JP4288862B2 - Method for producing prenyl alcohol - Google Patents

Description

【００３５】
PCR後、アガロースゲル電気泳動により、目的の位置（3.2kbp）に断片が確認されたので、その3.2kbpDNA断片をTAクローニング可能なpT7Blue Tベクター(Novagen, Madison, WI)にクローニングし、これをpT7HMG1とした。クローニングしたHMG-CoA還元酵素遺伝子の塩基配列を決定した。その結果、配列番号４の塩基配列及び配列番号５のアミノ酸配列を確認した。決定された塩基配列は、GenBankの配列に示した塩基配列と一部異なる変異型遺伝子となっていた（図2A）。このように、Taq DNAポリメラーゼ等の忠実性（fidelity）の低いDNAポリメラーゼを用いたPCR(polymerase chain reaction)で野性型DNAを増幅させたDNA断片に生じる塩基の置換変異を「PCRエラー」という。このPCRエラーを含んだ変異型HMG-CoA還元酵素のアミノ酸配列（配列番号５）をコードする遺伝子をHMG1'とする。
【００３６】
(2) HMG1'発現用プラスミドpYES-HMG1の作製
上述のように作製したpT7HMG1をBamHI、SalI、ScaI処理してPCRエラーによる変異型HMG-CoA還元酵素をコードする遺伝子HMG1'を取り出し、これをpYES2(Invitrogen, Carlsbad, CA)のBamHI-XhoI部位に導入した。得られた組換えベクターをpYES-HMG1とした。ベクター内の塩基配列決定を行ったところ、配列番号４に示す塩基配列であることを確認した。なお、pYES2は、複製起点として酵母２μmDNAのori、及びガラクトースで誘導可能なGAL1転写プロモーターをもつ酵母発現用シャトルベクターである（図３）。
【００３７】
(3) 欠失型HMG1'発現用プラスミドpYHMGxxyの作製
HMG-CoA還元酵素触媒部位と考えられている部分より上流の領域をコードする塩基配列を欠損させた欠失型HMG-CoA還元酵素遺伝子発現ベクターを作製するため、(2)で作製したpYES-HMG1を鋳型として、PCR法でベクター部分とともにHMG1'コード領域の一部分を欠失させた断片を調製した。得られた断片をKlenow酵素で平滑末端にした後、セルフライゲーションにより再び環化し、E. coli JM109へ形質転換させ、プラスミドDNAを調製した。プライマーとして使用した合成DNA配列とその組合せを表１に示した。
【００３８】
【表１】

【００３９】
得られたプラスミドDNA内のHMG1上流、下流のアミノ酸の読み枠がずれていないことと、その結合部位近辺にPCRエラーによるアミノ酸置換が起きていないことを373A DNA sequencer (Perkin Elmer, Foster City, CA) で確認した。その結果、結合部位近辺にPCRエラーによるアミノ酸置換がなく、読み枠がずれずに遺伝子を欠失する事のできた以下のプラスミドを得た。欠失型HMG1遺伝子は、欠失のパターンに従ってΔ02y（yは任意の作業番号を表す）のように記載し、Δ02yを含むpYES2ベクターを例えばpYHMG026と記すこととする（他の欠失体も同様）（図2B）。
【００４０】
HMG1Δ02y：配列番号６
HMG1Δ04y：配列番号７
HMG1Δ05y：配列番号８
HMG1Δ06y：配列番号９
HMG1Δ07y：配列番号10
HMG1Δ08y：配列番号11
HMG1Δ10y：配列番号12
HMG1Δ11y：配列番号13
HMG1Δ12y：配列番号14
HMG1Δ13y：配列番号15
ベクター：pYHMG026, pYHMG027, pYHMG044, pYHMG045, pYHMG062, pYHMG063, pYHMG065, pYHMG076, pYHMG081, pYHMG083, pYHMG085, pYHMG094, pYHMG100, pYHMG106, pYHMG107, pYHMG108, pYHMG109, pYHMG112, pYHMG122, pYHMG123, pYHMG125, pYHMG133。このうち、ベクターpYHMG122を本発明の試験に使用した。
【００４１】
(4)pYES-HMG1導入組換え体の作製
予備実験においてFOHを生産する可能性のあることが認められた酵母Saccharomyces cerevisiae A451株にベクターpYHMG122を導入することによって、組換え体のpYHMG122/A451を得た。宿主A451株へのベクターpYHMG122の導入は、Current Protocols in Molecular Biology, John Wiley & Sons, Inc., pp.13.7.1-13.7.2に記載の酢酸リチウム法、又は、Frozen-EZ Yeast Transformation II (Zymo Research, Orange, CA) を用いた方法で導入した（手順はキット同封の説明書に従った）。
【００４２】
２．微生物の培養方法
（１）Saccharomyces cerevisiae ATCC64031株の培養方法
・ジャーファメンター培養
エルゴステロール（20mg/L）を含むYM broth (200ml/500ml-バッフル付き三角フラスコ)にSaccharomyces cerevisiae ATCC64031株を一白金耳植菌し、２６℃、１３０ｒｐｍ、２日間培養しPre-Cultureとした。これより菌液５０ｍｌをファーメンターに無菌的に植菌し、以下の培養条件で培養した。
【００４３】
培養装置：MSJ-U 10L培養装置（丸菱バイオエンジ）
Working Volume：5L
培養温度：26℃
通気量：１vvm
アジテーション：300rpm
ｐH：４Ｎ水酸化ナトリウム溶液及び２Ｎ塩酸溶液を用い、以下のパラメーターでｐＨを比例制御
Proportional Band 1.00
Non Sensitive Band 0.15
Control Period 16 Sec
Full Stroke 1 Sec
Minimun Stroke 0 Sec
培地：６％グルコースを含むYM培地（Difco社製）
0.1または1％ 大豆油
0.1％ アデカノールLG109（旭電化）
4mg/L エルゴステロール（ナカライ）
（この時、エルゴステロール添加によってエタノール100mg/L、タージトール（ナカライ）100mg/Lが持ち込まれる）
【００４４】
（２）遺伝子導入酵母遺伝子導入酵母pYHMG122/A451の培養方法
・ジャーファメンター培養
以下のPre-Culture培地 （200ml/500ml-バッフル付き三角フラスコ）に組換え酵母pYHMG122/A451を一白金耳植菌し、３０℃、１３０ｒｐｍ、２日間培養した。次に培養液に含まれているグルコースを完全に除くため、遠心（1500ｇ、５分、４℃）及び滅菌した生理食塩水による洗浄を３回繰り返した後、ジャーファメンターに50ｍｌを植菌（１％）した。ジャーファメンター培養は(1)のATCC64031株と同様の培養条件で行った。
【００４５】
Pre-Culture培地：
CSM-URA（BIO 101 Inc.製）
DOB（BIO 101 Inc.製）
ファーメンター培地：
５％ ガラクトース
YNB without Amino Acids（Difco製）
1％ 大豆油（ナカライ）
0.1％アデカノールLG109（旭電化）
【００４６】
３．抽出方法
（１）ファルネソール及びゲラニルゲラニオールの分別抽出
▲１▼ 上清画分からのファルネソール及びゲラニルゲラニオールの抽出
培養液2.5mlを18Φmm×125mm試験管に入れ、ベックマン社製遠心分離器GP centrifugeで1000rpm、５分遠心し、上清を新しい18Φmm×125mm試験管に移した。６mMの塩化マグネシウムを含むトリス塩酸緩衝液（pH8.0）0.5ml、大腸菌アルカリフォスファターゼ（宝酒造製）５μl（２ユニット）を加え、65℃で30分加熱した。氷上で十分に冷却してからペンタン２ml、メタノール１mlを加え、十分に混合後、ベックマン社製遠心分離GP centrifugeで1000rpm、５分遠心し、上清を別の新しい試験管に移した。ドラフト内でペンタン・メタノール溶媒を蒸発させた後、300μlのペンタンに再溶解し、GC/MS用バイアル瓶に詰めた。
【００４７】
▲２▼ 菌体画分からのファルネソール及びゲラニルゲラニオールの抽出
1) バクテリアの場合
液体培養液10mlを50mlコーニングチューブに入れ、ベックマン製冷却遠心機（Avant J25-I）で6000rpm、５分遠心分離し菌体を集めた。菌体を脱イオン水0.5mlに懸濁させた後、10mlスピッツ管に移し、東海電機社製超音波細胞破砕機UCW-201で破砕した（条件：10℃、1分破砕−30秒停止を20分間繰り返す）。18Φmm×125mm試験管に移し、６mMの塩化マグネシウムを含むトリス塩酸緩衝液（pH8.0）0.5mlを加え、上述の▲１▼と同様にフォスファターゼ処理、抽出を行った。
【００４８】
2) 酵母・糸状菌の場合
液体培養液2.5mlを18Φmm×125mm試験管に入れ、ベックマン社製遠心分離器GP centrifugeで1000rpm、５分遠心し菌体を集めた。６mMの塩化マグネシウムを含むトリス塩酸緩衝液（pH8.0）0.5mlを加え菌体を懸濁させた後、破砕用ガラスチューブに移した。等量のガラスビーズ（シグマ社製 acid washed 425Φ-600μm）を加え、安井機械社製Multi-Beads Shocker MB-200で破砕した（条件：室温、2500rpm、20分）。18Φmm×125mm試験管に全量を移し、上述の▲１▼と同様にフォスファターゼ処理、抽出を行った。
なお、上述の各処理において、フォスファターゼ処理を行わない系は酵素処理工程を割愛した。
【００４９】
（２）ファルネソール及びゲラニルゲラニオールの全抽出
培養液２ｍｌに１.２５ｍｌメタノール（ナカライ）と２ｍｌのペンタン（ナカライ）を加え十分にボルテックスした後、試験管ごと遠心分離した（ベックマン社製CP、1500rpm、５分、室温）。ペンタン層を注意深くピペットマンまたはパスツールピペットでGC/MS用バイアル瓶に移した。この時、内部標準としての10μlウンデカノール溶液（1mg/ml-エタノール）をバイアル瓶にあらかじめ入れておいた。室温に長期保存するとペンタンが揮発するため、分析するまでアルミキャップで封印し−８０℃保存した。
【００５０】
４．分析方法
（１）ファルネソール及びゲラニルゲラニオールの分析
培養上清及び菌体画分のゲラニルゲラニオール及びファルネソールをGC/MSで分析した。カラムのバックグランド低減のため以下の分析条件で分析した。
【００５１】

