WO2016013671A1

WO2016013671A1 - Algae for producing long-chain fatty acids, and method for producing long-chain fatty acids using same

Info

Publication number: WO2016013671A1
Application number: PCT/JP2015/071156
Authority: WO
Inventors: 河野　重行; 誠和山▲崎▼; 修平大田; 絵梨香鴻▲巣▼; 毅竹下; 知子阿部; 裕介風間
Original assignee: 国立大学法人東京大学; 国立研究開発法人理化学研究所
Priority date: 2014-07-25
Filing date: 2015-07-24
Publication date: 2016-01-28
Also published as: JPWO2016013671A1; JP6721807B2

Abstract

　A long-chain fatty acid raw material derived from organisms that accumulate large amounts of fatty acids and permit a stable supply was developed, and a method for producing specific long-chain fatty acids easily, inexpensively, and in large quantities using the same is provided. Provided is a cysteine-requiring variant of Chlorella algae that accumulates large amounts of long-chain fatty acids intracellularly when cultured in a nutrient stress medium containing cysteine or the like.

Description

Long chain fatty acid producing algae and method for producing long chain fatty acid using the same

The present invention relates to a Chlorellaceae alga capable of controlling the intracellular content of long-chain fatty acids by the medium composition and a method for producing long-chain fatty acids using the algae.

Long chain fatty acids obtained by hydrolysis of fats and oils derived from animals and plants are industrially important raw materials, and are used for various applications such as foods, chemical products such as cosmetics or dyes, or biofuels such as biodiesel. Has been.

Long-chain fatty acids generally have different chemical properties depending on the number of carbon atoms and the degree of saturation. For example, erucic acid, an unsaturated fatty acid having 22 carbon atoms, is used as a starting material for various high-value-added derivatives, although there are restrictions on food consumption due to the effects on the human body due to ingestion. It is a high long chain fatty acid (Non-Patent Documents 1 to 4). For example, in addition to being used as a surfactant and a lubricant, it is added to oil paints as a polymerization agent. In addition, it can be converted to behenic acid by hydrogenation, and it is also used as a raw material for food as a shortening. Furthermore, docosanol, a derivative of erucic acid, has been reported to suppress stomatitis caused by herpes virus (Non-Patent Documents 5 and 6) and has been approved by the US Food and Drug Administration (FDA). In addition, octylododecyl erucate is added to cosmetics and hairdressing products. Erucic acid is abundant in rapeseed seeds and is industrially refined from rapeseed oil. However, in recent years, canola seeds, which are low erucic acid-containing rapeseed varieties, occupy most of the rapeseed planting area, and the raw material supply of erucic acid is decreasing.

Therefore, there is an urgent need to develop a raw material for erucic acid to replace rapeseed. However, oils and fats derived from plant seeds usually contain a large amount of long-chain fatty acids other than erucic acid, and for industrial use, fatty acids other than erucic acid must be separated. Therefore, in order to produce erucic acid efficiently and in large quantities, plant species and varieties having a uniform fatty acid composition and a high yield of erucic acid are required at the raw material stage. However, the plant species and varieties having such properties are very limited, and it is difficult to obtain a yield equal to or higher than that of rapeseed with less labor and cost. Furthermore, development of new varieties with a large amount of erucic acid accumulation requires a great deal of time and effort. In addition, raw materials such as rapeseed have limited cultivatable environments and cultivated land. On the other hand, expansion of cultivated land is accompanied by problems such as an increase in cultivation management burden and environmental destruction.

In recent years, hydrocarbon-producing algae has attracted attention in order to solve the above problems. Hydrocarbon-producing algae are microalgae having the ability to biosynthesize long-chain aliphatic hydrocarbons in the process of assimilating nutrient sources. Many hydrocarbon-producing algae can be cultured in large quantities in a culture pond or the like without requiring special fertilization or temperature control. In addition, since the long-chain fatty acid produced is derived from atmospheric carbon dioxide immobilized by photosynthesis, it has the advantage that it can lead to reduction of greenhouse gases and prevention of global warming. Furthermore, since it does not compete with edible vegetable oils and fats, a stable raw material supply is possible.

However, hydrocarbon-producing algae generally have a problem that the amount of long-chain fatty acids accumulated in the cell is small and the yield is low. In addition, algae that accumulate a large amount of terpene hydrocarbons in cells, such as Botryococcus braunii, have a problem that it takes time to grow and the biomass production efficiency is low.

An object of the present invention is to develop and provide a biological material having a high content of a specific long chain fatty acid and high biomass production efficiency. Another object is to develop and provide a method for producing a specific long-chain fatty acid easily, stably and in large quantities using the raw material organism.

In order to solve the above-mentioned problems, the present inventors have focused on Chlorellaceae algae which are a group of hydrocarbon-producing algae. Chlorellaceae algae have high biomass production efficiency because they are easy to culture and have a fast growth rate. Furthermore, it has the property of accumulating neutral lipids when cultured under sulfur source deficient conditions (Mizuno, Y., et al., 2013, Bioresour. Technol., 129: 150-155. Doi: 10.1016 / j. biortech. 2012.11.030). Although the details of this accumulation mechanism have not been clarified, the present inventors assumed a relationship with the metabolism of sulfate ion (SO ₄ ²⁻ ). In other words, mutant strains that are unable to biosynthesize their metabolite cysteine (cysteine-requiring mutant strain) by losing the metabolic pathway of sulfate ions are always deficient in sulfur sources in normal growth media that do not contain cysteine. It was predicted that lipids would accumulate in the cells without becoming a sulfur source deficient condition. Therefore, we used Parachlorella kessleri to carry out mutagenesis treatment using heavy ion beam as a mutagen to create a cysteine-requiring mutant. As a result, Pk 3A7 strain (or Srp1 strain; hereinafter referred to as 3A7 strain) and Pk YY7 strain (or Srp2 strain; hereinafter referred to as YY7 strain) which are cysteine-requiring mutants are as follows: As expected, lipids accumulated in the cells under non-cysteine conditions. In particular, strain 3A7 was found to accumulate erucic acid in excess of 60% of the total lipid content, and YY7 strain accumulated oleic acid in excess of 60% of the total lipid content, and up to about 16% of nervonic acid. On the other hand, in the medium supplemented with cysteine, both strains grew in the same manner as the wild type and accumulated starch in the cells. Based on these results, the new knowledge that cysteine mutants of Chlorellaceae algae grow and accumulate starch in cysteine-added medium and constitutively accumulate long-chain fatty acids in cells in cysteine-free medium. Got. This means that cell growth and intracellular accumulation of long chain fatty acids can be easily switched by medium exchange. This invention is based on the said knowledge and provides the following.
(1) A cysteine-requiring mutant of the Chlorellaceae algae that increases the production of long-chain fatty acids in the absence of cysteine.
(2) The mutant strain according to (1), wherein the Chlorella family algae is a Chlorella algae or a Parachlorella algae.
(3) The mutant strain according to (2), wherein the Parachlorella algae is P. kessleri represented by international accession numbers FERM BP-22268 and FERM BP-22288.
(4) Mutagenesis process in which chlorellaceae algae are treated with mutagen, chlorellae algae after mutagenesis process are applied to a plate of cysteine-added medium, and colony formation process in which the cells are cultured until colonies are formed. Cultivated chlorellaceae from single colonies grown on cysteine-added medium plate and cysteine-free medium plate, respectively, grown on cysteine-added medium plate, and grown on cysteine-free medium plate Candidate strain selection step of selecting a non-strain as a candidate strain, a confirmation culture step of culturing the candidate strain again on a plate of cysteine-added medium and a plate of cysteine-free medium, and a candidate strain that does not grow on a plate of cysteine-free medium Select cysteine as a cysteine-requiring mutant Long chain fatty method producing productivity Chlorella family algae including sexual mutant selection process.
(5) The production method according to (4), wherein the mutagen is an alkylating agent or radiation.
(6) The production method according to (5), wherein the radiation is a heavy ion beam.
(7) In a pre-culture step and a pre-culture step of culturing the cysteine-requiring mutant strain according to any one of (1) to (3) from a logarithmic growth phase to a stationary phase at 15 to 35 ° C. in a cysteine-added medium. A method for producing a long chain fatty acid comprising a stress applying step of culturing the obtained cells in a nutrient stress applying medium for 10 to 30 days.
(8) The production method according to (7), further comprising an extraction step of extracting long-chain fatty acids from the cells after the stress application step by a physical method and / or a chemical method.
(9) The production method according to (7) or (8), wherein the nutrient stress imparting medium is a cysteine-free medium.
(10) The production method according to any one of (7) to (9), wherein erucic acid is produced using a mutant of P. kessleri represented by the international accession number FERM BP-22268 described in (3).
(11) The oleic acid and nervonic acid are produced using a mutant of P. kessleri represented by the international accession number FERM BP-22288 described in (3), according to any one of (7) to (9) Production method.

