KR101839996B1 - Method for mass production of sesquiterpene from Polyporus brumalis - Google Patents

Abstract

The present invention relates to a method for culturing winter oyster mushroom in a culture medium containing magnesium to produce a gene encoding isopentenyl diphosphate isomerase, a gene encoding farnesyl diphosphate synthase, The present invention relates to a method for mass-producing sesquiterpene at a high yield by increasing the expression amount of a gene encoding sesquiterpene synthase.

Description

Method for producing sesquiterpene from Polyporus brumalis from winter mushroom {

The present invention relates to a method for producing sesquiterpene from winter umbrella, and more particularly, to a method for producing isopentenyl diphosphate isomerase It is possible to mass-produce sesquiterpene at a high yield by increasing the expression level of a gene encoding a farnesyl diphosphate synthase, a gene encoding a farnesyl diphosphate synthase, and a gene encoding a sesquiterpene synthase How to do it.

Microorganisms produce various secondary metabolites, and wood-borne fungi belonging to the mushroom class are known to parasitize on trees and produce various secondary metabolites.

Winter umbrella mushroom ( Polyporus Brumalis has the ability to biosynthesize certain sesquiterpene (eudesmol) components from the mycelium. Udesmol, which belongs to the sesquiterpene compound, is a terpene component biosynthesized in existing plants. It has anti-fungal activity against harmful fungi as well as anti-inflammatory (anti-inflammatory) ) Is known to be an important physiologically active substance.

At present, about 16,500 secondary metabolites isolated from microorganisms are isolated from actinomycetes (62%), fungi (20%) and bacteria (18%), As a result, the development of new resources is urgently required.

There are about 1,500 kinds of mushrooms native to Korea, but a few of them are used as edible or medicinal mushrooms.

However, some biologically active substances such as antibiotic, antitumor, and antivirus have been found from some mushrooms, and they have potential as agricultural resources even if they are not edible.

Fungi, including mushrooms, synthesize, secrete, and accumulate various constituents during the growth process. The primary metabolites are proteins, polysaccharides, organic acids, vitamins, fats and other nutrients. Secondary metabolites include anticancer substances, antibiotics, terpenoids, and toxins. .

Studies on the physiologically active substances contained in mushrooms are still unexplored in comparison with plants, but they can be used as important resources for creating new materials because of their abundant resources as microorganisms.

On the other hand, the extraction method of β-eudesmol, which is a kind of sesquiterpene, from Korean ginger (Korean J. Food & Nutr. Vol.13, No. 1, 66-70 (2000) A composition for treating cardiovascular diseases and cancer containing the extracted sesquiterpene lactone compound (Korean Patent Laid-Open No. 10-2004-0070985), hyperlipemia including sesquiterpene derivatives extracted from actual horsetail, fatty liver, diabetes or obesity (Korean Patent No. 10-0905419), and the like.

However, in order to produce the components in such plants, the sesquiterpene compounds according to the prior art are derived from plants. In order to produce such components, leaf extraction from plants, essential oil extraction by steam distillation, separation by chromatography principle, purification, etc. However, since the amount of the sesquiterpene-based compound contained in the plant is small, there is a limitation in use.

It is required to develop a mass production method of Sesquiterpene such as Yudesmol from mushroom.

DISCLOSURE OF THE INVENTION It is an object of the present invention to provide a method for the production of sesquiterpene, which comprises culturing the mycelium of mushroom, especially winter oyster mushroom under specific conditions, Completed.

Accordingly, it is an object of the present invention to provide a method for mass-producing sesquiterpene by culturing it in a medium containing specific components from winter umbrella.

In order to achieve the above-mentioned object, the present invention provides a method for culturing winter umbrella mushroom in a culture medium containing magnesium to produce a gene encoding isopentenyl diphosphate isomerase, farnesyl diphosphate isomerase diphosphate synthase and a gene encoding a sesquiterpene synthase in a cell, which comprises the step of increasing the expression level of a gene encoding sesquiterpene synthase.

The present invention also provides a sesquiterpene produced by the above method.

The mass production method of sesquiterpene according to the present invention is advantageous in that it can efficiently mass-produce sesquiterpene from a mycelial culture of winter mushroom by culturing in a culture medium containing magnesium, under specific culture conditions.

Given the extremely rare research on secondary metabolites produced from existing white rot fungi, it has value as a new microbial resource for the production of natural products.

