The isolation of piericidin A produced by a new Streptomyces massiliensis strain and purification method

Abstract

The present invention relates to a method for mass-producing piericidin A, a representative inhibitor of mitochondrial complex I, from a novel Streptomyces massiliensis 213DD-128 strain (accession number KCTC15467BP) and a method for isolating and purifying it. Specifically, the invention developed a culture method for mass-producing piericidin A from the Streptomyces massiliensis 213DD-128 strain (accession number KCTC15467BP) isolated from sediment collected from the East Sea, and a method for more economically isolating and purifying it from the culture extract using simple open reverse-phase column chromatography and liquid-liquid extraction without using expensive research equipment. Piericidin A isolated from the above strain is a representative inhibitor of mitochondrial complex I and has anticancer, antibacterial, and insecticidal activity, so it can be used in the development of related pharmaceuticals and insecticides for the treatment of cancer or autoimmune diseases, promotion of hair growth, and suppression of bacterial plant diseases.

Description

Technology Field

[0001] The present invention relates to a method for isolating and purifying piericidin A produced from a novel Streptomyces maciriensis strain, and more specifically, to a novel Streptomyces massiliensis The present invention relates to a culture method for mass-producing piericidin A, a representative inhibitor of mitochondrial complex I, from strain 213DD-128 (accession number KCTC15467BP), and a method for economically isolating and purifying it from the culture extract. Background Technology

[0003] Mitochondria are organelles that perform major cellular functions, including metabolism, signal transduction, apoptosis, cell cycle regulation, and differentiation. In particular, the production of ATP, the cellular energy source essential for the survival of organisms, takes place in the electron transport chain (ETC) located in the inner mitochondrial membrane. The electron transport chain consists of a total of five protein complexes, from I to V; by transferring electrons with higher energy levels to the next complex, ATP is ultimately produced in Complex V by utilizing the proton gradient formed on the inner mitochondrial membrane. As mitochondria play such a crucial role in the physiological functions of cells, research is underway on the development of treatments for various diseases using inhibitors that target the protein complexes of the mitochondrial electron transport chain.

[0004] Inhibitors acting on mitochondrial ETC complexes I to V include metformin, rotenone, epiberberine, piericidin A (inhibitor of complex I), sodium malonate and tenoyl trifluoroacetone (inhibitor of complex II), antimycin A (inhibitor of complex III), potassium cyanide and sodium azide (inhibitor of complex IV), and oligomycin (inhibitor of complex V), each acting by targeting different complexes.

[0005] Piericidin A is known to be primarily produced by actinomycetes, particularly those of the genus Streptomyces. Piericidin A has a 4-pyridinol core structure connected to methylated polyketide side chains, and because it is structurally similar to coenzyme Q and acts as an antagonist to coenzyme Q, it is known as a representative NADH-ubiquinone oxidoreductase (complex I) inhibitor in the mitochondrial electron transport chain.

[0006] Because piericidin A exhibits anticancer, antibacterial, and insecticidal activity, it can be utilized in the development of therapeutic agents or insecticides for cancer treatment, treatment of autoimmune diseases, promotion of hair growth, and suppression of bacterial plant diseases.

[0007] Therefore, the inventors [discovered] a novel Streptomyces masiliensis ( Streptomyces massiliensis ) A strain of 213DD-128 was isolated, and the present invention was completed by providing a culture method, isolation and purification method for mass-producing piericidin A from the strain. Prior art literature

[0009] Republic of Korea Published Patent Application No. 10-2015-0041786 (April 17, 2015) Republic of Korea Registered Patent Application No. 10-1587483 (January 15, 2016)

[0010] Xuefeng Zhou, William Fenical. The Journal of Antibiotics 2016, 69, 582-593. [DOI: https: / / doi.org / 10.1038 / ja.2016.71] The problem to be solved

[0011] The present invention is a novel invention characterized by producing piericidin A. Streptomyces massiliensis It is to provide the 213DD-128 strain (accession number KCTC15467BP).

[0012] In addition, the present invention Streptomyces massiliensis The present invention provides a method for mass-producing piericidin A using the 213DD-128 strain (accession number KCTC15467BP).

[0013] In addition, the present invention Streptomyces massiliensis The strain 213DD-128 (accession number KCTC15467BP) provides a method for isolating and purifying piericidin A from an extract of a large culture medium.

[0014] The technical problems that the invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the invention belongs from the description below. means of solving the problem

[0016] The present invention provides a novel solution to the above problems. Streptomyces massiliensis Strain 213DD-128 (accession number KCTC15467BP) is provided.

[0017] The above strain is characterized by producing piericidin A.