This specification includes the disclosure of Japanese Patent Application No. 2014-152376, which is the basis of the priority of the present application.

The cysteine-requiring mutant of the Chlorellaceae algae of the present invention can switch growth and intracellular accumulation of starch and intracellular accumulation of long-chain fatty acids depending on the presence or absence of cysteine in the medium. Utilizing this property, long-chain fatty acids can be efficiently produced using the mutant strain.

According to the method for producing long-chain fatty acids of the present invention, the growth and intracellular accumulation of long-chain fatty acids in cysteine-requiring mutant strains of Chlorellaceae algae are controlled by exchanging media with different compositions for cysteine. It is possible to easily and efficiently produce chain fatty acids with few steps.

The growth curves of P. kessleri wild strain (WT) and 3A7 strain in Cys medium (sulfur (S) low concentration cysteine (cys) -added medium) are shown. The culture conditions are light: dark = 12 hr: 12 hr, and light intensity (photon flux density) is 150 μmol · m ⁻² · s ⁻¹ . The growth curves of a wild strain (WT) of P. kessleri and a 3A7 strain in a TAP medium (a medium containing no S-containing cys) are shown. The culture conditions are light: dark = 12 hr: 12 hr, and light intensity is 150 μmol · m ⁻² · s ⁻¹ . The relationship between the culture time and the dry weight of cells when P. kessleri wild strain (WT) and 3A7 strain were cultured in TAP medium and STAP medium (medium without S-deficient cys) is shown. Total per cell when P. kessleri wild strain (WT) and 3A7 strain were cultured in TAP medium, STAP medium, TAP + cys medium (medium supplemented with S containing cys) and Cys medium (medium supplemented with S low concentration cys) for 10 days The amount of lipid is shown. The composition ratio of the lipid (long-chain fatty acid) accumulated in the cells of the wild strain (WT) of P.7kessleri and the 3A7 strain is shown. The main culture was performed for 8 days in the medium shown on the horizontal axis of the figure. Fatty acids were identified using gas chromatography mass spectrometry (GC-MS). The composition ratio of the lipid (long-chain fatty acid) accumulated in the cells of the wild strain (WT) of P.7kessleri and the 3A7 strain is shown. The main culture was carried out for 10 days in the medium shown on the horizontal axis of the figure. The fatty acid was identified using GC-MS. P. 培養 kessleri wild strains (a, b), 3A7 strains (c, d) and YY7 strains (e, f) were pre-cultured in Cys medium (medium supplemented with S low concentration cys), then transferred to TAP medium. The composition ratio (%) (a, c, e) of lipid (long chain fatty acid) accumulated in the cells when cultured and the amount of each lipid per cell are shown. The horizontal axis represents the number of days that have elapsed since the start of main culture.

1. 1. Chlorella family algae cysteine-requiring mutant 1-1. Outline | summary The 1st aspect of this invention is a cysteine requirement mutant of Chlorellaceae algae. The mutant strain of the present invention increases intracellular long-chain fatty acid accumulation by culturing in the absence of cysteine. Therefore, long-chain fatty acids can be produced easily and efficiently using the mutant strain of the present invention.
1-2. Structure The Chlorellaceae alga cysteine-requiring mutant of the present invention is composed of an auxotrophic mutant having an abnormality in the cysteine metabolism system of Chlorellaceae algae.

"Chlorellaceae algae" is a green algal plant belonging to the Trebouxiophyceae Chlorellales. They are immobile unicellular organisms (including colonies), and many species live floating in water. The Chlorellaceae algae include, for example, Chlorella algae, Parachlorella algae, and the like. Specific examples of Chlorella algae include C. pyrenoidosa (C. pyrenoidosa), C. sorokiniana (C. solokiniana), C. lobophora (C. robophora), C. vulgaris (C. bulgaris). Moreover, P. レ kessleri is mentioned as a specific example of Parachlorella algae.

Chlorellaceae are hydrocarbon-producing algae that can biosynthesize long-chain fatty acids in a pathway that assimilates starch biosynthesized by photosynthesis. It also has the property of accumulating long chain fatty acids in cells under sulfate ion (SO ₄ ²⁻ ) deficiency conditions.

Chlorellaceae algae preferred cysteine-requiring mutant of the present invention is not particularly limited as long as it is a species having the above properties, but is easy to culture, has a short doubling time, can be cultured in large quantities, and Species with high biomass production efficiency are preferred. A preferred example is P.lerkessleri.

