JP2014212732A - Ricinoleic acid-producing yeast - Google Patents
Ricinoleic acid-producing yeast Download PDFInfo
- Publication number
- JP2014212732A JP2014212732A JP2013092915A JP2013092915A JP2014212732A JP 2014212732 A JP2014212732 A JP 2014212732A JP 2013092915 A JP2013092915 A JP 2013092915A JP 2013092915 A JP2013092915 A JP 2013092915A JP 2014212732 A JP2014212732 A JP 2014212732A
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- JP
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- Prior art keywords
- ricinoleic acid
- gene
- yeast
- cpfah12
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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Abstract
Description
本発明は、リシノール酸を高産生し得る酵母の形質転換体、および該酵母を培養してリシノール酸を産生する方法に関する。 The present invention relates to a yeast transformant capable of producing ricinoleic acid at a high level, and a method for producing ricinoleic acid by culturing the yeast.
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. Accordingly, the present inventors have focused on the fact that the fission yeast Schizosaccharomyces pombe (hereinafter, S. pombe) has a high oleic acid content of about 80% as a fatty acid composition. 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、リシノール酸をより高産生することが可能なリシノール酸産生酵母、該リシノール酸産生酵母の製造方法、および該リシノール酸産生酵母を用いた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 ricinoleic acid-producing yeast capable of producing ricinoleic acid at a higher level, a method for producing the ricinoleic acid-producing yeast, and a method for producing ricinoleic acid using the ricinoleic acid-producing yeast. It is in.
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に、さらに脂質のエステル結合の切断活性を有する加水分解酵素をコードする遺伝子を導入することにより、ＦＡＨ１２遺伝子の導入により引き起こされた増殖阻害が抑制し得ること、および、該形質転換体を培養すると、産生されたリシノール酸の少なくない量が培地中に分泌されることを見出し、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 have encoded a hydrolase having a cleavage activity of a lipid ester bond in a yeast transformant into which a FAH12 (Fatty acid hydroxylase) gene has been introduced. The growth inhibition caused by the introduction of the FAH12 gene can be suppressed, and when the transformant is cultured, a small amount of the produced ricinoleic acid is secreted into the medium. As a result, the present invention has been completed.
本発明に係るリシノール酸産生酵母、リシノール酸産生酵母の製造方法、およびリシノール酸の製造方法は前記［１］〜［９］である。
［１］酵母を宿主とし、ＦＡＨ１２遺伝子と、脂質のエステル結合の切断活性を有する加水分解酵素をコードする遺伝子が導入された形質転換体であることを特徴とする、リシノール酸産生酵母。
［２］前記加水分解酵素が、フォスフォリパーゼＡ２活性またはトリアシルグリセロールリパーゼ活性を有する前記［１］のリシノール酸産生酵母。
［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。
［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。
［５］前記加水分解酵素をコードする遺伝子が、シゾサッカロミセス・ポンベ（Schizosaccharomyces pombe）のｐｌｇ７遺伝子である前記［１］〜［４］のいずれかのリシノール酸産生酵母。
［６］酵母を宿主とし、遺伝子工学的方法により、ＦＡＨ１２遺伝子と、脂質のエステル結合の切断活性を有する加水分解酵素をコードする遺伝子とを、同時にまたは順次組み込むことを特徴とする、リシノール酸産生酵母の製造方法。
［７］ＦＡＨ１２遺伝子が導入された酵母の形質転換体に、前記加水分解酵素をコードする遺伝子を導入する前記［６］のリシノール酸産生酵母の製造方法。
［８］前記［１］〜［５］のいずれかのリシノール酸産生酵母を培養した後、培養された該リシノール酸産生酵母または培養上清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。
［９］前記リシノール酸産生酵母の培養を、１０〜３５℃で行う前記［８］のリシノール酸の製造方法。
The ricinoleic acid-producing yeast, the method for producing ricinoleic acid-producing yeast, and the method for producing ricinoleic acid according to the present invention are the above [1] to [9].
[1] A ricinoleic acid-producing yeast characterized in that the yeast is a transformant into which a FAH12 gene and a gene encoding a hydrolase having a lipid ester bond cleavage activity have been introduced.
[2] The ricinoleic acid-producing yeast according to [1], wherein the hydrolase has phospholipase A2 activity or triacylglycerol lipase activity.
[3] The ricinoleic acid-producing yeast according to [1] or [2], wherein the yeast is a yeast belonging to the genus Schizosaccharomyces.
[4] The ricinoleic acid-producing yeast of any one of [1] to [3], wherein the FAH12 gene is derived from Ricinus communis, Lesquerella fendleri, or Claviceps purpurea.
[5] The ricinoleic acid-producing yeast according to any one of [1] to [4], wherein the gene encoding the hydrolase is a plg7 gene of Schizosaccharomyces pombe.
[6] Production of ricinoleic acid, wherein yeast is used as a host, and the FAH12 gene and a gene encoding a hydrolase having a lipid ester bond cleavage activity are incorporated simultaneously or sequentially by genetic engineering methods Yeast production method.
[7] The method for producing a ricinoleic acid-producing yeast according to [6], wherein the gene encoding the hydrolase is introduced into a yeast transformant into which the FAH12 gene has been introduced.
[8] After culturing the ricinoleic acid-producing yeast according to any one of [1] to [5], ricinoleic acid is obtained from the cultured ricinoleic acid-producing yeast or the culture supernatant. Production method.
[9] The method for producing ricinoleic acid according to [8], wherein the ricinoleic acid-producing yeast is cultured at 10 to 35 ° C.
本発明に係るリシノール酸産生酵母は、ＦＡＨ１２の発現によりリシノール酸を産生し得るにもかかわらず、ＦＡＨ１２遺伝子のみが導入された形質転換体よりも増殖能が高い。さらに、該リシノール酸産生酵母により産生されたリシノール酸のうちの少なくない量が培地中に分泌される。このため、該リシノール酸産生酵母を培養する本発明に係るリシノール酸の製造方法により、より大量のリシノール酸を簡便に製造できる。 Although the ricinoleic acid-producing yeast according to the present invention can produce ricinoleic acid by expression of FAH12, it has a higher growth ability than a transformant into which only the FAH12 gene has been introduced. Furthermore, not less than the amount of ricinoleic acid produced by the ricinoleic acid-producing yeast is secreted into the medium. Therefore, a larger amount of ricinoleic acid can be easily produced by the method for producing ricinoleic acid according to the present invention in which the ricinoleic acid-producing yeast is cultured.
［リシノール酸産生酵母］
本発明に係るリシノール酸産生酵母は、酵母を宿主とし、ＦＡＨ１２遺伝子と、脂質のエステル結合の切断活性を有する加水分解酵素をコードする遺伝子が導入された形質転換体である。ＦＡＨ１２遺伝子の導入により、酵母にリシノール産生能を付与できる。また、ＦＡＨ１２の発現により増殖阻害が引き起こされるが、ＦＡＨ１２遺伝子と共に特定の活性を有する加水分解酵素遺伝子を導入することにより、ＦＡＨ１２による増殖阻害が緩和され、ＦＡＨ１２遺伝子を単独で導入した形質転換体よりも、増殖能を高められる。
[Ricinolic acid-producing yeast]
The ricinoleic acid-producing yeast according to the present invention is a transformant in which a yeast is used as a host and the FAH12 gene and a gene encoding a hydrolase having a lipid ester bond cleavage activity are introduced. By introducing the FAH12 gene, the ability to produce ricinol can be imparted to yeast. Moreover, growth inhibition is caused by the expression of FAH12. By introducing a hydrolase gene having a specific activity together with the FAH12 gene, the growth inhibition by FAH12 is alleviated, and from the transformant in which the FAH12 gene is introduced alone. Can also increase the proliferation ability.
