JP5881003B2 - Method for culturing ricinoleic acid-producing yeast - Google Patents
Method for culturing ricinoleic acid-producing yeast Download PDFInfo
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- JP5881003B2 JP5881003B2 JP2011253226A JP2011253226A JP5881003B2 JP 5881003 B2 JP5881003 B2 JP 5881003B2 JP 2011253226 A JP2011253226 A JP 2011253226A JP 2011253226 A JP2011253226 A JP 2011253226A JP 5881003 B2 JP5881003 B2 JP 5881003B2
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Description
本発明は、酵母によりリシノール酸を産生させる方法に関する。より詳細には、ＦＡＨ１２遺伝子が導入された酵母の形質転換体を培養することにより、該形質転換体にリシノール酸を産生させる方法に関する。 The present invention relates to a method for producing ricinoleic acid by yeast. More specifically, the present invention relates to a method for producing ricinoleic acid by culturing a yeast transformant into which the FAH12 gene has been introduced.
現在、ウレタン製品等の化成品の原料は、専ら石油資源に頼っている。地球温暖化対策により、石油資源に代わる代替材料が模索されており、リシノール酸が注目されている。リシノール酸は、トウゴマ（Ricinus communis）を絞ったひまし油の８０〜９０％を占めており、ひまし油から精製したものが使用されている。しかし、トウゴマにはリシンという猛毒の蛋白質が含まれており、取り扱いに注意を要する上、植物資源であるため、安定供給には不安がある。そこで、ひまし油以外からリシノール酸を安全かつ安定して製造する方法の開発が望まれている。 Currently, raw materials for chemical products such as urethane products rely exclusively on petroleum resources. As a countermeasure against global warming, an alternative material to replace petroleum resources is being sought, and ricinoleic acid has attracted attention. Ricinoleic acid accounts for 80-90% of castor oil squeezed castor bean (Ricinus communis), and refined from castor oil is used. However, castor bean contains a highly toxic protein called ricin, which requires careful handling and is a plant resource. Therefore, development of a method for producing ricinoleic acid safely and stably from other than castor oil is desired.
リシノール酸は、鎖長が炭素数１８であり、不飽和結合が１つと１２位に水酸基を持つ脂肪酸であり、Δ１２−ヒドロキシラーゼにより、オレイン酸の１２位に水酸基を導入することによって生産できる。野生型の酵母はΔ１２−ヒドロキシラーゼを有していないため、リシノール酸を産生することはできない。そこで、本発明者らは、分裂酵母シゾサッカロミセス・ポンベ（Schizosaccharomyces pombe、以下、Ｓ．ポンベ）が、脂肪酸組成としてオレイン酸の含有量が８割程度と高いことに着目して、Ｓ．ポンベに異種生物由来のΔ１２−ヒドロキシラーゼ遺伝子を導入し、リシノール酸の生産を試みた。この結果、Ｓ．ポンベの形質転換体においてリシノール酸が生産されることが確認されたが、リシノール酸の生産量が多くなるほど宿主の増殖が遅くなることも分かった（非特許文献１参照。）。 Ricinoleic acid is a fatty acid having a chain length of 18 carbon atoms, one unsaturated bond and a hydroxyl group at the 12-position, and can be produced by introducing a hydroxyl group at the 12-position of oleic acid with Δ12-hydroxylase. Since wild-type yeast does not have Δ12-hydroxylase, it cannot produce ricinoleic acid. Therefore, the present inventors have focused on the fact that the fission yeast Schizosaccharomyces pombe (hereinafter referred to as S. pombe) has a high fatty acid composition with an oleic acid content of about 80%. A Δ12-hydroxylase gene derived from a different organism was introduced into Pombe to try to produce ricinoleic acid. As a result, S.E. Although it was confirmed that ricinoleic acid was produced in the transformant of pombe, it was also found that as the amount of ricinoleic acid produced increased, the growth of the host slowed (see Non-Patent Document 1).
そこで本発明の目的は、リシノール酸産生酵母を、増殖性とリシノール産生性のバランスをとりながら効率よく培養する方法、およびリシノール酸の製造方法を提供することにある。 Accordingly, an object of the present invention is to provide a method for efficiently cultivating ricinoleic acid-producing yeast while maintaining a balance between growth and ricinol productivity, and a method for producing ricinoleic acid.
本発明者等は、前記課題を解決すべく鋭意検討した結果、ＦＡＨ１２(Fatty acid hydroxylase）遺伝子が導入された酵母の形質転換体を、比較的高温で培養することにより、増殖阻害を抑制し得ること、また、高温培養により充分に培養させた後、温度シフトし、より低温で培養することによって、該形質転換体内のリシノール酸含有量を高められることを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors can suppress growth inhibition by culturing yeast transformants into which a FAH12 (Fatty acid hydroxylase) gene has been introduced at a relatively high temperature. In addition, it was found that the content of ricinoleic acid in the transformant can be increased by shifting the temperature after culturing sufficiently by high-temperature culture and then culturing at a lower temperature, thereby completing the present invention. .
本発明のリシノール酸産生酵母の培養方法、およびリシノール酸の製造方法は下記［１］〜［５］である。
［１］ＦＡＨ１２遺伝子が導入された酵母の形質転換体を、３５℃以上４０℃未満で、培養液のＯＤ６００が０．５〜５．５になるまで培養する高温培養工程と、
前記高温培養工程後、前記形質転換体を３５℃未満で培養する低温培養工程と、
を有することを特徴とするリシノール酸産生酵母の培養方法。
［２］前記低温培養工程における前記形質転換体の培養温度が、１０〜３３℃である前記［１］のリシノール酸産生酵母の培養方法。
［３］前記酵母がシゾサッカロミセス（Schizosaccharomyces）属酵母である前記［１］または［２］のリシノール酸産生酵母の培養方法。
［４］前記ＦＡＨ１２遺伝子が、トウゴマ（Ricinus communis）、レスクェレラ・フェンドレリ（Lesquerella fendleri）または麦角菌（Claviceps purpurea）由来である前記［１］〜［３］のいずれかのリシノール酸産生酵母の培養方法。
［５］ＦＡＨ１２遺伝子が導入された酵母の形質転換体を前記［１］〜［４］のいずれかのリシノール酸産生酵母の培養方法により培養した後、得られた形質転換体から、リシノール酸を取得することを特徴とするリシノール酸の製造方法。
The method for culturing ricinoleic acid-producing yeast and the method for producing ricinoleic acid of the present invention are the following [1] to [5].
[1] A high-temperature culture step of culturing a yeast transformant into which the FAH12 gene has been introduced at 35 ° C. or higher and lower than 40 ° C. until the OD 600 of the culture solution is 0.5 to 5.5,
A low temperature culture step of culturing the transformant at less than 35 ° C. after the high temperature culture step;
A method for culturing ricinoleic acid-producing yeast, comprising:
[2] The method for culturing ricinoleic acid-producing yeast according to [1], wherein the transformant is cultured at a temperature of 10 to 33 ° C. in the low-temperature culturing step.
[3] The method for cultivating a ricinoleic acid-producing yeast according to [1] or [2], wherein the yeast is a genus Schizosaccharomyces.
[4] The method of cultivating a ricinoleic acid-producing yeast according to any one of [1] to [3], wherein the FAH12 gene is derived from Ricinus communis, Lesquerella fendleri, or Claviceps purpurea. .
[5] After culturing by the method of culturing any of ricinoleic acid producing yeast of the transformants of yeast F AH12 gene is introduced [1] to [4], from the transformant obtained, ricinoleic acid To obtain ricinoleic acid.
本発明のリシノール酸産生酵母の培養方法により、リシノール酸産生酵母を、増殖性とリシノール酸生産性のバランスをとりながら効率よく培養できる。
また、該形質転換体を用いた本発明のリシノール酸の製造方法により、産生されたリシノール酸を簡便に取得できる。
By the method for culturing ricinoleic acid-producing yeast of the present invention, ricinoleic acid-producing yeast can be cultured efficiently while maintaining a balance between growth and ricinoleic acid productivity.
In addition, the produced ricinoleic acid can be easily obtained by the method for producing ricinoleic acid of the present invention using the transformant.
［ＦＡＨ１２遺伝子が導入された酵母の形質転換体（リシノール酸産生酵母）］
本発明におけるリシノール酸産生酵母は、ＦＡＨ１２遺伝子が導入された酵母の形質転換体である。酵母に導入するＦＡＨ１２遺伝子は、オレイン酸の１２位の炭素原子に水酸基を導入し得るΔ１２−ヒドロキシラーゼ活性を有する酵素をコードする遺伝子であれば、特に限定されず、微生物由来であってもよく、植物由来であってもよい。具体的には、例えば、トウゴマのＦＡＨ１２（ＲｃＦＡＨ１２）（GenBank ID.No.： AAC49010.1）をコードする遺伝子（ＲｃＦＡＨ１２遺伝子）、レスクェレラ・フェンドレリのＦＡＨ１２（ＬｆＦＡＨ１２）（GenBank ID.No.： AAC32755.1）をコードする遺伝子（ＬｆＦＡＨ１２遺伝子）、麦角菌のＦＡＨ１２（ＣｐＦＡＨ１２）（GenBank ID.No.： ACF37070.1）をコードする遺伝子（ＣｐＦＡＨ１２遺伝子）、フィサリア・リンドヘイメリ（Physaria lindheimeri）のｏｌｅａｔｅ １２−ｈｙｄｒｏｘｙｌａｓｅ（GenBank ID.No.：ABQ01458.1）をコードする遺伝子、アスペルギルス・ニガー（Aspergillus niger）のｕｎｎａｍｅｄ ｐｒｏｔｅｉｎ ｐｒｏｄｕｃｔ（GenBank ID.No.：CAK37451.1）をコードする遺伝子、が挙げられる。なお、GenBankはＮＣＢＩ（National Center for Biotechnology Information）のデータベースである。本発明では、ＲｃＦＡＨ１２遺伝子、ＬｆＦＡＨ１２遺伝子またはＣｐＦＡＨ１２遺伝子が好ましく、Δ１２−ヒドロキシラーゼ活性がより高いため、ＣｐＦＡＨ１２遺伝子がより好ましい。
[Transformant of yeast introduced with FAH12 gene (ricinoleic acid-producing yeast)]
The ricinoleic acid-producing yeast in the present invention is a yeast transformant into which the FAH12 gene has been introduced. The FAH12 gene to be introduced into yeast is not particularly limited as long as it is a gene encoding an enzyme having Δ12-hydroxylase activity capable of introducing a hydroxyl group into the 12th carbon atom of oleic acid, and may be derived from a microorganism. It may be derived from a plant. Specifically, for example, a gene (RcFAH12 gene) encoding FAH12 (RcFAH12) of castor bean (GenBank ID. No .: AAC49010.1), FAH12 (LfFAH12) of Resquerella Fendrel (GenBank ID. No .: AAC32755. 1) gene encoding (LfFAH12 gene), ergot FAH12 (CpFAH12) (GenBank ID. No .: ACF37070.1) encoding gene (CpFAH12 gene), Physaria lindheimeri oleate 12-hydroxylase A gene encoding (GenBank ID. No .: ABQ01458.1) and a gene encoding an unnamed protein product (GenBank ID. No .: CAK37451.1) of Aspergillus niger. GenBank is a database of NCBI (National Center for Biotechnology Information). In the present invention, the RcFAH12 gene, the LfFAH12 gene or the CpFAH12 gene is preferable, and since the Δ12-hydroxylase activity is higher, the CpFAH12 gene is more preferable.
（酵母）
ＦＡＨ１２遺伝子が導入される宿主は、酵母であれば特に限定されず、出芽酵母(Saccharomyces cerevisiae、以下、Ｓ．セレビシエ)をはじめとするサッカロミセス（Saccharomyces）属酵母、シゾサッカロミセス（Schizosaccharomyces）属酵母、クリュイベロミセス（Kluyveromyces）属酵母、ピキア（Pichia）属酵母、カンジダ（Candida）属酵母が挙げられる。中でも、オレイン酸含有量が高いことから、シゾサッカロミセス属酵母であることが好ましい。シゾサッカロミセス属酵母としては、たとえば、Ｓ．ポンベ、シゾサッカロミセス・ジャポニカス（Schizosaccharomyces japonicus）、シゾサッカロミセス・オクトスポラス（Schizosaccharomyces octosporus）が挙げられ、Ｓ．ポンベが好ましい。
(yeast)
The host into which the FAH12 gene is introduced is not particularly limited as long as it is a yeast. Saccharomyces yeasts including Saccharomyces cerevisiae (hereinafter, S. cerevisiae), yeasts belonging to the genus Schizosaccharomyces, Examples include yeasts belonging to the genus Kluyveromyces, yeasts belonging to the genus Pichia, and yeasts belonging to the genus Candida. Of these, yeasts belonging to the genus Schizosaccharomyces are preferred because of their high oleic acid content. Examples of Schizosaccharomyces yeasts include S. cerevisiae. Pombe, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus, and S. cerevisiae. Pombe is preferred.
また、宿主とする酵母は、野生型であってもよく、用途に応じて特定の遺伝子を欠失または失活させた変異型であってもよい。特定の遺伝子を欠失または失活させる方法としては、公知の方法を用いられる。具体的には、Latour法（Nucleic Acids Research誌、第34巻、e11ページ、2006年；国際公開第２００７／０６３９１９号パンフレット等に記載）を用いることにより遺伝子を欠失させられる。また、変異剤を用いた突然変異分離法（酵母分子遺伝学実験法、１９９６年、学会出版センター）や、ＰＣＲ（ポリメラーゼ連鎖反応）を利用したランダム変異法（PCR Methods Application誌、第2巻、28-33ページ、1992年）等により遺伝子の一部に変異を導入することにより該遺伝子を失活させられる。特定遺伝子を欠失または失活させたシゾサッカロミセス属酵母宿主としては、たとえば、国際公開第２００２／１０１０３８号、国際公開第２００７／０１５４７０号等に記載されている。
また、特定の遺伝子の削除または不活性化を行う部分はＯＲＦ（オープンリーディングフレーム）部分であってもよく、発現調節配列部分であってもよい。特に好ましい方法は、構造遺伝子のＯＲＦ部分をマーカー遺伝子に置換するＰＣＲ媒介相同組換え法（Yeast誌、第14巻、943-951ページ、1998年）による削除または不活性化の方法である。
Moreover, the yeast used as a host may be a wild type or a mutant type in which a specific gene is deleted or inactivated depending on the application. As a method for deleting or inactivating a specific gene, a known method can be used. Specifically, the gene can be deleted by using the Latour method (Nucleic Acids Research, Vol. 34, e11 page, 2006; described in International Publication No. 2007/063919 pamphlet, etc.). In addition, mutation isolation method using mutation agent (Yeast Molecular Genetics Experimental Method, 1996, Society Press Center) and random mutation method using PCR (Polymerase Chain Reaction) (PCR Methods Application, Volume 2, 28-33, 1992), etc., the gene can be inactivated by introducing a mutation into a part of the gene. Examples of yeasts belonging to the genus Schizosaccharomyces from which a specific gene has been deleted or inactivated are described in, for example, WO 2002/101038 and WO 2007/015470.
