WO2020175568A1

WO2020175568A1 - Composition for feed

Info

Publication number: WO2020175568A1
Application number: PCT/JP2020/007801
Authority: WO
Inventors: 太志原
Original assignee: Ａｇｃ株式会社
Priority date: 2019-02-28
Filing date: 2020-02-26
Publication date: 2020-09-03
Also published as: JPWO2020175568A1; JP7480771B2

Abstract

The present invention provides: a composition for a feed, said composition being obtained using a unicellular microorganism as a starting material and appropriately usable as an early stage feed for fishes and crustaceans; a method for raising fishes or crustaceans with the use of the composition for a feed, etc. The present invention pertains to a composition for a feed, said composition containing aggregates of a unicellular microorganism selected from among a yeast, a lactic acid bacterium and a microalga and the unicellular microorganism in a non-aggregated state, characterized in that, in an optical microscopic image of the composition, the ratio of the total area of aggregates having a diameter within a range of 20-1200 μm to the total area of the unicellular microorganism is 25% or greater.

Description

\¥0 2020/175568 1 卩 (: 17 2020 /007801 Clarification

Title of invention: Composition for feed

Technical field

[0001] The present invention relates to a feed composition, which is made of a single cell microorganism as a raw material and is used as a feed for a juvenile fish or a juvenile crustacean.

Background technology

[0002] The most difficult part of aquaculture of fish and crustaceans is the rearing of larvae at the early stage of rearing. It is difficult for larvae and juveniles to ingest a mixed feed for adult fish, and the problem of reduced survival rate due to poor feeding is often a problem. It is important that the size of feeds for larvae of fish or juveniles of crustaceans, so-called initial feeds, is appropriate. Initial feeds that are too small are not recognized as feed and are not fed. On the other hand, too large initial feed settles quickly and is difficult to feed, and it does not enter the mouth of larvae and is not fed. Thus, both oversized and undersized feeds are unsuitable as initial feeds.

[0003] Generally, as an initial feed, Shiomizutsubo rotifer (hereinafter referred to as "rotifer") and artemia are mainly used. Rotifers generally have a back shell length of 100 to 340 001, while Artemia have a size of 400 to 100 001, which are commonly used as initial feed. Has been done. Due to the small size of the larvae that start feeding, it is difficult to cultivate the fish species for which rotifers are not suitable for initial feeding. In addition, rotifers need to be prepared at the time of use and are relatively difficult to mass-produce, and Artemia also needs to be prepared at the time of use, and the production cost is relatively high. Therefore, there is a need for the development of an initial feed for fish species for which conventional rotifers and artemia are not suitable. In addition, there is a need for the development of an initial feed that is easier to manufacture than the conventional initial feed and does not require preparation at the time of use.

[0004] It is known that single-cell microorganisms such as yeast form various aggregates, and the mechanism of aggregation has been gradually elucidated. For example, Saccharomyces budding yeast Flocculation Protein Family (F LO), Epit he U al Adhesin Family (EPA), Agg lut ini n-1 i ke Sequence Protein Fam are genes involved in aggregation in Saccharomyces cerevisiae and Candida. ily (ALS) is known (Non-Patent Documents 1 to 3). Also, in the fission yeast Schizosaccharomyces pombe (hereinafter referred to as S. It is known that nonsexual aggregation is promoted by deleting the transferase pvg 1 (Pyr uvy I transferase 1) gene or introducing a mutation that reduces or inactivates the enzymatic activity of P vg 1. (Non-Patent Documents 4 to 5, Patent Document 1).

Prior art documents

Patent literature

[0005] Patent Document 1: Patent No. 59543 15 Publication

Patent Document 2: International Publication No. 201 4/030644

Non-patent literature

[0006] Non-Patent Document 1: Wi Uaert, Journal of Fungi, 2018, vo L.4(4), 119, doi: 10.

3390/ j of 4040119

Non-Patent Document 2: Verstrepen et al., Applied Microbiology and Biotechnolog y, 2003, vol.61, p.197-205.

Non-Patent Document 3: Hoyer and Cota, Frontiers in Microbiology, 2016, vo L.7, Art i c Le 280.

Non-Patent Document 4: Matsuzawa et al., FEMS Yeast Research, 2013, vo L.13(2013 ), p.259-266.

Non-Patent Document 5: Yor i tsune et al., FEBS Letters, 2013, vo L.587, p.917-921 Non-Patent Document 6: MuLvihi LL et al., Molecular Biology of the CeU, 1999, v 〇 U0, p. 2771-2785.

Non-Patent Document 7: Partow et al., Yeast, 2010, vo L.27, p.955-964. \¥0 2020/175568 3 ((17 2020/007801)

Problems to be Solved by the Invention

[0007] Single-cell microorganisms such as yeast are relatively easy to cultivate in a large amount and are preferable as a biological feed. However, the individual cells are too small to be suitable as an initial feed for fish or crustaceans. Even if it is a single-celled microorganism that is relatively easy to cultivate in large quantities, it produces spoilage microbes that cause so-called spoilage amines such as indoles and ketones, pathogenic microorganisms that cause infectious diseases, and toxins that are harmful compounds. The microorganisms that cause food poisoning are also unsuitable as initial feed.

