JP2014226048A - Method for producing aromatic compounds - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing 2-acetyl-3,4-dihydroxy-5-methoxybenzeneacetic acid or 2-acetyl-3,5-dihydroxy-4-methoxybenzeneacetic acid by using a microorganism.SOLUTION: Provided is a method for producing a chemical compound represented by the following Formula (1) or (2), in which a fungus strain of the genus Penicillium is cultured in a medium containing a glycerin to produce the chemical compound represented by the Formula (1) or (2) in the culture.

Description

  The present invention relates to a method for producing an aromatic compound using a microorganism.

In recent years, the production of useful substances using microorganisms has attracted attention as a method for producing substances that can reduce the environmental burden. At present, the production methods of aliphatic compounds such as ethanol and butanol by microorganisms are relatively developed, while the production of aromatic compounds lags considerably. Examples of aromatic compounds synthesized using microorganisms include compounds obtained by converting aromatic amino acids and their derivatives as raw materials, decomposition products obtained from lignin contained in wood as raw materials, and the like. Are known. However, there are still many problems such that the types of aromatic compounds that can be produced are still small and the desired compounds cannot be produced with high purity.
Aromatic compounds are widely used not only as chemical raw materials but also as UV absorbers, antibacterial agents, herbicides and the like. For example, 2-acetyl-3,4-dihydroxy-5-methoxybenzeneacetic acid is used as a herbicide (see Patent Document 1 and Non-Patent Documents 1 to 3) and a pollen formation inhibitor (see Non-Patent Document 4) as 2-acetyl. -3,5-dihydroxy-4-methoxybenzeneacetic acid has been proposed for use as an antibacterial agent (see Non-Patent Document 5), an antioxidant (see Non-Patent Document 6), and the like.
So far, 2-acetyl-3,4-dihydroxy-5-methoxybenzeneacetic acid and 2-acetyl-3,5-dihydroxy-4-methoxybenzeneacetic acid have been used with fungi such as Penicillium janthiellum , Nimnya alternantherae , Curularia siddiqui In addition, there is a report of biosynthesis in a medium containing glucose (see Non-Patent Documents 7 and 8), sucrose (see Non-Patent Document 6), or malto extract (see Non-Patent Document 4).

  Further, in the production of substances by microorganisms, attempts have been made to use by-products and waste as raw materials. If such by-products and wastes are discarded as they are, they can lead to environmental pollution, and the processing requires a corresponding cost. If these can be used as raw materials for substance production, secondary effects such as reduction of environmental burden and reduction of processing costs can be obtained. For example, biodiesel fuel whose production volume has been increasing in recent years is manufactured from fats and oils, but glycerin and the like are by-produced in the manufacturing process.

Chinese Patent Application No. 1843115 Specification

Huanan Nongye Daxue Xuebao, (2010), 31, p.26-31 Gaodeng Xuexiao Huaxue Xuebao, (2010), 31, p.2010-2014 Gaodeng Xuexiao Husxue Xuebao, (2006), 27, p.1485-1487 Agric. Biol. Chem., (1991), 55, p.1137-1138 Agric. Biol. Chem., (1984), 48, p.1899-900 Agric. Biol. Chem., (1982), 46, p.2369-2371 Tetrahedron, (1963), Vol. 19, p.117-122 Tetrahedron, (1962), Vol.18, p.433-436

The present invention relates to a process for producing 2-acetyl-3,4-dihydroxy-5-methoxybenzeneacetic acid or 2-acetyl-3,5-dihydroxy-4-methoxybenzeneacetic acid using fungi belonging to the genus Penicillium. It is an issue to provide. Moreover, this invention makes it a subject to provide the Penicillium genus fungi used for the said method.

In view of the above problems, the present inventors have intensively studied microorganisms that can use glycerin as a raw material and can synthesize aromatic compounds. As a result, penicillium as a microorganism capable of producing 2-acetyl-3,4-dihydroxy-5-methoxybenzeneacetic acid and 2-acetyl-3,5-dihydroxy-4-methoxybenzeneacetic acid using glycerin as a carbon source. The Penicillium sp. KSM-F26 strain belonging to the genus was found.
The present invention has been completed based on this finding.

