JP2016054703A - Lauric acid production method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a lauric acid production method using diacylglycerol acyl transferase derived from algae, and to provide a transformant for use in the method.SOLUTION: The invention provides a lauric acid production method comprising the following steps (1) and (2): (1) a step of carrying out co-introduction of a gene encoding acyl ACP thioesterase and a gene encoding the following protein (A) or (B) into a host to obtain a transformant: (A) a protein consisting of an amino acid sequence represented by a specific sequence having diacylglycerol acyl transferase activity derived from algae; (B) a protein consisting of an amino acid sequence having 81% or more identity with the specific amino acid sequence in (A), and having diacylglycerol acyl transferase activity; (2) a step of collecting lauric acid from the obtained transformant.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing lauric acid using diacylglycerol acyltransferase.

  Lauric acid is a fatty acid used as a surfactant or chemical base, and is mainly produced from coconut oil and palm kernel oil. However, coconut and oil palm, which are raw materials for palm oil and palm kernel oil, have limited cultivation areas, and it is difficult to ensure a sufficient amount. Therefore, it is desired to produce lauric acid from raw materials other than coconut and oil palm.

Triacylglycerol is a compound in which three molecules of fatty acid are ester-bonded to one molecule of glycerol. There are various types of fatty acids that bind to glycerol, and various triacylglycerol compounds are produced depending on the combination of fatty acids that bind. In vivo, triacylglycerol mainly acts as an energy storage substance and is accumulated in animal adipose tissue and plant seeds. In addition, it is known that algae have a large amount of lipid accumulation such as triacylglycerol compared to other organisms.
In vivo, triacylglycerol is synthesized by transferring an acyl group from diacylglycerol to acyl-CoA derived from a fatty acid. This transfer reaction involves diacylglycerol acyltransferase (DGAT). DGAT exists in mammals, plants, fungi, and the like, and it is known that there are two types, DGAT1 and DGAT2. Among algae, for example, Chlamydomonas reinhardtii is known to have DGAT1 and four types of DGAT2 (DGAT2-1, 2-2, 2-3, 2-4). Among these, it has been reported that DGAT2-1 to 2-3 increase the amount of mRNA under nitrogen-deficient conditions (Non-patent Document 1).

Attempts have also been made to produce triacylglycerol and fatty acids using DGAT. For example, a method for producing triacylglycerol using Chlamydomonas reinhardtii in which DGAT2 is overexpressed has been proposed (Patent Document 1).
There are various types of fatty acyl-CoA as a substrate for DGAT, and it is considered that DGAT determines what kind of carbon number / unsaturation acyl group is introduced into diacylglycerol. Many of the DGATs reported so far are those that selectively introduce a long-chain acyl group. For example, a method has been proposed in which the amount of the ultralong- chain polyunsaturated fatty acid in the plant is changed by introducing the DGAT gene of Talassiosira psuedonana into the plant (Patent Document 2). Patent Document 3 describes DGAT derived from Nannochloropsis oculata, suggesting the possibility of being involved in the production of C18 and C22 long-chain or ultralong- chain polyunsaturated fatty acids. On the other hand, there is almost no report about DGAT that selectively introduces a medium chain acyl group.

International Publication No. 2011/156520 International Publication No. 2009/085169 International Publication No. 2011-161093

Rachel Miller et al., "Changes in Transcript Abundance in Chlamydomonas reinhardtii following Nitrogen Deprivation Predict Diversion of Metabolism", Plant Physiology, 2010, Vol.154, p.1737-1752

  The present invention relates to a method for producing lauric acid using algae-derived diacylglycerol acyltransferase. The present invention also relates to the provision of a transformant in which a diacylglycerol acyltransferase gene derived from algae is introduced to produce a triacylglycerol having a high lauric acid content and has an improved lauric acid producing ability.

The present inventors have studied algae-derived diacylglycerol acyltransferase and found a diacylglycerol acyltransferase that selectively incorporates lauric acid into diacylglycerol from algae belonging to the genus Nannochloropsis . And when the host was transformed using this, it discovered that the lauric acid content in a triacylglycerol improved significantly in a transformant. The present invention has been completed based on these findings.

That is, this invention relates to the manufacturing method (henceforth "the manufacturing method of this invention") of lauric acid including the process of following (1) and (2).
(1) A step of obtaining a transformant by co-introducing a gene encoding an acyl-ACP thioesterase and a gene encoding the following protein (A) or (B) into a host (A) represented by SEQ ID NO: 1 A protein comprising the amino acid sequence (B) a protein comprising the amino acid sequence having 81% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having diacylglycerol acyltransferase activity (2) transformant obtained For collecting lauric acid from sewage

  The present invention also relates to a transformant obtained by co-introducing a gene encoding an acyl-ACP thioesterase and a gene encoding the protein (A) or (B) (hereinafter referred to as “the present invention”). Also referred to as “transformant”.

  In addition, the present invention improves the lauric acid content in the triacylglycerol produced by the transformant, including the step of introducing the gene encoding the protein (A) or (B) into the host for transformation. It relates to the method of making it.

  The transformant of the present invention has a diacylglycerol acyltransferase gene introduced therein and can produce triacylglycerol with a high content of lauric acid. The production method of the present invention using the transformant is excellent in the productivity of lauric acid or triacylglycerol using this as a constituent fatty acid.

The figure which shows the result of carrying out TLC expansion | deployment of the lipid component extracted from BY4741 strain (WT strain), (DELTA) 4 strain, + DGA1 strain, + NoDGAT2-8 strain, and + AtDGAT strain cultured in the induction | guidance | derivation culture medium (YPG) in Example 1. It is.

In the present invention, lipids include simple lipids, complex lipids, and derived lipids. Specifically, in addition to neutral fats such as triacylglycerol, hydrocarbons such as fatty acids, aliphatic alcohols, and alkanes. , Wax, ceramide, phospholipid, glycolipid, sulfolipid and the like.
In the present invention, in the notation of fatty acids and acyl groups constituting fatty acids, Cx: y means that the number of carbon atoms is x and the number of double bonds is y, and Cx is a fatty acid having x number of carbon atoms. Or an acyl group.
In the present invention, the medium chain in the medium chain fatty acid or the medium chain acyl-ACP means that the acyl group has 6 to 14 carbon atoms.

1. Diacylglycerol acyltransferase The diacylglycerol acyltransferase used in the present invention is a protein having the amino acid sequence represented by SEQ ID NO: 1, and a protein functionally equivalent to the protein. Specifically, the diacylglycerol acyltransferase used in the present invention includes the following protein (A) or (B).
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 1 (B) A protein comprising an amino acid sequence having 81% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having diacylglycerol acyltransferase activity

The protein consisting of the amino acid sequence of SEQ ID NO: 1 is a diacylglycerol acyltransferase derived from Nannochloropsis oculata , an algae belonging to the genus Nannochloropsis .
Diacylglycerol acyltransferase (diglyceryl acyltransferase, DGAT) is an enzyme involved in the final steps of triacylglycerol synthesis. In plants and algae, various acyl-ACPs are synthesized in a fatty acid synthesis system in chloroplasts, and free fatty acids are excised from the acyl-ACPs by the enzyme acyl-ACP thioesterase. Free fatty acids are converted to acyl-CoA and subjected to a triacylglycerol synthesis system. In the triacylglycerol synthesis system, the acyl group of acyl-CoA is introduced into the glycerol skeleton. In the final stage, the acyl group is transferred from acyl-CoA to diacylglycerol to synthesize triacylglycerol. An enzyme that catalyzes this reaction is DGAT.
Thus, triacylglycerol has three acyl groups (fatty acid residues) introduced into the glycerol skeleton, but the types of acyl groups (number of carbon atoms and degree of unsaturation) to be introduced are diverse. It is considered that it is the acyltransferase that works in each reaction step that determines what carbon number and degree of unsaturation to introduce into the glycerol skeleton. That is, at the final stage of triacylglycerol synthesis, it is considered that the type of fatty acyl-CoA that binds to diacylglycerol changes depending on the substrate specificity of DGAT.

The protein (A) or (B) has diacylglycerol acyltransferase activity. In the present invention, “diacylglycerol acyltransferase activity” of a protein refers to an activity of acylating diacylglycerol (DAG) to convert it into triacylglycerol (TAG).
The protein having diacylglycerol acyltransferase activity can be confirmed by a system using a triacylglycerol synthetic gene-deficient strain. Examples of the triacylglycerol synthetic gene-deficient strain include the yeast Δdga1, Δlro1, Δare1, and Δare2 strains (Δ4 strain) used in the examples. If the gene encoding the target protein is expressed in the deficient strain and the production of triacylglycerol is confirmed, it can be determined that the protein has diacylglycerol acyltransferase activity.

