JP6619229B2 - Arachidonic acid producing polyketide synthase and use thereof - Google Patents

Description

  In the present invention, the polyketide synthase protein complex producing arachidonic acid (hereinafter referred to as ARA), the DNA encoding the protein constituting the protein complex, and the activity of the protein complex are enhanced from the host organism. And a method for producing ARA or an ARA-containing composition using the organism.

  ARA is cis-5,8,11,14 eicosatetraenoic acid (20: 4) and belongs to the (n-6) family of long-chain polyunsaturated fatty acids (hereinafter referred to as PUFA). ARA is the main precursor of eicosanoids, a group that includes prostaglandins, thromboxanes and leukotrienes. Humans lack Δ-5 desaturase action and cannot convert fatty acid 18: 2 (n-6) (linoleic acid) to ARA. ARA is an essential fatty acid for humans.

As a method for producing ARA, a fermentation method using a filamentous fungus Mortierella alpina is known (Non-patent Documents 1 and 2). In addition, a method for producing ARA using a chain elongation enzyme and a desaturase in a microorganism or plant that cannot originally produce ARA and starting from fatty acids biosynthesized by the microorganism or plant is known. (Patent Document 1, Non-Patent Document 3).

  On the other hand, for docosahexaenoic acid (hereinafter referred to as DHA) and eicosapentaenoic acid (hereinafter referred to as EPA) belonging to PUFA other than ARA, acetyl CoA is used as a starting material in addition to the above-described pathway using fatty acid as a starting material. As described above, a pathway produced by a single complex enzyme called PUFA-polyketide synthase (hereinafter, polyketide synthase is referred to as PKS) is known (patent document 2, non-patent document 4), and PUFA-PKS is expressed. There have been reports that microorganisms produced DHA or EPA.

For example, it has been reported that Escherichia coli expressing EPA-PKS derived from microorganisms belonging to the genus Shewanella produced EPA (Non-Patent Documents 4 to 7). It has been reported that Escherichia coli expressing DHA-PKS derived from a microorganism belonging to the genus Moritella has produced DHA (Non-patent Document 8). Furthermore, a part of a domain of EPA-PKS derived from a microorganism belonging to the genus Shewanella is replaced with a corresponding domain of DHA-PKS derived from a microorganism belonging to the genus Moritella, and is expressed in Escherichia coli. It has been reported that EPA, which is a product, has been converted to DHA (Non-patent Document 9).

Thus, there are reports on the production of DHA or EPA by recombinant organisms expressing PUFA-PKS, but there is no report on the production of ARA by recombinant organisms expressing ARA-PKS.
In the first place, for ARA-PKS, only the gene encoding PKS involved in the production of ARA and EPA is estimated in the genomic base sequence of Cycloflexus torquis (Non-patent Document 10). . It is not specified whether the PKS is actually PKS involved in the production of ARA and EPA (Non-patent Document 10). That is, as for ARA-PKS, there is no report on those whose functions have been investigated.

Special table 2008-518628 US Pat. No. 6,140,486

Biotechnol. Lett. (2005), Vol.27, p731-735 Biopro. Biosyst. Eng. (2009), Vol. 32, p117-122 Transgenic Res. (2011), 1-9 Science (2001), Vol.293, p290-293 Biotechnol. Bioprocess Eng. (2006), Vol. 11, p510-515 Methods in Enzymology (2009), Vol.459, p79-96 J. AM. CHEM. SOC. (2008), Vol.130, p6336-6337 FEBS Lett. (2006), Vol.580, p4423-4429. FEMS Microbiol. Lett. (2009), Vol.295, p170-176 PLoS ONE (2011), Vol.6 (5), art.no.e20146

  An object of the present invention is to provide a complex of ARA-PKS protein, a DNA encoding a protein constituting the complex of the protein, an organism in which the activity of the complex of the protein is enhanced from a host organism, and the organism An object of the present invention is to provide a method for efficiently producing an ARA-containing composition containing no EPA or other PUFAs including EPA.

The present invention relates to the following (1) to (17).
(1) A protein complex selected from the group consisting of [1] to [4] below.
[1] A β-ketoacyl-acyl carrier protein synthase (hereinafter referred to as KS) domain, malonyl-CoA: acyl carrier protein acyltransferase (hereinafter referred to as MAT) domain, and acyl carrier protein (hereinafter referred to as ACP) domain. A protein having a ketoreductase (hereinafter referred to as KR) domain, a KS domain, a chain elongation factor (hereinafter referred to as CLF) domain, an acyltransferase (hereinafter referred to as AT) domain, and a FabA-like β-hydroxyacyl- A protein having an ACP dehydratase (hereinafter referred to as DH) domain, a protein having an enoyl ACP-reductase (hereinafter referred to as ER) domain, and a protein having a phosphopantethein transferase (hereinafter referred to as PPT) domain, and Starting from acetyl-CoA A protein having an activity to produce ARA as a protein [2] a protein having a KS domain, a MAT domain, an ACP domain and a KR domain, a protein having an AT domain, a protein having a KS domain, a CLF domain, and a DH domain, ER A protein having a domain and a protein having a PPT domain and having an activity of producing ARA using acetyl CoA as a starting substrate [3] a protein having a KS domain, a MAT domain, an ACP domain, and a KR domain A protein having a KS domain and an AT domain, a protein having a KS domain, a CLF domain, and a DH domain, a protein having an ER domain, and a protein having a PPT domain, and producing ARA using acetyl CoA as a starting substrate Active protein complex [4] KS domain, MAT domain, ACP ARA consisting of a protein having a main and KR domain, a protein having a KS domain, a CLF domain, an AT domain and an ER domain, a protein having a DH domain and an ER domain, and a protein having a PPT domain, and using acetyl CoA as a starting substrate A complex of proteins having the activity of producing (2) one or more of the domains described in (1) above are domains selected from the group consisting of [1] to [9] below (1) A protein complex according to 1.
[1] A KS domain having the amino acid sequence represented by SEQ ID NO: 2 or 4, or consisting of an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 2 or 4, and KS activity A MAT domain having the amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 6, and having MAT activity A homologous domain [3] consisting of an ACP domain having the amino acid sequence represented by SEQ ID NO: 8, or an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 8, and ACP Homologous domain having the function of [4] KR domain having the amino acid sequence represented by SEQ ID NO: 10, or amino acid represented by SEQ ID NO: 10 A CLF domain consisting of an amino acid sequence having at least 95% identity with an acid sequence and having an amino acid sequence represented by SEQ ID NO: 12, or a homologous domain having KR activity [5], or represented by SEQ ID NO: 12 A homologous domain consisting of an amino acid sequence having at least 95% identity with the amino acid sequence and having a function as CLF [6] AT domain having the amino acid sequence represented by SEQ ID NO: 14, or represented by SEQ ID NO: 14 A DH domain comprising an amino acid sequence having at least 95% identity with the amino acid sequence and having a homologous domain having AT activity [7] SEQ ID NO: 16 or 18, or SEQ ID NO: 16 Or an amino acid sequence having at least 95% identity with the amino acid sequence represented by 18, And a homologous domain having DH activity [8] consisting of an ER domain having the amino acid sequence represented by SEQ ID NO: 20, or an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 20, And a homologous domain having ER activity [9] a PPT domain having an amino acid sequence represented by SEQ ID NO: 22, 24, 26, 28 or 30, or an amino acid represented by SEQ ID NO: 22, 24, 26, 28 or 30 A protein comprising an amino acid sequence having at least 95% identity with the sequence and having a PPT activity and having a homologous domain (3) KS domain, MAT domain and ACP domain has the amino acid sequence represented by SEQ ID NO: 32 Or an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 32 And a homologous protein having an activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having an amino acid sequence represented by each of SEQ ID NOs: 34, 36, 38 and 22 The protein complex according to [1] of 1).
(4) the protein having the KR domain comprises a protein having the amino acid sequence represented by SEQ ID NO: 34, or an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 34; [1] of the above (1), which is a homologous protein having an activity of producing ARA using acetyl CoA as a starting substrate in cooperation with a protein having the amino acid sequence represented by each of SEQ ID NOs: 32, 36, 38 and 22. ] The protein complex described in the above.
(5) The protein having the KS domain, CLF domain, AT domain and DH domain has at least 95% identity with the protein having the amino acid sequence represented by SEQ ID NO: 36 or the amino acid sequence represented by SEQ ID NO: 36 A homologous protein having an activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having the amino acid sequence represented by SEQ ID NOs: 32, 34, 38 and 22 A protein complex according to [1] of (1) above.
(6) A protein having an ER domain is a protein having an amino acid sequence represented by SEQ ID NO: 38 or 40, or an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 38 or 40 And a homologous protein having the activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having the amino acid sequence represented by each of SEQ ID NOs: 32, 34, 36 and 22 (1) ) Of the protein complex according to [1].
(7) The protein having the PPT domain is a protein having an amino acid sequence represented by SEQ ID NO: 22, 24, 26, 28 or 30, or any one represented by SEQ ID NO: 22, 24, 26, 28 or 30 Acetyl CoA as a starting substrate in cooperation with a protein comprising an amino acid sequence having at least 95% identity with the amino acid sequence and having the amino acid sequence represented by each of SEQ ID NOs: 32, 34, 36 and 38 The protein complex according to [1] of (1), which is a homologous protein having an activity of producing ARA.
(8) DNA encoding a protein constituting the complex of the protein according to any one of (1) to (7) above.
(9) DNA encoding a protein having a KS domain, a MAT domain, and an ACP domain is a DNA having a base sequence represented by SEQ ID NO: 31 or a base sequence complementary to the base sequence represented by SEQ ID NO: 31 Activity to produce ARA using acetyl CoA as a starting substrate in cooperation with a protein having the amino acid sequence represented by each of SEQ ID NOs: 34, 36, 38 and 22 The DNA according to (8) above, which is a DNA encoding a homologous protein having
(10) A DNA encoding a protein having a KR domain is stringent with a DNA having a base sequence represented by SEQ ID NO: 33 or a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 33 A homologous protein that hybridizes under conditions and has the activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having the amino acid sequence represented by each of SEQ ID NOs: 32, 36, 38, and 22 The DNA according to (8) above,
(11) A DNA encoding a protein having a KS domain, a CLF domain, an AT domain, and a DH domain is complementary to the DNA having the base sequence represented by SEQ ID NO: 35 or the base sequence represented by SEQ ID NO: 35 Hybridizes with DNA consisting of a base sequence under stringent conditions, and cooperates with a protein having the amino acid sequence represented by each of SEQ ID NOs: 32, 34, 38 and 22 to produce ARA using acetyl CoA as a starting substrate. The DNA according to (8) above, which is a DNA encoding a homologous protein having an activity to be produced.
(12) DNA having a base sequence represented by SEQ ID NO: 37 or 39, or a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 37 or 39, wherein the DNA encoding a protein having an ER domain And has an activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having the amino acid sequence represented by each of SEQ ID NOs: 32, 34, 36, and 22 under stringent conditions. The DNA according to (8) above, which is a DNA encoding a homologous protein.
(13) DNA encoding a protein having a PPT domain is represented by DNA having a base sequence represented by SEQ ID NO: 21, 23, 25, 27 or 29, or SEQ ID NO: 21, 23, 25, 27 or 29 In cooperation with a protein having an amino acid sequence represented by each of SEQ ID NOs: 32, 34, 36 and 38. The DNA according to (8) above, which encodes a homologous protein having an activity of producing ARA using acetyl CoA as a starting substrate.
(14) An organism in which the activity of the protein complex according to any one of (1) to (7) above is enhanced from the host organism.
(15) The organism can produce ARA using acetyl-CoA in the cell obtained as a starting substrate obtained by transforming the host organism with the DNA according to any one of (8) to (13) above. The organism according to (14) above, which is an organism.
(16) The organism according to (14) or (15) above, wherein the host organism is a microorganism or a plant.
(17) The organism according to any one of (14) to (16) above is cultured or cultivated in a medium, and an ARA-containing composition is generated and accumulated in the culture or cultivated product, and the culture or cultivated product A method for producing an ARA or ARA-containing composition, which comprises collecting an ARA or an ARA-containing composition from a product.

  By using the ARA-PKS of the present invention, an ARA or ARA-containing composition, which has conventionally been required to be produced through many intermediates in a reaction involving two types of chain extender and unsaturated enzyme, is obtained from acetyl CoA. It becomes possible to produce ARA-PKS which is a single complex enzyme, and it is possible to establish a production method of ARA or an ARA-containing composition with fewer by-products.

