JP2020058321A - Algae variant-containing food/drink, animal feed, or prey - Google Patents

Abstract

To provide a food/drink, an animal feed, or a prey, in which an algae variant with excellent digestibility is used.SOLUTION: There are provided a food/drink, an animal feed, or a prey, containing algae in which expression of at least one protein selected from a group consisting of a cellulose synthase and a polyketide synthase involved in algaenan synthesis is reduced or lost.SELECTED DRAWING: None

Description

  The present invention relates to a food or drink containing an algal variant, a feed, or a feed, and a method for producing these.

Algae such as Chlorella and Nannochloropsis are attracting attention as health foods due to their rich fatty acid content, and supplements containing these algae are known.

Algae are also used as food for zooplankton.
In seed production of many fish, a zooplankton rotifers (Brachionus plicatilis, Brachionus plicatilis sp. Complex) and Artemia (Artemia) is utilized as the initial feed of the larval and juvenile fish. The n-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are essential components for the growth of larvae and are fed for the purpose of nutritive enhancement of rotifer and artemia. As a method for enhancing nutrition, there are methods in which microorganisms or microcapsules artificially added with EPA or DHA, fish oil, or the like, or microorganisms capable of biosynthesizing EPA or DHA are used.
Nannochloropsis, for example, is a microalgae that can biosynthesize EPA and has been widely used for nutritional enhancement of rotifer and artemia for many years.

  However, algae are covered with hard cell walls and extracellular layers. Therefore, the digestibility is poor, and it is necessary to physically or chemically destroy the cell wall when producing foods using algae. Furthermore, when algae are used as baits for zooplankton, it is said that artemia is not sufficiently fortified in an untreated state because it cannot be digested by artemia with relatively undeveloped masticatory organs. Therefore, it is described in Patent Documents 1 and 2 that it is possible to fortify rotifer and artemia by feeding a feed for zooplankton containing Nannochloropsis subjected to physical treatment or enzyme treatment. A method of physically treating the cell wall of chlorella has also been proposed (Patent Document 3), and chlorella having improved digestibility is sold.

  Thus, a method for physically or chemically improving the digestibility of algae has been reported, but there is no knowledge on a method for biologically or genetically improving the digestibility of algae.

International Publication No. 2015/159700 International Publication No. 2005/027651 JP-A-5-68536

TECHNICAL FIELD The present invention relates to the provision of foods and drinks, feeds, and feeds, which contain algae variants with excellent digestibility.
The present invention also relates to the provision of the modified algae.

Nannochloropsis has been studied in molecular biology and is capable of homologous recombination and genome editing (Oliver Kilian et al., PNAS, 2011 December, 108 (52) p.21265-21269; Qintao Wang). et al., the Plant Journal, 2016 August, 88, p.1071-1081). Under such circumstances, the present inventors have made extensive studies in view of the above problems.
The algae are covered with cell walls, and some of the algae have a hard shell-like structure called the alginane layer. Therefore, it has been necessary to physically or chemically destroy the cell wall when using algae as foods, drinks, feeds, and feeds.

Therefore, the inventors have developed a gene (hereinafter, “cellulose synthase gene” or “CES”) that encodes an enzyme that synthesizes cellulose that is a component of cell wall (hereinafter, also referred to as “cellulose synthase” or “CES”). (Also referred to as "gene") or a gene encoding a polyketide synthase (hereinafter, also referred to as "PKS") that is considered to be involved in the construction of the alginan layer (hereinafter, also referred to as "polyketide synthase gene" or "PKS gene"). Algae were produced by destroying the algae, and it was found that the algal variant had excellent digestibility.
Furthermore, they have found that even when the modified alga is ingested as a diet for zooplankton, it is useful as a diet having excellent digestibility.
The present invention has been completed based on these findings.

  The present invention, food and drink, feed, containing algae (hereinafter also referred to as "algal variant") in which expression of at least one protein selected from the group consisting of CES and PKS involved in alginane synthesis is reduced or lost. Or regarding food.

The present invention also relates to the modified algae.
Furthermore, the present invention relates to a method for producing the algal variant.
Furthermore, the present invention relates to a method for growing zooplankton, which comprises ingesting the algal variant.

The modified alga of the present invention has improved digestibility. Therefore, according to the present invention, it is possible to provide an algal variant having excellent digestibility.
Further, by using the algal modified product, a food, drink, feed or feed containing the algal modified product can be provided.

In the drawing-substitute photograph of FIG. 1, (A) is a wild-type Nannochloropsis oceanica NIES-2145 strain administered daily to Artemia as a diet, and one week after the start of the test, at the end of the test (B) is a culture medium at the end of the test when one week has passed since the start of the test, when Nanenochloropsis ocenica NIES-2145 strain in which CESA1 gene has been deleted in Artemia is daily administered. And (C) is a culture medium at the end of the test when one week has passed from the start of the test after daily administration of Nannochloropsis oceanica NIES-2145 strain in which PKS3 gene has been deleted to Artemia. , (D) were treated with Artemia by daily administration of Nannochloropsis oceanica NIES-2145 strain in which both CESA1 gene and PKS3 gene were deleted, and it was said that one week had passed since the start of the test. Of a culture solution at the end of the study.

In the present specification, the identity of the nucleotide sequence and the amino acid sequence is calculated by the Lipman-Pearson method (Science, 1985, vol.227, p.1435-1441). Specifically, it is calculated by using the homology analysis (Search homology) program of the genetic information processing software Genetyx-Win and setting the Unit size to compare (ktup) to 2.
In the present specification, the "stringent conditions" include, for example, Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell., Cold Spring Harbor Laboratory Press]. For example, in a solution containing 6 × SSC (1 × SSC composition: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhardt and 100 mg / mL herring sperm DNA. The conditions are such that the probe is kept at 65 ° C. for 8 to 16 hours to hybridize.
Furthermore, in the specification, the term "upstream" of a gene refers to a region subsequent to the 5'side of the gene or region captured as a target, not the position from the translation start point. On the other hand, the term "downstream" of a gene refers to a region 3'side of the gene or region captured as a target.

In the present invention, an alga in which the expression of at least one protein selected from the group consisting of CES and PKS involved in alginane synthesis is reduced or lost is used as the algal variant. In addition, it is preferable that the algal variant of the present invention have both reduced or lost expression of PKS involved in CES and alginane synthesis.
The algal variant used in the present invention may be a natural alga in which the expression of at least one protein selected from the group consisting of CES and PKS involved in alginane synthesis is reduced or lost, and the expression of the protein It is also possible to use algae with reduced or lost.
The reduction or loss of expression of PKS involved in CES or alginane synthesis in such algae can be confirmed by analyzing the gene encoding the protein or the base sequence around it.

Said alga in which the expression of PKS involved in CES or alginan synthesis is reduced or lost, the cellulose synthesis or the alginane synthesis is inhibited without dying or significantly impairing the viability. As a result, in such algae, the construction of the cell wall or alginane layer derived from cellulose is inhibited and the digestibility is improved. Here, "digestibility" refers to the ease of decomposition by digestive enzymes and the ease of decomposition in vivo when ingested as food, feed or feed. For example, it is said that the digestibility that the digestibility obtained by the method as shown in the examples described below is improved with respect to the wild strain is improved, and the algal variant of the present invention has excellent digestibility, so It is easily absorbed as nutrition in the body.
Such an improved algae modified algae, including food and drink, feed or pet food, by using as a feed, or by blending them, good digestive food and drink, feed, pet food, or, Food can be provided.

Algae cells are covered with a structure called a cell wall, and cellulose is the main component. Cellulose is synthesized by a cellulose synthase complex composed of a plurality of subunits. It is considered that subunit A (hereinafter referred to as “CESA”) is responsible for the active site of this complex. Therefore, by reducing or abolishing the expression of CES (preferably any one of the subunits constituting CES, preferably CESA) in algae, cell wall construction is inhibited by inhibition of cellulose synthesis and digestion Excellent algae can be obtained.
The “cellulose synthase” in the present invention refers to an enzyme (or complex) composed of a protein expressed from the CES gene, which is involved in the biosynthesis of cellulose.
Further, in the present specification, “cellulose synthesis activity” (hereinafter, also referred to as “CES activity”) means an activity that catalyzes a cellulose synthesis reaction.

  In addition, some algae have a cell wall containing a persistent alginane outside the cell wall derived from cellulose. By having an alginane layer in addition to the cell wall derived from cellulose, algae have acquired a very hard shell-like structure. No information has been accumulated on this alginane biosynthesis, but it is suggested that PKS is involved (see Scholz et al., Eukaryot Cell, 2014 Nov 13 (11) 1450-64). Therefore, it is considered that by reducing or eliminating the expression of PKS involved in alginan synthesis, the alginan synthesis is inhibited, the construction of the alginane layer is inhibited, and as a result, algae having excellent digestibility can be obtained. .