“A cysteine-requiring mutant” is an auxotrophic mutant having an abnormality in the cysteine metabolic system as described above. A wild strain of Chlorellaceae, a photoautotrophic organism, biosynthesizes L-cysteine (2-Amino-3-mercaptopropanoic acid) (referred to as “cysteine” in this specification) via the sulfate ion metabolic pathway it can. The cysteine-requiring mutant strain of the present invention corresponds to a mutant strain lacking the ability to biosynthesize cysteine due to a defect in a metabolic pathway from sulfate ion to cysteine. Any mutant strain that cannot grow on a cysteine-free medium may be used, and it does not matter which of the metabolic pathways is abnormal.

In the present specification, the term “Chlorellaceae algal cysteine-requiring mutant” (herein often referred to as “cys mutant”) refers to a cysteine-requiring mutant of Chlorellaceae algae. The cys mutant strain is characterized in that it cannot grow on a medium not containing cysteine but can grow on a medium containing cysteine. Specific examples of the cys mutant include a cysteine-requiring mutant of P. kessleri, 3A7 strain indicated by international accession number FERM BP-22268, or YY7 strain indicated by international accession number FERM BP-22288. P. kessleri 3A7 stock with international deposit number FERM BP-22268 was issued on April 8, 2014 by the Patent Biological Depositary Center of the National Institute of Technology and Evaluation (2 Kazusa Kamashizu, Kisarazu City, Chiba Prefecture 292-0818, Japan) Stocks deposited domestically in Room -5-8 (120) were deposited at the center, an international depositary organization, on July 14, 2015. In addition, P. kessleri YY7 stock with international deposit number FERM BP-22288 was issued on July 14, 2015 by the International Depositary Agency, National Institute of Technology and Evaluation, Patent Biological Depositary Center (Chiba, Japan 292-0818, Japan). It is deposited in Kazusa-Kamazu 2-5-8 120, Kisarazu City.

“Long-chain fatty acid” (long-chain aliphatic hydrocarbon or higher fatty acid) usually means a fat-soluble fatty acid represented by the general formula R-COOH. In the present specification, the long-chain fatty acid means a fatty acid having 10 to 30 carbon atoms, in which R represents an alkyl group having 9 ≦ C ≦ 29 in the general formula. Preferably, it is a fatty acid having 20 to 30 carbon atoms and showing an alkyl group of 19 ≦ C ≦ 29. In this specification, the shape of the carbon chain of the long chain fatty acid is not limited. It may be linear or branched. In the present specification, the long-chain fatty acid includes both a saturated fatty acid having no double bond in the carbon chain and an unsaturated fatty acid having one or more double bonds. Specific examples of long-chain fatty acids that are the subject of the present invention include C10: 0 (10 carbon atoms; 0 double bonds, the same shall apply hereinafter) capric acid (decanoic acid) and C12: 0 (carbon) for saturated fatty acids. Number 12: Number of double bonds: 0, lauric acid (dodecanoic acid) of C14: 0, myristic acid (tetradecanoic acid) of C14: 0, pentadecylic acid (pentadecanoic acid) of C15: 0, palmitic acid of C16: 0 (hexadecane) Acid), C17: 0 heptadecanoic acid (margaric acid), C18: 0 stearic acid (octadecanoic acid), C20: 0 arachidic acid (icosanoic acid), C22: 0 behenic acid (docosanoic acid), C24: 0 Lignoceric acid (tetracosanoic acid), C26: 0 serotic acid (hexacosanoic acid), C28: 0 montanic acid (octaconic acid), and C30: 0 melicic acid (triacontanoic acid). For unsaturated fatty acids, C16: 1 palmitoleic acid (hexadecenoic acid), C18: 1 oleic acid (octadecenoic acid), C18: 2 linoleic acid (octadecadienoic acid), C18: 3 linolenic acid (Octadecatrienoic acid), C20: 1 gadoleic acid (icosaenoic acid), C20: 4 arachidonic acid (icosatetraenoic acid), C20: 5 eicosapentaenoic acid (EPA), C22: 1 erucic acid (docosaenoic acid) C22: 5 docosapentaenoic acid (DPA), C22: 6 docosahexaenoic acid (DHA), C24: 1 nervonic acid (tetracosaenoic acid), and the like.

The cys mutant of the present invention has the property of biosynthesizes the long-chain fatty acid and accumulates it in a large amount in the cell by culturing under nutrient stress conditions. As used herein, “nutrient stress” refers to sulfur stress or cysteine stress. Specifically, sulfur source deficiency stress, low concentration sulfur stress, low concentration cysteine stress, and cysteine deficiency stress are applicable. For example, in the case of the P. kessleri 3A7 strain, C22 is added as a long chain fatty acid by culturing in a cysteine-free medium that imparts cysteine-deficient stress, preferably a sulfur-containing cysteine-free medium such as a TAP medium described later. : 1 erucic acid can be accumulated at a content of 60% or more of the total lipid content in the cell. Further, in the case of the P. kessleri YY7 strain, by culturing in a cysteine-free medium that imparts cysteine-deficient stress, preferably a sulfur-containing cysteine-free medium such as a TAP medium described later, the total amount of intracellular lipids It is possible to accumulate C18: 1 oleic acid at a content of 60% or more, and C24: 1 nervonic acid at a maximum of about 16%.
1-3. Creation of Chlorellaceae Algae Cysteine-Required Mutant The Chlorellaceae algae cysteine-requiring mutant of the present invention may be isolated from nature, but may be isolated from nature. It can also be artificially produced by subjecting algae to a sudden induction treatment and then culturing in a medium with and without cysteine added.

When artificially producing a cysteine-requiring mutant strain, the type of Chlorellaceae that are suddenly induced is not particularly limited, but a species that is easy to culture, has a short doubling time, and has high biomass production efficiency is preferable. For example, a seed having an increased cell weight per day (g / L / day) of 0.1 g or more, preferably 1 g or more. Examples of such species include C. pyrenoidosa (2.90-3.64 g / L / day), C. sorokiniana (0.23-1.473g / L / day), P.Pkessleri (1.29 g / L / day), and the like. It is done. In addition, the higher the production amount of long-chain fatty acids, the more preferable. For example, a seed having a long-chain fatty acid production amount (g / L / day) in 1 L per day of 0.05 g or more, preferably 0.1 g or more. Examples of such species include C. vulgaris (0.3 g / L / day), P. kessleri (0.48 to 0.59 g / L / day), and the like. Therefore, P. kessleri is a particularly preferable species as the chlorellaceae for producing cysteine-requiring mutants of the present invention. It is preferable to use cells collected from the medium during the logarithmic growth phase as the chlorellaceae used for the sudden induction treatment. Chlorellaceae algae are cultured in accordance with the general culture method for freshwater chlorella (or freshwater microalgae) in principle (see: Preservation of Microorganisms, 1977, edited by Yoshio Sonei, Tokyo University Press). Just do it.

A known mutagen may be used as the mutagen for the sudden induction treatment. For example, a mutagen and radiation are mentioned.