In addition, the part where a specific gene is deleted or inactivated may be an ORF (open reading frame) part or an expression regulatory sequence part. A particularly preferred method is a method of deletion or inactivation by PCR-mediated homologous recombination method (Yeast, Vol. 14, pages 943-951, 1998) in which the ORF portion of the structural gene is replaced with a marker gene.
さらに宿主となる酵母には、形質転換体を選択するためのマーカーを有するものを用いることが好ましい。たとえば、ある遺伝子が欠落していることにより特定の栄養成分が生育に必須である宿主を使用することが好ましい。目的遺伝子配列を含むベクターにより形質転換をして形質転換体を作製する場合、ベクターにこの欠落している遺伝子（栄養要求性相補マーカー）を組み込んでおくことにより、形質転換体は宿主の栄養要求性が消失する。この宿主と形質転換体の栄養要求性の相違により、両者を区別して形質転換体を得られる。
たとえば、オロチジン５’−リン酸デカルボキシラーゼ遺伝子（ｕｒａ４遺伝子）が欠失または失活してウラシル要求性となっている酵母を宿主とし、ｕｒａ４遺伝子（栄養要求性相補マーカー）を有するベクターにより形質転換した後、ウラシル要求性が消失したものを選択することにより、ベクターが組み込まれた形質転換体を得られる。宿主において欠落により栄養要求性となる遺伝子は、形質転換体の選択に用いられるものであればｕｒａ４遺伝子には限定されず、イソプロピルリンゴ酸デヒドロゲナーゼ遺伝子（ｌｅｕ１遺伝子）等であってもよい。
Furthermore, it is preferable to use yeast having a marker for selecting a transformant as a host yeast. For example, it is preferable to use a host in which a specific nutritional component is essential for growth because a certain gene is missing. When a transformant is produced by transforming with a vector containing the target gene sequence, the transformant can be auxotrophic of the host by incorporating this missing gene (auxotrophic complementary marker) into the vector. Sex disappears. Due to the difference in auxotrophy between the host and the transformant, a transformant can be obtained by distinguishing both.
For example, transformation is performed with a vector having a ura4 gene (an auxotrophic complementary marker) using a yeast in which orotidine 5′-phosphate decarboxylase gene (ura4 gene) has been deleted or inactivated to become uracil-requiring as a host. Thereafter, a transformant in which the vector is incorporated can be obtained by selecting those that have lost the requirement for uracil. The gene that becomes auxotrophic due to deletion in the host is not limited to the ura4 gene as long as it is used for selection of transformants, and may be an isopropylmalate dehydrogenase gene (leu1 gene) or the like.
（発現カセット）
本発明におけるリシノール酸産生酵母は、ＦＡＨ１２遺伝子をコードする領域を含む構造遺伝子配列（ＦＡＨ１２遺伝子配列）、並びに該遺伝子を発現させるためのプロモーター配列およびターミネーター配列を含む発現カセットを、酵母に導入することにより作製できる。ＦＡＨ１２遺伝子配列としては、麦角菌等由来のＦＡＨ１２をコードする遺伝子をそのまま用いてもよいが、宿主として用いる酵母内での発現量を増大させるために、前記遺伝子配列を、宿主での高発現遺伝子において使用頻度の高いコドンに改変することが好ましい。
(Expression cassette)
In the ricinoleic acid-producing yeast of the present invention, a structural gene sequence (FAH12 gene sequence) containing a region encoding the FAH12 gene and an expression cassette containing a promoter sequence and a terminator sequence for expressing the gene are introduced into the yeast. Can be produced. As the FAH12 gene sequence, a gene encoding FAH12 derived from ergot bacteria or the like may be used as it is, but in order to increase the expression level in yeast used as a host, the gene sequence is expressed as a highly expressed gene in the host. It is preferable to modify the codon to be frequently used.
発現カセットとは、蛋白質を発現するために必要なＤＮＡの組み合わせであり、該蛋白質をコードする構造遺伝子と宿主である酵母内で機能するプロモーターとターミネーターとを含む。
前記発現カセットには、さらに、５’−非翻訳領域、３’−非翻訳領域のいずれか１つ以上が含まれていてもよい。好ましい発現カセットは、ＦＡＨ１２遺伝子配列、プロモーター、ターミネーター、５’−非翻訳領域、３’−非翻訳領域を全て含む発現カセットである。さらに、栄養要求性相補マーカー等の遺伝子を有していてもよい。
An expression cassette is a combination of DNAs necessary for expressing a protein, and includes a structural gene encoding the protein, a promoter functioning in yeast as a host, and a terminator.
The expression cassette may further include any one or more of 5′-untranslated region and 3′-untranslated region. A preferred expression cassette is an expression cassette containing all of the FAH12 gene sequence, promoter, terminator, 5′-untranslated region, and 3′-untranslated region. Furthermore, you may have genes, such as an auxotrophic complementary marker.
プロモーターとターミネーターは、宿主である酵母内で機能してＦＡＨ１２を発現できるものであればよい。酵母内で機能するプロモーターとしては、酵母が本来有するプロモーター（転写開始活性が高いものが好ましい）や酵母が本来有しないプロモーター（ウイルス由来のプロモーターなど）を使用できる。プロモーターはベクター内に２種以上存在していてもよい。
シゾサッカロミセス属酵母が本来有するプロモーターとしては、たとえば、アルコールデヒドロゲナーゼ遺伝子プロモーター、チアミンの代謝に関与するｎｍｔ１遺伝子プロモーター、グルコースの代謝に関与するフルクトース−１、６−ビスホスファターゼ遺伝子プロモーター、カタボライト抑制に関与するインベルターゼ遺伝子のプロモーター（国際公開第９９／２３２２３号参照）、熱ショック蛋白質遺伝子プロモーター（国際公開第２００７／２６６１７号参照）などが挙げられる。
シゾサッカロミセス属酵母が本来有しないプロモーターとしては、たとえば、特開平５−１５３８０号公報、特開平７−１６３３７３号公報、特開平１０−２３４３７５号公報に記載されている動物細胞ウイルス由来のプロモーターが挙げられ、ｈＣＭＶプロモーター、ＳＶ４０プロモーターが好ましい。
シゾサッカロミセス属酵母内で機能するターミネーターとしては、シゾサッカロミセス属酵母が本来有するターミネーターやシゾサッカロミセス属酵母が本来有しないターミネーターを使用できる。ターミネーターはベクター内に２種以上存在していてもよい。
ターミネーターとしては、たとえば、特開平５−１５３８０号公報、特開平７−１６３３７３号公報、特開平１０−２３４３７５号公報に記載されているヒト由来のターミネーターが挙げられ、ヒトリポコルチンＩのターミネーターが好ましい。
The promoter and terminator are not particularly limited as long as they function in yeast as a host and can express FAH12. As a promoter that functions in yeast, a promoter inherent in yeast (preferably having a high transcription initiation activity) or a promoter inherent in yeast (such as a virus-derived promoter) can be used. Two or more promoters may be present in the vector.
Examples of promoters inherent to Schizosaccharomyces yeasts include alcohol dehydrogenase gene promoter, nmt1 gene promoter involved in thiamine metabolism, fructose-1, 6-bisphosphatase gene promoter involved in glucose metabolism, and catabolite repression. Invertase gene promoters (see International Publication No. 99/23223), heat shock protein gene promoters (see International Publication No. 2007/26617), and the like.
Examples of promoters that Schizosaccharomyces yeast originally does not include promoters derived from animal cell viruses described in JP-A-5-15380, JP-A-7-163373, and JP-A-10-234375. HCMV promoter and SV40 promoter are preferable.
As the terminator that functions in the yeast belonging to the genus Schizosaccharomyces, the terminator inherent in the yeast belonging to the genus Schizosaccharomyces or the terminator not inherent in the yeast belonging to the genus Schizosaccharomyces can be used. Two or more terminators may be present in the vector.
Examples of the terminator include human-derived terminators described in JP-A-5-15380, JP-A-7-163373, and JP-A-10-234375, and human lipocortin I terminator is preferable. .
（ベクター）
本発明におけるリシノール酸産生酵母は、具体的には、前記発現カセットを含むベクターにより宿主である酵母を形質転換することにより得られる。形質転換に用いるベクターは、環状ＤＮＡ構造または線状ＤＮＡ構造を有するベクターに、該発現カセットを組み込むことにより製造できる。該発現カセットが、宿主の細胞内で染色体外遺伝子として保持される形質転換体を作製する場合には、該ベクターは、宿主細胞内で複製されるための配列、即ち、自律複製配列（Autonomously Replicating Sequence: ARS）を含むプラスミドであることが好ましい。一方で、該発現カセットが、宿主細胞の染色体中に組み込まれた形質転換体を作製する場合には、該ベクターは、線状ＤＮＡ構造であり、かつＡＲＳを有していないものとして、宿主細胞へ導入されることが好ましい。たとえば、該ベクターは、線状ＤＮＡからなるベクターであってもよく、宿主への導入時に、線状ＤＮＡに切り開くための制限酵素部位を備える環状ＤＮＡ構造のベクターであってもよい。該ベクターがＡＲＳを有するプラスミドの場合、ＡＲＳ部分を削除して線状ＤＮＡ構造、またはＡＲＳ部分を開裂させることによりＡＲＳの機能を失活させた線状ＤＮＡ構造とした後、宿主へ導入できる。
(vector)
Specifically, the ricinoleic acid-producing yeast in the present invention can be obtained by transforming a host yeast with a vector containing the expression cassette. A vector used for transformation can be produced by incorporating the expression cassette into a vector having a circular DNA structure or a linear DNA structure. When the expression cassette produces a transformant that is maintained as an extrachromosomal gene in the host cell, the vector may contain a sequence for replication in the host cell, that is, an autonomously replicating sequence. Sequence: ARS) is preferred. On the other hand, when producing a transformant in which the expression cassette is integrated into the host cell chromosome, the vector is assumed to have a linear DNA structure and no ARS. It is preferable to be introduced into. For example, the vector may be a vector composed of linear DNA, or a vector having a circular DNA structure having a restriction enzyme site for cleavage into linear DNA upon introduction into a host. When the vector is a plasmid having ARS, it can be introduced into the host after forming a linear DNA structure by deleting the ARS part or a linear DNA structure in which the function of ARS is inactivated by cleaving the ARS part.
該ベクターは、形質転換体を選択するためのマーカーを有することが好ましい。該マーカーとしては、たとえば、ｕｒａ４遺伝子（栄養要求性相補マーカー）、イソプロピルリンゴ酸デヒドロゲナーゼ遺伝子（ｌｅｕ１遺伝子）が挙げられる。 The vector preferably has a marker for selecting a transformant. Examples of the marker include ura4 gene (auxotrophic complementary marker) and isopropylmalate dehydrogenase gene (leu1 gene).
リシノール酸産生酵母の製造に用いられる前記発現カセットを含むベクターは、たとえば、外来構造遺伝子を導入するためのクローニングサイトを備える公知の発現ベクター中の該クローニングサイトに、ＦＡＨ１２遺伝子配列を挿入することによって製造できる。また、公知の発現ベクターに、ＦＡＨ１２遺伝子配列を含む発現カセットを組み込むことによっても製造できる。 The vector containing the expression cassette used for the production of ricinoleic acid-producing yeast is obtained by, for example, inserting the FAH12 gene sequence into the cloning site in a known expression vector having a cloning site for introducing a foreign structural gene. Can be manufactured. It can also be produced by incorporating an expression cassette containing the FAH12 gene sequence into a known expression vector.
（形質転換体の製造方法）
本発明におけるリシノール酸産生酵母は、前記発現カセットを、染色体中に有するか、または、染色体外遺伝子として有する. 発現カセットを染色体中に有するとは、宿主細胞の染色体中の１カ所以上に発現カセットが組み込まれていることであり、染色体外遺伝子として有するとは、発現カセットを含むプラスミドを細胞内に有するということである。形質転換体の継代培養が容易であることから、該発現カセットを染色体中に有することが好ましい。
(Method for producing transformant)
The ricinoleic acid-producing yeast in the present invention has the expression cassette in the chromosome or as an extrachromosomal gene. The expression cassette in the chromosome means an expression cassette at one or more positions in the chromosome of the host cell. And having as an extrachromosomal gene means having a plasmid containing an expression cassette in the cell. Since the subculture of the transformant is easy, it is preferable to have the expression cassette in the chromosome.
形質転換方法は、公知の形質転換方法をいずれも用いられる。該形質転換方法としては、たとえば、酢酸リチウム法、エレクトロポレーション法、スフェロプラスト法、ガラスビーズ法など従来周知の方法や、特開２００５−１９８６１２号公報記載の方法が挙げられる。また、市販の酵母形質転換用キットを用いてもよい。 Any known transformation method can be used as the transformation method. Examples of the transformation method include conventionally known methods such as lithium acetate method, electroporation method, spheroplast method, glass bead method, and the method described in JP-A-2005-198612. A commercially available yeast transformation kit may also be used.