（ＦＡＨ１２遺伝子）
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、たとえば、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。
(FAH12 gene)
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 of castor bean (RcFAH12) (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.
（加水分解酵素およびこれをコードする遺伝子）
酵母に導入する加水分解酵素をコードする遺伝子は、脂質のエステル結合の切断活性を有する加水分解酵素をコードする遺伝子である。より詳細には、該加水分解酵素は、脂質のエステル結合を加水分解し、アシル基を遊離させる酵素活性を有する。該加水分解酵素としては、たとえば、グリセロリン脂質やトリアシルグリセリドを基質とするリパーゼ等が挙げられる。本発明に係るリシノール酸産生酵母を製造するために酵母に遺伝子を導入する加水分解酵素としては、フォスフォリパーゼＡ２（ＰＬＡ２）活性、フォスフォリパーゼＡ１（ＰＬＡ１）活性、またはトリアシルグリセロールリパーゼ（ＴＧリパーゼ）活性を少なくとも有する加水分解酵素が好ましく、ＰＬＡ２活性またはＴＧリパーゼ活性を有する加水分解酵素がより好ましい。なお、以降、該加水分解酵素を「加水分解酵素Ａ」、該加水分解酵素をコードする遺伝子を「加水分解酵素Ａ遺伝子」ということがある。
(Hydrolyzing enzyme and gene encoding it)
A gene encoding a hydrolase to be introduced into yeast is a gene encoding a hydrolase having a lipid ester bond cleavage activity. More specifically, the hydrolase has an enzyme activity that hydrolyzes an ester bond of a lipid and releases an acyl group. Examples of the hydrolase include lipase using glycerophospholipid or triacylglyceride as a substrate. Examples of the hydrolase that introduces a gene into yeast for producing the ricinoleic acid-producing yeast according to the present invention include phospholipase A2 (PLA2) activity, phospholipase A1 (PLA1) activity, or triacylglycerol lipase (TG). Lipase) is preferably a hydrolase having at least activity, and more preferably a hydrolase having PLA2 activity or TG lipase activity. Hereinafter, the hydrolase may be referred to as “hydrolase A”, and the gene encoding the hydrolase may be referred to as “hydrolase A gene”.
酵母に導入する加水分解酵素Ａ遺伝子の由来は特に限定されず、微生物由来であってもよく、植物由来であってもよく、動物由来であってもよい。酵母内における発現のしやすさの点から、微生物由来の遺伝子が好ましく、酵母由来の遺伝子がより好ましい。本発明に係るリシノール酸産生酵母としては、酵母由来であって、ＰＬＡ２活性、ＰＬＡ１活性、またはＴＧリパーゼ活性を少なくとも有する加水分解酵素をコードする遺伝子が導入されていることが好ましく、Ｓ．ポンベ由来のＰＬＡ２活性、ＰＬＡ１活性、またはＴＧリパーゼ活性を少なくとも有する加水分解酵素をコードする遺伝子が導入されていることがより好ましい。Ｓ．ポンベ由来の該遺伝子としては、たとえば、ｐｌｇ７遺伝子（ＳＰＢＣ１０６．１１ｃ）、ｐｔｌ２遺伝子（ＳＰＡＣ１７８６．０１ｃ）、およびＳＰＡＣ４Ａ８．１０遺伝子が挙げられる。ｐｌｇ７はＰＬＡ２活性を有する酵素であり、ｐｔｌ２はＴＧリパーゼ活性を有する酵素である。また、ＳＰＡＣ４Ａ８．１０遺伝子がコードする蛋白質ＦＹＧ１３０＿ｆ０は、そのアミノ酸配列からリパーゼであると推定されていた。 The origin of the hydrolase A gene to be introduced into yeast is not particularly limited, and may be derived from microorganisms, from plants, or from animals. From the viewpoint of easy expression in yeast, a microorganism-derived gene is preferable, and a yeast-derived gene is more preferable. The ricinoleic acid-producing yeast according to the present invention is preferably derived from a yeast and introduced with a gene encoding a hydrolase having at least PLA2 activity, PLA1 activity, or TG lipase activity. More preferably, a gene encoding a hydrolase having at least Pombe-derived PLA2 activity, PLA1 activity, or TG lipase activity has been introduced. S. Examples of the gene derived from Pombe include the plg7 gene (SPBC106.11c), the ptl2 gene (SPAC178.01c), and the SPAC4A8.10 gene. plg7 is an enzyme having PLA2 activity, and ptl2 is an enzyme having TG lipase activity. The protein FYG130_f0 encoded by the SPAC4A8.10 gene was estimated to be a lipase from its amino acid sequence.
なお、Ｓ．ポンベの染色体の全塩基配列は、サンガー研究所のデータベース「ＧｅｎｅＤＢ」に「Schizosaccharomyces pombe Gene DB（http://www.genedb.org/genedb/pombe/）」として、収録され、公開されている。本明細書記載のＳ．ポンベの遺伝子の配列データは前記データベースから遺伝子名または系統名で検索して、入手できる。 S. The entire base sequence of the pombe chromosome is recorded and disclosed as “Schizosaccharomyces pombe Gene DB (http://www.genedb.org/genedb/pombe/)” in the database “GeneDB” of the Sanger Institute. As described in S.A. The sequence data of the pombe gene can be obtained by searching from the database by gene name or strain name.
（酵母）
ＦＡＨ１２遺伝子等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。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 or the like 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 , Kluyveromyces genus yeast, Pichia genus yeast, Candida genus yeast. Since it has a high oleic acid content, it is preferably a yeast belonging to the genus Schizosaccharomyces. 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, WO2002 / 101038 and WO2007 / 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、ベクターに該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。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 the 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 the two.
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）
本発明に係るリシノール酸産生酵母は、ＦＡＨ１２遺伝子の発現カセットと加水分解酵素Ａ遺伝子の発現カセットとを、酵母に導入することにより作製できる。ＦＡＨ１２遺伝子の発現カセットは、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。加水分解酵素Ａ遺伝子の発現カセットは、加水分解酵素Ａ遺伝子をコードする領域を含む構造遺伝子配列（加水分解酵素Ａ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。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。加水分解酵素Ａ遺伝子として、宿主とは別種の生物由来の遺伝子を用いる場合にも同様である。
(Expression cassette)
The ricinoleic acid-producing yeast according to the present invention can be prepared by introducing an expression cassette of FAH12 gene and an expression cassette of hydrolase A gene into yeast. The expression cassette of the FAH12 gene includes a structural gene sequence (FAH12 gene sequence) including a region encoding the FAH12 gene, and a promoter sequence and a terminator sequence for expressing the gene. The hydrolase A gene expression cassette includes a structural gene sequence (hydrolase A gene sequence) including a region encoding the hydrolase A gene, and a promoter sequence and a terminator sequence for expressing the gene. 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. The same applies to the case of using a gene derived from a different organism from the host as the hydrolase A gene.
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遺伝子配列または加水分解酵素Ａ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 or hydrolase A 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等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 and the like. As a promoter that functions in yeast, a promoter inherent in yeast (preferably one having 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 promoter (see International Publication No. 99/23223), heat shock protein gene promoter (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）
本発明に係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 according to 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遺伝子配列等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遺伝子配列等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。また、ＦＡＨ１２遺伝子の発現カセットと加水分解酵素Ａ遺伝子の発現カセットは、それぞれ別個のベクターに組み込んでもよく、両者を１のベクターに組み込んでもよい。 The vector containing the expression cassette used for the production of ricinoleic acid-producing yeast is, for example, inserting a FAH12 gene sequence or the like 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. The FAH12 gene expression cassette and the hydrolase A gene expression cassette may be incorporated into separate vectors, or both may be incorporated into one vector.