That is, the present invention, culturing the fungi belonging to the Penicillium (Penicillium) genus in a medium containing glycerol, to produce a compound represented by the following formula in the culture (1) or Formula (2), the following equation (1 Or a method for producing a compound represented by formula (2).

The present invention also relates to Penicillium sp. KSM-F26 strain (NITE P-1475).
Furthermore, the present invention relates to a fungus belonging to the genus Penicillium , having a base sequence of the following (b) as part of a β-tubulin gene sequence, and from the glycerin, the above formula (1) or formula ( It relates to a fungus having the ability to produce the compound represented by 2).
(B) a nucleotide sequence having 93% or more identity with the nucleotide sequence represented by SEQ ID NO: 1

  According to the production method of the present invention, the compound represented by the formula (1) or the formula (2) can be efficiently produced. Moreover, according to this invention, the microorganisms used suitably for the said method can be provided. The compound represented by the above formula (1) or formula (2) obtained by the method of the present invention is useful as a raw material for chemical substances, or as a herbicide, a pollen formation inhibitor, an antibacterial agent, an antioxidant and the like. is there.

It is a molecular phylogenetic tree created based on the β-tubulin gene sequence. The lower left line in the figure indicates the scale bar, and the number located at the branch of the system branch indicates the bootstrap value (indicating 50% or more). T at the end of the bacterial name indicates a strain derived from that type, and NT indicates a strain derived from a neotype. Moreover, alphabets and numbers indicate accession numbers. It is a figure which shows the result of the HPLC analysis of the culture-medium supernatant of Penicillium sp KSM-F26 strain | stump | stock in Example 2. It is a figure which shows the time-dependent change of the productivity of 2-acetyl-3,4-dihydroxy-5-methoxybenzeneacetic acid by Penicillium sp KSM-F26 strain in Example 3. In Example 3, it is a figure which shows a time-dependent change of the productivity of 2-acetyl-3,5-dihydroxy-4-methoxybenzeneacetic acid by Penicillium sp KSM-F26 strain.

In the production method of the present invention, fungi belonging to the genus Penicillium are cultured in a medium containing glycerin, and a compound represented by the following formula (1) or formula (2) is produced in the culture.
The compound produced by the method of the present invention is represented by the following formula (1) or formula (2).

  The compound of the formula (1) is 2-acetyl-3,4-dihydroxy-5-methoxybenzeneacetic acid (common name: vulculic acid), and the compound of the formula (2) is 2-acetyl-3,5-dihydroxy-4 -Methoxybenzeneacetic acid (common name: curvulic acid). In the present invention, the compound represented by the formula (1) or (2) includes a salt thereof.

The microorganism used in the method of the present invention is a fungus belonging to the genus Penicillium (hereinafter referred to as Penicillium genus), and has the ability to synthesize a compound represented by the formula (1) or formula (2) from glycerin. I just need it.
As a specific strain, it is preferable to use Penicillium sp. KSM-F26 strain, which was isolated from the soil and named by the present inventors. The acquisition process of Penicillium sp. KSM-F26 strain will be described in detail in Examples below.
Penicillium sp. KSM-F26 strain has the following mycological properties.

(A) Culture properties Growth in Difco ^™ YM Agar medium (BD): good growth in 30 days at 7 ° C. 2. 2. Growth on agar medium for separation: good growth at 30 ° C. for 6 days Growth in potato dextrose agar medium “DAIGO” (manufactured by Nippon Pharmaceutical Co., Ltd.): 25 ° C-27 ° C, good growth in 3 days

(B) Morphological properties The morphology after culturing at 25 ° C. for 14 days in PDA (potato dextrose agar “DAIGO” (manufactured by Nippon Pharmaceutical Co., Ltd.)) is shown.
(1) Macroscopic observation Grayish green to gray, velvety, partially producing tan to reddish brown soluble pigment in the medium (2) Microscopic observation Standing up from vegetative mycelium and directly from the tip of the branched branch Monocytic to two-wheeled Penicillus is formed, and spheres to subspheres are formed in one cell, and the surface is formed by concatenating fiaro-type conidia with fine spines.