純度検定にFID-GCで分析した条件は以下のとおりである。なお、内部標準が異なるためファルネソール及びゲラニルゲラニオール全抽出操作でウンデカノールを添加する前のペンタン溶液を分析した。
【００５２】

【００５３】
（２）菌体量測定（O.D.）
培養液１００μｌを生理食塩水で希釈し、分光光度計によりO.D.600nmを測定した。
【００５４】
（３）菌体量測定（重量）
105℃、２時間減圧乾燥した50ｍｌチューブ（コーニング社製）を精密天秤で重量測定した（Ａ）。培養液10mlを同チューブに入れて遠心分離し（4300ｇ、10分）上清を取り除きチューブの重量を測定した（Ｂ）。次に菌体ペレットを減圧乾燥し（80℃、3時間）再びチューブ重量を測定した（Ｃ）。培養液１L当たりの菌体量は以下の式により算出した。
湿菌体重量（g/L-broth）＝１００ × （Ｂ−Ａ）
乾燥菌体重量（g/L-broth）＝１００ × （Ｃ−Ａ）
なお、O.D.660nmと菌体重量との関係は以下のようであった。
１O.D.660nm ＝ ２.１４ｇ/Ｌ-湿菌体重量
１O.D.660nm ＝ ０.３０５ｇ/Ｌ-乾燥菌体重量
【００５５】
（４）菌体数測定
培養液１００μｌを生理食塩水で１〜２０倍に希釈し、血球計（林理化学）で細胞数を計数した。0.06mm四方（最少のグリッド９つ分）の菌数を４平均し、以下の式から培養液1L当たりの菌数を算出した。
菌数（ 10⁹cell/L-broth）＝0.444×（0.06mm四方の菌体数）×希釈倍率
【００５６】
（５）エタノール、酢酸、グリセロール、コハク酸量測定
F-キット（JKインターナショナル製）を用い、添付マニュアルに従いエタノール、酢酸、グリセロール、コハク酸、ピルビン酸を分析した。
【００５７】
〔実施例1〕プレニルアルコール生産に及ぼす培養時のｐＨ制御の影響
（１）中性pH制御の影響
Saccharomyces cerevisiae ATCC64031株を用いて、プレニルアルコール生産における培養液のpH制御の影響を検討した。
酵母の生育は通常酸性領域であるので、本実施例では、まず、培養液のpHを中性に制御し、得られるFOH（全抽出）、培養液中のグルコース、エタノール、酢酸、グリセロール、コハク酸の変動を調べた。その結果を図４に示す。
【００５８】
図４から明らかなように、培養液をpH７に制御した場合、FOHの生産量は培養時間とともに増加し、培養後150時間でFOH量は約160mg/Lに達した（図４右パネル）。一方、培養液のpHを制御しなかった場合、FOHの生産量は培養後150時間で約90mg/Lに達するものの、その後の生産量に増加傾向は見られなかった（図４左パネル）。従って、プレニルアルコールの生産において、培養開始時から対数増殖期にかけてpHを中性付近に制御することが有効であることが確認された。これは、培養液をpH7.0付近に制御することによって微生物の増殖が低下し、糖の分解によって生じるアセチルCoA、NADPHが微生物の増殖に使用されなくなり、プレノール合成の原料に使用されるようになると考えられる。
【００５９】
（２）弱アルカリ性pH制御の影響
さらに制御pH域を弱アルカリ性のpH８に設定し、FOHの生産性について、フォスファターゼ処理の効果も含めて検討した。また、pHの制御を培養経過途中から開始しても効果があるか否かについて、培養後46〜61時間をpH7.0、61〜70時間をpH8.0、70〜84.5時間をpH9.0に制御して試験した。その結果を図５に示す。
【００６０】
培養液をpH７に一段階pH制御した場合、FOHの生産量は培養時間とともに増加するのに対し（図５中パネル）、弱アルカリ性のpH8に一段階pH制御した場合、FOHの生産はほとんど確認されなかった（図５下パネル）。また、滅菌後pH6.0〜6.5となっている培地に対して、培養開始時にはpH制御を行わず、対数増殖期後の46時間以降に弱アルカリ性に制御した場合、培養時間の経過に伴いFOHの生産量が約36mg/Lに達した。
【００６１】
さらに、pH７〜pH８の範囲における制御効果を確認するために、制御pH値を4N水酸化ナトリウムで7.0、7.5、8.0に設定し試験した。なお、pH8.0については、培養開始時からpH8.0に制御してもFOHの生産が認められないことが前記試験から明らかであることから、培養開始時から対数増殖期にかけて（培養開始から約42時間）はpH７に制御し、その後、静止期に入ってからpHを8.0へ変更して制御した。また、併せて培養時間に伴う平均細胞数の変化についても調べた。その結果を図６に示す。
【００６２】
培養液を一段階pH制御した場合、pH7.0のときもpH7.5のときも培養時間の経過とともにFOHの生産量が増加しpH制御効果が認められた（図６、上及び中パネル）。一方、培養開始時から対数増殖期にかけてpH7.0に制御し（培養開始から約42時間）、その後、静止期に入ってからpHを8.0に変更して制御する二段階pH制御法の場合には、pH7.0における一段階pH制御法と同様のFOH生産性が得られた（図６、下パネル）。また、この場合、平均細胞数がpH7.0における一段階pH制御法よりも少ないため、細胞1個当たりのFOH生産性が高いことが明らかになった。プレニルアルコール生産では、生産物取得後、培養微生物は廃棄物として処理されるので工業生産的には培養微生物量は少ないほど好ましい。従って、培養開始時から対数増殖期にかけてpH7.0に制御した後、静止期以降にpH値を弱アルカリ性に変更して制御する二段階pH制御法は、FOHの生産性および培養微生物量の観点から非常に有用であると考えられる。
【００６３】
また、前記試験において、培養時間の経過に対する各種テルペノイド（ネロリドール、ファルネソール、ゲラニルゲラニオール、スクアレン）の生産量、培養液中のグルコース、エタノール、酢酸、グリセロール、コハク酸量、菌数の変化を調べた結果を表２に示す。
【００６４】
【表２】

【００６５】
FOHの生産に関しては、pH７における一段階pH制御法を行った場合とpHを静止期後に７から８に変更して制御する二段階pH制御法を行った場合に、ほぼ同量のFOH生産量が確認され、pH7.5における一段階pH制御法を行った場合は、その生産量は若干低下していた。また、ゲラニルゲラニオール（GGOH）の生産に関しては、pH７における一段階pH制御法を行った場合、pH7.5における一段階pH制御法を行った場合、およびpHを静止期後に７から８に変更して制御する二段階pH制御法を行った場合のいずれの場合も、ほぼ同量のGGOH生産量が確認された。なお、培養経過に伴う微生物菌数当たりのFOH生産効率を比較した場合、最も効率的であったのは二段階pH制御法を行った場合であり、次いでpH７における一段階pH制御法を行った場合とpH7.5における一段階pH制御法を行った場合とがほぼ等しいという結果であった。
【００６６】
（３）フォスファターゼ処理の影響
ファルネソールはフォスファターゼがファルネシルニリン酸に作用して生成する。そこで、プレニルアルコール生産のpH制御において、フォスファターゼ処理が菌体画分および培養液画分のFOH生産量にどのような影響を及ぼすか検討した。
すなわち、ATCC64031株をジャーファメンターで培養する際、pHを制御しない場合、およびpHを7.0に制御して培養する場合において、フォスファターゼ処理効果を培養液および菌体について培地組成を変えて検討した。その結果を表３及び表４に示す。
【００６７】
【表３】