It is a figure which shows 6 open reading frames (henceforth Orf) of ARA-PKS system derived from Aureospila marina, and the domain structure of this PKS system. The gas chromatogram of the fatty acid extracted from colon_bacillus | E._coli which introduce | transduced ARA-PKS is shown.

1 Protein complex of the present invention
1.1 About the complex of the protein of the present invention The complex of the protein of the present invention includes the following [1] a protein having a KS domain, a MAT domain and an ACP domain, a protein having a KR domain, a KS domain, a CLF domain, an AT domain, and A complex of a protein having a DH domain, a protein having an ER domain, and a protein having a PPT domain, and having an activity of producing ARA using acetyl CoA as a starting substrate,
[2] From proteins having KS domain, MAT domain, ACP domain and KR domain, proteins having AT domain, proteins having KS domain, CLF domain and DH domain, proteins having ER domain, and proteins having PPT domain A complex of a protein having an activity to produce ARA using acetyl CoA as a starting substrate,
[3] A protein having a KS domain, a MAT domain, an ACP domain, and a KR domain, a protein having a KS domain and an AT domain, a protein having a KS domain, a CLF domain, and a DH domain, a protein having an ER domain, and a PPT domain A protein complex having an activity of producing ARA using acetyl CoA as a starting substrate, and [4] a protein having a KS domain, a MAT domain, an ACP domain, and a KR domain, a KS domain, a CLF domain, A complex of a protein having an AT domain and an ER domain, a protein having a DH domain and an ER domain, and a protein having a PPT domain, and having an activity of producing ARA using acetyl CoA as a starting substrate;
A protein complex selected from the group consisting of

In the present specification, a “domain” is a part consisting of a continuous amino acid sequence in a protein, and is a region having a specific biological activity or function in the protein.
As long as the protein complex of any one of the above [1] to [4] has an activity of producing ARA using acetyl CoA as a starting substrate, the protein constituting the protein complex of the present invention is physically May be bonded to each other or separated.

In the KS domain, MAT domain, ACP domain, KR domain, CLF domain, AT domain, DH domain, ER domain, and PPT domain, one or more proteins having the domain cooperate to form ARA using acetyl CoA as a starting substrate. As long as it produces, each domain is not limited, For example, the following examples can be given.
Examples of the KS domain include the KS domain of known PUFA-PKS and the KS domain of Table 1, preferably the KS domain of Table 1.

As used herein, the term "known PUFA-PKS" preferably, Shewanella sp (Shewanella), Koruweria genus (Colwellia), Desaru phosphatidyl drumstick Lum genus (Desulfatibacillum), Cyclo off Lexus genus (Psychroflexus), Schizochytrium (Schizochytrium ), Nostoc sp (Nostoc), micro-cis infantis species (Microcystis), Saccharopolyspora genus (Saccharopolyspora), Jiobakuta genus (Geobacter), plan ECTS My genus (Planctomyces), Desaru follower genus (Desulfococcus), the genus Sorangium (Sorangium), Renibakuteriumu genus (Renibacterium), Chichinofaga genus (Chitinophaga), Gureobakuta genus (Gloeobacter), Azotobacter species (Azotobacter), the genus Rhodococcus (Rhodococcus), Soribakuta genus (Solibacter), Desaru follower genus (Desulfobacterium) , Clostridium (Clostridium), Thraustochytrium (Thraustochytrium) genus Ulkenia (Ulkenia) genus, Japo carbonothioyl thorium (Japonochytrium) species, Au lunch Oki thorium (Aurantiochytrium) species, to a genus selected from the group consisting of Moritera (Moritella) genus The PUFA-PKS originally possessed by the microorganism to which it belongs is more preferably Shewanella pealeana ATCC 700345, Shewanella oneidensis MR-1, Colwellia psychrerythraea 34H, Desulfatibacillum alkenivorans AK-01, Psychroflexus torquis ATCC 700755, Schizochytrium sp. ATCC 20888, Nostoc punctiforme PCC 73102, Microcystis aeruginosa NIES -843, Saccharopolyspora erythraea NRRL 2338, Geobacter bemidjiensis Bem, Planctomyces limnophilus DSM3776, Desulfococcus oleovorans Hxd3, Sorangium cellulosum 'So ce 56', Renibacterium salmoninarum ATCC 33209, Chitinophaga pinensis DSM 2588, Gloeobacter violaceus PCC 7421 , Azotobacter vine landii DJ, Rhodococcus erythropolis PR4, Candidatus Solibacter usitatus Ellin6076, Desulfobacterium autotrophicum HRM2, and Clostridium thermocellum ATCC 27405, Schizochytrium minutum, Schizochytrium sp. S31 ATCC 20888, Schizochytrium sp. S8 ATCC 20889, Schizochytrium sp. LC-RM ATCC 18915, Schizochytrium sp from. SR21, Schizochytrium aggregatum ATCC 28209, Schizochytrium limacinum IFO 32693, Thraustochytrium sp. 23B ATCC 20891, Thraustochytrium striatum ATCC 24473, Thraustochytrium aureum ATCC 34304, Thraustochytrium roseum ATCC 28210, Ulkenia sp. BP-5601, and Japonochytrium sp. L1 ATCC 28207 A microorganism selected from the group consisting of PUFA-PKS originally possessed by Shewanella pealeana ATCC 700345, Shewanella oneidensis MR-1, Colwellia psychrerythraea 34H, Moritella marina MP-1, Schizochytrium sp. ATCC 20888 PUFA-PKS originally possessed by a microorganism selected from the group consisting of (Non-Patent Documents 9 and 10).

Examples of the MAT domain include the MAT domain of known PUFA-PKS and the MAT domain of Table 1, preferably the MAT domain of Table 1.
Examples of the ACP domain include the ACP domain of known PUFA-PKS and the ACP domain in Table 1, and preferably the ACP domain in Table 1.
Examples of the KR domain include the KR domain of known PUFA-PKS and the KR domain of Table 1, preferably the KR domain of Table 1.

Examples of the CLF domain include the CLF domain of known PUFA-PKS and the CLF domain of Table 1, preferably the CLF domain of Table 1.
As an example of AT domain, the AT domain which known PUFA-PKS has and the AT domain of Table 1 can be mentioned, Preferably the AT domain of Table 1 can be mentioned.
Examples of DH domains include the DH domains of known PUFA-PKS and the DH domains in Table 1, preferably the DH domains in Table 1.

Examples of the ER domain include the ER domain of known PUFA-PKS and the ER domain of Table 1, preferably the ER domain of PUFA-PKS derived from a microorganism belonging to the genus Shuwanella and the ER domain of Table 1. Can include the ER domains possessed by PUFA-PKS derived from Shewanella oneidensis MR-1 and the ER domains shown in Table 1.
Examples of the PPT domain include a PPT domain possessed by a known PUFA-PKS, a PPT domain possessed by a microorganism belonging to a group selected from the group consisting of Bacillus genus, Schwanella genus, Escherichia genus, Corynebacterium genus, and PPT domain in Table 1 Preferably, a PPT domain derived from a microorganism selected from the group consisting of Bacillus subtilis 168, Shewanella oneidensis MR-1, Escherichia coli W3110, Corynebacterium glutamicum ATCC13032 and the PPT domain of Table 1 can be mentioned.

In the protein complex of the present invention, one or more domains selected from the group consisting of KS domain, MAT domain, ACP domain, KR domain, CLF domain, AT domain, DH domain, ER domain, and PPT domain are represented by It is preferably a domain selected from one domain.
In PUFA-PKS, it is possible to convert PUFA as a product by exchanging a part of the domain (Non-patent Document 9). Therefore, according to the method described in Non-Patent Document 9, a group consisting of KS domain, MAT domain, ACP domain, KR domain, CLF domain, AT domain, DH domain, ER domain, and PPT domain possessed by known PUFA-PKS A PKS capable of producing ARA can be produced by substituting one or more selected domains with the domains shown in Table 1.

As used herein, a “homology domain” is a domain that is considered to have the same evolutionary origin as the original domain due to similarity in structure and function to the original domain, It refers to the domain of an existing organism.
Examples of such homologous domains have at least 95%, preferably 97%, more preferably 98%, most preferably 99% or more identity with each domain listed in Table 1. The homologous domain which consists of an amino acid sequence and can produce ARA by using acetyl-CoA as a starting substrate by cooperating with each domain described in Table 1 can be mentioned.

  In this specification, cooperating means that when a certain protein coexists with other proteins, a specific reaction is performed as one body, and in this specification, in particular, a KS domain, a MAT domain, an ACP domain, and a KR domain. , KS domain, CLF domain, AT domain, DH domain, ER domain, and PPT domain are divided into several proteins, and when these proteins coexist, It means that ARA is produced as a starting material, and that when a certain domain coexists with another domain, ARA is produced using acetyl-CoA as a starting material together with the other domain.

In the above, coexistence includes both the case where the protein is contained in the same protein as the other domain, and the case where the protein coexists when the other domain is contained in the other protein.
The identity of amino acid sequences and nucleotide sequences is determined by the algorithm BLAST [Pro. NAT domain l. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzymol., 183, 63 (1990)] by Karlin and Altschul. Can be determined. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)]. When the base sequence is analyzed by BLASTN based on BLAST, the parameters are, for example, Score = 100 and wordlength = 12. In addition, when an amino acid sequence is analyzed by BLASTX based on BLAST, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known.

  The homologous domain of each domain can produce ARA using acetyl-CoA as a starting material in cooperation with other domains, for example, a protein complex consisting of the proteins represented by SEQ ID NOs: 32, 34, 36, 38 and 22 In this method, a complex of the protein in which the original domain corresponding to the homologous domain is substituted with the homologous domain is prepared, and this can be confirmed by producing ARA using acetyl CoA as a starting material.

Specifically, a recombinant DNA having a DNA encoding a protein complex substituted with the homologous domain described above by a method described later, and a microorganism that does not produce ARA with the recombinant DNA, such as an acyl CoA described later It can be confirmed by culturing a microorganism obtained by transforming Escherichia coli BW25113 strain in which fadE encoding dehydrogenase is disrupted in a medium and detecting ARA accumulated in the culture by gas chromatography.
1.2 Examples of proteins constituting the protein complex of the present invention Examples of proteins constituting the protein complex of the present invention include the proteins constituting the protein complex of [1] to [4] above. Preferably, the protein constituting the protein complex of [1] above can be mentioned.
1.2.1 Protein having KS domain, MAT domain, and ACP domain Examples of the protein having the KS domain, MAT domain, and ACP domain of [1] above include, for example, (a) the amino acid sequence represented by SEQ ID NO: 32 Protein (hereinafter also referred to as OrfA protein), (b) at least 95%, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more of the amino acid sequence represented by SEQ ID NO: 32. Homology having an activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having an amino acid sequence having identity and having an amino acid sequence represented by each of SEQ ID NOs: 34, 36, 38 and 22. In the amino acid sequence represented by protein or (c) SEQ ID NO: 32, 1-20, preferably 1-10, most preferably 1-5 amino acids Production of ARA using acetyl-CoA as a starting substrate in cooperation with a protein comprising an amino acid sequence deleted, substituted, inserted or added and having the amino acid sequence represented by each of SEQ ID NOs: 34, 36, 38 and 22 And muteins having the activity of

A homologous protein is a protein whose structure and function are similar to those of the original protein, so that the gene encoding the protein is considered to have the same evolutionary origin as the gene encoding the original protein. It refers to the protein of living organisms.
A mutated protein refers to a protein obtained by artificially deleting or substituting amino acid residues in the original protein, or inserting or adding amino acid residues into the protein.

In the above mutant protein, an amino acid is deleted, substituted, inserted or added in any position in the same sequence, 1 to 20, preferably 1 to 10, most preferably 1 to 5 amino acids. May be deleted, substituted, inserted or added.
The amino acid to be deleted, substituted, inserted or added may be a natural type or a non-natural type. Natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L -Methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.

Examples of amino acids that can be substituted with each other are shown below. Amino acids contained in the same group can be substituted for each other.
Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid group C: asparagine, glutamine group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid group E: proline, 3 -Hydroxyproline, 4-hydroxyproline F group: serine, threonine, homoserine G group: phenylalanine, tyrosine
DNA encoding the OrfA protein (hereinafter referred to as OrfA) is a DNA having the base sequence represented by SEQ ID NO: 31.