The diacylglycerol acyltransferase (A) has a property of preferentially incorporating a C12: 0 acyl group (lauroyl group) into diacylglycerol, as will be described in Examples below. That is, it is a diacylglycerol acyltransferase exhibiting substrate specificity for lauroyl-CoA.
Regarding the lauroyl-CoA specificity of diacylglycerol acyltransferase, for example, a cell can be used under the conditions in which a diacylglycerol acyltransferase gene and an acyl-ACP thioesterase gene that produces acyl-CoA are introduced into a host cell and these transgenes are expressed. And then analyzing the change in fatty acid composition in the host cell or culture medium using a method such as gas chromatography analysis, or mixing purified diacylglycerol acyltransferase protein with acyl-CoA and diacylglycerol The triacylglycerol is isolated from the reacted solution by thin layer chromatography, etc., and the change in fatty acid composition in the triacylglycerol is analyzed using a method such as gas chromatography analysis. It can be confirmed by that method.

From the viewpoint of improving the productivity of lauric acid, the protein (B) is also preferably a protein having lauroyl-CoA-specific diacylglycerol acyltransferase activity. From this viewpoint, the identity with the amino acid sequence represented by SEQ ID NO: 1 in (B) is preferably 85% or more, more preferably 90% or more, and further preferably 95% or more. Preferably, it is 96% or more, 97% or more, 98% or more, or 99% or more.
In the present invention, the identity of amino acid sequences and base sequences is calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)). Specifically, it is calculated by performing analysis with a unit size to compare (ktup) of 2 using a homology search program of genetic information software Genetyx-Win (software development).

As the amino acid sequence of the protein of (B), an amino acid sequence in which a mutation is introduced into the amino acid sequence of SEQ ID NO: 1, that is, one or several amino acids in the amino acid sequence of SEQ ID NO: 1 are deleted, substituted, inserted or added. Also preferred are amino acid sequences. From the viewpoint of diacylglycerol acyltransferase activity, one or several mutant amino acids are preferably 1 to 50, more preferably 1 to 20, more preferably 1 to 10, and more preferably 1 to 5 More preferably, 1 to 2 is particularly preferable.
In the present specification, “addition” of an amino acid or base includes addition of an amino acid or base to one end and both ends of the sequence.

There is no restriction | limiting in particular about the acquisition method of the protein mentioned above, It can obtain by the chemical or genetic engineering method etc. which are performed normally. For example, a protein derived from a natural product can be obtained by isolation and purification from Nannochloropsis oculata. In addition, protein synthesis may be performed by chemical synthesis, or a recombinant protein may be produced by a gene recombination technique. When producing a recombinant protein, the diacylglycerol acyltransferase gene described later can be used.
Examples of the method for introducing mutation such as deletion, substitution, insertion, addition, etc. into the amino acid sequence include a method of introducing mutation into the base sequence encoding the amino acid sequence. A method for introducing a mutation into the base sequence will be described later.

2. Diacylglycerol acyltransferase gene The diacylglycerol acyltransferase gene used in the present invention is a gene encoding the protein (A) or (B).
An example of a gene encoding the amino acid sequence shown in SEQ ID NO: 1 is the base sequence shown in SEQ ID NO: 2. The base sequence shown in SEQ ID NO: 2 is an example of a base sequence of a gene encoding a wild-type diacylglycerol acyltransferase derived from Nannochloropsis oculata.

Specific examples of the gene encoding the protein (A) or (B) include the following genes (a) or (b), but the present invention is not limited thereto.
(A) DNA comprising the base sequence represented by SEQ ID NO: 2
(B) a DNA encoding a protein comprising a base sequence having 81% or more identity with the base sequence represented by SEQ ID NO: 2 and having diacylglycerol acyltransferase activity

  As shown in the Examples described later, the gene (a) consisting of DNA is a gene encoding lauroyl-CoA specific diacylglycerol acyltransferase. The gene comprising the DNA of (b) is also preferably a gene encoding a lauroyl-CoA specific diacylglycerol acyltransferase. From this viewpoint, in (b), the identity with the base sequence represented by SEQ ID NO: 2 is preferably 85% or more, more preferably 90% or more, and 95% or more. More preferably, it is 96% or more, 97% or more, 98% or more, or 99% or more.

  In addition, as the base sequence of (b), one or several bases are deleted, substituted, inserted, or added in the base sequence obtained by introducing a mutation into the base sequence of SEQ ID NO: 2, that is, the base sequence of SEQ ID NO: 2. Also preferred are base sequences. From the viewpoint of diacylglycerol acyltransferase activity, one or several mutant bases are preferably 1-20, more preferably 1-10, even more preferably 1-5, and more preferably 1-3. More preferably, 1 to 2 is particularly preferable.

  Examples of methods for introducing mutations such as deletion, substitution, insertion and addition into the base sequence include site-specific mutagenesis. As a specific method for introducing site-specific mutation, a method using a Splicing overlap extension (SOE) PCR reaction (Horton et al., Gene 77, 61-68, 1989), an ODA method (Hashimoto-Gotoh et al. , Gene, 152, 271-276, 1995), Kunkel method (Kunkel, TA, Proc. Natl. Acad. Sci. USA, 1985, 82, 488). Also, commercially available kits such as Site-Directed Mutagenesis System Mutan-SuperExpress Km Kit (Takara Bio), Transformer TM Site-Directed Mutagenesis Kit (Clonetech), KOD-Plus-Mutagenesis Kit (Toyobo) may be used. it can. Moreover, after giving a random gene mutation, the target gene can also be obtained by performing enzyme activity evaluation and gene analysis by an appropriate method.

  The method for obtaining the diacylglycerol acyltransferase gene is not particularly limited, and can be obtained by ordinary genetic engineering techniques. For example, based on the amino acid sequence shown in SEQ ID NO: 1 or the base sequence shown in SEQ ID NO: 2, a diacylglycerol acyltransferase gene can be obtained by artificial synthesis. For the artificial synthesis of genes, for example, services such as Invitrogen can be used. It can also be obtained by cloning from Nannochloropsis oculata, for example, by the method described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press (2001)]. Can do.

3. Acyl-ACP thioesterase The transformant of the present invention is co-introduced with a gene encoding the protein (A) or (B) above and a gene encoding another enzyme involved in the synthesis of triacylglycerol or lauric acid. It is preferable that Examples of other enzymes involved in the synthesis of triacylglycerol and lauric acid include lysophosphatidic acid acyltransferase, β-ketoacyl-ACP synthase, and acyl-ACP thioesterase.
Lysophosphatidic acid acyltransferase is an enzyme that catalyzes an intermediate reaction in triacylglycerol synthesis, and phosphatidic acid is synthesized by transferring an acyl group from a specific acyl-CoA to lysophosphatidic acid.
β-ketoacyl-ACP synthase is an enzyme that catalyzes the decarboxylation condensation reaction of malonyl ACP and acyl ACP in the fatty acid synthesis pathway. In the fatty acid synthesis pathway, carbon chain elongation reaction is repeated using malonyl-ACP to synthesize acyl-ACPs (complexes of acyl groups and ACPs that are fatty acid residues) of various chain lengths. β-ketoacyl-ACP synthase is one of the factors responsible for chain length control of acyl groups (how far the chain length of acyl-ACP is extended) in this pathway.
Acyl-ACP thioesterase is an enzyme that hydrolyzes the thioester bond of acyl-ACP synthesized in a fatty acid synthesis system to produce free fatty acid. By the action of acyl-ACP thioesterase, fatty acid synthesis on ACP is completed, and the cut fatty acid is subjected to synthesis of triacylglycerol and the like.
By introducing genes encoding these enzymes together with the DGAT gene into the host, it is possible to further improve the triacylglycerol productivity and the lauric acid productivity of the transformant.

In particular, the transformant of the present invention is preferably co-introduced with a gene encoding acyl-ACP thioesterase.
The acyl-ACP thioesterase used in the present invention may be a protein having acyl-ACP thioesterase activity. In the present invention, “having acyl-ACP thioesterase activity” means having an activity of hydrolyzing the thioester bond of acyl-ACP.

The acyl-ACP thioesterase has a plurality of acyl-ACP thioesterases having different reaction specificities depending on the number of carbon atoms and the number of unsaturated bonds of the acyl group (fatty acid residue) constituting the substrate acyl-ACP. It is known that it is an important factor that determines the fatty acid composition in vivo.
As described above, since the protein (A) is a diacylglycerol acyltransferase that selectively incorporates a lauroyl group (C12: 0) into diacylglycerol, the acyl-ACP thioesterase to be co-introduced is also a medium chain acyl. A thioesterase specific for ACP (hereinafter referred to as “medium chain specific acyl-ACP thioesterase”) is preferable, and a thioesterase specific for lauroyl-ACP (hereinafter also referred to as “lauroyl-ACP specific thioesterase”) Is more preferable. In the present invention, the “medium chain acyl-ACP-specific” acyl-ACP thioesterase is an acyl-ACP thioesterase having an activity of selectively hydrolyzing a thioester bond of acyl-ACP having 6 to 14 carbon atoms. . By using such an acyl-ACP thioesterase, the productivity of lauric acid can be further improved.

