Patent Application
20170202762
The present invention relates to a method for producing a mycosporine-like amino acid using a microorganism, a mycosporine-like amino acid produced by the method, and an UV-absorbing agent comprising the mycosporine-like amino acid.
It is known that ultraviolet rays (UV) induce acute skin reactions such as erythema, skin aging, and skin cancer. The ultraviolet rays contained in sunlight are classified, based on the wavelength, into three types: UV-A (320 nm-400 nm), UV-B (280 nm-320 nm) and UV-C (200-280 nm). Among them, UV rays having an effect on living bodies are UV-A and UV-B; whereas UV-C is harmless since it normally cannot pass through the atmosphere.
It is known that UV-B is a main cause of sunburn in the outdoors and has relatively larger energy than UV-A. When UV-B is absorbed by skin layer, it reaches the stratum corneum and the epidermis, and causes acute skin pigmentation such as spots and freckles. UV-B is also known to cause immunosuppression involved in aging and onset of skin cancer.
UV-A has a longer wavelength than UV-B and the energy is lower; however, UV-A penetrates into a further deeper portion of the skin than UV-B and is known to reach the dermis. As a result, not only acute skin pigmentation such as spots and freckles but also reduction in elasticity of the dermis (actinic elastosis) is caused, with the result that early skin aging such as wrinkles and sagging is induced. Furthermore, in recent years, it has been found that UV-A also causes immunosuppression and is involved in precancerous skin lesion and onset of skin cancer.
UV-B varies in amount depending upon e.g., season, weather and latitude; whereas the amount of UV-A reached the surface of the earth is constant throughout the year. Therefore, it is important to protect the skin also from UV-A.
UV protectors currently available in the market are divided into UV-absorbing agents and UV scattering agents. The UV-absorbing agents convert the UV energy into heat energy and release it, and include, for example, an organic compound such as 4-tert-butyl-4′-methoxydibenzoylmethane. The UV scattering agents contain an inorganic particle such as titanium oxide (TiO2) and zinc oxide (ZnO), and when the UV scattering agent is applied to the skin, the inorganic particles present on the skin reflect UV rays and serve as a UV barrier.
The UV-absorbing agents have problems: (1) the UV-absorbing agents are easily decomposed by light and have poor stability, (2) molecular excitation occurs and accelerates production of melanin to cause itchiness and allergy, and (3) a bad image is given to the users due to chemically synthesized substances. The UV scattering agents have the problems: (1) skin likely looks white when applied and the user tends to feel heavy in the skin, (2) generation of active oxygen is induced, and (3) pores of skin are closed and skin respiration may be inhibited. Because of these problems, development of a naturally derived safe UV-absorbing substance has been highly expected.
Mycosporine-like amino acids (hereinafter, referred to as MAAs) are natural UV-A absorbing substances, which are known to be present in a wide variety of aquatic organisms such as coral, red algae, fish viscera and microalgae. Among others, shinorine is known to be the most effective natural UV-A absorbing substance. Chemical synthesis of an MAA has been tried; however it requires long and complicated processes (Patent Document 1). Also, production of an MAA by light irradiation of cyanobacteria has been tried; however, the production amount of MAA is extremely low (Non-Patent Document 1). Further, extraction of MAAs from natural products such as laver, algae and shellfish has been tried; however, a sufficient yield has not been obtained in these cases (Patent Document 2 and Non-Patent Documents 2 to 4). Furthermore, extraction and production from natural products are easily influenced by climate conditions and mostly unstable, and thus, it is difficult to obtain MAAs stably in a large amount. In the meantime, microbial biosyntheses of MAAs have been tried; however, these processes require disruption of cells and extraction with an organic solvent in order to obtain biosynthesized MAAs from the microbial cells (Patent Document 3 and Non-Patent Documents 5 and 6). Thus, the operation becomes complicated and an operation for removing debris derived from microbial cells is further required.
Patent Document 1: WO02/39974
Patent Document 2: JP2013-518871A
Patent Document 3: JPI-6-62878A
Non-Patent Document 1: World J. Microbiol. Biotechnol. (2008) 24: 3111-3115
Non-Patent Document 2: Marine Biology 108, 157-166 (1991)
Non-Patent Document 3: Photomedicine and Photobiology (2002), 24, 39-42
Non-Patent Document 4: Tetrahedron : Letters (1979), 3181-3182
Non-Patent Document 5: FEMS Yeast Res. (2011), 11: 52-59
Non-Patent Document 6: J. Bacteriol. (2011), 193 (21) : 5923-5928
The problems to be solved by the present invention include providing a method of producing a naturally derived safe UV-absorbing substance stably and in a large amount.
The inventors have established a method of biosynthesizing an MAA by using a microorganism extracellularly producing the MAA, and obtaining the MAAs in a large amount from the extracellular culture solution. The inventors found that a naturally derived MAA can be more relatively easily and stably produced by this method than conventional methods, and have completed the present invention.