【００６８】
【表４】

【００６９】
表３のpHを制御しなかった場合及びpHを7.0に制御した場合において、培養液及び菌体のフォスファターゼ処理の有無によるFOH生産量を比較すると、フォスファターゼ処理によるFOH生産量の顕著な相違は認められなかった。一方、表４では、pHを7.0に制御して培養した場合に、フォスファターゼ処理した菌体画分のFOH生産量が培養時間の経過とともに顕著に増大していた。表３と表４との培養条件の違いは、添加する大豆油の濃度の0.1％と１％である。従って、pHを制御して培養する方法において、適当な濃度の大豆油等の油脂を含有する培地での培養でフォスファターゼ処理を組み合わせることによってFOH生産量を高めることが可能である。
【００７０】
［実施例２］組換え微生物によるプレニルアルコール生産におけるpH制御の影響
ファルネソール、ゲラニルゲラニオール生産組換え微生物によって当該物質を生産する場合、ファルネソールの生産性が低く、また、ファルネソールとの分離が容易でない異性体のネロリドール、ゲラニルリナロールが同時に生成されてしまう。この副生成物のネロリドール、ゲラニルリナロールはファルネソール等との分離が容易でないため、これら副生成物を生じる微生物によるファルネソール生産は量産化には適さないという問題があり、これは、微生物の培養経過とともにｐＨが低下し、ファルネシル二リン酸やゲラニルゲラニル二リン酸の酸加水分解が起こり、反応中間体のカルボニムカチオンの安定性から転位反応産物であるネロリドールやゲラニルリナロールが優先的に生産されるためであると考えられる。
【００７１】
そこで、ネロリドール及びゲラニルリナロールを生成するHMG-CoA還元酵素遺伝子導入酵母pYHMG122/A451を用いて、培養時のpH制御の効果を検討した。すなわち、pYHMG122/A451を5%ガラクトース、1%大豆油、0.1%アデカノール含有ＹＮＢ培地で培養し、144時間目に終濃度5%のグルコースを添加した。この際、pHは4NのNaOHおよび2NのHClで7.0に制御した。対照はpH制御無しとした。その結果を図７及び表５に示す。
【００７２】
【表５】

【００７３】
図７及び表５より、pH制御を行わずpYHMG122/A451を培養した場合、FOH量は培養開始から157時間で122.7mg/Lであり、FOH生産の増加が確認された。また、これと同時に副生成物のネロリドールの生産も増加した（図７上パネル）。一方、pH7.0で制御して培養した場合、FOH量は培養開始から157時間で145.7mg/Lであり、FOH生産は増加するが、副生成物のネロリドールの生産はほとんど抑制された（図７下パネル）。従って、ネロリドール等の副生成物を生成する微生物を用いてプレニルアルコールを生産する場合、pHを中性〜弱アルカリ性へ制御することによって副生成物の生成を抑制し、プレニルアルコールの純度を高めることができることが確認された。
【００７４】
【発明の効果】
本発明により、プレニルアルコールを効率的に製造する方法が提供される。本発明によれば、pHを中性付近から弱アルカリ性の範囲内に制御した培地で生物学的に活性なプレニルアルコールを生産する微生物を培養することにより、効率よく大量にプレニルアルコールを入手することができる。また、本発明の方法をプレニルアルコール生産遺伝子導入微生物に適用した場合、プレニルアルコール生産時に生成されるネロリドール、ゲラニルゲラニオール等の副生成物の生成が抑制され、プレニルアルコールの純度を高めることができる。
【００７５】
【配列表】