In the present invention, as the acyl-ACP thioesterase, a known acyl-ACP thioesterase or a protein functionally equivalent to them can be used. The acyl-ACP thioesterase to be used can be appropriately selected according to the type of the host.
Specifically, Umbellularia californica acyl-ACP thioesterase (GenBank AAA34215.1); Cuphea calophylla subsp. Mesostemon acyl-ACP thioesterase (GenBank ABB71581); Cocos nucifera acyl-ACP thioesterase (CnFatB3: Jing et al. BMC Biochemistry 2011, 12:44); Cinnamomum camphora acyl-ACP thioesterase (GenBank AAC49151.1); Myristica fragrans acyl-ACP thioesterase (GenBank AAB71729); Myristica fragrans acyl-ACP thioesterase (GenBank AAB71730); Cuphea lanceolata acyl- ACP thioesterase (GenBank CAA54060); Cuphea hookeriana acyl-ACP thioesterase (GenBank Q39513); Ulumus americana acyl-ACP thioesterase (GenBank AAB71731); Sorghum bicolor acyl-ACP thioesterase (GenBank EER87824); Sorghum bicolor acyl-ACP Thioesterase (GenBank EER88593); Cocos nucifera acyl-ACP thioesterase (see CnFatB1: Jing et al. BMC Biochemistry 2011, 12:44); Cocos nucifera acyl-ACP thioesterase (CnFatB2: Jing et al. BMC Biochemistry 2011, 12 Cuphea viscosissima acyl-ACP thioesterase (CvFatB1: Jing et al. BMC Biochemistry 2011, 12:44); Cuphea viscosissima acyl-ACP thioesterase (CvFatB2: Jing et al. BMC Biochemistry 2011, 12:44) Cuphea viscosissima acyl-ACP thioesterase (CvFatB3: see Jing et al. BMC Biochemistry 2011, 12:44); Elaeis guineensis acyl-ACP thioesterase (GenBank AAD42220); Desulfovibrio vulgaris acyl-ACP thioesterase (GenBank ACL08376) Bacteriodes fragilis acyl-ACP thioesterase (GenBank CAH09236); Parabacteriodes distasonis acyl ACP thioesterase (GenBank ABR43801); Bacteroides thetaiotaomicron acyl-ACP thioesterase (GenBank AAO77182); Clostridium asparagiforme acyl-ACP thioesterase (GenBank EEG55387); Bryanthella formatexigens acyl-ACP thioesterase (GenBank EET61113); Geobacillus sp. Thioesterase (GenBank EDV77528); Streptococcus dysgalactiae acyl-ACP thioesterase (GenBank BAH81730); Lactobacillus brevis acyl-ACP thioesterase (GenBank ABJ63754); Lactobacillus plantarum acyl-ACP thioesterase (GenBank CAD63310); Anaerococcus tetradius (GenBank EEI82564); Bdellovibrio bacteriovorus acyl -ACP thioesterase (GenBank CAE80300); Clostridium thermocellum acyl -ACP thioesterase (GenBank ABN54268) Nannochloropsis oculata acyl -ACP thioesterase (SEQ ID NO: 7, gene nucleotide sequence encoding it: SEQ ID NO: 8); Nannochloropsis gaditana acyl -ACP thioesterase (SEQ ID NO: 9, the gene of the nucleotide sequence encoding it: SEQ ID NO: 10 ); Nannochloropsis granulata acyl-ACP thioesterase (SEQ ID NO: 11, nucleotide sequence of the gene encoding this: SEQ ID NO: 12); Symbiodinium microadriaticum acyl-ACP thioesterase (SEQ ID NO: 13, nucleotide sequence of the gene encoding this: sequence) No. 14), etc.
Moreover, as a protein functionally equivalent to these, the amino acid sequence of any of the acyl-ACP thioesterases described above is 50% or more, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, Even more preferably, a protein comprising an amino acid sequence having 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity and having acyl-ACP thioesterase activity can also be used.

Among the acyl-ACP thioesterases mentioned above, a medium chain specific acyl-ACP thioesterase is more preferable. Specifically, Umbellularia californica acyl-ACP thioesterase (SEQ ID NO: 3, nucleotide sequence of gene encoding the same: sequence) No. 4), Cocos nucifera acyl-ACP thioesterase (SEQ ID NO: 5, nucleotide sequence of the gene encoding it: SEQ ID NO: 6), Nannochloropsis oculata acyl-ACP thioesterase (SEQ ID NO: 7), Nannochloropsis gaditana acyl-ACP thioesterase (SEQ ID NO: 9), Nannochloropsis granulata acyl-ACP thioesterase (SEQ ID NO: 11), Symbiodinium microadriaticum acyl-ACP thioesterase (SEQ ID NO: 13), or the amino acid sequence of these acyl-ACP thioesterases, preferably 50% or more, preferably 7 % Or more, more preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, And proteins having medium chain specific acyl-ACP thioesterase activity.
Furthermore, Umbellularia californica acyl-ACP thioesterase (SEQ ID NO: 3), Symbiodinium microadriaticum acyl-ACP thioesterase (SEQ ID NO: 13), or the amino acid sequences of these acyl-ACP thioesterases having high specificity for lauroyl-ACP 50% or more, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, still more preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the same More preferred is a protein consisting of an amino acid sequence having a property and having acyl-ACP thioesterase activity.
The sequence information of these acyl-ACP thioesterases and the genes encoding them can be obtained from, for example, the National Center for Biotechnology Information (NCBI).

When the amino acid sequence of each acyl-ACP thioesterase described above has a chloroplast translocation signal sequence, the signal sequence may be deleted and introduced into the host. For example, when a foreign acyl-ACP thioesterase gene is introduced into a host, it is preferable to use a DNA comprising a base sequence in which the sequence of the region corresponding to the chloroplast transition signal sequence is deleted from the base sequence of the gene.
Examples of the acyl-ACP thioesterase chloroplast transition signal sequence include the amino acid sequence at positions 1 to 83 of SEQ ID NO: 3, or a region corresponding thereto.
In the present specification, the “corresponding position” or “corresponding region” in an amino acid sequence or base sequence is the maximum homology to a conserved amino acid residue present in each amino acid sequence by comparing the target amino acid sequence with a reference sequence. Can be determined by aligning the sequences to give The alignment can be performed using known algorithms, the procedures of which are known to those skilled in the art. For example, the alignment can be performed manually based on the above-mentioned Lippman-Person method or the like, but the Clustal W multiple alignment program (Thompson, JD et al, (1994) Nucleic Acids Res. 22, p. 4673-4680) with default settings. Clustal W is, for example, the European Bioinformatics Institute (EBI, [www.ebi.ac.uk/index.html]) and the Japan DNA Data Bank (DDBJ, [DDBJ, [ www.ddbj.nig.ac.jp/Welcome-j.html]).

The acyl-ACP thioesterase used in the present invention is more preferably the following protein (C) or (D).
(C) A protein comprising the amino acid sequence from position 84 to position 382 of SEQ ID NO: 3 (D) An amino acid sequence having 70% or more identity with the amino acid sequence of the protein (C), and an acyl-ACP thioesterase Active protein

  In the above (D), from the viewpoint of acyl-ACP thioesterase activity, the identity with the amino acid sequence of the protein of (C) is preferably 80% or more, more preferably 85% or more, 90 % Or more, more preferably 95% or more, particularly preferably 96% or more, 97% or more, 98% or more, or 99% or more.

  In addition, as the amino acid sequence of the protein of (D), an amino acid sequence obtained by introducing a mutation into the amino acid sequence of the protein of (C), that is, one or several amino acids are deleted or replaced in the amino acid sequence of the protein of (C) Also preferred are amino acid sequences inserted, or added. From the viewpoint of acyl-ACP thioesterase activity, one or several mutant amino acids are preferably 1 to 50, more preferably 1 to 20, more preferably 1 to 10, and 1 to 5 Even more preferred is 1-2, especially preferred.

As an example of the gene encoding the protein (C) or (D), a gene comprising the following DNA (c) or (d) can be mentioned.
(C) DNA comprising a base sequence from position 250 to position 1149 of SEQ ID NO: 4
(D) DNA comprising a nucleotide sequence having 70% or more identity with the DNA sequence of (c) and encoding a protein having acyl-ACP thioesterase activity

In the above (d), from the viewpoint of acyl-ACP thioesterase activity, the identity with the DNA base sequence of (c) is preferably 80% or more, more preferably 85% or more, and 90% More preferably, it is more preferably 95% or more, and particularly preferably 96% or more, 97% or more, 98% or more, or 99% or more.
In addition, as the base sequence of (d), a base sequence obtained by introducing mutation into the DNA base sequence of (c), that is, one or several bases in the DNA base sequence of (c) is deleted, substituted, or inserted. Or an added base sequence is also preferred. From the viewpoint of acyl-ACP thioesterase activity, one or several mutant bases are preferably 1-20, more preferably 1-10, even more preferably 1-5, and 1-3 Even more preferred is 1-2, especially preferred.