That is, the present invention provides:
(1) A method for producing a mycosporine-like amino acid, comprising the steps of:
culturing a microorganism extracellularly producing the mycosporine-like amino acid,
separating the microbial cell and extracellular culture solution, and
recovering the mycosporine-like amino acid from the extracellular culture solution;
(2) The method according to (1), further comprising a step of purifying the recovered mycosporine-like amino acid;
(3) The method according to (1) or (2), wherein the microorganism is a microorganism belonging to Escherichia coli, yeast, actinomycetes, microalgae or labyrinthulea;
(4) The method according to (3), wherein the microorganism is an actinomycete;
(5) The method according to (4), wherein the actinomycete belongs to the genus Streptomyces, the genus Actinosynnema, the genus Pseudonocardia or the genus Corynebacterium;
(6) The method according to (3), wherein the microorganism is labyrinthulea and belongs to the genus Aurantiochytrium;
(7) The method according to (3), wherein the microorganism is yeast and belongs to the genus Saccharomyces;
(8) The method according to any one of (1) to (7), wherein the microorganism comprises heterologous mycosporine-like amino acid biosynthetic enzyme genes;
(9) The method according to (8), wherein the mycosporine-like amino acid biosynthetic enzyme genes are composed of amir_4256, amir_4257, amir_4258 and amir_4259 genes derived from Actinosynnema mirum;
(10) The method according to (8), wherein a codon of at least one gene of the mycosporine-like amino acid biosynthetic enzyme genes is modified for a microorganism into which the gene is to be introduced;
(11) The method according to (9) or (10), wherein the microorganism is Streptomyces avermitilis MA-4680 (NITE accession number: NBRC 14893), Streptomyces lividans 1326 (NITE accession number: NBRC 15675), Corynebacterium glutamicum ATCC13032 (NITE accession number: NBRC 12168), Aurantiochytrium sp. SAM2179 (FERM BP-5601), Escherichia coli JM109 or Saccharomyces cerevisiae YPH499XW;
(12) A mycosporine-like amino acid represented by Formula 1:
(13) The mycosporine-like amino acid according to (12), produced by the method according to any one of (1) to (11);
(14) A UV-absorbing composition comprising a mycosporine-like amino acid produced by the method according to any one of (1) to (11) or the mycosporine-like amino acid according to (12) or (13) and a component acceptable for cosmetics, quasi-drugs or pharmaceuticals; and
(15) A composition for preventing at least one symptom or disease selected from the group consisting of acute skin reactions, skin aging and skin cancer, comprising a mycosporine-like amino acid produced by the method according to any one of (1) to (11) or the mycosporine-like amino acid according to (12) or (13) and a component acceptable for cosmetics, quasi-drugs or pharmaceuticals.
According to the present invention, it is possible to produce an MAA more easily than a complicated conventional chemical synthesis having many steps. It is also possible to obtain an MAR in a larger amount than conventional methods of obtaining MAAs from a natural product such as laver and shellfish. Thus, an MAA can be stably produced. In addition, since an MAA can be obtained from extracellular culture solution, a purification step is simplified compared to methods of obtaining MAAs by disruption of a microbial cell. As a result, a highly purified MAA can be quickly obtained in a high yield.
In one aspect, the present invention provides a method for producing an MAA comprising the steps of: culturing a microorganism extracellularly producing the MAA; separating the microbial cell and extracellular culture solution; and recovering the MAA from the extracellular culture solution.
According to the method of the present invention, an MAA is obtained from a culture supernatant of a microorganism. Thus, the MAA can be obtained in a larger amount than conventional methods using a natural product. In addition, the method of the present invention does not require a step of disrupting a microbial cell. Thus, the time and cost can be reduced compared to conventional methods of extracting MAAs by disrupting natural products such as algae. In a method comprising a disrupting step, contaminants resulting from the disruption are carried in subsequent processes and complicate the following purification processes. However, in the method of the present invention, such carry-in of contaminants can be prevented, with the result that the subsequent purification processes can. be simplified. As a result, the time and cost required for purification can be both reduced.
In the present invention, the term “mycosporine-like amino acid (hereinafter referred to as MAA)” is a general term of a compound having a cyclohexenone or cyclohexeneimine skeleton (optionally having a substituent) to which amino acid(s) is/are bound.
Examples of the MAA include, but are not limited to, shinorine (the following Formula 2), porphyra-334 (the following Formula 3), asterina-330 (the following Formula 4), palythene (the following Formula 5), palythine (the following Formula 6), mycosporine-glycine (the following Formula 7), mycosporine-glycine:valine (the following Formula 8) and mycosporine serinol (the following Formula 9).
As used herein, examples of the “microorganism (microbe)” include, but are not limited to, bacteria such as actinomycetes, Escherichia coli and Bacillus subtilis; fungi such as molds and yeast; microalgae such as cyanobacteria; and labyrinthulea.
The term “actinomycete” refers to a gram-positive bacterium belonging to the Actinobacteria. Examples of the “actinomycete” include, for example, the genus Streptomyces such as Streptomyces lividans, Streptomyces violaceoruber, Streptomyces coelicolor, Streptomyces avermitilis and Streptomyces griseus; the genus Actinosynnema such as Actinosynnema pretiosum and Actinosynnema mirum; the genus Pseudonocardia such as Pseudonocardia autotrcphica, Pseudonocardia thermophila; and the genus Corynebacterium such as Corynebacterium glutamicum. The actinomycetes can be separated from e.g., soil, or obtained from microorganism depositary institutions.
The “yeast” includes ascosporogenous yeast, basidiosporogenous yeast, and yeast belonging to the imperfect fungi. Examples of the yeast include, for example, the genus Saccharomyces such as Saccharomyces cerevisiae; the genus Schizosaccharomyces such as Schizosaccharomyces pombe; the genus Phaffia such as Phaffia rhodozyma; the genus Kluyveromyces such as Kluyvercmyces marxianus; the genus Yarrowia such as Yarrowia lipolytica; the genus Pichia such as Pichia stipitis; and the genus Candida such as Candida utilis. The yeast can be separated from e.g., plants, animals and soil, or obtained from microorganism depositary institutions.
