JP7412753B2 - Microorganisms with improved α-linolenic acid production ability or their cultures - Google Patents

Description

The present invention relates to microorganisms with improved α-linolenic acid production ability or cultures thereof.

A technique for producing α-linolenic acid by improving microorganisms of the genus Yarrowia using genetic recombination technology has been reported (Patent Document 1). Furthermore, a method for producing feed by treating food waste using Aspergillus microorganisms has been reported (Patent Document 2).

Patent No. 4916886 Patent No. 4671436

Djousse L et al, Hypertension. 45, 368-373, 2005. Kazuyoshi Yazawa Journal of the Society of Food Science and Technology, 43, 1, 1231-1237, 1996 Kirubakaran A et al. Poult. Sci. 90, 1, 147-156, 2011. Corino C et al. Meat Sci. 98, 4, 679-688, 2014.

In recent years, interest in environmental issues has increased internationally, and expectations are high for the realization of a resource recycling society. It is estimated that 28 million tons of food waste is generated annually in Japan, and there is a need to develop technology for its effective use. In particular, it is necessary to effectively utilize food waste containing starch, such as cooked rice. In addition, the health functionality of omega-3 fatty acids is attracting attention, and there is a demand for the production of foods containing a large amount of α-linolenic acid, one of them.

As a result of intensive studies to solve the above problems, the present inventors have found that by self-cloning, the ability to produce α-linolenic acid, which is originally inherent in Aspergillus microorganisms, can be safely used as food or feed. It was revealed that it can be improved. Self-cloning refers to the introduction of DNA from microorganisms of the same genus and species as the host, either directly or in combination, into the cells of the host. Microorganisms improved through self-cloning do not need to undergo safety screening regarding genetic recombination, so they can be quickly put to industrial use without incurring any costs. By culturing this self-cloning strain of Aspergillus microorganisms using food waste, it is possible to effectively utilize food waste and to produce microorganism cells or their culture containing a large amount of α-linolenic acid. . By using microbial cells or cultures thereof containing a large amount of α-linolenic acid as feed, it is possible to produce livestock or marine products containing a large amount of α-linolenic acid and having high health functionality. Furthermore, microbial cells or cultures thereof containing a large amount of α-linolenic acid can also be used directly as foods with high health functionality.

That is, the present invention includes the following.
[1] In a microorganism that belongs to filamentous fungi and contains carbohydrate metabolic pathway and fatty acid desaturase genes, at least one nucleic acid derived from a microorganism of the same genus and species as the host is introduced from outside the cell, and the introduced nucleic acid is A microorganism or a culture thereof having an improved ability to produce α-linolenic acid, which highly expresses at least one fatty acid desaturase gene.
[2] The microorganism of [1] or a culture thereof, wherein the microorganism is a food-safe or feed-safe microorganism.
[3] The microorganism of [1] or [2] or a culture thereof, wherein the microorganism is a microorganism belonging to the genus Aspergillus.
[4] The highly expressed fatty acid desaturase gene has a polynucleotide sequence that has 70% or more identity to the amino acid sequence of SEQ ID NO: 2 and encodes an amino acid sequence that has fatty acid desaturation activity. The microorganism or culture thereof according to any one of [1] to [3], including:
[5] The highly expressed fatty acid desaturase gene has a polynucleotide sequence that has 70% or more identity to the amino acid sequence of SEQ ID NO: 9 and encodes an amino acid sequence that has fatty acid desaturation activity. The microorganism or culture thereof according to any one of [1] to [3], including:
[6] Any one of the microorganisms [1] to [5], or its microorganism, in which the expression of a fatty acid desaturase gene derived from a microorganism of the same genus and species as the host is controlled by a promoter derived from a microorganism of the same genus and species as the host. Culture.
[7] The microorganism of [6] or a culture thereof, wherein the promoter is the TEF1 promoter.
[8] Food or feed containing the microorganism or culture thereof according to any one of [1] to [7].
[9] A composition for fermenting an edible substance, comprising the microorganism according to any one of [1] to [7] or a culture thereof.
[10] A method for producing food or feed containing a high content of α-linolenic acid, including the following:
(i) the step of contacting the edible substance with the microorganism or its culture according to any one of [1] to [7] or the composition of [9]; and (ii) the step of fermenting the edible substance.
[11] The method of [10], wherein the edible substance is food or its waste.
[12] The method of [10] or [11], wherein the edible substance contains carbohydrates.
[13] The method according to any one of [10] to [12], wherein the edible substance contains at least one of starch and lactose.
[14] The method of any one of [10] to [12], wherein the edible substance includes cooked rice.
[15] The method according to any one of [10] to [12], wherein the edible substance contains whey.
[16] Method for producing α-linolenic acid, including the following:
(i) the step of contacting the carbohydrate source with the microorganism or its culture according to any one of [1] to [7] or the composition of [9]; and (ii) the step of fermenting the carbohydrate source .
[17] The method of [16], wherein the carbohydrate source contains at least one of starch and lactose.
[18] A method for producing livestock or marine products, which comprises feeding and raising animals (excluding humans) with the feed of [8] to increase the content of α-linolenic acid.

According to the present invention, it is possible to produce microbial cells or cultures thereof that contain a high content of α-linolenic acid and can be safely used as food or feed. Microbial cells containing high α-linolenic acid can be produced from starch or food waste containing starch and used as food or feed.

FIG. 1 is a list of nucleotide sequences that have been found to be homologous to the nucleotide sequence of the fatty acid desaturase gene of SEQ ID NO: 1, and are listed in descending order of homology from the top. Homology is indicated in the "Query Cover" and "Per. Ident" columns. FIG. 2 is a list of amino acid sequences that have been found to be homologous to the amino acid sequence of the fatty acid desaturase gene of SEQ ID NO: 2, and are listed in descending order of homology from the top. Homology is indicated in the "Query Cover" and "Per. Ident" columns. FIG. 3 is a gas chromatograph analyzing the fatty acid composition of the bacterial cells of the Aspergillus oryzae RIB40 strain and the fatty acid desaturase gene self-cloning strain. FIG. 4 is a list of amino acid sequences that were found to be homologous to the amino acid sequence of fatty acid desaturase of SEQ ID NO: 9, and are listed in descending order of homology from the top. Homology is indicated in the "Query Cover" and "Per. Ident" columns. FIG. 5 is a gas chromatograph analyzing the fatty acid composition of the bacterial cells of the Aspergillus kawachi strain and its fatty acid desaturase gene-introduced strain.

The present invention will be explained in detail below.

The present invention relates to a microorganism belonging to filamentous fungi or a culture thereof, which has an improved ability to produce α-linolenic acid. By culturing such microorganisms, microbial cells with a high content of α-linolenic acid can be produced. More specifically, the microorganism of the present invention or a culture thereof belongs to a filamentous fungus and contains a carbohydrate metabolic pathway and a fatty acid desaturase gene, into which at least one nucleic acid has been introduced from outside the cell, A microorganism or a culture thereof in which the expression level of a fatty acid desaturase gene is increased by at least one of the introduced nucleic acids compared to before introduction (wild strain). The introduced nucleic acid is not particularly limited, but includes, for example, a fatty acid desaturase gene derived from a microorganism of the same genus and species as the host (e.g., a fatty acid desaturase gene present in the genome of the microorganism), a promoter, and a transcription factor. , and nucleic acids derived from microorganisms of the same genus and species as other hosts involved in the production, accumulation, and secretion of α-linolenic acid by microorganisms. The nucleic acid derived from a microorganism of the same genus and species that is introduced into a microorganism may be a nucleic acid extracted directly from the microorganism, or a nucleic acid obtained by causing another microorganism (e.g., Escherichia coli) to produce a nucleic acid derived from a microorganism of the same genus and species. Nucleic acids may be artificially synthesized by methods known in the field of genetic engineering.