[0008] An object of the present invention is to use a single-cell microorganism as a raw material, which is suitable as an initial feed for fish and crustaceans, and a method for raising fish or crustaceans using the feed composition. Is provided.

Means for solving the problem

[0009] The present inventor regulates the aggregating ability of the single-cell microorganisms by substituting the promoter of a gene associated with the aggregation of the single-cell microorganisms or introducing a mutation into the gene, thereby cultivating fish larvae or shellfish. The present invention has been completed based on the finding that it can be adjusted to a clump of a size that can be ingested by juveniles of the class and that the obtained clump is suitable as an initial feed for fish or crustaceans.

[0010] That is, the present invention provides the following [1] to [15].

[1] A composition comprising an aggregate of unicellular microorganisms selected from yeast, lactic acid bacteria, and microalgae; and the unicellular microorganisms that are not aggregated,

The total area of the single-celled microorganisms in the optical microscope image of the composition, the ratio of the total area of aggregates having a diameter within the range of 20 to 120 is characterized by being 25% or more. , A composition for feed.

[2] The feed composition according to [1], wherein the ratio of the total area of aggregates having a diameter within the range of 20 to 100 is 30% or more.

[3] The composition for feed according to [1] or [2], wherein the single cell microorganism is yeast.

[4] Transformation of the single-celled microorganism in which a gene involved in aggregation is modified 20/175568 4 卩 (: 171? 2020 /007801

The composition for feed according to any one of [1] to [3], which is the body.

[5] The gene involved in aggregation is a gene involved in aggregation promotion, and the modification of the gene is replacement with a promoter having a stronger transcriptional strength than the promoter of the endogenous gene. ] The composition for feeds of.

[6] The veterinary composition according to [4] or [5], wherein the transformant is a yeast transformant.

[7] The feed composition according to [5], wherein the transformant is a Schizosaccharomyces sylvaticus transformant, and the gene involved in promoting endogenous aggregation is the 93 2 gene.

[8] The composition for feed according to [5], wherein the transformant is a transformant of Saccharomyces cerevisiae, and the gene involved in the promotion of endogenous aggregation is !_ 0 gene.

[9] a gene a gene involved in the aggregation is involved in aggregation-inhibiting, modifications of those said gene is a deletion of endogenous the gene or promoter ^_ evening of the gene endogenous has ^_ transfer strength than are substitutions weak to promote ^_ evening ^_, feed composition [4].

[10] The composition for feed according to [9], wherein the transformant is a transformant of Schizosaccharomyces cylinder, and the gene involved in the suppression of endogenous aggregation is the V91 gene. ..

[11] The feed composition according to any one of [1] to [10], which contains docosahexaenoic acid or eicosapentaenoic acid in an amount of 0.1% by mass or more based on the dry weight of the composition.

[1 2] A method for breeding fish or crustaceans, which comprises feeding the composition for feed according to any one of [1] to [11] to a juvenile fish or a juvenile crustacean as a feed.

[1 3] A microbial aggregate comprising an aggregate of single-cell microorganisms selected from yeast, lactic acid bacteria, and microalgae and the single-cell microorganisms that are not aggregated,

The ratio of the total area of aggregates having a diameter in the range of 20 to 120 to the total area of single-cell microorganisms in the optical microscope image of the microorganism aggregate is 2 〇 2020/175568 5 卩 (: 171-1? 2020 /007801

Use of the microbial aggregate in an amount of 5% or more in feed.

[14] Replacing a gene involved in aggregation of single-celled microorganisms with a promoter having a transcriptional strength different from that of the endogenous gene, deleting the gene, or adding the foreign gene to the genome A method for preparing an aggregate of unicellular microorganisms, wherein the size of an aggregate of unicellular microorganisms is adjusted within a desired range by incorporating it.

[15] Rearing of fish or crustaceans by feeding the larvae of fish or juveniles of crustaceans with the aggregate prepared by the method for preparing aggregates of unicellular microorganisms of [1 4] as food. Method.

Effect of the invention

[001 1] The feed composition according to the present invention contains a relatively large amount of aggregates of unicellular microorganisms having a diameter within the range of 20 to 120. Therefore, the feed composition is suitable as a feed for larvae of fish or juveniles of crustaceans (larvae, etc.).

MODE FOR CARRYING OUT THE INVENTION

[0012] A feed composition according to the present invention is a composition containing an aggregate of single cell microorganisms selected from yeast, lactic acid bacteria, and microalgae, and the single cell microorganisms that are not aggregated, and having a diameter of 2 Include a sufficient amount of aggregates in the range of 0 to 1200 with respect to the total amount of unicellular microorganisms in the composition. Larvae of fish and juveniles of crustaceans cannot be ingested because unaggregated unicellular microorganisms are too small to be recognized as food by larvae. In addition, because agglomerates that are too large in diameter exceed the mouth size of larvae or have a high sedimentation rate, it is difficult to ingest larvae. The feed composition according to the present invention contains a unicellular microorganism as an aggregate having a size that can be efficiently ingested by larvae of fish and juveniles of crustacean, and therefore, as an initial feed of fish or crustacean. It is suitable.