In the algal variant of the present invention, the expression of PKS involved in CES or alginane synthesis is reduced or lost, so that cellulose synthesis or alginane synthesis is inhibited. Therefore, in the algal variant of the present invention, the construction of the cell wall or alginane layer derived from cellulose is inhibited, and thus the algal variant of the present invention has excellent digestibility.
It should be noted that the reduction or loss of the expression of PKS involved in CES or alginane synthesis in the modified alga can be confirmed, for example, by the method described in Examples below.

The modified alga of the present invention has a digestibility improved by 2 times or more, more preferably 3 times or more, and more preferably 4 times or more as compared with the wild strain. Is more preferable.
When quantitatively evaluating digestibility, for example, as shown in the Examples below, wild strains and algal variants were treated with digestive enzymes such as amylase, pepsin, and pancreatin and leaked into the culture solution before and after the treatment. It can be evaluated by calculating the digestibility by measuring the amount of fatty acids and dividing the digestibility of the obtained algal variant by the digestibility of the wild strain.

Since the algal variant of the present invention is excellent in digestibility, by using the algal variant of the present invention as a raw material, a food, drink, feed or feed having excellent digestibility can be obtained.
When the algal variant of the present invention is compounded and applied to food and drink, feed, pet food, etc., it is provided in a form suitable for food or beverage, for example, in the form of granules, granules, tablets, capsules, paste, etc. be able to. Further, the food and drink, in addition to general food and drink, if necessary, beauty food, food for the sick, food with nutritional function, food for functional health such as food for specific health or food with functional claims, and food and drink in the form of functional food and drink. can do. When used as a feed, the algae modified product itself can be used.
Examples of blending into foods and drinks include processed wheat flour foods, processed rice foods, confectionery, beverages, dairy products, seasonings, processed meat storage foods, processed seafood foods, cooking oils and the like. In addition, tablets (tablets), tablet foods such as capsules, concentrated liquid foods, natural liquid foods, semidigestive nutritional foods, component nutritional foods, drink nutritional foods, and other oral nutritional foods, enteral nutritional foods, functional foods, etc. It may be in the form.
Examples of the feed, pet food, or feed include feed for small animals used for rabbits, rats, mice, etc., pet food used for dogs, cats, birds, squirrels, etc., feed feed for aquatic products such as fish or shellfish. .
These foods and drinks, feeds, pet foods and the like contain the algal modified product of the present invention or an extract or crushed product of the algae, and food ingredients such as sweeteners, colorants, antioxidants and vitamins. It can be prepared according to a conventional method by appropriately combining additives such as fragrances and minerals, proteins, lipids, sugars, carbohydrates and dietary fibers.

Furthermore, the algal variant of the present invention has the property of easily destructing cells physically or chemically because the expression of PKS involved in CES or alginane synthesis is reduced or lost, and thus has excellent digestibility. It can be used as a diet for zooplankton.
The zooplankton referred to in the present invention refers to rotifer, artemia, daphnia, and the like which are generally used as a feed in seed production.
There are no particular restrictions on the method of feeding zooplankton. The modified alga of the present invention preferably uses living cells rather than dead cells. In addition, the algal variant of the present invention can be administered as it is without treatment of living cells, or can be pulverized with a homogenizer and then dried to be administered in the form of powder. It is also possible to administer reshaped powder. The method for feeding an alga variant of the present invention can be given at one time at the start of zooplankton culture, but from the viewpoint of hygiene, it is possible to administer an alga variant in an amount that the zooplankton can ingest daily. preferable.

As the algae used in the present invention, from the viewpoint that a gene recombination technique has been established, algae of the genus Chlamydomonas ( Chlamydomonas ), algae of the genus Chlorella, algae of the genus Phaeodactylum , or a true eye spot alga Algae belonging to the group A, and those belonging to the true eye spot algae are more preferable. Examples of algae belonging to authentic methene Motsuna, Nannochloropsis genus algae, Monodopushisu (Monodopsis) genus algae, Bisukeria (Vischeria) genus algae, Kurorobotorisu (Chlorobotrys) genus algae, Goniokurorisu (Goniochloris) etc. Is mentioned. Of these, algae of the genus Nannochloropsis are preferred. Specific examples of algae belonging to the genus Nannochloropsis include Nannochloropsis oculata , Nannochloropsis oculata , Nannochloropsis gaditana , Nannochloropsis salina , and Nannochloropsis salina . Examples thereof include Nannochloropsis limnetica , Nannochloropsis granulata and Nannochloropsis sp. Of these, Nannochloropsis oceanica or Nannochloropsis gadiana is preferable, and Nannochloropsis oceanica is more preferable.
Algae such as Nannochloropsis oceanica can be obtained from a preservation organization such as a private or public research institute. For example, the Nannochloropsis oceanica NIES-2145 strain can be obtained from the National Institute for Environmental Studies (NIES).

As the algae medium, a medium based on natural seawater or artificial seawater may be used, or a commercially available culture medium may be used. Specific examples of the medium include f / 2 medium, ESM medium, Daigo IMK medium, L1 medium, MNK medium and the like. Among them, f / 2 medium, ESM medium or Daigo IMK medium is preferable, f / 2 medium or Daigo IMK medium is more preferable, and f / 2 medium is further preferable. To promote the growth of algae, nitrogen sources, phosphorus sources, metal salts, vitamins, trace metals and the like can be appropriately added to the medium.
The amount of algae inoculated into the medium can be appropriately selected and is preferably 1 to 50% (vol / vol) per medium, more preferably 1 to 10% (vol / vol) from the viewpoint of viability. The culture temperature is not particularly limited as long as it does not adversely affect the growth of algae, but is usually in the range of 5 to 40 ° C. From the viewpoint of promoting the growth of algae, the temperature is preferably 10 to 35 ° C, more preferably 15 to 30 ° C.
Further, it is preferable that the culture of algae is performed under light irradiation so that photosynthesis can be performed. The light irradiation may be performed under conditions that allow photosynthesis, and may be artificial light or sunlight. As the light intensity at the time of light irradiation, from the viewpoint of promoting the growth of algae, preferably in the range of 1 to 4,000 μmol / m ² / s, more preferably 10 to 2,500 μmol / m ² / s, and more preferably Is in the range of 100 to 2,500 μmol / m ² / s, more preferably in the range of 200 to 2,500 μmol / m ² / s, more preferably in the range of 250 to 2,500 μmol / m ² / s, and more preferably 300. ˜2,500 μmol / m ² / s. In addition, the interval of light irradiation is not particularly limited, but from the same viewpoint as described above, it is preferably performed in a light-dark cycle, and in 24 hours, the light period is preferably 8 to 24 hours, more preferably 10 to 18 hours, More preferably, it is 12 hours.
Further, it is preferable that the algae be cultured in the presence of a gas containing carbon dioxide so that photosynthesis can be performed or in a medium containing a carbonate such as sodium hydrogen carbonate. The concentration of carbon dioxide in the gas is not particularly limited, but from the viewpoint of growth promotion, it is preferably 0.03 (similar to atmospheric conditions) to 10%, more preferably 0.05 to 5%, further preferably 0.1. ˜3%, more preferably 0.3 to 1%. The concentration of the carbonate is not particularly limited, but when sodium hydrogen carbonate is used, for example, from the viewpoint of promoting growth, 0.01 to 5 mass% is preferable, more preferably 0.05 to 2 mass%, and further preferably 0.1. Is 1% by mass.
The culture time is not particularly limited, and the culture may be performed for a long time (for example, about 150 days). From the viewpoint of promoting the growth of algae and reducing the production cost, the culture period is preferably 3 to 90 days, more preferably 7 to 30 days, and further preferably 14 to 21 days. The culture may be aeration stirring culture, shaking culture, or static culture, and shaking culture is preferable from the viewpoint of improving aeration.

The CES whose expression is reduced or lost in the present invention is not particularly limited as long as it is a protein (enzyme) exhibiting CES activity.
In the present invention, CES whose expression is reduced or lost can be selected as a candidate by, for example, Blast analysis based on genomic information and annotated with CES. Alternatively, the amino acid sequence analyzed by Blastp and those annotated with CES can also be selected as candidates. Furthermore, the protein may be confirmed by culturing algae in which the gene encoding the selected protein is disrupted or deleted and examining the effect on the cellulosic layer of the cell wall.

Examples of preferable CES in the present invention include the following protein (A) or (B).

(A) A protein having the amino acid sequence represented by SEQ ID NO: 1.
(B) A protein consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of the protein (A) and having CES activity.

  The protein (A) consisting of the amino acid sequence of SEQ ID NO: 1 is a protein that constitutes subunit A of a cellulose synthase complex derived from the Nanochloropsis oceanica NIES-2145 strain, which is an alga belonging to the genus Nannochloropsis (hereinafter , "CESA1"). The protein comprising the amino acid sequence of SEQ ID NO: 1 of the present invention (protein (A) above) has CES activity.