In the present invention, the microorganism used to produce α-linolenic acid is a microorganism belonging to filamentous fungi. In the present invention, the microorganism used to produce α-linolenic acid is preferably a food-safe or feed-safe microorganism. Examples of microorganisms that are food safe or feed safe include microorganisms that have been used in food production or feed. Among such microorganisms, microorganisms of the genus Aspergillus include, more specifically, Aspergillus oryzae, Aspergillus niger, Aspergillus luchuensis, and Aspergillus ryukyuensis. Aspergillus luchuensis var kawachii, Aspergillus kawachii, Aspergillus sojae, Aspergillus tamari tamari), Aspergillus awamori, Aspergillus glaucus Can be mentioned. Among these, Aspergillus oryzae (eg, Aspergillus oryzae RIB40 strain) and Aspergillus kawachi (eg, Aspergillus kawachi NBRC4308 strain) are preferred.

Microorganisms belonging to filamentous fungi, especially microorganisms of the genus Aspergillus, are generally not microorganisms that have a high ability to produce α-linolenic acid, so even if fatty acid desaturase genes are present, their expression levels are generally low. low. Therefore, the combination of nucleic acids to be introduced preferably includes a fatty acid desaturase gene derived from a microorganism of the same genus and species as the host, and a promoter that increases the expression of such a gene. This greatly increases the α-linolenic acid production ability that microorganisms originally have. As the promoter, it is preferable to use a promoter derived from a microorganism of the same genus and species as the host. As a result, the modified microorganisms do not need to undergo safety screening regarding genetic recombination, and can be used for food or feed purposes. Modification of a microorganism using a genomic region (eg, promoter, coding region, 3' untranslated region) derived from a microorganism of the same genus and species is sometimes referred to as "self-cloning."

Self-cloning in the present invention refers to the characteristics of a host (in recombinant DNA technology, a living cell into which DNA is transferred; the same applies hereinafter) using only the DNA of a microorganism that belongs to the same taxonomic species as the host. It is about changing. Safety reviews regarding the use of genetically modified organisms as food and feed, and their release into the environment require careful preparation of large amounts of experimental data, and the lengthy review process requires enormous costs. is necessary. However, laws and regulations [(1) Procedures for safety review of recombinant DNA technology applied foods and additives (excerpt) (Ministry of Health and Welfare Notification No. 233 of 2000) https://www. mhlw. go. jp/file/06-Seisakujouhou-11130500-Shokuhinanzenbu/1_11. pdf, (2) Ministerial Ordinance on Ingredient Standards for Feed and Feed Additives http://www. famic. go. jp/ffis/feed/hourei/sub1_seibunkikaku. html, (3) Enforcement Regulations of the Act on Securing Biological Diversity through Regulations on the Use of Genetically Modified Organisms (2003 Ministry of Finance, Ministry of Education, Culture, Sports, Science and Technology, Ministry of Health, Labor and Welfare, Ministry of Agriculture, Forestry and Fisheries, Ministry of Economy, Trade and Industry, Ministry of the Environment Ordinance) No. 1) http://www. env. go. jp/press/files/jp/108458. As shown in [PDF], microorganisms improved through self-cloning can be used industrially without undergoing a safety review for genetically modified organisms, and there is no need to incur the cost of a safety review. I don't. Furthermore, when genetically modified microorganisms are used industrially, it is necessary to carefully sterilize them after culturing, which requires a great deal of cost. On the other hand, microorganisms improved through self-cloning can be treated as non-genetically modified microorganisms, so they do not require the cost of sterilization.

Self-cloning includes, for example, replacing or inserting the promoter region of the fatty acid desaturase gene on the microbial genome with a promoter derived from a microorganism of the same genus and species as the host, or inserting a promoter region downstream of the promoter on the microbial genome into the host. Replacement or insertion of a fatty acid desaturase gene derived from a microorganism of the same genus and species as the host, or a gene expression cassette that combines a promoter derived from a microorganism of the same genus and species as the host and a fatty acid desaturase gene derived from a microorganism of the same genus and species as the host. (optionally to which a selectable marker gene may be attached) is introduced into microorganisms. Examples of self-cloning methods include homologous recombination, genome editing (eg, CRISPR-Cas9, TALEN), and vector introduction.

Examples of highly expressed fatty acid desaturase genes include genes encoding ω-3 desaturases such as FAD3 and D15D. Examples of the fatty acid desaturase gene include a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 2, or a polynucleotide sequence that has 70% or more identity to the amino acid sequence of SEQ ID NO: 2 and that encodes the amino acid sequence of SEQ ID NO: 2. Examples include genes that include polynucleotide sequences that encode amino acid sequences that have activity. Furthermore, a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9, or a polynucleotide sequence encoding an amino acid sequence having 70% or more identity to the amino acid sequence of SEQ ID NO: 9 and having fatty acid desaturation activity. Examples include genes containing nucleotide sequences. Such identity is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more. % or more, 97% or more, 98% or more, 99% or more, and 99.5% or more. The fatty acid desaturase gene may be a gene encoding a fatty acid desaturase (eg, FAD3 protein) corresponding to the species of microorganism used. Examples of fatty acid desaturation activity include ω-3 desaturase activity, and specifically, for example, activity to convert linoleic acid (C18:2) to α-linolenic acid (C18:3).

In introducing the fatty acid desaturase gene, modifications may be made for more efficient production of α-linolenic acid using known genetic engineering techniques. For example, a high production amount has been obtained by introducing multiple copies when Yarrowia yeast produces eicosapentaenoic acid (Xue Z et al, Nature Biotechnology, 31, 8, 734-740, 2013).

The promoter may be selected depending on the purpose; for example, a high expression promoter, that is, a promoter with a large expression amount can be selected. By using a high expression promoter, the expression level of the fatty acid desaturase gene can be improved. Other examples of promoters include promoters that are specifically expressed under specific conditions. This makes it possible to strictly control the timing and conditions for producing α-linolenic acid. Specific examples of promoters include, but are not limited to, the following: TEF1 promoter; α-amylase gene and glucoamylase gene promoters (Yoji Hata et al., Jokyo, 93, 12, 922-931 , 1998, Toshiki Minetoki, Chemistry and Biology, 38, 12, 831-838, 2000); promoters of superoxide desmutase gene, cytochrome P-450 gene, catalase gene, ATPase gene, or histone gene (patent no. 3792467); an artificial promoter that overlaps the base sequence called region III that Aspergillus oryzae originally has (Toshiki Minetoki, Chemistry and Biology, 38, 12, 831-838, 2000).

Improvements by self-cloning in the present invention can be achieved, for example, by providing in cells a gene expression cassette that combines a promoter derived from a microorganism of the same genus and species as the host and a fatty acid desaturase gene. For example, in the case of Aspergillus, the TEF1 promoter may be used as the expression promoter. In order to select strains in which the expression cassette has been introduced into the cells, a marker gene derived from an organism of the same genus and species as the host is connected to a high-expression promoter and the expression cassette for the fatty acid desaturase gene, and then introduced into the host. It's okay. For example, a pyrithiamine resistance gene or orotidine 5'-phosphate decarboxylase gene (URA3 gene) may be used as the marker gene. In addition, when using a marker gene derived from a species other than Aspergillus, it should be introduced into the host together with a high expression promoter and a fatty acid desaturase gene expression cassette, and then the marker gene introduced by homologous recombination or Cre recombinase should be inserted into the genome. It may be removed from

In the present invention, the α-linolenic acid producing ability of microorganisms may be further improved by known genetic engineering techniques such as introducing a nucleic acid other than the aliphatic desaturase gene as described below.
It is known that lipid production can be increased by increasing the expression level of genes on the biosynthetic pathway from carbohydrates to fatty acids. In the case of Yarrowia yeast, it has been reported that lipid production increases by increasing the expression levels of the acetyl-CoA acyltransferase gene and the diacylglycerol acyltransferase gene (Tai M et al, Metabolic Engineering, 15, 1-9, 2013). In the case of Aspergillus bacteria, it has been reported that lipid biosynthesis is increased by increasing the expression levels of acetyl-CoA acyltransferase gene, palmitoyl ACP thioesterase, acetyl-CoA citrate lyase, and fatty acid synthase genes (Tamano K et al, Applied Microbiology and Biotechnology, 97, 269-281, 2013). By combining enhancement of expression of these genes, the production amount of α-linolenic acid can be further increased.

