JP4401471B2 - Method for producing isoprenoid compound by microorganism - Google Patents

Description

【０１３９】
ＣｏＱ８の生成量は、コントロール株DH5α／pEG400と比較し、DH5α／pADO-1、DH5α／pDXS-1およびDH5α／pXSE-1では有意に高かった。特に、実施例１で取得したＤＮＡを全て導入したDH5α／pADO-1において最も高い生産性が得られた。
【０１４０】
（２）Ｍ９培地を１０ｍｌ入れた試験管に、上記（１）で取得したE. coli DH5α／pDXS-1またはE. coli DH5α／pEG400をそれぞれ植菌し、３０℃で７２時間振盪培養した。
培養終了後、上記（１）と同様の方法により形質転換体によるＣｏＱ８の生産量を算定した。
結果を第２表に示す。
【０１４１】
【表２】

【０１４２】
ＣｏＱ８の生成量は、コントロール株DH5α／pEG400と比較し、DH5α／pDXS-1では有意に高かった。
（３） 組換え大腸菌によるＣｏＱ８の生産
実施例１で取得したプラスミドｐＥＧＹＭ１またはコントロールとしてｐＥＧ４００をE. coli DH5α株に導入し、１００μｇ／ｍｌ濃度のスペクチノマイシンに抵抗性を示す形質転換体E. coli DH5α／pEGYM1およびE. coli DH5α／pEG400を各々取得した。
【０１４３】
グルコース１％、ビタミンＢ₁ １００ｍｇ／ｌ、ビタミンＢ₆ １００ｍｇ／ｌ、ｐ−ハイドロキシ安息香酸５０ｍｇ／ｌ添加したＬＢ培地を１０ｍｌ入れた試験管にこれら形質転換体を植菌し、３０℃で７２時間振盪培養した。
培養終了後、上記（１）と同様の方法により形質転換体によるＣｏＱ８の生産量を算定した。
結果を第３表に示す。
【０１４４】
【表３】

【０１４５】
ＣｏＱ８の生成量は、コントロール株DH5a／pEG400と比較し、DH5a／pEGYM1では有意に高かった。
【０１４６】
実施例３ 組換え大腸菌によるメナキノンー８(ＭＫ−８)の生産
（１）スペクチノマイシンを１００μｇ／ｍｌ添加したＴＢ培地〔バクトトリプトン(ディフコ社製）１２ｇ、酵母エキス（ディフコ社製）２４ｇ、グリセロール５ｇを水９００ｍｌに溶解し、ＫＨ₂ＰＯ₄を０．１７Ｍ、Ｋ₂ＨＰＯ₄を０．７２Ｍ含有する水溶液を１００ｍｌ加えて調製した培地〕を１０ｍｌ入れた試験管に、実施例２（１）で取得した、E. coli DH5α／pADO-1またはE. coli DH5α／pEG400をそれぞれ植菌し、３０℃で７２時間振盪培養した。
【０１４７】
培養終了後、実施例２（１）のＣｏＱ８の定量法と同様の方法によりＭＫ−８を定量し、形質転換体によるＭＫ−８の生産量を算定した。
結果を第４表に示す。
【０１４８】
【表４】

【０１４９】
ＭＫ−８の生産量は、コントロール株DH5α／pEG400と比較して、DH5α／pADO-1では有意に高かった。
（２）実施例２（１）で取得したE. coli DH5α／pDXS-1またはE. coli DH5α／pEG400を、上記（１）と同様の方法で培養し、形質転換体によるＭＫ−８の生産量を算定した。
結果を第５表に示す。
【０１５０】
【表５】

【０１５１】
ＭＫ−８の生産量は、コントロール株DH5α／pEG400と比較して、DH5α／pDXS-1では有意に高かった。
【０１５２】
実施例4 Erwinia carotovoraによるＣｏＱ８の生産