形質転換を行った後、通常は得られた形質転換体を選抜する。選抜方法としては、たとえば、以下に示す方法が挙げられる。前記栄養要求性マーカーにより形質転換体を選択できる培地によりスクリーニングし、得られたコロニーから複数を選択する。選抜した形質転換体に対してパルスフィールドゲル電気泳動法によるゲノム解析を行うことにより、染色体に組み込まれたベクターの数や発現カセットの数を調べられる。 After transformation, the obtained transformant is usually selected. Examples of the selection method include the following methods. Screening is performed with a medium capable of selecting transformants using the auxotrophic marker, and a plurality of colonies obtained are selected. The number of vectors integrated in the chromosome and the number of expression cassettes can be examined by performing genome analysis by pulse field gel electrophoresis on the selected transformants.
［リシノール酸産生酵母の培養方法］
酵母にＦＡＨ１２を発現させた場合、通常の培養温度（３０℃程度）では増殖阻害がかかるが、比較的高温で培養した場合には増殖阻害がかからない。高温条件下では、ＦＡＨ１２の発現が抑制されるか、または発現したＦＡＨ１２のΔ１２−ヒドロキシラーゼ活性が失活若しくは低下するためと推察される。本発明では、リシノール酸産生酵母をまず高温で培養した後、培養温度を低温にシフトさせることによって、該酵母内でリシノールを産生させる。はじめに高温で培養することにより、温度シフト後にもＦＡＨ１２による増殖阻害の影響を抑制しつつ、ＦＡＨ１２によるリシノール酸産生を促進し得る。
すなわち、本発明のリシノール酸産生酵母の培養方法は、ＦＡＨ１２遺伝子が導入された酵母の形質転換体を、３５℃以上４０℃未満で、培養液のＯＤ６００が０．５〜５．５になるまで培養する高温培養工程と、前記高温培養工程後、前記形質転換体を３５℃未満で培養する低温培養工程とを有することを特徴とする。
[Method of culturing ricinoleic acid-producing yeast]
When FAH12 is expressed in yeast, growth inhibition takes place at a normal culture temperature (about 30 ° C.), but growth inhibition does not take place when cultured at a relatively high temperature. It is presumed that under high temperature conditions, the expression of FAH12 is suppressed, or the Δ12-hydroxylase activity of expressed FAH12 is inactivated or decreased. In the present invention, ricinoleic acid-producing yeast is first cultured at a high temperature, and then the culture temperature is shifted to a low temperature to produce ricinol in the yeast. By first culturing at a high temperature, ricinoleic acid production by FAH12 can be promoted while suppressing the effects of growth inhibition by FAH12 even after a temperature shift.
That is, in the method for culturing ricinoleic acid-producing yeast of the present invention, a yeast transformant introduced with the FAH12 gene is at 35 ° C. or higher and lower than 40 ° C., and the OD 600 of the culture solution is 0.5 to 5.5. And a high-temperature culture step of culturing the transformant at a temperature of less than 35 ° C. after the high-temperature culture step.
酵母の培養温度があまりに高い場合には、温度ストレスにより増殖がかえって低下する。リシノール酸産生酵母を３５℃以上４０℃未満で培養することにより、過度の高温ストレスを受けることなく、ＦＡＨ１２による増殖阻害作用を充分に抑制し得る。高温培養工程における培養温度は、３５〜３９℃が好ましく、３５〜３８℃がより好ましい。 When the culture temperature of yeast is too high, growth is reduced by temperature stress. By culturing ricinoleic acid-producing yeast at 35 ° C. or higher and lower than 40 ° C., the growth inhibitory action by FAH12 can be sufficiently suppressed without being subjected to excessive high temperature stress. The culture temperature in the high temperature culture step is preferably 35 to 39 ° C, more preferably 35 to 38 ° C.
低温培養工程における培養温度を、高温培養工程よりも低い温度、すなわち３５℃未満にすることで、増殖阻害作用が抑制されつつ、ＦＡＨ１２によるリシノール酸産生が促進される。但し、酵母の培養温度があまりに低い場合には、温度ストレスにより増殖がかえって低下する。そこで、低温培養工程における培養温度は、１０〜３３℃が好ましく、１５〜３３℃がより好ましく、２０〜３０℃がさらに好ましい。 By setting the culture temperature in the low-temperature culture step to a temperature lower than that in the high-temperature culture step, that is, less than 35 ° C., production of ricinoleic acid by FAH12 is promoted while the growth inhibitory action is suppressed. However, if the yeast culture temperature is too low, growth is reduced by temperature stress. Therefore, the culture temperature in the low temperature culture step is preferably 10 to 33 ° C, more preferably 15 to 33 ° C, and still more preferably 20 to 30 ° C.
低温培養工程移行後にも、リシノール酸産生酵母が充分に増殖でき、培養液当たりのリシノール酸産生量を改善し得るため、酵母の増殖がある程度盛んな状態、特に対数増殖期にある段階で、高温培養工程から低温培養工程へ移行することが好ましい。このため、本発明においては、形質転換体の培養液のＯＤ６００が５．５以下の時点で、好ましくは５以下の時点で、より好ましくは４，４以下の時点で、高温培養工程から低温培養工程へ移行する。また、高温培養工程から低温培養工程へ移行する時点における形質転換体の培養液のＯＤ６００は、０．５以上であり、０．５５以上であることがより好ましく、１．４以上であることがさらに好ましい。特に、リシノール酸産生酵母を、３５℃以上４０℃未満で、培養液のＯＤ６００が１．４〜４．４になるまで培養した後、当該培養液の温度を低温にシフトさせてさらに培養を続けることにより、より大量のリシノール酸を産生できる。培養液及びその希釈液のＯＤ６００は、例えば、分光光度計を使用して常法により測定できる。 Even after the transition to the low-temperature culture process, the ricinoleic acid-producing yeast can be sufficiently grown and the ricinoleic acid production amount per culture solution can be improved, so that the yeast is proliferated to some extent, particularly in the logarithmic growth phase. It is preferable to shift from the culture process to the low temperature culture process. Therefore, in the present invention, when the OD 600 of the culture medium of the transformant is 5.5 or less, preferably 5 or less, more preferably 4 or 4 or less, the high temperature culture step is performed at a low temperature. Move to culture process. Moreover, the OD 600 of the culture solution of the transformant at the time of transition from the high temperature culture step to the low temperature culture step is 0.5 or more, more preferably 0.55 or more, and 1.4 or more. Is more preferable. In particular, after culturing ricinoleic acid-producing yeast at 35 ° C. or higher and lower than 40 ° C. until the OD 600 of the culture solution becomes 1.4 to 4.4, the temperature of the culture solution is shifted to a low temperature and further cultured. By continuing, larger amounts of ricinoleic acid can be produced. The OD 600 of the culture solution and its diluted solution can be measured by a conventional method using, for example, a spectrophotometer.
本発明のリシノール酸産生酵母の培養方法では、高温培養工程および低温培養工程のいずれでも、リシノール酸産生酵母は、培養温度以外は一般的な酵母の培養方法と同様にして培養できる。たとえば、培養液には、宿主とした酵母の培養に用いられる公知の培養培地を用いることができ、宿主が資化しうる炭素源、窒素源、無機塩類等を含有し、宿主の培養を効率良く行えるものであればよい。培養液としては、天然培地を用いてもよく、合成培地を用いてもよい。 In the method for cultivating ricinoleic acid-producing yeast of the present invention, ricinoleic acid-producing yeast can be cultured in the same manner as in general yeast culturing methods except for the culture temperature in both the high-temperature culture step and the low-temperature culture step. For example, a known culture medium used for culturing yeast as a host can be used as the culture solution, and it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host, so that the host can be cultured efficiently. Anything that can be done. As the culture solution, a natural medium or a synthetic medium may be used.
炭素源としては、たとえばグルコース、フルクトース、スクロース等の糖が挙げられる。
窒素源としては、たとえばアンモニア、塩化アンモニウム、酢酸アンモニウム等の無機酸または無機酸のアンモニウム塩、ペプトン、カザミノ酸が挙げられる。
無機塩類としては、たとえばリン酸マグネシウム、硫酸マグネシウム、塩化ナトリウムが挙げられる。
Examples of the carbon source include sugars such as glucose, fructose, and sucrose.
Examples of the nitrogen source include inorganic acids such as ammonia, ammonium chloride, and ammonium acetate, ammonium salts of inorganic acids, peptone, and casamino acids.
Examples of inorganic salts include magnesium phosphate, magnesium sulfate, and sodium chloride.
培養には公知の細胞培養方法を用いることができ、たとえば振盪培養、攪拌培養等により行える。
培養は、回分培養であってもよく、連続培養であってもよい。また、培養時間は適宜決定できる。
A known cell culture method can be used for the culture, for example, shaking culture, stirring culture, or the like.
The culture may be batch culture or continuous culture. Further, the culture time can be determined as appropriate.
［リシノール酸の製造方法］
本発明のリシノール酸の製造方法は、本発明のリシノール酸産生酵母の培養方法により培養されたＦＡＨ１２遺伝子が導入された酵母の形質転換体から、リシノール酸を取得することを特徴とする。
培養されたリシノール酸産生酵母からのリシノール酸の抽出・精製は、溶剤抽出法等の一般的に脂肪酸の抽出・精製に使用される公知の手法により実施できる。例えば、リシノール酸産生酵母を培養した後、得られた培養液を遠心分離して菌体を回収し、該菌体をメタノール中でグラスビーズ等を用いて破砕しながら、クロロホルム／メタノール（１：２）を加えて抽出し、その後精製することにより、リシノール酸を精製できる。
また、リシノール酸は、ガスクロマトグラフィ法等の公知の手法で検出および定量できる。
[Method for producing ricinoleic acid]
The method for producing ricinoleic acid of the present invention is characterized in that ricinoleic acid is obtained from a transformant of yeast introduced with the FAH12 gene cultured by the method for culturing ricinoleic acid-producing yeast of the present invention.
Extraction and purification of ricinoleic acid from the cultured ricinoleic acid-producing yeast can be carried out by a known method generally used for extraction and purification of fatty acids such as a solvent extraction method. For example, after cultivating ricinoleic acid-producing yeast, the obtained culture solution is centrifuged to recover the cells, and the cells are crushed with glass beads or the like in methanol / chloroform (1: The ricinoleic acid can be purified by adding 2) and extracting, followed by purification.
Ricinoleic acid can be detected and quantified by a known method such as gas chromatography.
以下、実施例等を示して本発明を詳細に説明する。ただし、本発明は以下の記載によっては限定されない。
なお、以下の実施例等において、酵母の培養液および該希釈液のＯＤ値は、分光光度計ＵＶ１６００（島津製作所製）を用いて測定した。
Hereinafter, the present invention will be described in detail with reference to examples and the like. However, the present invention is not limited by the following description.
In the following Examples and the like, the OD values of the yeast culture solution and the diluted solution were measured using a spectrophotometer UV1600 (manufactured by Shimadzu Corporation).
［酵母］
以下の実施例等において、Ｓ．ポンベは、ロイシンおよびウラシル要求株ＡＲＣ０１０（遺伝子型：ｈ− 、ｌｅｕ１−３２、ｕｒａ４−Ｄ１８）（国際公開第２００７／０１５４７０号パンフレット参照。）を用いた。また、Ｓ．セレビシエは、ＢＹ４７４１（遺伝子型：ＭＡＴａ ｈｉｓ３Δ１ ｌｅｕ２Δ０ ｍｅｔ１５Δ０ ｕｒａ３Δ０）を用いた。
Ｓ．ポンベは、プラスミドを維持するために必要な選択圧に依存して、ＥＭＭＳまたはＥＭＭＳドロップアウト培地中で培養した（Alfa et al., 1993 ,“A laboratory course manual.”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.）。ＥＭＭＳは通常の培養時に使用され、窒素が制限された最小培地（ＥＭＭ−Ｃ／Ｎ３）がリシノール酸産生には使用された。発明者らによる従前の実験結果から、ＥＭＭ−Ｃ／Ｎ３中で培養したほうが脂肪酸含有量が高くなることが示唆されていたためである。ＥＭＭ培地は、０．５％（ｗ／ｖ）の塩化アンモニウム、２％（ｗ／ｖ）のグルコースを含有していた。一方、ＥＭＭ−Ｃ／Ｎ３は、塩化アンモニウムの濃度は０．１％にまで低減されており、グルコース濃度が１０％にまで増大していた。ｎｍｔ１プロモーターの下、遺伝子発現を抑制するために、１５μＭ（５μｇ／ｍＬ）のチアミンを使用した。
Ｓ．セレビシエは、プラスミドを維持するために必要な選択圧に依存して、最小合成培地（ＳＤ）、合成完全培地（ＳＣ）または合成完全ドロップアウト培地中で培養した（Sherman, et.al., 1986, “Methods in yeast genetics. ”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.）。最小合成培地またはＳＣは、０．１７％（ｗ／ｖ）のアミノ酸およびアンモニウム塩不含有イーストニトロジェンベース（Bactoyeast nitrogen base、Ｄｉｆｃｏ社製）、２％（ｗ／ｖ）のグルコース、および０．５％（ｗ／ｖ）の硫酸アンモニウムを含有していた。窒素が制限された最小培地（ＮＳＤ）では、硫酸アンモニウム含有量が０．１％にまで低減されており、グルコース濃度が１０％にまで増大していた(Yazawa, et.al., 2007,“Heterologous production of dihomo-gamma-linolenic acid in yeast Saccharomyces cerevisiae.”, Appl Env Microbiol, vol.73, pp.6965-6971)。
[yeast]
In the following examples, etc. Pombe is leucine and uracil auxotroph ARC010 (genotype: h -, leu1-32, ura4- D18) (. WO 2007/015470 pamphlet reference) was used. S. As the cerevisiae, BY4741 (genotype: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was used.
S. Pombe was cultured in EMMS or EMMS dropout media depending on the selection pressure required to maintain the plasmid (Alfa et al., 1993, “A laboratory course manual.”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). EMMS was used during normal culture, and a minimal medium with limited nitrogen (EMM-C / N3) was used for ricinoleic acid production. This is because the results of previous experiments by the inventors suggested that the fatty acid content was higher when cultured in EMM-C / N3. The EMM medium contained 0.5% (w / v) ammonium chloride, 2% (w / v) glucose. On the other hand, in EMM-C / N3, the concentration of ammonium chloride was reduced to 0.1%, and the glucose concentration was increased to 10%. 15 μM (5 μg / mL) thiamine was used to repress gene expression under the nmt1 promoter.