[0013] In the present invention and the specification of the present application, the diameter of a single cell microorganism is the equivalent circle area diameter of each aggregate obtained by image analysis from an optical microscope image, that is, the diameter of a circle having the same area from the area of the aggregate. It means the value converted into. Is it an optical microscope image? 〇 2020/175568 6 卩 (：171? 2020 /007801

The area of each of these aggregates and the equivalent diameter of the circular area can be measured by general image analysis using image analysis software such as "○! 396".

[0014] In the present invention and the specification of the present application, "with respect to the total amount of unicellular microorganisms in the composition for feed, the diameter is within the range of ₁ to ₂ (X _{! And} is the natural number satisfying ₁ < ₂₎ The percentage of aggregates in () is the total area of single-celled microorganisms (total area of aggregates and non-aggregated single-celled microorganisms included in the optical microscopic image obtained by measuring the diameter of aggregates). ), the ratio (%) of the total area of aggregates whose diameter is within the target numerical range. Hereinafter, the “ratio of aggregates having a diameter within the range of ₁ to ₂ with respect to the total amount of single-celled microorganisms in the feed composition” may be represented as “[¾ (乂₁ _ 乂₂ )”. The “number of particles of single-celled microorganisms” means the total number of aggregates and the number of unaggregated single-celled microorganisms. In order to obtain a more reliable result, the ratio is preferably measured for the number of single cell microorganisms of 1,000 or more, more preferably 2000 or more. It is also preferable to measure from an optical microscope image captured for two or more different visual fields.

[0015] The proportion of aggregates [8 (20-1 200)] in which the diameter of the feed composition according to the present invention is within the range of 20 to 1200 is preferably 25% or more, and more preferably 30% or more. It is preferably 35% or more, more preferably 45% or more, still more preferably 50% or more. From the viewpoint of obtaining a sample that matches the mouth size of juvenile fish and juvenile crustaceans and has an appropriate sedimentation rate, it is preferable to appropriately adjust the ratio of aggregates. Specifically, it is preferable to adjust the ratio of [8 (20-1 00)] and [[¾ (20-1 200) ]. For example, when it is difficult for the larvae and the like to ingest a relatively large agglomerate, such as when the larvae of the fish to which the composition for feed is given and the mouth of the larvae of the crustaceans are small,

(20-1 200)], it is possible to adjust it higher. On the other hand, when the mouth size of juvenile fish and juvenile crustaceans is relatively large, it is difficult to recognize smaller agglomerates as bait in juvenile fish.

(20-1 200) ], it is possible to adjust it lower. As an example in consideration of the good intake efficiency by the larvae of fish or the larvae of crustaceans and the sedimentation rate, for example, the diameter of the feed composition according to the present invention is in the range of 20 to 1 O Ogm. The ratio [R d (20-1 00)] of the agglomerates in is preferably 30% or more, more preferably 40% or more, still more preferably 50% or more.

[0016] The unicellular microorganism contained in the feed composition of the present invention may be any of yeast, lactic acid bacterium, and microalgae, and may contain two or more kinds of unicellular microorganisms. Examples of the yeast include S. bomb, Schizosaccharomyces octosporus, Schizozaccharomyces japonicns, and Schizosaccharomyces japonicns. ): Saccharomyces eluipoides, Saccharomyces rouxii, Saccharomyces yeast, Saccharomyces roasted varus uvarum, Saccharomyces evarium var. Albicans (Candida albicans), Candida glabrata (Candida glabrata), Candida utilis, Candida pseudotropical is (Candida pseudotropical is) Candida yeasts: Pichia pastoris (Pi chi a pastor is) _% Pichia kluyveri Pichia yeasts: Brettanomyces bruxeUensis, Brettanomvces naardenensis Bretanomyces yeasts: Torulopsis rupis is Uris Toru lops is Candida's yeast: Mycotoru la japonica, Mycotoru la lipolytica Mycotorula: Torraspora-Delburtuki (Toru laspora de Ibruecki la la) (Rhodotoru la rubra) is mentioned in the genus Ordotora. Examples of the lactic acid bacterium include Bifidobacterium Lon gum, Bifidobacterium animalis _% Bifidobacterium bifidum, Bif idobacter i um Lact is Bacteria: Lactobaci L Lus ac idophi Lus

Lactobaci L Lus amylovorus _N Lactobaci L Lus brevis Lactobaci L Lus casei Lactobaci L Lus del brueckii Lactobaci L Lus del brueckii Lactobaci L Lus del brueckii Lactobaci L Lus del brueckii Lactobaci L Lus gasseri, Lactobacillus helveticus _N Lactobacillus paracasei, Lactobacillus pentosus L Las ctobaci L Lus pentosus rhus bacillus molus Lactobacillus spp.): Enterokotsukasu belonging to the genus of Enterokotsukasu faecalis (Enterococc us faecalis): Rakutokotsukasu lactis (Lact ococcus Lact is) _N Rakutokotsukasu Ruff Ino lactate chair (Lactococcus raf fi no Lact is) N Rakutokotsukasu · Lactococcus spp. of Lactococcus plantarum can be mentioned. Examples of the microalgae include Eug Lena graci Lis _N Chlorella (ChLoreLLa), Spirulina (Arthrospira genus), Icadamo (Acutodesmus dimorphus), Chlamydomonas (Ch Lamydom onas re i nhardt ii; J. (Synechococcus e Longatus) Among them, yeast is preferable because gene modification technology and mass culture technology have been established, and it is relatively easy to prepare aggregates of a desired size. Further, fission yeast and budding yeast are more preferable because of their historical background as fermented foods and their safety as feeds is ensured.