【００７６】
【配列表フリーテキスト】
配列番号２：合成DNA
配列番号３：合成DNA
配列番号16：合成DNA
配列番号17：合成DNA
配列番号18：合成DNA
配列番号19：合成DNA
配列番号20：合成DNA
配列番号21：合成DNA
配列番号22：合成DNA
配列番号23：合成DNA
配列番号24：合成DNA
配列番号25：合成DNA
配列番号26：合成DNA
【図面の簡単な説明】
【図１】メバロン酸経路関連酵素の代謝経路を示す図である。
【図２】欠失型HMG1遺伝子の構築図及び置換変異のパターンを示す図である。
【図３】プラスミドpYES2を示す図である。
【図４】 Saccharomyces cerevisiae ATCC64031株によるFOH生産におけるpH制御効果を示す図である。
【図５】 Saccharomyces cerevisiae ATCC64031株によるFOH生産における中〜弱アルカリ性域のpH制御効果を示す図である。
【図６】 Saccharomyces cerevisiae ATCC64031株によるFOH生産における中〜弱アルカリ性域のpH制御効果と平均細胞数を示す図である。
【図７】遺伝子導入酵母pYHMG122/A451株によるFOH生産におけるpH制御効果を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing prenyl alcohol.
[0002]
[Prior art]
In vivo terpenoid (isoprenoid) synthesis is geranyl diphosphate, a linear prenyl diphosphate by condensing isopentenyl diphosphate (IPP, C5) sequentially to an allylic diphosphate substrate. (Geranyl diphosphate (GPP, C10)), farnesyl diphosphate (Fernesyl diphosphate (FPP, C15)), geranylgeranyl diphosphate (GGPP, C20)) is biosynthesized (FIG. 1). In FIG. 1, characters enclosed in a frame represent enzymes. hmgR represents hydroxymethyl glutaryl-CoA (HMG-CoA) reductase, GGPS represents GGPP synthase, and FPS represents FPP synthase.
[0003]
Among the prenyl diphosphates, FPP is the most important biosynthetic intermediate, and a huge variety of terpenoids such as steroids including ergosterol (provitamin D2), quinone (vitamin K, VK) Synthetic precursors such as chains, sesquiterpenes, squalene (SQ), anchor molecules of farnesylated proteins, natural rubber.
[0004]
GGPP is also an important biosynthetic intermediate in vivo: retinol (vitamin A, VA), β-carotene (provitamin A), phylloquinone (vitamin K1, VK1), tocopherols (vitamin E, VE), It is essential for biosynthesis of compounds including geranylgeranylated protein anchor molecule, chloroplast side chain, gibberellin, archaic ether type lipid.
[0005]
FPP, farnesol (FOH, C15) and geranylgeraniol (GGOH, C20), one of the prenyl alcohols that are alcohol derivatives of GGPP, are known as aromatic substances in essential oils used as perfumes. It is also an important substance as a starting material for the synthesis of compounds including the above-mentioned vitamins useful as pharmacologically active substances (FIG. 1).
[0006]
FOH is currently synthesized by chemical synthesis methods other than being prepared in small quantities from natural products such as essential oils. FOH synthesized by chemical synthesis generally has the same carbon skeleton, but a double bond is obtained as a mixture of (E) -type (trans type) and (Z) -type (cis type) . The (all-E) -type (E, E) -FOH or (E) -NOH is synthesized in the metabolic pathway of the organism and has industrial utility value. In order to obtain (E, E) -FOH or (E) -NOH in a pure form, purification using column chromatography or precision distillation is required. However, the precision distillation of FOH, a thermally unstable allyl alcohol, is difficult. In addition, purification by column chromatography requires a large amount of solvent packing material, and each fraction eluted in sequence must be collected while being analyzed, and the solvent must be removed. Not suitable for industrial implementation. Therefore, a biosynthetic method of (E, E) -FOH (hereinafter referred to as “FOH”) is desired from the features such as the control of formation of (E)-and (Z) -geometric isomers and the repeated structure of reaction products. Is not established. The FOH synthesis substrate is supplied through the mevalonate pathway in the cells of Saccharomyces cerevisiae, for example, the budding yeast, but squalene synthesis is also possible using HMG-CoA reductase, which is considered to be the key enzyme. It is only known that the performance increases (Japanese Patent Laid-Open No. 5-192184; Donald et al. (1997) Appl. Environ. Microbiol. 63, 3341-3344). In addition, even when a special budding yeast squalene synthetase gene-deficient strain that has acquired sterol uptake capacity is cultured, only 1.3 mg of FOH per liter of culture solution is known (Chambon et al. 1990) Curr. Genet. 18, 41-46), (E) -NOH (hereinafter referred to as “NOH”).
[0007]
GGOH is also currently synthesized by chemical synthesis (see, for example, JP-A-8-133999). However, compared to FOH and NOH, which have shorter carbon chains, chemical synthesis of GGOH requires more steps and costs more. In addition, GGOH synthesized by chemical synthesis generally has the same carbon skeleton, but the double bond is a mixture of (E) -type (trans type) and (Z) -type (cis type). can get. (E, E, E) -GGOH (hereinafter abbreviated as (all-E) -GGOH) is synthesized in the metabolic pathway of organisms and has industrial utility value. In order to obtain all-E-GGOH in a pure form, purification using column chromatography or precision distillation is required. However, precision distillation of GGOH, a thermally unstable allyl alcohol, is difficult. In addition, purification by column chromatography requires a large amount of solvent packing material, and each fraction eluted in sequence must be collected while being analyzed, and the solvent must be removed. Not suitable for industrial implementation. Therefore, a biosynthetic method of (all-E) -GGOH is desired but not established from the features such as control of formation of (E)-and (Z) -geometric isomers and repeated structure of reaction products. Regarding the biosynthesis of GGOH, in JP-A-9-238692, the production of 0.66 to 3.25 mg per liter culture solution is reported by plant cell culture, but it is not suitable for industrialization. Biosynthesis methods that are more practical than industrial GGOH preparations from natural products such as essential oils and more suitable for industrialization, for example, biosynthesis methods by microbial culture Is not known at all.
[0008]
In order to solve the above problems, the present applicant has developed a method for producing prenyl alcohol by culturing a recombinant transformed with a recombinant DNA for expression containing a prenyl diphosphate synthase gene. Patent applications are being filed.
In other words, FOH has been widely used in the fermentation industry since ancient times, and it is capable of synthesizing prenyl diphosphate via the mevalonate pathway or DXP pathway, and can utilize various techniques related to genetic recombination, such as unicellular eukaryotes (especially Yeast) or prokaryotes (eg bacteria, especially Escherichia coli) were used as experimental materials to develop a prenyl alcohol production system by gene transfer of an enzyme involved in prenyl diphosphate synthesis. In order to construct a system for artificially expressing a gene for an enzyme involved in prenyl diphosphate synthesis in yeast (eg, HMG-CoA reductase gene) in a host cell, a constitutive expression type or an inducible expression type transcription promoter is included, An expression shuttle vector with various auxotrophic markers is prepared, and the target gene or its mutant gene is incorporated into this and introduced into various host cells, and a method for obtaining NOH or FOH from the culture is established. did. In Escherichia coli, there is a method to obtain FOH after introducing a gene of an enzyme involved in prenyl diphosphate synthesis (for example, FPP synthase gene or IPPΔ-isomerase gene) into a host cell using an existing vector and dephosphorylating the culture. Established.
[0009]
Similarly, GGOH has been widely used in the fermentation industry since ancient times, and it can synthesize prenyl diphosphate via the mevalonate pathway or DXP pathway, and can use various techniques related to genetic recombination, such as unicellular eukaryotes ( A prenyl alcohol production system was developed by introducing genes for enzymes involved in the synthesis of prenyl diphosphate using, in particular, yeast) or prokaryotes (eg, bacteria, particularly E. coli) as experimental materials. As a result, genes of enzymes involved in prenyl diphosphate synthesis in yeast (mevalonate pathway-related enzyme genes such as HMG-CoA reductase gene, IPPΔ isomerase gene, various prenyl diphosphate synthase genes, or their genes) In order to construct a system that artificially expresses mutant or fusion genes) in host cells, an expression shuttle vector containing a constitutive expression or inducible expression transcription promoter and having various auxotrophic markers can be prepared. Then, the gene of interest or its mutant gene was incorporated into this, and this was introduced into a host cell to establish a method for obtaining prenyl alcohol (especially GGOH) from the culture. In bacteria, especially Escherichia coli, an enzyme vector involved in prenyl diphosphate synthesis (for example, FPP synthase mutant gene or IPPΔ-isomerase gene) is introduced into a host cell using an existing vector, and the culture is dephosphorylated. A method to obtain GGOH was established.
[0010]
[Problems to be solved by the invention]
An object of this invention is to provide the manufacturing method of a novel prenyl alcohol.
[0011]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventor has increased the productivity of prenyl alcohol by culturing prenyl alcohol-producing microorganisms under various pH controls, and is also produced during prenyl alcohol production. It was found that the production of by-products such as nerolidol and geranyl linalool can be suppressed, and the present invention has been completed.
[0012]
That is, the present invention is a method for producing prenyl alcohol, wherein prenyl alcohol is collected from a culture obtained after culturing a prenyl alcohol-producing microorganism in a medium controlled within a pH range of 6.5 to 8.0. is there.
Furthermore, the present invention provides a prenyl alcohol-producing microorganism that is cultured in a medium controlled to a pH of 6.5 to 8.0 to a logarithmic growth phase, and further cultured in a culture controlled to a pH of 7.5 to 9.0. And prenyl alcohol is collected from the resulting culture.
[0013]
Examples of prenyl alcohol include farnesol or geranylgeraniol.
Furthermore, the present invention is a prenyl alcohol-producing microorganism.
The present invention is described in detail below.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is a method for producing prenyl alcohol by culturing prenyl alcohol-producing bacteria under pH control.
[0015]
1. Microorganisms usable in the present invention
Any microorganism that can produce prenyl alcohol can be used as the microorganism that can be used in the present invention. For example, in the case of a natural microorganism, examples of such cells include the following.
(1) Saccharomyces genus;
Saccharomyces cerevisiae ATCC 12341, IFO 0565, IFO 0222, IFO 0216, ATCC 9080, IFO 1346, ATCC 204660, IFO 0538, IFO 0210, IFO 0262, ATCC64031, Saccharomyces ellipsoideus Kyoto University Conservation Strain 4102, Saccharomyces sake association 2 IFO 0252, Saccharomyces kluyveri IFO 1892, Saccharomyces unisporus IFO 0215, Saccharomyces logos Kyoto University Conservation Strain 4101, Saccharomyces bayanus IFO 0539, IFO 0613, Saccharomyces 0259
(2) genus Saccharomycopsis;
Saccharomycopsis fibuligera IFO 0106, IFO 1665, IFO 1774, IFO 0107
(3) Saccharomycodes genus;
Saccharomycodes ludwigii IFO 0339
(4) Genus Schizosaccharomyces;
Schizosaccharomyces octosporus IAM 4842, Schizosaccharomyces pombe IFO 0346, IFO 0358, Schizosaccharomyces octosporus IAM 4842
(5) Genus Wickerhamia;
Wickerhamia fluorescens IFO 1116
(6) Genus Debaryomyces;
Debaryomyces hansenii IFO 0023, Debaryomyces hansenii var.fabryi IFO 0794, Debaryomyces castellii IFO 1359, Debaryomyces vanrijiae var.vanrijiae JCM 2169
(7) Hansenula genus;
Hansenula polymorpha Kyoto University stock 4327
(8) Hanseniaspora genus;
Hanseniaspora valbyensis IFO 0115
(9) The genus Lypomyces;
Lypomyces starkeyi IFO 0678
(10) Pichia genus;
Pichia membranaefaciens IFO 0128, Pichia aganobii Kyoto University Conservation strain 4261, Pichia naganishii IFO 1670, Pichia silvicola IFO 0807, Pichia anomala IFO 0118, IFO 0569, IFO 0707, IFO 0963
(11) Genus Kloeckera;
Kloeckera japonica IFO 0151
(12) Genus Candida;
Candida krusei IFO 0013, Candida kefyr IFO 0706, Candida tenuis IFO 0716, Candida solani IFO 0762, Candida glabrata IFO 0005, IFO 0622, Candida albicans IFO 1060, IFO 0579, Candida zeylanoides IFO 0719, Candida zeylano IFO 0719, Candida zeylanoides IFO 0719, Candida , Candida stellata IFO 0701, Candida utilis IFO 0626
(13) Genus Zygosaccharomyces;
Zygosaccharomyces rouxii IFO 0487, IFO 0686, Zygosaccharomyces japanicus IFO 0595, Zygosaccharomyces fermentati IFO 0021
(14) Genus Ogataea;
Ogataea glucozyma IFO 1472, Ogataea polymorpha IFO 1475
(15) Genus Kuraishia;
Kuraishia capsulata IFO 0974
(16) Genus Komagataella;
Komagataella pastoris IFO 0948
(17) Genus Yarrowia;
Yarrowia lopolytica IFO 0717
(18) Williopsis genus;
Williopsis saturnus var.saturnus IFO 0125, IFO 0941
(19) Nakazawaea genus;
Nakazawaea holstii IFO 0980
(20) Genus Kluyveromyces;
Kluyveromyces marxianus IFO 0617, IFO 0288, Kluyveromyces thermotolerans IFO 0662, Kluyveromyces lactis IFO 0648
(21) genus Torulaspora;
Torulaspora delbrueckii IFO 0422
(22) Genus Cryptococcus);
Cryptococcus humicolus IFO 1527
(23) genus Mucor
Mucor javanicus IFO 4570
(24) Genus Citeromyces
Citeromyces matritensis IFO 0954
(25) genus Waltomyces
Waltomyces lipoder IFO 0673
(26) The genus Bacillus;
Bacillus amyloliquefaciens IFO 3022, Bacillus pumilus IFO 3030
(27) Staphylococcus genus;
Staphylococcus epidermidis IFO 3762
(28) Genus Pseudomonas;
Pseudomonas sp. 876, Kyoto University preservation stock
(29) Micrococcus genus;
Micrococcus luteus IFO 3067
(30) Exiguobacterium genus;
Exiguobacterium acetylicum IFO 12146
(31) genus Nocardia;
Norcadia asteroides IFO 3384, Norcadia fusca IFO 14340
(32) The genus Streptomyces;
Streptomyces gardneri IFO 12865
[0016]
Of these, the Saccharomyces cerevisiae ATCC64031 strain is particularly preferable. The microorganism may be a recombinant into which a gene involved in increasing prenyl alcohol productivity is introduced. A recombinant is obtained by introducing the recombinant DNA of the present invention into a host so that an HMG-CoA reductase gene or the like (including various mutant forms; the same shall apply hereinafter unless otherwise specified) can be expressed. be able to. Here, the host is not particularly limited as long as it can produce prenyl alcohol, but Escherichia coli or yeast is preferable. Recombinant DNA containing promoter, HMG-CoA reductase gene, etc. and terminator is introduced into fungi including unicellular eukaryotic microorganisms such as yeast, prokaryotes such as E. coli, animal cells, plant cells, etc. to obtain recombinants be able to. Examples of the fungi include Myxomycota, algal fungi (Phycomycetes), ascomycota (Ascomycota), basidiomycota, and incomplete fungi (Fungi Imperfecti). As fungi, those of single-cell organisms are well known as yeasts important for industrial use, and examples include ascomycete ascomycete yeast, basidiomycetous basidiomycete yeast or incomplete fungal incomplete fungal yeast. Examples of yeast include ascomycetous yeast, particularly fission yeast such as Saccharomyces cerevisiae (known as baker's yeast), Candida utilis, and Pichia pastris. Shizosaccharomyces pombe and the like are used. The strain of yeast is not particularly limited as long as prenyl alcohol can be produced. In the case of S. cerevisiae, for example, the following strains: A451, EUG8, EUG12, EUG27, YPH499, YPH500, W303-1A, W303-1B, AURGG101 and the like can be mentioned. As a method for introducing recombinant DNA into yeast, for example, an electroporation method, a spheroplast method, a lithium acetate method, or the like can be employed.
[0017]
A451 (ATCC200589, MATα can1 leu2 trp1 ura3 aro7)
YPH499 (ATCC76625, MATa ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ 200 leu2-Δ 1, Stratagene, La Jolla, CA)
YPH500 (ATCC76626, MATα ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ 200 leu2-Δ 1, Stratagene)
W303-1A (MATa leu2-3 leu2-112 his3-11 ade2-1 ura3-1 trp1-1 can1-100)
W303-1B (MATα leu2-3 leu2-112 his3-11 ade2-1 ura3-1 trp1-1 can1-100)
AURGG101 (A451, aur1 :: AUR1-C)
EUG8 (A451, ERG9p :: URA3-GAL1p)
EUG12 (YPH499, ERG9p :: URA3-GAL1p)
EUG27 (YPH500, ERG9p :: URA3-GAL1p)
[0018]
Prokaryotes include archaea and bacteria. Examples of archaea include methane producing bacteria such as Metanobacterium, halophilic bacteria such as Halobacterium, and thermoacidophilic bacteria such as Sulfolobus. Bacteria include various gram-negative bacteria or gram-positive bacteria with high industrial or academic value, such as Escherichia genus such as Escherichia coli, Bacillus subutilis, Bacillus brevis, etc. Pseudomonas genus such as Bacillus, Pseudomonas putida, Agrobacterium genus such as Agrobacterium tumefaciens and Agrobacterium rhizogenes, Corynebacterium glutamicum (Corynebacterium glutamicum) And Actinomycetes such as Lactobacillus plantarum, Lactobacillus genus such as Lactobacillus plantarum, Actinomyces genus and Streptmyces.
[0019]
When a bacterium such as Escherichia coli is used as a host, it is preferable that the recombinant DNA is autonomously replicable in the cell, and at the same time is composed of a transcription promoter, an SD sequence as a ribosomal RNA binding region, and the gene of the present invention. . A transfer terminator or the like can be appropriately inserted. Moreover, the gene which controls a promoter may be contained. Examples of E. coli include, but are not limited to, BL21, DH5α, HB101, JM101, MBV1184, TH2, XL1-Blue, Y-1088 and the like. Any transcription promoter may be used as long as it can be expressed in a host such as Escherichia coli. For example trp promoter, lac promoter, P _L Promoter, P _R A promoter derived from E. coli or phage, such as a promoter, is used. An artificially designed and modified promoter such as a tac promoter may be used. The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. For example, it is carried out by a method using calcium ions, an electroporation method, or a method using other commercially available kits.
[0020]
Whether or not a gene has been introduced into a host cell can be confirmed by PCR, Southern blot hybridization, or the like. For example, DNA is prepared from a recombinant, PCR is performed by designing introduced DNA-specific primers. After that, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., and stained with ethidium bromide, SYBR Green solution, etc. The introduced DNA is confirmed by detecting it as a single band or peak. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product.
[0021]
When prenyl alcohol is produced using the transgenic microorganism obtained as described above, by-products nerolidol and geranylgeraniol may be produced simultaneously with the production of FOH and GGOH. This is because the pH of the microorganisms decreases with the progress of microbial culture, and acid hydrolysis of farnesyl diphosphate and geranylgeranyl diphosphate occurs. Is preferentially generated. However, when the pH control of the present invention is carried out for the production of prenyl alcohol using these gene-introduced microorganisms, the production of these by-products is suppressed and the purity of the obtained prenyl alcohol is improved.
[0022]
2. PH control during culture
In the present invention, prenyl alcohol-producing microorganisms are cultured in a medium whose pH is controlled within the range of 6.5 to 8.0, whereby the productivity and purity of prenyl alcohol can be increased. In general, during culture, the pH of the medium fluctuates with the lapse of culture time. In the present invention, it is preferable to control the pH during culture to pH 6.5 to 8.0, for example, around pH 7.0 (for example, pH 7.0 to 7.5). ) Is most preferable. In order to produce prenyl alcohol at a high production rate, it is preferable to control the pH value by making the fluctuation value of the pH value as small as possible.
Further, the control of the pH value can be performed in a multi-step manner in which the pH value is changed after the logarithmic growth phase.
[0023]
A preferred pH control method of the present invention is as follows.
(1) A culture medium in which prenyl alcohol-producing bacteria are cultured in a pH 6.5-8.0 range from the beginning of the culture to the logarithmic growth phase and continue to be controlled in the pH 6.5-8.0 range after the stationary phase. (Hereinafter referred to as “one-step pH control method”). Generally, the time from the start of culture of prenyl alcohol-producing yeast to the logarithmic growth phase is about 42 to 46 hours.
[0024]
(2) The prenyl alcohol-producing bacterium is cultured in a medium controlled within the range of pH 6.5 to 8.0 from the beginning of the culture to the logarithmic growth phase. After the stationary phase, the pH value is controlled from the start of the culture to the logarithmic growth phase. The medium is cultured in a medium having a higher pH value and controlled in the range of pH 7.5 to 9.0 (hereinafter referred to as “two-stage pH control method”). Preferably, prenyl alcohol-producing bacteria are cultured in a medium controlled within the range of pH 6.5 to 7.0 from the start of culture to the logarithmic growth phase, and after the stationary phase, the pH value controlled from the start of culture to the logarithmic growth phase The medium is cultured in a medium having a higher pH value and controlled in the range of pH 7.5 to 8.0. When this two-stage pH control method is carried out, the prenyl alcohol production amount almost the same as that of the one-step pH control method is obtained, and the average number of cells of the cultured microorganisms is reduced. Increase. Since prenyl alcohol-producing bacteria are treated as waste after cultivation, the smaller the amount of cultured microorganisms, the better for industrial production. Therefore, a two-stage pH control method is useful as a method for increasing the amount of prenyl alcohol produced by suppressing the amount of cultured microorganisms. In the case of culturing that also serves the purpose of microbial growth, a one-step pH control method is useful.
[0025]
3. Reagents used for pH control
For the pH control of the medium, reagents used for adjusting the pH of the microorganism medium, for example, inorganic or organic acids, alkaline solutions, and the like can be used, and the type thereof is not particularly limited. Specifically, examples of the acid include hydrochloric acid, nitric acid, acetic acid, oxalic acid, and phosphoric acid, and examples of the alkaline solution include sodium hydroxide, potassium hydroxide, and ammonia. The pH of the medium of the present invention can be appropriately controlled by combining these pH adjusting reagents. Specifically, a combination of sodium hydroxide and hydrochloric acid is preferable. The concentration and addition amount of the acid and alkali solution to be used are not particularly limited, but it is preferably set appropriately within a range not departing from the pH control range of 6.5 to 8.0 of the present invention. Moreover, the present invention can realize the high production of prenyl alcohol according to the present invention by setting various control pH values, pH value fluctuation ranges, combinations of pH adjusting reagents, and pH control steps.
[0026]
4). Production of prenyl alcohol
In the present invention, prenyl alcohol can be obtained by culturing a prenyl alcohol-producing microorganism (including a transgenic microorganism) and collecting it from the culture. “Culture” means any of cultured cells, cultured cells themselves, or disrupted cells or cells, in addition to the culture supernatant. Examples of the prenyl alcohol include those having 15 carbon atoms such as farnesol (FOH) and those having 20 carbon atoms such as geranylgeraniol (GGOH). The prenyl alcohol accumulates in the culture either alone or as a mixture. The method for culturing the microorganism of the present invention is carried out according to a usual method.
[0027]
The medium for cultivating the microorganism may be a natural medium or a synthetic medium as long as it contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the microorganism and can be cultured efficiently. Good. Examples of the carbon source include carbohydrates such as glucose, galactose, fructose, sucrose, raffinose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol. Examples of nitrogen sources include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, or other nitrogen-containing compounds. Others include peptone, meat extract, corn steep liquor, and various amino acids. Etc. Examples of the inorganic substance include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. The culture is usually performed at 26 ° C to 36 ° C under aerobic conditions such as shaking culture or aeration and agitation culture, preferably at 30 ° C when S. cerevisiae is used as a host and at 37 ° C when E. coli is used as a host. , For a predetermined time. The culture may be performed by adding an antibiotic such as ampicillin, chloramphenicol, or aureobasidin A to the medium as necessary.
[0028]
When cultivating a recombinant into which an expression vector using an inducible transcription promoter as a promoter is cultured, an inducer may be added to the medium as necessary. For example, when the GAL1 promoter is used, galactose can be used as a carbon source. Further, when culturing a microorganism (for example, E. coli) transformed with an expression vector having a transcription promoter inducible with isopropyl-β-D-thiogalactopyranoside (IPTG), IPTG can be added to the medium.
[0029]
In the present invention, prenyl alcohol production efficiency can be increased by further adding terpenoids, fats and oils, surfactants and the like to the medium. The following can be illustrated as these additives.
Terpenoids: squalene, tocopherol, IPP, DMAPP
Fats and oils: soybean oil, fish oil, almond oil, olive oil
Surfactant: Taditol, Triton X-305, Span 85, Adecanol LG109 (Asahi Denka), Adecanol LG294 (Asahi Denka), Adecanol LG295S (Asahi Denka), Adecanol LG297 (Asahi Denka), Adecanol B-3009A (Asahi Denka), Adeka Pronic L-61 (Asahi Denka)
The fat and oil concentration is 0.01% or more, preferably 1 to 3%, the surfactant concentration is 0.005% to 1%, preferably 0.05 to 0.5%, and the terpenoid concentration is 0.01% or more, preferably 1 to 3%. is there.
[0030]
When prenyl alcohol is produced in the cells or cells after the culture, the target prenyl alcohol is collected by crushing the cells or cells by performing a homogenizer treatment or the like. Alternatively, the cells may be directly extracted with an organic solvent or the like without disrupting the cells. Alternatively, when the prenyl alcohol of the present invention is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, prenyl alcohol can be collected from the culture by extraction with an organic solvent or the like, and can be isolated and purified using various chromatographies and the like as necessary.
[0031]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
In addition, the microorganisms used, culture methods, extraction methods, analysis methods, etc. common to the following examples will be described in advance.
[0032]
1. Microorganisms used
In this example, prenyl alcohol-producing microorganism, Saccharomyces cerevisiae ATCC64031 strain, and HMG-CoA reductase gene-introduced yeast, pYHMG122 / A451 were used.
pYHMG122 / A451 was prepared as follows.
[0033]
(1) Cloning of HMG-CoA reductase gene (HMG1 'gene) by PCR
S. cerevisiae HMG1 ′ gene cloning was performed as follows.
Based on the information of S. cerevisiae HMG1 gene (accession number M22002) (ME Basson, et al., Mol. Cell. Biol. 8, 3797-3808 (1988): SEQ ID NO: 1) in GenBank A primer for the nucleotide sequence corresponding to the N-terminal and C-terminal of the protein is prepared and used as a template for a yeast cDNA library (derived from Clontech No.CL7220-1, S. cerevisiae DBY746) PCR was performed.
N-terminal primer (primer 1): 5'-ATG CCG CCG CTA TTC AAG GGA CT-3 '(SEQ ID NO: 2)
C-terminal primer (primer 2): 5'-TTA GGA TTT AAT GCA GGT GAC GG-3 '(SEQ ID NO: 3)
PCR was performed in the following reaction solution for 30 cycles, with one cycle consisting of denaturation at 94 ° C for 45 seconds, annealing at 55 ° C for 1 minute, and extension at 72 ° C for 2 minutes.
[0034]