実施例１で取得したプラスミドｐＤＸＳ−１またはコントロールとしてｐＥＧ４００をErwinia carotovora IFO-3380株に導入し、１００μｇ／ｍｌ濃度のスペクチノマイシンに抵抗性を示す形質転換体IFO-3380／pDXS-1およびIFO-3380／pEG400を取得した。
【０１５３】
スペクチノマイシンを１００μｇ／ｍｌ添加したＬＢ培地を１０ｍｌ入れた試験管にこれら形質転換体を植菌し、３０℃で７２時間振盪培養した。
培養終了後、実施例２（１）と同様の方法により形質転換体によるＣｏＱ８の生産量を算定した。
結果を第６表に示す。
【０１５４】
【表６】

【０１５５】
ＣｏＱ８の生成量は、コントロール株IFO-3380／pEG400と比較し、IFO-3380／pDXS-1では有意に高かった。
【０１５６】
実施例５：Erwinia uredovoraによるユビキノンおよびカロテノイドの生産
実施例１で取得したプラスミドpUCYM-1、pQEDXS-1、pQEYM-1またはコントロールとしてpUC19およびpQE30をエレクトロポレーション法によりErwinia uredovor a DSM-30080株に導入し、１００μｇ／ｍｌ濃度のアンピシリンに抵抗性を示す形質転換体E. uredovora DSM-30080／pUCYM-1、E. uredovora DSM-30080／pQEDXS-1、E. uredovora DSM-30080／pQEYM-1、E. uredovora DSM-30080／pUC19およびE. uredovora DSM-30080／pQE30を取得した。
【０１５７】
アンピシリン１００μｇ／ｍｌ、グルコース１％、ビタミンＢ₁ １００ｍｇ／ｌ、ビタミンＢ₆ １００ｍｇ／ｌ、ｐ−ハイドロキシ安息香酸５０ｍｇ／ｌ添加したＬＢ培地を１０ｍｌ入れた試験管にこれら形質転換体を植菌し、３０℃で７２時間振盪培養した。
【０１５８】
培養終了後、実施例２（１）と同様の方法により形質転換体によるＣｏＱ８の生産量を算定した。
カロテノイド色素の生産量は、実施例２（１）と同様の方法により得られた２−ブタノール層を分光光度計を用い、４５０ｎｍの吸光度を測定することにより算出した。
結果を第７表に示す。
【０１５９】
【表７】

【０１６０】
ＣｏＱ８の生産量およびカロテノイド色素の生産量ともに、コントロール株DSM-30080/pUC19と比較し、DSM-30080/pUCYM-1では有意に高かった。
同様に、ＣｏＱ８の生産量およびカロテノイド色素の生産量ともに、コントロール株DSM-30080/pQE30と比較し、DSM-30080/pQEYM-1およびDSM-30080/pQEDXS-1では有意に高かった。
【０１６１】
実施例６ 光合成細菌Rhodobacter sphaeroidesからのイソプレノイド化合物の生合成に関与する蛋白質をコードするＤＮＡの取得
（１）R．sphaeroidesからのＤＸＳ遺伝子の取得
大腸菌で見出されたDXS遺伝子配列を用いて、他生物種で保存されているDXSホモログをgenbankより検索した。その結果、Haemophilus influenzae(P45205)、Rhodobacter capsulatus(P26242)、Bacillussubtilis(P54523)、Synechocystis sp. PCC6803(P73067)およびMycobacteriun tuberculosis(O07184)等でホモログが見出された。これら配列を比較して、高度に保存されているアミノ酸配列を選択した。保存アミノ酸配列に対応する塩基配列をR. sphaeroidesのコドン使用頻度を考慮にいれて設計し、センスプライマーとして配列番号３２および配列番号３３に記載の塩基配列を有するＤＮＡ断片を、アンチセンスプライマーとして配列番号３４を有するＤＮＡ断片をＤＮＡ合成機を用いて合成した。