Within the OrfA protein, there is one KS domain, one MAT domain, and four ACP domains.
OrfA was compared to a known sequence in a BLAST search. At the nucleic acid level, OrfA has no significant homology with any known base sequence. At the amino acid level, the sequence with the highest degree of homology with the OrfA protein was: 54 for the 1574 amino acid residues of unidentified eubacterium SCB49 polyunsaturated fatty acid synthase pfaA (accession number EDM45380) And was 54% identical for 1557 amino acid residues of the Psychroflexus torquis ATCC 700755 multidomain β ketoacyl synthase (accession number EAS69828).

The first domain in the OrfA protein is a KS domain, also referred to herein as an OrfA-KS domain. This domain extends from the origin between about position 1 and about position 40 of SEQ ID NO: 31 (OrfA) to the end point between about position 1400 and position 1500 of SEQ ID NO: 31. The DNA encoding the OrfA-KS domain has a base sequence represented by SEQ ID NO: 1 (positions 1 to 1500 of SEQ ID NO: 31). The amino acid sequence containing the KS domain extends from the origin between about positions 1 and 14 of SEQ ID NO: 32 to the end point between positions 466 and 500 of SEQ ID NO: 32. The OrfA-KS domain has an amino acid sequence represented by SEQ ID NO: 2 (positions 1 to 500 of SEQ ID NO: 32). It is noted that the OrfA-KS domain contains an active site motif: DXAC * (* acyl binding site C ₂₁₅ ).

The second domain in the OrfA protein is the MAT domain, also referred to herein as the OrfA-MAT domain. The domain extends from an origin between about positions 1700 and 1800 of SEQ ID NO: 31 (OrfA) to an end point between about positions 2500 and 2600 of SEQ ID NO: 31. The DNA encoding the OrfA-MAT domain has a base sequence represented by SEQ ID NO: 5 (positions 1700 to 2700 of SEQ ID NO: 31). The amino acid sequence containing the MAT domain extends from the origin between about position 565 and about position 600 of SEQ ID NO: 32 (OrfA protein) to the end point between position 865 and about position 900 of SEQ ID NO: 32. The OrfA-MAT domain has an amino acid sequence represented by SEQ ID NO: 6 (positions 565 to 900 of SEQ ID NO: 32). It is noted that the OrfA-MAT domain contains an active site motif: GHS * XG (* acyl binding site S ₆₈₃ ). The active motif has the amino acid sequence represented by SEQ ID NO: 41.

Domains 3 to 6 of the OrfA protein are four tandem ACP domains.In this specification, OrfA-ACP (the first domain of the sequence is OrfA-ACP1, the second domain is OrfA-ACP2, the third domain is OrfA-ACP3 and the fourth domain are also called OrfA-ACP4). The first OrfA-ACP1 extends from about position 3381 to about position 3612 of SEQ ID NO: 31 (OrfA). The DNA encoding OrfA-ACP1 has a base sequence represented by SEQ ID NO: 7 (positions 3381 to 3612 of SEQ ID NO: 31). OrfA-ACP1 extends from about position 1127 to about position 1204 of SEQ ID NO: 32. OrfA-ACP1 has an amino acid sequence represented by SEQ ID NO: 8 (positions 1127 to 1204 of SEQ ID NO: 32). It is noted that OrfA-ACP1 contains an active site motif: LGIDS * (* pantethein binding motif S ₁₁₆₆ ). The active motif has the amino acid sequence represented by SEQ ID NO: 42.

The base and amino acid sequences of all four ACP domains are highly conserved and therefore the sequences for each domain are not represented herein by individual sequence identifiers. However, based on the information disclosed herein, one of ordinary skill in the art can readily determine a sequence that includes each of the other three ACP domains.
All four ACP domains together span the region of OrfA from SEQ ID NO: 31 to about position 3621 to about 4632 and correspond to amino acids from about position 1127 to about position 1544 of SEQ ID NO: 32. DNA encoding the entire ACP region including all four domains has the base sequence represented by SEQ ID NO: 43. The region represented by SEQ ID NO: 43 includes linker segments between individual domains. The repeat interval for the four domains is approximately every 340 bases of SEQ ID NO: 43 (the actual number of amino acids measured between adjacent active site serines ranges from 112 to 114 amino acids. Is). Each of the four ACP domains contains a pantethein binding motif LGIDS * (having the amino acid sequence represented by SEQ ID NO: 42), and S * is a pantethein binding site serine (S). The pantethein binding site serine (S) is located near the center of each domain sequence. For the amino acid sequence represented by SEQ ID NO: 32, the position of the active site serine residue (ie, pantethein binding site) for each of the four domains is as follows: ACP1 = S ₁₁₆₆ ; ACP2 = S ₁₂₈₀ ; ACP3 = S ₁₃₉₂ ; and ACP4 = S ₁₅₀₆ . Given that the average size of each domain is about 85 amino acids excluding the linker, about 110 amino acids including the linker, and that the active site serine is approximately in the middle of the domain, those skilled in the art The location of each of the four ACP domains can be easily determined.
1.2.2 Protein having KR domain Examples of the protein having the KR domain of [1] above include (a) a protein having the amino acid sequence represented by SEQ ID NO: 34 (hereinafter also referred to as OrfB protein), (b). ) Consisting of an amino acid sequence having at least 95% or more, preferably 97% or more, more preferably 98% or more, most preferably 99% identity with the amino acid sequence represented by SEQ ID NO: 34, and SEQ ID NO: 32, A homologous protein having an activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having the amino acid sequence represented by each of 36, 38 and 22, or (c) an amino acid sequence represented by SEQ ID NO: 34 1 to 20, preferably 1 to 10, and most preferably 1 to 5 amino acid sequences are deleted, substituted, inserted or added, and arranged. Can be exemplified mutation protein having an activity to produce ARA acetyl CoA as the starting substrate in cooperation with the protein having the amino acid sequence represented by each number 32, 36, 38 and 22.

The description regarding the homologous protein and the mutant protein is the same as in 1.2.1 above.
DNA encoding the OrfB protein (hereinafter referred to as OrfB) is a DNA having the base sequence represented by SEQ ID NO: 33.
There is one KR domain within the OrfB protein.
OrfB was compared with known sequences in a BLAST search. At the nucleic acid level, OrfB has no significant homology with any known base sequence. At the amino acid level, the sequence with the highest degree of homology with the OrfB protein was: 51 for the 802 amino acid residues of unidentified eubacterium SCB49 polyunsaturated fatty acid synthase pfaA (accession number EDM45379) And 50% identical for the 802 amino acid residues of Psychroflexus torquis ATCC 700755 hypothetical protein (accession number EAS69829).

The KR domain in the OrfB protein is also referred to herein as the OrfB-KR domain. This domain extends from the origin at about position 550 of SEQ ID NO: 33 to the end point at about position 1630 of SEQ ID NO: 33. The DNA encoding the OrfB-KR domain has a base sequence represented by SEQ ID NO: 9 (positions 550 to 1630 of SEQ ID NO: 33). The amino acid sequence containing the KR domain extends from the origin of about position 184 of SEQ ID NO: 34 (OrfB protein) to the end of position 543 of SEQ ID NO: 34. The OrfB-KR domain has an amino acid sequence represented by SEQ ID NO: 10 (positions 184 to 543 of SEQ ID NO: 34) herein.
1.2.3 Protein having KS domain, CLF domain, AT domain and DH domain The protein having KS domain, CLF domain, AT domain and DH domain of [1] above is, for example, (a) represented by SEQ ID NO: 36. (B) an amino acid sequence represented by SEQ ID NO: 36, at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably ARA is produced using acetyl-CoA as a starting substrate in cooperation with a protein having an amino acid sequence having an identity of 99% or more and having an amino acid sequence represented by each of SEQ ID NOs: 32, 34, 38 and 22. In the homologous protein having activity, or (c) the amino acid sequence represented by SEQ ID NO: 36, 1 to 20, preferably 1 to 10, most preferably 1 to 5 Acetyl CoA is started in cooperation with a protein having an amino acid sequence in which one amino acid is deleted, substituted, inserted or added and having the amino acid sequence represented by each of SEQ ID NOs: 32, 34, 38 and 22. Examples of the substrate include mutant proteins having an activity of producing ARA.

The description regarding the homologous protein and the mutant protein is the same as in 1.2.1 above.
DNA encoding the OrfC protein (hereinafter also referred to as OrfC) is a DNA having the base sequence represented by SEQ ID NO: 35.
Within the OrfC protein, there is one KS domain, one CLF domain, one AT domain, and two DH domains.

  OrfC was compared with known sequences in a BLAST search. At the nucleic acid level, OrfC has no significant homology with any known base sequence. At the amino acid level, the sequence with the highest degree of homology with the OrfC protein was: 58% identical for 2249 amino acid residues of unidentified eubacterium SCB49 hypothetical protein (accession number EDM45378); And 57% identical for 2250 amino acid residues of Psychroflexus torquis ATCC 700755 hypothetical protein (accession number EAS69830).

The first domain in the OrfC protein is a KS domain, also referred to herein as an OrfC-KS domain. This domain extends from an origin between about position 1 to about position 43 of SEQ ID NO: 35 (OrfC) to an end point between about position 1332 to about position 1350 of SEQ ID NO: 35. The DNA encoding the OrfC-KS domain has a base sequence represented by SEQ ID NO: 3 (positions 1 to 1350 of SEQ ID NO: 35). The amino acid sequence containing the KS domain extends from the origin between about position 1 to about position 15 of SEQ ID NO: 36 (OrfC protein) to the end point between about position 444 to about position 450 of SEQ ID NO: 36. The OrfC-KS domain has an amino acid sequence represented by SEQ ID NO: 4 (positions 1 to 450 of SEQ ID NO: 36). It is noted that the OrfC-KS domain contains an active site motif: DXAC * (* acyl binding site C ₁₉₅ ).

  The second domain in the OrfC protein is a CLF domain, also referred to herein as an OrfC-CLF domain. This domain extends from the origin between about position 1354 to about 1366 of SEQ ID NO: 35 (OrfC) to the end point between about 2400 to about 2460 of SEQ ID NO: 35. The DNA encoding the OrfC-CLF domain has a base sequence represented by SEQ ID NO: 11 (positions 1354 to 2460 of SEQ ID NO: 35). The amino acid sequence containing the CLF domain extends from the origin between about position 452 to about 456 of SEQ ID NO: 36 (OrfC protein) to the end point between about position 800 and about 820 of SEQ ID NO: 36. The OrfC-CLF domain has an amino acid sequence represented by SEQ ID NO: 12 (positions 452 to 820 of SEQ ID NO: 36). It is noted that the OrfC-CLF domain contains a KS domain active site motif that does not contain an acyl-linked cysteine.

The third domain in the OrfC protein is an AT domain, also referred to herein as an OrfC-AT domain. This domain extends from an origin between about position 2488 to about position 2938 of SEQ ID NO: 35 (OrfC) to an end point between about position 3990 to about position 4050 of SEQ ID NO: 35. The DNA encoding the OrfC-AT domain has a base sequence represented by SEQ ID NO: 13 (positions 2488 to 4050 of SEQ ID NO: 35) in the present specification. The amino acid sequence containing the AT domain extends from the origin between about position 830 to about 980 of SEQ ID NO: 36 (OrfC protein) to the end point between about position 1330 to about 1350 of SEQ ID NO: 36. The OrfC-AT domain has an amino acid sequence represented by SEQ ID NO: 14 (positions 830 to 1350 of SEQ ID NO: 36). It is noted that the OrfC-AT domain contains an active site motif of GxS * xG (* acyl binding site S ₁₀₉₄ ) that exhibits the properties of acyltransferases.

  The fourth domain in the OrfC protein is a DH domain, also referred to herein as OrfC-DH domain 1. This is one of the two DH domain domains in the OrfC protein and is therefore named DH domain 1. This domain extends from an origin between about positions 5008 to about 5068 of SEQ ID NO: 35 (OrfC) to an end point between about positions 5490 to about 5550 of SEQ ID NO: 35. The DNA encoding ORFC-DH domain 1 has a base sequence represented by SEQ ID NO: 15 (positions 5008 to 5550 of SEQ ID NO: 35). DH domain 1 extends from the origin between about positions 1670 and 1690 of SEQ ID NO: 36 (OrfC protein) to the end point between about positions 1830 and 1850 of SEQ ID NO: 36. OrfC-DH domain 1 has an amino acid sequence represented by SEQ ID NO: 16 (positions 1670 to 1850 of SEQ ID NO: 36).