[0035]
After PCR, the fragment was confirmed at the target position (3.2 kbp) by agarose gel electrophoresis. Therefore, the 3.2 kbp DNA fragment was cloned into a pT7Blue T vector (Novagen, Madison, WI) capable of TA cloning, and this was pT7HMG1 It was. The base sequence of the cloned HMG-CoA reductase gene was determined. As a result, the nucleotide sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 5 were confirmed. The determined base sequence was a mutated gene partially different from the base sequence shown in the GenBank sequence (FIG. 2A). Thus, a substitution mutation of a base that occurs in a DNA fragment obtained by amplifying wild-type DNA by PCR (polymerase chain reaction) using a low-fidelity DNA polymerase such as Taq DNA polymerase is called “PCR error”. The gene encoding the amino acid sequence of the mutant HMG-CoA reductase containing the PCR error (SEQ ID NO: 5) is designated as HMG1 ′.
[0036]
(2) Construction of HMG1 'expression plasmid pYES-HMG1
The pT7HMG1 prepared as described above was treated with BamHI, SalI, and ScaI to extract a gene HMG1 ′ encoding a mutant HMG-CoA reductase due to PCR error, and this was extracted from the BamHI-XhoI site of pYES2 (Invitrogen, Carlsbad, Calif.) Introduced. The obtained recombinant vector was designated as pYES-HMG1. When the nucleotide sequence in the vector was determined, it was confirmed to be the nucleotide sequence shown in SEQ ID NO: 4. PYES2 is a yeast expression shuttle vector having the yeast 2 μm DNA ori as a replication origin and the GAL1 transcription promoter inducible with galactose (FIG. 3).
[0037]
(3) Construction of deletion-type HMG1 'expression plasmid pYHMGxxy
In order to construct a deletion-type HMG-CoA reductase gene expression vector that lacks the base sequence encoding the region upstream of the portion considered to be the HMG-CoA reductase catalytic site, pYES- constructed in (2) was used. Using HMG1 as a template, a fragment was prepared by deleting a part of the HMG1 ′ coding region together with the vector portion by PCR. The resulting fragment was blunt-ended with Klenow enzyme, then recirculated by self-ligation and transformed into E. coli JM109 to prepare plasmid DNA. The synthetic DNA sequences used as primers and their combinations are shown in Table 1.
[0038]
[Table 1]