S. S. cerevisiae was cultured in minimal synthetic medium (SD), synthetic complete medium (SC) or synthetic complete dropout medium (Sherman, et.al., 1986), depending on the selection pressure required to maintain the plasmid. , “Methods in yeast genetics.”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Minimal synthetic medium or SC is 0.17% (w / v) amino acid and ammonium salt-free yeast nitrogen base (Bifoyeast nitrogen base, Difco), 2% (w / v) glucose, and 0. It contained 5% (w / v) ammonium sulfate. In nitrogen-limited minimal medium (NSD), the ammonium sulfate content was reduced to 0.1% and the glucose concentration was increased to 10% (Yazawa, et.al., 2007, “Heterologous production of dihomo-gamma-linolenic acid in yeast Saccharomyces cerevisiae. ”, Appl Env Microbiol, vol.73, pp.6965-6971).
［プラスミド構築］
ＲｃＦＡＨ１２遺伝子、ＬｆＦＡＨ１２遺伝子、およびＣｐＦＡＨ１２遺伝子のコード領域を含む酵母の形質転換用プラスミドを構築した。ＤＮＡ操作の標準的な技術は、SambrookおよびRussellの方法（“Molecular cloning: A laboratory manual. 3rd ed.”, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.）に準じて行った。また、プラスミド構築は、大腸菌ＤＨ５α（Ｆ−ｅｎｄＡ１、ｈｓｄＲ１７、ｓｕｐＥ４４、ｔｈｉ−１、ｒｅｃＡ１、ｇｙｒＡ９６、ｒｅｌＡ１、Δ（ａｒｇＦ−ｌａｃＺ）Ｕ１６９、Φ８０、Δ（ｌａｃＺ）Ｍ１５）株を用いて行った。当該ＤＨ５α株は、アンピシリンまたはカナマイシンを添加したＬＢ培地（Luria-Bertani broth）中で培養した。
[Plasmid construction]
A yeast transformation plasmid containing the coding regions of the RcFAH12 gene, the LfFAH12 gene, and the CpFAH12 gene was constructed. Standard techniques for DNA manipulation were performed according to the method of Sambrook and Russell (“Molecular cloning: A laboratory manual. 3rd ed.”, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Plasmid construction was performed using E. coli DH5α (F-endA1, hsdR17, supE44, thi-1, recA1, gyrA96, relA1, Δ (argF-lacZ) U169, Φ80, Δ (lacZ) M15). The DH5α strain was cultured in LB medium (Luria-Bertani broth) supplemented with ampicillin or kanamycin.
ＣｐＦＡＨ１２遺伝子のＤＮＡ配列（Meesapyodsuk and Qiu, 2008, Plant Physiology, vol.147, pp.1325−1333）は、Ｓ．ポンベにおいて好ましい使用頻度のコドンに改変した後、ＧＥＮＥＡＲＴ社（Regensburg, Germany）に依頼して化学合成した。合成されたＣｐＦＡＨ１２のＯＲＦは、５’末端と３’末端にそれぞれ人工的に付加されていた制限酵素部位を使用して、大腸菌のプラスミドｐＭＫ−ＣｐＦＡＨ１２から、制限酵素NcoIおよびSalIで二重消化したフラグメントとして切り出し、ｐＬ２４２８−９（略してｐＳＬ１０−ＣｐＦＡＨ１２）を構築するために、マルチクローニングサイトの制限酵素AarIおよびSalIで二重消化したプラスミドｐＳＬ１０に組み込んだ。合成されたプラスミドをｐＳＬ１０−ＣｐＦＡＨ１２とした。ｐＳＬ１０は、ｌｅｕ１マーカーを有する、染色体へ組み込まれるタイプのプラスミドであり、ｐＳＬ６プラスミド（Alimjan et al., Appl Microbiol Biotechnol, 2010, vol.85, pp.667−677）のｈＣＭＶプロモーター（ｈＣＭＶ−ｐ）をｎｍｔ１プロモーターに置換し、ＬＰＩターミネーター（ＬＰＩ−ｔｅｒ．）をｉｎｖ１ターミネーターに置換したプラスミドである。ｐＳＬ１０の塩基配列を配列番号１に示す。Ｓ．ポンベにｐＳＬ１０−ＣｐＦＡＨ１２を導入した形質転換体中では、チアミン非存在時には、ｎｍｔ１プロモーターの制御下でＣｐＦＡＨ１２が発現したが、チアミン存在時には発現は抑制された。
ｐＳＬ１０−ＣｐＦＡＨ１２（ｐＬ２４２８−９）と同様にして、ＬｆＦＡＨ１２遺伝子のＯＲＦを組み込んだｐＬ２４２７−８ (略してｐＳＬ１０−ＬｆＦＡＨ１２)を構築した。
図１に、ｐＳＬ１０−ＣｐＦＡＨ１２およびｐＳＬ１０−ＬｆＦＡＨ１２のベクターの構築手順を模式的に示した。なお、同様な手法によりｐＳＬ１０−ＲｃＦＡＨ１２も作製できる。
The DNA sequence of the CpFAH12 gene (Meesapyodsuk and Qiu, 2008, Plant Physiology, vol. 147, pp. 1325-1333) After modification to a codon having a preferred frequency of use in Pombe, it was chemically synthesized at the request of GENEART (Regensburg, Germany). The synthesized CpFAH12 ORF was double digested with the restriction enzymes NcoI and SalI from the plasmid pMK-CpFAH12 of E. coli using the restriction enzyme sites artificially added to the 5 ′ and 3 ′ ends, respectively. In order to construct pL2428-9 (abbreviated as pSL10-CpFAH12), it was incorporated into a plasmid pSL10 that was double-digested with restriction enzymes AarI and SalI at the multiple cloning site. The synthesized plasmid was designated as pSL10-CpFAH12. pSL10 is a chromosomally integrated plasmid having a leu1 marker and is the hCMV promoter (hCMV-p) of the pSL6 plasmid (Alimjan et al., Appl Microbiol Biotechnol, 2010, vol. 85, pp. 667-677). Is replaced with the nmt1 promoter, and the LPI terminator (LPI-ter.) Is replaced with the inv1 terminator. The base sequence of pSL10 is shown in SEQ ID NO: 1. S. In the transformant in which pSL10-CpFAH12 was introduced into pombe, CpFAH12 was expressed under the control of the nmt1 promoter when thiamine was not present, but the expression was suppressed when thiamine was present.
In the same manner as pSL10-CpFAH12 (pL2428-9), pL2427-8 (abbreviated as pSL10-LfFAH12) incorporating the ORF of the LfFAH12 gene was constructed.
FIG. 1 schematically shows a procedure for constructing vectors of pSL10-CpFAH12 and pSL10-LfFAH12. Note that pSL10-RcFAH12 can also be produced by a similar method.
また、ｐＭＫ−ＣｐＦＡＨ１２から、制限酵素HindIIIおよびPvuIIで二重消化したフラグメントとしてＣｐＦＡＨ１２を切り出し、マルチクローニングサイトの制限酵素NdeIで消化したプラスミドｐＲＥＰ１に組み込んだ。合成されたプラスミドをｐＬ２４１４−１（略してｐＲＥＰ１−ＣｐＦＡＨ１２）とした。ｐＲＥＰ１は、Ｓ．セレビシエのＬＥＵ２マーカーを有する染色体外に存在するタイプのプラスミドである。Ｓ．ポンベにｐＲＥＰ１−ＣｐＦＡＨ１２を導入した形質転換体中では、チアミン非存在時には、ｎｍｔ１プロモーターの制御下でＣｐＦＡＨ１２が発現したが、チアミン存在時には発現は抑制された。
ｐＬ２３９０−１（略してｐＲＥＰ１−ＲｃＦＡＨ１２）は、ｐＧＡ４−ＲｃＦＡＨ１２Ｈ６を制限酵素NcoIおよびEcl136IIで二重消化したフラグメントとしてＲｃＦＡＨ１２を切り出し、マルチクローニングサイトの制限酵素NdeIで消化したプラスミドｐＲＥＰ１に組み込んだ。ｐＬ２３９１−６３（略してｐＲＥＰ１−ＬｆＦＡＨ１２）は、ｐＭＡ−ＬｆＦＡＨ１２を制限酵素SmaIおよびPvuIIで二重消化したフラグメントとしてＬｆＦＡＨ１２を切り出し、マルチクローニングサイトの制限酵素NdeIで消化したプラスミドｐＲＥＰ１に組み込んだ。
図２に、ｐＲＥＰ１−ＲｃＦＡＨ１２、ｐＲＥＰ１−ＬｆＦＡＨ１２、およびｐＲＥＰ１−ＣｐＦＡＨ１２のベクターの構築手順を模式的に示した。
Further, CpFAH12 was excised from pMK-CpFAH12 as a fragment double digested with restriction enzymes HindIII and PvuII and incorporated into plasmid pREP1 digested with restriction enzyme NdeI at the multiple cloning site. The synthesized plasmid was designated as pL2414-1 (abbreviated as pREP1-CpFAH12). pREP1 is the S.P. It is an extrachromosomal plasmid having a L. cerevisiae LEU2 marker. S. In the transformant in which pREP1-CpFAH12 was introduced into pombe, CpFAH12 was expressed under the control of the nmt1 promoter when thiamine was not present, but the expression was suppressed when thiamine was present.
pL2390-1 (abbreviated as pREP1-RcFAH12) was obtained by excising RcFAH12 as a fragment obtained by double digesting pGA4-RcFAH12H6 with restriction enzymes NcoI and Ecl136II and incorporating it into plasmid pREP1 digested with restriction enzyme NdeI at the multiple cloning site. pL2391-63 (pREP1-LfFAH12 for short) was obtained by excising LfFAH12 as a fragment obtained by double digesting pMA-LfFAH12 with restriction enzymes SmaI and PvuII, and incorporating it into plasmid pREP1 digested with restriction enzyme NdeI at the multicloning site.
FIG. 2 schematically shows a procedure for constructing vectors of pREP1-RcFAH12, pREP1-LfFAH12, and pREP1-CpFAH12.
また、ｐＭＫ−ＣｐＦＡＨ１２から、制限酵素EcoRIおよびXhoIで二重消化したフラグメントとしてＣｐＦＡＨ１２を切り出し、ｐＬ２３７９−２を構築するために、マルチクローニングサイトの制限酵素EcoRIおよびXhoIで二重消化したプラスミドＹＥｐ３５２ＧＡＰに組み込んだ。Ｓ．セレビシエにｐＬ２３７９−２（略してＹＥｐ３５２−ＣｐＦＡＨ１２）を導入した形質転換体中では、解糖系のＧＡＰＤＨプロモーターの制御下でＣｐＦＡＨ１２が発現した。
図３に、ＹＥｐ３５２−ＣｐＦＡＨ１２のベクターの構築手順を模式的に示した。なお、同様な手法によりＹＥｐ３５２−ＲｃＦＡＨ１２も作製できる。
Also, CpFAH12 was excised from pMK-CpFAH12 as a fragment double digested with restriction enzymes EcoRI and XhoI, and incorporated into plasmid YEp352GAP double digested with restriction enzymes EcoRI and XhoI at the multiple cloning site to construct pL2379-2. It is. S. In a transformant in which pL2379-2 (YEp352-CpFAH12 for short) was introduced into S. cerevisiae, CpFAH12 was expressed under the control of the glycolytic GAPDH promoter.
FIG. 3 schematically shows the procedure for constructing the YEp352-CpFAH12 vector. YEp352-RcFAH12 can also be produced by the same method.
［形質転換体の作製］
酵母細胞は、Ｆｒｏｚｅｎ−ＥＺ Ｙｅａｓｔ Ｔｒａｎｓｆｏｒｍａｔｉｏｎ ＩＩ ｋｉｔ (Ｚｙｍｏ Ｒｅｓｅａｒｃｈ, ＣＡ， ＵＳＡ)を用いて、製造者により推奨されるプロトコールに従って形質転換した。
たとえば、ＣｐＦＡＨ１２遺伝子を染色体中に組み込む形で導入したＳ．ポンベの形質転換体の作製は、以下のようにして行った。すなわち、酵母の第２染色体のｌｅｕ１座にｐＳＬ１０−ＣｐＦＡＨ１２を組み込むため、ｐＳＬ１０−ＣｐＦＡＨ１２は制限酵素BsiWIによって消化し、得られた線状プラスミドをＳ．ポンベＡＲＣ０１０−１株に導入し、ｌｅｕ１＋の形質転換体を選抜した。
一方で、ＣｐＦＡＨ１２遺伝子を染色体外遺伝子として導入したＳ．ポンベの形質転換体は、ｐＲＥＰ１−ＣｐＦＡＨ１２をそのままＳ．ポンベＡＲＣ０１０−１株に導入し、ＬＥＵ２＋の形質転換体（すなわち、ロイシン要求性のない形質転換体）を選抜することによって作製した。
ＲｃＦＡＨ１２遺伝子またはＬｆＦＡＨ１２遺伝子を染色体中または染色体外に導入したＳ．ポンベの形質転換体は、ＣｐＦＡＨ１２遺伝子を染色体中または染色体外に導入したＳ．ポンベの形質転換体と同様にして作製した。
[Production of transformants]
Yeast cells were transformed with the Frozen-EZ Yeast Transformation II kit (Zymo Research, CA, USA) according to the protocol recommended by the manufacturer.
For example, S. cerevisiae having the CpFAH12 gene incorporated into the chromosome is introduced. Pombe transformants were prepared as follows. That is, in order to incorporate pSL10-CpFAH12 into the leu1 locus of the second chromosome of yeast, pSL10-CpFAH12 is digested with the restriction enzyme BsiWI. It was introduced into the Pombe ARC010-1 strain and a leu1 + transformant was selected.
On the other hand, S. cerevisiae having introduced the CpFAH12 gene as an extrachromosomal gene. As a transformant of Pombe, pREP1-CpFAH12 was used as it was as S. cerevisiae. It was introduced into the Pombe ARC010-1 strain and prepared by selecting a transformant of LEU2 + (that is, a transformant having no leucine requirement).
S. cerevisiae having the RcFAH12 gene or the LfFAH12 gene introduced into the chromosome or extrachromosomally. The Pombe transformant was transformed into S. cerevisiae having the CpFAH12 gene introduced into the chromosome or extrachromosomally. It was produced in the same manner as the Pombe transformant.