（リシノール酸産生酵母の製造方法）
本発明に係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 ricinoleic acid-producing yeast)
The ricinoleic acid-producing yeast according to the present invention has the expression cassette in the chromosome or as an extrachromosomal gene. Having an expression cassette in the chromosome means that the expression cassette is incorporated at one or more positions in the chromosome of the host cell, and having as an extrachromosomal gene means having a plasmid containing the expression cassette in the cell. That is. 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.
本発明に係るリシノール酸産生酵母は、宿主となる酵母に、ＦＡＨ１２遺伝子の発現カセットと加水分解酵素Ａ遺伝子の発現カセットを同時に導入して、両遺伝子を同時に組み込むことにより製造してもよく、両発現カセットを順次導入して製造してもよい。たとえば、まず、酵母にＦＡＨ１２遺伝子の発現カセットを導入し、ＦＡＨ１２遺伝子が組み込まれた形質転換体を得、これに加水分解酵素Ａ遺伝子の発現カセットを導入することによって、加水分解酵素Ａ遺伝子がさらに組み込まれた形質転換体を製造できる。 The ricinoleic acid-producing yeast according to the present invention may be produced by simultaneously introducing an expression cassette for the FAH12 gene and an expression cassette for the hydrolase A gene into a yeast serving as a host, and incorporating both genes simultaneously. You may manufacture by introducing an expression cassette one by one. For example, first, an expression cassette of the FAH12 gene is introduced into yeast to obtain a transformant in which the FAH12 gene is incorporated, and the hydrolase A gene expression cassette is further introduced into the transformant. An integrated transformant can be produced.
（リシノール酸産生酵母の培養方法）
本発明に係るリシノール酸産生酵母は、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、宿主の培養を効率よ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。
(Cultivation method of ricinoleic acid-producing yeast)
The ricinoleic acid-producing yeast according to the present invention can be cultured in the same manner as a general yeast culture method. For example, a known culture medium used for culturing yeast as a host can be used for the culture solution, and it contains a carbon source, 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、撹拌培養により行える。
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 or stirring culture.
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］
本発明に係るリシノール酸産生酵母では、産生されたリシノール酸のうちの少なくない量が培地中に分泌される。このため、該リシノール酸産生酵母の培養上清（培養後の液体培地）から、リシノール酸を容易に回収できる。該リシノール酸産生酵母では、菌体外へ分泌される脂肪酸の大部分がリシノール酸であるため、培養上清から回収することにより、菌体から回収するよりも純度の高いリシノール酸を容易に得られる。また、産生された全てのリシノール酸が菌体外に分泌されるわけではなく、培養後のリシノール酸産生酵母からもリシノール酸は回収できる。
[Method for producing ricinoleic acid]
In the ricinoleic acid-producing yeast according to the present invention, a small amount of the produced ricinoleic acid is secreted into the medium. For this reason, ricinoleic acid can be easily recovered from the culture supernatant of the ricinoleic acid-producing yeast (liquid medium after culture). In the ricinoleic acid-producing yeast, most of the fatty acids secreted outside the cells are ricinoleic acid, and therefore, by collecting from the culture supernatant, ricinoleic acid having a higher purity than that collected from the cells can be easily obtained. It is done. Further, not all produced ricinoleic acid is secreted outside the cells, and ricinoleic acid can be recovered from the ricinoleic acid-producing yeast after culture.
該リシノール酸産生酵母は、ＦＡＨ１２遺伝子導入による増殖阻害が抑制されており、１０〜３５℃の範囲内でリシノール酸を分泌しつつ良好に増殖できる。特に、増殖とリシノール酸の産生効率とのバランスがよいことから、リシノール酸回収のための培養温度は、１５〜３０℃が好ましく、１８〜２８℃がより好ましく、２０〜２５℃がさらに好ましい。 The ricinoleic acid-producing yeast has suppressed growth inhibition due to the introduction of the FAH12 gene, and can grow well while secreting ricinoleic acid within the range of 10 to 35 ° C. In particular, the culture temperature for recovering ricinoleic acid is preferably 15 to 30 ° C, more preferably 18 to 28 ° C, and even more preferably 20 to 25 ° C because the balance between growth and ricinoleic acid production efficiency is good.
培養上清や菌体からのリシノール酸の回収は、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。たとえば、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除去し、得られた培養上清にクロロホルム／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、該遠心分離により回収された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。
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。
Recovery of ricinoleic acid from the culture supernatant and cells 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 culturing ricinoleic acid-producing yeast, the resulting culture solution is centrifuged to remove the cells, and the resulting culture supernatant is extracted with chloroform / methanol (1: 2) and then purified. By doing so, ricinoleic acid can be purified. In addition, ricinoleic acid can be purified by adding and extracting chloroform / methanol (1: 2) while disrupting the cells recovered by centrifugation using glass beads or the like in methanol. .
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).
（脂肪酸含有量の測定）
以下の実施例等において、リシノール酸をはじめとする脂肪酸含有量は、ガスクロマトグラフ分析によるKainouらの方法（Kainou et al., Yeast, 2006, 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の測定試料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。
なお、菌体中のリシノール酸量を測定する場合には、遠心分離により回収された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）を加えて抽出したクロロホルム層を測定試料とする。また、培養上清中のリシノール酸量を測定する場合には、遠心分離により回収された培養上清にクロロホルム／メタノール（１：２）を加えて抽出したクロロホルム層を測定試料とする。
(Measurement of fatty acid content)
In the following examples and the like, the content of fatty acids including ricinoleic acid was determined according to the method of Kainou et al. (Kainou et al., Yeast, 2006, vol.23, pp.605-612) by gas chromatographic analysis. It was. 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). The measurement was performed by administering 0.2 μL of a measurement sample 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).
When measuring the amount of ricinoleic acid in the bacterial cells, extract the cells collected by centrifugation by adding chloroform / methanol (1: 2) while crushing the bacterial cells in methanol using glass beads. The obtained chloroform layer is used as a measurement sample. When measuring the amount of ricinoleic acid in the culture supernatant, a chloroform layer obtained by adding chloroform / methanol (1: 2) to the culture supernatant recovered by centrifugation is used as a measurement sample.
［参考例１］
まず、ＣｐＦＡＨ１２株（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）を製造した。
[Reference Example 1]
First, the CpFAH12 strain (S. pombe transformant in which the CpFAH12 gene was incorporated into the chromosome) was produced.
（酵母）
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。
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., “A laboratory course manual.”, 1993, 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。
(yeast)
S. Pombe is leucine and uracil auxotroph ARC010 (genotype: h -, leu1-32, ura4- D18) (. WO 2007/015470 pamphlet reference) was used.
S. Pombe was cultured in EMMS or EMMS dropout medium depending on the selection pressure required to maintain the plasmid (Alfa et al., “A laboratory course manual.”, 1993, 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.
（ＣｐＦＡＨ１２遺伝子導入のためのプラスミド構築）
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。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 for CpFAH12 gene introduction)
A yeast transformation plasmid containing the coding region of 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). Moreover, 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, Plant Physiology, 2008, 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。
図１に、ｐＳＬ１０−ＣｐＦＡＨ１２のベクターの構築手順を模式的に示した。
The DNA sequence of CpFAH12 gene (Meesapyodsuk and Qiu, Plant Physiology, 2008, 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.
FIG. 1 schematically shows a procedure for constructing a vector of pSL10-CpFAH12.
（ＣｐＦＡＨ１２株の作製）
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。
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（ＣｐＦＡＨ１２遺伝子が染色体中に組み込まれた形質転換体）を選抜し、これをＣｐＦＡＨ１２株とした。
(Preparation of CpFAH12 strain)
Yeast cells were transformed with the Frozen-EZ Yeast Transformation II kit (Zymo Research, CA, USA) according to the protocol recommended by the manufacturer.
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 (transformant in which the CpFAH12 gene was integrated into the chromosome) was selected, and this was designated as the CpFAH12 strain.