Fatty acid biosynthesis requires large amounts of NADPH. Therefore, it has been reported that lipid production can be increased by increasing the expression level of genes in the pentose phosphate cycle that supply NADPH (Tamano K et al, Bioscience Biotechnology and Biochemistry, 80, 9, 1829-1835 , 2016).

Generally, palmitic acid is synthesized by fatty acid synthase, and then converted to stearic acid by chain elongation enzyme. Stearic acid undergoes a reaction by Δ9 desaturase and is converted by oleic acid. Oleic acid undergoes a reaction by Δ12 desaturase and is converted to linoleic acid. Linoleic acid undergoes a reaction by Δ15 (or omega-3) desaturase and is converted to α-linolenic acid. For example, in Xue Z et al, Nature Biotechnology, 31, 8, 734-741, 2013, eicosapentaenoic acid It has been reported that it can be synthesized in large quantities. In this way, the amount of α-linolenic acid produced can be increased by increasing the expression levels of the chain elongase, Δ9 desaturase, and Δ12 desaturase genes necessary for the biosynthesis of α-linolenic acid. can be increased.

Biosynthesized fatty acids are generally accumulated within the bacterial cells as triacylglycerols, but they can be secreted and produced outside the bacterial cells by disrupting specific genes. In the case of Aspergillus bacteria, it has been reported that fatty acids are secreted outside the bacterial body by disruption of the acyl-CoA synthase gene (Tamano K et al, Bioscience Biotechnology and Biochemistry, 80, 9, 1829-1835, 2016) . By combining these gene disruptions, it is possible to secrete and produce α-linolenic acid outside the bacterial cell, further increasing the production amount.

It is known that fatty acids accumulated in living organisms are broken down into acetyl-CoA through a metabolic pathway called β-oxidation. In the case of yeast of the Yarrowia genus, fatty acid degradation is suppressed by disrupting the acyl-CoA oxidase gene involved in fatty acid degradation and the PEX10 gene involved in controlling peroxisomes, which are the site of β-oxidation. It has been reported that it becomes possible to produce (Ledesma-Amaro R et al, Progress in Lipid Research, 61, 40-50, 2016). Therefore, the production amount of α-linolenic acid can be further increased by destroying or reducing the expression level of genes involved in fatty acid decomposition.

When Aspergillus bacteria grow using edible substances as a nutritional source, they secrete various degrading enzymes such as amylase, glucoamylase, β-galactosidase, and protease to the outside of the bacteria to degrade the edible substances. Therefore, by increasing the expression level of the genes for these degrading enzymes, the decomposition of the nutrient source will proceed quickly, and the amount of α-linolenic acid produced in a predetermined period of time can be further increased.

In the present invention, α-linolenic acid refers to 9,12,15-octadecatrienoic acid, and the form contained in cells is an ester form contained in triacylglycerol or phospholipid, or a free fatty acid. There may be.

Since the microorganism of the present invention includes a carbohydrate metabolic pathway and a fatty acid synthesis pathway, by culturing it in the presence of carbohydrate-containing substances such as starch and lactose, it can metabolize carbohydrates such as starch and lactose and produce α - Produces C18 fatty acids (eg linoleic acid (C18:2)) which are precursors of linolenic acid. Then, linoleic acid (C18:2) is converted to α-linolenic acid (C18:3) by the action of fatty acid desaturase enhanced by self-cloning, and α-linolenic acid is added to the microorganism or its culture. Accumulates in high content.
In this specification, carbohydrates may be any sugars that can be assimilated by microorganisms, and their molecular weight is not particularly limited. In the case of trisaccharide or more, it may be a straight chain or a branched chain. For example, monosaccharides (e.g., xylose, arabinose, glucose, galactose), disaccharides (e.g., lactose, sucrose, maltose, trehalose, cellobiose), oligosaccharides (e.g., maltooligosaccharides, isomaltooligosaccharides), polysaccharides (e.g., starch) , cellulose, agarose, carrageenan, pectin, glucomannan, amylose, amylopectin, glycogen), sugar alcohols (e.g., erythritol, lactitol, maltitol, mannitol, sorbitol, xylitol), proteoglycans (e.g., glycosaminoglucan, galactosamino) glycans), glycoproteins, and glycolipids. Among these, starch and lactose are preferred.

By culturing the microorganism of the present invention, microbial cells with a high content of α-linolenic acid can be prepared. In particular, microbial cells with a high content of α-linolenic acid can be prepared from food waste, more preferably food waste containing carbohydrates such as starch and lactose.

In the Aspergillus microorganism improved by self-cloning in the present invention, α-linolenic acid can preferably account for 10% or more of the total fatty acids contained in the microorganism. More preferably, α-linolenic acid can account for 15% or more of the total fatty acids contained in the bacterial cells. More preferably, α-linolenic acid can account for 20% or more of the total fatty acids contained in the bacterial cells.

When the microorganism of the present invention is a microorganism of the genus Aspergillus, the strain of the microorganism of the genus Aspergillus can be cultured according to known culture conditions for microorganisms of the genus Aspergillus. Furthermore, waste containing carbohydrates can be used as a nutrient source. For example, food waste containing starch such as cooked rice (leftover rice) can be used as a nutrient source for cultivation. Food waste containing lactose, such as whey containing lactose, can also be cultured as a nutrient source. During culture, the medium may be liquid or solid.

The medium used for culturing Aspergillus microorganisms for the purpose of producing α-linolenic acid contains a carbon source. Carbon sources include, but are not limited to, the carbohydrates defined above (eg, starch, glucose, sucrose, lactose, cellobiose, arabinose, maltose, glycerol, triacylglycerol) and free fatty acids. Furthermore, carbohydrates such as starch and lactose, or food waste containing them, can be used as a culture medium either directly or by mixing them with other substances.

In addition to the carbon source, the medium usually contains a nitrogen source and inorganic salts, and may further contain vitamins, amino acids, trace elements, etc. as necessary. As the inorganic salts, for example, sodium salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, etc. can be used. In one preferred embodiment, the medium contains at least soluble starch, sodium nitrate, potassium chloride, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate. However, the medium composition is not particularly limited as long as it is suitable for culturing microorganisms.

Cultivation can be carried out under culture conditions commonly used for the microorganism of the present invention. For example, Aspergillus microorganisms can be cultured at 4°C to 40°C, preferably 20°C to 37°C. The pH of the medium for culturing Aspergillus microorganisms is preferably 3 to 10.

In order to produce α-linolenic acid, the microorganism of the present invention is preferably cultured in a medium containing carbohydrates (e.g., starch, lactose) with aeration and stirring, typically for several hours to 10 hours. days, preferably 24 hours or more.

By culturing the microorganism of the present invention as described above, the above-mentioned α-linolenic acid is produced and accumulated in the microbial cells. Furthermore, by culturing the microorganism of the present invention, a culture containing α-linolenic acid secreted by the microorganism can also be obtained.

The present invention also provides a food or feed containing the above-mentioned microorganism or a culture thereof. The type of food or feed of the present invention is not particularly limited, and can be applied to a wide range of foods or feeds to which it is desired to add value by increasing the content of α-linolenic acid.

The present invention also provides a composition for fermenting an edible substance, comprising the above-mentioned microorganism or a culture thereof. By applying the composition for fermenting an edible substance of the present invention to an edible substance and fermenting it, a food or feed containing a large amount of α-linolenic acid can be produced.
The composition for fermenting edible substances is so-called koji, and may contain the above-mentioned microorganisms or cultures thereof, and may further contain optional components (for example, preservatives, fermentation accelerators, etc.). The method for producing the composition for fermenting edible substances is not particularly limited, but may be a conventional method for producing koji.
The edible substance may be anything that can serve as a fermentation raw material for microorganisms, and is not particularly limited, and may be, for example, food or feed, raw materials thereof (so-called food materials), and waste. Preferably, the edible substance contains carbohydrates. Among these, starch-based foods (e.g., cooked rice, bread, noodles, beans, and other processed foods containing starch), starch-based feed, their raw materials, and waste (e.g., leftovers, byproducts from manufacturing) are More preferred. Lactose-based foods (e.g., processed foods containing lactose, such as milk, skim milk, whey, cheese, yogurt, butter, and other dairy products), lactose-based feeds, their raw materials, and waste (e.g., leftovers, manufacturing byproducts) ) is also more preferable. When producing feed, the edible substance may be waste of food or its raw materials, preferably starch-based food waste or lactose-based food waste.