[0017] The type of unicellular microorganisms contained in the feed composition according to the present invention can be selected according to the species of fish or crustaceans to which the feed composition is given and the degree of growth thereof. For example, in order to adjust a wide range of suitable sizes of feed composition according to the degree of growth of larvae or juveniles, yeast having a knowledge of genes involved in aggregation promotion is preferable, and S. cerevisiae, which has a lot of knowledge, is particularly preferable. Is preferred. Furthermore, in addition to the above, there is knowledge of genes involved in aggregation inhibition. 〇 2020/175 568 9 卩 (: 171-1? 2020 /007801

A cylinder is particularly preferred.

[0018] The aggregate of unicellular microorganisms contained in the feed composition according to the present invention may be formed by sexual aggregation or may be formed by nonsexual aggregation. In the case of lactic acid bacteria, flocs composed of extracellular polymeric substances and bacterial cells are also included in the aggregate.

[0019] The size of the aggregate of single-celled microorganisms can be adjusted to a preferred size by, for example, adjusting the expression level of a gene involved in aggregation or the intensity of activity. The expression level and activity intensity of genes involved in aggregation are regulated by modifying the promoter of the gene, modifying by introducing an amino acid mutation in the protein encoded by the gene, modifying the copy number of the gene. be able to. Specifically, incorporation of a promoter of a gene involved in agglutination promoter, transcription strength than Promo ^_ evening ^_ having the gene of the endogenous replaces strong promotion ^_ evening ^_, or pre SL exogenous gene into the genome .. In the transformant thus obtained, aggregation is promoted and the proportion of aggregates having a preferable size is increased. Furthermore, the deletion of genes you involved in aggregation inhibitor, or a promoter of the gene, also by transferring strength than the promoter having the gene of the endogenous replaces weak promoter ^_ evening _ aggregation promotes Transformants with an increased proportion of aggregates of the desired size are obtained. In addition, the size of the aggregate of single-celled microorganisms may be adjusted by combining these.

[0020] Examples of genes involved in aggregation promotion include !_〇 1 gene, !_〇 10 gene, 1_〇 11 gene (all derived from Saccharomyces cerevisiae), and 8 !_ 3 1 gene (Candida). ·Albicans origin), Minamihachi 1 gene (Candida glabrata origin), 93 3 2 genes (3. cylinder origin), and homologues of these genes. The genes involved in aggregation-inhibiting, For example, ₉ 1 gene (3. From bomb), and these homologous genes are exemplified up.

[0021] When the promoter of a gene involved in aggregation is modified, the promoter of the above-mentioned endogenous gene is used as a promoter of another gene originally possessed by the same species. 〇 2020/175568 10 units (：171? 2020/007801

It may be replaced with a lomotor or a promoter derived from another species.

[0022] For example, when the unicellular microorganism is a cylinder, the strength of the transcriptional strength of each promoter depends on the endogenous promoter of a gene involved in aggregation promotion such as the 93 2 gene <the II II 1 gene. Promoter <

(Non-patent document 6, Patent document 2)

.. For this reason, the endogenous promoter of the gene involved in aggregation promotion should be the promoter of the 1^0141 gene, the promoter of the IIII01 gene, the promoter of the IIII2 gene, or the promoter of the II39 gene. Although the degree of enhancement was different in each of the replaced transformants, the expression efficiency of genes involved in aggregation promotion was enhanced, 3. The aggregation of cylinders was promoted, and the proportion of large aggregates increased. That is, by replacing the promoter of a gene involved in aggregation promotion with a promoter having an appropriate transcriptional strength, it is possible to regulate the strength of the aggregation ability of single-cell microorganisms and increase the proportion of aggregates of a desired size.

[0023] When the unicellular microorganism is Saccharomyces cerevisiae, the strength of transcription of each promoter depends on the endogenous promoter <gene <1 gene promoter <◦ <1 gene promoter or transcription of genes involved in aggregation promotion. The order of the promoters is 1 (Non-Patent Document 7). Therefore, in transformants in which the endogenous promoters of the genes involved in aggregation promotion were replaced with the PYK^ gene promoter, PGK^ gene promoter or Ding 1 gene promoter, the degree of enhancement varied, but The efficiency of expression of genes involved in promotion is enhanced, the aggregation of S. cerevisiae is promoted, and the proportion of large aggregates is increased. That is, also in S. cerevisiae, the promoter of the gene involved in the promotion of aggregation can be replaced with a promoter having an appropriate transcriptional strength to control the strength of the aggregation ability of a single-cell microorganism, and the aggregation mass of a desired size can be controlled. Can be increased.