（ＣｐＦＡＨ１２株の増殖能）
ＣｐＦＡＨ１２株の各培養温度における増殖能を調べた。
具体的には、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、それぞれチアミン不含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）またはチアミン不含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２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、チアミン不含有プレート（図中、「Ｎｏ Ｔｈｉａｍｉｎｅ」）では、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、Ｓ．ポンベにＣｐＦＡＨ１２を発現させることにより、３５℃以下では増殖能が低下することが確認された。
(Proliferation ability of CpFAH12 strain)
The growth ability of CpFAH12 strain at each culture temperature was examined.
Specifically, CpFAH12 strain and Transformants in which pSL10 and pREP2 were introduced into the pombe (control strain) were respectively stored in thiamine-free EMM-C / N3-URA, LEU at 37 ° C. overnight until the OD 600 of the culture reached about 4. Cultured. 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 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 the thiamine-free plate (“No Thiamine” in the figure), the CpFAH12 strain was clearly less proliferative than the control strain except when cultured at 37 ° C. for 4 days. That is, S.M. By expressing CpFAH12 in pombe, it was confirmed that the growth ability was reduced at 35 ° C. or lower.
［実施例１］
ＦＡＨ１２遺伝子が導入された形質転換体における増殖阻害を解除し得る遺伝子を探索するために、Ｓ．ポンベのｃＤＮＡライブラリーを導入し、リシノール酸産生能を保持しつつ増殖阻害が抑制される遺伝子を調べた。なお、本実施例ではＳ．ポンベのｃＤＮＡライブラリーを用いたが、その他の酵母のｃＤＮＡライブラリーや酵母以外の生物種のｃＤＮＡライブラリーを用いてもよい。
[Example 1]
In order to search for a gene that can release growth inhibition in a transformant into which the FAH12 gene has been introduced, A pombe cDNA library was introduced, and genes whose growth inhibition was suppressed while maintaining the ability to produce ricinoleic acid were examined. In this embodiment, S.I. Although the pombe cDNA library is used, other yeast cDNA libraries or cDNA libraries of biological species other than yeast may be used.
（ｃＤＮＡライブラリーの導入）
Ｓ．ポンベのｃＤＮＡライブラリーは、ｃＤＮＡライブラリーの構築のための標準的な技術である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。Ｓ．ポンベのｃＤＮＡは、図３に示すプラスミドｐＲＥＰ４２のｎｍｔ４１プロモーターの下流のクローニングサイトに挿入した。
具体的には、まず、Ｓ．ポンベから定法によりｍＲＮＡを調製し、ｏｌｉｇｏｄＴにNotI部位を付加したプライマーを用いてｃＤＮＡを作成し、得られた２本鎖ＤＮＡの５’末端にSalIアダプターを付加した。これにより、一方の端にNotI部位を有し、他端にSalI部位を有するｃＤＮＡを得た。該ｃＤＮＡを制限酵素SalIとNotIで二重消化して得たフラグメントを、ｐＲＥＰ４２のXhoIとNotI 部分に挿入し、ｃＤＮＡライブラリーを作製した。
(Introduction of cDNA library)
S. The Pombe cDNA library is a standard technique for construction of cDNA libraries, Sambrook and Russell's method (“Molecular cloning: A laboratory manual. 3rd ed.”, 2001, Cold Spring Harbor Laboratory Press, Cold Spring). Harbor, NY). S. The pombe cDNA was inserted into the cloning site downstream of the nmt41 promoter of the plasmid pREP42 shown in FIG.
Specifically, first, S.I. MRNA was prepared from pombe by a conventional method, cDNA was prepared using a primer in which NotI site was added to oligoT, and a SalI adapter was added to the 5 ′ end of the resulting double-stranded DNA. As a result, cDNA having a NotI site at one end and a SalI site at the other end was obtained. A fragment obtained by double digesting the cDNA with restriction enzymes SalI and NotI was inserted into the XhoI and NotI portions of pREP42 to prepare a cDNA library.
（ＣｐＦＡＨ１２株へのＳ．ポンベのｃＤＮＡライブラリーの導入）
ＣｐＦＡＨ１２株のコンピテントセルに、該ｃＤＮＡライブラリーの各クローンをそれぞれ形質転換し、得られた形質転換体群を、チアミンを含まないＥＭＭＳ−ＬｅｕＵｒａプレート（アガー平板培地）上に播き、３０℃で培養した。該培養条件下では、形質転換体内でＣｐＦＡＨ１２が発現するため、ＣｐＦＡＨ１２株の増殖は阻害されるが、約２０万個の形質転換体が培養可能であった。該形質転換体の中から、増殖能が回復しており、普通サイズのコロニーを形成した形質転換体を６０株得た。この６０株をそれぞれ培養し、前記のガスクロマトグラフ分析によりリシノール酸の産生量を測定したところ、２株のみがｃＤＮＡ導入前のＣｐＦＡＨ１２株と同程度のリシノール酸を生産し、他の株ではリシノール酸の生産は見られなかった。
該２株からプラスミドを回収し、挿入されているｃＤＮＡの塩基配列を解読したところ、２株共にｐｌｇ７遺伝子配列と相同であることが確認された。
(Introduction of S. pombe cDNA library into CpFAH12 strain)
Each clone of the cDNA library was transformed into a competent cell of CpFAH12 strain, and the obtained transformant group was plated on an EMMS-LeuUra plate (Agar plate medium) not containing thiamine at 30 ° C. Cultured. Under the culture conditions, CpFAH12 was expressed in the transformant, so that growth of the CpFAH12 strain was inhibited, but about 200,000 transformants could be cultured. From the transformants, 60 transformants that had recovered their growth ability and formed normal-sized colonies were obtained. Each of these 60 strains was cultured, and the amount of ricinoleic acid produced was measured by the gas chromatographic analysis. As a result, only two strains produced ricinoleic acid of the same level as the CpFAH12 strain before introduction of cDNA, and the other strains produced ricinoleic acid. Production was not seen.
When plasmids were collected from the two strains and the nucleotide sequence of the inserted cDNA was decoded, both strains were confirmed to be homologous to the plg7 gene sequence.
（ＣｐＦＡＨ１２株にｐｌｇ７遺伝子を導入した形質転換体の作製）
ＣｐＦＡＨ１２株における増殖能の回復が、ｐｌｇ７遺伝子によることを再確認するため、該２株から回収されたプラスミド（ｐＲＥＰ４２−ｐｌｇ７）を再度ＣｐＦＡＨ１２株に形質転換して、ｐｌｇ７遺伝子を導入したＣｐＦＡＨ１２株の形質転換体（ＣｐＦＡＨ１２／ｐｌｇ７株）を得た。
(Preparation of a transformant in which the plg7 gene is introduced into CpFAH12 strain)
In order to reconfirm that the recovery of the growth ability in the CpFAH12 strain is due to the plg7 gene, the plasmid (pREP42-plg7) recovered from the two strains was transformed again into the CpFAH12 strain, and the CpFAH12 strain into which the plg7 gene was introduced was transformed. A transformant (CpFAH12 / plg7 strain) was obtained.