Figure 1 shows the product analysis (GC MS) results on MgSO4-free medium.
Figure 2 shows a mass pattern of two peaks (RT: 26.90 min and RT: 35.51 min).
Fig. 3 shows the result of analysis of the product (GC MS) in the MgSO4-containing medium.
Figure 4 shows a mass pattern of two peaks (RT: 35.70 min and RT: 39.90 min).
Figure 5 shows the mevalonic acid path.
FIG. 6A shows the results of comparing the expression levels of 9 genes on the 5th day of culture on two conditions (magnesium-containing medium, magnesium-free medium), and FIG. 6B shows the results of comparing the expression amounts of 9 genes on two conditions (magnesium- FIG. 6C shows the results of comparing the expression levels of 9 genes on the 12th day of culture on two conditions (magnesium-containing medium, magnesium-containing medium) will be.

The present invention relates to a method for culturing winter oyster mushroom in a culture medium containing magnesium to produce a gene encoding isopentenyl diphosphate isomerase, a gene encoding farnesyl diphosphate synthase, The present invention relates to a method for producing sesquiterpene, which comprises increasing the expression level of a gene encoding sesquiterpene synthase.

In the method for producing sesquiterpene of the present invention, the isopentenyl diphosphate isomerizing enzyme is characterized by being Enzyme 7 of the following mevalonic acid pathway.

[Mevalonic acid pathway]

In the method for producing sesquiterpene of the present invention, the parnesyl diphosphate synthase is characterized by being Enzyme 8 of the mevalonic acid pathway.

In the method for producing sesquiterpene of the present invention, the sesquiterpene synthase is an Enzyme 9 of the mevalonic acid pathway.

In the method for producing sesquiterpene of the present invention, the culture medium may contain 10 g of glucose, 0.2 g of ammonium tartrate, 0.5 g of magnesium sulfate, and 0.1 g of calcium chloride per liter of distilled water.

In the method for producing sesquiterpene of the present invention, the medium may further contain growth factors such as minor elements and vitamins selected from manganese, iron, cobalt, zinc, copper and the like in addition to the above components .

In the method for producing sesquiterpene of the present invention, the pH of the medium may be 3.5 to 4.5.

In the method of producing sesquiterpene of the present invention, the step of culturing the winter umbrella mushroom is carried out by inoculating mycelium of winter mushroom pre-cultured in a potato dextrose agar (PDA) medium into the medium, . ≪ / RTI >

In the method for producing sesquiterpene of the present invention, the PDA medium is generally known as a medium suitable for growing fungi such as fungi, and particularly a medium rich in carbon such as potatoes.

In general, a fungus-like fungus can be cultivated using a shaker incubator or a stationary incubator. The stationary culture is a method of growing microorganisms while keeping it still. The shaking culture is an efficient method of supplying oxygen As a method for increasing the oxygen concentration, there is an advantage that the oxygen supply can be smoothly performed as compared with the stationary culture.

In the method for producing sesquiterpene of the present invention, the culturing can be carried out at a temperature of 25 to 30 DEG C and 50 to 150 rpm for 5 to 15 days.

In the method for producing sesquiterpene of the present invention, the culturing can be carried out for 12 days at a temperature of 28 DEG C and 100 rpm.

The method for producing sesquiterpene of the present invention may further comprise, after the end of the step of culturing the winter umbrella mushroom, adding an organic solvent to the mycelial culture of the winter umbrella mushroom, extracting it by shaking, and concentrating it.

In the method for producing sesquiterpene of the present invention, after adding sodium chloride to the mycelium culture solution, an organic solvent may be added to shake the extract, in order to increase the extraction efficiency of sesquiterpene.

      In the method for producing sesquiterpene of the present invention, the organic solvent may be ethyl acetate, but the organic solvent is not limited thereto, and the organic solvent may be varied as required.

The present invention also relates to sesquiterpene produced by the process.

Sesquiterpenes are a component of linseed and cedar essential oils and are an antimicrobial activity against dermatophytes and respiratory track bacteria.

In addition, sesquiterpene is a sesquiterpene alcohol compound containing an alcohol functional group and studies on various physiological activities have been reported, and it is also known that it exhibits antitumor activity.

On the other hand, sesquiterpene is known to be a major active ingredient of cedar or sclerotin essential oil and exhibits antifungal activity. It is known that it exhibits antimicrobial activity against Trichophytons strains belonging to skin fungi (dermatophytes) which induce athlete's foot and cause ringworm have.