The term “microalgae” refers to algae having microscopic structure except sea algae which is multicellular organism. The term “algae” refer to all the oxygenic photosynthetic organisms except moss plants, ferns and seed plants principally living on the ground. The algae include various unicellular organisms and multicellular organisms. For example, the algae include sea algae; Cyanobacteria belonging to prokaryotes; and Glaucophyta, Rhodophyta (red algae) and Chlorophyta belonging to eukaryotes. The microalgae also include a group body formed of a plurality of cells. The microalgae are not always living in water and those living in soil or on body surfaces of animals are included. Examples of the “Cyanobacteria” include, for example, Anabaena variabilis, Nostoc punctiforme, Nostoc linckia, Nostoc commune, Nostoc verrucosum and Nostoc muscorum. The Cyanobacteria can be separated from nature, or obtained from microorganism depositary institutions.
The “labyrinthulea” is an amoeboid eukaryote included in stramenopiles. Examples of the labyrinthulea include, for example, the genus Aurantiochytrium, the genus Schizochytrium, the genus Thraustochytrium and the genus Ulkenia. The labyrinthulea can be separated from nature such as seaweeds and land plants, or obtained from microorganism depositary institutions.
As used herein, the term “microorganism extracellularly producing a mycosporine-like amino acid (microbes that produce MAA on the outside of bacterial cells)” refers to a microorganism having ability to biosynthesize and extracellularly produce an MAA, for example, a microorganism having an MAA biosynthetic enzyme gene. Such a “microorganism extracellularly producing a MAA” may be a wild-type or an artificially mutated strain. Examples of an artificially mutation treatment include genetic recombination, UV irradiation, X-ray irradiation and treatment with a mutagenesis agent. Furthermore, the microorganism extracellularly producing a MAA may be a spontaneous mutant. The “microorganism extracellularly producing the MAA” includes a microorganism having a homologous or heterologous MAA biosynthetic enzyme gene. For example, a microorganism into which a heterologous MAA biosynthetic enzyme gene is introduced by genetic recombination may be used. For introducing a heterologous gene into a microorganism, a method widely known in the art may be used.
In the case where a homologous or heterologous MAA biosynthetic enzyme gene is introduced into a microorganism by genetic recombination, for example, a promoter, a 5′ untranslated region (UTR), a marker gene for transformant selection, a 3′ untranslated region (UTR), or a part thereof may be introduced together with the gene. In this case, a promoter etc. widely known to those skilled in the art as those used in the microorganism may be used. Furthermore, a codon of the gene may be appropriately modified depending upon the codon usage in the microorganism into which the MAA biosynthetic enzyme gene is be introduced. Codon usage in a microorganism can be checked by those skilled in the art, for example, by using Codon Usage Database (http://www.kazusa.or.jp/codon/) of Kazusa DNA Research Institute. Alternatively, codon usage can be checked by using the gene sequence design program, GeneOptimizer (R) provided by GENEART AG, etc. Further, based on the information of codon usage thus obtained, the codon of a subject gene can be optimized by a usual practice.
Examples of the mycosporine-like amino acid biosynthetic enzyme gene include, but are not, limited to, amir_4256 (SEQ ID NO: ami_4257 (SEQ ID NO: 2), amir_4258 (SEQ ID NO: 3) and amir_4259 (SEQ ID NO: 4) genes derived from Actinosynnema mirum DSM43827; pseP1_010100031425 (SEQ ID NO: 5), pseP1_010100031430 (SEQ ID NO: 6), pseP1_010100031435 (SEQ ID NO: 7) and pseP1_010100031440 (SEQ ID NO: 8) genes derived from Pseudonocardia sp. P1l; Ava3855, Ava3856, Ava3857 and Ava3858 genes derived from Anabaena variabilis ATCO29413 (see, Balskus EP et al., Science, (2010), 329: 1633-1656); and mysA, mysB, mysC and mysD genes derived from Nostoc punctiforme ATCC29133 (see, JOURNAL OF BACTERIOLOGY, November 2011, Vol. 193, No. 21, p. 5923-5928). For example, in an embodiment where a labyrinthulea is used, codon-optimized amir_4256 (SEQ ID NO: 9), codon-optimized amir_4257 (SEQ ID NO: 10), codon-optimized amir_4258 (SEQ ID NO: 11) and codon-optimized amir_4259 (SEQ ID NO: 12) genes are used as the mycosporine-like amino acid biosynthetic enzyme genes.
In an embodiment of the present invention, the microorganism used herein is a microorganism belonging to Escherichia coli, yeast, actinomycetes, microalgae or labyrinthulea. For example, the microorganism used herein is a microorganism belonging to Escherichia coil, yeast, actinomycetes, or labyrinthulea.
In an embodiment where an actinomycete is used, a microorganism belonging to the genus Streptomyces, the genus Actinosynnema, the genus Pseudonocardia or the genus Corynebacterium is used. In other embodiment, a microorganism belonging to the genus Streptomyces or the genus Corynebacterium is used. In another embodiment, Streptomyces lividans, Streptomyces avermitilis or Corynebacterium glutamicum is used. In yet another embodiment, an actinomycete comprising amir_4256, amir_4257, amir_4258 and amir_4259 genes derived from Actinosynnema mirum is used. For example, Streptomyces avermitilis MA-4680 (NITE accession number: NBRC 14893, deposited with the Patent Microorganisms Depositary (NPMD), National Institute of Technology and Evaluation (NITE), located at 2-5-8, Kazusakamatari, Kisarazu-shi, Chiba, Japan); Streptomyces lividans 1326 (NITE accession number: NBRC 15675, deposited with the Patent Microorganisms Depositary (NPMD), National Institute of Technology and Evaluation (NITE), located at 2-5-8, Kazusakamatari, Kisarazu-shi, Chiba, Japan); or Corynebacterium giutamicum ATCC13032 (NITE accession number: NBRC 12168, deposited with the Patent Microorganisms Depositary (NPMD), National Institute of Technology and Evaluation (MITE), located at 2-5-8, Kazusakamatari, Kisarazu-shi, Chiba, Japan), which comprises amir_4256 (SEQ ID NO: 1), amir_4257 (SEQ ID NO: 2), amir_4258 (SEQ ID NO: 3) and amir_4259 (SEQ ID NO: 4) genes derived from Actinosynnema mirum, is used.