Sudden induction by a mutagenic agent can be achieved by contacting a chlorellaceae algae with a mutagen agent solution for a predetermined time. As the mutagenic agent, an alkylating agent such as ethyl methanesulfonate (EMS) or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) can be used. The concentration and contact time of the mutagenic agent may be appropriately determined in consideration of the type of mutagenic agent, the type of Chlorellaceae algae, the survival rate, and the like. For specific methods, see the methods described in Green, GreenMR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Good.

Sudden induction by radiation can be achieved by irradiating Chlorellaceae algae with radiation for a predetermined time. Examples of radiation include alpha rays, beta rays, gamma rays, X rays, ultraviolet rays, and heavy ion beams (for example, ¹² C, ¹⁴ N, ²⁰ Ne, ⁴⁰ Ar, and ⁵⁶ Fe). Generally, radiation has a higher mutation rate as LET (Linear Energy Transfer) increases. LET refers to the energy (keV / mm) that is lost (given to the target) on average per unit length along the range when radiation passes through a substance. LET varies depending on the radiation quality. For example, the LET of gamma rays and X-rays is as low as 0.2 to 2 keV / mm, and the LET of heavy ion beams is as high as 23 to 4000. In order to obtain an appropriate mutation rate, a low LET radiation quality requires a larger amount of absorbed dose, but on the other hand, the higher the absorbed dose, the lower the survival rate. Therefore, sudden induction by radiation may be determined as appropriate in accordance with the radiation quality. In the case of gamma rays and X-rays, DNA single strand breaks and point mutations can be induced with a semi-lethal dose. In the case of heavy ion beams, suitable mutations can be induced in the dose range that has no effect on the fertility and survival rate, or at a very low dose range (Ota et al., 2013, Bioresour. Technol. 149: 432-438).

The cells after the sudden induction treatment are washed and then applied to a plate of cysteine-added medium, and under appropriate growth conditions (for example, the same conditions as described in the preculture step in the long-chain fatty acid production method of the second aspect described later) (Under conditions). Colonies may be formed to a size of 2 mm to 5 mm in diameter. Then, transfer the colonies to a plate of cysteine-free medium such as TAP medium using a cellulose filter or sterilized cloth to take a replica, or pick up from cells derived from the same colony with a spatula or platinum ear etc. and add cysteine And transplanted into the co-located compartment on the plate of non-supplemented medium. After culturing again under appropriate growth conditions, the strain grows on the cysteine-added plate to reconstitute colonies, but the strain that does not grow on the cysteine-free plate and cannot form colonies is selected as a candidate strain.

Candidate strains are taken from the grown plate of the stain-containing medium and re-implanted into the in-situ compartment on the plate with and without cysteine added medium. Thereafter, it is reconfirmed that it does not grow only on the cysteine-free medium plate. The Chlorellaceae algae obtained by the above method is used as the Chlorellaceae alga cysteine-requiring mutant of the present invention. If it is necessary to identify the long-chain fatty acids that cys mutants biosynthesize and accumulate, after culturing the cys mutants under nutrient stress conditions such as TAP medium, the intracellular long-chain fatty acids can be identified by known methods. Good. The method for identifying and quantifying long chain fatty acids is not particularly limited. For example, mass spectrometry or NMR can be used. Mass spectrometry includes high performance liquid chromatography mass spectrometry (LC-MS), high performance liquid chromatography tandem mass spectrometry (LC-MS / MS), gas chromatography mass spectrometry (GC-MS), and gas chromatography tandem mass spectrometry. Methods (GC-MS / MS), capillary electrophoresis mass spectrometry (CE-MS) and ICP mass spectrometry (ICP-MS). Any of these analysis methods are known in the art and may be performed according to these methods. For example, Iijima et al., The Plant Journal (2008) 54,949-962, Hirai et al. Proc Natl Acad Sci USA (2004) 101 (27) 10205-10210, Sato et al., The Plant Journal (2004) 40 ( 1) Refer to 151-163 or Shimizu et al., Proteomics (2005) 5,3919-3931. In the method for producing long-chain fatty acids of the second aspect, the long-chain fatty acids that each cys mutant strain biosynthesizes and accumulates in cells under nutrient stress conditions are preliminarily examined and converted into data. When a cys mutant having a uniform fatty acid composition and a high content of the desired long-chain fatty acid is required, it can be immediately selected from the group of cys mutants in the database. For example, the P. kessleri 3A7 strain can be used as a cys mutant having a high erucic acid content, and the P. kessleri YY7 strain can be used as a cys mutant having a high oleic acid or nervonic acid content.
1-4. Effect The cys mutant of the present invention is easy to culture, and biosynthesizes a large amount of long-chain fatty acids under nutrient stress conditions and accumulates in the cells. Therefore, by using the cys mutant of the present invention, it is possible to easily and inexpensively produce long chain fatty acids.

According to the method for producing a cys mutant of the present invention, a target cys mutant can be produced relatively easily without requiring a great deal of trial and error using the auxotrophy of cysteine as an index.
2. 2. Long chain fatty acid production method 2-1. Outline | summary The 2nd aspect of this invention is a long-chain-fatty-acid production method. The production method of the present invention produces long-chain fatty acids using the Chlorellaceae algal cysteine-requiring mutant described in the first aspect. According to the production method of the present invention, a long-chain fatty acid can be provided easily, inexpensively and stably.
2-2. Configuration of Production Method The long-chain fatty acid production method of the present invention includes a pre-culture step and a stress application step as essential steps, and an extraction step as a selection step. Hereinafter, each step will be specifically described.
(1) Pre-culture process The "pre-culture process" is an essential process in this production method in which the chlorellaceae algaline cysteine-requiring mutant strain described in the first aspect is cultured in a cysteine-added medium. The pre-culture process is aimed at the growth of cys mutants and the accumulation of intracellular starch. This is because cys mutants can hardly grow on the nutrient-stressed medium used in the next stressing step, and long-chain fatty acids are synthesized by converting starch accumulated in the cells, the amount of which is the amount of starch This is because they are in inverse proportion.

As used herein, “cysteine-added medium” refers to a growth medium for Chlorellaceae algae containing cysteine. Examples of such a growth medium include a cysteine medium in which cysteine is added to a low-concentration sulfur medium (in this specification, often referred to as “Cys medium”) and a medium in which cysteine is added to a basal growth medium.