（ＣｐＦＡＨ１２／ｐｌｇ７株の増殖能）
ＣｐＦＡＨ１２／ｐｌｇ７株を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。ＣｐＦＡＨ１２株に空ベクターｐＡＬ−ＫＳ（Tanaka et al., Mol Cell Biol, 2000, vol.20, pp.3459-3469）を導入した形質転換株（ＣｐＦＡＨ１２／ｖｅｃｔ株）を、比較対照とした。
まず、チアミン不含ＥＭＭ−Ｃ／Ｎ３中、３７℃で一晩前培養し、該前培養液の原液および１０倍希釈系列を、チアミン不含ＥＭＭ−Ｃ／Ｎ３のプレート（アガー平板培地）またはチアミン含有ＥＭＭ−Ｃ／Ｎ３のプレートに塗布し、３０℃で４日間培養した。
培養後の各プレート表面の写真図を図４に示す。図中、「＋Ｖｅｃｔｏｒ」はＣｐＦＡＨ１２／ｖｅｃｔ株の結果であり、「＋ｐｌｇ７」はＣｐＦＡＨ１２／ｐｌｇ７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、ＣｐＦＡＨ１２／ｖｅｃｔ株とＣｐＦＡＨ１２／ｐｌｇ７株は共に、前培養液の１００００倍希釈液をスポットした場合でもコロニーが形成された。一方で、チアミン不含有プレート（図中、「Ｎｏ Ｔｈｉａｍｉｎｅ」）では、ＣｐＦＡＨ１２／ｖｅｃｔ株では前培養液の原液と１０倍希釈液をスポットした場合にのみコロニーが形成されたのに対して、ＣｐＦＡＨ１２／ｐｌｇ７株では前培養液の１０００倍希釈液をスポットした場合でもコロニーが形成された。すなわち、ＣｐＦＡＨ１２／ｐｌｇ７株では、ＣｐＦＡＨ１２発現による増殖阻害が抑制されていた。
(Proliferation ability of CpFAH12 / plg7 strain)
CpFAH12 / plg7 strain was cultured in the presence and absence of thiamine, and the proliferation ability was examined. A transformant (CpFAH12 / vector strain) in which an empty vector pAL-KS (Tanaka et al., Mol Cell Biol, 2000, vol. 20, pp. 3459-3469) was introduced into the CpFAH12 strain was used as a comparative control.
First, precultured overnight at 37 ° C. in thiamine-free EMM-C / N3, and a stock solution of the preculture and a 10-fold dilution series were used as thiamine-free EMM-C / N3 plates (Agar plate medium) or It was applied to a plate of thiamine-containing EMM-C / N3 and cultured at 30 ° C. for 4 days.
A photograph of the surface of each plate after culture is shown in FIG. In the figure, “+ Vector” is the result of the CpFAH12 / vector strain, and “+ plg7” is the result of the CpFAH12 / plg7 strain. In the figure, the white part is a colony. On the thiamine-containing plate ("+ Thiamine" in the figure), both the CpFAH12 / vect strain and the CpFAH12 / plg7 strain formed colonies even when a 10,000-fold dilution of the preculture was spotted. On the other hand, in the thiamine-free plate ("No Thiamine" in the figure), the CpFAH12 / vect strain formed colonies only when spotting the stock solution of the preculture and the 10-fold diluted solution, whereas CpFAH12 In the / plg7 strain, colonies were formed even when a 1000-fold dilution of the preculture was spotted. That is, in the CpFAH12 / plg7 strain, growth inhibition due to CpFAH12 expression was suppressed.
また、ＣｐＦＡＨ１２／ｐｌｇ７株のチアミン非存在下における液体培地中の増殖能を調べた。ＣｐＦＡＨ１２／ｖｅｃｔ株と、ＣｐＦＡＨ１２遺伝子導入前のＡＲＣ０１０株にｐＲＥＰ２（空ベクター）を導入した形質転換株（Ｎｏ＿ＦＡＨ１２／ｖｅｃｔ株）、およびＡＲＣ０１０株に（ｐＲＥＰ４２−ｐｌｇ７）を導入した形質転換株（Ｎｏ＿ＦＡＨ１２／ｐｌｇ７株）を、比較対照とした。
具体的には、チアミンを含むＥＭＭ−Ｃ／Ｎ３中、３０℃で一晩前培養した後、チアミンを枯渇させた前培養液を、チアミン不含ＥＭＭ−Ｃ／Ｎ３の新しい培地で、ＯＤ６００が０．０５になるように希釈した後、３０℃で７２時間、撹拌培養した。６３０ｎｍの濁度（ＯＤ６３０）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５に増殖曲線を示す。図中、「Ｎｏ ＦＡＨ１２＋Ｖｅｃｔ」がＮｏ＿ＦＡＨ１２／ｖｅｃｔ株の結果を、「Ｎｏ ＦＡＨ１２＋ｐｌｇ７」がＮｏ＿ＦＡＨ１２／ｐｌｇ７株の結果を、「ＣｐＦＡＨ１２＋Ｖｅｃｔ」がＣｐＦＡＨ１２／ｖｅｃｔ株の結果を、「ＣｐＦＡＨ１２＋ｐｌｇ７」がＣｐＦＡＨ１２／ｐｌｇ７株の結果を、それぞれ示す。図中、ＯＤ６３０が培養時間経過により頭打ちになっているのは、検出限界値を超えたためである。酵母にｐｌｇ７遺伝子を導入しただけでは増殖能には影響しないが、ＣｐＦＡＨ１２株にｐｌｇ７遺伝子を導入することにより増殖能が顕著に改善された。つまり、ＣｐＦＡＨ１２による増殖抑制を解除するためには、ＰＬＡ２活性が重要であることが示された。
また、ＣｐＦＡＨ１２／ｐｌｇ７株を培養した培養液のみ白濁していた。該培養液を顕微鏡により検鏡したところ、油滴様のものが確認された。
In addition, the growth ability of the CpFAH12 / plg7 strain in a liquid medium in the absence of thiamine was examined. CpFAH12 / vect strain, a transformant in which pREP2 (empty vector) was introduced into the ARC010 strain before introduction of the CpFAH12 gene (No_FAH12 / vector strain), and a transformant in which (pREP42-plg7) was introduced into the ARC010 strain (No_FAH12 / plg7 strain) was used as a comparative control.
Specifically, after pre-culturing overnight at 30 ° C. in EMM-C / N3 containing thiamine, thiamin-depleted preculture was added to a new medium of thiamine-free EMM-C / N3 at an OD 600 Was diluted to 0.05 and then stirred and cultured at 30 ° C. for 72 hours. A growth curve in a liquid medium was drawn by monitoring the turbidity at 630 nm (OD 630 ) with an automatic detector (Bio-Plotter, manufactured by Toyo Sokki). FIG. 5 shows the growth curve. In the figure, “No FAH12 + Vect” is the result of the No_FAH12 / vect strain, “No FAH12 + plg7” is the result of the No_FAH12 / plg7 strain, “CpFAH12 + Vect” is the result of the CpFAH12 / vect strain, and “CpFAH12 + plg7” is the CpFAH12 / pg7 / pg7 strain. The results are shown respectively. In the figure, the reason why the OD 630 peaked with the lapse of the culture time is because the detection limit value was exceeded. The introduction of the plg7 gene into yeast does not affect the growth ability, but the introduction of the plg7 gene into the CpFAH12 strain significantly improved the growth ability. That is, it was shown that PLA2 activity is important in order to release the growth suppression by CpFAH12.
Moreover, only the culture solution which cultured CpFAH12 / plg7 strain was cloudy. When the culture solution was examined with a microscope, an oil droplet-like product was confirmed.