In an embodiment where labyrinthulea is used, a microorganism belonging to the genus Aurantiochytrium, the genus Schizochytrium, the genus Thraustochytrium or the genus Ulkenia is used. In other embodiment, a microorganism belonging to the genus Aurantiochytrium is used. In another embodiment, labyrinthulea comprising amir_4256 (SEQ ID NO: 1), amir_4257 (SEQ ID NO: 2), amir_4258 (SEQ ID NO: 3) and amir_4259 (SEQ ID NO: 4) genes derived from Actinosynnema mirum is used. In yet another embodiment, labyrinthulea comprising codon-modified (for labyrinthulea) amir_4256, amir_4257, amir_4258 and amir_4259 genes is used. In other embodiment, Aurantiochytrium sp. SAM2179 (FERM BP-5601) comprising codon-modified (for labyrinthulea) amir_4256, amir_4257, amir_4258 and amir_4259 genes is used (note that, this strain was deposited as Ulkenia sp. SAM2179, and the classification of this strain was changed at a later date after completion of genome sequencing).
In an embodiment where yeast is used, a microorganism belonging to the genus Saccharomyces, the genus Schizosaccharomyces, the genus Phaffia, the genus Kluyveromyces, the genus Yarrowia, the genus Pichia or the genus Candida is used. In other embodiment, a microorganism belonging to the genus Saccharomyces is used. In another embodiment, yeast having a xylose-assimilating gene is used. In yet another embodiment, yeast compressing amir_4256 (SEQ ID NO: 1), amir_4257 (SEQ ID NO: 2), amir_4258 (SEQ ID NO: 3) and amir_4259 (SEQ ID NO: 4) genes derived from Actinosynnema mirum DSM43827 is used. In other embodiment, Saccharomyces cerevisiae YPH499XW having a xylose-assimilating gene and comprising amir_4256 (SEQ ID NO: 1), amir_4257 (SEQ ID NO: 2), amir_4258 (SEQ ID NO: 3) and amir_4259 (SEQ ID NO: 4) genes derived from Actinosynnema mirum DSM43827 is used.
In an embodiment where Escherichia coli is used, Escherichia coli comprising amir_4256 (SEQ ID NO: 1), amir_4257 (SEQ ID NO: amir_4258 (SEQ ID NO: 3) and amir_4259 (SEQ ID NO: 4) genes derived from Actinosynnema mirum DSM43827 is used. In another embodiment, Escherichia coli JM109 comprising amir_4256 (SEQ ID NO: 1), amir_4257 (SEQ ID NO: 2), amir_4258 (SEQ ID NO: 3) and amir_4259 (SEQ ID NO: 4) genes derived from Actinosynnema mirum DSM43827 is used.
As used herein, the term, “microbial cell” refers to a cell of a microorganism. In addition, as used herein, the term “extracellular culture solution (fluid)” refers to a part obtained by moving the microbial cell from the culture solution obtained by culturing a microorganism. That is, the extracellular culture solution comprises various components contained in the culture medium used in culture and substances produced by a microorganism during the culture.
A method for separating a microbial cell and extracellular culture solution is appropriately selected by those skilled in the art. For example, a microbial cell and extracellular culture solution may be separated by subjecting the culture solution obtained by culturing a microorganism to centrifugation. Centrifugation conditions such as temperature, time and speed vary depending upon the type of microorganism and the conditions well-known to those skilled in the art may be used. Alternatively, a microbial cell and extracellular culture solution may be separated by filtering the culture solution obtained by culturing a microorganism by use of an appropriate filtration membrane. Alternatively, microbial cells may be aggregated with the help of an appropriate aggregating agent and thereafter subjected to centrifugation filtration.
Recovering an MAA extracellular culture solution refers to obtaining a liquid mainly containing an MAA by removing various components contained in the culture medium used in culture that are contained in the extracellular culture solution and substances except the MAA produced by a microorganism during culture. A method for recovering an MAA from extracellular culture solution is also appropriately selected by those skilled in the art. For example, an MAA can be recovered from extracellular culture solution by filtration with a membrane or by using an appropriate medium. The medium is appropriately selected by those skilled in the art. In a preferable embodiment, an aqueous solvent is used. Examples of the aqueous solvent include, but are not limited to, acidic, neutral or alkaline aqueous solution or an aqueous solution containing a salt.
In other aspect, the present invention provides the method as mentioned above further comprising a step of purifying the recovered MAA. For purification of MAA, a method for purifying a metabolite from a culture of microorganism that is well-known to those skilled in the art may be used. For example, a purified MAA may be obtained by extraction with an organic solvent, a treatment with activated carbon, gel filtration, ion exchange column chromatography, high performance liquid chromatography (HPLC), crystallization and/or electrodialysis.