(C) Physiological properties Growth temperature test (PDA medium, 3 days culture)
Grows at 27 ° C 30 ° C Grows at 37 ° C Grows at 45 ° C Grows without optimal growth temperature range: 20-35 ° C
Optimal growth pH range: 5-7

(D) Chemical taxonomic properties Penicillium sp. KSM-F26 strain has a base sequence represented by SEQ ID NO: 1 as a partial sequence of a base sequence encoding a β-tubulin gene. As a result of BLAST search against the international base sequence database using the base sequence represented by SEQ ID NO: 1, the highest 92.6% of the β-tubulin gene sequence of Penicillium daleae (CBS211.28T) Showed homology. In addition, as a result of molecular phylogenetic analysis using the base sequence represented by SEQ ID NO: 1 and the β-tubulin gene sequence of Penicillium spp., It is supported by Penicillium daleia (CBS211.28T) and a bootstrap value of 96%. A cluster was formed (FIG. 1).

  Methods for studying the evolution of organisms and genes based on molecular phylogenetic trees have been established as molecular phylogeny (see, for example, Shigeo Kimura, Introduction to Molecular Evolution Chemistry (Baifukan), pages 164-184, “7 Molecular Phylogenetic Trees”). "How to make and evaluation"). The molecular phylogenetic tree based on the base sequence of the β-tubulin gene is based on the β-tubulin gene base sequence of the target microorganism, the β-tubulin gene of the known microorganism presumed to be the same species or similar to the same microorganism from the mycological properties. It can be obtained by calculating multiple alignments and evolutionary distances together with the base sequence of the tubulin gene and creating a phylogenetic tree based on the obtained values. The base sequence of a known microbial β-tubulin gene used to create a molecular phylogenetic tree can also be obtained by an identity search of an existing database. Here, the evolution distance refers to the total number of mutations per locus (sequence length) between certain genes.

The properties shown above support that the KSM-F26 strain isolated by the present inventors is a fungus belonging to the genus Penicillium, and that the closest species is Penicillium daleia. However, since there is no conventional species that perfectly matches the physiological and scientific properties and the results of the base sequence analysis of the β-tubulin gene, it was identified as a new species of the genus Penicillium, and Penicillium sp .).
Penicillium SP KSM-F26 strain was deposited with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2-5-8, Kazusa Kamashizu, Chiba Prefecture) on December 4, 2012. -1475.
The Penicillium sp. KSM-F26 strain has the ability to produce the compound represented by the formula (1) or (2) from glycerin, as demonstrated in the examples described later.

Furthermore, in the production method of the present invention, a Penicillium sp. KSM-F26 strain mutant or a related fungus as a Penicillium genus fungus, which produces a compound represented by Formula (1) or Formula (2) from glycerin. It is also preferable to use fungi having ability. Specifically, it is a Penicillium genus, has a base sequence of the following (a) as part of the β-tubulin gene sequence, and is represented by the formula (1) or (2) from glycerin. Fungi having the ability to produce compounds can be preferably used.

(A) a nucleotide sequence having 89% or more identity with the nucleotide sequence represented by SEQ ID NO: 1

In the above (a), the nucleotide sequence identity is preferably 90% or more, more preferably 93% or more, still more preferably 95% or more, and even more preferably 97% or more.
In the present invention, the identity of the base sequence is calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)). Specifically, it is calculated by performing analysis with a unit size to compare (ktup) of 2 using a homology search program of genetic information software Genetyx-Win (software development).

Further, Penicillium sp. KSM-F26 strain as a related bacterium, Penicillium sp. KSM-F26 strain and a related bacterium, Penicillium daleia and its related bacterium can be preferably used. More preferred is Penicillium daleia.
Penicillium daleia related bacteria include penicillium having a base sequence having 93% or more identity with the partial base sequence of the penicillium dareya β-tubulin gene shown in SEQ ID NO: 4 as part of the β-tubulin gene sequence Genus fungi. The identity with the base sequence shown in SEQ ID NO: 4 is preferably 95% or more, more preferably 97% or more, and further preferably 99% or more.