The protein (B) consists of an amino acid sequence having 60% or more identity with the amino acid sequence of the protein (A), and has CES activity.
In general, an amino acid sequence encoding an enzyme protein does not necessarily show enzymatic activity unless the entire region of the sequence is conserved, and there is also a region that does not affect the enzymatic activity even if the amino acid sequence changes. It is known to exist. In such a region that is not essential for enzyme activity, the original activity of the enzyme can be maintained even if a mutation such as amino acid deletion, substitution, insertion or addition is introduced. The proteins defined in the present invention also include proteins in which the CES activity is retained and the amino acid sequence is partially mutated.

  From the viewpoint of CES activity in the protein (B), the identity with the amino acid sequence of the protein (A) is preferably 65% or more, more preferably 70% or more, more preferably 75% or more, and 80% or more. More preferably, 85% or more is more preferable, 90% or more is more preferable, 92% or more is more preferable, 94% or more is more preferable, 96% or more is more preferable, 98% or more is further preferable, 99% or more is further preferable.

  Further, as the protein (B), one or a plurality (for example, 1 or more and 300 or less, preferably 1 or more and 263 or less, and more preferably 1 or more and 225 or less) in the amino acid sequence of the protein (A). , More preferably 1 or more and 188 or less, more preferably 1 or more and 150 or less, more preferably 1 or more and 112 or less, more preferably 1 or more and 75 or less, more preferably 1 or more and 60 or less. , More preferably 1 or more and 45 or less, more preferably 1 or more and 30 or less, more preferably 1 or more and 15 or less, and still more preferably 1 or more and 7 or less) amino acid deletion, substitution, insertion Alternatively, a protein that has been added and has CES activity can be mentioned.

In order to reduce or eliminate the expression of the protein (A) or (B), it is also preferable to suppress the expression of the gene (CES gene) encoding the protein (A) or (B) or delete the gene. . Examples of the gene encoding the protein (A) or (B) include the gene consisting of the following DNA (a) or (b).

(A) A DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2.
(B) A DNA which comprises a base sequence having 60% or more identity with the base sequence of the DNA (a) and which encodes a protein having CES activity.

  The DNA (a) consisting of the nucleotide sequence represented by SEQ ID NO: 2 is a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1, and is the CESA1 gene derived from Nannochloropsis oceanica NIES-2145 strain.

  From the viewpoint of CES activity, the DNA (b) preferably has 65% or more identity, more preferably 70% or more, more preferably 75% or more, and 80% or more with the base sequence of the DNA (a). More preferably, 85% or more is more preferable, 90% or more is more preferable, 92% or more is more preferable, 94% or more is more preferable, 96% or more is more preferable, 98% or more is further preferable, 99% or more is further preferable.

In addition, as the DNA (b), one or a plurality (for example, 1 or more and 903 or less, preferably 1 or more and 790 or less, more preferably 1 or more and 677 or less) in the base sequence represented by SEQ ID NO: 2 is used. , More preferably 1 or more and 564 or less, more preferably 1 or more and 451 or less, more preferably 1 or more and 338 or less, more preferably 1 or more and 225 or less, more preferably 1 or more and 180 or less. , More preferably 1 or more and 135 or less, more preferably 1 or more and 90 or less, more preferably 1 or more and 45 or less, and further preferably 1 or more and 22 or less) bases are deleted, substituted or inserted. , Or a gene added thereto and encoding a protein having CES activity is also preferable.
Further, as the DNA (b), a gene that hybridizes with a DNA having a nucleotide sequence complementary to the DNA (a) or (b) under stringent conditions and encodes a protein having CES activity is also preferable.

In addition, PKS whose expression is reduced or lost in the present invention is not particularly limited as long as it is a protein (enzyme) involved in alginane synthesis.
In the present invention, PKS whose expression is reduced or lost can be selected as a candidate by, for example, Blast analysis based on genomic information and annotated PKS. Alternatively, the amino acid sequence can be analyzed by Blastp, and those annotated with PKS can be selected as candidates. Further, the protein may be confirmed by culturing algae in which the gene encoding the selected protein is disrupted or deleted and examining the influence of the alginane layer of the cell wall.

The preferred PKS in the present invention includes the following protein (C) or (D).

(C) A protein consisting of the amino acid sequence represented by SEQ ID NO: 3.
(D) A protein consisting of an amino acid sequence having 60% or more identity with the amino acid sequence of the protein (C) and involved in arginane synthesis.

  The protein (C) consisting of the amino acid sequence of SEQ ID NO: 3 is one kind of PKS (hereinafter, also referred to as “PKS3”) derived from the Nanochloropsis oceanica NIES-2145 strain, which is an alga belonging to the genus Nannochloropsis. . The protein having the amino acid sequence of SEQ ID NO: 3 of the present invention (the protein (C)) is involved in alginane synthesis.

The protein (D) is an amino acid sequence having 60% or more identity with the amino acid sequence of the protein (C), and is a protein involved in arginane synthesis.
In general, an amino acid sequence encoding an enzyme protein does not necessarily show enzymatic activity unless the entire region of the sequence is conserved, and there is also a region that does not affect the enzymatic activity even if the amino acid sequence changes. It is known to exist. In such a region that is not essential for enzyme activity, the original activity of the enzyme can be maintained even if a mutation such as amino acid deletion, substitution, insertion or addition is introduced. The protein defined in the present invention also includes a protein in which the function involved in the arginane synthesizing property is retained and the amino acid sequence is partially mutated.

  In the protein (D), the identity with the amino acid sequence of the protein (C) is preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, and 86% or more. Is more preferable, 90% or more is more preferable, 92% or more is more preferable, 94% or more is more preferable, 96% or more is more preferable, 98% or more is more preferable, 99% or more is further preferable.

  Further, as the protein (D), one or a plurality (for example, 1 or more and 1203 or less, preferably 1 or more and 1053 or less, more preferably 1 or more and 902 or less) in the amino acid sequence of the protein (C). , More preferably 1 or more and 752 or less, more preferably 1 or more and 601 or less, more preferably 1 or more and 421 or less, more preferably 1 or more and 300 or less, more preferably 1 or more and 240 or less. , More preferably 1 or more and 180 or less, more preferably 1 or more and 120 or less, more preferably 1 or more and 60 or less, and still more preferably 1 or more and 30 or less) amino acid deletion, substitution, insertion Alternatively, a protein that has been added and is involved in alginane synthesis can be mentioned.

In order to reduce or eliminate the expression of the protein (C) or (D), it is also preferable to suppress the expression of the gene (PKS gene) encoding the protein (C) or (D) or delete the gene. . Examples of the gene encoding the protein (C) or (D) include the gene consisting of the following DNA (c) or (d).

(C) A DNA having the nucleotide sequence represented by SEQ ID NO: 4.
(D) A DNA comprising a nucleotide sequence having 60% or more identity with the nucleotide sequence of the DNA (c) and encoding a protein involved in alginane synthesis.

  The DNA (c) consisting of the nucleotide sequence represented by SEQ ID NO: 4 is a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 3, and is a PKS3 gene derived from Nannochloropsis oceanica NIES-2145 strain.

  In the DNA (d), the identity with the base sequence of the DNA (c) is preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, and 86% or more. Is more preferable, 90% or more is more preferable, 92% or more is more preferable, 94% or more is more preferable, 96% or more is more preferable, 98% or more is more preferable, 99% or more is further preferable.

In addition, as the DNA (d), one or a plurality (for example, 1 or more and 3612 or less, preferably 1 or more and 3160 or less, more preferably 1 or more and 2709 or less) in the base sequence represented by SEQ ID NO: 4 is used. , More preferably 1 or more and 2257 or less, more preferably 1 or more and 1806 or less, more preferably 1 or more and 1264 or less, more preferably 1 or more and 903 or less, more preferably 1 or more and 722 or less. , More preferably 1 or more and 541 or less, more preferably 1 or more and 361 or less, more preferably 1 or more and 180 or less, and further preferably 1 or more and 90 or less) bases are deleted, substituted, or inserted. , Or a gene that encodes a protein that is added and is involved in arginane synthesis is also preferable.
Further, as the DNA (d), a gene which hybridizes with a DNA having a nucleotide sequence complementary to the DNA (c) or (d) under stringent conditions and which encodes a protein involved in arginane synthesis is also preferable. .

A method for reducing or abolishing the expression of PKS involved in CES or alginane synthesis is described.
In the present invention, the method for reducing or eliminating the expression of PKS involved in CES or alginane synthesis can be appropriately selected from ordinary methods. For example, a method of deleting (partial or full length) a CES gene or a PKS gene involved in arginane synthesis, a method of down-regulating a CES gene or a PKS gene involved in arginane synthesis, a CES gene or a PKS involved in arginane synthesis. Examples thereof include a method of modifying a gene promoter, a method of utilizing a genome editing technique such as antisense and promoter competition, and the like. Among them, the expression of CES or PKS involved in alginan synthesis is reduced or lost by deleting the PKS gene involved in CES gene or alginan synthesis or down-regulating the PKS gene involved in CES gene or alginane synthesis. Preferably.
Further, in place of the method of reducing or losing the expression of PKS involved in CES or alginan synthesis, a method of inactivating the PKS gene involved in CES gene or alginan synthesis, CES or PKS protein itself involved in alginan synthesis A method for weakening the function of PKS protein involved in CES or alginane synthesis by introducing a deletion or mutation, adding a drug, etc. can be appropriately selected. The effect obtained by these methods is equivalent to the effect obtained by the method of reducing or eliminating the expression of PKS involved in CES or alginane synthesis.