［実施例２］
ＣｐＦＡＨ１２／ｐｌｇ７株のリシノール産生能を調べた。
ＣｐＦＡＨ１２／ｐｌｇ７株、ＣｐＦＡＨ１２／ｖｅｃｔ株、およびＮｏ＿ＦＡＨ１２／ｖｅｃｔ株を、チアミン不含ＥＭＭ−Ｃ／Ｎ３中、３７℃で一晩前培養した前培養液を、グルコース濃度２質量％または１０質量％のチアミン不含ＥＭＭ−Ｃ／Ｎ３でＯＤ６００が２．０になるように希釈した後、２０℃で５日間、撹拌培養した。ＯＤ６００を手動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。また、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、培養液のグルコース濃度と、酵母を除去した培養上清の濁度を、Ｋｌｅｔｔ−Ｓｕｍｍｅｒｓｏｎフォトメトリックカロリメーターを使用してＫｌｅｔｔ法により測定した。測定結果を図６に示す。ｔＣｐＦＡＨ１２／ｖｅｃｔ株を培養した場合には、培養時間が経過しても培養液のＯＤ６００はほとんど上昇せず、グルコース濃度の低下も少なく、培養上清の濁度は、Ｎｏ＿ＦＡＨ１２／ｖｅｃｔ株よりは高かったものの、視認できるほどの白濁はなかった〔図６（Ｂ）および（Ｅ）〕。ＣｐＦＡＨ１２／ｐｌｇ７株を培養した場合には、Ｎｏ＿ＦＡＨ１２／ｖｅｃｔ株よりはやや緩やかであるものの、培養時間の経過と共に培養液のＯＤ６００は上昇し、グルコース濃度は低下し、さらに、Ｎｏ＿ＦＡＨ１２／ｖｅｃｔ株とは異なり、培養上清の濁度は経時的に顕著な上昇が確認され、これは油滴様構造の量に依存することも確認された〔図６（Ａ）および（Ｃ）〕。この傾向は、グルコース濃度２質量％の培地よりも、グルコース濃度１０質量％の培地のほうが顕著であった。
[Example 2]
The ricinol-producing ability of CpFAH12 / plg7 strain was examined.
CpFAH12 / plg7 strain, CpFAH12 / vector strain, and No_FAH12 / vector strain were precultured overnight at 37 ° C. in thiamine-free EMM-C / N3 at a glucose concentration of 2% by mass or 10% by mass. After dilution with thiamine-free EMM-C / N3 so that the OD 600 was 2.0, the mixture was cultured with stirring at 20 ° C. for 5 days. Growth curves in liquid media were drawn by manually monitoring OD 600 . In addition, every day after the start of main culture, a part of the culture solution is sampled, and the glucose concentration of the culture solution and the turbidity of the culture supernatant from which the yeast has been removed are measured using a Klett-Summerson photometric calorimeter. And measured by the Klett method. The measurement results are shown in FIG. When the tCpFAH12 / vect strain was cultured, the OD 600 of the culture broth hardly increased and the glucose concentration did not decrease even when the culture time passed, and the turbidity of the culture supernatant was lower than that of the No_FAH12 / vect strain. Although it was high, there was no cloudiness enough to be visually recognized (FIGS. 6B and 6E). When the CpFAH12 / plg7 strain was cultured, although slightly slower than the No_FAH12 / vect strain, the OD 600 of the culture increased with the passage of the culture time, the glucose concentration decreased, and the No_FAH12 / vector strain was further reduced. In contrast, it was confirmed that the turbidity of the culture supernatant increased remarkably over time, and this depended on the amount of oil droplet-like structures [FIGS. 6 (A) and (C)]. This tendency was more remarkable in the medium having a glucose concentration of 10% by mass than in the medium having a glucose concentration of 2% by mass.
５日間の培養終了後の培養上清を回収し、脂肪酸（ＦＡ）の含有量と組成を測定した。測定結果を図７に示す。図７中、「Ｃ１８：１−ＯＨ」はリシノール酸を、「Ｃ１８：２ｎ−６」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。また、「ＣｐＦＡＨ１２＋ｐｌｇ７ １０％Ｇｌｃ」はＣｐＦＡＨ１２／ｐｌｇ７株をグルコース濃度１０質量％の培地で培養した結果を、「ＣｐＦＡＨ１２＋ｐｌｇ７ ２％Ｇｌｃ」はＣｐＦＡＨ１２／ｐｌｇ７株をグルコース濃度２質量％の培地で培養した結果を、「ＣｐＦＡＨ１２＋Ｖｅｃｔｏｒ １０％Ｇｌｃ」はＣｐＦＡＨ１２／ｖｅｃｔ株をグルコース濃度１０質量％の培地で培養した結果を、それぞれ示す。ｐｌｇ７を発現させたＣｐＦＡＨ１２／ｐｌｇ７株では、ＣｐＦＡＨ１２のみを発現させたＣｐＦＡＨ１２／ｖｅｃｔ株よりも培養上清に分泌される脂肪酸の量が顕著に増大しており、かつ、分泌された脂肪酸全量の大部分がリシノール酸であることが確認された。特に、ＣｐＦＡＨ１２／ｐｌｇ７株をグルコース濃度１０質量％の培地で培養した場合には、培養上清中に７４．５ｍｇ／Ｌ以上の脂肪酸が分泌されており、その７０％程度がリシノール酸であった。 The culture supernatant after 5 days of culture was collected, and the content and composition of fatty acid (FA) were measured. The measurement results are shown in FIG. In FIG. 7, “C18: 1-OH” represents ricinoleic acid, “C18: 2n-6” represents linolenic acid, and “Others” represents the total of other fatty acids. “CpFAH12 + plg7 10% Glc” is the result of culturing the CpFAH12 / plg7 strain in a medium with a glucose concentration of 10% by mass, and “CpFAH12 + plg7 2% Glc” is the result of culturing the CpFAH12 / plg7 strain in a medium with a glucose concentration of 2% by mass. “CpFAH12 + Vector 10% Glc” indicates the results of culturing the CpFAH12 / vector strain in a medium having a glucose concentration of 10% by mass, respectively. In the CpFAH12 / plg7 strain expressing plg7, the amount of fatty acid secreted into the culture supernatant is significantly increased compared to the CpFAH12 / vector strain expressing only CpFAH12, and the total amount of secreted fatty acids is large. The part was confirmed to be ricinoleic acid. In particular, when CpFAH12 / plg7 strain was cultured in a medium having a glucose concentration of 10% by mass, 74.5 mg / L or more of fatty acid was secreted into the culture supernatant, and about 70% was ricinoleic acid. .
さらに、１日間培養して得た菌体を回収し、細胞内のリシノール酸含有量を調べた。
具体的には、まず、ＣｐＦＡＨ１２／ｐｌｇ７株の菌体とＣｐＦＡＨ１２／ｖｅｃｔ株の菌体について、それぞれメタノールを添加して菌体を破砕した後、クロロホルム／メタノール（１：２）を加えて脂質を抽出し、クロロホルム層を脂質画分として回収した。次いで、該脂質画分を薄層クロマトグラフィー（ＴＬＣ）により分離し、リン脂質（ＰＬ）以外の脂質（遊離脂肪酸（ＦＡＡ）を含む。）中、およびリン脂質中のリシノール酸含有割合（％）を、ガスクロマトグラフ分析によって測定した。ＴＬＣは、具体的には、該脂質画分の１／３量を、ＴＬＣガラスプレート（ｓｉｌｉｃａ ｇｅｌ ６０ ｐｌａｔｅ、Ｍｅｒｃｋ社製）にスポットし、クロロホルム混合溶媒［クロロホルム：アセトン：メタノール：酢酸：水＝５０：２０：１０：１０：５（容量比）］で展開させた。次いで、同じＴＬＣガラスプレートに、該脂質画分の全残量をスポットし、ヘキサン混合溶媒［ヘキサン：ジエチルエーテル：酢酸＝８０：４０：１（容量比）］で展開させた。次いで、該ＴＬＣプレートをプリムリンで染色し、各バンドをプレートより掻き取り、ガスクロマトグラフ分析にかけた。
Furthermore, the microbial cells obtained by culturing for one day were collected, and the ricinoleic acid content in the cells was examined.