[0024] To adjust the proportion of aggregates of single-celled microorganisms, foreign genes involved in aggregation may be introduced into the genome. For example, 3! 〇 2020/175568 11 卩(: 171-1? 2020/007801

The offspring may be introduced into the 3. cylinder, and the 93 genes derived from the cylinder may be introduced into the 3. cerevisiae.

[0025] Modifications of genes involved in aggregation include replacement of promoters, introduction of mutations, deletion of the above genes, and insertion of foreign genes. These can be performed alone or in combination, and can be performed by a genetic engineering method. Insertion of an exogenous gene may be introduced as an extrachromosomal gene or may be introduced into the chromosome. Examples of the genetic engineering method include, for example, JP-A-5_15380, WO 95/09914, JP-A-10-234375, JP-A-2000-262284, and JP-A-2005-1. The methods described in Japanese Patent Publication No. 986 12 and International Publication No. 201 0/087344 can be used.

[0026] The gene modified in order to improve the aggregating ability of the unicellular microorganism contained in the feed composition according to the present invention may be one kind or two or more kinds. For example, two or more kinds of genes involved in aggregation promotion may be modified, or both the genes involved in aggregation promotion and the genes involved in aggregation inhibition may be modified.

[0027] A microbial aggregate comprising the aggregates of single-cell microorganisms contained in the feed composition according to the present invention and the single-cell microorganisms that are not aggregated is obtained by culturing the single-cell microorganisms. The unicellular microorganism can be cultured in the same manner as the naturally occurring microorganism of the same species. When the above-mentioned single cell microorganism is cultured, a part thereof aggregates to form an aggregate, and the rest remains as a single cell without aggregation. The microbial aggregate consisting of aggregates and single cells obtained by culturing is used as a raw material for feed.

[0028] Cultivation of a single-cell microorganism with modified agglutination ability is performed in a culture medium containing a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the single-cell microorganism to be cultured. As the culture medium, known culture media used for culturing microorganisms and modified media thereof can be appropriately used. As the culture medium, a natural medium or a synthetic medium may be used. It is also possible to use a solution such as krill extract added to the culture medium.

[0029] Examples of carbon sources include sugars such as glucose, fructose, and sucrose. Can be mentioned.

Examples of the nitrogen source include ammonia, ammonium sulfide, inorganic acid of ammonium acetate or ammonium salt of inorganic acid, peptone and casamino acid.

Examples of the inorganic salts include magnesium phosphate, magnesium sulfate, and sodium chloride.

Specifically, a nutrient medium such as YPD medium (MDRose et al., “Methods in Yes Genetics”, Cold Spring Harbor Laboratory Press (1990)) or a minimal medium such as MB medium (K. Okazaki et a. , Nucleic AcidsRes., vo 1.18, p.6485-6 489 (1990)) can be used.

[0030] A known yeast culture method can be used for the culture, and for example, shake culture and agitation culture can be performed.

The culture temperature is preferably 23 to 37 ^° C. Also, the culture time can be appropriately determined.

Further, the culture may be batch culture or continuous culture.

[0031] The feed composition according to the present invention may consist of only the microbial aggregates of the single-cell microorganisms described above, or may contain other components. Examples of the other components include amino acids, peptides, proteins, nucleic acids, sugars, lipids, fatty acids, vitamins and minerals. In the case of a feed composition for a living organism that requires growth such as DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid), DHA or EPA in the feed composition according to the present invention may be used in terms of the dry weight of the composition. , Preferably 0.1% by mass or more.

[0032] The feed composition according to the present invention is added to a cage of a fish or crustacean farm like any other feed. In particular, since the feed composition according to the present invention is easily ingested even by juvenile fish or juvenile crustaceans, it is preferable to feed the composition for feeding to a farm for raising these.

Example

[0033] Hereinafter, the present invention will be described in detail with reference to Examples. However, the present invention is as follows. 〇 2020/175 568 13 卩 (: 171-1? 2020 /007801

It is not limited depending on the description.

[0034] [Example 1]

3. Genes involved in the aggregation of cylinders were modified to produce strains with different aggregation ability.

[0035] <Deletion of V91 gene>

3. To remove the V91 gene from the cylinder of the bomb, a ratool fragment for gene deletion was prepared by gene synthesis. The rattle fragment was a fragment consisting of a region homologous to the genome of recombination and the I·! 34 gene sequence of an auxotrophic marker that can be processed. When the target gene region is deleted from the genome, no traces of foreign alien sequences are left in the genome.

[0036] After transforming 3. Pombe 8 [¾ (3 0 3 2 strain (Patent Document 1)] with the obtained rattle fragment for V 91 gene deletion, V 9 1 gene was deleted.The obtained V 9 1 deleted strain was named 丨 ◦ 7 86 strain,

The cells were inoculated into a test tube containing and cultured at 30° for 3 days with shaking. The obtained culture solution was mixed with a 50% glycerol solution at a ratio of 1:1 and frozen and stored as a glycerol stock at 180°.

[0037] <Replacement of 9 3 † 2 promoter>

In order to replace the promoter of the galactose-specific cell aggregation protein 93 3 gene, a replacement ratwort fragment was prepared by gene synthesis. The replacement rattle fragment consists of the sequence to be replaced by a region homologous to the genome of the recombination target to be integrated, and the gene sequence of the auxotrophic marker that can be processed (34 gene sequence). Two bomber fragments for replacement of two gene promoters.