The fact that a protein has an acyl-ACP thioesterase activity or a medium chain specific acyl-ACP thioesterase activity is, for example, a fusion gene in which an acyl-ACP thioesterase gene is linked downstream of a promoter that functions in a host cell such as E. coli. Is introduced into a host cell deficient in the fatty acid degradation system, cultured under conditions where the introduced acyl-ACP thioesterase gene is expressed, and changes in the fatty acid composition in the host cell or culture solution are analyzed by gas chromatography analysis, etc. It can be confirmed by using and analyzing.
In addition, a fusion gene in which an acyl-ACP thioesterase gene is linked downstream of a promoter that functions in a host cell such as E. coli is introduced into the host cell, and the cells are cultured under conditions such that the introduced acyl-ACP thioesterase gene is expressed. Later, the method of Yuan et al. (Yuan L, Voelker TA, Hawkins DJ. “Modification of the substrate specificity of an acyl-acyl carrier protein thioesterase by protein engineering” Proc Natl Acad Sci US A. 1995 Nov 7; 92 (23), p.10639-10643), by carrying out reactions using various acyl-ACPs as substrates, the acyl-ACP thioesterase activity can be measured.

  Acyl-ACP thioesterase and methods for obtaining the genes encoding them are not particularly limited, and can be obtained by chemical or genetic engineering techniques that are usually performed. For example, it can be artificially synthesized based on each amino acid or base sequence. Moreover, you may isolate from a living body according to a normal method. Gene cloning can be performed, for example, by the method described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press (2001)].

4). Transformant (recombinant)
The transformant of the present invention is obtained by introducing a gene encoding the protein (A) or (B) into a host, preferably a gene encoding the protein (A) or (B) Co-introduced with a gene encoding acyl-ACP thioesterase. In the transformant, the ability to produce triacylglycerol (triacylglycerol having a lauroyl group) containing lauric acid as a constituent fatty acid is significantly improved as compared with the host. Therefore, the amount of lauric acid obtained from triacylglycerol increases. Therefore, lauric acid can be efficiently produced by using the transformant.
The productivity of the lauric acid and triacylglycerol of the host and transformant, and the composition of the fatty acid constituting the triacylglycerol can be measured by the method used in the examples.

  The transformant of the present invention can be obtained by introducing a gene encoding the protein (A) or (B) into a host by an ordinary genetic engineering method. Specifically, an expression vector or gene expression capable of expressing in a host cell a gene encoding the protein (A) or (B), preferably a gene comprising the DNA (a) or (b). It can be produced by preparing a cassette, introducing it into a host cell, and transforming the host cell.

  A transformant cotransfected with a gene encoding an acyl-ACP thioesterase, preferably a medium chain specific acyl-ACP thioesterase, can also be produced in the same manner.

The host of the transformant is not particularly limited, and microorganisms, plants, or animals can be used. In the present invention, the microorganism includes algae and microalgae. From the viewpoint of production efficiency and availability of lauric acid, the host is preferably a microorganism or a plant, and more preferably a microorganism.
The microorganism may be either prokaryotic or eukaryotic.
As prokaryotes, microorganisms belonging to the genus Escherichia or microorganisms belonging to the genus Bacillus can be used.
As eukaryotic microorganisms, it can be used yeasts and filamentous fungi such as, Sakkamaisesu (Saccharomyces) genus Kluyveromyces (Kluyveromyces) genus Schizosaccharomyces (Schizosaccharomyces) genus Zygosaccharomyces (Zygosaccharomyces) genus Yarrowia (Yarrowia) genus Hansenula (Hansenula) spp, Pichia (Pichia) sp., Candida (Candida) spp., or it is preferable to use a Moruchiera (Mortierella) microorganisms belonging to the genus. Among them, Saccharomyces cerevisiae (Saccharomyces cerevisiae), Yarrowia lipolytica (Yarrowia lipolytica), red yeast (Rhodosporidium toruloides), or Moruchiera alpina (Mortierella alpina), more preferably, Saccharomyces cerevisiae (Saccharomyces cerevisiae) is more preferred.

Further, as the microorganism, microalgae are also preferable. The microalgae, from the viewpoint of gene recombination techniques has been established, Chlamydomonas (Chlamydomonas) algae belonging to the genus Chlorella (Chlorella) algae belonging to the genus faecium Oda Kuti ram (Phaeodactylum) algae belonging to the genus or Nan'nokuroro Algae belonging to the genus Psys is preferred, and algae belonging to the genus Nannochloropsis is more preferred. Specific examples of algae belonging to the genus Nannochloropsis include Nannochloropsis oculata , Nannochloropsis gaditana , Nannochloropsis salina , Nannochloropsis oceanica , Nannochloropsis atomus , Nannochloropsis maculata , Nannochloropsis granulata , or Nannochloropsis sp. , Etc. Among these, from the viewpoint of lauric acid productivity, Nannochloropsis oculata or Nannochloropsis gaditana is preferable, and Nannochloropsis oculata is more preferable.
The plant body is preferably Arabidopsis thaliana , rapeseed, coconut palm, palm, coffea, sunflower, soybean, corn, rice, sunflower, camphor, or jatropha, more preferably Arabidopsis thaliana , from the viewpoint of high lipid content in the seed. preferable.

As a host vector for an expression vector, a gene encoding the protein (A) or (B) or an acyl-ACP thioesterase gene can be introduced into the host, and the gene can be expressed in the host cell. Any vector may be used. For example, a vector having an expression regulatory region such as a promoter or terminator according to the type of host to be introduced, and a vector having a replication origin or a selection marker can be used. Further, it may be a vector that autonomously grows and replicates outside the chromosome, such as a plasmid, or a vector that is integrated into the chromosome.
As specific vectors, when a microorganism is used as a host, for example, pBluescript (pBS) II SK (-) (Stratagene), pSTV vector (Takara Bio), pUC vector (Takara Shuzo) ), PET vectors (Takara Bio), pGEX vectors (GE Healthcare), pCold vectors (Takara Bio), pHY300PLK (Takara Bio), pUB110 (Mckenzie, T. et al. (1986), Plasmid 15 (2); p.93-103), pBR322 (Takara Bio), pRS403 (Stratagene), or pMW218 / 219 (Nippon Gene).
When using algae as a host, for example, pUC19 (manufactured by Takara Bio Inc.), P66 (Chlamydomonas Center), P-322 (Chlamydomonas Center), pPha-T1 (Yangmin Gong, Xiaojing Guo, Xia Wan, Zhuo Liang, Mulan Jiang, “Characterization of a novel thioesterase (PtTE) from Phaeodactylum tricornutum”, Journal of Basic Microbiology, 2011 December, Volume 51, p.666-672.), Or pJET1 (manufactured by Cosmo Bio). In particular, when the host is an algae belonging to the genus Nannochloropsis, pPha-T1 or pJET1 is preferably used. When the host is an algae belonging to the genus Nannochloropsis, the literature description of Oliver Kilian, et al., Proceedings of the National Academy of Sciences of the United States of America, Dec 27; 108 (52), 2011. With reference to the above method, a host can also be transformed with a DNA fragment comprising the gene, promoter and terminator of the present invention. Examples of the DNA fragment include a PCR amplified DNA fragment and a restriction enzyme cleaved DNA fragment.
When a plant cell is used as a host, for example, a pRI vector (manufactured by Takara Bio Inc.), a pBI vector (manufactured by Clontech), or an IN3 vector (manufactured by Implanta Innovations) can be mentioned. In particular, when the host is Arabidopsis thaliana, pRI vectors or pBI vectors are preferably used.

The type of expression regulatory region such as a promoter or terminator used in an expression vector or gene expression cassette and the type of selectable marker are not particularly limited, and may be appropriately selected and used depending on the type of host into which a commonly used promoter or marker is introduced. Can do.
Specific promoters include, for example, lac promoter, trp promoter, tac promoter, trc promoter, T7 promoter, SpoVG promoter, cauliflower mozil virus 35 SRNA promoter, housekeeping gene promoter (eg, tubulin promoter, actin promoter, ubiquitin promoter) Etc.), rapeseed-derived Napin gene promoter, plant-derived Rubisco promoter, or promoter of violaxanthin / chlorophyll a-binding protein gene derived from the genus Nannochloropsis.
Selectable markers include antibiotic resistance genes (ampicillin resistance gene, chloramphenicol resistance gene, erythromycin resistance gene, neomycin resistance gene, kanamycin resistance gene, spectinomycin resistance gene, tetracycline resistance gene, blasticidin S resistance And drug resistance genes such as genes, bialaphos resistance genes, zeocin resistance genes, paromomycin resistance genes, or hygromycin resistance genes. Furthermore, it is also possible to use a gene defect associated with auxotrophy as a selection marker gene.

In addition, the acyl-ACP thioesterase gene expression vector preferably has a chloroplast translocation signal sequence. The chloroplast transition signal sequence is preferably a chloroplast transition signal sequence that functions in a host cell. For example, when an algae belonging to the genus Nannochloropsis is used as a host, a chloroplast transition signal sequence (SEQ ID NO: 18) derived from Nannochloropsis oculata can be used. The chloroplast transit signal sequence is preferably incorporated upstream of the acyl-ACP thioesterase gene.