  Penicillium fungi used in the production method of the present invention can be obtained from, for example, glycerol-utilizing bacteria. As an example, these assimilating bacteria are separated into an agar medium for isolation (bactopeptone 0.5%, yeast extract 0.1%, iron phosphate 0.01%, glycerin 10%, Daigo artificial seawater SP 1.8%, A combination of the above-mentioned mycological properties (cultural properties, morphological properties, physiological properties, chemical taxonomic properties) of Penicillium sp. KSM-F26 strain cultured in agar 2% (pH 6.5)) By screening a colony, Penicillium spp. Having the ability to produce the compound represented by the formula (1) or formula (2) can be found.

As a second aspect, the present invention provides Penicillium sp. KSM-F26 strain, which is a novel microorganism that can be used in the above production method. Furthermore, as a mutant or a related bacterium of Penicillium sp. KSM-F26 strain, it is a Penicillium genus, having a base sequence of the following (b) in a part of a β-tubulin gene sequence, Provided is a fungus having the ability to produce a compound represented by 1) or formula (2).

(B) a nucleotide sequence having 93% or more identity with the nucleotide sequence represented by SEQ ID NO: 1

In the above (b), the identity of the base sequence is preferably 95% or more, more preferably 97% or more, and further preferably 99% or more.

  In the production method of the present invention, the aforementioned Penicillium sp. May be used alone or in combination of two or more.

Penicillium fungi are cultured in a medium containing glycerin as a carbon source.
The medium can be inoculated by a normal method, for example, a method of inoculating the medium with Penicillium fungi suspended in physiological saline, a method of inoculating the medium with Penicillium fungi directly on a platinum loop, etc. .
The culture may be appropriately selected from aeration and agitation culture, shaking culture, stationary culture and the like according to the type of medium used.

As the medium, those commonly used for culturing Penicillium fungi can be used, and either a liquid medium or a solid medium may be used. As a specific example of the medium, for example, GPY medium can be mentioned.
The content of glycerin in the medium may be an amount sufficient for the Penicillium fungus to produce the compound represented by Formula (1) or Formula (2). By changing the amount of glycerin added, the production amount of the compound represented by the formula (1) or the formula (2) can be adjusted. From the viewpoint of compound productivity, glycerol is preferably contained in an amount of 1 g to 200 g, more preferably 25 g to 100 g, with respect to 1 L of the medium. Glycerin may be added stepwise into the medium.

The above-mentioned glycerin can be obtained from commercial products or chemical synthesis.
Further, glycerin produced as a by-product in the industrial production process can also be used. For example, glycerin produced as a by-product produced during biofuel production can be used. By using by-products, secondary effects such as reduction of energy required for the treatment of surplus substances and reduction of environmental pollution due to disposal can be obtained. Furthermore, since by-products can be obtained at low cost, the cost can be kept low. When using a by-product, separation or purification may be performed as appropriate before adding it to the medium.

In addition to glycerin, other carbon sources, nitrogen sources, inorganic salts, organic nutrients, inorganic substances, surfactants, or antifoaming agents that are normally required for the growth of microorganisms are added to the medium as needed. can do.
Examples of carbon sources other than glycerin include maltose, sucrose, cellobiose, fructose, xylose, and soluble starch.
Examples of the nitrogen source include ammonium sulfate compounds and ammonia compounds.
Examples of inorganic salts include iron, magnesium, manganese, zinc, cobalt, nickel and the like.
Examples of organic nutrient sources include yeast extract, peptone, beef extract, fish extract and the like.
The amount of nitrogen source or the like added to the medium is not particularly limited, and may be appropriately adjusted based on the amount usually used for culturing Penicillium fungi. Carbon sources other than glycerin are preferably added in an amount of about 0.1 g to 100 g with respect to 1 L of the medium. Each of the nitrogen source and the organic nutrient source is preferably added in an amount of about 0.1 g to 20 g with respect to 1 L of the medium. These medium components such as a nitrogen source can be added to the medium as necessary.