Examples of the composition of the Cys medium include NH ₄ Cl 40 mg, CaCl ₂ · 2H ₂ O 5.1 mg, MgCl ₂ · 6H ₂ O 25.3 mg, K ₂ HPO ₄ 11.9 mg, KH ₂ PO ₄ 6.03 mg, Hutner's trace elements 0.1 mL , Acetic acid 0.1 mL, Tris (hydroxymethyl) aminomethane 242 mg, Distilled water 99.8 mL (pH 6.5-6.8) (where Hutner's trace elements: Na ₂ EDTA · 2H ₂ O 5g, ZnSO ₄ · 7H ₂ O 2.2g, H ₃ BO ₃ 1.14g, MnCl ₂・ 4H ₂ O 506mg, FeSO ₄・ 7H ₂ O 499mg, CoCl ₂・ 6H ₂ O 161mg, CuSO ₄・ 5H ₂ O 157mg, (NH ₄ ) ₆ Mo ₇ O ₂₄・ 4H ₂ O 110 mg, distilled water 100 mL), and cysteine added at a final concentration of 1 μg / mL. The composition of the basic growth medium is not particularly limited as long as it is a medium in which wild-type Chlorellaceae can grow. For example, TAP medium containing only sulfuric acid (S) as a sulfur source (NH ₄ Cl 40 mg, CaCl ₂ · 2H ₂ O 5.1 mg, MgSO ₄ · 7H ₂ O 10 mg, K ₂ HPO ₄ 11.9 mg, KH ₂ PO ₄ 6.03 mg , Hutner's trace elements (described above) 0.1 mL, Acetic acid 0.1 mL, Tris (hydroxymethyl) aminomethane 242 mg, Distilled water 99.8 mL) (pH 6.5 to 6.8). The amount of cysteine added may be 500 ng / mL to 100 μg / mL. Preferably, it is 1 μg / mL.

The culture temperature may be any temperature at which the cys mutant can grow. Usually, it is in the range of 15 to 35 ° C., preferably 18 to 30 ° C., more preferably 20 to 28 ° C., same as wild type Chlorellaceae.

Chlorellaceae algae undergo photosynthesis, so the culture is performed under exposure. The photon flux density at the time of exposure may be in the range of 5 to 150 μmol · m ⁻² · s ⁻¹ , preferably 25 to 100 μmol · m ⁻² · s ⁻¹ . The photon flux density is a value obtained by dividing the total number of photons contained in the surface irradiated with light for 1 second by the light receiving area. The exposure may be continuous irradiation or intermittent irradiation having a light and dark period. In the case of intermittent irradiation, the light-dark period is not particularly limited. For example, the light period is 8 hours: the dark period is 16 hours (referred to as “8hr: 16 hr”, the same shall apply hereinafter) to 16 hr: 8 hours. Good. The range of 10hr: 14hr to 14hr: 10hr is preferable. Usually, it may be performed at 12hr: 12hr. Light of 600 to 750 nm and light of 400 to 500 nm are effective for photosynthesis of Chlorellaceae algae. Accordingly, the light source is not particularly limited as long as it can emit these wavelength spectrum lights contributing to photosynthesis. In addition to sunlight, fluorescent lamps, mercury lamps, metal halide lights, and the like can be given. In addition, in the case of a light source that emits monochromatic light having a spectrum peak at a specific wavelength, such as an LED (Light Emitting Diode), a plurality of types of LEDs having a spectrum peak in the range of the wavelength spectrum may be combined. You may use as a light source of this process.

The culture period of this step is performed until the cys mutant inoculated in the cysteine-added medium reaches the logarithmic growth phase to the stationary phase. The logarithmic growth phase is preferably a late logarithmic growth phase in which a high cell growth rate is maintained and a large number of cells can be collected. The stationary phase is a period from the extremely slow increase in growth rate to 0, and the early stationary phase in which the appearance rate of dead cells and debilitated cells is relatively low is preferable. The specific culture period varies depending on the cys mutant, medium, culture temperature, etc. For example, in the case of P. kessleri 3A7 strain, it is 2-14 days under conditions of 18-25 ° C. in Cys medium. Preferably 6 to 12 days.

In this step, the medium may be agitated as necessary throughout the pre-culture period, or regularly or irregularly. Any stirring means may be used. For example, a stirring device such as a stirring rod may be used, or the culture medium may be stirred by reversing, rotating, or vibrating the culture tank.

Moreover, you may add a carbon dioxide to a culture medium as needed.
(2) Stress Application Process The “stress application process” is an essential process in the production method in which cells of the cys mutant obtained in the pre-culture process are cultured in a nutrient stress application medium. The purpose of this process is to biosynthesize long-chain fatty acids in the cells and then accumulate them by transferring cys mutants that have grown in the pre-culture process and accumulated starch in the cells under nutrient stress conditions. And

As described above, the nutrient stress corresponds to sulfur source deficiency stress, low-concentration cysteine stress, and cysteine deficiency stress. Sulfur source deficiency stress can be imparted by culturing cys mutants in the absence of a sulfur source (S) such as sulfate ion (SO ₄ ²⁻ ). Moreover, low concentration cysteine stress can be imparted by culturing the cys mutant in a medium having a cysteine concentration lower than that required for growth and / or proliferation of the cys mutant. Cysteine deficiency stress can be imparted by culturing the cys mutant in the absence of cysteine. In this step, preferable nutritional stress is low-concentration cysteine stress or cysteine deficiency stress, and more preferable nutritional stress is cysteine deficiency stress.

“Nutritional stress imparting medium” refers to a medium that imparts nutritional stress to the cys mutant. For example, if the nutrient stress is a sulfur source deficient stress, a sulfur source deficient medium (S deficient medium) is applicable. Specifically, for example, a ZnSO ₄ and FeSO ₄ of Hutner's trace elements in TAP medium each substituted with ZnCl ₂ and FeCl _2, STAP medium (Takeshita et al which completely removed the sulfate ions from the medium., 2014, Bioresour Technol. 158: 127-34.). Further, when the nutritional stress is a low concentration cysteine stress, a low concentration cysteine medium (low Cys medium) is applicable. Specifically, a Cys medium having a cysteine concentration of 0.001 to 0.01 μg / mL or less is applicable. If the nutrient stress is cysteine deficiency stress, a cysteine-free medium, for example, a TAP medium containing only the above-mentioned sulfate ion as a sulfur source is applicable.

In this step, after collecting the cells of the cys mutant from the culture solution after the pre-culture step, the cells may be planted in a nutrient stressed medium. Cell recovery can be achieved by centrifuging or filtering the culture solution by a conventional method to remove the medium. For example, a method of recovering the cell slurry after centrifuging the culture solution for an appropriate time with a centrifuge and a method of removing the culture solution using an appropriate filter having a pore size smaller than the cell size of the culture solution can be mentioned. The collected cells may be washed once or more with a buffer or physiological saline having an osmotic pressure comparable to that of water or a medium as necessary.