Each of the strains and the 7 8 6 strains was transformed, and then treated with Eighty-eight to replace the 93 2 promoter.

[0038] 8 [¾ 0 0 3 2 strain as a host and 9 3 x 2 promoter replaced with |-| 〇 1 1 4 1 promoted strain

did.丨 ◦ 7 8 6 strain as host The strain in which the 3 2 promoter was replaced with the 01 4 1 promoter was designated as 801 39 strains. In the same manner, using the 786 strain as the host, the strain with the 101 promoter replaced was the I 799 strain, the strain with the I 02 promoter was the I 800 strain, and the strain with the 39 promoter was the I strain. ◦ Named each 798 shares. Table 1 shows the presence or absence of deletion of the promoter and V91 gene of the 93 genes in each strain.

[0039] [Table 1]

[0040] Each of these promoter-replaced strains was inoculated into a test tube containing each of the culture medium 3 (51_) and shake-cultured at 30 ^° C for 3 days. The obtained culture solutions were mixed with 50% glycerol solution at a ratio of 1:1 (volume ratio), and frozen and stored as glycerol stock at 180°.

[0041] <Culture of bacterial cells>

Glycerol stock of each strain (1) was added to each of the 3 culture media (5

1_) was inoculated into a test tube. For the shaking culture, a shaking incubator XV-1 6 [¾_3 manufactured by Takasaki Kagaku Kikai Co., Ltd. was used. The test tube was cultivated with shaking at 30° for 3 days, and the obtained culture broth 3 !_ was transferred to a Sakaro flask containing the Kakuguchi culture medium 150 1 _ containing the krill extract.

[0042] The Sakaro flask was shake-cultured at 30° for 3 days, and the resulting culture solution (each 150

!_) with a centrifuge 7780 11 (<11 manufactured by Mitsuhoha Co., Ltd.) 4800 X 9, 1 It was centrifuged under the condition of 0 minutes. After centrifugation, the supernatant was discarded and the bacterial cells were collected.

[0043] <Production of dried body>

The bacterial cells collected by centrifugation were frozen at 80 ^° C and freeze-dried using a freeze dryer L ABC ONCO (Asahi Life Science). The internal pressure of the device was kept below 0.1 33 mbar and dried briefly. The resulting dried product contained 0.2 to 0.5 mass% DHA per dry weight.

[0044] <Microscopic observation>

A sample was prepared by adding 0.1 g of each of the obtained yeast dry bodies to R 0 (R ev er s e O s mo s s s) water (1 mL) and suspending them. The samples were observed with a light microscope CKC-T R 2 (manufactured by Olympus Co., Ltd.) at a magnification of 40 and photographed by using a digital camera U-CMAD3 (manufactured by Olympus Co., Ltd.) to obtain an image file.

[0045] <Image analysis>

The image file of the obtained micrograph was analyzed by the image analysis software Imag e J distributed by Nat i o n a l In n st i t u t e sof H e a It h (N I H). Specifically, first, the image file was binarized by the Ma ke b i n a r y process. Next, the scale was set by the Set-scaIe treatment, and finally the area value data of each individual particle (including both aggregates and non-aggregated unicellular microorganisms) was obtained by the Analyzeparticles treatment. .. Table 2 shows the image analysis results for each strain. In the table, "particles" are particles identified as one region in the image, and include both aggregates and non-aggregated single-cell microorganisms. In other words, the "total number of particles" is the total number of aggregates and the number of unicellular microorganisms that have not aggregated. Also, "total area value" is the sum of the area values of all particles, and "1 200 ³ d ³ 20" is the value of [particle diameter (diameter equivalent to circular area) of 20 Mm or more and 1 200 Mm or less]. [Total area value] / [Total area value of all particles] (%), "100 ³ d ³ 20" is [diameter (circle area equivalent diameter

) Is more than 20 Mm and less than 100 Mm total area value] / [total surface of all particles] 〇2020/175568 16 卩(: 171-1?2020/007801

Product value] (%), “20>” is [total area value of particles whose diameter (circular area equivalent diameter) is less than 20] / [total area value of all particles] (%). The upper row of each strain shows the results of the first trial, and the lower row shows the results of the second trial.

[0046] [Table 2]

[0047] As shown in Table 2,

Compared to strain, ₉ 1 gene has been deleted viii [¾_〇 1 38 strain and, I ◦ 786 strain further transfer strength promoters 93 NOTE 2 gene was replaced with strong promoters, eight [¾_〇 1 39 Strains, 10 799 shares, 丨 ◦ 800 shares, and 丨 ◦ 798 strains had a significant decrease in the proportion of small particles with a diameter of less than 20. It is speculated that this is because the aggregation ability was improved and the aggregation was promoted, and the number of non-aggregated yeasts decreased. In particular, comparing the ∨ 786 strain, 8 [¾ 〇 1 39 strain, 10 799 strain, 1 800 strain, and I ◦ 798 strain, the strain with stronger transcriptional strength of the 93 chi 2 gene promoter showed that The proportion of particles with diameters over 100 is high, and the genes involved in aggregation are 〇 2020/175 568 17 卩(：171? 2020/007801

The size of the aggregate could be controlled by controlling the transcription strength of the promoter. Among them,

丨 ◦ 7 8 6 shares, and 8

The large proportion of aggregates of a size (diameter 20 to 100) easily ingested by juvenile fish or juvenile crustaceans was particularly suitable as an initial feed. On the other hand, the 丨 ◦ 799 strain, 丨 ◦ 800 strain, and 丨 ◦ 798 strain are aggregates with a diameter of 100 to 1 200 0.01, which is about the same size as rotifer and artemia. It had many lumps and was suitable as a substitute feed for rotifer and artemia.