Hereinafter, the present invention will be described in more detail by way of examples. However, the scope of the present invention is not limited to the following examples.

<Examples>

In the following examples, the mechanism by which the sesquiterpene component is produced from winter umbrella is understood, and the transcript expressed from the mycelium is analyzed to confirm the genetic information related to the production.

In particular, the produced and non-generated conditions of the sesquiterpene components were studied and differentially expressed genes (DEGs) were performed under the two conditions.

1. Materials and Methods

(One). Culture of strain

Winter umbrella mushroom ( Polyporus Brumalis was cultivated in a national institute of forest science and pre-cultured in PDA (Potato Dextrose agar) medium for 10 days. Then, only the cells were scraped off and homogenized with sterilized distilled water (homogenizing).

The homogenized (milled) bacterial body fluid was precultured, and the mycelial amount was inoculated to 100 ml of the medium on the basis of the preculture, and the mixture was cultured for 5 days, 7 days, and 12 days while shaking at 28 rpm at 100 rpm.

The cultivation of the strain was performed under two conditions (containing MgSO ₄ and MgSO ₄ ), and the composition of the culture medium under the two conditions is shown in Table 1 below.

(2). Analysis of metabolites

For the analysis of useful metabolites produced from winter umbrella hyphae, cultured medium was separated into mycelium and culture medium by centrifugation. The culture medium was again extracted with an organic solvent (ethyl acetate).

The culture broth separated for each incubation days was completely concentrated using an evaporator, and the final volume was adjusted to 2 ml to prepare samples for component analysis.

Each component was analyzed by gas chromatography (GC MS) suitable for volatile matter analysis. The analytical conditions were 260 ℃ for the injector and 280 ℃ for the detector. The mass pattern of the detected peaks was compared with the National Institute of Standards Technology 2005 library.

(3). Terpene synthesis-related gene expression analysis

      (3-1). DNA extraction

Each finely ground sample was mixed with 300 ml of DNA extraction buffer (Tris-HCl, NaCl, EDTA, SDS, distilled water) and vortexed. The supernatant was separated by centrifugation (13,000 rpm, 15 min, 4 ° C).

The separated supernatant was mixed with 250 [mu] l of phenol-chloroform-isoamyl alcohol. This procedure was repeated one more time and the separated supernatant was mixed with 500 μl of 95% ethanol. The mixed solution was centrifuged to remove ethanol, RNA was removed with RNAse, and the final DNA was obtained by incubation at 37 DEG C for 30 minutes.

(3-2). RNA extraction

The culture obtained from the culture of the strain (1) and the separated mycelium were used for gene expression analysis. The mycelia recovered per culture days were ground using liquid nitrogen, and RNA was isolated using an RNA extraction kit (NucleoSpin @ RNA Plant kit).

350 μl of lysis buffer was added to dissolve the cell wall of the mycelia, and the RNA was precipitated by adding 350 μl of 70% ethanol to the solution by using a NucleoSipn Filter. This was passed through a NucleoSpin RNA Plant Column to bind the RNA to the silica membrane of the column.

And then desalted on a silica thin film through a column in which RNA was bound with 350 의 of MDB (Membrane Desalting Buffer).

To dissociate the DNA, 95 μl of rDNase reaction mixture was added to the column, allowed to stand at room temperature for 15 minutes, and then the column was washed three times with 200 μl, 600 μl and 250 μl using a washing buffer.

After the alcohol component of the washing solution was completely evaporated using a centrifugal separator, 40 μl of RNase-free water was added to the column, and the RNA was recovered.

The RNA thus isolated was quantitated using Experion ™ RNA StdSens Chips.

(3-3). Fabrication of library for de novo sequencing of RNA

For optimal library production, gDNA was added to the g-tube and made into 40-kb size with AMPure PB magnetic beads (Beckman Coulter Inc., Brea, CA, USA). The final concentrations of gDNA and RNA were measured with a NanoDrop spectrophotometer and Qubit fluorometer, and approximately 200 ng / μl was loaded into the field-inversion gel.

A total of 10 μl of the library was prepared using PacBio DNA Template Prep Kit 1.0 (for 310 kb).

Sequencing was performed using the third-generation sequencing SMRT technology. In the third-generation sequencing, the PCR amplification process is omitted, and DNA single molecules are directly sequenced to identify sequences.