The culture conditions in this step may be performed according to the pre-culture step with respect to the culture temperature, light and dark period, and the like. The medium can be stirred and carbon dioxide added. However, the culture period is 10 to 30 days, preferably 5 to 15 days. In the case of the P. kessleri 3A7 strain and the YY7 strain, it is 14 to 28 days under conditions of 20 to 28 ° C. in a TAP medium. In the 3A7 strain, erucic acid is biosynthesized intracellularly by this process, and in the YY7 strain, oleic acid and nervonic acid are biosynthesized and accumulated in large quantities.
(3) Extraction process The "extraction process" is an extraction process for extracting long-chain fatty acids from the cells of the cys mutant strain after the stress application process. This step is a selection step and may be performed as necessary. The purpose of this step is to extract and recover long-chain fatty acids that are biosynthesized by cys mutants in the stressing step and accumulated in the cells.

In this step, cys mutant cells are first recovered from the culture solution after the stressing step. The recovery method may be the same as the method described in the stress applying step. Thereafter, if necessary, the cells may be washed once or more with water or a buffer or physiological saline having an osmotic pressure comparable to that of the medium.

The long-chain fatty acid extraction method used in this step can be any method known in the art for extracting hydrocarbons from hydrocarbon-producing algae such as Chlorellaceae algae. Chlorellaceae algae usually have a strong cell wall. Therefore, in order to efficiently extract long-chain fatty acids from cells, it is preferable to treat the recovered cys mutants by physical methods and / or chemical methods.

Examples of physical methods include an extraction method using a crushing method, an ultrasonic method, or a combination thereof.

The “crushing method” is a method in which a physical pressure is applied to cells to crush the cell wall and extract long-chain fatty acids in the cells. In the crushing method, for example, first, the collected cells are physically ground using a bead mill or a grind mill to obtain a crushed liquid. Specific conditions and methods may be referred to the mill instruction manual. Thereafter, the protein in the crushing solution is denatured and removed as necessary, and the crushing solution is centrifuged as it is or after adding an organic solvent or the like, and then an oil phase (organic solvent phase) containing a long-chain fatty acid is recovered. The organic solvent used may be a low-polar or non-polar water-immiscible medium, such as an aliphatic or alicyclic hydrocarbon having 6 to 10 carbon atoms such as n-hexane or n-heptane, or benzene. An aromatic hydrocarbon having 6 to 10 carbon atoms is preferable, and hexane or n-heptane is more preferable.

The “ultrasonic method” is a method in which cell walls are crushed by ultrasonic waves to extract long-chain fatty acids in the cells. In the ultrasonic method, for example, first, the collected cells are suspended in an appropriate solution such as water or a buffer, and then the cells are crushed at an appropriate oscillation frequency and output using an ultrasonic crusher (sonicator). Specific conditions and methods may be referred to the instruction manual for the ultrasonic crushing apparatus. Then, the protein in a crushing liquid is denatured and removed as needed, and the method of collect | recovering long chain fatty acids from the crushing solution similarly to the extraction method by the said crushing is mentioned.

Examples of the chemical method include an enzyme method.

The “enzymatic method” is a method in which a cell wall is lysed by an enzyme to extract intracellular long-chain fatty acids. In the enzyme method, for example, a cocktail of Cellulase R-10, Hemicellulase, Lysozyme, α-Mannosidase, and β-Mannosidase may be used. In the case of enzyme treatment, cells are first treated under the optimum conditions for the enzyme used. Thereafter, if necessary, the protein in the enzyme treatment solution is denatured and removed, and the long chain fatty acid is recovered from the remaining solution in the same manner as the extraction method by crushing.
(4) Separation step The method for recovering or purifying the long-chain fatty acid dissolved in the organic solvent may be any method known in the art and is not particularly limited. For example, a method of saponification with an esterification or alkali or the like and fractionating may be mentioned. Further, the invention disclosed in JP-A-8-9981 can be used.
2-3. Effect According to the method for producing a long-chain fatty acid of the present invention, it is possible to produce by simply changing the cell culture medium without requiring the vegetable oil / fat hydrolysis step performed by the conventional method. Since long-chain fatty acids can be efficiently produced with few steps, production costs can be reduced, and long-chain fatty acids can be provided at low cost.

In addition, since the amount of starch accumulated in cells and the amount of accumulated long-chain fatty acids are in a trade-off relationship, the amount of long-chain fatty acids accumulated in the cells can be adjusted by adjusting the culture period in the medium for accumulating each. Can be controlled.

Furthermore, since the method for producing long-chain fatty acids of the present invention uses chlorellaceae algae as a raw material, it is not affected by the planting amount or production amount of a plant for fats and oils, and does not cause any competition problems with edible vegetable oil. Can be secured stably.

<Example 1: Isolation of cysteine-requiring mutant of Chlorellaceae algae>
(the purpose)
Chlorellaceae algae are subjected to mutagenesis to isolate cysteine-requiring mutants.
(Method)
A. Cultivation of Chlorellaceae Algae P. kessleri is a freshwater unicellular green algae with a diameter of 5 to 10 μm, and belongs to a slightly larger class as Chlorellaceae. Wild strains grow easily on TAP medium and are extremely easy to culture. Moreover, since the oil content is relatively higher than other chlorellaceae and the growth rate is fast, the biomass production efficiency is also high. Therefore, a wild strain (WT) of P. kessleri was used as a Chlorellaceae for isolation of cysteine-requiring mutants. P. kessleri wild strain (N-2152 strain) obtained from National Institute for Environmental Studies (Tsukuba; Japan) is inoculated into 40 mL of TAP medium, and the light intensity is 150 μmol · m ⁻² · s ⁻¹ , 12hr: 12hr Light and dark periods were given, and static culture was performed at 23 ° C. for 1 to 2 weeks. This culture solution was seeded in 100 mL of TAP medium and cultured again for 5 days under the same conditions.
B. Mutagenesis treatment Divide 200 mL of the culture solution into 8 tubes (Rikaken) and irradiate the culture solution with RIBF (RI-Beam Factory) (RIKEN; Wako; Japan) to P. kessleri. Mutation induction treatment was performed. For the irradiation beam, ⁵⁶ Fe ions (LET = 645 keV / μm) were used. Strains after irradiation with heavy ions were applied to 500 Cys-1.5% agar media (Cys plates), and the cells were cultured at 23 ° C. for 14 days until a colony was formed at a light / dark period of 12 hr: 12 hr.
C. Selection of Cysteine-Required Mutant Strains Single colonies were scraped from the plate with a sterile toothpick and inoculated on agar plates (Cys plate and TAP plate, respectively) containing Cys medium and TAP medium. The two plates used at this time are sectioned by 192 squares, and addresses are given to the sections. A total of about 3,500 single colonies were inoculated (duplicate) in duplicate on the same address section of two agar plates. The light intensity was 50 μmol · m ⁻² · s ⁻¹ , a 12 hr: 12 hr light-dark period was given, and the cells were statically cultured at 23 ° C. for about 10 days.