【０１６２】
R．sphaeroides KY4113株（FERM-P4675）の染色体ＤＮＡを鋳型として、上記プライマーと、Expand^TM High-Fidelity PCR System(ベーリンガー・マンハイム社製)を用い、ＤＮＡThermal Cycler(パーキンエルマージャパン社製)でＰＣＲを行った。
ＰＣＲは、９４℃で４０秒間、６０℃で４０秒間、７２℃で１分間からなる反応工程を１サイクルとして、３０サイクル行った後、７２℃で７分間反応させる条件で行い、目的とするＤＮＡ断片を取得した。このＤＮＡ断片をDIG DNA Labeling Kit（ベーリンガー・マンハイム社製）を用いてＤＩＧ標識した。
【０１６３】
R. sphaeroidesのＤＸＳ遺伝子全長を取得するため、ＫＹ４１１３株のゲノムライブラリーを作成した。ＫＹ４１１３株をＬＢ培地で一晩培養し、染色体ＤＮＡを抽出した。制限酵素Ｓａｕ３ＡＩで部分消化してショ糖密度勾配超遠心法により４〜６ｋｂのＤＮＡ断片を精製した。該ＤＮＡ断片とＢａｍＨＩ消化したベクターｐＵＣ１９をLigation Pack(ニッポンジーン社製)を用いて連結し、大腸菌ＤＨ５αに形質転換した。形質転換体をアンピシリン１００μｇ／ｍｌを含むＬＢプレートに塗布し、約１００００個のコロニーを得た。
【０１６４】
上記方法で取得したＤＩＧ標識ＤＮＡ断片をプローブとして、コロニーハイブリダイゼーション法によるスクリーニングをしたところ、２種類のＤＮＡ断片が検出された。各々をシークエンスしたところ、それぞれのＤＮＡ断片から他生物種の既知ＤＸＳ遺伝子と相同性の高いＯＲＦが見出された。配列番号２６に示すアミノ酸配列をＤＸＳ１、配列番号２８に示すアミノ酸配列をＤＸＳ２と命名した。
【０１６５】
（２）大腸菌ＤＸＳ遺伝子欠損株を用いた相補性確認
▲１▼ 大腸菌ＤＸＳ遺伝子欠損株の取得
E. coli W3110株（ATCC14948）を、ＬＢ液体培地に植菌し、対数増殖期まで培養した。培養後、得られた培養液より遠心分離により菌体を取得した。
該菌体を、０．０５Ｍトリスーマレイン酸緩衝液（ｐＨ６．０）で洗浄後、菌体濃度が１０⁹細胞／ｍｌになるように同緩衝液に懸濁した。
【０１６６】
該懸濁液にＮＴＧを終濃度が６００ｍｇ／ｌになるように加え、室温で２０分間保持して変異処理を行った。
得られた変異処理菌体を１−デオキシキシルロース０．１％を含むＭ９最少寒天培地〔モレキュラー・クローニング第二版〕プレートに塗布して培養した。１−デオキシキシルロースは、J. C. S. Perkin Trans I, 2131-2137 (1982)に記載の方法に準じて化学合成した。
【０１６７】
１−デオキシキシルロース０．１％を含むＭ９最少寒天培地上で生育してきたコロニーを、Ｍ９最少寒天培地と１−デオキシキシルロースを０．１％含むＭ９最少寒天培地にレプリカし、１−デオキシキシルロースを０．１％含むＭ９最少寒天培地では生育できるが、Ｍ９最少寒天培地では生育できない、１−デオキシキシルロース要求性を示す株を目的の変異株として選択した。
【０１６８】
該方法で選択され、取得された変異株をＭＥ１株と命名した。
該ＭＥ１株にｐＤＸＳ−１を導入したところ、１−デオキシキシルロース要求性を相補したことから、ＭＥ１株はＤＸＳ遺伝子が欠損した株であると判断した。
【０１６９】
（３）ＤＸＳ１およびＤＸＳ２の相補性試験