[0018] The present invention Streptomyces massiliensis The present invention provides a method for mass-producing piericidin A, characterized by comprising the steps of: inoculating a strain 213DD-128 (accession number KCTC15467BP) into a culture medium for seed culture; and mass-culturing the seed-cultured strain in a fermenter.

[0019] The step of culturing the above species is characterized by first culturing in 90 to 110 mL of liquid medium, and then secondarily culturing in 600 to 800 mL of liquid medium.

[0020] The above seed culture step is characterized by shaking culture at a temperature of 27 to 29°C for 4 to 6 days.

[0021] The above mass culture step is characterized by culturing in a liquid medium of 60 to 80 L to which an antifoaming agent has been added.

[0022] The above mass cultivation step is characterized by culturing with an air inflow of 15 to 25 L / min (LPM), a temperature of 27 to 29℃, and stirring at 40 to 60 rpm for 19 to 21 days.

[0023] In addition, the present invention is said to Streptomyces massiliensis A step of obtaining a crude extract by extracting a culture medium mass-cultured using strain 213DD-128 (accession number KCTC15467BP) with ethyl acetate and concentrating under reduced pressure; a step of obtaining a fraction by eluting the crude extract using vacuum column chromatography with a solvent serially diluted by mixing methanol and water; a step of selecting a fraction containing a large amount of piericidin A by concentrating the eluted fraction under reduced pressure; and a step of dissolving the selected fraction in a small amount of methanol and mixing it with water (H2O), and then n -Heptane( n -heptane), n -Hexane( n A step of primary purification by sequentially fractionating with solvents of -hexane), chloroform, and ethyl acetate; to the primary purified fraction n The present invention provides a method for isolating and purifying piericidin A, characterized by including the step of adding heptane and refractioning with a mixture of water and methanol for secondary purification.

[0024] The method of separation and purification above is characterized by having a yield of piericidin A eluted from the above method of separation and purification of 20 to 30%. Effects of the invention

[0026] The present invention is a novel Streptomyces massiliensis The present invention relates to a method for mass-producing piericidin A, a representative inhibitor of mitochondrial complex I, from strain 213DD-128 (accession number KCTC15467BP) and a method for isolating and purifying it, which allows for the isolation and purification of piericidin A more economically using simple open reverse-phase column chromatography and liquid-liquid extraction without using expensive research equipment.

[0027] In addition, isolated and purified piericidin A is effective in treating cancer or autoimmune diseases, promoting hair growth, and suppressing bacterial plant diseases, so it can be used in the development of related pharmaceuticals and insecticides. Brief explanation of the drawing

[0029] Figure 1 is cultured according to Example 1 Streptomyces massiliensis This is an image showing a colony of strain 213DD-128 (accession number KCTC15467BP). FIG. 2 is separated according to Example 1. Streptomyces massiliensis This is a schematic diagram showing the genetic location of strain 213DD-128 (accession number KCTC15467BP). FIG. 3 is according to Example 1 and Example 2 Streptomyces massiliensis This is a schematic diagram showing the process of isolating and purifying piericidin A from the culture medium of the 213DD-128 strain (accession number KCTC15467BP) in large quantities. Figure 4 shows the isolation and purification process of piericidin A according to Example 1 and Example 2. 1It was analyzed using the H NMR spectrum. Specific details for implementing the invention

[0030] The terms used in this specification will be briefly explained, and the invention will be described in detail.

[0031] The terms used in this invention have been selected based on currently widely used general terms while considering their functions within the invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall context of the invention.

[0032] When a part of a specification is described as “comprising” a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0033] Embodiments of the present invention are described below with reference to the attached drawings so that those skilled in the art can easily implement them. However, the present invention may be embodied in various different forms and is not limited to the embodiments described herein.

[0034] Specific details regarding the problem to be solved by the present invention, the means for solving the problem, and the effects of the invention are included in the embodiments and drawings described below. The advantages and features of the present invention, and the methods for achieving them, will become clear by referring to the embodiments described below in detail together with the accompanying drawings.

[0035] Hereinafter, the present invention will be described in more detail with reference to the attached drawings.

[0037] The present invention is a novel Streptomyces massiliensisStrain 213DD-128 (accession number KCTC15467BP) is provided.

[0038] The above strain may produce piericidin A.

[0039] The present invention Streptomyces massiliensis The present invention provides a method for mass-producing piericidin A, characterized by comprising the steps of: inoculating a strain 213DD-128 (accession number KCTC15467BP) into a culture medium for seed culture; and mass-culturing the seed-cultured strain in a fermenter.

[0040] The step of culturing the above species is preferably to perform a primary culture in 90 to 110 mL of liquid medium followed by a secondary culture in 600 to 800 mL of liquid medium, but it is more preferable to perform a primary culture in 100 mL of liquid medium followed by a secondary culture in 700 mL of liquid medium.