The present invention also provides a method for producing food or feed containing a high content of α-linolenic acid. Such methods include:
(i) contacting the edible substance with the above-mentioned microorganism or culture thereof; and (ii) fermenting the edible substance.

Step (i) is a step of preparing an environment for fermentation to be carried out in step (ii). As a method for bringing the edible substance into contact with the above-mentioned microorganism or its culture, for example, a method of adding the edible substance and the microorganism or its culture to a container simultaneously or sequentially, and stirring and mixing as necessary. Examples include, but are not particularly limited to. The edible substances are as described above. The amounts of the edible substance and the microorganism or culture thereof to be used are not particularly limited, as long as they can be fermented. The edible substance may be one type of edible substance or a combination of two or more types of edible substances.

Step (ii) is a step of performing fermentation under the fermentation environment prepared in step (i). Fermentation conditions are not particularly limited, but conditions are preferred that allow the microorganisms used to carry out fermentation and produce α-linolenic acid. After fermentation in step (ii), the fermented product contains α-linolenic acid, so it is purified as necessary and subjected to other necessary processing as a final product to produce the desired α-linolenic acid-containing food or feed. can be obtained.

The present invention also provides a method for producing α-linolenic acid. Such methods include:
(i) contacting a carbohydrate source (e.g., a starch source or a lactose source) with a microorganism or culture thereof as described above; and (ii) a carbohydrate source (e.g., a starch source or a lactose source); The process of fermenting (source).

Both (i) and (ii) are as described above, except that a carbohydrate source (e.g., starch source or lactose source) is used instead of the edible substance, and the product is α-linolenic acid. It is similar to the manufacturing method of food or feed. The carbohydrate source may be any substance containing carbohydrates, and preferably contains at least one of starch and lactose. As the carbohydrate source, the above-mentioned edible substances are preferred. After the fermentation in step (ii), the fermented product contains α-linolenic acid, so it is purified as necessary and subjected to other necessary processing as a final product to obtain α-linolenic acid of desired purity. I can do it.

In the method of the present invention, the Aspergillus cells thus produced are preferably recovered by centrifugation or filtration, and can be used as food or feed. For example, Aspergillus oryzae is used in the production of sake, miso, and soy sauce, and is a safe microorganism that can be used as food. Aspergillus microorganisms improved through the above self-cloning can also be used as part of foods in this way. Furthermore, Aspergillus ryukyuensis is used in the production of awamori, and is also used to produce feed by decomposing food waste (Patent Document 2). Furthermore, Aspergillus kawachi is used in the production of shochu and is a safe microorganism that can be used as food. Aspergillus microorganisms improved through the above self-cloning can also be used as feed in this way.

α-linolenic acid in the cells of Aspergillus genus may be extracted from the cells and used. Furthermore, the entire culture can be used as food or feed without separating the bacterial cells from the culture (eg, culture solution).

Ingestion of foods containing α-linolenic acid can be expected to suppress hypertension (Non-Patent Document 1). In addition, when humans ingest α-linolenic acid as food, it acts as a precursor to eicosapentaenoic acid and docosahexaenoic acid, so it has the effect of reducing neutral fat in the blood, which is known as the health effect of these fatty acids. can also be expected (Non-Patent Document 2). Furthermore, it has been revealed that α-linolenic acid is accumulated in livestock products by feeding animals with feed containing a large amount of α-linolenic acid (Non-patent Documents 3 and 4). Livestock products high in α-linolenic acid have already been produced and sold by growing animals using α-linolenic acid as feed. Similarly, by using the Aspergillus microorganisms improved through self-cloning as feed, α-linolenic acid is transferred into the animal's tissues, resulting in livestock and fisheries products with high health functionality. (including processed meat and dairy products).

Hereinafter, the present invention will be explained in more detail using Examples. However, the technical scope of the present invention is not limited to these Examples.

[Example 1] Selection of fatty acid desaturase gene involved in the production of α-linolenic acid Aspergillus oryzae RIB40 strain (NBRC100959 strain) was used as a microorganism belonging to the genus Aspergillus. Many genomes of the RIB40 strain have been registered in Genbank. In this information, multiple fatty acid desaturase genes were registered, and some were registered as "delta-15 desaturase", which means that it is directly involved in the biosynthesis of α-linolenic acid. did not exist. Therefore, among the multiple fatty acid desaturase genes present in the genome of the RIB40 strain, the gene registered as Locus tag AO090010000714 of "SC010" (fatty acid desaturase gene of SEQ ID NO: 1) was injected into α-linolene. We decided to conduct research on the assumption that it is involved in acid production.

Using the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Information, we performed a homology search for SEQ ID NO: 1 with the amino acid sequence registered in the Genbank nr database. Highly homologous to SEQ ID NO: 1 The registered sequences that showed sex are as shown in the list in Figure 1. Query cover and Per. The only sequence that completely matched SEQ ID NO: 1 with both Idents being 100% was the sequence of the RIB40 strain itself. In addition, the base sequence that showed the highest homology with SEQ ID NO: 1 other than the RIB40 strain sequence itself was registered as "delta-12 desaturase", which is not involved in the production of α-linolenic acid. However, it could not be easily inferred that the gene of SEQ ID NO: 1 is involved in the production of α-linolenic acid.

The amino acid sequence encoded by the gene of SEQ ID NO: 1 is SEQ ID NO: 2. A BLAST search was performed on SEQ ID NO: 2 against Genebank's nr database, but as shown in Figure 2, Query cover and Per. The only sequence that completely matched with both Idents at 100% was the sequence of the RIB40 strain itself. In addition, it was confirmed that the amino acid sequence matches 90% or more (Query cover 100% and Per.Ident 90% or more) with the sequence registered as "delta-12 desaturase" which does not contribute to the production of α-linolenic acid. -No homology with the sequence registered as "delta-15 desaturase" involved in the production of linolenic acid was found. From this, it could not be easily inferred that the protein of SEQ ID NO: 2 is involved in the production of α-linolenic acid.

[Example 2] Preparation of DNA fragment containing fatty acid desaturase gene expression cassette (composition of medium for culturing Aspergillus microorganisms)
A YPD medium (YPD liquid medium) was prepared by dissolving 1% yeast extract (powder; Difco Laboratories), 2% peptone (Difco Laboratories), and 2% D-glucose (Wako Pure Chemical Industries) in pure water, and used it for subsequent implementation. Used in the example.

(Preparation of Aspergillus microorganism-derived promoter and fatty acid desaturase gene genomic DNA fragment)
YPD liquid medium was added to a plastic tube, RIB40 strain was cultured, and genomic DNA was extracted from the resulting bacterial cells. ISOPLANT (Nippon Gene Co., Ltd.) was used to extract genomic DNA. Using the obtained genomic DNA as a template, PCR was performed with primers consisting of the nucleotide sequences of SEQ ID NOs: 4 and 5, and the TEF1 promoter region, which has been reported as a high expression promoter of Aspergillus oryzae, was amplified to obtain a PCR fragment (Kitamoto N et al. Appl. Microbiol. Biotechnol 50, 85-92 (1998)).

Similarly, PCR was performed using genomic DNA as a template and primers consisting of the nucleotide sequences of SEQ ID NO: 6 and 7, and the fatty acid desaturase gene of SEQ ID NO: 1 and its 3' untranslated region were amplified to obtain a PCR fragment. .