Construction of an expression vector used for transformation by incorporating a gene encoding the protein (A) or (B) or an acyl-ACP thioesterase gene into the vector by a usual method such as restriction enzyme treatment or ligation. Can do.
The transformation method is not particularly limited as long as it is a method capable of introducing a target gene into a host. For example, a method using calcium ions, a general competent cell transformation method (J. Bacterial. 93, 1925 (1967)), a protoplast transformation method (Mol. Gen. Genet. 168, 111 (1979)), electro Polation method (FEMS Microbiol. Lett. 55, 135 (1990)) or LP transformation method (T. Akamatsu and J. Sekiguchi, Archives of Microbiology, 1987, 146, p.353-357; Taguchi, Bioscience, Biotechnology, and Biochemistry, 2001, 65, 4, p.823-829) and the like can be used. When the host is an algae belonging to the genus Nannochloropsis, transformation can also be performed using the electroporation method described in Randor Radakovits, et al., Nature Communications, DOI: 10.1038 / ncomms1688, 2012. When the host is a plant, methods using Agrobacterium (CR Acad. Sci. Paris. Life Science 316, 1194 (1993) etc.), particle gun method (BioRad PDS-1000 / He etc.) etc. can be used. it can.

  Selection of the transformant introduced with the target gene fragment can be performed by utilizing a selection marker or the like. For example, the drug resistance gene acquired by the transformant as a result of introducing a vector-derived drug resistance gene into the host cell together with the target DNA fragment at the time of transformation can be used as an indicator. The introduction of the target DNA fragment can also be confirmed by PCR method using a genome as a template.

5. Next, lauric acid or triacylglycerol containing lauric acid as a constituent component is produced using the transformant obtained above.
The method for producing lauric acid of the present invention comprises a step of collecting lauric acid from a transformant into which a gene encoding the protein (A) or (B) and a gene encoding acyl-ACP thioesterase are co-introduced. . From the viewpoint of improving the productivity of lauric acid, the step may include a step of culturing the transformant under appropriate conditions to obtain a culture, and a step of collecting lauric acid from the obtained culture. preferable. In addition, culture | cultivating a transformant in this invention means culture | cultivating and growing microorganisms, a plant body, an animal body, and those cells and structures | tissues, and cultivating a plant body in soil etc. is also included. The culture includes not only a culture solution but also a transformant after culturing.
The step of collecting lauric acid from the transformant preferably includes a step of collecting triacylglycerol from the transformant and a step of collecting a fatty acid mixture containing lauric acid from the obtained triacylglycerol. In the transformant of the present invention, lauric acid is mainly obtained as triacylglycerol containing lauric acid as a constituent component. By subjecting this triacylglycerol to a treatment such as hydrolysis, a fatty acid mixture containing lauric acid can be obtained. Further lauric acid may be isolated from the fatty acid mixture.
From the above viewpoint, the present invention can provide a method for producing triacylglycerol, which includes the step of collecting triacylglycerol from the transformant of the present invention. The step preferably includes a step of culturing the transformant under appropriate conditions to obtain a culture, and a step of collecting triacylglycerol from the obtained culture.

The culture conditions for the transformant can be appropriately selected depending on the host of the transformant, and the culture conditions usually used for the host can be used.
From the viewpoint of production efficiency of lauric acid or triacylglycerol, glycerol, acetic acid, malonic acid, or the like may be added to the medium as a precursor involved in the fatty acid biosynthesis system, for example.

  As an example, in the case of a transformant using yeast as a host, culturing in a YPG medium or an SD medium at 25 to 32 ° C. for 0.5 to 1 day can be mentioned.

When the host for transformation is algae, the following medium and culture conditions can be used.
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, and MNK medium. Among these, from the viewpoint of improving the productivity of lauric acid and triacylglycerol and the concentration of nutrient components, f / 2 medium, ESM medium, or Daigo IMK medium is preferable, f / 2 medium, or Daigo IMK medium is more preferable, and f A / 2 medium is more preferable. In order to promote the growth of algae and improve the productivity of lauric acid and triacylglycerol, a nitrogen source, a phosphorus source, a metal salt, vitamins, trace metals and the like can be appropriately added to the medium.
The amount of algae inoculated into the medium is not particularly limited, but is preferably 1 to 50% (vol / vol) and more preferably 1 to 10% (vol / vol) from the viewpoint of growth. Although culture | cultivation temperature will not be restrict | limited especially if it is a range which does not have a bad influence on the growth of algae, Usually, it is the range of 5-40 degreeC. From the viewpoint of promoting the growth of algae, improving the productivity of lauric acid and triacylglycerol, and reducing the production cost, the temperature is preferably 10 to 35 ° C, more preferably 15 to 30 ° C.
Moreover, it is preferable to culture algae under light irradiation so that photosynthesis is possible. The light irradiation may be performed under conditions that allow photosynthesis, and may be artificial light or sunlight. The illuminance at the time of light irradiation is preferably in the range of 100 to 50000 lux, more preferably in the range of 300 to 10000 lux, and still more preferably 1000 from the viewpoint of promoting the growth of algae and improving the productivity of lauric acid and triacylglycerol. It is in the range of ~ 6000 lux. Further, the light irradiation interval is not particularly limited, but from the same viewpoint as described above, it is preferably performed in a light-dark cycle, and the light period is preferably 8-24 hours, more preferably 10-18 hours, More preferably, it is 12 hours.
Moreover, it is preferable to perform culture | cultivation of algae in the presence of the gas containing carbon dioxide so that photosynthesis is possible, or the culture medium containing carbonates, such as sodium hydrogencarbonate. The concentration of carbon dioxide in the gas is not particularly limited, but from the viewpoint of promoting growth and improving the productivity of lauric acid or triacylglycerol, it is preferably 0.03 (same as atmospheric conditions) to 10%, more preferably 0.0. It is 05-5%, More preferably, it is 0.1-3%, More preferably, it is 0.3-1%. The concentration of the carbonate is not particularly limited. For example, when sodium bicarbonate is used, it is preferably 0.01 to 5% by mass, more preferably 0.05 from the viewpoint of promoting growth and improving the productivity of lauric acid and triacylglycerol. It is -2 mass%, More preferably, it is 0.1-1 mass%.
The culture time is not particularly limited, and may be performed for a long period (for example, about 150 days) so that algal bodies that accumulate lauric acid and triacylglycerol at a high concentration can grow at a high concentration. From the viewpoint of promoting the growth of algae, improving the productivity of lauric acid and triacylglycerol, and reducing the production cost, the culture period is preferably 3 to 90 days, more preferably 3 to 30 days, and further preferably 7 to 30. Days. In addition, culture | cultivation may be any of aeration stirring culture, shaking culture, or stationary culture, and a shaking culture is preferable from a viewpoint of an improvement in aeration.

Next, triacylglycerol is collected from the culture or transformant. This triacylglycerol contains a large amount of lauric acid as a constituent fatty acid. Triacylglycerol may be collected as a mixture with other lipid components.
Lauric acid is preferably collected from the triacylglycerol thus obtained or a lipid component containing the same. Lauric acid may be collected as a mixture with other fatty acids. Since the triacylglycerol obtained by the method of the present invention is rich in lauric acid, the fatty acid mixture obtained from the triacylglycerol is also rich in lauric acid.
As a method for collecting triacylglycerol or a lipid containing the same from a culture or a transformant, a method usually used for isolating lipid components in a living body, for example, filtration from a culture or a transformant. , Centrifugal separation, cell disruption, gel filtration chromatography, ion exchange chromatography, chloroform / methanol extraction method, hexane extraction method, ethanol extraction method, and the like, and methods for isolating and recovering lipid components. In the case of larger scale, triacylglycerol is obtained by performing general purification such as degumming, deoxidation, decolorization, dewaxing and deodorization after oil is recovered from the culture or transformant by pressing or extraction. Can do.
In order to obtain a fatty acid component from triacylglycerol, a usual method can be used. For example, a fatty acid can be obtained by hydrolyzing triacylglycerol. Specifically, a method of treating at a high temperature of about 70 ° C. in an alkaline solution, a method of treating with lipase, a method of decomposing using high-pressure hot water, etc. Isolation of lauric acid from the fatty acid mixture can also be performed by a usual method such as distillation.

  By using the production method of the present invention, it is possible to efficiently produce lauric acid and triacylglycerol containing this as a constituent.

  The lauric acid and triacylglycerol obtained by the production method of the present invention and the transformant are used as food, as well as emulsifiers such as cosmetics, detergents such as soaps and detergents, fiber treatment agents, hair rinse agents, disinfectants, It can be used as a preservative.

  In relation to the above-described embodiment, the present invention further discloses the following method and transformant.