In the present invention, a culture medium may be neutralized, if necessary. As a neutralizer, those known in the art may be used, including for example, a carbonate such as calcium carbonate, magnesium carbonate, sodium carbonate and sodium hydrogen carbonate; a hydroxide such as sodium hydroxide, potassium hydroxide, calcium hydroxide and magnesium hydroxide; ammonia; quicklime, limestone and hydrated lime. In one embodiment, the neutralizer used in the present invention is a carbonate such as calcium carbonate, magnesium carbonate and sodium hydrogen carbonate. The neutralizer may be added to a culture medium before or during culture. The neutralizer may be added continuously or intermittently. The addition amount of neutralizer can be readily determined by measuring pH of the culture medium. The pH of the culture medium can be measured by a conventionally known method, for example, by a pH meter.
In the present invention, culture medium and other culture conditions (e.g., temperature, time, pH, stirring or not) for culturing a microorganism can be appropriately selected by those skilled in the art depending upon the type of the microorganism to be cultured.
In the case where an actinomycete is used, for example, a semi-synthetic medium for an actinomycete (6% glucose, 0.2% NaCl, 0.05% K2HPO4, 0.01% MgSO4.7H2O, 0.2% (NH4)2SO4, 0.2% yeast extract, 0.005% FeSO4.7H2O, 0.005% MnSO4.4H2O, 0.005% ZnSO4.7H2O, 0.5% CaCO3), TSB culture medium (0.25% glucose, 1.7% pancreatic digest of casein, 0.3% papaic digest of soybean, 0.5% NaCl, 0.25% K2HPO4), or SYN culture medium (0.7% casamino acid, 0.2% yeast extract, 0.264% (NH4)2SO4, 0. 38% KH2PO4, 0.556% K2HPO4, 0.1% MgSO4.7H2O, 0.0064% CuSO4.5H2O, 0.0011% FeSO4.7H2O, 0.0079% MnSO4.4H2O, 0.0015% ZnSO4.7H2O, 0.5% CaCO3) may be used. In the case where Escherichia coli is used, for example, LB culture medium, 2×YT culture medium, NZY culture medium, M9 culture medium, SOC culture medium, or YPD culture medium may be used. In the case where yeast is used, for example, SD culture medium, YPD culture medium or YPAD culture medium may be used.
The above culture medium may be appropriately modified in order to improve the culture state of a microorganism. For example, a culture medium may be modified by increasing the initial concentration of glucose in the culture medium or by adding Trace element solution (x200) (CuSO4.5H2O 64 mg, FeSO4.7H2O 11 mg, MnSO4.4H2O 79 mg, ZnSO4.7H2O 15 mg/50 mL).
In another aspect, the present invention provides an MAA produced by the aforementioned method of the present invention. The MAA produced by the present invention is not limited to MAAs whose structures are already determined but an MAA whose structure is newly determined may be included. In one embodiment, the former MAA may he shinorine, porphyra-334, palythine, mycosporine serinol or mycosporine glycine, or any combination thereof. Examples of the MAA having new structure include mycosporine-glycine-alanine (the following Formula 10). Accordingly, in one embodiment, the present invention provides mycosporine-glycine-alanine. In other embodiment, the present invention provides mycosporine-glycine-alanine produced by the aforementioned method of the present invention.
As a method for identifying an MAA, a conventionally known method may be used. For example, high-performance liquid chromatography-time-of-flight mass spectrometry (HPLC-TOFMS) may be used. Similarly, as a method for identifying a novel MAA, a conventionally known method may be used. For example, a novel MAA may be identified by using High Resolution Mass Spectrometry (HR-MS) and Nuclear Magnetic Resonance (NMR) in combination. Alternatively, an MAA may be identified by using HPLC based on the retention time and the results of a UV spectrum. Alternatively, a novel MAA may be identified by measuring a UV absorption spectrum and accurate mass by using HPLC apparatus equipped with a photodiode array detector and an HR-MS detector.
The present invention further provides a UV-absorbing composition comprising an effective amount of MAA produced by the method of the present invention or mycosporine-glycine-alanine as mentioned above as an active ingredient and another component acceptable for cosmetics, quasi-drugs or pharmaceuticals. The UV-absorbing composition of the present invention can be used not only in the fields of cosmetics and pharmaceuticals but also as a paint composition and another coating agent. For example, in the case where the UV-absorbing composition of the present invention is applied to human skin, the composition may comprise the MAA produced by the method of the present invention in an amount of about 0.05 to 10 wt %, an oil phase medium (about 5 to 40 wt %), an emulsifier (about 1 to 10 wt %), a small amount of an auxiliary agent and an aqueous phase medium such as water.
The present invention further provides a composition, for preventing at least one symptom or disease selected from the group consisting of acute skin reactions, skin aging and skin cancer, comprising an MAA produced by the method of the present invention or mycosporine-glycine-alanine as mentioned above and a component acceptable for cosmetics, quasi-drugs or pharmaceuticals. The composition comprises an effective amount of MAA produced by the method of the present invention as an active ingredient.
The composition of the present invention as mentioned above may be prepared in the form that ordinary cosmetics or pharmaceuticals for skin application may take, such as cream, lotion, paste, ointment, emulsion (oil-in-water emulsion, water-in-oil emulsion, multiple emulsion, microemulsion, PET-emulsion, Pickering emulsion), gel (hydrogel, alcohol gel), suspension, foam, spray, tablet or powder.