Specifically, first, with respect to the cells of the CpFAH12 / plg7 strain and the cells of the CpFAH12 / vector strain, methanol was added to disrupt the cells, and then chloroform / methanol (1: 2) was added to obtain lipids. Extraction was performed, and the chloroform layer was collected as a lipid fraction. The lipid fraction is then separated by thin layer chromatography (TLC), and the ricinoleic acid content (%) in lipids other than phospholipids (PL) (including free fatty acids (FAA)) and in phospholipids Was measured by gas chromatographic analysis. Specifically, TLC spotted 1/3 amount of the lipid fraction on a TLC glass plate (silica gel 60 plate, manufactured by Merck) and mixed with a chloroform mixed solvent [chloroform: acetone: methanol: acetic acid: water = 50: 20: 10: 10: 5 (volume ratio)]. Subsequently, the total remaining amount of the lipid fraction was spotted on the same TLC glass plate, and developed with a hexane mixed solvent [hexane: diethyl ether: acetic acid = 80: 40: 1 (volume ratio)]. The TLC plate was then stained with primulin, and each band was scraped from the plate and subjected to gas chromatographic analysis.
測定結果を図８に示す。図中、「Ｖｅｃｔｏｒ」はＣｐＦＡＨ１２／ｖｅｃｔ株の結果を、「ｐｌｇ７」はＣｐＦＡＨ１２／ｐｌｇ７株の結果をそれぞれ示す。ＣｐＦＡＨ１２／ｐｌｇ７株では、リン脂質中におけるリシノール酸含有割合（図中、「ＰＬ」）が、ＣｐＦＡＨ１２／ｖｅｃｔ株よりも顕著に減少していることが示された。該結果から、ＣｐＦＡＨ１２／ｐｌｇ７株では、リシノール酸がリン脂質として蓄積された後、ｐｌｇ７によって切り出され、細胞外に排出されることを示唆しており、リシノール酸が排出されることで細胞増殖抑制が解除されたものと推測された。 The measurement results are shown in FIG. In the figure, “Vector” indicates the result of the CpFAH12 / vector strain, and “plg7” indicates the result of the CpFAH12 / plg7 strain. In the CpFAH12 / plg7 strain, it was shown that the ricinoleic acid content ratio (“PL” in the figure) in the phospholipid was significantly reduced compared to the CpFAH12 / vector strain. This result suggests that in CpFAH12 / plg7 strain, ricinoleic acid is accumulated as phospholipid and then excised by plg7 and discharged to the outside of the cell. Was estimated to have been lifted.
［実施例３］
ＣｐＦＡＨ１２／ｐｌｇ７株の培養上清の脂肪酸組成分析を、ＴＬＣにより調べた。
チアミン不含ＥＭＭ−Ｃ／Ｎ３中、３７℃で一晩前培養した前培養液を、前培養と同種の新しい培地で、ＯＤ６００が２．０になるように希釈した後、２０℃で５日間、撹拌培養した。培養後の培養液を遠心分離して培養上清を回収し、クロロホルム／メタノール（１：２）を加えて脂質を抽出し、クロロホルム層を脂質画分として回収した。回収した脂質画分を、実施例２と同様にしてＴＬＣで展開した。ＴＬＣの展開結果を図９に示す。レーン１には各脂質［トリアシルグリセリド（ＴＧ）、オレイン酸（ＯＡ）、ジアシルグリセロール（ＤＧ）、モノアシルグリセロール（ＭＧ）、ホスファチジルエタノールアミン（ＰＥ）、ホスファチジルコリン（ＰＣ）、リゾホスファチジルコリン（ＬＰＣ）、ホスファチジルイノシトール（ＰＩ）］の標準品を、レーン２にはＣｐＦＡＨ１２／ｐｌｇ７株の培養上清の脂質画分を、レーン３にはひまし油を、レーン４にはリシノール酸の標準品を、それぞれ展開した。その結果、ＣｐＦＡＨ１２／ｐｌｇ７株の培養上清中の脂肪酸の大部分がリシノール酸であった。また、ひまし油は、その大部分がリシノール酸であるが、標準品のリシノール酸とは異なる移動度の位置に展開していたのに対して、ＣｐＦＡＨ１２／ｐｌｇ７株の培養上清の脂質画分中のリシノール酸は、標準品と同じ移動度であった。すなわち、ＣｐＦＡＨ１２／ｐｌｇ７株の培養上清から、純度の高いリシノール酸が回収できることが示唆された。
[Example 3]
The fatty acid composition analysis of the culture supernatant of the CpFAH12 / plg7 strain was examined by TLC.
A pre-culture solution pre-cultured overnight at 37 ° C. in thiamine-free EMM-C / N3 was diluted with a new medium of the same type as the pre-culture so that the OD 600 would be 2.0, and then 5 ° C. at 20 ° C. The culture was stirred for days. The culture broth after the culture was centrifuged to collect the culture supernatant, chloroform / methanol (1: 2) was added to extract lipids, and the chloroform layer was recovered as a lipid fraction. The collected lipid fraction was developed by TLC in the same manner as in Example 2. The development result of TLC is shown in FIG. Lane 1 shows each lipid [triacylglyceride (TG), oleic acid (OA), diacylglycerol (DG), monoacylglycerol (MG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysophosphatidylcholine (LPC). , Phosphatidylinositol (PI)], lane 2 with the lipid fraction of the culture supernatant of CpFAH12 / plg7 strain, lane 3 with castor oil, lane 4 with ricinoleic acid standard did. As a result, most of the fatty acids in the culture supernatant of the CpFAH12 / plg7 strain were ricinoleic acid. Castor oil, which is mostly ricinoleic acid, was developed at a position of mobility different from that of standard ricinoleic acid, whereas in the lipid fraction of the culture supernatant of CpFAH12 / plg7 strain The ricinoleic acid had the same mobility as the standard product. That is, it was suggested that highly pure ricinoleic acid can be recovered from the culture supernatant of the CpFAH12 / plg7 strain.
［実施例４］
ＣｐＦＡＨ１２／ｐｌｇ７株の培養時間に伴うリシノール酸の蓄積状況を調べた。
ＣｐＦＡＨ１２／ｐｌｇ７株とＣｐＦＡＨ１２／ｖｅｃｔ株について、チアミン不含ＥＭＭ−Ｃ／Ｎ３中、３７℃で一晩前培養した前培養液を、グルコース濃度１０質量％のチアミン不含ＥＭＭ−Ｃ／Ｎ３でＯＤ６００が２になるように希釈した後、２０℃で１１日間、撹拌培養した。本培養開始から３、５、７、１１日後に培養液の一部をサンプリングし、培養上清中および細胞内の脂肪酸の含有量と組成を測定した。測定結果を図１０に示す。図中、「Ｖｅｃｔｏｒ」はＣｐＦＡＨ１２／ｖｅｃｔ株の結果を、「ｐｌｇ７」はＣｐＦＡＨ１２／ｐｌｇ７株の結果をそれぞれ示す。「Ｃ１８：１−ＯＨ」、「Ｃ１８：２ｎ−６」、および「Ｏｔｈｅｒｓ」は図７と同様である。両株とも、リシノール酸の含有割合は、細胞内で５０〜６０％であり、培養上清で７０％以上であった。特にＣｐＦＡＨ１２／ｐｌｇ７株では、培養上清中には細胞内と同程度の量のリシノール酸が排出されており、培養上清から大量のリシノール酸を容易に取得できた。本実施例は試験管レベルの実験であるが、本発明に係るリシノール酸の製造方法をジャー培養槽等を用いて行うことにより、より大量のリシノール酸の製造が可能になることが示された。
[Example 4]
The accumulation state of ricinoleic acid with the culture time of the CpFAH12 / plg7 strain was examined.
