JP6085882B2 - カロテノイド生合成遺伝子発現による乳酸菌の環境ストレス耐性向上技術 - Google Patents
カロテノイド生合成遺伝子発現による乳酸菌の環境ストレス耐性向上技術 Download PDFInfo
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Description
乳酸菌の一部にはカロテノイドを生産するものがあることが知られている。例えば、C30カロテノイドを著量生産する乳酸菌(Lactobacillus plantarum)が報告されているが(非特許文献2)、カロテノイドの環境ストレス低減作用については何ら検討されていない。乳酸菌以外のカロテノイドを微生物のストレス耐性向上に用いた例としては、枯草菌においてスタフィロコッカス・アウレウス(Staphylococcus aureus)由来のカロテノイド生合成遺伝子を発現させることにより抗酸化性が付与されたという報告がある(特許文献5;非特許文献3)。また、乳酸菌についても、スタフィロコッカス・アウレウス(Staphylococcus aureus)由来のカロテノイドをカロテノイド非生産乳酸菌であるストレプトコッカス・ピオジェネス(Streptococcus pyogenes)で発現させることにより抗酸化性が向上したことが報告される(非特許文献4)。しかしながら、この例では、病原菌由来のカロテノイド遺伝子を病原性連鎖球菌である乳酸菌で発現させた病原菌同士の特殊な例であることから、属を超えて利用できる汎用性の高い乳酸菌の環境ストレス耐性付与技術を確立するには至っていない。
(1) エンテロコッカス・ギルバス(Enterococcus gilvus)由来のcrtN遺伝子とcrtM遺伝子を含み、かつ乳酸菌の環境ストレス耐性を向上させる活性を有するカロテノイド生合成遺伝子。
(2) 以下の(a)〜(d)のいずれかに示すDNAを含む、(1)に記載のカロテノイド生合成遺伝子。
(a) 配列番号1に示す塩基配列からなるDNA
(b) 配列番号1に示す塩基配列に対して80%以上の相同性を有する塩基配列からなり、かつ乳酸菌の環境ストレス耐性向上活性を有するタンパク質をコードするDNA
(c) 配列番号1に示す塩基配列において1若しくは数個の塩基が欠失、置換、若しくは付加された塩基配列からなり、かつ乳酸菌の環境ストレス耐性向上活性を有するタンパク質をコードするDNA
(d) 配列番号1に示す塩基配列からなるDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ乳酸菌の環境ストレス耐性向上活性を有するタンパク質をコードするDNA
(3)乳酸菌において機能しうるプロモーター領域に連結された(1)または(2)に記載の遺伝子を含む組換えベクター。
(4) (3)に記載の組換えベクターを導入し、環境ストレス耐性が向上した乳酸菌。
(5) (3)に記載の組換えベクターを乳酸菌に導入することを特徴とする、環境ストレス耐性が向上した乳酸菌の製造方法。
(6) (1)または(2)に記載の遺伝子を乳酸菌菌体内で発現させることにより、乳酸菌に環境ストレス耐性を付与する方法。
(実施例1)カロテノイド生合成遺伝子のクローニングと乳酸菌への導入
乳酸菌の分離源として牛乳を用いた。1%スキムミルクを含む標準プレートカウント寒天培地培地(OXOID コード:CM0463)に牛乳希釈液を添加し、37℃で48時間静置培養した。培養終了後、形成されたコロニーの中から、黄色のコロニーを形成した乳酸菌の1菌株を分離した。本菌株について、16S rDNA 配列解析を行なったところ、Enterococcus gilvusに近縁な種と分類された。本菌株をCR1株と命名した。
カロテノイドを生産する細菌(Lactobacillus plantarum, Enterococcus casseliflavus, Enterococcus gallinarum, Carnobacterium sp. AT7, Bacillus sp. NRRL B-14911, Eubacterium limosum)のカロテノイド生合成遺伝子をNCBIデータベースより取得し、保存配列を参考に、下記の縮合プライマーcrtN-FおよびcrtN-Rを作成した。