ＫＹ４１１３株由来の、配列番号２７からなるＤＸＳ１をコードするＤＮＡ断片、および配列番号２９からなるＤＸＳ２をコードするＤＮＡ断片を各々ベクターｐＵＣ１９のｌａｃプロモーター下流に連結したプラスミドを構築した。
【０１７０】
これら構築したプラスミドをそれぞれＭＥ１株に導入したところ、ＤＸＳ１およびＤＸＳ２いずれもＭＥ１株の１−デオキシキシルロース要求性を相補した。
以上のことから、R. sphaeroidesはピルビン酸とグリセルアルデヒド３−リン酸から１−デオキシ−Ｄ−キシルロース５−リン酸を生成する反応を触媒する活性を有するＤＸＳ１およびＤＸＳ２のＤＸＳ遺伝子を2つ持つことが判明した。
【０１７１】
（４）R.sphaeroides由来メチルエリスリトール要求性相補遺伝子の取得実施例１（２）▲１▼で得られた大腸菌メチルエリスリトール要求性変異株ＭＥ７株を、メチルエリスリトール０．１％含むＬＢ液体培地に植菌して対数増殖期まで培養した後、遠心分離して菌体を回収した。
【０１７２】
該菌体を１０％グリセロールを含む１ｍＭＨＥＰＥＳ水溶液で２回洗浄して培地成分を可能な限り除去した。
該洗浄菌体に、実施例６（１）で作成したR.sphaeroides KY4113株のゲノムライブラリーから抽出したプラスミドを、常法に従い、エレクトロポレーションにより導入した。
【０１７３】
導入後、アンピシリン１００μｇ／ｌを含むＬＢ寒天培地に塗布し、３７℃で一晩培養した。
生育してきたコロニーを釣菌し、ＬＢ液体培地に植菌して培養し、該培養菌体よりプラスミドを抽出した。
【０１７４】
抽出したプラスミドをＭＥ７株に再導入したところ、メチルエリスリトールを含まない培地でも生育したことから、抽出したプラスミドにR.sphaeroides由来のメチルエリスリトール要求性を相補するＤＮＡ断片が含まれていることを確認した。
本ＤＮＡ断片の塩基配列を決定した結果、配列番号３１からなる、大腸菌yaeMと相同性の高いアミノ酸配列をコードするＤＮＡ配列が見出された。
【０１７５】
実施例７ 組換え光合成細菌によるユビキノンー１０（ＣｏＱ１０）の生産
実施例６で取得した配列番号２７からなるＤＮＡ断片ＤＸＳ１および配列番号２９からなるＤＮＡ断片ＤＸＳ２の上流に、KY4113株由来のglnBプロモーターを連結し、広宿主域ベクターpEG400に挿入して作成したプラスミドをpRSDX-1およびpRSDX-2と命名した。また、yaeMとＤＸＳ１をタンデムに連結し、glnBプロモーター下流に連結したプラスミドを構築し、pRSYMDX1と命名した。これらプラスミドを、エレクトロポレーション（Bio-Rad社製）によりR．sphaeroides KY4113株に導入した。
【０１７６】
導入後、１００μｇ／ｍｌ濃度のスペクチノマイシンを含むＬＢ寒天培地に塗布し、３０℃で３日間培養した。
生育してきたコロニーを１００μｇ／ｍｌ濃度のスペクチノマイシンを含むＬＢ培地に植菌して一晩培養後、培養菌体を遠心分離により回収した。
【０１７７】
得られた菌体よりプラスミド抽出（Qiagen社製）し、各々導入したプラスミドを保持していることを確認した。このようにして得られた形質転換体をKY4113/pRSDX-1、KY4113/pRSDX-2、KY4113/pRSYMDX1およびKY4113/pEG400と命名した。
種培地〔グルコース２％、ペプトン１％、酵母エキス１％、ＮａＣｌ０．５％（ｐＨ７．２、ＮａＯＨで調整）〕を５ｍｌ入れた試験管に各形質転換体を一白金耳植菌し、３０℃で２４時間培養した。
【０１７８】