The second domain in the OrfC protein is a DH domain, also referred to herein as OrfC-DH domain 2. This is the second of the two DH domains in the OrfC protein and is therefore named DH domain 2. This domain extends from the origin between about position 6328 to about position 6418 of SEQ ID NO: 35 (OrfC) to the end of SEQ ID NO: 35. The DNA encoding OrfC-DH domain 2 has a base sequence represented by SEQ ID NO: 17 (positions 6328 to 6726 of SEQ ID NO: 35). DH domain 2 extends from the origin between about position 2110 to about position 2140 of SEQ ID NO: 36 (OrfC protein) to the end of SEQ ID NO: 36. OrfC-DH domain 2 has an amino acid sequence represented by SEQ ID NO: 18 (positions 2110 to 2241 of SEQ ID NO: 36).
1.2.4 Protein Having ER Domain As the protein having the ER domain of [1] above, for example, (a) a protein having an amino acid sequence represented by SEQ ID NO: 38 or 40 (an amino acid sequence represented by SEQ ID NO: 38) (B) the amino acid sequence represented by SEQ ID NO: 38 or 40 and at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably ARA is produced using acetyl-CoA as a starting substrate in cooperation with a protein comprising an amino acid sequence having an identity of 99% or more and having an amino acid sequence represented by each of SEQ ID NOs: 32, 34, 36 and 22. A homologous protein having activity, or (c) 1-20, preferably 1-10, most preferably in the amino acid sequence represented by SEQ ID NO: 38 or 40 Acetyl in cooperation with a protein comprising an amino acid sequence in which 1 to 5 amino acids are deleted, substituted, inserted or added, and having an amino acid sequence represented by each of SEQ ID NOs: 32, 34, 36 and 22 Mention may be made of mutant proteins having the activity of producing ARA using CoA as a starting substrate.

The description regarding the homologous protein and the mutant protein is the same as in 1.2.1 above.
DNA encoding the OrfD protein (hereinafter also referred to as OrfD) is a DNA having the base sequence represented by SEQ ID NO: 37.
There is one ER domain within the OrfD protein.
OrfD was compared with known sequences in a BLAST search. At the nucleic acid level, OrfD has no significant homology with any known base sequence. At the amino acid level, the sequence with the highest degree of homology to the OrfD protein is as follows: unidentified eubacterium SCB49 2-nitropropane dioxygenase (accession number EDM45377), 65% identical for 515 amino acid residues And 66% identical for the 515 amino acid residues of the Psychroflexus torquis ATCC 700755 hypothetical protein (accession number EAS69831).

The ER domain in the OrfD protein is also referred to herein as the OrfD-ER domain. The DNA encoding this domain extends from the origin between about position 208 and about position 238 of SEQ ID NO: 37 (OrfD) to the end point between about position 1455 and about position 1485 of SEQ ID NO: 37. The DNA encoding the OrfD-ER domain has a base sequence represented by SEQ ID NO: 19 (positions 208 to 1485 of SEQ ID NO: 37). The ER domain extends from the origin between about position 70 and about position 80 of SEQ ID NO: 38 (OrfD protein) to the end point between position 485 and about position 495 of SEQ ID NO: 38. The OrfD-ER domain has an amino acid sequence represented by SEQ ID NO: 20 (positions 70 to 495 of SEQ ID NO: 38).
1.2.5 Protein Having PPT Domain Examples of the protein having the PPT domain of [1] above include (a) a protein having an amino acid sequence represented by SEQ ID NO: 22, 24, 26, 28 or 30 (SEQ ID NO: 22 (Hereinafter also referred to as OrfE protein), (b) at least 95% or more of any amino acid sequence represented by SEQ ID NO: 22, 24, 26, 28 or 30; Is a protein comprising an amino acid sequence having an identity of 97% or more, more preferably 98% or more, most preferably 99% or more, and having the amino acid sequences represented by SEQ ID NOs: 32, 34, 36 and 38, respectively. A homologous protein having the activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with, or (c) SEQ ID NO: 22, 24, 26, 28 or 30 Any amino acid sequence represented, consisting of an amino acid sequence in which 1 to 20, preferably 1 to 10, most preferably 1 to 5 amino acids have been deleted, substituted, inserted or added; Mention may be made of mutant proteins having the activity of producing ARA using acetyl CoA as a starting substrate in cooperation with proteins having the amino acid sequences represented by numbers 32, 34, 36 and 38, respectively.

The description regarding the homologous protein and the mutant protein is the same as in 1.2.1 above.
DNA encoding the OrfE protein (hereinafter referred to as OrfE) is DNA having the base sequence represented by SEQ ID NO: 21.
OrfE protein functions as a PPT of the ARA-producing PKS system.
OrfE was compared to known sequences in a standard BLAST search. At the nucleic acid level, OrfE has no significant homology with any known base sequence. At the amino acid level, the sequence with the highest degree of homology to the OrfE protein is: 37 for the 203 amino acid residues of 4 'phosphopantetheinyl transferase (accession number EAY28494) of Microscilla marina ATCC 23134 And 58% identical for 171 amino acid residues of Kordia algicida OT-1 putative phosphopanthetteinyl transferase accession number EDP94250).
2 DNA of the present invention
The DNA of the present invention is a DNA encoding a protein constituting the complex of the protein of the present invention.
2.1 Examples of DNA of the present invention
2.1.1 DNA encoding a protein with KS domain, MAT domain and ACP domain
Examples of DNA encoding a protein having a KS domain, a MAT domain, and an ACP domain include (a) a DNA encoding the protein described in 1.2.1 above, and (b) a base sequence represented by SEQ ID NO: 31 DNA, (c) hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 31 and represented by SEQ ID NOs: 34, 36, 38 and 22, respectively. DNA encoding a homologous protein having an activity of producing ARA using acetyl CoA as a starting substrate in cooperation with a protein having an amino acid sequence, or (d) at least 95% or more of the base sequence represented by SEQ ID NO: 31, preferably Consists of nucleotide sequences having identity of 97% or more, more preferably 98% or more, most preferably 99% or more, and SEQ ID NOs: 34, 36, 38 and 22 DNA encoding the homologous protein having an activity to produce ARA acetyl CoA as the starting substrate in cooperation with the protein having the amino acid sequence represented by respectively, can be mentioned.

  In the above, hybridizing is a process in which DNA hybridizes to DNA having a specific base sequence or a part of the DNA. Accordingly, the DNA having the specific base sequence or a part of the base sequence of the DNA is useful as a probe for Northern or Southern blot analysis, or DNA of a length that can be used as an oligonucleotide primer for PCR analysis. May be. Examples of DNA used as a probe include at least 100 bases, preferably 200 bases or more, more preferably 500 bases or more. DNA used as a primer is at least 10 bases, preferably 15 bases or more. Can give you DNA.

  Methods for DNA hybridization experiments are well known, for example, Molecular Cloning 2nd edition, 3rd edition (2001), Methods for GenER domain al and Molecular BactEriology, ASM Press (1994), Immunology methods manual, Academic In addition to the description in press (Molecular), hybridization conditions can be determined according to a number of other standard textbooks and experiments can be performed.

  Furthermore, DNA that hybridizes under stringent conditions can also be obtained by following the instructions attached to a commercially available hybridization kit. Examples of commercially available hybridization kits include a random primed DNA labeling kit (Roche Diagnostics) that prepares a probe by a random prime method and performs hybridization under stringent conditions.

  The above stringent conditions include, for example, a DNA-immobilized filter and probe DNA, 50% formamide, 5 × SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate ( pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / l denatured salmon sperm DNA, incubated overnight at 42 ° C., for example, 0.2 × SSC at about 65 ° C. Conditions for washing the filter in a solution can be given.

The various conditions described above can also be set by adding or changing a blocking reagent used to suppress the background of the hybridization experiment. The addition of the blocking reagent described above may be accompanied by a change in hybridization conditions in order to adapt the conditions.
The DNA that can hybridize under the stringent conditions described above includes, for example, at least the base sequence represented by SEQ ID NO: 31 when calculated based on the above parameters using a program such as BLAST and FASTA described above. Examples thereof include DNA having a base sequence having an identity of 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more.
2.1.2 DNA encoding a protein having a KR domain
Examples of DNA encoding a protein having a KR domain include: (a) DNA encoding the protein described in 1.2.2 above, (b) DNA having the base sequence represented by SEQ ID NO: 33, (c) sequence A protein that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by No. 33 and has an amino acid sequence represented by each of SEQ ID Nos. 32, 36, 38, and 22; DNA that encodes a homologous protein having the activity of producing ARA using acetyl-CoA as a starting substrate in cooperation, or (d) at least 95% or more, preferably 97% or more, with the base sequence represented by SEQ ID NO: 33 Preferably, the amino acid comprises a nucleotide sequence having identity of 98% or more, most preferably 99% or more, and represented by each of SEQ ID NOs: 32, 36, 38 and 22 DNA encoding the homologous protein having an activity to produce ARA acetyl CoA as the starting substrate in cooperation with the protein having the sequence, and the like.

The description about hybridization and stringent conditions is the same as 2.1.1 above.
The DNA that can hybridize under the above stringent conditions includes, for example, at least the base sequence represented by SEQ ID NO: 33 when calculated based on the above parameters using a program such as BLAST and FASTA described above. Examples thereof include DNA having a base sequence having an identity of 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more.
2.1.3 DNA encoding a protein having KS domain, CLF domain, AT domain and DH domain
Examples of DNA encoding a protein having a KS domain, a CLF domain, an AT domain, and a DH domain include (a) DNA encoding the protein described in 1.2.3 above, (b) a base represented by SEQ ID NO: 35 (C) hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 35, and each of SEQ ID NOS: 32, 34, 38 and 22 A DNA encoding a homologous protein having an activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having the amino acid sequence represented by: (d) at least 95% or more of the base sequence represented by SEQ ID NO: 35 Consisting of a base sequence having an identity of preferably 97% or more, more preferably 98% or more, most preferably 99% or more, and SEQ ID NOs: 32, 34, 38 and And a DNA encoding a homologous protein having an activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having an amino acid sequence represented by each of 22 and 22.

The description about hybridization and stringent conditions is the same as 2.1.1 above.
The DNA that can hybridize under the above stringent conditions includes, for example, at least the base sequence represented by SEQ ID NO: 35 when calculated based on the above parameters using a program such as BLAST and FASTA described above. Examples thereof include DNA having a base sequence having an identity of 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more.
2.1.4 DNA encoding a protein having an ER domain
Examples of DNA encoding a protein having an ER domain include (a) DNA encoding the protein described in 1.2.4 above, (b) DNA having the base sequence represented by SEQ ID NO: 37 or 39, (c ) It hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 37 or 39, and is represented by SEQ ID NO: 32, 34, 36 and 22, respectively. A DNA encoding a homologous protein having an activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having (d) a base sequence represented by SEQ ID NO: 37 or 39, preferably at least 95% or more, preferably 97% or more, more preferably 98% or more, most preferably 99% or more of the nucleotide sequence, and each of SEQ ID NOs: 32, 34, 36 and 22 DNA, encoding the protein in cooperation with homologous protein having an activity to produce ARA acetyl CoA as the starting substrate and having an amino acid sequence represented in the like.

The description about hybridization and stringent conditions is the same as 2.1.1 above.
The DNA that can hybridize under the above stringent conditions includes, for example, the base sequence represented by SEQ ID NO: 37 or 39 when calculated based on the above parameters using a program such as the above BLAST and FASTA. And DNA having a nucleotide sequence having at least 95%, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more.
2.1.5 DNA encoding a protein having a PPT domain
Examples of DNA encoding a protein having a PPT domain include (a) DNA encoding the protein described in 1.2.5 above, (b) a nucleotide sequence represented by SEQ ID NO: 21, 23, 25, 27 or 29 (C) hybridizes under stringent conditions with a DNA comprising a base sequence complementary to any one of the base sequences represented by SEQ ID NO: 21, 23, 25, 27 or 29, and SEQ ID NO: DNA encoding a homologous protein having an activity of producing ARA using acetyl CoA as a starting substrate in cooperation with a protein having an amino acid sequence represented by each of 32, 34, 36 and 38, or (d) SEQ ID NO: 21 , 23, 25, 27 or 29 and at least 95% or more, preferably 97% or more, more preferably 98% or more, most preferably Producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein comprising a base sequence having an identity of 99% or more and having an amino acid sequence represented by each of SEQ ID NOs: 32, 34, 36 and 38 Examples thereof include DNA encoding a homologous protein having activity.