<1> A method for producing lauric acid, comprising the following steps (1) and (2).
(1) A step of obtaining a transformant by co-introducing a gene encoding an acyl-ACP thioesterase and a gene encoding the following protein (A) or (B) into a host (A) represented by SEQ ID NO: 1 (B) 81% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 96% or more with the amino acid sequence represented by SEQ ID NO: 1 A protein having an amino acid sequence having 97% or more, 98% or more, or 99% or more identity and having diacylglycerol acyltransferase activity (2) A step of collecting lauric acid from the obtained transformant

<2> The production method according to <1>, wherein the acyl-ACP thioesterase is a medium-chain acyl-ACP-specific acyl-ACP thioesterase.
<3> The production method according to <1> or <2>, wherein the acyl-ACP thioesterase is a protein of the following (C) or (D).
(C) A protein comprising the amino acid sequence from position 84 to position 382 of SEQ ID NO: 3 (D) The amino acid sequence of the protein of (C) is 70% or more, preferably 80% or more, more preferably 85% or more, still more preferably Is an amino acid sequence having 90% or more, more preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity, and a protein having acyl-ACP thioesterase activity <4 > The production method according to any one of <1> to <3>, wherein the host is a microorganism or a plant.
<5> The production method according to <4>, wherein the microorganism is yeast.
<6> the yeast is Saccharomyces cerevisiae (Saccharomyces cerevisiae), <5> The method according to claim.
<7> The production method according to <4>, wherein the microorganism is a microalgae.
<8> The production method according to <7>, wherein the microalgae are algae belonging to the genus Nannochloropsis .
<9> The transformant according to <8>, wherein the alga belonging to the genus Nannochloropsis is Nannochloropsis oculata .
<10> The step (2) includes a step of collecting triacylglycerol from the transformant, and a step of collecting a fatty acid mixture containing lauric acid from the obtained triacylglycerol. <1> to <9> The manufacturing method in any one of.

<11> A transformant obtained by co-introducing a gene encoding an acyl-ACP thioesterase and a gene encoding the protein (A) or (B) into a host.

<12> The transformant according to <11>, wherein the acyl-ACP thioesterase is a medium-chain acyl-ACP-specific acyl-ACP thioesterase.
<13> The transformant according to <11> or <12>, wherein the acyl-ACP thioesterase is the protein of (C) or (D).
<14> The transformant according to any one of <11> to <13>, wherein the host is a microorganism or a plant.
<15> The transformant according to <14>, wherein the microorganism is yeast.
<16> the yeast is Saccharomyces cerevisiae (Saccharomyces cerevisiae), <15> transformant according to claim.
<17> The transformant according to <14>, wherein the microorganism is a microalgae.
<18> The transformant according to <17>, wherein the microalgae are algae belonging to the genus Nannochloropsis .
<19> The transformant according to <18>, wherein the algae belonging to the genus Nannochloropsis is Nannochloropsis oculata .

<20> A method for producing triacylglycerol, comprising the step of collecting triacylglycerol from the transformant according to any one of <11> to <19>.

<21> Improving the lauric acid content in the triacylglycerol produced by the transformant, including the step of introducing a gene encoding the protein (A) or (B) into the host and transforming the host. Method.

<22> Use of the transformant according to any one of <11> to <19> for producing lauric acid.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.

Medium composition f / 2 medium (75 mg NaNO ₃ , 6 mg NaH ₂ PO ₄ .2H ₂ O, 0.5 μg vitamin B12, 0.5 μg biotin, 100 μg thiamine, 10 mg Na ₂ SiO ₃ .9H ₂ O, 4.4 mg Na ₂ EDTA · 2H ₂ O, 3.16 mg FeCl ₃ .6H ₂ O, 12 μg FeCl ₃ .6H ₂ O, 21 μg ZnSO ₄ .7H ₂ O, 180 μg MnCl ₂ .4H ₂ O, 7 μg CuSO ₄ .5H ₂ O, 7 μg Na ₂ MoO ₄ .2H ₂ O / artificial seawater 1L)

Example 1 Verification of substrate specificity of nanatachloropsis oculata-derived DGAT Acquisition of DGAT gene derived from Nannochloropsis oculata After culturing Nannochloropsis oculata N-2145 strain in f / 2 medium, 10% inoculation was carried out in 50 mL of f / 2 medium, and carbon dioxide at 25 ° C. Culturing was carried out for 6 days in an artificial weather machine at a concentration of 0.3%. After the cultivation, the collected sample was crushed with a multi-bead shocker, and RNA was purified using RNeasy Plant Mini Kit (Qiagen). A cDNA library was prepared from the obtained total RNA using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen).
Using this cDNA as a template, a gene fragment consisting of the base sequence shown in SEQ ID NO: 2 was obtained by PCR reaction using primers 1 and 2 shown in Table 1-1. The resulting gene was named NoDGAT2-8 gene .

2. Preparation of a strain lacking the triacylglycerol synthesis gene A strain lacking all the triacylglycerol synthesis gene of yeast ( Saccharomyces cerevisiae ) was obtained by the following procedure. The triacylglycerol synthesis gene targets the DGA1, LRO1, ARE1, and ARE2 genes with reference to literature (Line Sandager, et al., J Biol Chem. Feb 22; 277 (8): 6478-82, 2002). did. The gene deletion was performed by a general technique by homologous recombination using an amino acid marker gene as a DNA fragment.

Colonies of Saccharomyces cerevisiae S288C strain were cultured for 24 hours under conditions of 5 ml of YPD medium (Yeast extract 1%, Peptone 2%, D-Glucose 2%), 30 ° C., 160 rpm. Gentolic DNA was extracted and purified from the obtained bacterial cells using Gen Toru-kun (for yeast) High Recovery (manufactured by Takara Bio Inc.).

A plasmid was constructed to disrupt the triacylglycerol synthesis gene. Using the genomic DNA of Saccharomyces cerevisiae obtained above as a template, a PCR fragment using primers 3 and 4 shown in Table 1-1 was used to obtain a gene fragment in the region from 350 bp upstream to 250 bp downstream of the ORF of the HIS3 gene. 6, or a gene fragment in the region of 500 bp upstream of the ARE1 gene or the region of 500 bp downstream was obtained by PCR reaction using primers 7 and 8. Further, the plasmid vector pUC19 (manufactured by Takara Bio Inc.) was used as a template, the pUC vector was amplified by PCR reaction using primers 9 and 10 shown in Table 1-1, and the template was obtained by treatment with restriction enzyme Dpn I (manufactured by Toyobo Co., Ltd.). Digestion was performed. These fragments were purified using High Pure PCR Product Purification Kit (Roche Applied Science) and then fused using In-Fusion HD Cloning Kit (Clontech) to obtain ΔARE1 plasmid.
Similarly, by PCR reaction using primers 11 and 12 shown in Table 1-1, a gene fragment in the region of 700 bp upstream to 500 bp downstream of the ORF of the LEU2 gene was subjected to PCR using primers 13 and 14 or primers 15 and 16 By the reaction, gene fragments in the region of 500 bp upstream or 500 bp downstream of the ARE2 gene were obtained. The obtained gene fragment was connected to the pUC vector in the same manner as the ΔARE1 plasmid to obtain the ΔARE2 plasmid.
Subsequently, by PCR reaction using primers 17 and 18 shown in Table 1-1, a gene fragment in the region of 300 bp upstream to 300 bp downstream of ORF of the MET15 gene was converted to PCR using primers 19 and 20 or primers 21 and 22. By the reaction, gene fragments in the region of 500 bp upstream or 500 bp downstream of the LRO1 gene were obtained. The obtained gene fragment was connected to the pUC vector in the same manner as the ΔARE1 plasmid to obtain the ΔLRO1 plasmid.
Using the ΔARE1 plasmid as a template, a gene fragment was obtained by PCR reaction using primers 5 and 8 shown in Table 1-1. Using this, the Saccharomyces cerevisiae BY4741ΔDGA1 strain (ΔDGA1 :: kanMX his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) (Yeast Knock Out Strain Collection: Open Biosystems) was transformed by the lithium acetate method. The obtained strain was transformed in the same manner as described above using a gene fragment obtained by PCR reaction using the primers 13 and 16 shown in Table 1-1 using ΔARE2 plasmid as a template. The obtained strain was further transformed with a gene fragment obtained by PCR reaction using primers 19 and 22 using ΔLRO1 plasmid as a template. As a result, Saccharomyces cerevisiae Δdga1, Δlro1, Δare1, and Δare2 strains (hereinafter referred to as yeast Δ4 strain) were obtained.