  The pH of the medium may be adjusted as appropriate, but is preferably in the range of 2 to 10 and more preferably in the range of 5 to 7 from the viewpoint of growth and compound productivity.

The culture condition is not particularly limited as long as it can grow Penicillium fungi and can produce the compound represented by Formula (1) or Formula (2).
The culture is preferably performed under aerobic conditions.
The culture temperature may be within a temperature range where Penicillium spp. Can grow, preferably 4 ° C. or lower and 37 ° C. or lower, more preferably 25 ° C. or higher and 30 ° C. or lower.
The culture time can be appropriately selected according to the type of medium used and the concentration of the carbon source. From the viewpoint of compound productivity, the nutrient source of the medium is utilized to the maximum, and the production and accumulation amount of the compound represented by the formula (1) or formula (2) produced in the medium has reached the maximum. It is preferable to terminate the culture at the time. Considering these points, the culture time is preferably 24 hours or more, and preferably about 14 days or less. In addition, the production amount of the compound represented by the formula (1) or the formula (2) in the culture can be measured by a usual method such as high performance liquid chromatography or LC-MS.

After the culture, the compound represented by the formula (1) or the formula (2) produced in the culture is isolated and purified. In the present invention, the culture includes a medium (solid medium, liquid culture solution, etc.) and cultured cells.
In particular, since the compound represented by the formula (1) or (2) is synthesized and then accumulated outside the microbial cells, the microbial cells are removed after the culture and the culture solution is recovered. It is preferable to isolate and purify the compound.

  Isolation and purification of the target compound can be carried out by means usually used in the production of substances by microorganisms. For example, operations such as filtration, centrifugation, vacuum concentration, ion exchange or adsorption chromatography, solvent extraction, distillation, crystallization and the like can be mentioned, and these can be used alone or in combination as appropriate. After these operations, further operations such as purification may be performed.

  By the production method of the present invention, the compound represented by the formula (1) or the formula (2) can be produced using glycerin as a carbon source. The obtained compound represented by the formula (1) or formula (2) is useful as a raw material for various chemical substances, or as a herbicide, a pollen formation inhibitor, an antibacterial agent, an antioxidant and the like.

  Regarding the embodiment described above, the present invention further discloses the following production method, strain and fungus.

<1> A fungus belonging to the genus Penicillium is cultured in a medium containing glycerin, and the compound represented by the formula (1) or the formula (2) is produced in the culture, the formula (1) or the formula The manufacturing method of the compound represented by (2).

<2> The production method according to <1>, wherein the fungus belonging to the genus Penicillium is Penicillium daleae or a related fungus.
<3> The method according to <1> or <2>, wherein the fungus belonging to the genus Penicillium is a fungus having the following base sequence (a) in part of the β-tubulin gene sequence.
(A) a base having the identity of 89% or more, preferably 90% or more, more preferably 93% or more, still more preferably 95% or more, and even more preferably 97% or more with the base sequence represented by SEQ ID NO: 1 Sequence <4> The fungus belonging to the genus Penicillium is Penicillium sp. KSM-F26 strain (NITE P-1475), according to any one of <1> to <3> Production method.

<5> Penicillium sp. KSM-F26 strain (NITE P-1475).
<6> A fungus belonging to the genus Penicillium , having a base sequence of the following (b) as part of the β-tubulin gene sequence, and from glycerin in the above formula (1) or (2) Fungi having the ability to produce the compounds represented.
(B) a nucleotide sequence having 93% or more, preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more identity with the nucleotide sequence represented by SEQ ID NO: 1

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.