得られた培養液０．５ｍｌを、ユビキノンー１０生産培地（廃糖蜜４％、グルコース２．７％、コーンスチープリカー４％、硫酸アンモニウム０．８％、リン酸１カリウム０．０５％、リン酸２カリウム０．０５％、硫酸マグネシウム・７水和物０．０２５％、硫酸第一鉄・７水和物３ｍｇ／ｌ、チアミン８ｍｇ／ｌ、ニコチン酸８ｍｇ／ｌ、トレースエレメント１ｍｌ／ｌを含む培地をｐＨ９に調整後、炭酸カルシウム１％を添加してオートクレーブ滅菌したもの）を５ｍｌ入れた試験管に添加し、３０℃で５日間振盪培養した。
培養終了後実施例２（１）のＣｏＱ８の定量法と同様の方法により、形質転換体によるＣｏＱ１０の生産量を算定した。結果を表８に示す。
【０１７９】
【表８】

【０１８０】
ＣｏＱ１０生成量は、コントロール株KY4113/pEG400と比較しKY4113/pRSDX-1、KY4113/pRSDX-2およびKY4113/pRSYMDX1では有意に高かった。
【０１８１】
実施例８ yaeM遺伝子がコードする酵素の活性測定
（１）yaeM遺伝子の高発現化
ｙａｅＭ遺伝子を十分発現させるような組換え体プラスミドをＰＣＲ法〔Science，230，1350 (1985)〕を用いて下記方法により構築した。
配列番号２４に示した配列を有するセンスプライマーおよび配列番号２５に示した配列を有するアンチセンスプライマーをＤＮＡ合成機を用いて合成した。
【０１８２】
該センスプライマーおよびアンチセンスプライマーの５'末端にはそれぞれＢａｍＨＩの制限酵素サイトを付加させた。
染色体ＤＮＡを鋳型として、これらプライマーおよびTaq DNA polymerase（ベーリンガー社製）を用い、DNA Thermal Cycler（パーキンエルマージャパン社製）でＰＣＲを行うことによりｙａｅＭ遺伝子を増幅した。
【０１８３】
ＰＣＲは、９４℃で３０秒間、５５℃で３０秒間、７２℃で２分間からなる反応工程を１サイクルと３０サイクル行った後、７２℃で７分間反応させる条件で行った。
増幅されたＤＮＡ断片およびpUC118（宝酒造社製）を制限酵素ＢａｍＨＩで消化後、各々のＤＮＡ断片をアガロースゲル電気泳動によって精製した。
【０１８４】
これら精製された両断片を混合した後エタノール沈殿を行い、得られたＤＮＡ沈殿物を５μｌの蒸留水に溶解し、ライゲーション反応を行うことにより組換え体ＤＮＡを取得した。
該組換え体ＤＮＡがｙａｅＭ遺伝子であることをＤＮＡ配列を決定することによって確認した。
【０１８５】
該組換え体からプラスミドを抽出し、制限酵素制限酵素ＢａｍＨＩで消化後、アガロースゲル電気泳動を行いＢａｍＨＩ処理ｙａｅＭ遺伝子含有ＤＮＡ断片を取得した。
ｐQE30（QIAGEN社製）を制限酵素ＢａｍＨＩで消化後、アガロースゲル電気泳動を行いＢａｍＨＩ処理pQE30断片を取得した。
【０１８６】
上記で取得されたＢａｍＨＩ処理ｙａｅＭ遺伝子含有ＤＮＡ断片をＢａｍＨＩ消化pQE30断片と混合した後、エタノール沈殿を行い、得られたＤＮＡ沈殿物を５μｌの蒸留水に溶解し、ライゲーション反応を行うことにより組換え体ＤＮＡを取得した。
該組換え体ＤＮＡを用い、E. Coli JM109株を常法に従って形質転換後、該形質転換体をアンピシリン１００μｇ／ｍｌを含むＬＢ寒天培地に塗布し、３７℃で一晩培養した。
【０１８７】
上記と同様の方法で、該大腸菌よりプラスミドを単離した。
上記同様、該方法により単離したプラスミドを各種制限酵素で切断して構造を調べ、塩基配列を決定することにより、目的のＤＮＡ断片が挿入されているプラスミドであることを確認した。このプラスミドをpQEDXRと命名した。
【０１８８】
（２） ｙａｅＭ遺伝子産物の活性測定
(i)ｙａｅＭ遺伝子産物の精製