[0039]
The 373A DNA sequencer (Perkin Elmer, Foster City, CA) shows that the reading frame of the amino acids upstream and downstream of HMG1 in the obtained plasmid DNA is not shifted and that no amino acid substitution due to PCR error has occurred in the vicinity of the binding site. ) Confirmed. As a result, the following plasmid was obtained in which there was no amino acid substitution due to a PCR error in the vicinity of the binding site, and the gene could be deleted without shifting the reading frame. Deletion type HMG1 gene is described as Δ02y (y represents an arbitrary work number) according to the deletion pattern, and pYES2 vector containing Δ02y is described as, for example, pYHMG026 (the same applies to other deletions) (Figure 2B).
[0040]
HMG1Δ02y: SEQ ID NO: 6
HMG1Δ04y: SEQ ID NO: 7
HMG1Δ05y: SEQ ID NO: 8
HMG1Δ06y: SEQ ID NO: 9
HMG1Δ07y: SEQ ID NO: 10
HMG1Δ08y: SEQ ID NO: 11
HMG1Δ10y: SEQ ID NO: 12
HMG1Δ11y: SEQ ID NO: 13
HMG1Δ12y: SEQ ID NO: 14
HMG1Δ13y: SEQ ID NO: 15
Vector: pYHMG026, pYHMG027, pYHMG044, pYHMG045, pYHMG062, pYHMG063, pYHMG065, pYHMG076, pYHMG081, pYHMG083, pYHMG085, pYHMG094, pYHMG100, pYHMG106, pYHMG106, pYHMG106 Of these, vector pYHMG122 was used in the test of the present invention.
[0041]
(4) Preparation of recombinant pYES-HMG1
Recombinant pYHMG122 / A451 was obtained by introducing the vector pYHMG122 into the yeast Saccharomyces cerevisiae A451 strain, which was found to produce FOH in a preliminary experiment. The vector pYHMG122 was introduced into the host strain A451 by the lithium acetate method described in Current Protocols in Molecular Biology, John Wiley & Sons, Inc., pp. 13.7.1-13.7.2, or Frozen-EZ Yeast Transformation II ( Zymo Research, Orange, CA) was used (in accordance with the instructions included in the kit).
[0042]
2. Microbial culture method
(1) Culture method of Saccharomyces cerevisiae ATCC64031 strain
・ Jar fermenter culture
One platinum ear of Saccharomyces cerevisiae ATCC64031 strain was inoculated into YM broth (200 ml / 500 ml-conical flask with baffle) containing ergosterol (20 mg / L), and cultured at 26 ° C., 130 rpm for 2 days to prepare Pre-Culture. From this, 50 ml of the bacterial solution was aseptically inoculated into a fermenter and cultured under the following culture conditions.
[0043]
Culture device: MSJ-U 10L culture device (Maruhishi Bio-Engineering)
Working Volume: 5L
Culture temperature: 26 ℃
Airflow: 1vvm
Agitation: 300rpm
pH: Proportional control of pH using 4N sodium hydroxide solution and 2N hydrochloric acid solution with the following parameters
Proportional Band 1.00
Non Sensitive Band 0.15
Control Period 16 Sec
Full Stroke 1 Sec
Minimun Stroke 0 Sec
Medium: YM medium containing 6% glucose (Difco)
0.1 or 1% soybean oil
0.1% Adecanol LG109 (Asahi Denka)
4mg / L Ergosterol (Nacalai)
(At this time, 100mg / L of ethanol and 100mg / L of Taditol (Nacalai) are brought in by adding ergosterol)
[0044]
(2) Transgenic yeast Transgenic method of transgenic yeast pYHMG122 / A451
・ Jar fermenter culture
One platinum ear of the recombinant yeast pYHMG122 / A451 was inoculated into the following Pre-Culture medium (conical flask with 200 ml / 500 ml-baffle) and cultured at 30 ° C., 130 rpm for 2 days. Next, in order to completely remove glucose contained in the culture solution, centrifugation (1500 g, 5 minutes, 4 ° C.) and washing with sterilized physiological saline were repeated three times. 1%). Jar fermenter culture was performed under the same culture conditions as the ATCC 64031 strain of (1).
[0045]
Pre-Culture medium:
CSM-URA (manufactured by BIO 101 Inc.)
DOB (manufactured by BIO 101 Inc.)
Fermenter medium:
5% galactose
YNB without Amino Acids (Difco)
1% soybean oil (Nacalai)
0.1% Adecanol LG109 (Asahi Denka)
[0046]
3. Extraction method
(1) Fractionated extraction of farnesol and geranylgeraniol
(1) Extraction of farnesol and geranylgeraniol from the supernatant fraction
2.5 ml of the culture solution was put into an 18Φ mm × 125 mm test tube, centrifuged at 1000 rpm for 5 minutes with a centrifuge GP centrifuge manufactured by Beckman, and the supernatant was transferred to a new 18Φ mm × 125 mm test tube. 0.5 ml of Tris-HCl buffer (pH 8.0) containing 6 mM magnesium chloride and 5 μl (2 units) of Escherichia coli alkaline phosphatase (Takara Shuzo) were added and heated at 65 ° C. for 30 minutes. After sufficiently cooling on ice, 2 ml of pentane and 1 ml of methanol were added. After thorough mixing, the mixture was centrifuged at 1000 rpm for 5 minutes with a centrifuge GP centrifuge manufactured by Beckman, and the supernatant was transferred to another new test tube. After evaporating the pentane / methanol solvent in a fume hood, it was redissolved in 300 μl of pentane and filled in a GC / MS vial.
[0047]
(2) Extraction of farnesol and geranylgeraniol from the bacterial cell fraction
1) In case of bacteria
10 ml of the liquid culture solution was put into a 50 ml Corning tube and centrifuged at 6000 rpm for 5 minutes with a Beckman cooling centrifuge (Avant J25-I) to collect the cells. The cells were suspended in 0.5 ml of deionized water, transferred to a 10 ml Spitz tube, and crushed with an ultrasonic cell crusher UCW-201 manufactured by Tokai Electric Co., Ltd. (Condition: 10 ° C, 1 min disruption-30 sec stop Repeat for 20 minutes). The sample was transferred to an 18Φ mm × 125 mm test tube, 0.5 ml of Tris-HCl buffer (pH 8.0) containing 6 mM magnesium chloride was added, and phosphatase treatment and extraction were performed in the same manner as in the above (1).
[0048]
2) For yeast and filamentous fungi
A liquid culture solution (2.5 ml) was placed in an 18Φ mm × 125 mm test tube and centrifuged at 1000 rpm for 5 minutes in a centrifuge GP centrifuge manufactured by Beckman, and the cells were collected. After adding 0.5 ml of Tris-HCl buffer (pH 8.0) containing 6 mM magnesium chloride to suspend the cells, the cells were transferred to a crushing glass tube. An equal amount of glass beads (acid washed 425Φ-600 μm manufactured by Sigma) was added and crushed with Multi-Beads Shocker MB-200 manufactured by Yasui Machinery Co., Ltd. (conditions: room temperature, 2500 rpm, 20 minutes). The entire amount was transferred to an 18Φ mm × 125 mm test tube, and phosphatase treatment and extraction were performed in the same manner as in the above (1).
In each of the above treatments, the system that does not perform the phosphatase treatment omits the enzyme treatment step.
[0049]
(2) Total extraction of farnesol and geranylgeraniol
After 1.25 ml of methanol (Nacalai) and 2 ml of pentane (Nacalai) were added to 2 ml of the culture solution and vortexed sufficiently, the whole test tube was centrifuged (CP from Beckman, 1500 rpm, 5 minutes, room temperature). The pentane layer was carefully transferred to a GC / MS vial with a Pipetteman or Pasteur pipette. At this time, 10 μl undecanol solution (1 mg / ml ethanol) as an internal standard was placed in a vial in advance. Since pentane volatilizes when stored for a long time at room temperature, it was sealed with an aluminum cap and stored at −80 ° C. until analysis.
[0050]
4). Analysis method
(1) Analysis of farnesol and geranylgeraniol
The geranylgeraniol and farnesol of the culture supernatant and the bacterial cell fraction were analyzed by GC / MS. Analysis was performed under the following analysis conditions to reduce the background of the column.
[0051]