Example 1. Acquisition and identification of Penicillium sp. KSM-F26 strain (1) Acquisition of strain Add an appropriate amount of soil (Okinawa soil) to 5 mL of 0.85% (w / v) saline, and stir and let stand After that, the upper suspension was taken out. GPY agar medium (10% (w / v) glycerin (Wako Pure Chemical Industries), 0.5% (w / v) Bacto Peptone (BD)), 0.1 % (W / v) Yeast Extract (manufactured by BD), 0.01% (w / v) iron phosphate (Wako Pure Chemical Industries), 1.8% (w / v) Daigo artificial seawater SP (Wako Pure Chemical Industries, Ltd.) Industrial) and 2.0% (w / v) agar (Wako Pure Chemical Industries)) were smeared in an appropriate amount and cultured at 30 ° C. for 2 to 14 days. The grown strain was monocolonized and named KSM-F26 strain.

(2) Identification of strains As a result of observation of the properties and morphology of the colonies, the KSM-F26 strain is grey-green to gray and velvety, and forms a colony that produces a yellow-brown to red-brown soluble pigment in the medium. It is formed by linking unicellular fiaro-type conidia that are finely spiky and spherical to subspherical to raw to two-wheeled Penicillus, and is relatively similar to the characteristics of Penicillium daleae It was confirmed.
Then, using the chromosomal DNA of the KSM-F26 strain as a template, and using the oligonucleotide consisting of the base sequence of SEQ ID NO: 2 and the oligonucleotide consisting of the base sequence of SEQ ID NO: 3 shown in Table 1 as a primer pair, a PCR reaction according to a conventional method And the DNA of the region encoding the β-tubulin gene was amplified. The base sequence of the obtained DNA amplified fragment was analyzed, and the partial sequence (SEQ ID NO: 1: 476 bases) of the β-tubulin gene of the KSM-F26 strain was determined.

When the BLAST homology search was performed with respect to the international base sequence database using the analyzed base sequence, high sequence identity was shown between the β-tubulin gene sequences derived from a plurality of Penicillium fungi. In particular, the β-tubulin gene sequence of Penicillium daleia strain CBS211.28 showed the highest identity of 92.6%.
Next, as a result of the homology search, molecular phylogenetic analysis based on the β-tubulin gene sequence was performed with the fungus belonging to the genus Penicillium or its asexual generation, Eupenicillium ( Eupenicillium ). . The results are shown in FIG.
As a result of molecular phylogenetic analysis, as shown in FIG. 1, the KSM-F26 strain formed a cluster supported by Penicillium darea and 96% bootstrap value.

From these results, the KSM-F26 strain was identified as Penicillium sp., A fungus belonging to the genus Penicillium, which is a related species of Penicillium daleia.

Example 2 Identification of substances produced by Penicillium sp. KSM-F26 strain Using glycerin as a carbon source, substances produced by Penicillium sp. KSM-F26 strain were identified.
1. Culture and HPLC analysis of cultures GPY medium adjusted to pH 6.5 (5% (w / v) glycerin, 0.5% (w / v) Bacto Peptone, 0.1% (w / v) Yeast Extract, 0 .01% (w / v) iron phosphate, 1.8% (w / v) Daigo artificial seawater SP) were inoculated with 1 platinum ear of Penicillium sp. KSM-F26, and cultured with shaking for 6 days (30 ° C. 250 rpm). Thereafter, the culture solution was centrifuged at 12,000 rpm for 20 minutes, and the supernatant fraction was fractionated.
Analysis of the compounds contained in the culture supernatant was performed with Hitachi LaChrom Elite apparatus (HITACHI) using Acclaim Surfactant (registered trademark, manufactured by DIONEX) as a column. A culture supernatant sample is appropriately diluted with a 0.1% (v / v) formic acid aqueous solution to prepare a 0.1% formic acid aqueous solution and a 0.1% formic acid aqueous solution containing 50% acetonitrile. By mixing the aqueous solution, HPLC was performed under a concentration gradient condition (shown in Table 2) from 0.1% formic acid aqueous solution to 0.1% formic acid aqueous solution containing 35% acetonitrile, and Corona CAD ™ charged particles Analysis was performed using a detector (ESA) and a UV detector (HITACHI). The HPLC was performed at a flow rate of 0.5 mL / min, a column temperature; 30 ° C., and a sample injection amount: 10 μL. All analysis samples were used after being filtered through DISMIC-13CP Cellulose Acetate 0.2 μm (manufactured by ADVANTEC).
The result of HPLC is shown in FIG.
In addition, using a GPY medium as a control sample, HPLC analysis was performed in the same manner as described above.