Examples of the component that can be comprised in the composition of the present invention as mentioned above and acceptable for cosmetics, quasi-drugs or pharmaceuticals include auxiliaries and additives of ordinary cosmetics, quasi-drugs or pharmaceuticals, for example, a preservative such as benzalkonium chloride, benzethonium chloride, hexamethonium chloride, butyl alcohol, benzyl alcohol, alkyl parabens such as methyl paraben or propyl paraben, catechol, resorcinol, cyclohexanol and m-cresol; an antioxidant such as ascorbic acid and methionine; a buffer such as phosphoric acid, citric acid and other organic acids; an emulsifier such as a sorbitan ester, Tween (R), silicon polyol, potassium stearate and an ethoxylated fatty acid ester; an emulsion stabilizer; anionic, cationic, nonionic or amphoteric polymer; a chelating agent such as EDTA; oil phase medium (a hydrocarbon-based oil such as a mineral oil, a paraffin wax, a natural oil, a silicone oil, a fatty acid ester such as isopropyl palmitate, a fatty alcohol such as stearyl alcohol); a thickener; a humectant; an emollient; a surfactant such as polyethylene glycol (PEG); an acidifying or basifying agent; a flavor; a fragrance; a dye; a coloring agent; or other components generally blended in cosmetics, quasi-drugs or pharmaceuticals.
The present invention will be described in detail and specifically by way of examples; however, they are provided for illustrative purposes only and should not be construed as limiting the present invention.
1. Production of MAA using Streptomyces
The present inventors produced MAAs using Streptomyces lividans and Streptomyces avermitilis. In addition, the present inventors compared the production amount of MAA with respect to the production in extracellular culture solution and in microbial cells (
1-1. Introduction of MAA Biosynthetic Enzyme Genes
1-1-1. Introduction of MAA Biosynthetic Enzyme Genes into Streptomyces lividans
As MAA biosynthetic enzyme genes, amir_4256 (SEQ ID NO: 1), amir_4257 (SEQ ID NO: 2), amir_4258 (SEQ ID NO: 3) and amir_4259 (SEQ ID NO: 4) genes derived from Actinosynnema mirum DSM43827 were used. These genes were ligated to a vector having pIJ101 ori under the control of PLD promoter (see, JP2002-51780A) to prepare a gene expression vector. Streptomyces lividans 1326 (NITE accession number: NBRC 15675) was transformed with the gene expression vector to obtain an MAA producing strain. Streptomyces lividans was transformed in accordance with a conventionally known method.
1-1-2. Introduction of MAA Biosynthetic Enzyme Genes into Streptomyces avermitilis
The aforementioned genes: amir_4256 (SEQ ID NO: 1), amir_4257 (SEQ ID NO: 2), amir_4258 (SEQ ID NO: 3) and amir_4259 (SEQ ID NO: 4), were introduced into Streptomyces avermitilis MA-4680 (NITE accession number: NBRC 14893) by homologous recombination obtain an MAA producing strain. The homologous recombination of Streptomyces avermitilis was carried out in accordance with a conventionally known method.
1-2. Pre-Culture of Streptomyces lividans and Streptomyces avermitilis
To 5 mL of AVM culture medium (see the following Table 1), a glycerol stock of a spore of Streptomyces lividans 1326 or Streptomyces avermitilis MA-4680 having the MAA biosynthetic enzyme genes, which was prepared in Section 1-1 above, was added. These actinomycetes were cultured at 28° C. and shaken at 160 rpm for 48 hours.
1-3. Main Culture of Streptomyces lividans and Streptomyces avermitilis
The pre-culture solution (an amount corresponding to 0.1%) was added to 50 mL of TSBt culture medium (see the following Tables 2 to 4) contained in a 500 mL baffled flask. Glucose was further added to the TSBt culture medium at the starting of the culture such that the initial concentration of glucose became 50 g/L. These strains were cultured at 28° C. and shaken at 160 rpm for two weeks.
1-4. Measurement of MAA
During the main culture, 1 mL of the culture solution was sampled at a predetermined time and the turbidity of the sampled culture solution was measured at 600 nm. The sampled culture solution was centrifuged at 14000 rpm for 20 minutes to separate extracellular culture solution and a precipitate (microbial cells). The separated extracellular culture solution was used as an extracellular culture solution-sample. Methanol (1 mL) was added to the separated microbial cells, and the microbial cells were disrupted by stirring and then subjected to centrifugation to recover the supernatant (microbial cell-sample). The production amounts of shinorine and porphyra-334 in each of the extracellular culture solution-samples and the diluted microbial cell-samples were measured by HPLC. The measurement conditions of HPLC are as shown in the following Table.
1-5. Results
The results of Streptomyces avermitilis MA-4680 are shown in
2. Production of Novel MAA (Mycosporine-Glycine-Alanine)
2-1. Streptomyces avermitilis producing an MAA was cultured in the same manner as in Example 1 for two weeks. The initial glucose concentration was set at 100 g/L. As a result, in the extracellular culture solution, mycosporine-glycine-alanine was produced as a novel MAA in addition to shinorine and porphyra-334. The production amount of mycosporine-glycine-alanine obtained was 25 mg/L. HPLC analysis was carried out in the conditions shown in Table 5, and as a result, the retention time was around 15 minutes.
2-2. Identification of Novel MAA (Mycosporine-Glycine-Alanine)
The novel MAA was identified as follows. Using an HPLC apparatus equipped with a photodiode array detector and an HR-MS detector, UV absorption spectrum and the accurate mass were measured. The measurement conditions of HPLC are as shown in the following Table.
The UV absorption spectrum of a peak eluted around minutes had an absorption maximum at around 333 nm and exhibited the same pattern as in shinorine and porphyra-334. The peak was ionized by ESI (fragmentor voltage of 200.0 V) and the accurate mass was measured by a TOF detector. The value of m/z ([M+H]) was 317.1338 (calculated value for C13H21N2O7+: 317.1349).
3. Production of MAA using Corynebacterium
The present inventors produced MAAs by using Corynebacterium glutamicum.