The DNA single molecule of the experimental group was annealed using SMRTbell Tel Plate and PacBio DNA / Polymerase Binding Kit P6. The finally generated SMRT cells were analyzed by a PacBio RS II (Pacific Biosciences) sequencing platform.

(3-4). DEGs analysis

In order to identify genes directly involved in the production of useful products, the difference in gene expression was confirmed by separating mycelium under conditions in which terpenes were generated and those in non-producing conditions.

Since a reference sequence is required to perform DEGs (differnetly expressed genes) analysis between each sample, trancriptome de novo assembly for total RNA of winter umbrella was performed first, and sequencing method PacBio SMRT sequencing analysis was performed.

Based on the results of RNA isoquencing of winter umbrella mushrooms obtained through Pacbio sequencing, gene expression was confirmed using Illumina hiseq for the analysis of DEGs between 6 samples.

Each sample fastq was subjected to referee mapping using the program included in the assembly cell package program of CLCBIO, and the mapping read count was calculated.

The rpkm is applied to the lead counts according to each sample to perform normalization. The method for obtaining the RPKM is expressed by Equation 1 below.

2. Results

(One). Analysis of products according to culture conditions

The secondary metabolic pathway of microorganisms can be affected by the surrounding environment. In this example, it was confirmed that the sesquiterpene components produced from winter umbrella were affected by the culture conditions.

After culturing mycelium in MgSO4-containing medium and magnesium-free medium, the culture broth was separated and analyzed for each day (5, 7 and 12 days). As a result, And showed distinct production differences.

First, magnesium non-treated conditioned medium products are shown in FIG. As a result of the analysis, peaks were detected at a retention time of 26.90 minutes and 35.51 minutes, but the mass pattern of the detected peaks revealed that propanoic acid and 1,1-dipentyl-2,13-ethylene were detected (See Fig. 2).

From these results, it was confirmed that sesquiterpene components were not detected in a culture condition containing no magnesium with a biterpene compound, and the sesquiterpene components were not detected even after 12 days of culture.

On the other hand, the peaks of eudesmane and eudesmol, which are components of sesquiterpene, were observed at 35.70 and 39.90 min in the magnesium-containing medium (see Figs. 3 and 4) , 7, and 12 days, respectively.

As a result, it was confirmed that metabolite production was varied depending on the presence or absence of magnesium element in the culture solution. It was confirmed from previous studies that the domain site of the important protein involved in the formation of sesquiterpene is activated in the presence of magnesium, confirming that magnesium plays an important role in the formation of sesquiterpene.

After the gene analysis, we confirmed whether the expression level of the genes involved in the production depends on the presence or absence of magnesium.

(2). DNA sequencing

Whole genome sequencing of the DNA of mycelium of winter umbrella mushroom showed that the size of the genome was about 56 Mb. The N50 was 11,627 and the read number was 676,520.

A total of 138 contigs were identified. The function of the gene was predicted by annotating the conic information to the blast. In this embodiment, since the focus is on the synthesis route of the terpene material, the related genes are confirmed.

The terpene component is originally known to be biosynthesized via a mevalonic acid (MEA) pathway as shown in FIG. The MEA pathway is derived from glycolysis, which is known as the primary metabolic pathway, and is known to be derived from acetyl CoA produced during the course of glucose.

Enzyme 1 to Enzyme 9 in the mevalonic acid pathway of FIG. 5 are shown in Table 2 below.

acetyl-CoA-acetyl transferase, hydroxymethylglutaryl-coenzyme A (hereinafter, referred to as &quot; coenzyme A &quot;) until the farnesyl pyrophosphate, the basic skeleton structure of the sesquiterpene component, Hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate decarboxylase, mevalonic acid 2 It is known that diphosphatemevalonate decarboxylase, isopentenyl-diphosphate isomerase, and farnesyl diphosphate synthase are involved.

These enzymes were identified by blast annotation in winter umbrella and similar gene sequences were identified in Dichomitus squalene and yeast, S accharomyces It was confirmed that the sequence was similar to that of S. cerevisiae .

In addition, in the case of the Udesmole structure, which is a main terpene compound produced from winter umbrella mushroom, a cyclic structure is formed by a cyclization reaction from a farnesyl pyro diphosphate, which is a precursor, sesquiterpene structure &Lt; / RTI &gt;

At this time, the action of terpenoids synthase, which removes the phosphate group from the precursor and induces cyclization of the structure, is required. In this embodiment, Gleophyllum it was predicted that a terpenoid synthase similar to that of trabeum was present. In other words, nine genes play an important role in the production of uidesol, and sequencing results suggest that all nine genes are present in winter mushroom.