［参考例１］
Ｓ．ポンベおよびＳ．セレビシエの脂肪酸含有量を測定した。
具体的には、Ｓ．ポンベの場合は、ＥＭＭ−Ｃ／Ｎ３（ロイシンおよび／またはウラシル無含有培地）中で、３０℃で一晩前培養し、該前培養物を新しいＥＭＭ−Ｃ／Ｎ３に接種し、４０ｒｐｍで振とう培養した。Ｓ．セレビシエの場合は、ＮＳＤ培地を用いた。
得られた培養物の総脂肪酸含有量は、ガスクロマトグラフ分析によるKainouらの方法（2006, Yeast, vol.23, pp.605-612）に準じて行った。具体的には、以下に示す通りである。まず、脂肪酸分析は、温度制御環境下（１６０〜２３４℃、昇温速度：４．５℃／ｍｉｎ）で、ＴＣ−７０キャピラリーカラム（３０ｍ×０．２５ｍｍ（内径）、ＧＬ Ｓｃｉｅｎｃｅｓ製）を備えたガスクロマトグラフ（ＧＣ２０１０、島津製作所製）に０．２μＬの培養液を投与して実施した。脂肪酸組成は、各ピークの面積に基づいて算出され、含有量は、標準としたヘプタデカン酸メチル（Ｃ１７：０)と比較することによって決定した。
[Reference Example 1]
S. Pombe and S. The fatty acid content of cerevisiae was measured.
Specifically, S.M. In the case of Pombe, pre-incubate overnight at 30 ° C. in EMM-C / N3 (leucine and / or uracil-free medium), inoculate the preculture into fresh EMM-C / N3 and shake at 40 rpm. Cultured at last. S. In the case of cerevisiae, NSD medium was used.
The total fatty acid content of the obtained culture was determined according to the method of Kainou et al. (2006, Yeast, vol. 23, pp. 605-612) by gas chromatographic analysis. Specifically, it is as shown below. First, the fatty acid analysis was equipped with a TC-70 capillary column (30 m × 0.25 mm (inner diameter), manufactured by GL Sciences) under a temperature control environment (160 to 234 ° C., heating rate: 4.5 ° C./min). This was carried out by administering 0.2 μL of the culture solution to a gas chromatograph (GC2010, manufactured by Shimadzu Corporation). The fatty acid composition was calculated based on the area of each peak, and the content was determined by comparison with standard methyl heptadecanoate (C17: 0).
測定結果を図４に示す。縦軸は全脂肪酸に対する各脂肪酸の含有量割合（％）である。Ｓ．セレビシエでは、オレイン酸（Ｃ１８：１）は３０％強含まれていたが、パルミトレイン酸（Ｃ１６：１）が約４５％と最も多かった。一方、Ｓ．ポンベでは、８０％強がオレイン酸（Ｃ１８：１）であり、その他の脂肪酸含有量はいずれも非常に少なかった。 The measurement results are shown in FIG. The vertical axis represents the content ratio (%) of each fatty acid relative to the total fatty acid. S. In S. cerevisiae, oleic acid (C18: 1) was more than 30%, but palmitoleic acid (C16: 1) was the highest at about 45%. On the other hand, S.M. In Pombe, over 80% was oleic acid (C18: 1), and the content of other fatty acids was very low.
［参考例２］
ｐＲＥＰ１、ｐＲＥＰ１−ＣｐＦＡＨ１２、ｐＲＥＰ１−ＲｃＦＡＨ１２、またはｐＲＥＰ１−ＬｆＦＡＨ１２を導入したＳ．ポンベの形質転換体を、それぞれチアミン存在下および非存在下で培養し、増殖能を調べた。
具体的には、まず、チアミン含有ＥＭＭ−Ｃ／Ｎ３−ＬＥＵ（ＥＭＭ−Ｃ／Ｎ３からロイシンが除去された培地）中、３０℃で一晩前培養した前培養液を、チアミン不添加のＥＭＭ−Ｃ／Ｎ３−ＬＥＵのプレート（アガー平板培地）またはチアミン含有ＥＭＭ−Ｃ／Ｎ３−ＬＥＵのプレートに塗布し、３０℃で４日間培養し、コロニーの形成の有無や、形成されたコロニーのサイズを調べた。
空ベクターであるｐＲＥＰ１を導入した形質転換体をチアミン含有プレートに塗布した場合に形成されたコロニーのサイズを基準として、その他の形質転換体の増殖能を評価した。評価結果を表１に示す。表１中、「＋＋＋」は生育が正常であり、「＋＋」は生育が若干悪く、「＋」は生育が悪く、「−」は増殖できなかったことを意味する。また、「遺伝子発現ＯＦＦ（チアミン有り）」はチアミン含有ＥＭＭ−Ｃ／Ｎ３−ＬＥＵプレートの結果を、「遺伝子発現ＯＮ（チアミン無し）」はチアミン不添加のＥＭＭ−Ｃ／Ｎ３−ＬＥＵプレートの結果を、それぞれ示す。
[Reference Example 2]
S. pREP1, pREP1-CpFAH12, pREP1-RcFAH12, or pREP1-LfFAH12 introduced S. Pombe transformants were cultured in the presence and absence of thiamine, respectively, and their proliferation ability was examined.
Specifically, first, a pre-culture solution pre-cultured overnight at 30 ° C. in thiamine-containing EMM-C / N3-LEU (a medium in which leucine has been removed from EMM-C / N3) was added to an EMM without addition of thiamine. -C / N3-LEU plate (Agar plate medium) or thiamine-containing EMM-C / N3-LEU plate, cultured at 30 ° C. for 4 days, presence or absence of colonies, size of formed colonies I investigated.
The growth ability of other transformants was evaluated on the basis of the size of the colonies formed when the transformants introduced with the empty vector pREP1 were applied to thiamine-containing plates. The evaluation results are shown in Table 1. In Table 1, “++” means normal growth, “++” means that growth is slightly bad, “+” means that growth is bad, and “−” means that growth is not possible. "Gene expression OFF (with thiamine)" is the result of thiamine-containing EMM-C / N3-LEU plate, and "Gene expression ON (without thiamine)" is the result of EMM-C / N3-LEU plate without thiamine added. Are shown respectively.
ＲｃＦＡＨ１２を発現させた場合には、外来蛋白質を発現させていない場合と同程度の増殖能であったが、ＬｆＦＡＨ１２を発現させた形質転換体は、ＲｃＦＡＨ１２を発現させた形質転換体よりも増殖能が低下しており、ＣｐＦＡＨ１２を発現させた形質転換体は非常に増殖能が低下していることがわかった。つまり、ＬｆＦＡＨ１２やＣｐＦＡＨ１２を発現させることによって、増殖能が低下した。 When RcFAH12 was expressed, the growth ability was comparable to that when no foreign protein was expressed, but the transformant expressing LfFAH12 was more proliferative than the transformant expressing RcFAH12. It was found that the transformant expressing CpFAH12 had a very low growth ability. That is, the growth ability decreased by expressing LfFAH12 and CpFAH12.
［参考例３］
ｐＲＥＰ１−ＬｆＦＡＨ１２を導入したＳ．ポンベの形質転換体を培養し、リシノール酸をはじめとする脂肪酸の含有量を調べた。
具体的には、まず、チアミン含有ＥＭＭ−Ｃ／Ｎ３−ＬＥＵ中、３０℃で一晩前培養した前培養液を、ＥＭＭ−Ｃ／Ｎ３−ＬＥＵ中に接種し、３０℃、４０ｒｐｍで５日間振とう培養した。得られた培養物の各脂肪酸の含有量を、参考例１と同様にしてガスクロマトグラフ分析によって測定した。測定の結果得られたチャート図を図５に示す。該形質転換体では、リシノール酸の標準サンプルと同じ位置にピークが検出された。すなわち、該形質転換体ではリシノール酸が生産されていることが確認できた。
[Reference Example 3]
p. REP1-LfFAH12 introduced S. p. Pombe transformants were cultured and the content of fatty acids including ricinoleic acid was examined.
Specifically, first, a preculture solution pre-cultured overnight at 30 ° C. in thiamine-containing EMM-C / N3-LEU was inoculated into EMM-C / N3-LEU, and then at 30 ° C. and 40 rpm for 5 days. Cultured with shaking. The content of each fatty acid in the obtained culture was measured by gas chromatographic analysis in the same manner as in Reference Example 1. FIG. 5 shows a chart obtained as a result of the measurement. In the transformant, a peak was detected at the same position as the standard sample of ricinoleic acid. That is, it was confirmed that ricinoleic acid was produced in the transformant.
［実施例１］
ＣｐＦＡＨ１２遺伝子を染色体中に組み込んだＳ．ポンベの形質転換体（以下、ＣｐＦＡＨ１２インテグラント株）の各培養温度における増殖能を調べた。
具体的には、ＣｐＦＡＨ１２インテグラント株にｕｒａ４マーカーを含む空ベクターｐＲＥＰ２を導入した形質転換体（ＣｐＦＡＨ１２株）と、Ｓ．ポンベにｐＳＬ１０およびｐＲＥＰ２を導入した形質転換体（コントロール株）を、それぞれチアミンが添加されていないＥＭＭ−Ｃ／Ｎ３−ＵＲＡ，ＬＥＵ中、３７℃で一晩、培養液のＯＤ６００が約４になるまで前培養した。該前培養液を同種の新しい培地で希釈し、さらに、１０倍希釈系列を調整し、各１０μＬずつを、１５μＭのチアミン含有ＥＭＭ−Ｃ／Ｎ３−ＵＲＡ，ＬＥＵプレート（発現抑制条件）またはチアミン不添加のＥＭＭ−Ｃ／Ｎ３−ＵＲＡ，ＬＥＵプレート（発現誘導条件）上にスポットした。該プレートを３７℃、３５℃、３０℃、２５℃、または２０℃で４〜９日間インキュベートした。
インキュベート後の各プレート表面の写真図を図６に示す。図中、「Ｃｏｎｔｒｏｌ」はコントロール株の結果であり、「ＣｐＦＡＨ１２」はＣｐＦＡＨ１２株の結果を示す。また、図中、白い部分がコロニーである。チアミン含有プレート（図中、「＋Ｔｈｉａｍｉｎｅ」）では、２０℃、９日間培養した場合のみ、ＣｐＦＡＨ１２株がコントロール株よりもやや増殖能が低かったものの、その他の培養条件では両形質転換体の増殖能に特段の差はなかった。一方、参考例２ではＣｐＦＡＨ１２を発現させた株ではほとんど増殖が観察されなかったにも関わらず、チアミン不含有プレート（図中、「Ｎｏ Ｔｈｉａｍｉｎｅ」）では、濃い濃度でスポットとした場合に、全ての培養条件でＣｐＦＡＨ１２株の増殖が確認できた。但し、３７℃、４日間培養した場合を除き、ＣｐＦＡＨ１２株のほうがコントロール株よりも明らかに増殖能が低かった。すなわち、ＣｐＦＡＨ１２の発現誘導条件下のＣｐＦＡＨ１２インテグラントの増殖能は培養温度に影響され、３７℃で前培養した後に培養温度を低下させた場合には、増殖能は低下するものの、増殖自体は可能であること、および３７℃ではほとんど低下しないことがわかった。
[Example 1]
S. cerevisiae having the CpFAH12 gene integrated into the chromosome. The growth ability of pombe transformants (hereinafter referred to as CpFAH12 integrant strain) at each culture temperature was examined.
Specifically, a transformant (CpFAH12 strain) in which an empty vector pREP2 containing a ura4 marker was introduced into a CpFAH12 integrant strain, A transformant in which pSL10 and pREP2 were introduced into a pombe (control strain) was placed in EMM-C / N3-URA, LEU without addition of thiamine overnight at 37 ° C., and the OD 600 of the culture solution was reduced to about 4. Pre-culture until complete. The preculture solution is diluted with a new medium of the same type, and a 10-fold dilution series is prepared. Each 10 μL is diluted with 15 μM thiamine-containing EMM-C / N3-URA, LEU plate (expression suppression condition) or thiamine-free. Spotted on added EMM-C / N3-URA, LEU plate (expression induction conditions). The plates were incubated at 37 ° C, 35 ° C, 30 ° C, 25 ° C, or 20 ° C for 4-9 days.
The photograph figure of each plate surface after incubation is shown in FIG. In the figure, “Control” is the result of the control strain, and “CpFAH12” is the result of the CpFAH12 strain. In the figure, the white part is a colony. On the thiamine-containing plate (“+ Thiamine” in the figure), only when cultured at 20 ° C. for 9 days, the CpFAH12 strain had a slightly lower growth ability than the control strain, but the growth ability of both transformants was observed under other culture conditions. There was no particular difference. On the other hand, in Reference Example 2, although almost no growth was observed in the strain expressing CpFAH12, in the thiamine-free plate (“No Thiamine” in the figure), all the spots were concentrated at a high concentration. The growth of the CpFAH12 strain was confirmed under the above culture conditions. However, except for the case of culturing at 37 ° C. for 4 days, the CpFAH12 strain was clearly less proliferative than the control strain. That is, the growth ability of CpFAH12 integrant under the CpFAH12 expression-inducing condition is affected by the culture temperature. If the culture temperature is lowered after pre-culture at 37 ° C, the growth ability is reduced, but the growth itself is possible. And at 37 ° C., it was found that there was almost no decrease.
［実施例２］
実施例１で用いたＣｐＦＡＨ１２株およびコントロール株の、３０℃または３７℃の液体培地中の増殖能を調べた。
具体的には、ＣｐＦＡＨ１２株およびコントロール株を、チアミン不添加のＥＭＭ−Ｃ／Ｎ３−ＵＲＡ，ＬＥＵ中、３７℃で一晩前培養した。該前培養物を、前培養と同種の新しい培地で、ＯＤ６００が０．０５になるように希釈した後、３０℃または３７℃で３６時間、攪拌培養した。６３０ｎｍの濁度を自動検出器（Ｂｉｏ−Ｐｌｏｔｔｅｒ、東洋測器製）でモニターすることにより、液体培地中の増殖曲線を描いた。図７に増殖曲線を示す。図７（Ａ）は３０℃で培養した結果であり、図７（Ｂ）は３７℃で培養した結果である。図中、三角がＣｐＦＡＨ１２株の増殖曲線を、円がコントロール株の増殖曲線を、それぞれ示す。３７℃で培養した場合よりも、３０℃で培養した場合には、ＣｐＦＡＨ１２株はコントロール株よりも明らかに増殖が抑制されていた。
[Example 2]
The growth ability of the CpFAH12 strain and the control strain used in Example 1 in a liquid medium at 30 ° C. or 37 ° C. was examined.