[0048] [Example 2]

3. Genes involved in cerevisiae aggregation were modified to produce strains with different aggregation abilities.

[0049] <Replacement of !_〇 1 promoter>

In order to replace the promoter of the aggregation protein !_○ 1 gene, the replacement rattle fragment was prepared by gene synthesis. The replacement rattle fragment is a sequence that you want to replace and integrate with a homologous region in the genome to be recombined, and a auxotrophic marker that can be processed. J

It consists of 3 gene sequences. The obtained !_〇1 gene was used as a replacement for the rattle fragment 3.

The 192 strain was transformed to replace the 1_O1 promoter.

[0050] <!_ 〇 10 Promoter replacement>

In order to replace the promoter of the aggregate protein 1_〇10 gene, a replacement ratool fragment was prepared by gene synthesis. The replacement rattle fragment is composed of a sequence to be replaced by a region homologous to the genome to be recombined and integrated, and II [¾3 gene sequence. The Latwool fragment for replacement of the obtained 1_○10 gene promoter was used to transform the 3. cerevisiae 8 [¾〇192 strain and replace the !_○10 promoter.

[0051] <!_〇 1 1 Promoter replacement>

In order to replace the promoter of the aggregated protein 1_〇 11 gene, a replacement ratool fragment was prepared by gene synthesis. Replacement Lat Wool 〇 2020/175 568 18 卩 (: 171-1? 2020 /007801

The fragment is composed of the sequence to be integrated by substituting the homologous region in the recombination target genome, and II [¾3 gene sequence. The Latwool fragment for replacement of the obtained 1_○ 1 1 gene promoter was used to transform 3 cerevisiae 8 [¾ 0 1 92 strain to replace the 1_○ 1 1 promoter.

[0052] Pyruvate kinase as the !_〇 1 promoter using the 8192 strain as a host.

The strain in which the promoter of the PYK ^ gene had been replaced was designated as Ũ 887 strain.

The strain with the _ 192 strain as the host and the !_ 〇 1 promoter replaced by the promoter of phosphoglycerate kinase ◦ <1 gene was used as the strain 890. Eight strains were used as hosts, and strains in which the !_○1 promoter was replaced with the promoter of the elongation factor Tingami 1 gene were designated as Ō 893 strains.

Strains in which the !_○ 10 promoter was replaced with the promoter of less than 1 gene were mainly set as the I 089 1 strain. Using the eight [¾ 〇 192 strain as a host, the !_ 〇 1 0 promoter

The strain in which the promoter of one gene was replaced was the Ÿ 888 strain.

Using the strain as the host, the strain in which the !_○ 1 0 promoter was replaced with the promoter of the Tingmi 1 gene was used as strain ◦ 894.

As the host, the strain in which the !_○ 11 promoter was replaced with the promoter of <1 gene was designated as I ◦ 889 strain.

As the host!_〇 1 1 Promoter

The strain in which the promoter of one gene had been replaced was used as the Ō 829 strain.

Using the strain as a host, the strain in which the !_○ 11 promoter was replaced with the promoter of the Tongmi 1 gene was used as the strain ◦ 895. Table 3 shows the promoters of !_○ genes of each strain.

[0053] [Table 3]

[0054] Each of these promoter-replaced strains was inoculated into a test tube containing each of the culture medium 3 (51_) and shake-cultured at 30 ^° C for 3 days. The obtained culture solutions were mixed with 50% glycerol solution at a ratio of 1:1 (volume ratio), and frozen and stored as glycerol stock at 180°.

[Cultivation of bacterial cells]

Glycerol stock of each strain (1) was added to each of the 3 culture media (5

1_) was inoculated into a test tube. In the same manner as in Example 1, the test tube was shake-cultured at 30 ^° 〇 for 3 days, and the obtained culture broth 3 !_ was added to the mouth culture medium 150 1 _ supplemented with the krill extract. I transferred to Sakaro Flask. Saka Lofrasco was shake-cultured at 30° for 3 days, and the obtained culture solution (150!_ each) was centrifuged in the same manner as in Example 1 and the supernatant was discarded to recover the bacterial cells. did.

The bacterial cells collected by centrifugation were frozen at 80°C and freeze-dried in the same manner as in Example 1 to obtain a dried product. The resulting dried product contained 0.2 to 0.5 mass% of mouth ! ^~ 18 per dry weight. 〇 2020/175 568 20 units (: 171-1?2020/007801

[0057] <Microscopic observation>

Each of the obtained yeast dry bodies (0.19) was suspended in water (1 !_). These samples were observed at 40x magnification using an optical microscope 〇<○1Cho [¾ 2 (manufactured by Olympus) and photographed using a digital camera 11-〇1\/1803 (manufactured by Olympus). , Got the image file.