Algal growth was confirmed visually, and a strain that grew on the Cys plate but not on the TAP plate was selected as a candidate strain for a cysteine-requiring mutant. Using a sterile toothpick from the corresponding address of the Cys plate, the candidate strain was inoculated into a 24-well titer plate (Iwaki) that had been preliminarily filled with Cys liquid medium, and the number of cells was increased by stationary culture for 2 weeks. Using the tip of a sterile toothpick handle, the culture solution was re-inoculated on a 24-well agar plate containing Cys medium and TAP medium. Again, a 12 hr: 12 hr light-dark period was given, and static culture was performed at 23 ° C. for 10 days. Thereafter, again, a strain that grew on the Cys plate but did not grow on the TAP plate was isolated as a cysteine-requiring mutant of the present invention.
(result)
As cysteine-requiring mutants of P. kessleri, 3A7 strain and YY7 strain were obtained. As mentioned above, P. kessleri 3A7 shares are under the international accession number FERM BP-22268, and YY7 stock is under the international accession number FERM BP-22288 on July 14, 2015. Deposited at the biological deposit center.
<Example 2: Verification of basic properties of P. kessleri cysteine-requiring mutant 3A7>
(the purpose)
The 3A7 strain, which is a cysteine-requiring mutant strain of P. kessleri obtained in Example 1, is examined for basic properties such as growth curve, cell dry weight and total lipid amount.
(Method)
A. Growth curve P. kessleri 3A7 strain is inoculated into 100mL of TAP medium and Cys medium respectively, light intensity is 150μmol · m ^-2 · s ^-1 , 12hr: 12hr light / dark period is given, and static culture at 23 ° C for 10 days did. Meanwhile, a part of the culture solution was collected every day, and the cell concentration was calculated using a particle size distribution analyzer (CDA-1000, Sysmex). As a positive control, a wild P. kessleri strain (WT: N-2152 strain) was used under the same conditions.
B. Dry weight and total lipids P. kessleri strain 3A7 is cultured in two types of media: TAP medium (medium without sulfur-containing cysteine) and STAP medium (medium without sulfur-deficient cysteine). The change in dry weight was examined. In addition, culturing in 4 types of media: TAP medium, STAP medium, TAP + cys medium (sulfur-containing cysteine-added medium) and Cys medium (low-concentration sulfur cysteine-added medium). Examined. The culture was performed at a light intensity of 150 μmol · m ⁻² · s ⁻¹ , a light / dark period of 12 hr: 12 hr, and statically cultured at 23 ° C. for 10 days. For verification of changes in dry weight, after 2 days, 4 days, 7 days and 10 days of culture, and for verification of changes in total lipid content, a portion of the culture solution after 3 days, 5 days, 7 days and 10 days of culture. Were collected.

The dry weight was measured as follows. 4 mL of the cell suspension was collected, and the supernatant was removed by centrifugation to obtain a cell pellet. The obtained cell pellet was resuspended in 1 mL of ethanol, placed in a pre-weighed aluminum petri dish, and dried at 105 ° C. for 3 hours. The difference in weight before and after drying was determined and defined as the dry weight. As a positive control, P. kessleri wild strain (WT: N-2152 strain) was used under the same conditions.

The total lipid amount was measured as follows. 10 mL of the cell suspension was collected, and the supernatant was removed by centrifugation to obtain a cell pellet. The obtained cell pellet was suspended in the order of 1.5 mL methanol and 5 mL MTBE, and the cells were disrupted by ultrasonic waves for 60 seconds. After crushing, the mixture was shaken at 150 rpm for 2 hours with a shaker (DOUBLE ： SHAKER NR-30: Taitec), and 1.5 mL of water was added to separate it into an aqueous layer and an oil layer. The upper oil layer was collected in a pre-weighed aluminum petri dish and dried overnight in a fume hood. The difference in weight before and after drying was determined as the dry weight. As a positive control, P. kessleri wild strain (WT: N-2152 strain) was used under the same conditions.

Further, after 10 days of culture, 1 mL of cell suspension was separated from each medium, and the supernatant was removed by centrifugation to obtain a cell pellet. 1 mM NileRed staining solution (in DMSO) was added to the obtained cell pellet, and the mixture was well stirred by vortexing for 4 minutes. The stained cells were observed using a fluorescence microscope (Olympus BX52). The total lipid amount per cell was calculated by dividing the measured total lipid amount by the number of cells. The method for measuring the total lipid amount was determined from the method described in the previous section, and the number of cells was determined from the same method as in the section “A. Growth curve”.
(result)
A. The results are shown in FIG. The 3A7 strain grew to the same extent as the wild strain on the Cys medium containing cysteine (FIG. 1A). On the other hand, in the TAP medium containing sulfur but not containing cysteine, the wild strain grew normally, but the 3A7 strain could hardly grow, confirming that it was a cysteine-requiring mutant (FIG. 1B).
B. The results of the culture time and dry weight of P. kessleri are shown in FIG. 2, and the amount of lipid accumulation per cell of P. kessleri cultured in each medium is shown in FIG.

2. From FIG. 2, it was revealed that the dry weight, that is, the biomass of the wild strain and the 3A7 strain was significantly reduced in the STAP medium under the stress without addition of sulfur-deficient cysteine. This result suggests that biomass declines because sulfur deficiency suppresses the growth of P. ikessleri.

FIG. 3 shows that in the case of the 3A7 strain, the amount of lipid accumulation per cell is remarkably increased when cultured in a cysteine-free medium such as TAP medium or STAP medium. In contrast, in the complete medium (TAP + cys medium and Cys medium) supplemented with cysteine, the amount of lipid accumulation per cell was low. On the other hand, in the case of the wild strain, the lipid accumulation amount per cell was high only when cultured in the STAP medium lacking sulfur.

A sulfur-deficient cysteine-free medium such as STAP medium is suitable in terms of the amount of lipid accumulation per cell, but the total lipid amount does not increase because the biomass is significantly reduced. Therefore, when the 3A7 strain of P. kessleri, a cysteine-requiring mutant of the present invention, is cultured in a cysteine-free medium such as TAP medium, the amount of lipid accumulation per cell and the amount of biomass and total lipid are high. It was found that the lipid production efficiency was the best because it could be maintained at the value.
<Example 3: Composition ratio of intracellular lipid in P. kessleri 3A7 strain>
(the purpose)
P. kessleri strain 3A7 identifies lipids that accumulate in cells and identifies changes in composition ratio due to medium.
(Method)
P. kessleri strain 3A7 was cultivated in 100 mL of Cys medium at a light intensity of 150 μmol · m ⁻² · s ⁻¹ , 12 hr: 12 hr, and incubated at 23 ° C. for 10 days (pre-culture step) . After culturing, the slurry was collected by centrifugation at 700 g for 10 minutes (LOW SPEED CENTRIFUGE LC-121: TOMY). After washing once with sterilized water, the slurry was transplanted to 100 mL of TAP medium, STAP medium, TAP + cys medium, and Cys medium, respectively, which were used as main culture. Again, the light intensity was 150 μmol · m ⁻² · s ⁻¹ , a 12 hr: 12 hr light / dark period was given, and the cells were left to stand at 23 ° C. for 10 days (stress application step).