Specifically, CpFAH12 strain and control strain were pre-cultured overnight at 37 ° C. in EMM-C / N3-URA, LEU without addition of thiamine. The preculture was diluted with a new medium of the same type as the preculture so that the OD 600 was 0.05, and then stirred at 30 ° C. or 37 ° C. for 36 hours. A growth curve in a liquid medium was drawn by monitoring the turbidity at 630 nm with an automatic detector (Bio-Plotter, manufactured by Toyo Sokki). FIG. 7 shows the growth curve. FIG. 7 (A) shows the result of culturing at 30 ° C., and FIG. 7 (B) shows the result of culturing at 37 ° C. In the figure, the triangle shows the growth curve of the CpFAH12 strain, and the circle shows the growth curve of the control strain. When cultured at 30 ° C., the growth of the CpFAH12 strain was clearly suppressed as compared to the control strain when cultured at 37 ° C.
［実施例３］
３７℃から２０℃または１５℃に培養温度をシフトさせるタイミングの、ＣｐＦＡＨ１２インテグラント株のリシノール酸産生に対する影響を評価した。
図８は、評価方法のスキーム図である。まず、ＣｐＦＡＨ１２インテグラント株を、チアミンが添加されていないＥＭＭ−Ｃ／Ｎ３−ＵＲＡ，ＬＥＵ中、３７℃で一日間前培養した。次いで、培養した細胞を、前培養と同種の新しい培地で、ＯＤ６００が０．０５になるように希釈した後、最初に３７℃で１日間（ＯＤ６００が５、対数増殖期後期）または２日間（ＯＤ６００が６、定常増殖期初期）培養し、その後２０℃または１５℃に培養温度をシフトさせて５日間培養した。対照として、前培養と同種の新しい培地で、ＯＤ６００が０．０５になるように希釈した後、温度シフトせずに３７℃で７日間培養した（図８の下段）。液体培養は全て、４０ｒｐｍで振とう培養した。
[Example 3]
The effect of the timing of shifting the culture temperature from 37 ° C. to 20 ° C. or 15 ° C. on the ricinoleic acid production of the CpFAH12 integrant strain was evaluated.
FIG. 8 is a schematic diagram of the evaluation method. First, the CpFAH12 integrant strain was precultured at 37 ° C. for one day in EMM-C / N3-URA, LEU without addition of thiamine. The cultured cells are then diluted with a new medium of the same type as the preculture so that the OD 600 is 0.05, and then initially at 37 ° C. for 1 day (5 OD 600 , late logarithmic growth phase) or 2 The cells were cultured for 5 days (OD 600 was 6, early in the stationary growth phase), and then cultured for 5 days by shifting the culture temperature to 20 ° C or 15 ° C. As a control, after diluting with a new medium of the same type as the preculture so that the OD 600 was 0.05, the cells were cultured at 37 ° C. for 7 days without a temperature shift (lower part of FIG. 8). All liquid cultures were cultured at 40 rpm with shaking.
本培養開始後１日経過ごとに、培養液の一部をサンプリングし、培養液のＯＤ６００、培養液１ｍＬ当たりの乾燥菌体重量（ＤＣＷ）、および培養液１ｍＬ当たりの各種脂肪酸の含有量を測定した。培養液のＯＤ６００は実施例２と同様にして、各種脂肪酸の含有量は参考例１と同様にして、それぞれ測定した。培養液のＯＤ６００および培養液１ｍＬ当たりの乾燥菌体重量の測定結果を図９（Ａ）に、各種脂肪酸の含有量の測定結果を図９（Ｂ）に、それぞれ示す。図９（Ｂ）中、「Ｃ１８：１−ＯＨ」はリシノール酸を、「Ｃ１８：２」はリノレン酸を、「Ｏｔｈｅｒｓ」はその他の脂肪酸の総和を、それぞれ示す。培養液のＯＤ６００および培養液１ｍＬ当たりの乾燥菌体重量は、培養温度の温度シフトによってはあまり差がなかった。しかし、各種脂肪酸の含有量は、温度シフトせずに３７℃で培養し続けた場合、３７℃で２日間培養した後に２０℃または１５℃にシフトした場合には、リシノール酸の含有量は非常に低かった。これに対して、３７℃で１日間培養した後に２０℃または１５℃にシフトした場合には、培養時間の経過とともにリシノール酸の含有量が顕著に増大していた。該結果から、培養温度が３７℃の場合、および増殖が定常状態になった後に低温へ温度シフトした場合には、リシノール酸が産生されないことが分かった。培養温度が３７℃の場合等でリシノール酸産生が行われないのは、ＣｐＦＡＨ１２が発現しないため、またはＣｐＦＡＨ１２の活性が非常に低下するためと推察される。 Every day after the start of the main culture, a part of the culture solution is sampled, and the OD 600 of the culture solution, the dry cell weight (DCW) per 1 mL of the culture solution, and the contents of various fatty acids per 1 mL of the culture solution. It was measured. The OD 600 of the culture solution was measured in the same manner as in Example 2, and the content of various fatty acids was measured in the same manner as in Reference Example 1. The measurement results of the OD 600 of the culture solution and the dry cell weight per 1 mL of the culture solution are shown in FIG. 9A, and the measurement results of the contents of various fatty acids are shown in FIG. 9B. In FIG. 9B, “C18: 1-OH” represents ricinoleic acid, “C18: 2” represents linolenic acid, and “Others” represents the total of other fatty acids. The OD 600 of the culture solution and the dry cell weight per 1 mL of the culture solution were not significantly different depending on the temperature shift of the culture temperature. However, the content of various fatty acids is very high when culturing at 37 ° C without temperature shift, or when culturing at 37 ° C for 2 days and then shifting to 20 ° C or 15 ° C. It was low. In contrast, when the culture was shifted to 20 ° C. or 15 ° C. after culturing at 37 ° C. for 1 day, the content of ricinoleic acid was remarkably increased as the culture time passed. From the results, it was found that ricinoleic acid was not produced when the culture temperature was 37 ° C. and when the temperature shifted to a low temperature after the growth reached a steady state. The reason that ricinoleic acid production is not performed when the culture temperature is 37 ° C. is presumed that CpFAH12 is not expressed or that the activity of CpFAH12 is greatly reduced.
さらに、図１０に、脂肪酸組成の経時的変化を示す。図１０（Ａ）は３７℃で１日間培養した後に２０℃にシフトして５日間培養した場合、図１０（Ｂ）は３７℃で２日間培養した後に２０℃にシフトして５日間培養した場合、図１０（Ｃ）は３７℃で７日間培養した場合の結果である。３７℃で２日間培養した後に２０℃にシフトした場合には、３７℃で７日間培養した場合と同様、脂肪酸組成はほとんど経時的変化がなかった。これに対して３７℃で１日間培養した後に２０℃にシフトした場合には、培養時間の経過と共にオレイン酸含有割合が低下し、逆にリシノール酸含有割合が増大した。その他の脂肪酸の含有割合は、ほとんど経時的変化がなかった。 Furthermore, FIG. 10 shows the change over time in the fatty acid composition. FIG. 10A shows a case of culturing for 1 day at 37 ° C. and then shifting to 20 ° C. for 5 days. FIG. 10B shows a case of culturing for 2 days at 37 ° C. and then shifting to 20 ° C. for 5 days. In this case, FIG. 10 (C) shows the results when cultured at 37 ° C. for 7 days. When shifting to 20 ° C. after culturing at 37 ° C. for 2 days, the fatty acid composition hardly changed over time, as in the case of culturing at 37 ° C. for 7 days. On the other hand, when it shifted to 20 degreeC after culture | cultivating for 1 day at 37 degreeC, the oleic acid content rate fell with progress of culture | cultivation time, and conversely the ricinoleic acid content rate increased. The content of other fatty acids hardly changed over time.
［実施例４］
３７℃から２０℃、２５℃または３０℃への培養温度シフトの、ＣｐＦＡＨ１２インテグラント株のリシノール酸産生に対する影響を評価した。
具体的には、図１１に示すように、まず、ＣｐＦＡＨ１２インテグラント株を、チアミンが添加されていないＥＭＭ−Ｃ／Ｎ３−ＵＲＡ，ＬＥＵ中、３７℃で一日間前培養した。次いで、培養した細胞を、前培養と同種の新しい培地で、ＯＤ６００が０．５になるように希釈した後、２０℃、２５℃または３０℃で１０日間、４０ｒｐｍで振とう培養した（図１１の上段）。対照として、前培養と同種の新しい培地で、ＯＤ６００が０．５になるように希釈した後、温度シフトせずに３７℃で１０日間、４０ｒｐｍで振とう培養した（図１１の下段）。
実施例３と同様に、本培養開始後１日経過ごとに、培養液の一部をサンプリングし、培養液のＯＤ６００、培養液１ｍＬ当たりの乾燥菌体重量（ＤＣＷ）、および培養液１ｍＬ当たりの各種脂肪酸の含有量を測定した。培養液のＯＤ６００および培養液１ｍＬ当たりの乾燥菌体重量の測定結果を図１２（Ａ）に、各種脂肪酸の含有量の測定結果を図１２（Ｂ）に、それぞれ示す。図１２（Ｂ）中、「Ｃ１８：１−ＯＨ」、「Ｃ１８：２」および「Ｏｔｈｅｒｓ」は図９（Ｂ）と同様である。
[Example 4]
The influence of the culture temperature shift from 37 ° C. to 20 ° C., 25 ° C. or 30 ° C. on the ricinoleic acid production of the CpFAH12 integrant strain was evaluated.
Specifically, as shown in FIG. 11, first, the CpFAH12 integrant strain was pre-cultured at 37 ° C. for one day in EMM-C / N3-URA, LEU without addition of thiamine. The cultured cells were then diluted with a new medium of the same type as the preculture so that the OD 600 was 0.5, and then cultured with shaking at 40 rpm for 10 days at 20 ° C., 25 ° C. or 30 ° C. (FIG. 11 top). As a control, after diluting with a new medium of the same type as the preculture so that the OD 600 was 0.5, the cells were cultured with shaking at 40 rpm at 37 ° C. for 10 days without a temperature shift (lower part of FIG. 11).
As in Example 3, a portion of the culture solution was sampled every day after the start of the main culture, and the OD 600 of the culture solution, the dry cell weight (DCW) per mL of culture solution, and per mL of culture solution The content of various fatty acids was measured. The measurement results of the OD 600 of the culture solution and the dry cell weight per 1 mL of the culture solution are shown in FIG. 12A, and the measurement results of the contents of various fatty acids are shown in FIG. In FIG. 12B, “C18: 1-OH”, “C18: 2”, and “Others” are the same as those in FIG. 9B.
最終的なリシノール酸産生量は、本培養を２０℃で実施した場合が、２５℃または３０℃で実施した場合よりも多くなることが確認された。しかし、本培養を２５℃で行うほうが、利点があると考えられる。細胞増殖は２５℃のほうが良好であるため、本培養開始４日目では、２５℃の培養条件のほうが２０℃よりもリシノール酸の産生量が高かったためである。具体的には、培養液当たりのリシノール酸産生量は、２０℃で１０日間培養後には１６３．２μｇ／ｍＬであったが、２０℃で４日間培養後には４８．２μｇ／ｍＬでしかなかった。これに対して２５℃で４日間培養後には１０８．８μｇ／ｍＬであり、該生産量は、２０℃で１０日間培養した場合の６６％であった。さらにコストの点から、冷却コストを低減でき、かつ培養日数を１０日間から４日間に短縮することで培養コストを低減できるため、細胞増殖は２０℃よりも２５℃で行うほうが好ましい。このように、冷却コストを低減し、培養時間を短縮することで製造コストを低減できるため、工業上の観点からは、２５℃培養のほうが２０℃培養よりも好ましい。 It was confirmed that the final production amount of ricinoleic acid was higher when the main culture was performed at 20 ° C than when it was performed at 25 ° C or 30 ° C. However, it is considered that it is advantageous to perform the main culture at 25 ° C. This is because the cell growth was better at 25 ° C., and on the fourth day from the start of the main culture, the amount of ricinoleic acid produced was higher under the culture conditions at 25 ° C. than at 20 ° C. Specifically, the amount of ricinoleic acid produced per culture broth was 163.2 μg / mL after 10 days of culture at 20 ° C., but only 48.2 μg / mL after 4 days of culture at 20 ° C. . On the other hand, after culturing at 25 ° C. for 4 days, it was 108.8 μg / mL, and the production amount was 66% when culturing at 20 ° C. for 10 days. Further, from the viewpoint of cost, it is preferable to perform cell growth at 25 ° C. rather than 20 ° C. because the cooling cost can be reduced and the culture cost can be reduced by shortening the culture days from 10 days to 4 days. Thus, since the manufacturing cost can be reduced by reducing the cooling cost and shortening the culture time, the culture at 25 ° C. is preferable to the culture at 20 ° C. from the industrial viewpoint.