（１）で作成したpQEDXRを常法によりpREP4を有するE. coli M15株(QIAGEN社製)に導入し、アンピシリン200μg/ml、カナマイシン25μg/mlに耐性を示すM15/pREP4+pQEDXR株を得た。
【０１８９】
M15/pREP4+pQEDXR株をアンピシリン２００μｇ／ｍｌ、カナマイシン２５μｇ／ｍｌを含むＬＢ液体培地１００ｍｌ中、３７℃で培養し、６６０ｎｍの濁度が０．８に達した時点でイソプロピルチオガラクトシドを終濃度０．２ｍＭになるように添加した。さらに３７℃で５時間培養した後、遠心分離（３０００ｒｐｍ、１０分間）によって培養上清を除いた。この菌体を１００ｍＭトリス塩酸緩衝液（ｐＨ８．０）６ｍｌに懸濁し、超音波破砕機(SONIFIER,BRANSON社製)を用いて氷冷しつつ破砕した。得られた菌体破砕液を遠心分離（１０，０００ｒｐｍ、２０分間、４℃）にかけ、上清を回収した。この細胞抽出液遠心上清をNi-NTAレジンカラム(QIAGEN社製)に通し、２０ｍｌの洗浄緩衝液〔１００ｍＭトリス塩酸（ｐＨ８．０）、５０ｍＭイミダゾール、０．５％ Tween２０〕で洗浄した。ついで溶出緩衝液〔１００ｍＭトリス塩酸（ｐＨ８．０）、２００ｍＭイミダゾール〕１０ｍｌを通塔し、溶出液を１ｍｌづつ分画した。
各分画について蛋白量を測定（ＢｉｏＲａｄ社の蛋白量定量キット使用)し、蛋白質を含む画分を精製蛋白画分とした。
【０１９０】
(ii) 基質１−デオキシ-D-キシルロース5-リン酸の調製
反応基質である１−デオキシ-D-キシルロース5-リン酸は以下のようにして調製した。１−デオキシ-D-キシルロース5-リン酸の検出は、ＨＰＬＣ〔カラム：Senshu pak NH2-1251-N(4.6 x 250mm、Senshu社製)、移動層：１００ｍＭ ＫＨ₂ＰＯ₄（ｐＨ３．５）〕によって１９５ｎｍの吸光度を測定する方法で行った。
【０１９１】
大腸菌のｄｘｓ遺伝子を高発現するプラスミドpQDXS-1を上記と同様にE.coli M15/pREP4株に導入し、M15/pREP4+pQDXS-1株を得た。該株を実施例８（２）(i)と同様に培養し、Ni-NTAレジンカラムを用いてｄｘｓ酵素蛋白質を精製した。
【０１９２】
該精製ｄｘｓ蛋白質を２０ｍｌの反応液〔１００ｍＭトリス塩酸（ｐＨ７．５）、１０ｍＭピルビン酸ナトリウム、３０ｍＭＤＬ−グリセルアルデヒド−３−リン酸、１．５ｍＭチアミンピロリン酸、１０ｍＭＭｇＣｌ₂、１ｍＭＤＬ−ジチオスレイトール〕に加え３７℃で保温した。
【０１９３】
１２時間反応した後、反応液を水で３００ｍｌに希釈し、活性炭カラム（２．２ｘ８ｃｍ）を通した後、Dowex 1-X8（Ｃｌ−型、３．５ｘ２５ｃｍ）に通塔し、１％食塩水で溶出した。溶出画分を濃縮後、Sephadex G-10（１．８ｘ１００ｃｍ）に通塔し、水で溶出した。１−デオキシ−Ｄ−キシルロース５−リン酸含有画分を凍結乾燥し、約５０ｍｇの白色粉末を得た。
該粉末が１−デオキシ-D-キシルロース5-リン酸であることをＮＭＲ分析(A-500、日本電子社製)で確認した。
【０１９４】
(iii) yaeM遺伝子産物の酵素活性測定
１００ｍＭトリス塩酸（ｐＨ７．５）、１ｍＭ ＭｎＣｌ _２ 、０．３ｍＭ ＮＡＤＰＨと実施例８（２）(i)で得たｙａｅＭ遺伝子産物を含む反応液１ｍｌに、上記のように合成した１−デオキシ−Ｄ−キシルロース５−リン酸０．３ｍＭ（終濃度）を加え、３７℃でインキュベートした。インキュベート中のＮＡＤＰＨの増減を３４０ｎｍの吸光を分光光度計(UV-160、島津社製)で測定する方法で追跡したところ、経時的にＮＡＤＰＨが減少することが分かった。
【０１９５】