For the CpFAH12 / plg7 strain and the CpFAH12 / vector strain, precultured overnight at 37 ° C. in thiamine-free EMM-C / N3 was OD with thiamine-free EMM-C / N3 having a glucose concentration of 10% by mass. After diluting so that 600 becomes 2, it was stirred and cultured at 20 ° C. for 11 days. A portion of the culture solution was sampled 3, 5, 7, and 11 days after the start of the main culture, and the fatty acid content and composition in the culture supernatant and in the cells were measured. The measurement results are shown in FIG. In the figure, “Vector” indicates the result of the CpFAH12 / vector strain, and “plg7” indicates the result of the CpFAH12 / plg7 strain. “C18: 1-OH”, “C18: 2n-6”, and “Others” are the same as those in FIG. In both strains, the content of ricinoleic acid was 50-60% in the cells and 70% or more in the culture supernatant. In particular, in the CpFAH12 / plg7 strain, ricinoleic acid in the same amount as that in the cells was discharged in the culture supernatant, and a large amount of ricinoleic acid could be easily obtained from the culture supernatant. Although this example is an experiment at a test tube level, it was shown that a larger amount of ricinoleic acid can be produced by performing the method for producing ricinoleic acid according to the present invention using a jar culture tank or the like. .
［実施例５］
ＣｐＦＡＨ１２／ｐｌｇ７株の増殖能に対する培養温度の影響を調べた。
Ｎｏ＿ＦＡＨ１２／ｖｅｃｔ株とＣｐＦＡＨ１２／ｖｅｃｔ株とＣｐＦＡＨ１２／ｐｌｇ７株について、チアミンを含むＥＭＭ−Ｃ／Ｎ３中、３０℃で一晩前培養した後、チアミンを枯渇させた前培養液を、グルコース濃度１０質量％のチアミン不含ＥＭＭ−Ｃ／Ｎ３でＯＤ６００が０．２になるように希釈した後、２０℃、２５℃または３０℃で１００時間撹拌培養した。実施例２と同様にして、ＯＤ６００をモニターした。描いた増殖曲線を図１１に示す。図中、「Ｎｏ ＦＡＨ１２＋Ｖｅｃｔ」がＮｏ＿ＦＡＨ１２／ｖｅｃｔ株の結果を、「ＣｐＦＡＨ１２＋Ｖｅｃｔ」がＣｐＦＡＨ１２／ｖｅｃｔ株の結果を、「ＣｐＦＡＨ１２＋ｐｌｇ７」がＣｐＦＡＨ１２／ｐｌｇ７株の結果を、それぞれ示す。２０℃、２５℃および３０℃のいずれにおいても、ＣｐＦＡＨ１２／ｖｅｃｔ株よりもＣｐＦＡＨ１２／ｐｌｇ７株のほうが培養液のＯＤ６００が高く、増殖能が高いことがわかった。すなわち、培養温度にかかわらず、ＣｐＦＡＨ１２発現による増殖抑制に対するｐｌｇ７による解除効果が観察された。
また、ＣｐＦＡＨ１２／ｐｌｇ７株を培養した培養液のみ白濁していた。該培養液を顕微鏡により検鏡したところ、油滴様のものが確認された。
[Example 5]
The influence of culture temperature on the growth ability of CpFAH12 / plg7 strain was examined.
No_FAH12 / vect strain, CpFAH12 / vect strain and CpFAH12 / plg7 strain were pre-cultured overnight at 30 ° C. in EMM-C / N3 containing thiamine, and then the thiamin-depleted preculture was treated with a glucose concentration of 10 mass. After diluting with an EMM-C / N3 containing no thiamin to an OD 600 of 0.2, the mixture was stirred and cultured at 20 ° C, 25 ° C or 30 ° C for 100 hours. OD 600 was monitored in the same manner as in Example 2. The drawn growth curve is shown in FIG. In the figure, “No FAH12 + Vect” indicates the result of the No_FAH12 / vect strain, “CpFAH12 + Vect” indicates the result of the CpFAH12 / vect strain, and “CpFAH12 + plg7” indicates the result of the CpFAH12 / plg7 strain. At 20 ° C., 25 ° C., and 30 ° C., it was found that the CpFAH12 / plg7 strain had a higher OD 600 of the culture solution and higher growth ability than the CpFAH12 / vector strain. That is, regardless of the culture temperature, the release effect by plg7 on the growth suppression by CpFAH12 expression was observed.
Moreover, only the culture solution which cultured CpFAH12 / plg7 strain was cloudy. When the culture solution was examined with a microscope, an oil droplet-like product was confirmed.
［実施例６］
ＣｐＦＡＨ１２／ｐｌｇ７株のリシノール酸産生能に対する培養温度の影響を調べた。
ＣｐＦＡＨ１２／ｖｅｃｔ株とＣｐＦＡＨ１２／ｐｌｇ７株について、チアミン不含ＥＭＭ−Ｃ／Ｎ３中、３７℃で一晩前培養した前培養液を、グルコース濃度１０質量％のチアミン不含ＥＭＭ−Ｃ／Ｎ３でＯＤ６００が２．０になるように希釈した後、２０℃、２５℃または３０℃で撹拌培養した。４日間培養後の培養液の培養上清の濁度（Ｋｌｅｔｔ値）を、実施例２と同様にして測定した。測定結果を図１２に示す。図中、「ｐｌｇ７」はＣｐＦＡＨ１２／ｐｌｇ７株の培養結果を、「Ｖｅｃｔ」はＣｐＦＡＨ１２／ｖｅｃｔ株の培養結果を、それぞれ示す。いずれの温度においても、ＣｐＦＡＨ１２／ｖｅｃｔ株よりもＣｐＦＡＨ１２／ｐｌｇ７株のほうが培養上清の濁度が明らかに高く、リシノール酸の分泌量が高かった。特に、３０℃で培養した場合よりも、２０〜２５℃で培養した場合のほうがＫｌｅｔｔ値が高く、より大量のリシノール酸が分泌されていた。
[Example 6]
The influence of culture temperature on the ricinoleic acid production ability of CpFAH12 / plg7 strain was examined.
For the CpFAH12 / vect and CpFAH12 / plg7 strains, the preculture was pre-cultured overnight at 37 ° C. in thiamine-free EMM-C / N3. After diluting so that 600 becomes 2.0, stirring culture was performed at 20 ° C, 25 ° C, or 30 ° C. The turbidity (Klett value) of the culture supernatant of the culture solution after 4 days of culture was measured in the same manner as in Example 2. The measurement results are shown in FIG. In the figure, “plg7” indicates the culture result of the CpFAH12 / plg7 strain, and “Vect” indicates the culture result of the CpFAH12 / vector strain. At any temperature, the turbidity of the culture supernatant was clearly higher in the CpFAH12 / plg7 strain than in the CpFAH12 / vect strain, and the secretion amount of ricinoleic acid was higher. In particular, the Klett value was higher when cultured at 20-25 ° C. than when cultured at 30 ° C., and a larger amount of ricinoleic acid was secreted.
本発明に係るリシノール酸産生酵母およびこれを用いた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。 The ricinoleic acid-producing yeast according to the present invention and the method for producing ricinoleic acid using the yeast can efficiently produce ricinoleic acid using a yeast transformant. Therefore, in particular, ricinoleic acid or castor oil is used as a raw material. It is preferably used in the field of manufacturing chemical products, paints / printing inks, cosmetics, pharmaceuticals, lubricants and the like.
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| CN114317540A (en) * | 2022-01-13 | 2022-04-12 | 内蒙古民族大学 | RcFAH12 gene promoter, deletion mutant thereof and application thereof |
| WO2023196923A1 (en) * | 2022-04-07 | 2023-10-12 | Checkerspot, Inc. | Ricinoleate oil production and uses thereof |
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| CN114317540A (en) * | 2022-01-13 | 2022-04-12 | 内蒙古民族大学 | RcFAH12 gene promoter, deletion mutant thereof and application thereof |
| CN114317540B (en) * | 2022-01-13 | 2023-07-18 | 内蒙古民族大学 | RcFAH12 gene promoter, deletion mutant thereof and application thereof |
| WO2023196923A1 (en) * | 2022-04-07 | 2023-10-12 | Checkerspot, Inc. | Ricinoleate oil production and uses thereof |
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