［実施例５］
３７℃から２０℃に培養温度をシフトさせる時点の酵母の増殖状態の、ＣｐＦＡＨ１２インテグラント株のリシノール酸産生に対する影響を評価した。
具体的には、図１３に示すように、まず、ＣｐＦＡＨ１２インテグラント株を、チアミンが添加されていないＥＭＭ−Ｃ／Ｎ３−ＵＲＡ，ＬＥＵ中、３７℃で一日間前培養した。次いで、培養した細胞を、前培養と同種の新しい培地で、ＯＤ６００が０．０１〜０．１６の間様々な値になるように希釈した５つの本培養液を調製後、３７℃で１日間、４０ｒｐｍで振とう培養した後、培養液の温度を２０℃にシフトし、シフト後さらに５日間、４０ｒｐｍで振とう培養した（図１３の上段）。５つの培養液の温度シフト時のＯＤ６００は、それぞれ、０．５５（培養液Ａ）、１．４（培養液Ｂ）、２．５（培養液Ｃ）、４．４（培養液Ｄ）および５．５（培養液Ｅ）であった。対照として、前培養と同種の新しい培地で、ＯＤ６００が０．０５になるように希釈した後、温度シフトせずに３７℃で６日間、４０ｒｐｍで振とう培養した（図１３の下段）。
実施例３と同様にして、本培養終了時点の培養液のＯＤ６００、培養液１ｍＬ当たりの乾燥菌体重量（ＤＣＷ）、および培養液１ｍＬ当たりの各種脂肪酸の含有量を測定した。温度シフト開始時点および培養終了時点の培養液のＯＤ６００、並びに培養終了時点の培養液１ｍＬ当たりの乾燥菌体重量の測定結果を図１４（Ａ）に、培養終了時点の培養液１ｍＬ当たりの各種脂肪酸の含有量の測定結果を図１４（Ｂ）に、それぞれ示す。図１４（Ｂ）中、「Ｃ１８：１−ＯＨ」、「Ｃ１８：２」および「Ｏｔｈｅｒｓ」は図９（Ｂ）と同様である。さらに、総脂肪酸含有量に対する各脂肪酸の含有量比（質量％）を表２に示す。図１４および表２の各測定値は、独立した２回の試行を行い、その平均値である。
[Example 5]
The influence of the yeast growth state at the time of shifting the culture temperature from 37 ° C. to 20 ° C. on the ricinoleic acid production of the CpFAH12 integrant strain was evaluated.
Specifically, as shown in FIG. 13, first, the CpFAH12 integrant strain was pre-cultured at 37 ° C. for one day in EMM-C / N3-URA, LEU without addition of thiamine. Next, after preparing five main culture solutions obtained by diluting the cultured cells with a new medium of the same type as the preculture so that the OD 600 becomes various values between 0.01 and 0.16, 1 After shaking culture at 40 rpm for one day, the temperature of the culture solution was shifted to 20 ° C., and after 5 days of shifting, shaking culture was performed at 40 rpm for further 5 days (the upper part of FIG. 13). The OD 600 at the time of temperature shift of the five culture solutions is 0.55 (culture solution A), 1.4 (culture solution B), 2.5 (culture solution C), and 4.4 (culture solution D), respectively. And 5.5 (culture medium E). As a control, after diluting with a new medium of the same type as the preculture so that the OD 600 was 0.05, the cells were cultured with shaking at 40 rpm for 6 days at 37 ° C. without temperature shift (lower part of FIG. 13).
In the same manner as in Example 3, the OD 600 of the culture solution at the end of the main culture, the dry cell weight (DCW) per mL of the culture solution, and the contents of various fatty acids per mL of the culture solution were measured. FIG. 14A shows the OD 600 of the culture solution at the start of the temperature shift and the end of the culture, and the measurement results of the dry cell weight per 1 mL of the culture at the end of the culture. The measurement results of the fatty acid content are shown in FIG. In FIG. 14B, “C18: 1-OH”, “C18: 2”, and “Others” are the same as those in FIG. 9B. Furthermore, Table 2 shows the content ratio (% by mass) of each fatty acid with respect to the total fatty acid content. Each measured value in FIG. 14 and Table 2 is an average value of two independent trials.
図１４（Ｂ）および表２に示す通り、培養液Ａ〜Ｅの全てでリシノール酸の産生が確認されたが、温度シフト時点の培養液のＯＤ６００が１．４〜４．４である培養液Ｂ〜Ｄが、培養液ＡおよびＥよりもリシノール酸産生量が多かった。該結果から、酵母が対数増殖期にある時点で温度シフトを行うことにより、より大量のリシノール酸を産生させ得ることが示唆された。 As shown in FIG. 14 (B) and Table 2, the production of ricinoleic acid was confirmed in all of the culture solutions A to E, but the culture solution at the time of temperature shift had an OD 600 of 1.4 to 4.4. Liquids B to D produced more ricinoleic acid than culture liquids A and E. From the results, it was suggested that a larger amount of ricinoleic acid can be produced by performing a temperature shift when the yeast is in the logarithmic growth phase.
［実施例６］
３７℃から２０℃に培養温度をシフトさせる時点の培養液の濃度の、ＣｐＦＡＨ１２インテグラント株のリシノール酸産生に対する影響を評価した。
具体的には、図１５に示すように、まず、ＣｐＦＡＨ１２インテグラント株を、チアミンが添加されていないＥＭＭ−Ｃ／Ｎ３−ＵＲＡ，ＬＥＵ中、３７℃で一日間前培養した。次いで、培養した細胞を、前培養と同種の新しい培地で、ＯＤ６００が０．０７になるように希釈した後、３７℃で培養液のＯＤ６００が２．５になるまで４０ｒｐｍで振とう培養した後、培養液を遠心分離処理して菌体を回収した後、遠心分離処理前の菌体濃度の１倍、２倍、または４倍となるように、元々の培地または新しい同種の培地に懸濁して培養液を調製した。該培養液の温度を２０℃にシフトし、シフト後さらに５日間、４０ｒｐｍで振とう培養した。
実施例３と同様にして、本培養終了時点の培養液のＯＤ６００、および培養液１ｍＬ当たりの各種脂肪酸の含有量を測定した。測定結果を図１６に示す。図１６中、「Ｃ１８：１−ＯＨ」、「Ｃ１８：２」および「Ｏｔｈｅｒｓ」は図９（Ｂ）と同様である。また、「１×Ｕｓｅｄ」、「２×Ｕｓｅｄ」および「４×Ｕｓｅｄ」は、元々の培地で菌体濃度が遠心分離処理前の菌体濃度の１倍、２倍、または４倍になるように調製した培養液の結果であり、「１×Ｆｒｅｓｈ」、「２×Ｆｒｅｓｈ」および「４×Ｆｒｅｓｈ」は、新しい培地で菌体濃度が遠心分離処理前の菌体濃度の１倍、２倍、または４倍になるように調製した培養液の結果である。
[Example 6]
The influence of the concentration of the culture solution at the time of shifting the culture temperature from 37 ° C. to 20 ° C. on the ricinoleic acid production of the CpFAH12 integrant strain was evaluated.
Specifically, as shown in FIG. 15, first, the CpFAH12 integrant strain was pre-cultured at 37 ° C. for one day in EMM-C / N3-URA, LEU without addition of thiamine. Next, the cultured cells are diluted with a new medium of the same type as the preculture so that the OD 600 becomes 0.07, and then cultured at 37 ° C. with shaking at 40 rpm until the OD 600 of the culture becomes 2.5. Thereafter, the culture solution is centrifuged to collect the cells, and then the original medium or a new medium of the same kind is added so that the concentration of the cells before centrifugation is 1, 2 or 4 times. The culture solution was prepared by suspending. The temperature of the culture solution was shifted to 20 ° C., and further cultured with shaking at 40 rpm for 5 days after the shift.
In the same manner as in Example 3, the OD 600 of the culture solution at the end of the main culture and the contents of various fatty acids per 1 mL of the culture solution were measured. The measurement results are shown in FIG. In FIG. 16, “C18: 1-OH”, “C18: 2”, and “Others” are the same as those in FIG. 9B. In addition, “1 × Used”, “2 × Used”, and “4 × Used” indicate that the cell concentration in the original medium is one, two, or four times the cell concentration before centrifugation. “1 × Fresh”, “2 × Fresh” and “4 × Fresh” are the results of the culture solution prepared in 1), and the cell concentration in the new medium is 1 or 2 times the cell concentration before centrifugation. Or the result of the culture solution prepared to be 4 times.
遠心分離処理後の培養液の調製に新しい培地を用いたほうが、元々の培地を用いるよりも細胞増殖速度が速く、よって培養液１ｍＬ当たりのリシノール酸産生量も多くなった。また、遠心分離処理後の菌体の濃縮倍率が大きいほど、培養液１ｍＬ当たりのリシノール酸産生量も多くなった。しかし、総脂肪酸全体に占めるリシノール酸の含有割合は、各培養液において特に大きな差はなかった。 The cell growth rate was faster when the new culture medium was used for the preparation of the culture solution after the centrifugation than when the original culture medium was used, and thus the amount of ricinoleic acid produced per 1 mL of the culture solution increased. Moreover, the larger the concentration factor of the cells after the centrifugation, the greater the amount of ricinoleic acid produced per 1 mL of the culture solution. However, the content of ricinoleic acid in the total fatty acids was not particularly different in each culture solution.
［実施例７］
３７℃から２０℃または３０℃への培養温度シフトの、実施例１で用いたＣｐＦＡＨ１２株およびコントロール株のリシノール酸産生に対する影響を評価した。
まず、ＣｐＦＡＨ１２株およびコントロール株をそれぞれ、チアミンが添加されていないＥＭＭ−Ｃ／Ｎ３−ＵＲＡ，ＬＥＵ中、３７℃で一日間前培養した。次いで、培養した細胞を、前培養と同種の新しい培地で、ＯＤ６００が０．５になるように希釈した後、２０℃、３０℃または３７℃に培養温度をシフトさせて４日間培養した。本培養開始後１日経過ごとに、培養液の一部をサンプリングし、参考例１と同様にして、培養液１ｍＬ当たりの各種脂肪酸の含有量を測定した。
本培養を２０℃で行った場合の各株のガスクロマトグラフ分析の結果のチャート図を図１７に示す。ＣｐＦＡＨ１２株では、リシノール酸の標準サンプルと同じ位置にピークが検出された。該ピークは、ＣｐＦＡＨ１２を発現していないコントロール株では観察されなかった。
培養液１ｍＬ当たりの各種脂肪酸の、総脂肪酸含有量に対する含有割合（％）の結果を図１８に示す。図１８（Ａ）はコントロール株の結果であり、図１８（Ｂ）はＣｐＦＡＨ１２株の結果である。ＣｐＦＡＨ１２を発現していないコントロール株では、本培養の温度に関わらず、脂肪酸組成は全てほぼ同様であった。これに対してＣｐＦＡＨ１２株では、３７℃で培養した場合にはコントロール株の結果とほぼ変わらなかったのに対して、２０℃および３０℃で培養した場合には、オレイン酸の含有割合の低下に伴い、リシノール酸の含有割合が増大していた。２０℃の場合には、培養日数が長くなるにつれてリシノール酸含有割合が増大していた。３０℃の場合には、培養日数が２日目でリシノール酸の含有割合はピークを迎えていた。
[Example 7]
The influence of the culture temperature shift from 37 ° C. to 20 ° C. or 30 ° C. on the ricinoleic acid production of the CpFAH12 strain and the control strain used in Example 1 was evaluated.
First, the CpFAH12 strain and the control strain were each pre-cultured at 37 ° C. for one day in EMM-C / N3-URA, LEU without addition of thiamine. Next, the cultured cells were diluted with a new medium of the same type as the preculture so that the OD 600 was 0.5, and then cultured for 4 days with the culture temperature shifted to 20 ° C., 30 ° C. or 37 ° C. A part of the culture solution was sampled every day after the start of the main culture, and the content of various fatty acids per 1 mL of the culture solution was measured in the same manner as in Reference Example 1.
FIG. 17 shows a chart of the results of gas chromatographic analysis of each strain when main culture is performed at 20 ° C. In CpFAH12 strain, a peak was detected at the same position as the standard sample of ricinoleic acid. The peak was not observed in a control strain that did not express CpFAH12.
The result of the content rate (%) with respect to the total fatty acid content of various fatty acids per 1 mL of the culture solution is shown in FIG. FIG. 18 (A) shows the results for the control strain, and FIG. 18 (B) shows the results for the CpFAH12 strain. In the control strain not expressing CpFAH12, the fatty acid composition was almost the same regardless of the temperature of the main culture. In contrast, the CpFAH12 strain did not differ substantially from the control strain when cultured at 37 ° C, whereas the oleic acid content decreased when cultured at 20 ° C and 30 ° C. Along with this, the content ratio of ricinoleic acid increased. In the case of 20 ° C., the content of ricinoleic acid increased as the number of days of culture increased. In the case of 30 ° C., the culture period was the second day, and the content of ricinoleic acid reached its peak.
［実施例８］
ｐＲＥＰ１、ｐＲＥＰ１−ＣｐＦＡＨ１２、ｐＲＥＰ１−ＲｃＦＡＨ１２、またはｐＲＥＰ１−ＬｆＦＡＨ１２を導入したＳ．ポンベの形質転換体を、それぞれチアミン存在下および非存在下で培養し、各培養温度における増殖能を調べた。
まず、各形質転換体をそれぞれ、チアミンが添加されていないＥＭＭ−Ｃ／Ｎ３−ＬＥＵ中、３７℃で一日間前培養した。次いで、該前培養液を同種の新しい培地で希釈し、さらに、１０倍希釈系列を調整し、各１０μＬずつを、１５μＭのチアミン含有ＥＭＭ−Ｃ／Ｎ３−ＬＥＵプレート（発現抑制条件）またはチアミン不添加のＥＭＭ−Ｃ／Ｎ３−ＬＥＵプレート（発現誘導条件）上にスポットした。該プレートを２０℃で１０日間、３０℃で５日間、または３７℃で５日間インキュベートした。インキュベート後の各プレート表面の写真図を図１９に示す。図中、白い部分がコロニーである。図中、「＋Ｔｈｉａｍｉｎｅ」がチアミン含有プレートであり、「Ｎｏ Ｔｈｉａｍｉｎｅ」がチアミン不含有プレートである。
チアミン不含有プレートでは、培養温度にかかわらず、ｐＲＥＰ１−ＣｐＦＡＨ１２を導入した形質転換体およびｐＲＥＰ１−ＬｆＦＡＨ１２を導入した形質転換体では、ｐＲＥＰ１を導入した形質転換体よりも増殖能が低い傾向が観察された。
[Example 8]
S. pREP1, pREP1-CpFAH12, pREP1-RcFAH12, or pREP1-LfFAH12 introduced S. Pombe transformants were cultured in the presence and absence of thiamine, and the growth ability at each culture temperature was examined.