3-1. Introduction of MAA Biosynthetic Enzyme Genes
3-1-1. Introduction of MAA biosynthetic enzyme genes into Corynebacterium glutamicum
As MAA biosynthetic enzyme genes, amir_4256 (SEQ ID NO: 1), amir_4257 (SEQ ID NO: 2), amir_4258 (SEQ ID NO: 3) and amir_4259 (SEQ ID NO: 4) genes derived from Actinosynnema mirum DSM43827 were used. These genes were ligated to a vector having pBL1 ori, under the control of a gapA promoter (see, Appl Microbiol Biotechnol (2008) 81: 291-301) to obtain a gene expression vector. Corynebacterium glutamicum ATCC13032 (NITE accession number: NBRC 12168, deposited with International Patent Organism Depositary (IPOD), National Institute of Technology and Evaluation (NITE), located at 2-5-8, Kazusakamatari, Kisarazu-shi, Chiba) was transformed with the gene expression vector by electroporation to obtain an MAA producing strain. The electroporation was carried out in accordance with a conventionally known method.
3-2. Pre-Culture of Corynebacterium glutamicum
To 50 mL of BHI culture medium (see Table 7), a glycerol stock of bacterium glutamicum ATCC13032 having the MAA biosynthetic enzyme genes, which was prepared in Section 3-1 above, was added. The Corynebacterium glutamicum was cultured at 30° C. and shaken at 180 rpm for 24 hours.
3-3. Main Culture of Corynebacterium glutamicum
The pre-culture solution (an amount corresponding to 3%) was added to 50 mL of BHI culture medium contained in a Sakaguchi flask. At the starting of the culture, 5 mL of 400 g/L sodium gluconate was further added to the BHI culture medium such that the initial concentration of sodium gluconate became 20 g/L. To adjust pH, 5 mL of 10% calcium carbonate solution was added. The strain was cultured at 30° C. and shaken at 160 rpm for 48 hours.
3-4. Measurement of MAA
During the main culture, 1 mL of the culture solution was sampled at a predetermined time and the turbidity of the sampled culture solution was measured at 600 nm. The sampled culture solution was centrifuged at 15000 rpm for 10 minutes to separate extracellular culture solution and a precipitate (microbial cells). The separated extracellular culture solution was filtered by using a membrane filter having a pore diameter of 0.2 μm and the filtrate was used as an extracellular culture solution-sample. The production amounts of shinorine and porphyra-334 in the extracellular culture solution-sample were measured by HPLC. The Measurement conditions of HPLC are the same as shown in Table 5.
3-5. Results
The results are shown in
4. Production of MAA using Yeast
The present inventors produced MAAs by using Saccharomyces cerevisiae.
4-1. Introduction of MAA Biosynthetic Enzyme Genes
4-1-1. Construction of YPH499XW
Cassettes separately expressing XYL1 (xylose reductase derived from Scheffersomyces stipitis)(SEQ ID NO: 13) XYL2 (xylitol dehydrogenase derived from Scheffersomyces stipitis)(SEQ ID NO: 14), and XKS1 (xylulokinase derived from Saccharomyces cerevisiae) (SEQ ID NO: 15) each encoding a xylose-assimilating gene, under the control of TDH3 promoter were digested with BssHII from plasmid pWX1X2XK (see, Appl Environ Microbiol. 2004 September 70 (9): 5407-14). These cassettes were ligated to pRS404 vector (ATCC accession number: ATCC 87515) digested with BssHII and having TRP1 selection marker to prepare a genomic integration vector pIWX1X2XK. The genomic integration vector pIWX1X2XK was treated with EcoRV. Saccharomyces cerevisiae YPH499 (Genetics 1969 May; 122 (1): 19-27) was transformed with the obtained fragments to obtain YPH499XW having xylose-assimilating ability. Saccharomyces cerevisiae was transformed in accordance with a conventionally known method.
4-1-2. Introduction of MAA Biosynthetic Enzyme Genes into Saccharomyces cerevisiae YPH499XW
MAA biosynthetic enzyme genes, amir 4256 (SEQ ID NO: 1) and amir 4257 (SEQ ID NO: 2) were ligated to pAT426 vector (FEMS Yeast Res. 2014 May; 14 (3): 399-411) having 2μ ori and URA3 selection marker such that these genes were expressed under the control of TDH3 promoter and ADH1 promoter to prepare a gene expression vector pAT426-amir4256-7. Furthermore, MAA biosynthetic enzyme genes, amir4258 (SEQ ID NO: 3) and amir4259 (SEQ ID NO: 4) were ligated to pAT425 vector (FEMS Yeast Res. 2014 May; 14 (3) : 399-411) having 2μ ori under the control of TDH3 promoter and ADH1 promoter to obtain a gene expression vector pAT425-amir4258-9. YPH499XW was transformed with these gene expression vectors pAT426-amir4256-7 and pAT425-amir4258-9 to obtain an MAA producing strain, i.e., YPH499XWMAA. Saccharomyces cerevisiae was transformed in accordance with a conventionally known method.
4-2. Pre-Culture of YPH499XWMAA
A glycerol stock of YPH499XWMAA having MAA biosynthetic enzyme genes, which was prepared in section 5-1 above, was cultured on SD-LUW agar medium (see, the following Table 8). Thereafter, the obtained colony was inoculated onto 5 mL of SX-LUW liquid medium (see, the following Table 9). The strain was cultured at 30° C. and shaken at 150 rpm for 13 days.
4-3. Main Culture of YPH499XWMAA
The pre-culture solution (an amount corresponding to 2%) was added to 100 mL of SD-LU liquid medium contained in a 300 mL baffled flask. The strain was cultured at 30° C. and shaken at 150 rpm for 48 hours.