The conditions analyzed by FID-GC for the purity test are as follows. Since the internal standards were different, the pentane solution before undecanol was added in the farnesol and geranylgeraniol total extraction operation was analyzed.
[0052]

[0053]
(2) Cell mass measurement (OD)
100 μl of the culture solution was diluted with physiological saline, and OD600 nm was measured with a spectrophotometer.
[0054]
(3) Bacterial mass measurement (weight)
A 50 ml tube (manufactured by Corning) dried under reduced pressure at 105 ° C. for 2 hours was weighed with a precision balance (A). 10 ml of the culture solution was placed in the same tube and centrifuged (4300 g, 10 minutes), the supernatant was removed, and the weight of the tube was measured (B). Next, the cell pellet was dried under reduced pressure (80 ° C., 3 hours), and the tube weight was measured again (C). The amount of cells per liter of the culture solution was calculated by the following formula.
Wet cell weight (g / L-broth) = 100 × (BA)
Dry cell weight (g / L-broth) = 100 × (CA)
The relationship between OD660nm and the cell weight was as follows.
1O.D.660nm = 2.14g / L-wet cell weight
1O.D.660nm = 0.305g / L-dry cell weight
[0055]
(4) Cell count measurement
100 μl of the culture solution was diluted 1 to 20 times with physiological saline, and the number of cells was counted with a hemocytometer (Hayashiri Chemical). The number of bacteria in 0.06 mm square (for the minimum 9 grids) was averaged, and the number of bacteria per liter of culture was calculated from the following formula.
Number of bacteria (10 ⁹ cell / L-broth) = 0.444 x (0.06 mm square cell count) x dilution factor
[0056]
(5) Measurement of ethanol, acetic acid, glycerol and succinic acid
Using F-kit (manufactured by JK International), ethanol, acetic acid, glycerol, succinic acid and pyruvic acid were analyzed according to the attached manual.
[0057]
[Example 1] Effect of pH control during culture on prenyl alcohol production
(1) Effect of neutral pH control
Using Saccharomyces cerevisiae ATCC64031 strain, the influence of pH control of the culture solution on prenyl alcohol production was examined.
Since the growth of yeast is usually in the acidic region, in this example, first, the pH of the culture solution is controlled to be neutral, and the resulting FOH (total extraction), glucose in the culture solution, ethanol, acetic acid, glycerol, The acid variation was examined. The result is shown in FIG.
[0058]
As apparent from FIG. 4, when the culture solution was controlled at pH 7, the production amount of FOH increased with the culture time, and the FOH amount reached about 160 mg / L after 150 hours of culture (FIG. 4, right panel). On the other hand, when the pH of the culture solution was not controlled, the production amount of FOH reached about 90 mg / L 150 hours after culturing, but there was no increase in the subsequent production amount (FIG. 4, left panel). Therefore, in the production of prenyl alcohol, it was confirmed that it was effective to control the pH to near neutral from the beginning of the culture to the logarithmic growth phase. This is because the growth of microorganisms is reduced by controlling the culture solution to around pH 7.0, and acetyl-CoA and NADPH generated by the decomposition of sugar are no longer used for the growth of microorganisms. It is considered to be.
[0059]
(2) Influence of weak alkaline pH control
Furthermore, the control pH range was set to weak alkaline pH 8, and the productivity of FOH was examined including the effect of phosphatase treatment. In addition, regarding whether or not the control of pH is effective even if it is started in the middle of the culture, pH 7.0 for 46 to 61 hours after culture, pH 8.0 for 61 to 70 hours, pH 9.0 for 70 to 84.5 hours And controlled to test. The result is shown in FIG.
[0060]
When the culture solution is pH-controlled to one step, the production of FOH increases with the incubation time (panel in Fig. 5), whereas when the pH is controlled to one step of weakly alkaline pH 8, the production of FOH is almost confirmed. Not (FIG. 5, lower panel). In addition, when pH is not controlled at the start of culture for medium that has become pH 6.0 to 6.5 after sterilization, if the pH is controlled to be weakly alkaline after 46 hours after the logarithmic growth phase, FOH increases with the passage of culture time. Production amount reached about 36mg / L.
[0061]
Furthermore, in order to confirm the control effect in the range of pH 7 to pH 8, the control pH value was set to 7.0, 7.5, 8.0 with 4N sodium hydroxide and tested. As for pH 8.0, it is clear from the above test that production of FOH is not observed even when the pH is controlled to 8.0 from the start of the culture. Therefore, from the start of the culture to the logarithmic growth phase (from the start of the culture). About 42 hours), the pH was controlled to 7, and then the pH was changed to 8.0 after entering the stationary phase. In addition, the change in the average number of cells with the culture time was also examined. The result is shown in FIG.
[0062]
When the pH of the culture solution was controlled at one step, FOH production increased with the passage of culture time at pH 7.0 and pH 7.5, and a pH control effect was observed (FIG. 6, upper and middle panels). . On the other hand, in the case of the two-stage pH control method, which is controlled to pH 7.0 from the beginning of the culture to the logarithmic growth phase (about 42 hours from the start of the culture) and then changed to 8.0 after entering the stationary phase. Obtained the same FOH productivity as the one-step pH control method at pH 7.0 (FIG. 6, lower panel). Moreover, in this case, since the average number of cells was smaller than that in the one-step pH control method at pH 7.0, it was revealed that the FOH productivity per cell was high. In prenyl alcohol production, since the cultured microorganisms are treated as waste after the product is obtained, the amount of cultured microorganisms is preferably as small as possible for industrial production. Therefore, the two-stage pH control method that controls the pH value from the beginning of culture to the logarithmic growth phase to pH 7.0 and then changes the pH value to weak alkalinity after the stationary phase is a viewpoint of FOH productivity and the amount of cultured microorganisms. It is considered very useful.
[0063]
In addition, in the above test, the production amount of various terpenoids (nerolidol, farnesol, geranylgeraniol, squalene), changes in the amount of glucose, ethanol, acetic acid, glycerol, succinic acid, and the number of bacteria in the culture solution were examined with the passage of the culture time. The results are shown in Table 2.
[0064]
[Table 2]