A method for reducing or eliminating the expression of PKS involved in CES or alginan synthesis by deleting or inactivating the CES gene or PKS gene involved in alginan synthesis will be described.
The method for deleting or inactivating the CES gene or the PKS gene involved in arginane synthesis can be appropriately selected from conventional methods. For example, a gene disruption method utilizing the homologous recombination ability of the algae itself, a transcription editing-like effector nuclease (Transcription activator-like effector nuclease, TALEN) or a crisper (CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat) genome editing technique. The CES gene or the PKS gene involved in arginane synthesis can be deleted or inactivated by a general method such as a method utilizing the above-mentioned method, a mutation introducing method utilizing a mutation or the like.
Specifically, a circular recombinant plasmid obtained by cloning into a DNA fragment or a suitable plasmid (vector) containing a part of the PKS gene involved in CES gene or alginan synthesis is incorporated into the cells of microalgae, Deletion of the CES gene or PKS gene on the genome by homologous recombination in a partial region of the PKS gene involved in CES gene or alginan synthesis, or inactivation of the PKS gene involved in CES gene or alginan synthesis by dividing them. Is possible.
In addition, the use of a mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine, a method for inducing mutations in the CES gene or PKS gene involved in arginane synthesis by irradiation with ultraviolet rays or gamma rays, CES gene Or a site-specific point mutation (eg, frameshift mutation, in-frame mutation, termination) in a PKS gene involved in alginan synthesis (eg, a site important for expressing CES or PKS involved in alginane synthesis). CES gene or a CES gene or a PKS gene involved in alginane synthesis is partially or entirely replaced with any other DNA fragment (for example, any selectable marker), etc. The PKS genes involved in alginane synthesis can be randomly inactivated.
In the present invention, it is preferable to delete or inactivate the CES gene on the genome or the PKS gene involved in arginane synthesis by homologous recombination.

When deleting or inactivating the PKS gene involved in CES gene or arginane synthesis by homologous recombination, in the algae, a plasmid for homologous recombination of PKS gene involved in CES gene or arginane synthesis or for homologous recombination Introduce the DNA cassette.
The homologous recombination plasmid or the DNA cassette for homologous recombination of the CES gene or the PKS gene involved in arginane synthesis, which is used herein, uses the whole or part of the PKS gene involved in CES gene or arginane synthesis as a target region. Then, constructing a plasmid or a DNA cassette having a base sequence homologous to a part of the upstream side of the genome encoding the target region, and a base sequence homologous to a part of the downstream side of the genome, this to the algae It is preferable to introduce.
Information on the upstream and downstream nucleotide sequences of the CES gene or PKS gene involved in arginane synthesis necessary for homologous recombination can be obtained, for example, from the National Center for Biotechnology Information (NCBI).

  In addition, it was confirmed that the CES gene or PKS gene involved in alginan synthesis was replaced with a homologous recombination plasmid or a DNA cassette for homologous recombination, and the CES gene or PKS gene involved in alginan synthesis was deleted or inactivated. In order to do so, the selectable marker incorporated into the homologous recombination plasmid or the homologous recombination DNA cassette can be appropriately selected from the commonly used selectable markers. Selectable markers that can be preferably used in the present invention include ampicillin resistance gene, chloramphenicol resistance gene, erythromycin resistance gene, neomycin resistance gene, kanamycin resistance gene, spectinomycin resistance gene, tetracycline resistance gene, blasticidin S. Examples include drug resistance genes such as resistance gene, bialaphos resistance gene, zeocin resistance gene, paromomycin resistance gene, and hygromycin resistance gene. Furthermore, genes related to auxotrophy can be used as marker genes. The selection marker can be introduced into the vector by a conventional method such as restriction enzyme treatment or ligation.

The homologous recombination plasmid or the DNA cassette for homologous recombination used to delete or inactivate the CES gene or the PKS gene involved in arginane synthesis is prepared using a commonly used plasmid (vector). be able to. Examples of plasmids that can be used include pUC18 (Takara Bio Inc.), pUC19 (Takara Bio Inc.), pUC118 (Takara Bio Inc.), P66 (Chlamydomonas Center), P-322 (Chlamydomonas Center), pPha-T1 ( Journal of Basic Microbiology, 2011, vol. 51, p. 666-672) or pJET1 (manufactured by Cosmo Bio). Particularly, pUC18, pPha-T1 or pJET1 is preferably used.
The size of the plasmid for homologous recombination or the DNA cassette for homologous recombination used for deletion or inactivation of the CES gene or the PKS gene involved in arginane synthesis should be determined in consideration of the efficiency of introduction into algae, the efficiency of homologous recombination, etc. , Can be set appropriately. For example, the upstream or downstream base sequence of the target region used as the homologous sequence is preferably 300 bp or more, and more preferably 500 bp or more. The upper limit is preferably 2.5 kbp, more preferably 2 kbp.

The transformation method for introducing the above-mentioned homologous recombination plasmid or homologous recombination DNA cassette into algae is a homologous recombination plasmid or homologous recombination DNA cassette of algae type or CES gene or PKS gene involved in alginan synthesis. Can be appropriately selected from ordinary methods according to the above. For example, transformation method using calcium ion, general competent cell transformation method, protoplast transformation method, electroporation method, LP transformation method, method using Agrobacterium, particle gun method and the like can be mentioned. . Further, in the present invention, transformation can also be performed using the electroporation method described in Randor Radakovits, et al., Nature Communications, DOI: 10.1038 / ncomms1688, 2012 and the like.
Further, referring to the method described in Proceedings of the National Academy of Sciences of the United States of America, 2011, vol. 108 (52), the upstream sequence of the target gene, promoter, selectable marker gene, terminator, and A host can also be transformed with a DNA fragment (gene disruption cassette) consisting of a sequence downstream of the gene of interest.

  Selection of algae in which the CES gene or the PKS gene involved in arginane synthesis is deleted or inactivated can be performed by using a selection marker or the like. For example, drug resistance acquired by algae as a result of introduction of a drug resistance gene into a host cell can be used as an index. It is also possible to confirm the introduction of the target DNA fragment by a PCR method using the genome as a template.

A method for down-regulating the CES gene or the PKS gene involved in arginane synthesis to reduce or eliminate the expression of PKS involved in CES or arginane synthesis is described.
Inhibition of expression of PKS gene involved in CES gene or alginan synthesis, or by identifying or activating or deleting a promoter located upstream of PKS gene involved in CES gene or alginan synthesis, CES gene or The expression level of the PKS gene involved in arginane synthesis is reduced (down-regulation of the CES gene or the PKS gene involved in arginane synthesis). When the expression level of the CES gene or the PKS gene involved in alginane synthesis decreases, the expression of CES involved in cellulose synthesis or alginane synthesis or the PKS protein involved in alginane synthesis is inhibited. As a result, the cell wall or alginan layer derived from cellulose is inhibited from being constructed in the alga. Therefore, by reducing the expression level of the CES gene or the PKS gene involved in alginane synthesis, it is possible to obtain algae having excellent digestibility.

  The method for down-regulating the CES gene or the PKS gene involved in arginane synthesis can be appropriately selected from conventional methods. For example, mutagenesis agents such as N-methyl-N'-nitro-N-nitrosoguanidine, mutations in the promoter and transcription / translation initiation region of the CES gene or PKS gene involved in alginane synthesis by irradiation with UV, gamma rays, etc. A method of inducing, a method of inserting any other DNA fragment (for example, any repressor, any selection marker, etc.) in the promoter sequence or transcription / translation initiation region of the CES gene or PKS gene involved in arginane synthesis, A method of replacing all or part of the promoter sequence and transcription / translation initiation region of the CES gene or the PKS gene involved in arginane synthesis with another arbitrary DNA fragment (for example, any repressor, any selection marker, etc.), Antisense method, RNA interference method, promoter competition and the like can be mentioned.

  The present invention relates to a nucleotide sequence homologous to a part of the upstream side of a region consisting of a nucleotide sequence of a PKS gene involved in CES gene or alginan synthesis and its upstream nucleotide sequence, and a nucleotide sequence involved in CES gene or alginan synthesis on a genome. A homologous recombination plasmid or homologous recombination of the CES gene or the PKS gene involved in arginane synthesis, which has a nucleotide sequence homologous to the downstream side of the region consisting of the nucleotide sequence of the PKS gene and its downstream nucleotide sequence. We also provide DNA cassettes. This plasmid for homologous recombination or the DNA cassette for homologous recombination can be suitably used for the production of algae in which the expression of PKS involved in CES or alginane synthesis is reduced or lost.