  As shown in FIG. 2, in the supernatant after culturing, there are two characteristic peaks not present in the control sample, peak 1 (elution time 19.0 minutes) and peak 2 (elution time 19.4 minutes). Was recognized.

2. Purification of Peak Component One platinum ear of Penicillium sp. KSM-F26 strain was inoculated into GPY medium adjusted to pH 6.5 and cultured with shaking (30 ° C., 250 rpm) for 7 days. The culture solution was centrifuged (12000 rpm, 20 minutes), and the supernatant fraction was filtered through a 0.45 μm filter.
The fractionated supernatant is appropriately concentrated, and 30 mL is applied to a column (Φ5.0 × 20 cm) packed with 90 cm ³ silica gel (Wakosil C-300) and stabilized with ultrapure water. And fraction 1 containing peak 1 and fraction 2 containing peak 2 were obtained.
Inertsil ODS-3 preparative column (manufactured by GL Sciences) stabilized with a 0.1% formic acid aqueous solution containing 20% acetonitrile as a mobile phase for purification after appropriately concentrating the obtained fraction 1. Using HPLC (AZURA (manufactured by KNAUER)) under conditions of a flow rate of 7.5 mL / min, a column temperature of 40 ° C., a sample injection amount of 200 μL, and an analysis time of 30 minutes, a UV (210 nm) detector (VISCTEK 270 DUAL DETECTER) (UV detector) (manufactured by Malvern)), and peak 1 was collected.
The fraction collected is the same as in 1. HPLC analysis under the same conditions as above confirmed that peak 1 was purified.
The same operation was performed on fraction 2, and peak 2 was collected. The fraction collected is the same as in 1. HPLC analysis under the same conditions as above confirmed that peak 2 was purified.

3. Identification of peak component Peak 1 was analyzed by NMR. First, a dried sample of peak 1 (17 mg) was dissolved in CD ₃ OD, and ¹ H NMR (600 MHz) and ¹³ C NMR (150 MHz) were measured with AV-600 (manufactured by BRUKER). The results are shown in Table 3.

  When the measurement result was analyzed, the compound of peak 1 was identified as 2-acetyl-3,4-dihydroxy-5-methoxybenzeneacetic acid represented by the following formula (1).

Peak 2 was analyzed by NMR. The dried sample of peak 2 (4.0 mg) was dissolved in CD ₃ OD, and ¹ H NMR (600 MHz) and ¹³ C NMR (150 MHz) were measured with AV-600 (manufactured by BRUKER). The results are shown in Table 4.

  When the measurement result was analyzed, the compound of peak 2 was identified as 2-acetyl-3,5-dihydroxy-4-methoxybenzeneacetic acid represented by the following formula (2).

Example 3 Examination of productivity of compound represented by formula (1) or formula (2) GPY medium adjusted to pH 6.5 (5% (w / v) glycerin, 0.5% (w / v) Bacto Peptone, 0.1% (w / v) Yeast Extract, 0.01% (w / v) iron phosphate, 1.8% (w / v) Daigo artificial seawater SP), and 1 platinum of Penicillium sp KSM-F26 strain Ear-inoculated and cultured with shaking at 250C (250 rpm). After culturing for a predetermined time, the culture solution was recovered and the cells were removed in the same manner as in Example 2, and 2-acetyl-3,4-dihydroxy-5-methoxybenzeneacetic acid (formula (1) above) in the culture supernatant was removed. The amount of product of 2-acetyl-3,5-dihydroxy-4-methoxybenzeneacetic acid (compound represented by the above formula (2)) was measured by HPLC. The measurement was performed by changing the culture time, and the change with time of the productivity was examined.
As a control, a sample obtained by culturing in the same manner as described above was prepared using a medium obtained by removing glycerin from the GPY medium, and the production amount over time was measured.