On the 8th and 10th days of culture, 30 mL of each culture solution of the main culture was collected to identify fatty acids in the culture solution. GCMS-QP2010 Plus (Shimadzu Corporation) was used for fatty acid identification. The column used is SP-2380 (Sigma Aldrich), and qualitative analysis is performed with the standard sample Supelco 37 Component FAME Mix 10 mg / mL in methylene chloride (varied), analytical standard (Sigma Aldrich), which is built into the MS instrument. Qualitative analysis was also performed by MS using the library. The ratio of the fatty acid was determined by an area percentage method inspected from the obtained peak area.
(result)
FIG. 4 shows the composition ratio of intracellular lipid of P. kessleri strain 3A7 in each culture solution after 8 days of culture and FIG. 5 after 10 days of culture. On the 8th and 10th days, the 3A7 strain was found to accumulate the most C22: 1 erucic acid in the cells when the main culture was performed in the TAP medium. The amount reached 63% on the 10th day of culture. The wild type of P. kessleri contained about 40% erucic acid on the 10th day of culture in the STAP medium lacking the sulfur source, but contained only about 10% in the TAP medium. The sulfur source is contained in various forms in the medium, and it is not easy to control the content of erucic acid by the sulfur source-deficient medium. On the other hand, since the wild type of Chlorellaceae algae can biosynthesize cysteine, cysteine is not added to a normal growth medium. Therefore, the 3A7 strain can easily control cell growth and intracellular accumulation of erucic acid by adding or not adding cysteine to the normal growth medium.
<Example 4: Composition ratio of intracellular lipid in P. kessleri YY7 strain>
(the purpose)
We will identify the lipids that P. kessleri strain YY7 accumulates in the cells, and verify the difference in lipid composition ratio from wild-type and other mutants.
(Method)
The basic operation is in accordance with the third embodiment. First, P. kessleri wild strain, 3A7 strain, and YY7 strain were each 100 mL of Cys medium, light intensity was set to 150 μmol · m ⁻² · s ⁻¹ , and 12 hr: 12 hr of light and dark period was given, and 10 ° C. at 23 ° C. The culture was allowed to stand for 1 day (preculture process). After culturing, the slurry was collected by centrifugation at 700 g for 10 minutes (LOW SPEED CENTRIFUGE LC-121: TOMY). After washing once with sterilized water, the slurry of each strain was transplanted into 100 mL of TAP medium, which was used as main culture. Again, the light intensity was set to 150 μmol · m ⁻² · s ⁻¹ , a 12 hr: 12 hr light / dark period was given, and the cells were statically cultured at 23 ° C. for 10 days (stress application step).

On the 0th, 2nd, 4th, 7th, and 10th days of the main culture, 30 mL of the culture solution was collected, and fatty acids in the culture solution were identified. GCMS-QP2010 Plus (Shimadzu Corporation) was used for fatty acid identification. The column used is SP-2380 (Sigma Aldrich), and qualitative analysis is performed with the standard sample Supelco 37 Component FAME Mix 10 mg / mL in methylene chloride (varied), analytical standard (Sigma Aldrich), which is built into the MS instrument. Qualitative analysis was also performed by MS using the library. The ratio of the fatty acid was determined by an area percentage method inspected from the obtained peak area.
(result)
The results are shown in FIG. a (wild strain), c (3A7 strain), and e (YY7 strain) show changes in the intracellular lipid composition ratio over time after the start of the main culture. Further, b (wild strain), d (3A7 strain), and f (YY7 strain) indicate changes over time in the amount of each lipid accumulated in the cells.

In the YY7 strain, C18: 1 oleic acid and C24: 1 nervonic acid could be stably obtained during the main culture period. On the 7th day of the main culture, the amount of oleic acid accumulated was found to be about 65% of the total intracellular lipid amount, and the amount of nervonic acid accumulated was about 16%. The 3A7 strain and the YY7 strain are both P. skessleri, and both accumulate oleic acid in the cells in the main culture, but the 3A7 strain does not accumulate nervonic acid at all, and the YY7 strain does not contain erucic acid. It became clear that it did not accumulate at all. This result suggests that even if the cysteine-requiring mutant strains of the present invention are derived from the same species, different long-chain fatty acids can be produced depending on the strain species.

All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims

A cysteine-requiring mutant of the Chlorellaceae algae that increases the production of long-chain fatty acids in the absence of cysteine.
The mutant according to claim 1, wherein the Chlorellaceae is a Chlorella algae or a Parachlorella algae.
The mutant strain according to claim 2, wherein the parachlorella algae is P. kessleri represented by international accession numbers FERM BP-22268 and FERM BP-22288.
A mutagenesis step in which a chlorellaceae algae is treated with a mutagen;
A colony forming step of applying the chlorellaceae algae after the mutagenesis step to a plate of a cysteine-added medium and culturing until colony formation;
Cysteine stress culture step of culturing chlorellaceae derived from a single colony formed in a plate shape on a plate of cysteine-added medium and a plate of cysteine-free medium,
A candidate strain selection step of selecting a strain that grows on a plate of cysteine-added medium and does not grow on a plate of cysteine-free medium,
Confirmation culture step of culturing the candidate strain again on a cysteine-added medium plate and a cysteine-free medium plate, and selecting a candidate strain that does not grow on the cysteine-free medium plate as a cysteine-requiring mutant A method for producing a long-chain fatty acid-producing chlorellaceae including a strain selection step.
The production method according to claim 4, wherein the mutagen is an alkylating agent or radiation.
The production method according to claim 5, wherein the radiation is a heavy ion beam.
A pre-culturing step of culturing the cysteine-requiring mutant strain according to any one of claims 1 to 3 in a cysteine-added medium from a logarithmic growth phase to a stationary phase, and a cell obtained in the pre-culturing step comprising a nutrient-stressed medium A method for producing a long chain fatty acid comprising a stress applying step of culturing for 10 to 30 days.
The production method according to claim 7, further comprising an extraction step of extracting long-chain fatty acids from the cells after the stress application step by a physical method and / or a chemical method.
The production method according to claim 7 or 8, wherein the nutrient-stressed medium is a cysteine-free medium.
The production method according to any one of claims 7 to 9, wherein erucic acid is produced using a mutant of P. kessleri represented by the international accession number FERM BP-22268 according to claim 3.
The production method according to any one of claims 7 to 9, wherein oleic acid and nervonic acid are produced using a mutant of P. kessleri represented by the international accession number FERM BP-22288 according to claim 3.