  Regarding the above-described embodiment, the present invention further discloses the following foods and drinks, methods for manufacturing foods and drinks, feeds, methods for producing feeds, feeds, and algae.

<1> A food, drink, feed or feed containing algae in which expression of at least one protein selected from the group consisting of CES and PKS involved in alginane synthesis is reduced or lost.

<2> The food, drink, feed or feed according to the above <1>, wherein the expression of the CES and the expression of PKS involved in the alginane synthesis are reduced or lost.
<3> By deleting or inactivating the gene encoding PKS involved in CES or alginan synthesis, or down-regulating the gene encoding PKS involved in CES or alginane synthesis, the CES or alginan The food, drink, feed, or feed as described in <1> or <2> above, wherein expression of PKS involved in synthesis is reduced or lost.
<4> In the algae, the digestibility is improved, preferably the digestibility is improved by 2 times or more, more preferably 3 times or more, further preferably 4 times or more as the digestion ratio, <1> to The food or drink, feed, or feed according to any one of <3>.

<5> The food or drink, feed, or feed according to any one of <1> to <4>, wherein the alga is an alga belonging to the true eye spot alga.
<6> The food, drink, feed or feed according to the item <5>, wherein the alga belonging to the true eye spot alga is an alga belonging to the genus Nannochloropsis.
<7> The algae belonging to the genus Nannochloropsis are Nannochloropsis oceanica, Nannochloropsis oculata, Nannochloropsis gadiana, Nannochloropsis salina, Nannochloropsis limnetica, Nannochloropsis granulata, An alga selected from the group consisting of Nannochloropsis sp., Preferably Nannochloropsis oceanica, Nannochloropsis gadiana, and more preferably Nannochloropsis oceanica, The food or drink according to the item <6>, Feed or feed.

<8> The food or drink, feed or feed according to any one of <1> to <7>, wherein the CES is the following protein (A) or (B).
(A) A protein having the amino acid sequence represented by SEQ ID NO: 1.
(B) The identity with the amino acid sequence of the protein (A) is 60% or more, preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85. % Or more, more preferably 90% or more, more preferably 92% or more, more preferably 94% or more, more preferably 96% or more, more preferably 98% or more, still more preferably 99% or more of the amino acid sequence. , And a protein having CES activity.
<9> The protein (B) has 1 or more amino acid sequences of the protein (A), preferably 1 or more and 300 or less, preferably 1 or more and 263 or less, and more preferably 1 or more and 225. 1 or less, more preferably 1 or more and 188 or less, more preferably 1 or more and 150 or less, more preferably 1 or more and 112 or less, more preferably 1 or more and 75 or less, more preferably 1 or more and 60. 1 or less, more preferably 1 or more and 45 or less, more preferably 1 or more and 30 or less, more preferably 1 or more and 15 or less, and still more preferably 1 or more and 7 or less amino acids deleted or substituted. The protein, which has been inserted or added and has CES activity, according to the item <8>.
<10> The food, drink, feed or feed according to any one of <3> to <9>, wherein the CES-encoding gene is a gene consisting of the following DNA (a) or (b).
(A) A DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2.
(B) The identity with the nucleotide sequence of the DNA (a) is 60% or more, preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85. % Or more, more preferably 90% or more, more preferably 92% or more, more preferably 94% or more, more preferably 96% or more, more preferably 98% or more, still more preferably 99% or more of a nucleotide sequence. And a DNA encoding a protein having CES activity.
<11> The DNA (b) has 1 or more, preferably 1 or more and 903 or less, preferably 1 or more and 790 or less, more preferably 1 or more and 677 in the base sequence of the DNA (a). Or less, more preferably 1 or more and 564 or less, more preferably 1 or more and 451 or less, more preferably 1 or more and 338 or less, more preferably 1 or more and 225 or less, more preferably 1 or more and 180. Or less, more preferably 1 or more and 135 or less, more preferably 1 or more and 90 or less, more preferably 1 or more and 45 or less, and still more preferably 1 or more and 22 or less, a base is deleted or substituted. DNA having a nucleotide sequence encoding a protein having CES activity, which is inserted, or added, or DNA having a nucleotide sequence complementary to the DNA (a). Hybridize under stringent conditions, and DNA consisting of a nucleotide sequence encoding a protein having a CES activity is, the <10> food product according to claim, feeds, or feed.
<12> The food, drink, feed or feed according to any one of <1> to <11>, wherein PKS involved in the alginane synthesis is the following protein (C) or (D).
(C) A protein consisting of the amino acid sequence represented by SEQ ID NO: 3.
(D) The amino acid sequence identity with the protein (C) is 60% or more, preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85. % Or more, more preferably 90% or more, more preferably 92% or more, more preferably 94% or more, more preferably 96% or more, more preferably 98% or more, still more preferably 99% or more of the amino acid sequence. , And a protein involved in arginane synthesis.
<13> The protein (D) has one or more amino acids in the amino acid sequence of the protein (C), preferably 1 or more and 1203 or less, preferably 1 or more and 1053 or less, and more preferably 1 or more and 902. Or less, more preferably 1 or more and 752 or less, more preferably 1 or more and 601 or less, more preferably 1 or more and 421 or less, more preferably 1 or more and 300 or less, more preferably 1 or more and 240. 1 or less, more preferably 1 or more and 180 or less, more preferably 1 or more and 120 or less, more preferably 1 or more and 60 or less, still more preferably 1 or more and 30 or less amino acid, and substituted. The food or drink, feed or feed according to the item <12>, which is a protein that is inserted or added and is involved in alginane synthesis.
<14> The food and drink, the feed according to any one of <3> to <13>, wherein the gene encoding PKS involved in the arginane synthesis is a gene consisting of the following DNA (c) or (d). , Or food.
(C) A DNA having the nucleotide sequence represented by SEQ ID NO: 4.
(D) The identity with the nucleotide sequence of the DNA (c) is 60% or more, preferably 65% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85. % Or more, more preferably 90% or more, more preferably 92% or more, more preferably 94% or more, more preferably 96% or more, more preferably 98% or more, still more preferably 99% or more of a nucleotide sequence. And a DNA encoding a protein involved in arginane synthesis.
<15> The DNA (d) has 1 or more, preferably 1 or more and 3612 or less, preferably 1 or more and 3160 or less, more preferably 1 or more and 2709 in the base sequence of the DNA (c). 1 or less, more preferably 1 or more and 2257 or less, more preferably 1 or more and 1806 or less, more preferably 1 or more and 1264 or less, more preferably 1 or more and 903 or less, and more preferably 1 or more and 722. Or less, more preferably 1 or more and 541 or less, more preferably 1 or more and 361 or less, more preferably 1 or more and 180 or less, and still more preferably 1 or more and 90 or less, the bases are deleted or substituted. Or a DNA complementary to the DNA (c), which comprises a nucleotide sequence inserted, or added, and encodes a protein involved in alginane synthesis. A DNA which hybridizes with the DNA under stringent conditions consists of a sequence, and DNA consisting of a nucleotide sequence encoding a protein involved in Arujinan synthetic, the <14> food product according to claim, feeds, or feed.

<16> The feed according to any one of <1> to <15>, wherein the feed is a feed for zooplankton.
<17> The feed according to the above item <16>, wherein the zooplankton is a rotifer or artemia.
<18> The feed according to the above <16> or <17>, characterized in that the feed is administered to zooplankton alive.

<19> A food or drink, a feed, or a feed containing cultivated algae selected from the group consisting of CES and PKS involved in alginane synthesis, in which the expression of at least one protein is reduced or lost, and containing the algae. Production method.
<20> The method according to <19>, wherein the expression of the CES and the expression of PKS involved in the alginane synthesis are reduced or lost, respectively.
<21> The gene encoding PKS involved in CES or alginan synthesis is deleted or inactivated, or the gene encoding PKS involved in CES or alginane synthesis is down-regulated to obtain the CES or alginan. The method according to <19> or <20> above, wherein expression of PKS involved in synthesis is reduced or lost.
<22> The method according to any one of <19> to <21>, wherein the digestibility of the algae is improved by 2 times or more, preferably 3 times or more, more preferably 4 times or more as a digestion rate. .

<23> The method according to any one of <19> to <22>, wherein the alga is an alga belonging to the true eye spot alga.
<24> The method according to the above item <23>, wherein the alga belonging to the true eye spot algae is an alga belonging to the genus Nannochloropsis.
<25> The algae belonging to the genus Nannochloropsis is Nannochloropsis oceanica, Nannochloropsis oculata, Nannochloropsis gadiana, Nannochloropsis salina, Nannochloropsis limnetica, Nannochloropsis granulata, The method according to the item <24>, which is an alga selected from the group consisting of Nannochloropsis sp., Preferably Nannochloropsis oceanica, Nannochloropsis gadiana, and more preferably Nannochloropsis oceanica.