First, each transformant was pre-cultured at 37 ° C. for one day in EMM-C / N3-LEU without addition of thiamine. Next, the preculture solution was diluted with a new medium of the same type, and a 10-fold dilution series was prepared, and 10 μL each was added to a 15 μM thiamine-containing EMM-C / N3-LEU plate (expression suppression condition) or thiamine-free solution. Spotted on added EMM-C / N3-LEU plate (expression induction conditions). The plates were incubated at 20 ° C. for 10 days, 30 ° C. for 5 days, or 37 ° C. for 5 days. A photograph of the surface of each plate after incubation is shown in FIG. In the figure, the white part is a colony. In the figure, “+ Thiamine” is a thiamine-containing plate, and “No Thiamine” is a thiamine-free plate.
In the thiamine-free plate, regardless of the culture temperature, the transformant introduced with pREP1-CpFAH12 and the transformant introduced with pREP1-LfFAH12 tended to have a lower growth ability than the transformant introduced with pREP1. It was.
［実施例９］
ＲｃＦＡＨ１２遺伝子を染色体外遺伝子として導入したＳ．ポンベの形質転換体（以下、ｐＲＥＰ１−ＲｃＦＡＨ１２導入株）およびＬｆＦＡＨ１２遺伝子を染色体外遺伝子として導入したＳ．ポンベの形質転換体（以下、ｐＲＥＰ１−ＬｆＦＡＨ１２導入株）の３０℃で培養した場合の脂肪酸組成を調べた。対照として、ｐＲＥＰ１を導入したＳ．ポンベの形質転換体（以下、ｐＲＥＰ１導入株）を３０℃で培養した場合の脂肪酸組成を調べた。
まず、各株をチアミンが添加されていないＥＭＭ−Ｃ／Ｎ３−ＬＥＵ中、３０℃で一日間前培養した。次いで、培養した細胞を、前培養と同種の新しい培地またはチアミン含有ＥＭＭ−Ｃ／Ｎ３−ＬＥＵで、ＯＤ６００が０．０５になるように希釈した後、３０℃で７日間、４０ｒｐｍで振とう培養した。参考例１と同様にして、培養終了後の培養液１ｍＬ当たりの各種脂肪酸の含有量を測定した。培養液１ｍＬ当たりの各種脂肪酸の総脂肪酸含有量に対する含有比率の結果を図２０に示す。図２０中、「ｐＲＥＰ１」、「ＲｃＦＡＨ１２」、および「ＬｆＦＡＨ１２」はそれぞれ、ｐＲＥＰ１導入株、ｐＲＥＰ１−ＲｃＦＡＨ１２導入株、およびｐＲＥＰ１−ＬｆＦＡＨ１２導入株をチアミンが添加されていないＥＭＭ−Ｃ／Ｎ３−ＬＥＵ中で本培養した結果を示し、「ｐＲＥＰ１＋Ｔｈｉ」、「ＲｃＦＡＨ１２＋Ｔｈｉ」、および「ＬｆＦＡＨ１２＋Ｔｈｉ」はそれぞれ、ｐＲＥＰ１導入株、ｐＲＥＰ１−ＲｃＦＡＨ１２導入株、およびｐＲＥＰ１−ＬｆＦＡＨ１２導入株をチアミン含有ＥＭＭ−Ｃ／Ｎ３−ＬＥＵ中で本培養した結果を示す。
[Example 9]
S. cerevisiae having introduced the RcFAH12 gene as an extrachromosomal gene. Pombe transformant (hereinafter referred to as pREP1-RcFAH12-introduced strain) and S. cerevisiae into which LfFAH12 gene was introduced as an extrachromosomal gene. The fatty acid composition when the pombe transformant (hereinafter referred to as pREP1-LfFAH12-introduced strain) was cultured at 30 ° C. was examined. As a control, S. pylori introduced with pREP1. The fatty acid composition when Pombe transformants (hereinafter referred to as pREP1-introduced strains) were cultured at 30 ° C. was examined.
First, each strain was pre-cultured at 30 ° C. for one day in EMM-C / N3-LEU without addition of thiamine. The cultured cells are then diluted with the same kind of new medium as in the pre-culture or thiamine-containing EMM-C / N3-LEU to an OD 600 of 0.05, and then shaken at 30 ° C. for 7 days at 40 rpm. Cultured. In the same manner as in Reference Example 1, the content of various fatty acids per 1 mL of the culture solution after completion of the culture was measured. The result of the content ratio with respect to the total fatty acid content of various fatty acids per 1 mL of culture solution is shown in FIG. In FIG. 20, “pREP1”, “RcFAH12”, and “LfFAH12” are respectively the pREP1-introduced strain, the pREP1-RcFAH12-introduced strain, and the pREP1-LfFAH12-introduced strain in EMM-C / N3-LEU to which thiamine is not added. The results of the main culture are shown in Fig. 1. "pREP1 + Thi", "RcFAH12 + Thi", and "LfFAH12 + Thi" represent pREP1-introduced strain, pREP1-RcFAH12-introduced strain, and pREP1-LfFAH12-introduced strain in thiamine-containing EMM-C / N3-LEU Shows the result of the main culture.
図２０に示すように、ｐＲＥＰ１−ＲｃＦＡＨ１２導入株およびｐＲＥＰ１−ＬｆＦＡＨ１２導入株では、チアミン含有培地ではｐＲＥＰ１導入株と同様、リシノール酸はほとんど含有されていなかったが、チアミンが添加されていない培地では、オレイン酸含有割合が低下し、リシノール酸の含有割合が上昇していた。すなわち、ＣｐＦＡＨ１２遺伝子を導入した形質転換体と同様、ＲｃＦＡＨ１２遺伝子またはＬｆＦＡＨ１２遺伝子を導入した形質転換体では、３０℃で培養した場合に、リシノール酸が産生されることがわかった。 As shown in FIG. 20, the pREP1-RcFAH12-introduced strain and the pREP1-LfFAH12-introduced strain contained almost no ricinoleic acid in the thiamine-containing medium as in the case of the pREP1-introduced strain, but in the medium to which no thiamine was added, The oleic acid content ratio decreased and the ricinoleic acid content ratio increased. That is, it was found that, similarly to the transformant introduced with the CpFAH12 gene, the transformant introduced with the RcFAH12 gene or the LfFAH12 gene produced ricinoleic acid when cultured at 30 ° C.
［参考例４］
ＣｐＦＡＨ１２遺伝子を染色体外遺伝子として導入したＳ．セレビシエの形質転換体の増殖能を調べた。
具体的には、該Ｓ．セレビシエにＹＥｐ３５２−ＣｐＦＡＨ１２を導入した形質転換体（ＣｐＦＡＨ１２株）と、Ｓ．セレビシエに空ベクターＹＥｐ３５２ＧＡＰを導入した形質転換体（コントロール株）を、それぞれＳＣ−ＵＲＡ中、３０℃で一晩前培養した。該前培養物を、前培養と同種の新しい培地で、ＯＤ６００が０．０５になるように希釈した後、２０℃または３０℃で攪拌培養した。６３０ｎｍの濁度を自動検出器（Ｂｉｏ−Ｐｌｏｔｔｅｒ、東洋測器製）でモニターすることにより、液体培地中の増殖曲線を描いた。それぞれ独立した試行を３回行った。図２１に増殖曲線を示す。図２１（Ａ）は３０℃で培養した結果であり、図２１（Ｂ）は２０℃で培養した結果である。図中、三角がＣｐＦＡＨ１２株の増殖曲線［ＹＥｐ３５２−ＣｐＦＡＨ１２−１、ＹＥｐ３５２−ＣｐＦＡＨ１２−２、ＹＥｐ３５２−ＣｐＦＡＨ１２−３］を、円がコントロール株の増殖曲線［ＹＥｐ３５２ＧＡＰ−１、ＹＥｐ３５２ＧＡＰ−２、ＹＥｐ３５２ＧＡＰ−３］を、それぞれ示す。いずれの培養温度においても、コントロール株よりもＣｐＦＡＨ１２株のほうが、明らかに増殖が抑制されていた。すなわち、ＣｐＦＡＨ１２株の増殖のダブリングタイムは、コントロール株よりも１．４倍長くなっていた。
ＣｐＦＡＨ１２遺伝子を染色体中に組み込んだＳ．セレビシエの形質転換体は、ＣｐＦＡＨ１２遺伝子を染色体中に組み込んだＳ．ポンベの形質転換体と同様、ＣｐＦＡＨ１２を発現させることによって増殖が抑制されていた。つまり、ＣｐＦＡＨ１２遺伝子をＳ．セレビシエに導入することによって、Ｓ．ポンベに導入した場合と同様、リシノール酸を産生できることが示唆された。
[Reference Example 4]
S. C. introduced the CpFAH12 gene as an extrachromosomal gene. The growth ability of the cerevisiae transformants was examined.
Specifically, the S.P. A transformant obtained by introducing YEp352-CpFAH12 into C. cerevisiae (CpFAH12 strain); Transformants obtained by introducing empty vector YEp352GAP into cerevisiae (control strains) were each pre-cultured overnight at 30 ° C. in SC-URA. The preculture was diluted with a new medium of the same type as the preculture so that the OD 600 was 0.05, and then stirred at 20 ° C. or 30 ° C. A growth curve in a liquid medium was drawn by monitoring the turbidity at 630 nm with an automatic detector (Bio-Plotter, manufactured by Toyo Sokki). Each independent trial was performed 3 times. FIG. 21 shows a growth curve. FIG. 21 (A) shows the result of culturing at 30 ° C., and FIG. 21 (B) shows the result of culturing at 20 ° C. In the figure, the triangle indicates the growth curve of the CpFAH12 strain [YEp352-CpFAH12-1, YEp352-CpFAH12-2, YEp352-CpFAH12-3], and the circle indicates the growth curve of the control strain [YEp352GAP-1, YEp352GAP-2, YEp352GAP-3. ] Are shown respectively. At any culture temperature, the growth of the CpFAH12 strain was clearly suppressed compared to the control strain. That is, the doubling time for the growth of the CpFAH12 strain was 1.4 times longer than that of the control strain.
S. cerevisiae having the CpFAH12 gene integrated into the chromosome. The S. cerevisiae transformant is a S. cerevisiae strain that has the CpFAH12 gene integrated into its chromosome. Like Pombe transformants, growth was suppressed by expressing CpFAH12. That is, the CpFAH12 gene was transformed into S. cerevisiae. By introducing it into S. cerevisiae, It was suggested that ricinoleic acid can be produced as in the case of introduction into pombe.
［実施例１０］
ＣｐＦＡＨ１２遺伝子を染色体外遺伝子として導入したＳ．セレビシエの形質転換体を培養し、リシノール酸の産生能を調べた。対照として、Ｓ．セレビシエにＹＥｐ３５２ＧＡＰを導入した形質転換体を用いた。
まず、各株をＮＳＤ−ＵＲＡ中、２０℃または３０℃で一日間前培養した。次いで、培養した細胞を、前培養と同種の新しい培地で、ＯＤ６００が０．０５になるように希釈した後、２０℃または３０℃で５日間、４０ｒｐｍで振とう培養した。参考例１と同様にして、培養終了後の培養液１ｍＬ当たりの各種脂肪酸の含有量を測定した。脂肪酸組成（培養液１ｍＬ当たりの各種脂肪酸の総脂肪酸含有量に対する含有割合）の結果を図２２に示す。図２２中、「ＣｐＦＡＨ１２」および「ｅｍｐｔｙ」はそれぞれ、ＣｐＦＡＨ１２を染色体外遺伝子として導入した形質転換体、およびＳ．セレビシエにＹＥｐ３５２ＧＡＰを染色体外遺伝子として導入した形質転換体を本培養した結果を示す。
図２２に示すように、ＣｐＦＡＨ１２遺伝子を導入したＳ．セレビシエを２０℃または３０℃で培養した場合に、リシノール酸が産生されることがわかった。
[Example 10]
S. C. introduced the CpFAH12 gene as an extrachromosomal gene. A cerevisiae transformant was cultured and examined for its ability to produce ricinoleic acid. As a control, S. A transformant in which YEp352GAP was introduced into S. cerevisiae was used.
First, each strain was pre-cultured in NSD-URA at 20 ° C. or 30 ° C. for one day. Next, the cultured cells were diluted with a new medium of the same type as the preculture so that the OD 600 was 0.05, and then cultured with shaking at 20 rpm or 30 ° C. for 5 days at 40 rpm. In the same manner as in Reference Example 1, the content of various fatty acids per 1 mL of the culture solution after completion of the culture was measured. The results of the fatty acid composition (content ratio of various fatty acids per 1 mL of culture solution to the total fatty acid content) are shown in FIG. In FIG. 22, “CpFAH12” and “empty” are respectively a transformant having CpFAH12 introduced as an extrachromosomal gene, and S. cerevisiae. The result of carrying out the main culture of the transformant which introduce | transduced YEp352GAP as an extrachromosomal gene in cerevisiae is shown.
As shown in FIG. 22, S. cerevisiae having the CpFAH12 gene introduced therein was introduced. When cerevisiae was cultured at 20 ° C. or 30 ° C., it was found that ricinoleic acid was produced.
本発明のリシノール酸産生酵母の培養方法およびリシノール酸の製造方法により、リシノール酸を、酵母の形質転換体を用いて効率よく生産できるため、特に、リシノール酸またはひまし油が原料として使用されている化成品、塗料・印刷インキ、化粧品、医薬品、潤滑油等の製造分野において好適に用いられる。 Since the ricinoleic acid-producing yeast culture method and ricinoleic acid production method of the present invention can efficiently produce ricinoleic acid using a yeast transformant, in particular, ricinoleic acid or castor oil is used as a raw material. It is suitably used in the production field of products, paints / printing inks, cosmetics, pharmaceuticals, lubricants and the like.
Claims (5)
前記高温培養工程後、前記形質転換体を３５℃未満で培養する低温培養工程と、
を有することを特徴とするリシノール酸産生酵母の培養方法。 A high-temperature culture step of culturing a yeast transformant into which the FAH12 gene has been introduced at 35 ° C. or more and less than 40 ° C. until the OD 600 of the culture solution is 0.5 to 5.5;
A low temperature culture step of culturing the transformant at less than 35 ° C. after the high temperature culture step;
A method for culturing ricinoleic acid-producing yeast, comprising:
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