(3). RNA results (DEGs analysis)

In order to determine whether the expected genes (Enzyme 1-9) present in the DNA sequence directly participate in the production of uidemole, the amount of gene expression at the RNA level was examined on the 5th day, 7th day and 12th day of the culture, The results are shown in Figs. 6A to 6C. Here, the high expression level means that the gene is highly related.

In particular, the culture conditions of the strain were set as a magnesium - containing medium and a magnesium - free medium, respectively, and then the association was confirmed by comparing the genes expressed in both mycelium.

As a result, it was confirmed that the gene (Enzyme 1 to 9) was relatively increased in the culture condition containing magnesium. This means that the expression level of terpene-related enzymes in the magnesium-containing medium is increased, and as a result, the result of this example means that the production of terpene is associated with the expression of genes (Enzyme 1 to 9) .

Therefore, it is shown that the involvement of terpene-related genes is higher when magnesium is contained in the culture medium. In particular, the expression of the gene in the magnesium environment was high because it was bound to the active site necessary for the activation of the related enzyme, and thus it was considered to be a cofactor for the activation of the enzyme.

FIGS. 6A to 6C show the order of the fold change values according to the culture medium containing or not containing magnesium according to the culture days.

6A to 6C, a list of genes having a large difference is shown as follows: sesquiterpene synthase, isopentenyl-diphosphate isomerase, farnesyl diphosphate synthase ) Are thought to be genes directly related to the production of uidesol.

Through this example, important culturing elements for the production of sesquiterpene were identified, and the changes in the amount of gene expression according to culture elements were confirmed.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the invention as disclosed in the accompanying claims. .

Since the method of producing sesquiterpene according to the present invention has an advantage that it can efficiently mass-produce sesquiterpene from a mycelial culture solution of winter umbrella mushroom by culturing in a culture medium containing magnesium, it is useful for the technical field to which the present invention belongs Lt; / RTI &gt;

Claims (10)

Winter oyster mushroom was cultured in a medium containing magnesium to produce a gene encoding isopentenyl diphosphate isomerase, a gene encoding farnesyl diphosphate synthase, and a gene encoding sesquiterpene synthase A method for producing sesquiterpene, which comprises increasing the expression level of a gene encoding an enzyme (sesquiterpene synthase). The method according to claim 1, wherein the isopentenyl diphosphate isomerase is Enzyme 7 of the following mevalonic acid pathway and the Enzyme 7 is guaranyl pyrophosphate using dimethylallyl pyrophosphate as a reaction substrate Production method of sesquiterpene.
[Mevalonic acid pathway]

2. The method according to claim 1, wherein the parnesyl diphosphate synthase is Enzyme 8 of the following mevalonic acid pathway, and Enzyme 8 is feronyl pyrophosphate using geranyl pyrophosphate as a reaction substrate. Production method of quiterpenes.
[Mevalonic acid pathway]

[Claim 2] The method according to claim 1, wherein the sesquiterpene synthase is Enzyme 9 of the mevalonic acid pathway, and Enzyme 9 is parathion pyrophosphate as a reaction substrate to produce uidesol Production method.
[Mevalonic acid pathway]

The method according to claim 1, wherein the culture medium comprises 10 g of glucose, 0.2 g of ammonium tartrate, 0.5 g of magnesium sulfate, and 0.1 g of calcium chloride per liter of distilled water. [3] The method of claim 1, wherein the step of culturing the winter umbrella includes the step of inoculating mycelium of winter mushroom pre-cultured in a potato dextrose agar (PDA) medium into a culture medium containing magnesium, followed by shaking culture &Lt; / RTI &gt; wherein the method comprises the steps of: The method for producing sesquiterpene according to claim 1, wherein the culturing is carried out at a temperature of 25 to 30 DEG C and 50 to 150 rpm for 5 to 15 days. 8. The method for producing sesquiterpene according to claim 7, wherein the culture is carried out at a temperature of 28 DEG C and 100 rpm for 12 days. 2. The method according to claim 1, further comprising, after the end of the step of culturing the winter umbrella mushroom, adding an organic solvent to the mycelial culture liquid of the winter mushroom, extracting it by shaking, and concentrating it. Production method. delete

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