<26> The method according to any one of <19> to <25>, wherein the CES is the protein (A) or (B).
<27> The method according to the item <26>, wherein the protein (B) is the protein defined in the item <9>.
<28> The method according to any one of <21> to <27>, wherein the gene encoding the CES is a gene composed of the DNA (a) or (b).
<29> The method according to <28>, wherein the DNA (b) is the DNA defined in the item <11>.
<30> The method according to any one of <19> to <29>, wherein the PKS is the protein (C) or (D).
<31> The method according to <30>, wherein the protein (D) is a protein defined in <13>.
<32> The method according to any one of <21> to <31>, wherein the gene encoding PKS is a gene composed of the DNA (c) or (d).
<33> The method according to <32>, wherein the DNA (d) is the DNA defined in the item <15>.

<34> An alga in which the expression of at least one protein selected from the group consisting of CES and PKS involved in alginane synthesis is reduced or lost.
<35> The algae according to <34>, wherein the expression of the CES and the expression of PKS involved in the alginane synthesis are reduced or lost, respectively.
<36> The gene encoding PKS involved in CES or alginan synthesis is deleted or inactivated, or the gene encoding PKS involved in CES or alginan synthesis is down-regulated to obtain the CES or alginan. The alga according to the item <34> or <35>, in which the expression of PKS involved in synthesis is reduced or lost.

<37> The alga according to any one of <34> to <36>, wherein the CES is the protein (A) or (B).
<38> The alga according to the item <37>, wherein the protein (B) is the protein defined in the item <5>.
<39> The alga according to any one of <36> to <38>, wherein the gene encoding the CES is a gene composed of the DNA (a) or (b).
<40> The algae according to <39>, wherein the DNA (b) is the DNA defined in <7>.
<41> The alga according to any one of <34> to <40>, wherein the PKS involved in the alginane synthesis is the protein (C) or (D).
<42> The algae according to the item <41>, wherein the protein (D) is the protein defined in the item <9>.
<43> The alga according to any one of <36> to <42>, wherein the gene encoding PKS involved in the alginan synthesis is a gene composed of the DNA (c) or (d).
<44> The alga according to the item <43>, wherein the DNA (d) is the DNA defined in the item <11>.

<45> The alga according to any one of <34> to <44>, wherein the alga belongs to the true eye spot alga.
<46> The alga according to the item <45>, wherein the alga belonging to the true eye spot alga is an alga belonging to the genus Nannochloropsis.
<47> The algae belonging to the genus Nannochloropsis is Nannochloropsis oceanica, Nannochloropsis oculata, Nannochloropsis gadiana, Nannochloropsis salina, Nannochloropsis limnetica, Nannochloropsis granulata, The alga according to the item <46>, which is an alga selected from the group consisting of Nannochloropsis sp., Preferably Nannochloropsis oceanica, Nannochloropsis gadiana, and more preferably Nannochloropsis oceanica.
<48> A method for producing algae, comprising preparing and culturing the algae according to any one of <34> to <47>.

<49> A method for growing zooplankton, which comprises feeding an alga in which the expression of at least one protein selected from the group consisting of CES and PKS involved in alginane synthesis is reduced or lost.
<50> The method according to <49>, wherein the expression of the CES and the expression of PKS involved in the arginane synthesis are reduced or lost, respectively.
<51> The gene encoding PKS involved in CES or alginan synthesis is deleted or inactivated, or the gene encoding PKS involved in CES or alginan synthesis is down-regulated to give CES or alginane. The method according to <49> or <50>, wherein the expression of PKS involved in synthesis is reduced or lost.

<52> The method according to any one of <49> to <51>, wherein the CES is the protein (A) or (B).
<53> The method according to <52> above, wherein the protein (B) is a protein defined in <5> above.
<54> The method according to any one of <51> to <53>, wherein the CES-encoding gene is a gene composed of the DNA (a) or (b).
<55> The method according to the item <54>, wherein the DNA (b) is the DNA defined in the item <7>.
<56> The method according to any one of <49> to <55>, wherein PKS involved in the arginane synthesis is the protein (C) or (D).
<57> The method according to <56>, wherein the protein (D) is the protein defined in <9>.
<58> The method according to any one of <51> to <57>, wherein the gene encoding PKS involved in the alginane synthesis is a gene consisting of the DNA (c) or (d).
<59> The method according to the above item <58>, wherein the DNA (d) is the DNA defined in the item <11>.
<60> The zooplankton is a rotifer or artemia, the feed according to any one of <49> to <59><61>, wherein the alga is used as a live feed. The method according to any one of <49> to <60>.

<62> Use of the alga according to any one of <33> to <48> as a food or drink, a feed, or a feed.

  Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited thereto. The base sequences of the primers used in this example are shown in Table 1.

Preparation Example 1 Preparation of CESA1 gene-disrupted strain in Nannochloropsis oceanica NIES-2145 strain (1) Construction of plasmid for expressing zeocin resistance gene A zeocin resistance gene (SEQ ID NO: 10) was artificially synthesized. A zeocin resistance gene fragment was amplified using the synthesized DNA fragment as a template and the primer pair of SEQ ID NO: 13 and SEQ ID NO: 14 shown in Table 1. In addition, PCR was performed using the genomic DNA of the Nanochloropsis oceanica NIES-2145 strain as a template and the primer pair of SEQ ID NO: 15 and SEQ ID NO: 16 and the primer pair of SEQ ID NO: 17 and SEQ ID NO: 18 shown in Table 1. , VCP1 promoter sequence (SEQ ID NO: 9) and VCP1 terminator sequence (SEQ ID NO: 11) were amplified. Further, PCR was carried out using the plasmid vector pUC19 (manufactured by Takara Bio Inc., SEQ ID NO: 38) as a template and using the primer pair of SEQ ID NO: 19 and SEQ ID NO: 20 shown in Table 1 to amplify the plasmid vector pUC19. Each of these four amplified fragments was treated with a restriction enzyme Dpn I (manufactured by Toyobo Co., Ltd.) and purified using High Pure PCR Product Purification Kit (manufactured by Roche Applied Science). Then, the obtained four fragments were fused using In-Fusion HD Cloning Kit (Clontech) to construct a zeocin resistant gene expression plasmid.
The expression plasmid comprises an insert sequence in which a VCP1 promoter sequence, a zeocin resistance gene, and a VCP1 terminator sequence are linked in this order, and a pUC19 vector sequence.

(2) Construction of plasmid for gene disruption Based on the genomic sequence information of Nannochloropsis oceanica NIES-2145 strain, BLAST analysis was performed to determine the CESA1 gene (SEQ ID NO: 2) as a CES candidate gene used in the present invention, or arginane synthesis. The PKS3 gene (SEQ ID NO: 4) was identified as a PKS candidate gene involved in A.
PCR was carried out using the genomic DNA of N. nanopiropsis oceanica NIES-2145 as a template and the primer pair of SEQ ID NO: 21 and SEQ ID NO: 22 and the primer pair of SEQ ID NO: 23 and SEQ ID NO: 24 shown in Table 1 to obtain CESA1. A homologous sequence upstream of the gene (SEQ ID NO: 5) and a homologous sequence downstream of the CESA1 gene (SEQ ID NO: 6) were amplified. Further, PCR was carried out using the plasmid vector pUC118 (manufactured by Takara Bio Inc., SEQ ID NO: 37) as a template and using the primer pair of SEQ ID NO: 25 and SEQ ID NO: 26 shown in Table 1 to amplify the plasmid vector pUC118. Further, PCR was performed using the above-mentioned zeocin resistance gene expression plasmid as a template and the primer pair of SEQ ID NO: 27 and SEQ ID NO: 28 to amplify a zeocin resistance gene expression cassette (VCP1 promoter sequence, zeocin resistance gene, VCP1 terminator sequence). . These four amplified fragments were fused in the same manner as described above to construct a CESA1 gene disruption plasmid.
The plasmid consists of a homologous sequence upstream of the CESA1 gene, a VCP1 promoter sequence, a zeocin resistance gene, a VCP1 terminator sequence, an insert sequence linked in the order of a homologous sequence downstream of the CESA1 gene, and the pUC118 vector sequence.