After electrophoresing these PCR fragments on agarose gel, the DNA fragments were excised from the gel and purified using QIAEX II Gel extraction kit (Qiagen Corporation). These purified DNA fragments were designated as "TEF1 promoter purified fragment" and "fatty acid desaturase gene/3' untranslated region purified fragment", respectively.

(Preparation of vector fragment)
Furthermore, pPTRI (Takara Bio Inc.) was used as a transformation vector for Aspergillus. pPTRI was cut with the restriction enzyme NdeI (Nippon Gene Co., Ltd.). After electrophoresing this cut fragment on an agarose gel, the DNA fragment was excised from the gel and purified using a QIAEX II Gel extraction kit. This was used as a pPTRI vector purified fragment.

(Composition of medium for expression cassette creation)
A medium containing 1% tryptone, 0.5% yeast extract, and 1% sodium chloride is used as an LB liquid medium, and to this is added 15 g of agar per liter of medium and ampicillin sodium, and LB agar with ampicillin containing ampicillin sodium at 100 μg/mL. Culture media were prepared and used in the following examples, respectively.

(Creation of DNA fragment containing expression cassette)
The three purified fragments of TEF1 promoter, fatty acid desaturase gene/3' untranslated region, and pPTRI vector were mixed with Gibson Assembly Master mix (New England Biolab Japan Co., Ltd.) and incubated at 50°C. The reaction was allowed to proceed for 30 minutes. This reaction solution was mixed with E. coli for transformation (NEB 5-alpha Competent E. coli (High Efficiency); New England Biolab Japan Co., Ltd.), and transformation treatment was performed according to the manual.

The transformed E. coli was cultured on an LB agar medium containing ampicillin for about 18 hours to obtain grown colonies. Using DNA extracted from this colony as a template for PCR, it was confirmed that an expression cassette consisting of the TEF1 promoter, fatty acid desaturase gene, and 3' untranslated region had been inserted into the pPTRI vector. This colony was cultured in LB liquid medium, and vector DNA was purified from the obtained E. coli cells using QIAprep Spin Miniprep Kit (Qiagen Co., Ltd.). It was confirmed by base sequence analysis and PCR that the sequence of the expression cassette consisting of the TEF1 promoter, fatty acid desaturase gene, and 3' untranslated region introduced into this vector DNA was the desired sequence. Furthermore, using the restriction enzyme MfeI (New England Biolab Japan Co., Ltd.), we extracted the expression cassette consisting of the TEF1 promoter, fatty acid desaturase gene, and 3' untranslated region, and the pyrithiamine resistance marker gene originally present on the vector. A DNA fragment containing the following was cut. This sample was electrophoresed on an agarose gel, the DNA fragment was cut out from the gel, and the DNA fragment was purified using a QIAEX II Gel extraction kit to obtain a purified DNA fragment containing the expression cassette. The base sequence of the DNA fragment containing the expression cassette is SEQ ID NO:3.

The TEF1 promoter, fatty acid desaturase gene, and 3' untranslated region contained in SEQ ID NO: 3 were each searched for homology with Aspergillus oryzae genomic DNA using BLAST. As a result, all of the constituent elements completely matched the registered base sequence derived from Aspergillus oryzae, and it was confirmed that all of them were derived from Aspergillus oryzae. The pyrithiamine-resistant gene contained in SEQ ID NO: 3 was obtained from a pyrithiamine-resistant mutant strain of Aspergillus oryzae (Japanese Patent Application Laid-Open No. 2000-308491), and it has been confirmed that the nucleotide sequence is derived from Aspergillus oryzae. Ta.

[Example 3] Introduction of an expression cassette into the genome of a microorganism belonging to the Aspergillus genus (composition of a medium for a microorganism belonging to the Aspergillus genus)
Per 1 liter of pure water, 6.0 g of sodium nitrate, 0.52 g of potassium chloride, 1.52 g of potassium dihydrogen phosphate, 10 g of glucose, 0.49 g of magnesium sulfate heptahydrate, 0.001 g of iron sulfate heptahydrate, A pH 6.5 medium containing 0.0088 g of zinc sulfate heptahydrate, 0.0004 g of copper sulfate pentahydrate, 0.0001 g of sodium tetraborate decahydrate, and 0.00005 g of hexaammonium heptamolybdate tetrahydrate. A Czapek-Dox (CD) medium was prepared, and a CD agar medium prepared by adding 20 g of agar per liter of CD medium was used in the following examples.

(Creation of self-cloning strain of fatty acid desaturase gene of Aspergillus microorganism)
Protoplasts of the host RIB40 strain were prepared according to the manual of pPTRI (Takara Bio Inc.), and the purified DNA fragment containing the expression cassette described in Example 2 was introduced into the RIB40 strain. The RIB40 strain into which the DNA fragment had been introduced was cultured on a CD agar medium containing pyrithiamine at a final concentration of 0.1 μg/ml. Colonies that were observed to grow on this pyrithiamine-containing CD agar medium were cultured on YPD medium. Next, the cultured cells were collected by centrifugation, genomic DNA was extracted using ISOPLANT, and an expression cassette containing the pyrithiamine resistance gene, TEF1 promoter, fatty acid desaturase gene, and 3' untranslated region was introduced into the genome. This was confirmed by PCR. Since the base sequences constituting the introduced DNA are all derived from the same Aspergillus oryzae as the host, this genetic modification can be considered as self-cloning. The obtained RIB40 strain is referred to as a "fatty acid desaturase gene self-cloning strain" (hereinafter referred to as a "self-cloning strain").

[Example 4] Cultivation of fatty acid desaturase gene self-cloning strain from starch A medium to which 30 g of soluble starch was added instead of 10 g of glucose in the above CD medium was used as a starch CD medium in the following examples. RIB40 strain was cultured on a CD agar medium, and spores were collected with a sterile cotton swab to prepare a spore suspension. In addition, the self-cloning strain was cultured on a CD agar medium containing pyrithiamine, and spores were similarly collected using a sterile cotton swab to collect a spore suspension. 50 mL of starch CD medium was prepared in a 300 mL bladed flask. The OD600 of the above spore suspension was measured, and it was inoculated into a starch CD medium so that the absorbance was 0.05. 30℃, 120r. p. The cells were cultured with shaking for 4 days at M, and a sufficient amount of bacterial cells were collected.

[Example 5] Measurement of lipid content of fatty acid desaturase gene self-cloning strain cultured on starch Lipids were extracted from the bacterial cells described in Example 4. After washing the bacterial cells with water, they were ground with a mortar and pestle. The ground product was freeze-dried and the dry weight was measured. To this dried product were added 2 ml of water, 2.5 ml of chloroform, and 5 ml of methanol, and the mixture was shaken for 2 hours. Furthermore, 2.5 mL of chloroform and 1 mL of water were added, and the mixture was centrifuged to collect a chloroform/methanol layer. The solvent was distilled off from this chloroform/methanol layer, and the weight of the remaining lipid was measured. As a result, as shown in Table 1, the lipid content was almost the same between the RIB40 strain and the self-cloning strain.

[Example 6] Fatty acid composition of fatty acid desaturase gene self-cloning strain cultured on starch The bacterial cells remaining from Example 4 were placed in a plastic tube together with beads, and treated with a bead crusher FastPrep (Thermo Electron). , crushed. The sample was treated at 60° C. for 1 hour using a centrifugal evaporator to remove moisture, and a sample for fatty acid composition analysis was obtained. To this was added 50 μL of a hexane solution (2 g/L) of heptadecanoic acid methyl ester as an internal standard, and then immediately added 500 μL of 0.5N sodium hydroxide methanol solution, mixed uniformly, and heated at 1200 r.p.m. p. The mixture was stirred for 1 hour at m. Furthermore, after adjusting the pH by adding 40 μL of sulfuric acid, 500 μL of hexane was added and stirred for 30 minutes. The hexane layer was collected and analyzed using a gas chromatograph analyzer. The gas chromatograph analyzer used was GC-2010 plus, manufactured by Shimadzu Corporation, equipped with a hydrogen flame ionization detector. 1 μL of the sample was injected in splitless mode. The analysis was started at 60°C in the vaporization chamber, the temperature was raised at 100°C/min, and the temperature was maintained at 240°C. The column used was DB-225 (30 m, 0.25 mm, 0.25 μm, Agilent Technologies), and the column oven was started at 50°C, ramped up at 30°C/min, and held at 200°C. Helium was flowed as a carrier gas at a linear velocity of 33 cm/min, and the analysis was completed in 20 minutes. The detector was used at 240°C and 25Hz. Hydrogen was flowed at 40 ml/min, air was flowed at 400 ml/min, and helium was used as the makeup gas at 30 ml/min.