[0058] <Image analysis>

The image file of the obtained micrograph was analyzed by image analysis software 1 01396" in the same manner as in Example 1 to obtain individual particles (including both aggregates and non-aggregated single cell microorganisms). Area value data was obtained. Table 4 shows the image analysis results for each strain. In the table, "particle", "total number of particles", "total area value", "1 200 ³ ³ 20", "100 ³ ³ 20", and "20>" mean the same as in Table 2. In addition, two independent trials were performed, the upper row of each strain shows the results of the first trial, and the lower row shows the results of the second trial.

[0059] [Table 4]

[0060] As shown in Table 4,

Compared with 1_〇 1 gene, !_〇

Promote the promoter of 10 gene or 1_〇 1 1 gene with strong transcription. 20/175568 21 卩 (: 171? 2020 /007801

The ratio of large particles with a diameter of 20 or more increased significantly in the 丨 888 strains, 丨〇 889 strains, 丨〇 893 strains, and 1 ◦ 891 strains that were replaced by Yuichi. It is speculated that this is because the aggregating ability was improved and the agglutination was promoted, and the number of non-aggregated yeasts decreased. In particular, in the 08989 and 0891 strains, the proportion of particles having a diameter of more than 100 is high, and the size of the aggregate can be regulated by controlling the transcription strength of the promoter of the gene involved in aggregation. Was confirmed.

The entire contents of the specification, claims and abstract of Japanese Patent Application No. 2019-035860 filed on Feb. 28, 2010 are cited herein as a disclosure of the specification of the present invention. , To be incorporated.

Claims

\¥0 2020/175568 22 卩(: 17 2020/007801 Claims

[Claim 1] Yeast, lactic acid bacteria, and aggregates of unicellular microorganisms selected from microalgae

A composition containing the non-aggregated unicellular microorganisms, wherein the total area of the unicellular microorganisms in an optical microscope image of the composition, the aggregate having a diameter within a range of 20 to 120 A feed composition, wherein the ratio of the total area of the lumps is 25% or more.

[Claim 2] The feed composition according to claim 1, wherein the ratio of the total area of the aggregates having the diameter within the range of 20 to 100 is 30% or more.

[Claim 3] The feed composition according to claim 1 or 2, wherein the unicellular microorganism is yeast.

[Claim 4] The feed composition according to any one of claims 1 to 3, wherein the unicellular microorganism is a transformant in which a gene involved in aggregation is modified.

[Claim 5] The gene involved in aggregation is a gene involved in aggregation promotion, and the modification of the gene is replacement with a promoter having a stronger transcriptional strength than the promoter of the endogenous gene. Item 4. The feed composition according to item 4.

6. The feed composition according to claim 4 or 5, wherein the transformant is a yeast transformant.

[Claim 7] The transformant is a transformant of Schizosaccharomyces cylinder.

The feed composition according to claim 5, wherein the gene involved in the promotion of endogenous aggregation is the 93 2 gene.

[Claim 8] The transformant is a Satsucaromyces cerevisiae transformant

The feed composition according to claim 5, wherein the gene involved in promoting endogenous aggregation is the !_ 0 gene.

[Claim 9] The gene involved in aggregation is a gene involved in aggregation inhibition, and the modification of the gene is a deletion of the endogenous gene, or a promoter contained in the endogenous gene. The composition for feed according to claim 4, which is also a substitution of a promoter having a low transcription strength. 〇 2020/175 568 23 卩 (：171? 2020 /007801

10. The feed composition according to claim 9, wherein the transformant is a Schizosaccharomyces cylinder cylinder transformant, and the gene involved in the suppression of endogenous aggregation is the V 91 gene. ..

[Claim 11] The feed composition according to any one of claims 1 to 10, which contains docosahexaenoic acid or eicosapentaenoic acid in an amount of 0.1% by mass or more based on the dry weight of the composition. ..

[Claim 12] A method for breeding fish or crustaceans, which comprises feeding the composition for feed according to any one of claims 1 to 11 as feed to the larvae of fish or juveniles of crustaceans.

[Claim 13] A microbial aggregate comprising an aggregate of single-cell microorganisms selected from yeast, lactic acid bacteria, and microalgae and the single-cell microorganisms that are not aggregated, wherein the single-cell microorganisms in an optical microscope image of the microorganism aggregate are Use of the microbial aggregate in feed, wherein the ratio of the total area of aggregates having a diameter within the range of 20 to 120 to the total area is 25% or more.

[Claim 14] A gene involved in the aggregation of a single-cell microorganism is replaced with a promoter having a transcriptional strength different from that of a promoter of the endogenous gene, the gene is deleted, or the foreign gene is replaced with a genome. A method for preparing an aggregate of unicellular microorganisms, which comprises adjusting the size of an aggregate of unicellular microorganisms within a desired range by incorporating the above into a.

[Claim 15] A fish or shellfish, which is bred by feeding to a fish larva or a crustacean juvenile the agglutinate prepared by the method for preparing an aggregate of single-cell microbes according to claim 14. Breeding method.