(3) Preparation of CESA1 gene-disrupted strain by homologous recombination Using the above-mentioned CESA1 gene-disrupted plasmid as a template, PCR was carried out using the primer pairs of SEQ ID NO: 21 and SEQ ID NO: 24 shown in Table 1 for CESA1 gene homologous recombination. A DNA fragment (homologous sequence upstream of CESA1 gene, VCP1 promoter sequence, zeocin resistance gene, VCP1 terminator sequence, homologous sequence downstream of CESA1 gene) was amplified. The amplified DNA fragment was purified using High Pure PCR Product Purification Kit (Roche Applied Science). For elution during purification, sterilized water was used instead of the elution buffer included in the kit.
About 10 ⁹ cells of Nannochloropsis oceanica NIES-2145 strain were washed with 384 mM sorbitol solution to completely remove salts, and used as host cells for transformation. About 500 ng of each of the DNA fragments amplified above was mixed into a host cell, and electroporation was performed under the conditions of 50 μF, 500Ω, and 2,200 v / 2 mm. f / 2 liquid medium _{(NaNO 3 75mg, NaH 2 PO} 4 · 2H 2 O 6mg, vitamin B12 0.5 [mu] g, Biotin 0.5 [mu] g, thiamine _{100μg, Na 2 SiO 3 · 9H} 2 O 10mg, Na 2 EDTA · 2H 2 O 4.4 mg, FeCl ₃ .6H ₂ O 3.16 mg, CoSO ₄ .7H ₂ O 12 μg, ZnSO ₄ 7H ₂ O 21 μg, MnCl ₂ 4H ₂ O 180 μg, CuSO ₄ 5H ₂ O 7 μg, Na ₂ MoO ₄ - at _2H 2 O 7 [mu] g / artificial seawater 1L) after the 24-hour recovery culture was applied on zeocin-containing f / 2 agar medium 2μg / mL, 25 ℃, 0.3 % CO 2 atmosphere, 12h The cells were cultured for 12 to 3 hours in a dark / light condition for 12 hours. The obtained colonies were selected as a CESA1 gene-disrupted strain (hereinafter, also referred to as "ΔCESA1 strain"). In order to confirm the disruption of the CESA1 gene by homologous recombination, PCR is performed using the primer pair of SEQ ID NO: 29 and SEQ ID NO: 14 shown in Table 1 to confirm that the target DNA fragment has been inserted into the CESA1 gene. I went there.

Preparation Example 2 Preparation of PKS3 gene-disrupted strain in Nannochloropsis oceanica NIES-2145 strain (1) Construction of plasmid for expressing paromomycin resistance gene Paromomycin resistance gene (SEQ ID NO: 12) was artificially synthesized. A DNA fragment of the paromomycin resistance gene was amplified using the synthesized DNA fragment as a template and the primer pair of SEQ ID NO: 30 and SEQ ID NO: 31 shown in Table 1. In addition, PCR was carried out using the zeocin resistance gene expression plasmid prepared in Preparation Example 1 as a template and the primer pairs of SEQ ID NO: 16 and SEQ ID NO: 17 shown in Table 1, and consisting of VCP1 promoter, VCP1 terminator and pUC19 vector sequence. The DNA fragment was amplified. These two DNA fragments were fused in the same manner as in Preparation Example 1 to construct a plasmid for paromomycin resistance gene expression.
The expression plasmid comprises an insert sequence in which a VCP1 promoter sequence, a paromomycin resistance gene, and a VCP1 terminator sequence are linked in this order, and a pUC19 vector sequence.

(2) Construction of plasmid for gene disruption Using the genomic DNA of Nannochloropsis oceanica NIES-2145 as a template, the primer pair of SEQ ID NO: 32 and SEQ ID NO: 33 and the primers of SEQ ID NO: 34 and SEQ ID NO: 35 shown in Table 1 PCR was performed using the pair to amplify the homologous sequence upstream of PKS3 gene (SEQ ID NO: 7) and the homologous sequence downstream of PKS3 gene (SEQ ID NO: 8). Further, using the plasmid vector pUC118 (manufactured by Takara Bio Inc.) as a template, PCR was carried out using the primer pairs of SEQ ID NO: 25 and SEQ ID NO: 26 shown in Table 1 to amplify the plasmid vector pUC118. Further, PCR was performed using the above paromomycin resistance gene expression plasmid as a template and the primer pair of SEQ ID NO: 27 and SEQ ID NO: 28 to amplify a paromomycin resistance gene expression cassette (VCP1 promoter sequence, paromomycin resistance gene, VCP1 terminator sequence). . These four amplified fragments were fused in the same manner as described above to construct a PKS3 gene disruption plasmid.
This plasmid consists of a homologous sequence upstream of the PKS3 gene, a VCP1 promoter sequence, a paromomycin resistance gene, a VCP1 terminator sequence, an insert sequence linked in the order of a homologous sequence downstream of the PKS3 gene, and the pUC118 vector sequence.

(3) Preparation of PKS3 gene-disrupted strain by homologous recombination Using the above-mentioned PKS3 gene-disrupted plasmid as a template, PCR was carried out using the primer pairs of SEQ ID NO: 32 and SEQ ID NO: 35 shown in Table 1 for PKS3 gene homologous recombination. The fragment (homologous sequence upstream of PKS3 gene, VCP1 promoter sequence, paromomycin resistance gene, VCP1 terminator sequence, homologous sequence downstream of PKS3) was amplified. The amplified DNA fragment was prepared and transformed in the same manner as in Preparation Example 1. After carrying out recovery culture for 24 hours in f / 2 liquid medium, it was applied to f / 2 agar medium containing 500 μg / mL paromomycin, and at 25 ° C., 0.3% CO ₂ atmosphere, under 12h / 12h light / dark conditions. Cultured for 2-3 weeks. The obtained colonies were selected as a PKS3 gene-disrupted strain (hereinafter, also referred to as "ΔPKS3 strain"). To confirm the destruction of the PKS3 gene by homologous recombination, PCR was performed using the primer pair of SEQ ID NO: 36 and SEQ ID NO: 14 shown in Table 1, and it was confirmed that the target DNA fragment was inserted into the PKS3 gene. I went by.

(4) Preparation of double-disrupted strain of CESA1 gene and PKS3 gene Using the CESA1 gene-disrupted strain prepared in Preparation Example 1 as a parent strain, transformation was carried out in the same manner as when the PKS3 gene-disrupted strain was prepared. After carrying out recovery culture for 24 hours in f / 2 liquid medium, it was applied to f / 2 agar medium containing 500 μg / mL paromomycin, and at 25 ° C., 0.3% CO ₂ atmosphere, under 12h / 12h light / dark conditions. Cultured for 2-3 weeks. The obtained colonies were selected as double disruption strains of CESA1 gene and PKS3 gene (hereinafter, also referred to as "ΔCESA1 / PKS3 strain"). To confirm the destruction of the PKS3 gene by homologous recombination, PCR was performed using the primer pair of SEQ ID NO: 36 and SEQ ID NO: 14 shown in Table 1, and it was confirmed that the target DNA fragment was inserted into the PKS3 gene. I went by.

Example 1 Evaluation of digestibility of gene-disrupted strain (1) Digestion treatment of each gene-disrupted strain As samples, ΔCESA1 strain prepared in Preparation Example 1, ΔPKS3 strain and ΔCESA1 / PKS3 strain prepared in Preparation Example 2, negative control strain The wild type algal cells (hereinafter, also referred to as “WT”) were used. The algal cells were inoculated in 50 mL of a medium (hereinafter, also referred to as “N15P5 medium”) in which the nitrogen concentration of the f / 2 medium was increased 15 times and the concentration of phosphorus was increased 5 times, and the alga was cultured at 25 ° C. in a 0.3% CO ₂ atmosphere. The cells were cultured for 3 weeks under 12h / 12h light / dark conditions.
Amylase (manufactured by Wako Pure Chemical Industries, Ltd.), pepsin (manufactured by Wako Pure Chemical Industries, Ltd.), and pancreatin (manufactured by Wako Pure Chemical Industries, Ltd.) were used as digestive enzymes.
1 mL of the culture solution was collected by centrifugation, and seawater was washed with 20 mM phosphate buffer pH 6.8. After suspending in a phosphate buffer, amylase was added to 0.48 μg / mL and shaken at 37 ° C. for 5 minutes. Subsequently, pepsin dissolved in 0.1N hydrochloric acid was added to 3 mg / mL, the pH was adjusted to 2.0 (using NaOH), and the mixture was shaken at 37 ° C. for 1 hour. After the reaction, the pH was adjusted to 5.7 with 1M sodium hydrogen carbonate solution, then pancreatin was added to 3 mg / mL, the pH was adjusted to 7.0 with NaOH, and the mixture was shaken at 37 ° C. for 2 hours. And digested. The sample after the digestion treatment was centrifuged at 15,000 rpm for 5 minutes to collect the supernatant.

(2) Quantification of amount of fats and oils and digestibility The fats and oils were extracted from the collected supernatant and the sample before digestion treatment by the following method.
After adding 50 μL of 1 mg / mL 7-pentadecanone (manufactured by Alfa Aesar) as an internal standard to 1 mL of the culture solution and the supernatant after digestion treatment, 0.5 mL of chloroform, 1 mL of methanol and 10 μL of 2N hydrochloric acid were added to the culture solution. After stirring vigorously for 30 minutes, add 0.5 mL of chloroform and 0.5 mL of 1.5% KCl, stir, centrifuge at 3,000 rpm for 15 minutes, and paste with a Pasteur pipette. The chloroform layer (lower layer) was collected at. Nitrogen gas was blown to the obtained chloroform layer to dry it, 0.7 mL of 0.5N potassium hydroxide / methanol solution was added, and the temperature was kept constant at 80 ° C. for 30 minutes. Subsequently, 1 mL of a 14% boron trifluoride solution (manufactured by SIGMA) was added, and the temperature was kept constant at 80 ° C. for 10 minutes. Then, 1 mL each of hexane and saturated saline was added, and the mixture was vigorously stirred and left standing at room temperature for 30 minutes, and then the hexane layer as the upper layer was recovered to obtain a fatty acid ester.