The description about hybridization and stringent conditions is the same as 2.1.1 above.
As the DNA that can hybridize under the above-mentioned stringent conditions, for example, when calculated based on the above parameters using a program such as BLAST and FASTA described above, SEQ ID NO: 21, 23, 25, 27, or 29 DNA having a nucleotide sequence having at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with any one of the nucleotide sequences represented by
2.2 Recombinant DNA having the DNA of the present invention
The DNA of the present invention includes recombinant DNA having the DNA of the present invention described in 2.1 above.

The recombinant DNA is incorporated into an expression vector containing the promoter at a position where the DNA of the present invention can autonomously replicate in a host organism or can be integrated into a chromosome and can transcribe the DNA. Recombinant DNA.
The host organism will be described later in 3.
When a prokaryotic organism such as a bacterium is used as a parent strain for the host organism, the recombinant DNA is a recombinant DNA composed of a promoter, a ribosome binding sequence, the DNA of the present invention described in 2 above, and a transcription termination sequence. It is preferable. A gene that controls the promoter may also be included.

It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases). In the recombinant DNA, a transcription termination sequence is not necessarily required for the expression of the DNA, but a transcription termination sequence is preferably arranged immediately below the structural gene.
When a microorganism belonging to the genus Escherichia is used as a parent strain, examples of expression vectors include pColdI (Takara Bio), pCDF-1b, pRSF-1b (all manufactured by Novagen), pMAL-c2x (New England Biolabs) PGEX-4T-1 (manufactured by GE Healthcare Bioscience), pTrcHis (manufactured by Invitrogen), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-30 (manufactured by Qiagen) , PET-3 (manufactured by Novagen), pKYP10 (JP 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 ( 1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82, 4306 (1985)], pBluescriptII SK (+), pBluescript II KS (-) (Stratagene), pTrS30 [Escherichia coli JM109 / pTrS30 (prepared from Ferm BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (Ferm BP-5408)], pTK31 [APPLIED AND ENVIRONMENTAL MICROBIO LOGY, 2007, Vol. 73, No. 20, p.6378-6385] pPAC31 (WO98 / 12343), pUC19 [Gene, 33, 103 (1985)], pSTV28 (manufactured by Takara Bio Inc.), pUC118 (Takara Bio Inc.) And pPA1 (Japanese Patent Laid-Open No. 63-233798).

Any promoter can be used as long as it functions in the cells of microorganisms belonging to the genus Escherichia. For example, trp promoter (P _trp ), lac promoter (P _lac ), P _L promoter, P _R promoter and P _SE promoter, may be used promoters derived from Escherichia coli, phage and the like. In addition, an artificially designed and modified promoter such as a promoter in which two Ptrps are connected in series, a tac promoter, a lacT7 promoter, and a let I promoter can also be used.

When a coryneform bacterium is used as a parent strain, examples of expression vectors include pCG1 (Japanese Patent Laid-Open No. 57-134500), pCG2 (Japanese Patent Laid-Open No. 58-35197), pCG4 (Japanese Patent Laid-Open No. 57-183799), pCG11 (special Kaisho 57-134500), pCG116, pCE54, pCB101 (all of which are JP-A-58-105999), pCE51, pCE52, pCE53 (all of which are Molecular and General Genetics, 196, 175 (1984)).
As the promoter in the case of using the above expression vector, any promoter can be used as long as it functions in cells of coryneform bacteria. For example, P54-6 promoter [Appl. Microbiol. Biotechnol., 53, 674-679 ( 2000)] can be used.

When a yeast strain is used as the parent strain, examples of the expression vector include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 and the like.
As the promoter in the case of using the above expression vector, any promoter can be used as long as it functions in yeast strain cells. For example, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter And promoters such as heat shock polypeptide promoter, MFα1 promoter, CUP 1 promoter, and the like.

When a plant is used as the host organism, the recombinant DNA is a recombinant DNA composed of a promoter, a 5 ′ untranslated region (translation enhancer), the DNA of the present invention described in 2 above, and a transcription termination sequence. It is preferable. A gene that controls the promoter may also be included. Such an expression vector may be any expression vector that functions in plant cells. For example, pRI 201-AN, pRI 201-ON, pRI 201-AN-GUS, and pRI 201-ON- GUS etc. (all manufactured by Takara Bio Inc.) can be mentioned.
3 Organism of the Present Invention The organism of the present invention is an organism in which the activity of the complex of the protein of the present invention 1 is enhanced from the host organism.

The host organism refers to the original organism that is the subject of genetic modification and transformation. When the original organism to be transformed by gene transfer is a microorganism, it is also called a parent strain or a host strain.
The host organism may be any organism, but examples include microorganisms or plants.
Microorganisms can include any bacteria, protozoa, microalgae, fungi, protozoa.

In particular, examples of microalgae include microorganisms of the order of the genus Aurelia. Examples of the microorganism of the order of the genus Ceratomycetes include microorganisms selected from the group consisting of the genus Thraustochytrium, Labyrinthuloides, Japonotitrium, Schizochytrium, and Aurantiochytrium.
Preferred species within these genera include, but are not limited to, Schizochytrium Minutsumu (Schizochytriumminutum), Schizochytrium sp (S31) (ATCC 20888), Schizochytrium sp (S8) (ATCC 20889), Schizochytrium sp ( (LC-RM) (ATCC 18915), Schizochytrium sp (SR21), Schizochytrium agregatum (Goldstein and Belsky) (ATCC 28209), Schizochytrium remacinum (Honda and Yokochi) (IFO 32693), Thraustochytrium sp (23B) (ATCC 20891), Thraustochytrium / Schneider (ATCC 24473), Thraustochytrium / Aureum (ATCC 34304), Thraustochytrium / Roseum (ATCC 28210), Urkenia SP BP-5601 , Japonochytrium sp. (L1) (ATCC 28207), Aurantiochytrium sp. OH4 ( FERM BP-11524).

  In particular, fungi include yeasts including Saccharomyces cerevisiae, Saccharomyces carlsbergensis, or other yeasts such as Candida, Kluyveromyces, or other fungi Examples thereof include filamentous fungi such as Aspergillus, Neurospora, and Penicillium.

In particular, as a bacterium, preferably, a microorganism belonging to a genus selected from the group consisting of Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, and Aureospila, most preferably, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli BL21 codon plus ( Stratagene), Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066, Corynebacterium ammoniagenes, Corynebacterium glutamicu Examples include microorganisms selected from the group consisting of m ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, Pseudomonas sp. D-0110, and Aureispira marina JCM23201.

  Plants used as host organisms include, but are not limited to, any higher plant and in particular consumable plants including crop plants and especially plants using their oils. Such plants can include, for example, canola, soybeans, rapeseed, flaxseed, corn, safflower, sunflower, and tobacco. Other useful plants are those plants known to produce compounds used as pharmaceuticals, seasonings, nutrients, functional food ingredients or cosmetic active agents, or these compounds / drugs Plants that have been genetically engineered to produce

  When the host organism originally has a DNA encoding the protein constituting the complex of the protein of the present invention of 1 above, the activity of the protein complex of the above 1 of the present invention is the host The organism enhanced from the organism is obtained by modifying (a) a gene encoding a protein constituting the complex of the protein of the present invention of 1 above, which is present on the chromosomal DNA of the host organism, i) a host An organism having an increased specific activity of the protein compared to the organism, and ii) an organism having an increased amount of transcription of the gene or production of the protein complex compared to the host organism, and (b) the recombination of 2.2 above By transforming a host organism with body DNA, an organism having an increased copy number of the gene than the host organism can be mentioned.

The organism of the above (a) i) is an organism in which the specific activity of the protein complex is enhanced. The organism is cultured or cultivated by the following method 6 and accumulated in the culture or the cultivated product. ARA can be confirmed by comparison with that of the host organism.
The organism of the above (a) ii) is an organism in which the transcription amount of the gene or the production amount of the protein complex is increased as compared with the host organism, and the organism of the above (b) is more than the host organism. For example, an organism having an increased copy number of the gene can be expressed by, for example, the amount of transcription of the DNA of the present invention by Northern blotting or the production of the complex of the protein of the present invention by Western blotting. This can be confirmed by comparison.

When the host organism does not have the DNA encoding the protein that constitutes the complex of the protein of the present invention described in 1 above, the organism of the present invention is (c) the host organism is transformed with the recombinant DNA described in 2.2 above. It is an organism capable of producing ARA using acetyl-CoA in cells obtained by transformation as a starting substrate.
The recombinant DNA is introduced as a plasmid capable of autonomous replication in the parent strain or is integrated into the chromosomal DNA of the parent strain.

  In the organism, when the host cell has a gene encoding a known PUFA-PKS, a protein or domain that determines the carbon chain length of the known PUFA-PKS in the known PUFA-PKS, A DNA encoding a protein or domain selected from the group consisting of a protein or domain that determines the number and arrangement of double bonds, and a protein or domain that reduces trans double bonds, and the corresponding domains described in Table 1 above Alternatively, it includes organisms in which the activity of the known PUFA-PKS is converted to the activity of producing ARA using acetyl-CoA as a starting substrate by substituting with a DNA encoding a protein having the domain.

  The replacement of the DNA encoding the protein or domain described above is a combination in which the DNA to be replaced in the host cell is replaced with a part of the corresponding DNA of the present invention and also has a part of the corresponding DNA of the present invention. The case where the recombinant DNA is incorporated into a region different from the gene encoding the known PUFA-PKS in the chromosomal DNA of the host cell or the case where the recombinant DNA exists in the host cell as a self-replicating plasmid is included.

As a method for confirming that the organism (c) has acquired the ability to produce ARA using acetyl-CoA in the cells of the host organism as a starting substrate, for example, the organism is cultured or cultivated by the following method 5: It can be confirmed by detecting ARA accumulated in the culture or culture by gas chromatography.
A method for transforming a host organism with recombinant DNA will be described later in 5.
4 Method for Acquiring DNA of the Present Invention The DNA of the present invention comprises, for example, a microorganism using a probe DNA that can be designed based on a base sequence selected from the group consisting of SEQ ID NOs: 31, 33, 35, 37, and 21, Preferably, Southern hybridization to a microorganism belonging to the genus Aureispira , more preferably, a chromosomal DNA library of Aureispira marina JCM23201, or a primer DNA that can be designed based on the nucleotide sequence is used. It can be obtained by PCR [PCR Protocols, Academic Press (1990)] using the chromosomal DNA of the microorganism as a template.

Aureospila Marina JCM23201 can be obtained from RIKEN BioResource Center, Japan.
Among the DNAs of the present invention, the DNA encoding the homologous protein described in 1 above is, for example, a sequence number selected from the group consisting of SEQ ID NOs: 31, 33, 35, 37 and 21 with respect to various gene sequence databases. Search for a base sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the represented base sequence, or against various protein sequence databases 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more of the amino acid sequence represented by SEQ ID NO: selected from the group consisting of SEQ ID NOs: 32, 34, 36, 38 and 22. Probe DNA that can be searched for amino acid sequences having identity and designed based on the base sequence or amino acid sequence obtained by the search Or it can acquire by the method similar to the method of acquiring said DNA using primer DNA and the microorganisms which have the said DNA.

