111…Gas cylinder 112...Gas flow path 1121…1st confluence point 113…Pressurizing pump 114…Modifier container 115…Modifier flow passage 116... Liquid delivery pump 12...Supercritical fluid extraction (SFE) section 121...Branch channel 122...Sample container 1221…Lid 1222…Bottom opening 1223…Lid opening 1224…First filter 1225...Cylindrical body 1226…Second filter 1231…First opening and closing valve 1232…Second on-off valve 1233…Third opening and closing valve 13…Supercritical Fluid Chromatograph (SFC) Section 131: Trap column 132: SFC analysis column 14...Mass spectrometry (MS) department 15...Mobile phase flow path 151...Branch point 152…Second confluence point 16...Back pressure control valve 171…Makeup container 172…Make-up channel 20...Object to be collected 21...Agar medium 211...Agar medium piece (part of agar medium) 22...Microbial colony 23...Pipette

Claims

1. A process for transferring components of metabolites of the microorganisms present in the microorganisms and the agar medium to the mobile phase by supplying a mobile phase containing carbon dioxide in a liquid state, a subcritical state, or a supercritical state to a container in which the microorganisms are cultured on the agar medium and the agar medium are contained; introducing the mobile phase into a column in which the metabolite components have been transferred; subjecting the metabolite components contained in the mobile phase passed through the column to mass spectrometry; A method for analyzing metabolites of a microorganism having the above structure.

2. Cultivating the microorganism on the agar medium outside the container; The microorganism is excised together with a portion of the agar medium; The microorganism is placed in a container together with a portion of the agar medium; A method for analyzing microbial metabolites according to claim 1.

3. The components of the metabolites of the microorganisms are fat-soluble components, The mobile phase is a mixture of carbon dioxide and ammonium formate in methanol; A method for analyzing microbial metabolites according to claim 1.

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