  The results are shown in FIGS. FIG. 3 shows the change over time in the productivity of 2-acetyl-3,4-dihydroxy-5-methoxybenzeneacetic acid, and FIG. 4 shows the change over time in the productivity of 2-acetyl-3,5-dihydroxy-4-methoxybenzeneacetic acid. Are shown respectively. The productivity of each compound was expressed as a relative value (%) with the production amount in a sample having 12 days of culture as 100.

Control samples cultured in a medium that does not contain glycerin as a carbon source produce both 2-acetyl-3,4-dihydroxy-5-methoxybenzeneacetic acid and 2-acetyl-3,5-dihydroxy-4-methoxybenzeneacetic acid Was not confirmed (not shown).
On the other hand, as is clear from FIGS. 3 and 4, it was confirmed that any of the above compounds was produced satisfactorily in a sample cultured in a medium containing glycerin as a carbon source.

Example 4 Production amount of compound represented by formula (1) or formula (2) 6 mL of GPY medium was charged into a test tube, 1 platinum ear of Penicillium sp. KSM-F26 was inoculated, and cultured at 30 ° C. for 12 days with shaking (250 rpm). )did. Thereafter, the culture solution was collected and the cells were removed in the same manner as in Example 2, and the amount of 2-acetyl-3,4-dihydroxy-5-methoxybenzeneacetic acid in the culture supernatant was measured by HPLC. .
As a control, a sample obtained by culturing in the same manner as described above was prepared using a medium obtained by removing glycerin from the GPY medium, and the amount of 2-acetyl-3,4-dihydroxy-5-methoxybenzeneacetic acid produced was measured. .
The results are shown in Table 5.

A test tube was charged with 10 mL of GPY medium, 1 platinum ear of Penicillium sp. KSM-F26 strain was inoculated, and cultured with shaking (250 rpm) at 30 ° C. for 12 days. Thereafter, the culture solution was collected and the cells were removed in the same manner as in Example 2, and the amount of 2-acetyl-3,5-dihydroxy-4-methoxybenzeneacetic acid produced in the culture supernatant was measured by HPLC. .
As a control, using a medium obtained by removing glycerin from the GPY medium, a sample cultured in the same manner as described above was prepared, and the amount of 2-acetyl-3,5-dihydroxy-4-methoxybenzeneacetic acid produced was measured .
The results are shown in Table 6.

  As is clear from Tables 5 and 6, samples cultured in GPY medium without the addition of glycerin include 2-acetyl-3,4-dihydroxy-5-methoxybenzeneacetic acid and 2-acetyl-3,5-dihydroxy-4. No production of methoxybenzeneacetic acid was confirmed.

From the above results, it was found that the compound represented by the formula (1) or the formula (2) can be produced by the production method of the present invention.

Claims (6)

A fungus belonging to the genus Penicillium is cultured in a medium containing glycerin, and a compound represented by the following formula (1) or formula (2) is produced in the culture. The following formula (1) or formula (2) The manufacturing method of the compound represented by these.

The method according to claim 1, wherein the fungus belonging to the genus Penicillium is Penicillium daleae or a related fungus. The production method according to claim 1 or 2, wherein the fungus belonging to the genus Penicillium is a fungus having the following base sequence (a) in part of the β-tubulin gene sequence.
(A) a nucleotide sequence having 89% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 The production method according to any one of claims 1 to 3, wherein the fungus belonging to the genus Penicillium is Penicillium sp. KSM-F26 strain (NITE P-1475). Penicillium sp. KSM-F26 strain (NITE P-1475). A fungus belonging to the genus Penicillium , having a base sequence of the following (b) in a part of the β-tubulin gene sequence and represented by the following formula (1) or (2) from glycerin Fungi with the ability to produce compounds.
(B) a nucleotide sequence having 93% or more identity with the nucleotide sequence represented by SEQ ID NO: 1

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