Among the DNAs of the present invention, the DNA encoding the mutant protein described in 1 above is represented by, for example, a base sequence represented by a sequence number selected from the group consisting of SEQ ID NOs: 31, 33, 35, 37 and 21. Can be obtained by subjecting the DNA to a template using error-prone PCR.
Alternatively, PCR [Gene, 77, 51 (1989) using a set of PCR primers each having a base sequence designed to introduce a target mutation (deletion, substitution, insertion or addition) at the 5 ′ end thereof. )] Can also obtain the DNA of [2] above. That is, first, a sense primer corresponding to the 5 ′ end of the DNA and an antisense primer corresponding to the sequence immediately before the mutation introduction site (5 ′ side) having a sequence complementary to the mutation sequence at the 5 ′ end. PCR is performed using the DNA as a template, and a fragment A (mutation is introduced at the 3 ′ end) from the 5 ′ end to the mutation introduction site of the DNA is amplified. Next, using the DNA as a template with a sense primer corresponding to the sequence immediately after the mutation introduction site (3 ′ side) having a mutation sequence at the 5 ′ end and an antisense primer corresponding to the 3 ′ end of the DNA PCR is performed to amplify the fragment B from the mutation introduction site to the 3 ′ end of the DNA in which the mutation is introduced at the 5 ′ end. When these amplified fragments are purified and mixed, and PCR is carried out without adding a template or primer, the sense strand of amplified fragment A and the antisense strand of amplified fragment B have the same mutation introduction site and thus hybridize. Then, the PCR reaction proceeds as a primer and template, and the DNA into which the mutation has been introduced is amplified.

The obtained DNA is cut as it is or with an appropriate restriction enzyme and incorporated into a vector by a conventional method. After the obtained recombinant DNA is introduced into a host cell, a commonly used nucleotide sequence analysis method such as By analyzing using the dideoxy method [Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)] or a base sequence analyzer such as 3700 DNA Analyzer (Applied Biosystems), The sequence can be determined. Examples of the host cell, e.g., Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49 Escherichia coli W3110, Escherichia coli NY49, Escherichia coli MP347, Escherichia coli NM522 and the like.

The above vectors include pBluescriptII KS (+) (Stratagene), pDIRECT [Nucleic Acids Res., 18, 6069 (1990)], pCR-Script Amp SK (+) (Stratagene), pT7Blue ( Novagen), pCR II (Invitrogen), and pCR-TRAP (Gen Hunter).
As a method for introducing recombinant DNA, any method can be used as long as it introduces DNA into a host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], protoplast method (Japanese Patent Laid-Open No. 63-248394), electroporation method [Nucleic Acids Res., 16, 6127 (1988)] and the like.

If the obtained DNA has a partial length as a result of determining the base sequence, the full-length DNA can be obtained by Southern hybridization or the like for a chromosomal DNA library using the partial length DNA as a probe. .
Further, based on the determined DNA base sequence, the target DNA can be prepared by chemical synthesis using a 8905 type DNA synthesizer manufactured by Perceptive Biosystems.

  Among the DNAs of the present invention, in DNA encoding known PUFA-PKS, a protein or domain that determines the carbon chain length of the PUFA-PKS, a protein or domain that determines the number and arrangement of cis double bonds, And a DNA encoding a protein or domain selected from the group consisting of a protein or domain that reduces a trans double bond, and a DNA encoding a corresponding domain or a protein having the domain described in Table 1 above. Thus, DNA encoding a protein that has acquired the activity of producing ARA using acetyl CoA as a starting substrate can be obtained using the following overlap extension PCR method (Gene 77: 61-68, 1989).

As known PUFA-PKS, PUFA-PKS which Shewanella oneidensis MR-1 has can be mentioned, for example. Shewanella oneidensis MR-1 is available as ATCC BAA1096 strain from the American Tissue Culture Collection (ATCC). The DNA encoding the PUFA-PKS can be obtained by a method similar to the method for obtaining the DNA of the present invention described above.

  Of the DNA encoding known PUFA-PKS, two DNA fragments located at both ends of the DNA to be replaced are each amplified by PCR. Next, a DNA encoding a corresponding domain described in Table 1 or a protein having the domain is amplified by PCR. In addition, a primer for amplifying each fragment is designed so that the base sequence of the linking portion of each fragment is complementary. Furthermore, these three fragments are mixed at an equimolar ratio and used as a PCR template, and when the three fragments are ligated, PCR amplification is performed using a primer that can amplify the whole fragment. The bound target DNA can be obtained.

Further, by repeating the above steps, it is also possible to obtain DNA substituted with a plurality of DNA fragments.
Further, a DNA having the base sequence represented by SEQ ID NO: 23, a DNA encoding the homologous protein, and a DNA encoding the mutant protein can be obtained by the same method as described above using the genomic DNA of Bacillus subtilis 168. Can be acquired.

A DNA having the base sequence represented by SEQ ID NO: 25, a DNA encoding the homologous protein, and a DNA encoding the mutant protein were prepared by the same method as described above using the genomic DNA of Shewanella oneidensis MR-1. Can be acquired.
DNA having the nucleotide sequence represented by SEQ ID NO: 27, DNA encoding the homologous protein, and DNA encoding the mutant protein are obtained by the same method as described above using the genomic DNA of Escherichia coli W3110. be able to.

In addition, DNA having the nucleotide sequence represented by SEQ ID NO: 29, DNA encoding the homologous protein, and DNA encoding the mutant protein were prepared in the same manner as described above using the genomic DNA of Corynebacterium glutamicum ATCC13032. Can be acquired.
The recombinant DNA having the DNA of the present invention can be prepared by inserting the DNA of the present invention obtained above, for example, downstream of the promoter of the appropriate expression vector described in 2.2 above.

Examples of such recombinant DNA include pSTV29-OrfA-OrfB, pMW219-OrfD-OrfC, pUC19-OrfE, pUC19-sfp, pUC19-SO1604, pUC19-entD, pUC19-cgl1980, which will be described later. it can.
(5) Method for constructing organism of the present invention Among the organisms of the present invention, the organism of (3) (a) i) described above is obtained by, for example, comparing the specific activity of the protein complex of the present invention with a usual mutation treatment method or It can be constructed by enhancing using a gene replacement method using a replacement DNA technique.

Examples of the mutation treatment method include a method using N-methyl-N′-nitro-N-nitrosoguanidine (NTG) (Microbial Experiment Manual, 1986, p. 131, Kodansha Scientific), ultraviolet irradiation method and the like. Can give.
As a gene replacement method using recombinant DNA technology, a DNA encoding the mutant protein of the present invention that can be obtained by the above method 4 is homologous to a gene encoding the protein present on the chromosomal DNA of the host organism. Examples of the replacement method include a recombinant method.

As the host organism, for example, the above-mentioned microorganism belonging to the genus Aureospira, preferably Aureispira marina JCM23201 can be mentioned.
Examples of the homologous recombination method include a method using a plasmid for homologous recombination that can be prepared by ligating with a plasmid DNA having a drug resistance gene that cannot autonomously replicate in the host organism to be introduced, and is frequently used in Escherichia coli. As a method using homologous recombination, a method of introducing recombinant DNA using a lambda phage homologous recombination system [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000) ] Can be given.

  In addition, a selection method that makes Escherichia coli susceptible to sucrose by Bacillus subtilis levanshuclase integrated on the chromosome together with the recombinant DNA, or the wild-type rpsL gene is incorporated into Escherichia coli having a mutant rpsL gene resistant to streptomycin. The chromosomal DNA of the parent strain using a selection method that makes Escherichia coli susceptible to streptomycin [Mol. Microbiol., 55, 137 (2005), Biosci. Biotechnol. Biochem., 71, 2905 (2007)] Microorganisms in which the above target region has been replaced with recombinant DNA can be obtained.

Among the organisms of the present invention, the organism of (3) (a) ii) described above is, for example, the amount of transcription of the gene encoding the protein constituting the protein complex of the present invention or the production amount of the protein, It can be constructed by enhancing using a mutation treatment method or a gene replacement method using a recombinant DNA technique.
Examples of the mutation treatment method include the above-described methods.

  The gene replacement method by the recombinant DNA technique comprises a transcription regulatory region and a promoter region of a gene encoding a protein constituting the protein complex of the present invention, for example, a base sequence of 200 bp upstream, preferably 100 bp upstream of the start codon of the protein. A gene encoding the protein present on the chromosomal DNA of the host organism and the homologous recombination described above after introducing the mutation into the DNA by subjecting the DNA having DNA to a mutation treatment in vitro or error-prone PCR, etc. A method of substitution using the method can be mentioned.

Further, by replacing the promoter region of the gene encoding the protein with a known strong promoter sequence, the transcription amount of the gene encoding the protein constituting the protein complex of the present invention or the production amount of the protein can be increased. An enhanced organism can be obtained.
As the host organism, for example, the above-mentioned microorganism belonging to the genus Aureospira, preferably Aureispira marina JCM23201 can be mentioned.

The organisms (b) and (c) above can be produced by the following method.
A host organism is transformed with the recombinant DNA that can be obtained by the above method 4.
As the host organism, when the organism of (3) (b) above is constructed, the microorganism belonging to the genus Aureospira of (3) above, preferably Aureispira marina JCM23201 can be mentioned. In the case of constructing the organism of (3) above, for example, host organisms other than the microorganism belonging to the genus Aureospira of 3 above can be mentioned.

  When the host organism is a microorganism, the method for introducing the recombinant DNA as a plasmid capable of autonomous replication in the parent strain is, for example, a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110. (1972)], protoplast method (JP-A 63-248394), electroporation method [Nucleic Acids Res., 16, 6127 (1988)] and the like.

When the host organism is a microorganism, the homologous recombination method described above can be used as a method for incorporating the recombinant DNA into the chromosomal DNA of the parent strain.
When the host organism is a plant, examples of the method for introducing the recombinant DNA as a plasmid capable of autonomous replication in the parent strain include a particle gun method and an electroporation method.

When the host organism is a microorganism, examples of a method for incorporating the recombinant DNA into the chromosomal DNA of the parent strain include a method using Agrobacterium.
It can be confirmed using the method 3 above that the organism obtained by the above method is the target microorganism.
6 Method for Producing ARA or ARA-Containing Composition of the Present Invention The method for producing the ARA-containing composition of the present invention is carried out by culturing the microorganism in a culture medium when the microorganism constructed by the above method 5 is used. ARA or an ARA-containing composition is produced and accumulated, and an ARA or ARA-containing composition is collected from the culture.

Examples of the ARA-containing composition include ARA-containing fats and oils or ARA-containing phospholipids, preferably ARA-containing fats and oils.
The culture of the microorganism can be obtained by inoculating the microorganism in an appropriate medium and culturing according to a conventional method.
As the medium, any known medium containing a carbon source, a nitrogen source, an inorganic salt, and the like can be used. Examples of the carbon source include carbohydrates such as glucose, fructose, and galactose, fats and oils such as oleic acid and soybean oil, glycerol, sodium acetate, and the like. These carbon sources can be used, for example, at a concentration of 20 to 300 g per liter of medium. According to a particularly preferred embodiment, after the initial carbon source has been consumed, culturing can be continued by feeding the carbon source. By culturing under such conditions, the amount of carbon source to be consumed can be increased, and the production amount of the ARA-containing composition can be increased.

Further, as the nitrogen source, organic nitrogen such as yeast extract, corn steep liquor, polypeptone, sodium glutamate, urea, or inorganic nitrogen such as ammonium acetate, ammonium sulfate, ammonium chloride, sodium nitrate, ammonium nitrate, ammonia can be used. .
As inorganic salt, potassium phosphate etc. can be used in combination as appropriate.

The medium containing the above components is preferably used after sterilizing by an autoclave after adjusting the pH within the range of 4.0 to 9.5 by adding an appropriate acid or base.
The culture temperature is generally 10 to 45 ° C, preferably 20 to 37 ° C. The culture temperature is preferably controlled to a culture temperature capable of producing the ARA-containing composition. The pH during culture is generally 3.5 to 9.5, preferably pH 4.5 to 9.5. The particularly preferable pH varies depending on the purpose, and is 5.0 to 8.0 in order to produce a large amount of fats and oils.

The culture period can be, for example, 2 to 7 days, and the culture can be performed by aeration stirring culture or the like.
A method for separating the culture solution and the microorganism from the culture can be performed by a conventional method known to those skilled in the art, and can be performed, for example, by a centrifugation method or filtration.
The ARA-containing composition can be obtained by crushing the microorganisms separated from the above culture with, for example, ultrasonic waves or dynomill, and then performing solvent extraction with, for example, chloroform, hexane, butanol or the like.

The ARA-containing composition produced by the above production method can be obtained by, for example, converting a short-chain fatty acid with a hydrolyzing enzyme such as a low-temperature solvent fractionation method [Zotaro Takahashi, Yukagaku, 40: 931-941 (1991)] or lipase. The ARA-containing composition having a high ARA content can be obtained by concentrating the ARA-containing composition by a method such as a method of free removal [Takahashi Kotaro, Yukagaku, 40: 931-941 (1991)].
ARA can be produced by separating and collecting ARA from the ARA-containing composition. For example, after preparing a mixed fatty acid containing ARA from an ARA-containing composition by a hydrolysis method, ARA is separated by, for example, a urea addition method, a cooling separation method, a high performance liquid chromatography method, or a supercritical chromatography method. Thus, ARA can be produced.