Gas chromatographs of the RIB40 strain and the self-cloning strain were as shown in FIG. Comparison with standard products revealed that the main fatty acids in each case were palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and α-linolenic acid ( C18:3). The peak area of each fatty acid was corrected using the theoretical correction factors described in AOCS Official Method Ce 1j-07, and the α-linolenic acid of RIB40 strain and a strain with high fatty acid desaturase gene expression were It was confirmed that the content ratio was as shown in Table 2. From this result, it was found that the ratio of α-linolenic acid to total fatty acids in the self-cloning strain was increased about 11 times compared to the RIB40 strain. Furthermore, it was revealed that the proportion of α-linolenic acid in the dry bacterial weight of the self-cloning strain was approximately 10 times greater than that of the RIB40 strain (Table 3).

[Example 7] Cultivation of fatty acid desaturase gene self-cloning strain from lactose A medium to which 30 g of lactose was added instead of 10 g of glucose in the above CD medium was used as a lactose CD medium and used in the following examples. RIB40 strain was cultured on a CD agar medium, and spores were collected with a sterile cotton swab to prepare a spore suspension. In addition, the self-cloning strain was cultured on a CD agar medium containing pyrithiamine, and spores were similarly collected using a sterile cotton swab to collect a spore suspension. 50 mL of lactose CD medium was prepared in a 300 mL bladed flask. The OD600 of the above spore suspension was measured, and the spore suspension was inoculated into a lactose CD medium so that the absorbance was 0.05. 30℃, 90r. p. The cells were cultured with shaking for 4 days at M, and a sufficient amount of bacterial cells were collected.

[Example 8] Fatty acid composition of fatty acid desaturase gene self-cloning strain cultured in lactose The bacterial cells obtained in Example 7 were placed in a plastic tube together with beads, and treated with a bead crusher FastPrep (Thermo Electron). , crushed. The sample was treated at 60° C. for 1 hour using a centrifugal evaporator to remove moisture, and a sample for fatty acid composition analysis was obtained. To this was added 50 μL of a hexane solution (2 g/L) of heptadecanoic acid methyl ester as an internal standard, and then immediately added 500 μL of 0.5N sodium hydroxide methanol solution, mixed uniformly, and heated at 1200 r.p.m. p. The mixture was stirred for 1 hour at m. Furthermore, after adjusting the pH by adding 40 μL of sulfuric acid, 500 μL of hexane was added and stirred for 30 minutes. The hexane layer was collected and analyzed using a gas chromatograph analyzer. The gas chromatograph analyzer used was GC-2010 plus, manufactured by Shimadzu Corporation, equipped with a hydrogen flame ionization detector. 1 μL of the sample was injected in splitless mode. The analysis was started at 60°C in the vaporization chamber, the temperature was raised at 100°C/min, and the temperature was maintained at 240°C. The column used was DB-225 (30 m, 0.25 mm, 0.25 μm, Agilent Technologies), and the column oven was started at 50°C, ramped up at 30°C/min, and held at 200°C. Helium was flowed as a carrier gas at a linear velocity of 33 cm/min, and the analysis was completed in 20 minutes. The detector was used at 240°C and 25Hz. Hydrogen was flowed at 40 ml/min, air was flowed at 400 ml/min, and helium was used as the makeup gas at 30 ml/min.

According to the results of gas chromatography, the major fatty acids are palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and α-linolenic acid (C18). :3) was confirmed. The peak area of each fatty acid was corrected using the theoretical correction factors described in AOCS Official Method Ce 1j-07, and the α-linolenic acid of RIB40 strain and a strain with high fatty acid desaturase gene expression were It was confirmed that the content ratio was as shown in Table 4. From this result, it was found that the ratio of α-linolenic acid to total fatty acids in the self-cloning strain was increased approximately 58 times compared to the RIB40 strain.

[Example 9] Selection of fatty acid desaturase gene involved in the production of α-linolenic acid in Aspergillus kawachi strain Aspergillus kawachii NBRC4308 strain (sometimes referred to as Aspergillus luchuensis mut. kawachii) as a microorganism of the genus Aspergillus (hereinafter Kawachi strain) ) was used. Many Kawachi strain genomes have been registered in Genbank. In this information, multiple fatty acid desaturase genes were registered, and some were registered as "delta-15 desaturase", which means that it is directly involved in the biosynthesis of α-linolenic acid. did not exist. Therefore, among the multiple fatty acid desaturase genes present in the genome of the Kawachi strain, Genbank Accession No. DF126480 Registered as Locus Tag AKAW_09486 of "Aspergillus kawachii IFO 4308 DNA, contig: scaffold00034, whole genome shotgun sequence" The gene (fatty acid desaturase gene of SEQ ID NO: 8) involved in the production of α-linolenic acid We decided to conduct our research assuming that.

The amino acid sequence (SEQ ID NO: 9) encoded by the gene of SEQ ID NO: 8 is given in Genbank Accession No. It was registered in the database as GAA91372 "oleate delta-12 desaturase". In addition, using the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Information, we performed a homology search for SEQ ID NO: 9 with the amino acid sequence registered in the Genbank nr database. , SEQ ID NO: 9 and The registered sequences that showed high homology were as shown in the list in Figure 4. All of the amino acid sequences with presumed functions were registered as "delta-12 desaturases" that are not directly involved in the production of α-linolenic acid. From this, it could not be easily inferred that the gene of SEQ ID NO: 8 and the amino acid sequence of SEQ ID NO: 9 are directly involved in the production of α-linolenic acid.

[Example 10] Preparation of DNA fragment containing fatty acid desaturase gene expression cassette (preparation of Aspergillus microorganism-derived promoter and fatty acid desaturase gene genomic DNA fragment)
A YPD liquid medium was added to a plastic tube, the Kawachi strain was cultured, and genomic DNA was extracted from the resulting bacterial cells. ISOPLANT (Nippon Gene Co., Ltd.) was used to extract genomic DNA. PCR was performed using the obtained genomic DNA as a template and primers consisting of the nucleotide sequences of SEQ ID NOs: 10 and 11, and the TEF1 promoter region of SEQ ID NO: 12 was amplified as a high expression promoter of Aspergillus kawachi to obtain a PCR fragment.

Similarly, PCR was performed using genomic DNA as a template and primers consisting of the nucleotide sequences of SEQ ID NO: 13 and 14, and the fatty acid desaturase gene of SEQ ID NO: 8 and its 3' untranslated region were amplified to obtain a PCR fragment. .

After electrophoresing these PCR fragments on agarose gel, the DNA fragments were excised from the gel and purified using QIAEX II Gel extraction kit (Qiagen Corporation). These purified DNA fragments were designated as "TEF1 promoter purified fragment" and "fatty acid desaturase gene/3' untranslated region purified fragment", respectively.

(Preparation of vector fragment)
Furthermore, pPTRI (Takara Bio Inc.) was used as a transformation vector for Aspergillus. pPTRI was cut with the restriction enzyme NdeI (Nippon Gene Co., Ltd.). After electrophoresing this cut fragment on an agarose gel, the DNA fragment was excised from the gel and purified using a QIAEX II Gel extraction kit. This was used as a pPTRI vector purified fragment.

(Creation of DNA fragment containing expression cassette)
The three purified fragments of TEF1 promoter, fatty acid desaturase gene/3' untranslated region, and pPTRI vector were mixed with Gibson Assembly Master mix (New England Biolab Japan Co., Ltd.) and incubated at 50°C. The reaction was allowed to proceed for 30 minutes. This reaction solution was mixed with E. coli for transformation (NEB 5-alpha Competent E. coli (High Efficiency); New England Biolab Japan Co., Ltd.), and transformation treatment was performed according to the manual.