4-4. Measurement of MAA
After culture was continued for 13 days, 4 mL of the culture solution was sampled and the turbidity of the sampled culture solution was measured at 600 nm. The sampled culture solution was centrifuged at 3,000 rpm for 5 minutes to separate extracellular culture solution and a precipitate (microbial cells). The separated extracellular culture solution was passed through a PTFE filter (0.45 μm) and the resultant solution was used as an extracellular culture solution-sample.
4-5. Results
The MAA concentration of the extracellular culture solution was measured. As a result, shinorine was extracellularly produced in an amount of 0.19 mg/L.
5. Production of MAA using Labyrinthulea
The present inventors produced MAAs using labyrinthulea.
5-1. Introduction of MAA Biosynthetic Enzyme Genes 5-1. Introduction of MAA Biosynthetic Enzyme Genes into Labyrinthulea
For Aurantiochytrium (Aurantiochytrium sp.) SAM2179 (FERM BP-5601), the codons the above genes, amir_4256 (SEQ ID NO: 1), amir_4257 (SEQ ID NO: 2), amir_4258 (SEQ ID NO: 3) and amir_4259 (SEQ ID NO: 4) were modified (SEQ ID NOs: 9 to 12,respectively), These genes thus modified were introduced into Aurantiochytrium SAM2179 by homologous recombination to obtain an MAA producing strain. Homologous recombination of the labyrinthulea was carried out in accordance with a conventionally known method.
5-2. Pre-Culture of Aurantiochytrium
To GY seawater medium plate containing 1.5% agar (see, the following Table 10), a glycerol stock of Aurantiochytrium SAM2179 having the MAA biosynthetic enzyme genes, which was prepared in Section 5-1 above, was added. The Aurantiochytrium was cultured at 28° C. for 2 days.
5-3. Main Culture of Aurantiochytrium
Aurantiochytrium pre-cultured in Section 5-2 above was cultured in 10 mL of GY seawater medium (see, the following Table 11) at 28° C. and shaken at 300 rpm for 5 days.
5-4. Measurement of MAA
After completion of the main culture, the culture solution was centrifuged at 15000 rpm for 10 minutes to separate extracellular culture solution and a precipitate (microbial cells). The separated extracellular culture solution was passed through a membrane filter having a pore diameter of 0.2 μm and the resultant solution was used as an extracellular culture solution-sample. The production amount shinorine in the extracellular culture solution sample was measured by HPLC. The measurement conditions of HPLC are the same as shown in Table 5.
5-5. Results
Shinorine was extracellularly produced in an amount of 1.5 mg/L.
6. Production of MAA using Escherichia coli
The present inventors produced MAAs using Escherichia cell.
6-1. Introduction of MAA Biosynthetic Enzyme Genes
6-1-1. Introduction of MAA Biosynthetic Enzyme Genes into Escherichia coli
As MAA biosynthetic enzyme genes, amir_4256 (SEQ ID NO: 1), amir_4257 (SEQ ID NO: 2), amir_4258 (SEQ ID NO: 3) and amir_4259 (SEQ ID NO: 4) were used. These genes were ligated to a vector pkk223-3 (GenBank No. M77749) having pBR322 ori under the control of tac promoter to prepare a gene expression vector. Escherichia coli JM109 (product code: 9052, manufactured by Takara Bio Inc.) was transformed with this gene expression vector to obtain an MAA producing strain. Escherichia coil was transformed in accordance with a conventionally known method.
6-2. Pre-Culture of Escherichia coli
To 5 mL of LB culture medium (see, the following Table 12), a glycerol stock of Escherichia coli JM109 having the MAA biosynthetic enzyme genes, which was prepared in Section 6-1 above, was added. The Escherichia coli was cultured at 37° C. and shaken at 160 rpm for 18 hours.
6-3. Main Culture of Escherichia coli
The pre-culture solution (an amount corresponding to 2%) was inoculated to 50 mL of LB culture medium contained in a 500 mL, baffled flask. Sodium gluconate was further added to the LB culture medium at the starting of the culture such that the initial concentration of sodium gluconate became 50 g/L (a final concentration of 50 g/L), and calcium carbonate was further added such that its final concentration became 0.5%. The strain was cultured at 30° C. and shaken at 160 rpm a week.
6-4. Measurement of MAA
Twenty four hours after initiation of the main culture, 1 mL of the culture solution was sampled and centrifuged at 14000 rpm for 10 minutes to separate extracellular culture solution and a precipitate (microbial cells). The separated extracellular culture solution was used as an extracellular culture solution-sample. The production amount of shinorine in the extracellular culture solution-sample was measured by HPLC. The measurement conditions of HPLC are the same as shown in Table 5.
6-5. Results
The concentration of MAA in the extracellular culture solution was measured. As a result, shinorine was extracellularly produced in an amount of 0.82 mg/L.
From the foregoing, it was demonstrated that an MAA can be produced by using a microorganism and recovered from extracellular culture solution. In addition, it was demonstrated that a novel MAA, i.e., mycosporine-glycine-alanine, can be obtained. According to the method of the present invention, MAAs can be obtained from extracellular culture solution. This is extremely advantageous in relation to the subsequent purification process.
According to the method of the present invention, an MAA can be obtained stably in a large amount by use of a microorganism. The MAA obtained in this way can be used as an active ingredient of a UV-absorbing composition. Accordingly, the present invention can be used in the fields of e.g., cosmetics and pharmaceuticals.
SEQ ID NO: 12: Codon-optimized amir_4259 for SAM2179
| Number | Date | Country | Kind |
|---|---|---|---|
| 2014-099647 | May 2014 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2015/063682 | 5/12/2015 | WO | 00 |