[0065]
As for FOH production, almost the same amount of FOH is produced when the one-step pH control method at pH 7 is used and when the two-step pH control method is used by changing the pH from 7 to 8 after the stationary phase. When the one-step pH control method at pH 7.5 was performed, the production amount was slightly reduced. In addition, regarding the production of geranylgeraniol (GGOH), when the one-step pH control method at pH 7, the one-step pH control method at pH 7.5, and the pH was changed from 7 to 8 after the stationary phase. In both cases, the same amount of GGOH production was confirmed. In addition, when comparing the FOH production efficiency per number of microorganisms with the progress of culture, the most efficient was the case where the two-step pH control method was performed, and then the one-step pH control method at pH 7 was performed. The results were almost equal to the case of the one-step pH control method at pH 7.5.
[0066]
(3) Effects of phosphatase treatment
Farnesol is produced by phosphatase acting on farnesyl diphosphate. Therefore, in the pH control of prenyl alcohol production, the effect of phosphatase treatment on the FOH production in the bacterial cell fraction and the culture broth fraction was examined.
That is, when culturing the ATCC64031 strain with a jar fermenter, the phosphatase treatment effect was examined by changing the medium composition of the culture solution and the cells when the pH was not controlled and when the pH was controlled at 7.0. The results are shown in Tables 3 and 4.
[0067]
[Table 3]

[0068]
[Table 4]

[0069]
When the pH in Table 3 was not controlled and the pH was controlled to 7.0, when the FOH production was compared with the presence or absence of phosphatase treatment of the culture medium and the cells, there was a marked difference in FOH production due to phosphatase treatment. I couldn't. On the other hand, in Table 4, when the culture was performed while controlling the pH to 7.0, the FOH production amount of the cell fraction treated with phosphatase increased significantly with the passage of the culture time. The difference in the culture conditions between Table 3 and Table 4 is 0.1% and 1% of the concentration of soybean oil to be added. Therefore, in the method of culturing while controlling pH, it is possible to increase the FOH production amount by combining phosphatase treatment with culturing in a medium containing fats and oils such as soybean oil at an appropriate concentration.
[0070]
[Example 2] Effect of pH control on prenyl alcohol production by recombinant microorganisms
When the substance is produced by farnesol and geranylgeraniol-producing recombinant microorganisms, the productivity of farnesol is low, and isomeric nerolidol and geranyl linalool that cannot be easily separated from farnesol are produced at the same time. Since these by-products nerolidol and geranyl linalool are not easily separated from farnesol, etc., there is a problem that farnesol production by microorganisms that produce these by-products is not suitable for mass production. At the same time, the pH drops and acid hydrolysis of farnesyl diphosphate and geranylgeranyl diphosphate occurs, leading to preferential production of rearrangement reaction products, nerolidol and geranyl linalool, due to the stability of the carbonium cation of the reaction intermediate. This is probably because of this.
[0071]
Accordingly, the effect of pH control during culture was examined using the HMG-CoA reductase gene-introduced yeast pYHMG122 / A451 that produces nerolidol and geranyl linalool. That is, pYHMG122 / A451 was cultured in a YNB medium containing 5% galactose, 1% soybean oil and 0.1% adecanol, and glucose having a final concentration of 5% was added at 144 hours. At this time, the pH was controlled at 7.0 with 4N NaOH and 2N HCl. The control was without pH control. The results are shown in FIG.
[0072]
[Table 5]

[0073]
From FIG. 7 and Table 5, when pYHMG122 / A451 was cultured without pH control, the FOH amount was 122.7 mg / L after 157 hours from the start of the culture, confirming an increase in FOH production. At the same time, production of the by-product nerolidol also increased (FIG. 7, upper panel). On the other hand, when the culture was controlled at pH 7.0, the FOH amount was 145.7 mg / L after 157 hours from the start of the culture, and the FOH production increased, but the production of the byproduct nerolidol was almost suppressed ( FIG. 7 lower panel). Therefore, when producing prenyl alcohol using microorganisms that produce by-products such as nerolidol, the production of by-products is suppressed by controlling the pH from neutral to weakly alkaline, and the purity of prenyl alcohol is increased. It was confirmed that it was possible.
[0074]
【The invention's effect】
The present invention provides a method for efficiently producing prenyl alcohol. According to the present invention, prenyl alcohol can be obtained efficiently and in large quantities by culturing a microorganism that produces biologically active prenyl alcohol in a medium whose pH is controlled in the range from near neutral to weakly alkaline. Can do. In addition, when the method of the present invention is applied to a prenyl alcohol-producing gene-introduced microorganism, the production of byproducts such as nerolidol and geranylgeraniol produced during prenyl alcohol production is suppressed, and the purity of prenyl alcohol can be increased. .
[0075]
[Sequence Listing]

[0076]
[Sequence Listing Free Text]
Sequence number 2: Synthetic DNA
Sequence number 3: Synthetic DNA
Sequence number 16: Synthetic DNA
Sequence number 17: Synthetic DNA
Sequence number 18: Synthetic DNA
Sequence number 19: Synthetic DNA
Sequence number 20: Synthetic DNA
SEQ ID NO: 21: synthetic DNA
Sequence number 22: Synthetic DNA
Sequence number 23: Synthetic DNA
Sequence number 24: Synthetic DNA
Sequence number 25: Synthetic DNA
SEQ ID NO: 26: synthetic DNA
[Brief description of the drawings]
FIG. 1 is a diagram showing the metabolic pathway of mevalonate pathway-related enzymes.
FIG. 2 is a diagram showing the construction of a deletion-type HMG1 gene and the pattern of substitution mutations.
FIG. 3 shows plasmid pYES2.
FIG. 4 is a diagram showing the pH control effect in FOH production by Saccharomyces cerevisiae ATCC64031 strain.
FIG. 5 is a graph showing the pH control effect in a medium to weak alkaline region in FOH production by Saccharomyces cerevisiae ATCC64031 strain.
FIG. 6 is a graph showing the pH control effect and average cell number in the medium to weak alkaline region in FOH production by Saccharomyces cerevisiae ATCC64031 strain.
FIG. 7 shows the pH control effect in FOH production by the transgenic yeast pYHMG122 / A451 strain.

Claims (6)

A method for producing farnesol , comprising culturing yeast having prenyl alcohol-producing ability in a medium controlled within a range of pH 7.0 to 7.5 , and collecting farnesol from the obtained culture. The production method according to claim 1 , wherein the pH is controlled before the logarithmic growth phase of the yeast having the prenyl alcohol-producing ability . After yeast having prenyl alcohol-producing ability is cultured in a medium controlled to a pH range of 6.5 to 7.0 until the logarithmic growth phase, the yeasts are further controlled to a pH range of 7.5 to 8.0. method for producing farnesol, characterized in that in cultured, collected farnesol from the resulting culture. The process according to any one of claims 1 to 3 for adjusting the control of the pH with sodium hydroxide. The said yeast is Saccharomyces yeast, The manufacturing method in any one of Claims 1-4 characterized by the above-mentioned. The method according to any one of claims 1 to 4, wherein the yeast is Saccharomyces cerevisiae.

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