The transformed E. coli was cultured on an LB agar medium containing ampicillin for about 18 hours to obtain grown colonies. Using DNA extracted from this colony as a template for PCR, it was confirmed that an expression cassette consisting of the TEF1 promoter, fatty acid desaturase gene, and 3' untranslated region had been inserted into the pPTRI vector. This colony was cultured in LB liquid medium, and vector DNA was purified from the obtained E. coli cells using QIAprep Spin Miniprep Kit (Qiagen Co., Ltd.). It was confirmed by base sequence analysis and PCR that the sequence of the expression cassette consisting of the TEF1 promoter, fatty acid desaturase gene, and 3' untranslated region introduced into this vector DNA was the desired sequence. Furthermore, using the restriction enzyme MfeI (New England Biolab Japan Co., Ltd.), we extracted the expression cassette consisting of the TEF1 promoter, fatty acid desaturase gene, and 3' untranslated region, and the pyrithiamine resistance marker gene originally present on the vector. A DNA fragment containing the following was cut. This sample was electrophoresed on an agarose gel, the DNA fragment was cut out from the gel, and the DNA fragment was purified using a QIAEX II Gel extraction kit to obtain a purified DNA fragment containing the expression cassette. The base sequence of the DNA fragment containing the expression cassette is SEQ ID NO: 15.

[Example 11] Introduction of an expression cassette into the genome of Aspergillus kawachi strain (Creation of a fatty acid desaturase gene-introduced strain of Aspergillus kawachi strain)
Protoplasts of the Kawachi strain as a host were prepared, and the purified DNA fragment containing the expression cassette described in Example 10 was introduced into the Kawachi strain. The Kawachi strain introduced with the DNA fragment was cultured on a CD agar medium containing pyrithiamine at a final concentration of 0.2 μg/ml. Colonies that were observed to grow on this pyrithiamine-containing CD agar medium were cultured on YPD medium. Next, the cultured cells were collected by centrifugation, genomic DNA was extracted using ISOPLANT, and an expression cassette containing the pyrithiamine resistance gene, TEF1 promoter, fatty acid desaturase gene, and 3' untranslated region was introduced into the genome. This was confirmed by PCR. The obtained Kawachi strain is called the "gene-transferred Kawachi strain."

[Example 12] Cultivation of Kawachi strain carrying a fatty acid desaturase gene from starch A normal Kawachi strain was cultured on a CD agar medium, and spores were collected with a sterile cotton swab to prepare a spore suspension. In addition, the transgenic Kawachi strain was cultured on a CD agar medium containing pyrithiamine, and spores were similarly collected using a sterile cotton swab to collect a spore suspension. 100 mL of starch CD medium was prepared in a 500 mL bladed flask. The OD600 of the above spore suspension was measured, and it was inoculated into a starch CD medium so that the absorbance was 0.05. 30℃, 90r. p. The cells were cultured with shaking for 4 days at M, and a sufficient amount of bacterial cells were collected.

[Example 13] Fatty acid composition of a fatty acid desaturase gene-introduced strain cultured on starch The bacterial cells remaining in Example 12 were placed in a plastic tube together with beads, and treated with a bead crusher FastPrep (Thermo Electron). It was crushed. The sample was treated at 60° C. for 1 hour using a centrifugal evaporator to remove moisture, and a sample for fatty acid composition analysis was obtained. To this was added 50 μL of a hexane solution (2 g/L) of heptadecanoic acid methyl ester as an internal standard, and then immediately added 500 μL of 0.5N sodium hydroxide methanol solution, mixed uniformly, and heated at 1200 r.p.m. p. The mixture was stirred for 1 hour at m. Furthermore, after adjusting the pH by adding 40 μL of sulfuric acid, 500 μL of hexane was added and stirred for 30 minutes. The hexane layer was collected and analyzed using a gas chromatograph analyzer. The gas chromatograph analyzer used was GC-2010 plus, manufactured by Shimadzu Corporation, equipped with a hydrogen flame ionization detector. 1 μL of the sample was injected in splitless mode. The analysis was started at 60°C in the vaporization chamber, the temperature was raised at 100°C/min, and the temperature was maintained at 240°C. The column used was DB-225 (30 m, 0.25 mm, 0.25 μm, Agilent Technologies), and the column oven was started at 50°C, ramped up at 30°C/min, and held at 200°C. Helium was flowed as a carrier gas at a linear velocity of 33 cm/min, and the analysis was completed in 20 minutes. The detector was used at 240°C and 25Hz. Hydrogen was flowed at 40 ml/min, air was flowed at 400 ml/min, and helium was used as the makeup gas at 30 ml/min.

The gas chromatographs of the Kawachi strain cells and the transgenic Kawachi strain were as shown in FIG. Comparison with standard products revealed that the major fatty acids in each case were palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and α-linolenic acid ( C18:3). The peak area of each fatty acid was corrected using the theoretical correction factors described in AOCS Official Method Ce 1j-07, and the content ratio of α-linolenic acid between Kawachi strain cells and the transgenic strain is shown in Table 5. It was confirmed that it was as shown. From this result, it was found that the ratio of α-linolenic acid to total fatty acids in the transgenic Kawachi strain was increased by about 5 times compared to the Kawachi strain. It was also revealed that the proportion of α-linolenic acid in the dry bacterial weight of the transgenic Kawachi strain was approximately five times greater than that of the Kawachi strain (Table 5).

[Example 14] Cultivation of the Kawachi strain carrying the fatty acid desaturase gene from lactose The Kawachi strain was cultured on a CD agar medium, and spores were collected with a sterile cotton swab to prepare a spore suspension. In addition, the transgenic strain was cultured on a CD agar medium containing pyrithiamine, and spores were similarly collected using a sterile cotton swab to collect a spore suspension. 100 mL of lactose CD medium was prepared in a 500 mL bladed flask. The OD600 of the above spore suspension was measured, and the spore suspension was inoculated into a lactose CD medium so that the absorbance was 0.05. 30℃, 90r. p. The cells were cultured with shaking for 4 days at M, and a sufficient amount of bacterial cells were collected.

[Example 15] Fatty acid composition of Kawachi strain with fatty acid desaturase gene introduced and cultured in lactose The bacterial cells obtained in Example 14 were placed in a plastic tube together with beads, and treated with a bead crusher FastPrep (Thermo Electron). , crushed. The sample was treated at 60° C. for 1 hour using a centrifugal evaporator to remove moisture, and a sample for fatty acid composition analysis was obtained. To this was added 50 μL of a hexane solution (2 g/L) of heptadecanoic acid methyl ester as an internal standard, and then immediately added 500 μL of 0.5N sodium hydroxide methanol solution, mixed uniformly, and heated at 1200 r.p.m. p. The mixture was stirred for 1 hour at m. Furthermore, after adjusting the pH by adding 40 μL of sulfuric acid, 500 μL of hexane was added and stirred for 30 minutes. The hexane layer was collected and analyzed using a gas chromatograph analyzer. The gas chromatograph analyzer used was GC-2010 plus, manufactured by Shimadzu Corporation, equipped with a hydrogen flame ionization detector. 1 μL of the sample was injected in splitless mode. The analysis was started at 60°C in the vaporization chamber, the temperature was raised at 100°C/min, and the temperature was maintained at 240°C. The column used was DB-225 (30 m, 0.25 mm, 0.25 μm, Agilent Technologies), and the column oven was started at 50°C, ramped up at 30°C/min, and held at 200°C. Helium was flowed as a carrier gas at a linear velocity of 33 cm/min, and the analysis was completed in 20 minutes. The detector was used at 240°C and 25Hz. Hydrogen was flowed at 40 ml/min, air was flowed at 400 ml/min, and helium was used as the makeup gas at 30 ml/min.