上記反応産物の構造を確認するため、以下のようにスケールを大きくして反応を行い、産物を単離した。１−デオキシ−Ｄ−キシルロース５−リン酸の濃度を０．１５ｍＭとした以外は上記と同じ組成の反応液２００ｍｌを、同様に３７℃で３０分間インキュベートした後、その全量を活性炭カラムに通し、通過液を水で１Ｌに希釈した後、Dowex 1-X8（Cl-型、３．５ｘ２０ｃｍ）カラムに通塔した。
【０１９６】
１％食塩水４００ｍｌで溶出し、Sephadex G-10（１．８ｘ１００ｃｍ）に通塔し、水で溶出した。溶出画分を凍結乾燥することで、反応産物を単離した。
ＨＲ−ＦＡＢＭＳ解析から単離された反応産物の分子式はＣ₅Ｈ₁₂Ｏ₇Ｐ［ｍ／ｚ２１５．０２７６（Ｍ−Ｈ）^-、△−４．５ｍｍｕ］と推定された。¹Ｈおよび¹³ＣＮＭＲ解析から、以下のケミカルシフトが得られた。
【０１９７】
¹H NMR(D₂O, 500 MHz):δ4.03(ddd, J=11.5, 6.5, 2.5 Hz, 1H), 3.84(ddd, J=11.5, 8.0, 6.5 Hz, 1H), 3.78(dd, J=80, 2.5 Hz, 1H), 3.60(d, J=12.0 Hz, 1H), 3.50(d, J=12.0 Hz, 1H), 1.15(s, ³H); ¹³C NMR(D₂O, 125 MHz):δ75.1(C-2), 74.8(C-3), 67.4(C-1), 65.9(C-4), 19.4(2-Me)
この反応産物をアルカリ性ホスファターゼ(宝酒造社製)で処理して得られる化合物を¹Ｈおよび¹³Ｃ ＮＭＲ解析して得られるケミカルシフトは、Tetrahedron Letter,38, 6184 (1997)に記載の方法で合成した２−Ｃ−メチル−Ｄ−エリスリトールのＮＭＲ解析で得られるケミカルシフトと完全に一致した。
【０１９８】
さらに前者の旋光度は［α］_D ²¹=＋６．０（ｃ=０．０５０，Ｈ₂Ｏ）で、報告されている〔Tetrahedron Letter, 38, 6184 (1997)〕２−Ｃ−メチル−Ｄ−エリスリトールの旋光度［α］_D ²⁵=＋７．０（ｃ=０．１３，Ｈ₂Ｏ）と一致した。
【０１９９】
これらの結果から、ｙａｅＭ遺伝子産物の反応産物は２−Ｃ−メチル−Ｄ−エリスリトール４−リン酸であることが明らかになった。即ち、ｙａｅＭ遺伝子産物はＮＡＤＰＨの消費を伴いながら１−デオキシ−Ｄ−キシルロース５−リン酸から２−Ｃ−メチル−Ｄ−エリスリトール４−リン酸を生じる活性を有することが判明した。この触媒活性に基づき、本酵素を１−デオキシ−Ｄ−キシルロース５−リン酸レダクトイソメラーゼと命名した。
【０２００】
(iv) １−デオキシ−Ｄ−キシルロース５−リン酸レダクトイソメラーゼの性質
実施例８（２）(iii)に記した１ｍｌ反応系を用いて、１−デオキシ−Ｄ−キシルロース５−リン酸レダクトイソメラーゼの酵素学的性質を調べた。なお１ｕとは１分間に１ｍｍｏｌのＮＡＤＰＨを酸化する活性と定義する。ＮＡＤＰＨをＮＡＤＨに置換した場合、活性は１／１００以下に低下した。
【０２０１】
１−デオキシ−Ｄ−キシルロース５−リン酸の代りに１−デオキシ−Ｄ−キシルロースを用いると全く反応は起らなかった。
ＳＤＳ−ＰＡＧＥ解析から、本酵素は４２ｋＤａポリペプチドから構成されていることが分かった。
反応系への金属添加効果を第9表に示した。
【０２０２】
【表９】

【０２０３】
ＭｎＣｌ₂存在下での１−デオキシ−Ｄ−キシルロース５−リン酸、ＮＡＤＰＨへのＫｍは、それぞれ２４９μＭ、７．４μＭだった。
反応温度の影響を図１に、反応ｐＨの影響を図２に示した。
【０２０４】
実施例９ ｙａｅＭ欠損変異株の作成と性質