[0041] The above seed culture step is preferably carried out by shaking culture at a temperature of 27 to 29°C for 4 to 6 days, but is more preferably carried out by shaking culture at a temperature of 28°C for 5 days.

[0042] In the above mass cultivation step, a fermenter with a capacity of 100 L is used, but this is not limited to this.

[0043] The above mass culture step is preferably performed in a liquid medium of 60 to 80 L with added antifoaming agent, but it is more preferable to perform the culture in a liquid medium of 70 L with added 0.05% antifoaming agent.

[0044] It is preferable to culture with an air inflow of 15 to 25 L / min (LPM), a temperature of 27 to 29℃, and stirring at 40 to 60 rpm for 19 to 21 days, but it is more preferable to culture with an air inflow of 20 L / min (LPM) and stirring at 50 rpm for 20 days at a temperature of 28℃.

[0045] The liquid medium for the above seed culture step and mass culture step may be a Bennett (BN) liquid medium containing glucose, tryptone, yeast extract, beef extract, glycerol, and artificial sea salt, but is not limited thereto.

[0046] In addition, the present invention comprises the steps of: obtaining a crude extract by extracting a culture medium mass-cultured according to the above-described culture method with ethyl acetate and concentrating under reduced pressure; obtaining a fraction by eluting the crude extract with a solvent diluted stepwise by mixing methanol and water using vacuum column chromatography; concentrating the eluted fraction under reduced pressure to select a fraction containing a large amount of piericidin A; dissolving the selected fraction in a small amount of methanol and mixing it with water (H2O), and then n -Heptane( n -heptane), n -Hexane( n A step of primary purification by sequentially fractionating with solvents of -hexane), chloroform, and ethyl acetate; to the primary purified fraction n The present invention provides a method for isolating and purifying piericidin A, characterized by including the step of adding heptane and refractioning with a mixture of water and methanol for secondary purification.

[0047] In the step of obtaining the above crude extract, the extraction of ethyl acetate may be performed twice, but is not limited thereto.

[0048] The above vacuum column chromatography was performed on ODS-A gel (YMC Gel ODS-A, 12 nm, S 75 μ It can be filled with m).

[0049] The solvent used to stepwise dilute the above crude extract by mixing methanol (methyl alcohol) and water may be methanol diluted to concentrations of 20%, 40%, 60%, 80%, and 100%, but is not limited thereto.

[0050] The step of concentrating the eluted fraction under reduced pressure to select the fraction containing a large amount of piericidin A 1 Selection can be made by confirming the amount of piericidin A contained via 1H NMR, but is not limited to this.

[0051] In the second purification step above, it is preferable that the water and methanol mixture be diluted with a 90% methanol solution.

[0052] The yield of piericidin A eluted by the above separation and purification method may be 20 to 30%.

[0054] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0056] <실시예 1> 해에리시니 A(piericidin A)의 다이생산

[0057] 1-1. new Streptomyces massiliensis 213DD-128 균주(기학번호 KCTC15467BP

[0058] Streptomyces massiliensisStrain 213DD-128 (accession number KCTC15467BP) was isolated from sediment at a depth of 127 m using a grab sampler during an exploration of the East Sea of ​​Korea aboard the Onnuri in 2021. The collected sample was transferred to a tube, and a portion of the sediment from the tube was transferred to a sterile plate and heat-treated at 60°C for 30 minutes. The sample was then scattered onto 10% Bennett solid medium and cultured in a 28°C BOD incubator. After approximately 3 weeks, the formed beige colonies were acclimatized on Bennett solid medium, named “213DD-128,” and stored in a -70°C ultra-low temperature freezer in a 40% glycerol solution. Strain 213DD-128 initially appeared as beige colonies, gradually darkening to brown and forming gray spores (Fig. 1). As a result of analyzing the 16S rRNA base sequence, Streptomyces maciriensis ( Streptomyces massiliensis By confirming a 99% match with ) (Fig. 2), the final “Streptomyces maciriensis 213DD-128( Streptomyces massiliensis It was named “213DD-128” and deposited at the National Institute of Bioscience and Biotechnology’s Center for Biological Resources in June 2023, receiving the “Deposit Number KCTC15467BP”.