Moreover, the ARA alkyl ester can be produced by separating and collecting the ARA alkyl ester from the ARA-containing composition.
The ARA alkyl ester is not particularly limited as long as it is an ARA alkyl ester, but preferably, an ARA methyl ester or an ARA ethyl ester, more preferably an ARA ethyl ester.

  In order to separate and collect an ARA alkyl ester from an ARA-containing composition, for example, after preparing a mixed fatty acid alkyl ester containing an ARA alkyl ester from an ARA-containing composition by an alcoholysis method, for example, urea addition method, cooling separation The ARA alkyl ester can be separated and collected by a method, a high performance liquid chromatography method or a supercritical chromatography method.

Further, in the method for producing the ARA-containing composition of the present invention, when using the plant constructed by the above method 5, the plant is cultivated in a medium, and the ARA-containing composition is produced and accumulated in the cultivated product. The manufacturing method of the ARA containing composition characterized by extract | collecting an ARA containing composition from this cultivation can be mentioned.
The plant is cultivated in a medium suitable for the growth of the plant. The medium can be, for example, soil, sand, a granular medium that supports the growth of any other roots (e.g. vermiculite, perlite, etc.), or hydroponics, and the growth of suitable light, water and higher plants Any growth medium for the plant containing the nutritional supplement is included.

The period for cultivating the plant can be, for example, 1 week to 12 months, preferably 2 weeks to 6 months. Depending on the growth of the plant, light, water, nutritional supplements and the like used for normal plant growth are provided.
After growing the cultivated product and pressing the cultivated product, it is extracted with water or the like to obtain a crude oil. From the crude oil, an ARA-containing composition, ARA, or ARA alkyl ester can be produced by the same method as described above.

  Examples are shown below, but the present invention is not limited to the following examples.

Acquisition of DNA of the Present Invention Chromosomal DNA of Aureispira marina JCM23201 strain was prepared by a conventional method, and genome base sequence was determined using Genome Sequencer FLX system (GS FLX +) (Roche Diagnostics). .
As a result, DNA containing the base sequence represented by SEQ ID NO: 44 was obtained, including DNA encoding the protein constituting the protein complex of the present invention.

  As shown in FIG. 1, 6 Orf can be identified in the base sequence of the DNA, and it was named OrfA, OrfB, OrfC, OrfD, OrfE, OrfR. Table 2 shows the base sequence number, the base sequence length (including the stop codon), the amino acid sequence number, the amino acid sequence length, and the position in SEQ ID NO: 44 in each Orf.

  Of the above Orf, OrfR was considered to encode a transcription factor and was not considered to belong to the ARA-PKS system.

Preparation of recombinant DNA containing the DNA of the present invention In order to express the complex of the protein of the present invention identified in Example 1 in E. coli, OrfA, OrfB, OrfC, OrfD and OrfE were placed on three plasmids. Each was cloned. First, each Orf was amplified by PCR using the “Orf amplification primer” shown in Table 3 using the genomic DNA of Aureospira Marina (JCM23201) obtained in Example 1 as a template. As an amplification enzyme, DNA polymerase manufactured by Takara Bio Inc., Primestar GXL was used. The start codons for OrfB and OrfE were TTG and GTG, respectively, but were replaced with ATG to improve the expression level.

In addition, the primer on the 5 ′ side of each Orf (SEQ ID NOs: 47, 49, 51, 53, and 55) is a lac promoter using overlap extension PCR (Gene 77: 61-68, 1989) in a later step. For ligation, a sequence complementary to a primer that amplifies the 3 ′ portion of the lac promoter is added.
In addition, a BamHI recognition site represented by GGATCC is added to the 3 ′ primer (SEQ ID NOs: 48, 50, 52, 54 and 56) of each Orf for cloning into a vector.

Further, the Sse8387I site represented by CCTGCAGG is added to the 3 ′ primer (SEQ ID NOS: 48 and 54) of OrfA and OrfD in addition to the BamHI site.
Next, the lac promoter to be linked to each Orf was amplified by PCR using the “Lac promoter amplification primer” shown in Table 3 using the genomic DNA of E. coli W3110 as a template.

These 3 ′ primers (SEQ ID NOs: 59, 60, 61, 62, and 63) were linked to each Orf using overlap extension PCR in a later step. A sequence complementary to the primer to be amplified is added.
In addition, since these 5 ′ primers are cloned into a vector, an EcoT22I recognition site represented by ATGCAT is added to SEQ ID NO: 57, and an Sse8387I recognition site represented by CCTGCAGG is added to SEQ ID NO: 58. Yes.

Next, overlap extension PCR was performed in order to link the DNA fragment containing the lac promoter obtained above and the DNA fragment containing OrfA to OrfE.
The obtained sequence in which the lac promoter was linked to each Orf was divided into three vectors and cloned. That is, OrfE is a multi-copy vector pUC19 (Gene, 33, 103 (1985), Genbank accession number: L09137), OrfA and OrfB are medium-copy vectors pSTV29 (manufactured by Takara Bio, http: //catalog.takara-bio. asp? unitid = U100003450), OrfC and OrfD were cloned into the low copy vector pMW219 (Nippon Gene, http://nippongene.com/pages/products/clomod/dna_vec/). Details are shown below.

OrfE and lac promoter-ligated DNA were subjected to restriction enzyme treatment with BamHI and EcoT22I, and BamHI and pUC19 treated with Sse8387I were mixed and ligated to obtain an OrfE expression vector pUC19-OrfE (Table 3).
OrfA and lac promoter-ligated DNA were treated with restriction enzymes with BamHI and EcoT22I, mixed with pSTV29 treated with BamHI and Sse8387I, and ligated to obtain an OrfA expression vector pSTV29-OrfA.

OrfB and lac promoter-ligated DNA were subjected to restriction enzyme treatment with BamHI and EcoT22I, mixed with pSTV29-OrfA treated with BamHI and Sse8387I, and ligated to obtain the OrfA and OrfB co-expression vector pSTV29-OrfA-OrfB (Table) 3).
OrfD and lac promoter-linked DNA were subjected to restriction enzyme treatment with BamHI and EcoT22I, mixed with pMW219 treated with BamHI and Sse8387I, and ligated to obtain an OrfD co-expression vector pMW219-OrfD.

  OrfC and lac promoter-linked DNA were restricted with BamHI and Sse8387I, mixed with pMW219-OrfD treated with BamHI and Sse8387I, and ligated to obtain OrfC and OrfD co-expression vector pMW219-OrfD-OrfC (Table) 3).

Construction of Escherichia coli strains expressing the DNA of the present invention Using pUC19-OrfE, pST29-OrfA-OrfB, and pMW219-OrfD-OrfC obtained in Example 2, E. coli, which is a host, was confirmed for ARA production in E. coli. In order to suppress degradation of ARA by E. coli, E. coli having a blocked fatty acid degradation pathway of the host was used. It is known that Escherichia coli in which fadE encoding acyl-CoA dehydrogenase is disrupted exhibits a phenotype that cannot assimilate fatty acids (J BactEriol. 2002 184 (13): 3759-64.).

Therefore, Escherichia coli in which fadE was disrupted was obtained from the E. coli 1 gene disruption library (Mol. Syst. Biol., 2: article number: 2006.0008) (BW25113ΔfadE :: FRT).
According to a conventional method, the E. coli was transformed with pUC19-OrfE, pST29-OrfA-OrfB and pMW219-OrfD-OrfC, and LB agar medium (Bactotrypton (Becton Dickinson)) 10 g / L, Yeast extract (Becton Dickinson) 5 g / L, sodium chloride 5 g / L (manufactured by Wako Pure Chemical Industries), agarose for microorganism culture (manufactured by Nacalai Tesque), ampicillin sodium 50 mg / L, kanamycin sulfate 10 mg / L as antibiotics, Selection was made with a medium (LB agar medium + Amp, Km, Cm) supplemented with chloramphenicol 20 mg / L (all manufactured by Wako Pure Chemical Industries, Ltd.) As a control, an empty vector in which each Orf was not cloned, pUC19 Strains transformed with pSTV29 and pMW219 were also constructed as controls by the same method.

  The strain thus obtained was named ΔfadE-EABDC strain. The control strain created at the same time was named ΔfadE-NNNNN strain.

Method for Producing ARA-Containing Composition of the Present Invention ΔfadE-EABDC strain and ΔfadE-NNNNN strain were applied to LB agar medium (Amp, Km, and Cm added) and pre-cultured at 30 ° C. for 24 hours. Thereafter, the cells were inoculated into a large test tube containing 5 ml of LB liquid medium (Amp, Km, and Cm added) and cultured at 25 ° C. for 24 hours. The cultured medium was further inoculated with 500 ul in a large test tube containing 5 ml of LB liquid medium (Amp, Km, and Cm added), and 100 mM IPTG (manufactured by Wako Pure Chemical Industries) was added. Incubated at 40 ° C. for 40 hours.

After completion of the culture, 4 ml of the culture solution was centrifuged at 9000 rpm for 15 minutes to recover the cells. Lipids were extracted from the cells according to the Bligh-Dyer method, dried under reduced pressure, and then fatty acid methylated using a fatty acid methylation kit (manufactured by Nacalai Tesque). The methylated fatty acid was finally dissolved in 500 ul of hexane. The obtained methylated fatty acid was analyzed using gas chromatography (GC) and gas chromatography mass spectrometry (GCMS). The analysis conditions are as follows.
GC analysis:
Equipment used: GC2010 manufactured by Shimadzu Corporation, column: HR-SS-10 (0.25 mm x 25 m) manufactured by Shinwa Chemical, carrier gas: helium (39.9 cm / sec), column temperature: 190 ° C, vaporization chamber temperature 250 ° C, Split injection (split ratio 60)
GCMS analysis:
Equipment used: GCMS-QP2010, manufactured by Shimadzu Corporation, column: HR-SS-10 (0.25 mm x 25 m), manufactured by Shinwa Kako, carrier gas: helium (39.9 cm / sec), column temperature: 190 ° C, vaporization chamber temperature 250 C, split injection (split ratio 60).

In the analysis, the obtained fatty acid methyl ester was injected into 2 ul GC and GCMS. As a standard, a mixed sample for qualification of fatty acid methyl ester (catalog code 1021-58209) manufactured by GL Sciences Inc. was used. This mixed sample contains arachidic acid, eicosenoic acid, ARA, EPA, and DHA.
The obtained gas chromatogram is shown in FIG. Only when the ARA-PKS system was introduced, a peak corresponding to ARA of the mixed sample was detected.

  Next, as a result of analyzing this peak using GCMS, it was confirmed that the obtained peak actually shows the same fragment pattern as ARA. From the above results, it was confirmed that by introducing the ARA-PKS system found in the present invention, ARA can be produced in Escherichia coli that does not originally have the ability to produce ARA.

Utilization of other types of PPT In order to identify the essential ARA of each ARA-PKS system Orf obtained in the present invention, a replacement experiment with other PUFA-PKS homologous Orf was performed.
First, an experiment was conducted to introduce a homologous gene for OrfE. OrfE is a gene that encodes PPT, as detailed above. PPT functions in the PUFA PKS system include catalyzing the attachment of phosphopantethein cofactors to the ACP domain. It is known that phosphopantethein cofactor adheres to apo-ACP to become holo-ACP and is activated (Lambalot RH et al., Chemistry and Biology, 3, 923 (1996)).

In order to amplify each gene described in Table 4, PCR was performed using the genomic DNA of the microorganism described in “Source of gene” in Table 4 as a template and “Gene amplification primer” as a primer.
Each plasmid described in Table 4 was prepared by the following method.
For pUC19-sfp, an overlap extension was obtained by (a) DNA containing the sfp gene amplified above and (b) a lac promoter amplified by PCR using the primers represented by the nucleotide sequences of SEQ ID NOs: 72 and 73. PUC19-sfp was obtained by ligation by PCR, treating the ligated DNA fragment with restriction enzymes SpeI and KpnI, and ligating to pUC19 treated with XbaI and KpnI.