3. 1. Introduction of DGAT gene into yeast Δ4 strain (1) Construction of DGAT gene expression plasmid Using the genomic DNA of Saccharomyces cerevisiae obtained in step 1 as a template, a gene fragment in the region from 300 bp upstream of the ORF of the URA3 gene to the ORF stop codon was obtained by PCR using primers 23 and 24 shown in Table 1-1. PCR using primers 29 and 30 and a gene fragment in the region of 200 bp downstream of the ORF of the CYC1 gene by PCR reaction using PCR, and a gene fragment in the region of 500 bp upstream of the ORF of the GAL1 gene by PCR reaction using primers 27 and 28 By the reaction, gene fragments in the region of 200 bp downstream of the ORF of the URA3 gene were obtained. Using the Saccharomyces cerevisiae genomic DNA as a template and the PCR reaction using primers 31 and 32 shown in Table 1-1, the yeast DGA1 gene (diacylglycerol acyltransferase gene derived from Saccharomyces cerevisiae ) sequence shown in SEQ ID NO: 15 was subjected to PCR. Acquired. The obtained gene fragment was connected to the pUC19 vector to obtain a yeast DGA1 gene expression plasmid pUC-URAm-GAL1p-DGA1.
Instead of the DGA1 gene, 1. A NoDGAT2-8 gene expression plasmid pUC-URAm-GAL1p-NoDGAT2-8 was obtained in the same manner as above except that the NoDGAT2-8 gene fragment derived from Nannochloropsis oculiata obtained in the above was used.
Further, the Arabidopsis thaliana-derived DGAT gene (hereinafter referred to as AtDGAT1 gene) represented by SEQ ID NO: 16 was obtained using a man-made gene commissioned synthesis service, and PCR reaction using primers 33 and 34 shown in Table 1-1, AtDGAT1 gene fragment was obtained. AtDGAT1 gene expression plasmid pUC-URAm-GAL1p-AtDGAT1 was constructed in the same manner as described above except that AtDGAT1 gene was used instead of DGA1 gene.
These plasmids are composed of an URA3 promoter sequence, a URA3 gene fragment, a CYC1 terminator sequence, a GAL1 promoter sequence, various DGAT gene sequences, and an insert sequence linked in the order of the URA3 terminator sequence, and a pUC19 vector sequence.

(2) Acquisition of transformant PCR was performed using the plasmid pUC-URAm-GAL1p-NoDGAT2-8 as a template and primers 23 and 28. Using the amplified DNA fragment, yeast strain Δ4 was transformed by the lithium acetate method to obtain + NoDGAT2-8 strain.
Similarly, + DGA1 strain was obtained using plasmid pUC-URAm-GAL1p-DGA1, and + AtDGAT1 strain was obtained using plasmid pUC-URAm-GAL1p-AtDGAT1.
Each transformed strain was inoculated with 1 platinum ear in 5 mL of YPD liquid medium and cultured with shaking at 30 ° C. for 1 day. The culture solution was centrifuged, and the cells were collected. The cells were added with 1% fatty acid-containing YPG (Yeast Extract 1%, Peptone 2%, D-Galactose 2%) liquid medium (Briji 58 (Sigma Aldrich)), and lauric acid, myristic acid, palmitic acid, stearic acid And 2 mM each of oleic acid and eicosapentaenoic acid, or 2 mM each of lauric acid, myristic acid, and palmitic acid). After culturing at 30 ° C. for 24 hours, the cells were collected by centrifugation, washed with water, and lyophilized.

(3) Extraction of lipids and analysis of constituent fatty acids Lyophilized cells were suspended in 8 mL of chloroform / methanol solution (chloroform: methanol = 2: 1) and subjected to thin layer chromatography to fractionate lipids. Thin layer chromatography was carried out under the conditions of silica gel 60 plate (manufactured by Merck) and developing solvent hexane: diethyl ether: formic acid = 42: 24: 0.3. Lipids were visualized by spraying with 0.01% primulin (dissolved in methanol) and UV irradiation. The results are shown in FIG.
Next, only the triacylglycerol (TAG) fraction is scraped, and the scraped silica particles are induced to methyl ester with KOH and 3-boron fluoride methanol, and 0.7 mL of 0.5N potassium hydroxide / methanol solution is added. And constant temperature at 80 ° C. for 30 minutes. Subsequently, 1 mL of 14% boron trifluoride solution (manufactured by SIGMA) was added, and the temperature was kept constant at 80 ° C. for 10 minutes. Thereafter, 1 mL each of hexane and saturated saline was added and stirred vigorously. After standing at room temperature for 30 minutes, the upper hexane layer was recovered to obtain a fatty acid methyl ester. The obtained fatty acid methyl ester was subjected to gas chromatography analysis to analyze the fatty acid composition. Gas chromatographic analysis was performed under the following conditions.

Capillary column: DB-1 MS 20 m × 100 μm × 0.10 μm (manufactured by J & W Scientific),
Mobile phase: high purity helium,
In-column flow rate: 0.3 mL / min,
Temperature rising program: 150 ° C. (0.5 minutes) → 20 ° C./minute→320° C. (2 minutes)
Equilibration time: 1 minute
Inlet: Split injection (split ratio: 75: 1),
Pressure 62.4 psi, 24.5 mL / min,
Injection volume 5 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 compared from the peak area of the waveform data obtained by gas chromatography analysis. The total amount of each fatty acid was defined as the total fatty acid amount, and the ratio of each fatty acid amount to the total fatty acid amount was calculated. The results are shown in Tables 2 and 3.
Table 2 shows the ratio of fatty acids in triacylglycerols (%) extracted from the + DGA1, + NoDGAT2-8, and + AtDGAT1 strains after culturing each transformed strain in a medium containing lauric acid (C12: 0). It is a result to show.
Table 3 shows triacylates obtained by culturing each transformed strain in a medium containing lauric acid, myristic acid and palmitic acid (C12: 0, C14: 0, C16: 0) and extracted from + DGA1 strain and + NoDGAT2-8 strain. It is a result which shows the fatty acid composition ratio (%) in glycerol.

As is clear from FIG. 1, a triacylglycerol (TAG) spot was confirmed in BY4741 strain (WT strain) in which the triacylglycerol synthesis gene was not deleted, but in the yeast Δ4 strain in which the DGAT gene was deleted, avian strain was detected. It can be seen that no spots of acylglycerol are seen and TAG synthesis is not possible.
On the other hand, in each strain in which the yeast DGA1 gene, AtDGAT1 gene, or NoDGAT2-8 gene was introduced into the yeast Δ4 strain, a TAG spot was observed and the TAG synthesis ability was restored. From these results, it was found that the NoDGAT2-8 gene encodes an enzyme having diacylglycerol acyltransferase activity, like the AtDGAT1 gene.
In addition, as is clear from Table 2, the strain introduced with the NoDGAT2-8 gene has an increased content of lauric acid (C12: 0) in triacylglycerol as compared to the strain introduced with the DGA1 gene or AtDGAT1 gene. Was. Further, as is apparent from Table 3, the strain into which the NoDGAT2-8 gene was introduced introduced lauric acid into triacylglycerol with particular priority even under conditions where a plurality of fatty acids were present.
From these results, it was found that the NoDGAT2-8 gene preferentially incorporated the C12: 0 acyl group into diacylglycerol to produce triacylglycerol.

Example 2 Production of Transformant Co-introduced with Acyl-ACP Thioesterase and DGAT, and Production of Lauric Acid by Transformant Construction of BTE gene and NoDGAT2-8 gene expression plasmid A gene encoding acyl-ACP thioesterase derived from Umbellularia californica shown in SEQ ID NO: 4 (hereinafter referred to as BTE gene) was artificially synthesized. Using the synthesized DNA fragment as a template, a BTE gene fragment was obtained by PCR reaction using primers 35 and 36 shown in Table 1-2. In this gene fragment, a region corresponding to the N-terminal 83 amino acids, which is a chloroplast transfer signal, was deleted.
From the complete cds sequence (Accession number: JF957601.1) of the VCP1 (violaxanthin / chlorophyll a binding protein) gene of Nannochloropsis sp. W2J3B strain registered in GenBank, the VCP1 promoter sequence (SEQ ID NO: 17 ), A VCP1 chloroplast transition signal sequence (SEQ ID NO: 18), and a VCP1 terminator sequence (SEQ ID NO: 19) were artificially synthesized. Using the synthesized DNA fragment as a template, PCR reaction was performed using primers 37 and 38, primers 39 and 40, and primers 41 and 42 shown in Table 1-2 to obtain a VCP1 promoter sequence, a VCP1 chloroplast transition signal sequence, and a VCP1 terminator. Each array was obtained. Moreover, plasmid vector pUC19 was amplified by PCR reaction using primers 9 and 10 shown in Table 1-1 using plasmid vector pUC19 (manufactured by Takara Bio Inc.) as a template.
The BTE gene fragment, VCP1 promoter sequence, VCP1 chloroplast translocation signal sequence, and VCP1 terminator sequence obtained as described above were obtained as described in 2. The plasmid pUC19 was fused with the plasmid vector pUC19 in the same manner as described above to construct a plasmid pUC-vcp1c-BTE for expression of the BTE gene Nannochloropsis. This plasmid consists of an insert sequence linked in the order of a VCP1 promoter sequence, a VCP1 chloroplast transfer signal sequence, a BTE gene fragment, and a VCP1 terminator sequence, and a pUC19 vector sequence.

Using the cDNA of Nannochloropsis oculata N2145 strain as a template, PCR reaction was performed using primers 43 and 44 shown in Table 1-2 to amplify the NoDGAT2-8 gene fragment. The NoDGAT2-8 gene fragment, VCP1 promoter sequence, and VCP1 terminator sequence were fused in the same manner as described above to construct a plasmid pUC-vcp1c-NoDGAT2-8 for expression of Nonochloropsis of the NoDGAT2-8 gene.