The obtained fatty acid ester was subjected to gas chromatography analysis. Gas chromatography analysis was performed under the following conditions.
<Gas chromatography conditions>
Analyzer: Agilent technology 7890A
Capillary column: DB-1 MS 30m × 200μm × 0.25μm (J & W Scientific)
Mobile bed: High-purity helium column flow rate: 1.0 mL / min
Temperature rising program: 100 ° C. (1 minute) → 10 ° C./min→300° C. (5 minutes), equilibration time: 1 minute Inlet port: split injection (split ratio: 100: 1)
Pressure 14.49psi, 104mL / min
Injection volume: 1 μL
Washing vial: Methanol / chloroform Detector temperature: 300 ° C

The fatty acid ester was identified by subjecting the sample to gas chromatograph mass spectrometry analysis under the same conditions.
The amount of methyl ester of each fatty acid was quantified from the peak area of the waveform data obtained by gas chromatography analysis. Correction was made between samples by comparing each peak area with the peak area of 7-pentadecanone, which is an internal standard, and the amount of each fatty acid was calculated, and the sum was made the total fatty acid amount (TFA). The "total fatty acid amount" in this example means the sum of C12: 0 amount, C14: 0 amount, C16: 1 amount, C16: 0 amount, C18: n amount, and C20: 5 amount. Cx: y represents a fatty acid having x carbon atoms and y double bonds. In addition, “n” of C18: n represents an integer of 0 to 5, and represents the total sum of C18: 0, C18: 1, C18: 2, C18: 3, C18: 4, and C18: 5 fatty acids.
The digestibility was calculated based on the total amount of fatty acids obtained. The digestibility was calculated by the following formula. The higher the digestibility, the more oil and fat in the cell leaks into the reaction solution, and the higher the digestibility value calculated by the following formula. In addition, as shown in Example 2 described later, no significant difference was found in the total fatty acid amount in each sample.

Digestibility (%) = total fatty acid amount in supernatant after digestion / total fatty acid amount before digestion × 100

Furthermore, based on the obtained digestibility, the digestibility (digestibility improvement rate) of each gene-disrupted strain when compared with the wild strain was calculated by the following formula.

Digestibility = Digestibility of gene-disrupted strain (%) / Digestibility of wild strain

The obtained digestibility and digestibility are shown in Table 2. In the table below, the wild strain is described as "WT", the gene-disrupted strain is expressed as "ΔCES" for the ΔCESA1 strain, "ΔPKS" for the ΔPKS3 strain, and "ΔCP" for the ΔCESA1 / PKS3 strain.

  As is clear from Table 2, each of the single gene-disrupted strains (ΔCESA1 strain and ΔPKS3 strain) showed higher digestibility (digestion ratio: 2.39 times, 2.66 times, respectively) compared to the wild type strain. The disrupted strain (ΔCESA1 / PKS3 strain) showed a higher digestibility (digestion rate: 4.12 times) than the wild strain.

Example 2 Feeding Test for Artemia Tetrabrine shrimp eggs (manufactured by Spectrum Brands Japan) were used as Artemia. As the sample to be fed, one prepared in the same manner as the algal cells used in Example 1 was used.
Table 3 shows the fatty acid composition (FA composition) and total fatty acid content (TFA) of the algal cells used for feeding. Further, the "fatty acid composition" means the ratio of the weight of each fatty acid to the total weight of fatty acids, which is the sum of free fatty acids and fatty acid residues contained in salts or ester compounds. The numerical values shown in Table 3 are average values of independent lines (3 lines).

  As shown in Table 3, it was clarified that there was no significant difference in the fatty acid composition and the total fatty acid amount in any of the samples.

Artemia eggs were sown in artificial seawater (Sea Life, manufactured by Marinetech), and after 24 hours, hatched Artemia larvae were collected and adjusted to 20 to 50 individuals / mL. For feeding, algal cells were added once a day as a feed to 200,000 cells / mL, and the animals were raised for 1 week.
After one week, the number of surviving and dying of Artemia was counted and the survival rate was calculated. The body length of Artemia after the test was measured by stereoscopic observation.
The results are shown in Table 4. The survival rate and body length are shown in the form of mean value ± standard deviation (SD) of independent line.

  As is clear from Table 4, each single gene-disrupted strain (ΔCESA1 strain and ΔPKS3 strain) had a higher survival rate and a longer body length than the wild strain (with feeding). Furthermore, the double gene disrupted strain (ΔCESA1 / PKS3 strain) showed more significant survival rate and body length than the wild strain. A photograph of the culture solution at the end of the test is shown in FIG. As is clear from FIG. 1, the culture solution in which the wild strain was added as a feed was suspended in green, indicating that the alga bodies of the wild strain remained in water. On the other hand, in the culture solution containing the disrupted strain as a feed, the tint was weakened, and it was confirmed that the fed disrupted strain, Nannochloropsis, was digested by Artemia.

  From the above, the algal variant of the present invention in which the expression of at least one PKS involved in CES and alginane synthesis is reduced or lost is excellent in digestibility, and a food, drink, feed or feed containing the same is excellent in digestibility. I understand. Further, it was shown that the modified alga of the present invention is useful as a feed for zooplankton.

Claims (15)

  A food, drink, feed, or feed containing algae in which expression of at least one protein selected from the group consisting of cellulose synthase and polyketide synthase involved in alginane synthesis is reduced or lost.   The food, drink, feed or feed according to claim 1, wherein the digestibility is improved.   The food, drink, feed or feed according to claim 1 or 2, wherein the alga is an alga belonging to the true eye spot alga. The food / beverage product, feedstuff, or feedstuff according to any one of claims 1 to 3, wherein the cellulose synthase is the following protein (A) or (B).
(A) A protein having the amino acid sequence represented by SEQ ID NO: 1.
(B) A protein comprising an amino acid sequence having 60% or more identity with the amino acid sequence of the protein (A) and having a cellulose synthesizing activity. The food, drink, feed or feed according to any one of claims 1 to 4, wherein the polyketide synthase is the following protein (C) or (D).
(C) A protein consisting of the amino acid sequence represented by SEQ ID NO: 3.
(D) A protein which is composed of an amino acid sequence having 60% or more identity with the amino acid sequence of the protein (C) and which is involved in arginane synthesis.   The feed according to any one of claims 1 to 5, wherein the feed is a zooplankton feed.   An alga in which expression of at least one protein selected from the group consisting of cellulose synthase and polyketide synthase involved in alginane synthesis is reduced or lost.   The alga according to claim 7, wherein the alga belongs to the true eye spot alga. The alga according to claim 7 or 8, wherein the cellulose synthase is the following protein (A) or (B).
(A) A protein having the amino acid sequence represented by SEQ ID NO: 1.
(B) A protein comprising an amino acid sequence having 60% or more identity with the amino acid sequence of the protein (A) and having a cellulose synthesizing activity. The algae according to any one of claims 7 to 9, wherein the polyketide synthase involved in the alginane synthesis is the following protein (C) or (D).
(C) A protein consisting of the amino acid sequence represented by SEQ ID NO: 3.
(D) A protein which is composed of an amino acid sequence having 60% or more identity with the amino acid sequence of the protein (C) and which is involved in arginane synthesis.   A method for producing an alga, which comprises preparing and culturing an alga in which expression of at least one protein selected from the group consisting of a cellulose synthase and a polyketide synthase involved in alginane synthesis is reduced or lost.   The method for producing an alga according to claim 11, wherein the alga is an alga belonging to the true eye spot alga. The method for producing algae according to claim 11 or 12, wherein the cellulose synthase is the following protein (A) or (B).
(A) A protein having the amino acid sequence represented by SEQ ID NO: 1.
(B) A protein comprising an amino acid sequence having 60% or more identity with the amino acid sequence of the protein (A) and having a cellulose synthesis activity. The method for producing algae according to any one of claims 11 to 13, wherein the polyketide synthase involved in the alginane synthesis is the following protein (C) or (D).
(C) A protein consisting of the amino acid sequence represented by SEQ ID NO: 3.
(D) A protein which is composed of an amino acid sequence having 60% or more identity with the amino acid sequence of the protein (C) and which is involved in arginane synthesis.   A method for growing zooplankton, which comprises feeding an alga in which expression of at least one protein selected from the group consisting of cellulose synthase and polyketide synthase involved in alginane synthesis is reduced or lost.