From the results of gas chromatography of the Kawachi strain and the transgenic Kawachi strain, the major fatty acids in both cases are palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2). ), α-linolenic acid (C18:3). The peak area of each fatty acid was corrected using the theoretical correction factors described in AOCS Official Method Ce 1j-07, and the content ratio of α-linolenic acid in Kawachi strain bacterial cells and transgenic Kawachi strain was determined in Table 6. It was confirmed that the results were as shown in . From this result, it was found that the ratio of α-linolenic acid to total fatty acids in the gene-transferred Kawachi strain was increased approximately 23 times compared to the normal Kawachi strain.

[Example 16] Evaluation of amino acid sequence identity In Examples 1 to 8, it was confirmed that the enzyme of SEQ ID NO: 2 of Aspergillus oryzae RIB40 strain is an enzyme that contributes to the production of α-linolenic acid. The amino acid sequences of this enzyme of SEQ ID NO: 2 and a similar enzyme possessed by the same strain were compared. For the evaluation, the alignment creation program Crustal omega was used. An alignment of two sequences to be compared was created, the number of completely matching amino acid residues was calculated, and the percentage obtained by dividing this by the length of the entire alignment was taken as the amino acid identity. First, a BLAST search was performed from information on the RIB40 strain registered in Genbank, and five proteins of the RIB40 strain that had high amino acid sequence homology with the enzyme of SEQ ID NO: 2 were found. Subsequently, the amino acid identity between these proteins and the enzyme of SEQ ID NO: 2 was calculated (Table 7).

Furthermore, in Examples 9 to 15, it was confirmed that the enzyme of SEQ ID NO: 9 of Aspergillus kawachii strain contributes to the production of α-linolenic acid. The amino acid sequences of this enzyme of SEQ ID NO: 9 and a similar enzyme possessed by the same strain were compared. First, a BLAST search was performed from information on the Kawachi strain registered in Genbank, and four proteins of the Kawachi strain that were highly homologous to the enzyme of SEQ ID NO: 9 were found. Next, amino acid identity with these proteins was evaluated (Table 8).

The enzyme of Aspergillus oryzae RIB40 strain (SEQ ID NO. 2) and the enzyme of Aspergillus kawachii strain (SEQ ID NO. 9), which were confirmed to contribute to the production of α-linolenic acid, have the same amino acid content as other enzymes in each strain. The gender was less than 40%. On the other hand, the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 9 had 78% identity. From this, it can be inferred that Aspergillus proteins having, for example, more than 70% amino acid sequence identity with SEQ ID NO: 2 or SEQ ID NO: 9 are enzymes that contribute to the production of α-linolenic acid. By searching for enzyme genes with high amino acid sequence homology to SEQ ID NO: 2 or SEQ ID NO: 9 using databases such as Genbank and highly expressing them, we can increase the production amount of α-linolenic acid in various strains of Aspergillus. can be increased.

The present invention can be used to produce microbial cells containing high α-linolenic acid content that can be safely used as food or feed.

SEQ ID NO: 1: Nucleotide sequence of fatty acid desaturase gene of Aspergillus oryzae RIB40 strain.
SEQ ID NO: 2: Amino acid sequence encoded by the fatty acid desaturase gene of Aspergillus oryzae RIB40 strain.
SEQ ID NO: 3: Gene expression cassette containing pyrithiamine resistance gene and TEF1 promoter, fatty acid desaturase gene, and 3' untranslated region.
SEQ ID NOs: 4 and 5: Primers for amplifying the Aspergillus oryzae TEF1 promoter region.
SEQ ID NOs: 6 and 7: Primers for amplifying the Aspergillus oryzae fatty acid desaturase gene and its 3' untranslated region.
SEQ ID NO: 8: Nucleotide sequence of fatty acid desaturase gene of Aspergillus kawachi strain.
SEQ ID NO: 9: Amino acid sequence encoded by the fatty acid desaturase gene of Aspergillus kawachi strain.
SEQ ID NOs: 10 and 11: Primers for amplifying the Aspergillus kawachi strain TEF1 promoter region.
SEQ ID NO: 12: Aspergillus kawachi strain TEF1 promoter region.
SEQ ID NOs: 13 and 14: Primers for amplifying the Aspergillus kawachi strain fatty acid desaturase gene and its 3' untranslated region.
SEQ ID NO: 15: Gene expression cassette containing the pyrithiamine resistance gene, TEF1 promoter, fatty acid desaturase gene, and 3' untranslated region introduced into Aspergillus kawachi strain.

Claims (18)

A microorganism that belongs to filamentous fungi and contains a carbohydrate metabolic pathway and a fatty acid desaturase gene, into which at least one nucleic acid derived from a microorganism of the same genus and species as the host is introduced from outside the cell, and at least one of the introduced nucleic acids Highly expresses the fatty acid desaturase gene,
The microorganism is a microorganism of the genus Aspergillus,
The highly expressed fatty acid desaturase gene contains a polynucleotide sequence that has 90% or more identity to the amino acid sequence of SEQ ID NO: 2 or 9 and encodes an amino acid sequence that has fatty acid desaturation activity. ,
A microorganism with improved α-linolenic acid production ability. The microorganism according to claim 1, wherein the microorganism is an Aspergillus microorganism that is food safe or feed safe. Claim 1 or 2, wherein the Aspergillus microorganism is Aspergillus oryzae, Aspergillus niger, Aspergillus ryukyuensis, Aspergillus ryukyuensis kawachi, Aspergillus kawachi, Aspergillus soya, Aspergillus tamari, or Aspergillus awamori. Microorganism according to 2. The microorganism according to any one of claims 1 to 3 , wherein expression of the fatty acid desaturase gene derived from a microorganism of the same genus and species as the host is controlled by a promoter derived from a microorganism of the same genus and species as the host. The microorganism according to claim 4 , wherein the promoter is the TEF1 promoter. A microorganism that belongs to filamentous fungi and contains a carbohydrate metabolic pathway and a fatty acid desaturase gene, into which at least one nucleic acid derived from a microorganism of the same genus and species as the host is introduced from outside the cell, and at least one of the introduced nucleic acids Highly expresses the fatty acid desaturase gene,
The microorganism is a microorganism of the genus Aspergillus,
The highly expressed fatty acid desaturase gene contains a polynucleotide sequence that has 90% or more identity to the amino acid sequence of SEQ ID NO: 2 or 9 and encodes an amino acid sequence that has fatty acid desaturation activity. ,
A method for producing a microorganism or a culture thereof having an improved ability to produce α-linolenic acid. Claim 6, wherein the Aspergillus microorganism is Aspergillus oryzae, Aspergillus niger, Aspergillus ryukyuensis, Aspergillus ryukyuensis kawachi, Aspergillus kawachi, Aspergillus soya, Aspergillus tamari, or Aspergillus awamori. Method described. A food or feed comprising the microorganism according to any one of claims 1 to 5 . A composition for fermenting an edible substance, comprising the microorganism according to any one of claims 1 to 5 , or a culture thereof. Process for producing food or feed containing high amounts of alpha-linolenic acid, including:
(i) contacting the edible substance with the microorganism according to any one of claims 1 to 5 or a culture thereof or the composition according to claim 9; and (ii) fermenting the edible substance. Process. 11. The method according to claim 10, wherein the edible material is food or its waste. 12. The method according to claim 10 or 11, wherein the edible substance comprises a carbohydrate. A method according to any one of claims 10 to 12, wherein the edible material comprises at least one of starch and lactose. A method according to any one of claims 10 to 12, wherein the edible substance comprises cooked rice. A method according to any one of claims 10 to 12, wherein the edible substance comprises whey. Method for producing alpha-linolenic acid, including:
(i) contacting the carbohydrate source with the microorganism according to any one of claims 1 to 5 , or a culture thereof , or the composition according to claim 9; and (ii) bringing the carbohydrate source into contact with The process of fermentation. 17. The method of claim 16, wherein the carbohydrate source comprises at least one of starch and lactose. A method for producing livestock or aquatic products, which comprises feeding animals (excluding humans) with the feed according to claim 8 and raising them to increase the content of α-linolenic acid.

Images