  Further, a zeocin resistance gene (SEQ ID NO: 20) was artificially synthesized. Using the synthesized DNA fragment as a template, PCR reaction was performed using primers 45 and 46 shown in Table 1-2 to obtain a zeocin resistant gene fragment. A zeocin resistant gene fragment, VCP1 promoter sequence, and VCP1 terminator sequence were fused in the same manner as described above to construct a plasmid pUC-vcp1c-ble for expression of zeocin resistant gene Nannochloropsis.

Paramomycin-resistant fertilizer (SEQ ID NO: 21) and a tubulin promoter sequence derived from the Nannochloropsis gaditana CCMP526 strain described in Randor Radakovits, et al., Nature Communications, DOI: 10.1038 / ncomms1688, 2012 22) was artificially synthesized. Using the synthesized DNA fragment as a template, PCR reaction was performed using primers 47 and 48 and primers 49 and 50 shown in Table 1-2 to obtain a paramomycin resistance gene and a tubulin promoter sequence, respectively. These amplified fragments were fused with the VCP1 terminator sequence obtained above and the amplified fragment of the plasmid vector pUC19 in the same manner to obtain a plasmid pUC-TUBp-aphVIII for expression of the paramomycin resistance gene Nannochloropsis.

2. Introduction of BTE gene expression fragment into Nannochloropsis
Using the plasmid pUC-vcp1c-BTE for BTE expression as a template, PCR reaction was performed using primers 51 and 52 shown in Table 1-2, and the VCP1 promoter sequence, VCP1 chloroplast translocation signal sequence, BTE gene fragment in the plasmid And a BTE gene expression fragment consisting of the VCP1 terminator sequence. Moreover, PCR reaction was performed using primers 51 and 52 using pUC-vcp1c-ble as a template to amplify a zeocin resistant gene expression fragment. Both amplified fragments were purified using High Pure PCR Product Purification Kit (Roche Applied Science). Note that sterilized water was used for elution during purification, not the elution buffer included in the kit.
About ¹⁰⁹ cells of Nannochloropsis Okyurata (Nannochloropsis oculata) NIES2145 strain, salt was removed completely by washing with sorbitol solution of 384mm, it was used as host cells transformed. About 500 ng of the BTE gene expression fragment and the zeocin resistance gene expression fragment amplified above were mixed in a host cell and electroporated under the conditions of 50 μF, 500Ω, and 2,200 v / 2 mm. After recovery culture in f / 2 liquid medium for 24 hours, it was applied to 2 μg / mL zeocin-containing f / 2 agar medium, and light and dark conditions at 25 ° C. and 0.3% CO ₂ atmosphere for 12 h / 12 h. Cultured for 2-3 weeks. From the obtained colonies, those containing a BTE gene expression fragment were selected by the PCR method.

3. Introduction of NoDGAT2-8 gene expression fragment into Nannochloropsis
Using NoDGAT2-8 gene expression plasmid pUC-vcp1c-NoDGAT2-8 as a template, PCR reaction was performed using primers 51 and 52 shown in Table 1-2, and the VCP1 promoter sequence in the plasmid, NoDGAT2-8 gene fragment, And a NoDGAT2-8 gene expression fragment consisting of the VCP1 terminator sequence. In addition, a PCR reaction was performed using pUC-TUBp-aphVIII as a template and primers 53 and 52 shown in Table 1-2 to amplify a fragment for paramomycin resistance gene expression. Both amplified fragments were purified using High Pure PCR Product Purification Kit (Roche Applied Science). Note that sterilized water was used for elution during purification, not the elution buffer included in the kit.

2. above. The Nannochloropsis Okyurata (Nannochloropsis oculata) strain BTE genes obtained was introduced in using about 10 ⁹ cells, to completely remove salts and washed with sorbitol solution of 384mm, it was used as host cells transformed. About 500 ng of the NoDGAT2-8 gene expression fragment and the paramomycin resistance gene expression fragment amplified above were mixed with the host cells, and electroporation was performed under the conditions of 50 μF, 500Ω, and 2,200 v / 2 mm. After 24 hours recovery culture in f / 2 liquid medium, it was applied to f / 2 agar medium containing 100 μg / mL of paramomycin, and light / dark conditions at 25 ° C. and 0.3% CO ₂ atmosphere for 12 h / 12 h. For 2 to 3 weeks. From the obtained colonies, those containing the NoDGAT2-8 gene expression fragment were selected by the PCR method.

4). 2. Culture of BTE gene and NoDGAT2-8 gene expression strain and evaluation of lipid productivity The strains selected in (1) were inoculated into 50 mL of a medium (hereinafter referred to as N15P5 medium) in which the nitrogen concentration of f / 2 medium was increased 15-fold and the phosphorus concentration was increased 5-fold, and the mixture was cultured at 25 ° C. under 0.3% CO ₂ atmosphere for 12 h. / 12h Shake culture for 4 weeks under light and dark conditions to prepare a preculture solution. 10 mL of the preculture solution was transferred to 40 mL of N15P5 medium, and cultured with shaking for 2 weeks at 25 ° C. in a 0.3% CO ₂ atmosphere under 12 h / 12 h light / dark conditions. As a negative control, 2. The same experiment was performed on the Nannochloropsis strain into which only the BTE gene obtained in (1) was introduced.

After adding 50 μL of 1 mg / mL 7-pentadecanone as an internal standard to 1 mL of the culture solution, 0.5 mL of chloroform, 1 mL of methanol and 10 μL of 2N hydrochloric acid were added to the culture solution, and the mixture was vigorously stirred and allowed to stand for 30 minutes. Then, 0.5 mL of chloroform and 0.5 mL of 1.5% KCl were added and stirred, and then centrifuged at 3,000 rpm for 15 minutes, and the chloroform layer (lower layer) was recovered with a Pasteur pipette. did. Nitrogen gas was blown into the resulting chloroform layer to dryness, 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 14% boron trifluoride solution (manufactured by SIGMA) was added, and the temperature was kept constant at 80 ° C. for 10 minutes. Thereafter, 1 mL each of hexane and saturated saline was added and stirred vigorously. After standing for 30 minutes at room temperature, the upper hexane layer was recovered to obtain a fatty acid ester.
The obtained fatty acid ester was subjected to gas chromatography analysis. Gas chromatographic conditions and identification of fatty acid esters were carried out in the same manner as in Example 1. Based on the results of gas chromatography analysis, the total fatty acid amount and fatty acid composition were calculated in the same manner as in Example 1. The results are shown in Table 4.
Table 4 shows the total fatty acid amount (μg / L) in the lipid extracted from the BTE gene-introduced strain, the BTE gene, and the NoDGAT2-8 gene-introduced strain, and the fatty acid composition (%) in the lipid. C18: n represents the total sum of C18: 0 to C18: 3 fatty acids.

As is clear from Table 4, the lauric acid content in the lipid was increased in the transformed strain into which the BTE gene and NoDGAT2-8 gene were introduced, compared to the transformed strain into which only the BTE gene was introduced.

Claims (10)

The manufacturing method of lauric acid including the process of following (1) and (2).
(1) A step of obtaining a transformant by co-introducing a gene encoding an acyl-ACP thioesterase and a gene encoding the following protein (A) or (B) into a host (A) represented by SEQ ID NO: 1 A protein comprising the amino acid sequence (B) a protein comprising the amino acid sequence having 81% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having diacylglycerol acyltransferase activity (2) transformant obtained For collecting lauric acid from sewage   The production method according to claim 1, wherein the acyl-ACP thioesterase is a medium-chain acyl-ACP-specific acyl-ACP thioesterase. The production method according to claim 1 or 2, wherein the acyl-ACP thioesterase is a protein of the following (C) or (D).
(C) a protein comprising the amino acid sequence from position 84 to position 382 of SEQ ID NO: 3 (D) comprising an amino acid sequence having 70% or more identity with the amino acid sequence of the protein of (C), and an acyl-ACP thioesterase Active protein   The manufacturing method of any one of Claims 1-3 whose said host is microorganisms or a plant body.   The production method according to claim 4, wherein the microorganism is yeast. Wherein the yeast is Saccharomyces cerevisiae (Saccharomyces cerevisiae), a manufacturing method of claim 5, wherein.   The production method according to claim 4, wherein the microorganism is a microalgae. The production method according to claim 7, wherein the microalgae are algae belonging to the genus Nannochloropsis . A transformant obtained by co-introducing a gene encoding an acyl-ACP thioesterase and a gene encoding the following protein (A) or (B) into a host.
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 1 (B) A protein comprising an amino acid sequence having 81% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having diacylglycerol acyltransferase activity A method for improving the content of lauric acid in triacylglycerol produced by a transformant, which comprises the step of introducing a gene encoding the protein (A) or (B) below into a host for transformation.
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 1 (B) A protein comprising an amino acid sequence having 81% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having diacylglycerol acyltransferase activity