crtN-F:HTNDSNTTYCARACVYTVTAYATHGG (配列番号2)
crtN-R:ATNGGNACNGGNGCNCCNGGRT (配列番号3)
crtN SEQ1:GAATCGAATGCAACGCCTCTAC (配列番号4)
crtN inv-nest2:GATTGGAATGAAGAGACCATTC (配列番号5)
crtMNproBamHI-F:ATA ggatcc AATGATTTACAATTATTAATTTCT(配列番号7)
crtMNproEcoRI-R:ATA gaattc TATTCAGTGTTGTTTGAACA(配列番号8)
(1) 過酸化水素耐性
野生株MG1363(WT)、空ベクター導入株MGpRH100(pRH100を導入したMG1363株)、カロテノイド生合成遺伝子導入株MGpRC(pRCを導入したMG1363株)を用いて、過酸化水素耐性を調べた。
128 mM H2O2溶液の代わりに2.5% Oxgallを用いる以外は、(1)と同様にして野生株MG1363(WT)、空ベクター導入株 MGpRH100(pRH100を導入したMG1363株)、カロテノイド生合成遺伝子導入株MGpRC(pRCを導入したMG1363株)を用いて、胆汁酸耐性を調べた。
128 mM H2O2溶液の代わりに生理食塩水(HClで生理食塩水のpHを2.0にしたもの)を用いる以外は(1)と同様にして野生株MG1363(WT)、空ベクター導入株 MGpRH100(pRH100を導入したMG1363株)、カロテノイド生合成遺伝子導入株MGpRC(pRCを導入したMG1363株)を用いて、酸耐性を調べた。その結果、カロテノイド生合成遺伝子導入株MGpRC は、酸耐性が向上することが確認できた(図6)。
前記野生株MG1363(WT)、空ベクター導入株MGpRH100(pRH100を導入したMG1363株)、カロテノイド生合成遺伝子導入株MGpRC(pRCを導入したMG1363株)をGM17液体培地で24時間培養後、菌体を遠心(8000 rpm, 10 min)により回収し、生理食塩水で2回洗浄した。洗浄した菌体に500μLのリゾチーム溶液(12 mg/mL)を加えて懸濁し、37℃で180分間静置した。コントロールとして、生理食塩水に懸濁した菌体を37℃で180分間静置した。180分後、菌体を遠心して生理食塩水で洗浄した後、GM17寒天培地に接種して、菌数を測定し、生存率(ストレス負荷後の菌数/生理食塩水中で180分放置後の菌数)を決定した。その結果、カロテノイド生合成遺伝子導入株MGpRC は、約4倍生存率が高くなり、リゾチーム耐性が向上することが確認できた(図4)。リゾチームは溶菌作用があることから細菌等による品質劣化防止を目的としてチーズ等の食品に使用されるが、本発明のカロテノイド生合成遺伝子導入株はリゾチーム耐性があるのでそのような食品にも使用できる。
Claims (5)
- 以下の(a)〜(d)のいずれかに示すDNAを含む、カロテノイド生合成遺伝子。
(a) 配列番号1に示す塩基配列からなるDNA
(b) 配列番号1に示す塩基配列に対して95%以上の相同性を有する塩基配列からなり、かつ乳酸菌の環境ストレス耐性向上活性を有するタンパク質をコードするDNA
(c) 配列番号1に示す塩基配列において1から20個の塩基が欠失、置換、若しくは付加された塩基配列からなり、かつ乳酸菌の環境ストレス耐性向上活性を有するタンパク質をコードするDNA
(d) 配列番号1に示す塩基配列からなるDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ乳酸菌の環境ストレス耐性向上活性を有するタンパク質をコードするDNA - 乳酸菌において機能しうるプロモーター領域に連結された請求項1に記載の遺伝子を含む組換えベクター。
- 請求項2に記載の組換えベクターを導入し、環境ストレス耐性が向上した乳酸菌。
- 請求項2に記載の組換えベクターを乳酸菌に導入することを特徴とする、環境ストレス耐性が向上した乳酸菌の製造方法。
- 請求項1に記載の遺伝子を乳酸菌菌体内で発現させることにより、乳酸菌に環境ストレス耐性を付与する方法。
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