[0060] 1-2. 해에리시니 A(piericidin A)의 다이생소다 다이니니다배양

[0061] Separated according to Example 1 Streptomyces massiliensisTo obtain a large amount of piericidin A from strain 213DD-128 (accession number KCTC15467BP), mass culture was performed. The medium used for both seed culture and mass culture was Bennett (BN) liquid medium of the same composition (1% glucose, 0.2% tryptone, 0.1% yeast extract, 0.1% beef extract, 0.5% glycerol, 3.2% artificial sea salt). The strain grown on Bennett solid medium was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of sterile BN liquid medium and cultured in a shaking incubator at 28°C and 120 rpm for 5 days. 1% of the culture medium from the first seed culture was re-inoculated into a 2 L Erlenmeyer flask containing 700 mL of sterile BN liquid medium and cultured in a shaking incubator at 28°C and 120 rpm for 5 days. For mass culture, the second seed culture medium was aseptically inoculated into 70 L of liquid medium prepared by adding 0.05% antifoaming agent to a 100 L fermenter and cultured for 20 days under conditions of 28°C, 50 rpm, and an airflow rate of 20 L / min (LPM).

[0063] <실시예 2> Separation and purification of 해에리시니 A(piericidin A).

[0064] According to Example 1 Streptomyces massiliensis The culture medium of the large-scale culture of strain 213DD-128 (accession number KCTC15467BP) was extracted twice with an equal volume of ethyl acetate (EtOAc, 70 L), and then concentrated under reduced pressure to obtain approximately 63 g of crude extract. A portion of the crude extract, approximately 5.94 g, was taken and subjected to ODS-A gel (YMC Gel ODS-A, 12 nm, S 75 μ Vacuum column chromatography was performed by packing the m). The solvent was a mixture of methanol (methyl alcohol, MeOH) and water (H2O), and elution was performed in stepwise (20, 40, 60, 80, and 100% MeOH). Each fraction was collected in sets of three and concentrated under reduced pressure, 1¹H NMR confirmed the 100% MeOH-1 fraction (2.6299 g, 44.3%) containing a large amount of piericidin A. It was confirmed that this fraction contains a large amount of piericidin A and a lipid portion, along with small amounts of some impurities; therefore, solvent fractionation was performed to remove these minor impurities. The first 100% MeOH fraction was dissolved in a small amount of methanol (MeOH), and water (H2O) and n -Heptane( n -heptane), n -Hexane( n It was fractionated in the order of -hexane), chloroform, and ethyl acetate (EtOAc), yielding 12.8 mg, 1.251 g, 0.541 g, 762.1 mg, and 125.7 mg, respectively. Among these, n - heptane and n - The hexane layer contains only piericidin A and lipid portions 1 It was confirmed via H NMR. To remove some remaining lipid portions n Fractionation was performed once more using a mixture of heptane and 90% methanol, yielding 283.4 mg and 1.5 g, respectively (Fig. 3). 1 As a result of confirming with the H NMR spectrum, it was confirmed that the 90% methanol layer was pure piericidin A (yield 25%) (Fig. 4).

[0066] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents. The scope of the invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and their equivalents should be interpreted as being included within the scope of the invention.

[0067] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resources Center (KCTC) Trustee Number: KCTC15467BP Date of Deposit: 2023-06-13

Claims

Claim 1 A new accession number KCTC15467BP characterized by producing piericidin A Streptomyces massiliensis 213DD-128 strain. Claim 2 delete Claim 3 Trustee number KCTC15467BP Streptomyces massiliensis A method for mass-producing piericidin A, characterized by comprising: a seed culture step of inoculating a 213DD-128 strain into 90 to 110 mL of liquid medium and performing a first shaking culture at a temperature of 27 to 29°C for 4 to 6 days, and then inoculating the strain cultured in the first shaking culture into 600 to 800 mL of liquid medium and performing a second shaking culture at a temperature of 27 to 29°C for 4 to 6 days; and a step of inoculating the seed cultured strain into a fermenter containing 60 to 80 L of liquid medium to which an antifoaming agent has been added, and mass-culturing while stirring at 40 to 60 rpm for 19 to 21 days at an air inflow rate of 15 to 25 L / min (LPM) and a temperature of 27 to 29°C. Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 A step of obtaining a crude extract by extracting a culture medium mass-cultured by the culture method of claim 3 with ethyl acetate and concentrating under reduced pressure; a step of obtaining a fraction by eluting the crude extract using vacuum column chromatography with a solvent diluted stepwise by mixing methanol and water; a step of selecting a fraction containing a large amount of piericidin A by concentrating the eluted fraction under reduced pressure; and a step of dissolving the selected fraction in a small amount of methanol and mixing it with water (H2O), and then n -Heptane( n -heptane), n -Hexane( n A step of primary purification by sequentially fractionating with solvents of -hexane), chloroform, and ethyl acetate; to the primary purified fraction n A method for separating and purifying piericidin A characterized by including the step of adding heptane and refractioning with a mixture of water and methanol for secondary purification, and a method for separating and purifying piericidin A characterized by the yield of piericidin A eluted by the method of separation and purification being 20 to 30%. Claim 9 delete

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