  For pUC19-SO1604, (a) a DNA containing the SO1604 gene amplified above and (b) a lac promoter amplified by PCR using the primers represented by the nucleotide sequences of SEQ ID NOs: 72 and 74 were overlap extension. PUC19-SO1604 was obtained by ligation by PCR, treating the ligated DNA fragment with restriction enzymes SpeI and KpnI, and ligating to pUC19 treated with XbaI and KpnI.

  For pUC19-entD, (a) a DNA containing the entD gene amplified above and (b) a lac promoter amplified by PCR using the primers represented by the nucleotide sequences of SEQ ID NOs: 72 and 75 were overlap extension. PUC19-entD was obtained by ligation by PCR, treating the ligated DNA fragment with restriction enzymes SpeI and KpnI, and ligating to pUC19 treated with XbaI and KpnI.

  For pUC19-cgl1980, (a) a DNA containing the cgl1980 gene amplified above and (b) a lac promoter amplified by PCR using the primers represented by the nucleotide sequences of SEQ ID NOs: 72 and 76 were overlap extension. PUC19-cgl1980 was obtained by ligating with PCR and treating the ligated DNA fragment with restriction enzymes SpeI and KpnI and ligating to pUC19 treated with XbaI and KpnI.

The pUC19-sfp, pUC19-SO1604, pUC19-entD, or pUC19-cgl1980 obtained as described above, and a strain obtained by removing only pUC19-OrfE from ΔfadE-EABDC (BW25113ΔfadE :: FRT / pSTV29-OrfA-OrfB pMW219-OrfD-OrfC strain).
The obtained strain was cultured and analyzed in the same manner as in Example 4 to examine whether ARA was produced. The results are shown in Table 5.

  As shown in the above results, it was revealed that various phosphopantetheinyltransferases can activate the ARA-PKS system.

  Utilization of other types of enoyl reductase Following OrfE, we conducted experiments to introduce homologous genes for OrfD. OrfD is a gene encoding a protein having an ER domain. An example of a gene having homology to OrfD is the SO1597 gene derived from Shiwanella oneidensis MR-1. This SO1597 gene and OrfD of arachidonic acid PKS of the present invention were exchanged.

  First, PCR was performed using the synthetic DNA represented by the nucleotide sequences of SEQ ID NOs: 77 and 78 as a primer using the genomic DNA of the Shiwanella oneidensis MR-1 strain as a template to prepare a DNA fragment containing the SO1597 gene. Simultaneously, PCR was carried out using E. coli genomic DNA as a template and the synthetic DNAs represented by SEQ ID NOs: 72 and 79 as primers to prepare DNA fragments containing the lac promoter. By carrying out overlap extension PCR by mixing these two fragments of DNA, a DNA fragment in which the SO1597 gene derived from the Shiwanella Oneidensis MR-1 strain was linked to the lac promoter was prepared. This DNA fragment was treated with restriction enzymes SpeI and KpnI and ligated to pUC19-SO1604 treated with XbaI and KpnI to obtain pUC19-SO1604-SO1597.

Next, in order to remove only OrfD from the ARA-PKS system, DNA ligated with the OrfC and lac promoter constructed in Example 2 was treated with restriction enzymes with BamHI and Sse8387I, and pMW219 treated with BamHI and Sse8387I was Takara Bio. Ligation was performed using a ligation kit LONG manufactured by KK to obtain an expression vector pMW219-OrfC containing only OrfC.
A strain obtained by removing pUC19-OrfE and pMW-OrfD-OrfC from ΔfadE-EABDC (BW25113ΔfadE :: FRT / pSTV29-OrfA-OrfB strain) was simultaneously transformed with the above-described two types of expression vectors.
The obtained strains BW25113ΔfadE :: FRT / pUC19-SO1604-SO1597, pSTV29-OrfA-OrfB, and pMW219-OrfC were cultured in the same manner as in Example 4, and fatty acids were analyzed.

As a result, the production of ARA was confirmed. Therefore, it was shown that OrfD can be replaced with SO1597 of the Shiwanella Oneidensis MR-1 strain.
An example of the experiment for replacing each Orf and domain has been shown, but by using the same method, innumerable combinations of Orf and domain that ARA can produce can be assumed, and the ARA-PKS according to the present invention provides for their use. I can do it.

  According to the present invention, ARA-PKS, DNA encoding the ARA-PKS constituent protein, organisms in which the activity of ARA-PKS is enhanced from the host organism, and EPA using the organism or other PUFAs including EPA It is possible to provide a method for efficiently producing an ARA-containing composition containing no ARA.

SEQ ID NO: 47—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 48—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 49—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 50—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 51—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 52—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 53—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 54—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 55—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 56—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 57—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 58—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 59—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 60—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 61—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 62—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 63—Description of artificial sequence: synthetic DNA
SEQ ID NO: 64—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 65—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 66—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 67—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 68—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 69—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 70—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 71—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 72—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 73—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 74—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 75—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 76—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 77—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 78—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 79—Description of artificial sequence: synthetic DNA

Claims (9)

a protein having a β-ketoacyl-acyl carrier protein synthase (hereinafter referred to as KS) domain, malonyl-CoA: acyl carrier protein acyltransferase (hereinafter referred to as MAT) domain, and acyl carrier protein (hereinafter referred to as ACP) domain; A protein having a ketoreductase (hereinafter referred to as KR) domain, a KS domain, a chain elongation factor (hereinafter referred to as CLF) domain, an acyltransferase (hereinafter referred to as AT) domain, and a FabA-like β-hydroxyacyl-ACP dehydratase ( DH), a protein having an enoyl ACP-reductase (hereinafter referred to as ER) domain, and a protein having a phosphopantethein transferase (hereinafter referred to as PPT) domain, and acetyl CoA As the starting substrate Arachidonic acid A complex of a protein having an activity of producing (hereinafter, referred to. ARA),
[1] A protein having a KS domain, a MAT domain, and an ACP domain, a protein having the amino acid sequence represented by SEQ ID NO: 32, or an amino acid having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 32 A homologous protein consisting of a sequence and having an activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having an amino acid sequence represented by each of SEQ ID NOs: 34, 36, 38 and 22;
[2] The protein having the KR domain consists of a protein having the amino acid sequence represented by SEQ ID NO: 34, or an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 34, and A homologous protein having an activity of producing ARA using acetyl CoA as a starting substrate in cooperation with a protein having an amino acid sequence represented by each of SEQ ID NOs: 32, 36, 38 and 22,
[3] A protein having a KS domain, a CLF domain, an AT domain, and a DH domain has at least 95% identity with a protein having the amino acid sequence represented by SEQ ID NO: 36 or the amino acid sequence represented by SEQ ID NO: 36 And a homologous protein having an activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having the amino acid sequence represented by each of SEQ ID NOs: 32, 34, 38 and 22. Yes,
[4] The protein having the ER domain is a protein having the amino acid sequence represented by SEQ ID NO: 38 or 40, or an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 38 or 40 And a homologous protein having an activity of producing ARA using acetyl CoA as a starting substrate in cooperation with a protein having the amino acid sequence represented by each of SEQ ID NOs: 32, 34, 36 and 22, and [5 ] A protein having a PPT domain is a protein having an amino acid sequence represented by SEQ ID NO: 22, 24, 26, 28 or 30, or any amino acid sequence represented by SEQ ID NO: 22, 24, 26, 28 or 30 And an amino acid sequence having at least 95% identity and each of SEQ ID NOs: 32, 34, 36 and 38 In a homologous protein having an activity to produce ARA acetyl CoA as the starting substrate in cooperation with the protein having the amino acid sequence represented by,
Protein complex. A DNA encoding a protein constituting the protein complex according to claim 1. KS domain, DNA encoding a protein having a MAT domain and ACP domains, DNA having the nucleotide sequence shown in SEQ ID NO: 31, or a base sequence represented by SEQ ID NO: 31 and at least 95% identity to And a homologous protein having an activity of producing ARA using acetyl CoA as a starting substrate in cooperation with a protein having an amino acid sequence represented by SEQ ID NOs: 34, 36, 38 and 22 The DNA according to claim 2, which is DNA to be treated. DNA encoding a protein having a KR domain consists of DNA having the base sequence represented by SEQ ID NO: 33, or a base sequence having at least 95% identity with the base sequence represented by SEQ ID NO: 33, and A DNA encoding a homologous protein having an activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having an amino acid sequence represented by each of SEQ ID NOs: 32, 36, 38 and 22. 2. DNA according to 2. DNA encoding a protein having a KS domain, a CLF domain, an AT domain, and a DH domain is at least 95% or more identical to DNA having the base sequence represented by SEQ ID NO: 35 or the base sequence represented by SEQ ID NO: 35 Homologous protein comprising a nucleotide sequence having a property and having an activity of producing ARA using acetyl CoA as a starting substrate in cooperation with a protein having an amino acid sequence represented by each of SEQ ID NOs: 32, 34, 38 and 22 The DNA according to claim 2, which is a DNA encoding the DNA. A DNA encoding a protein having an ER domain is a DNA having a base sequence represented by SEQ ID NO: 37 or 39, or a base sequence having at least 95% identity with the base sequence represented by SEQ ID NO: 37 or 39 And a DNA encoding a homologous protein having an activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having an amino acid sequence represented by each of SEQ ID NOs: 32, 34, 36 and 22. The DNA according to claim 2, wherein A DNA encoding a protein having a PPT domain is a DNA having a base sequence represented by SEQ ID NO: 21, 23, 25, 27 or 29, or any one represented by SEQ ID NO: 21, 23, 25, 27 or 29 A starting substrate for acetyl-CoA in cooperation with a protein comprising an amino acid sequence represented by each of SEQ ID NOs: 32, 34, 36 and 38, having a base sequence having at least 95% identity with the base sequence of The DNA according to claim 2, which is a DNA encoding a homologous protein having an activity to produce ARA as DNA encoding protein having KS domain, MAT domain and ACP domain, DNA encoding protein having KR domain, DNA encoding protein having KS domain, CLF domain, AT domain and DH domain, protein having ER domain A microorganism capable of producing ARA using acetyl-CoA in a cell as a starting substrate, obtained by transforming a host microorganism with DNA encoding a protein having a PPT domain, and
[1] A protein having a KS domain, a MAT domain, and an ACP domain, a protein having the amino acid sequence represented by SEQ ID NO: 32, or an amino acid having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 32 A homologous protein consisting of a sequence and having the activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having the amino acid sequence represented by each of SEQ ID NOs: 34, 36, 38 and 22;
[2] The protein having the KR domain consists of a protein having the amino acid sequence represented by SEQ ID NO: 34, or an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 34, and A homologous protein having an activity of producing ARA using acetyl CoA as a starting substrate in cooperation with a protein having an amino acid sequence represented by each of SEQ ID NOs: 32, 36, 38 and 22,
[3] A protein having a KS domain, a CLF domain, an AT domain, and a DH domain has at least 95% identity with a protein having the amino acid sequence represented by SEQ ID NO: 36 or the amino acid sequence represented by SEQ ID NO: 36 A homologous protein having an activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having the amino acid sequence represented by SEQ ID NOs: 32, 34, 38 and 22 Yes,
[4] The protein having the ER domain is a protein having the amino acid sequence represented by SEQ ID NO: 38 or 40, or an amino acid sequence having at least 95% identity with the amino acid sequence represented by SEQ ID NO: 38 or 40 And a homologous protein having an activity of producing ARA using acetyl-CoA as a starting substrate in cooperation with a protein having the amino acid sequence represented by each of SEQ ID NOs: 32, 34, 36 and 22, and
[5] A protein having a PPT domain is a protein having an amino acid sequence represented by SEQ ID NO: 22, 24, 26, 28 or 30, or any one represented by SEQ ID NO: 22, 24, 26, 28 or 30 Acetyl CoA as a starting substrate in cooperation with a protein comprising an amino acid sequence having at least 95% identity with the amino acid sequence and having the amino acid sequence represented by each of SEQ ID NOs: 32, 34, 36 and 38 A homologous protein having an activity to produce ARA,
A microorganism in which the activity of the protein complex according to claim 1 is enhanced from a host organism. An ARA- or ARA-containing composition comprising culturing the microorganism according to claim 8 in a medium, producing and accumulating an ARA-containing composition in the culture, and collecting the ARA or ARA-containing composition from the culture. A method for producing the composition.