（１）ｙａｅＭ欠損変異株の作成
１−デオキシ−Ｄ−キシルロース５−リン酸レダクトイソメラーゼが細胞の生育に必須か否かを調べるため、以下のようにしてその欠損変異株を作成した。
ｙａｅＭ遺伝子中に挿入するためのカナマイシン耐性遺伝子カセットを以下のようにして作成した。
【０２０５】
実施例１（２）▲２▼で得たプラスミドｐＭＥＷ４１を制限酵素ＢａｌＩで消化後アガロースゲル電気泳動し、ＢａｉＩＩ処理ＤＮＡ断片を取得した。
Ｔｎ５を制限酵素ＨｉｎｄＩＩＩとＳａｍＩで消化した後、DNA blunting kit（宝酒造社製）を用いて断片の末端を平滑化した。
得られた平滑化ＤＮＡ断片を先に作成したＢａｉＩＩ処理ｐＭＥＷ４１ＤＮＡ断片と混合した後、エタノール沈殿を行い、得られたＤＮＡ沈殿物を５μｌの蒸留水に溶解し、ライゲーション反応を行うことにより組換え体ＤＮＡを取得した。
【０２０６】
該組換え体ＤＮＡを用い、E. coli JM109株（宝酒造より購入）を常法に従って形質転換後、該形質転換体をアンピシリン１００μｇ／ｍｌとカナマイシン１５μｇ／ｍｌを含むＬＢ寒天培地に塗布し、３７℃で一晩培養した。
生育してきたアンピシリン耐性の形質転換体のコロニー数個について、アンピシリン１００μｇ／ｍｌとカナマイシン１５μｇ／ｍｌを含むＬＢ液体培地１０ｍｌで３７℃１６時間振盪培養した。
得られた培養液を遠心分離することにより菌体を取得した。
【０２０７】
該菌体より常法に従ってプラスミドを単離した。
該方法により単離したプラスミドを各種制限酵素で切断して構造を調べ、目的のＤＮＡ断片が挿入されているプラスミドであることを確認した。このプラスミドをpMEW41Kmと名づけた。
pMEW41Kmを用いて、相同組換えによる染色体上のyaeM遺伝子の破壊を行った。組換えの模式図を図３に示した。
【０２０８】
pMEW41Kmを制限酵素ＨｉｎｄＩＩＩとＳａｃＩで消化し、アガロースゲル電気泳動を行い直鎖状の断片を精製した。この断片を用いて常法に従って、大腸菌FS1576株を形質転換した。FS1576株は国立遺伝学研究所よりME9019株の名で入手可能である。該形質転換体をカナマイシン１５μｇ／ｍｌと２−Ｃ−メチル−Ｄ−エリスリトール１ｇ／ｌを含むＬＢ寒天培地に塗布し、３７℃で一晩培養した。
【０２０９】
生育してきたカナマイシン耐性コロニー数個について、カナマイシン１５μｇ／ｍｌと２−Ｃ−メチル−Ｄ−エリスリトール１ｇ／ｌを含むＬＢ液体培地１０ｍｌで３７℃１６時間振盪培養した。
得られた培養液を遠心分離することにより菌体を取得した。
該菌体より常法に従って染色体ＤＮＡを単離した。
【０２１０】
染色体ＤＮＡを制限酵素ＳｍａＩまたはＰｓｔＩで消化した。またFS1576株の染色体についても同様に処理した。常法に従って、これら制限酵素処理ＤＮＡをアガロースゲル電気泳動後、カナマイシン耐性遺伝子およびｙａｅＭ遺伝子をプローブとしたサザンハイブリダイゼーション解析に供した。その結果、カナマイシン耐性コロニーの染色体は図３に示した構造をとっており、目的どおりｙａｅＭ遺伝子がカナマイシン耐性遺伝子で分断破壊されていることが確かめられた。
【０２１１】
（２） ｙａｅＭ欠損変異株の性質
上記の手順で作成されたｙａｅＭ欠損株およびその親株であるＦＳ１５７６株を、ＬＢ寒天培地および２−Ｃ−メチル−Ｄ−エリスリトール１ｇ／ｌを含むＬＢ寒天培地に塗布し、３７℃で培養した。２日後の生育度合いを第１０表に示した。
【０２１２】
【表１０】

【０２１３】
ｙａｅＭ欠損変異株は２−Ｃ−メチル−Ｄ−エリスリトールを添加しない培地では生育できないため、２−Ｃ−メチル−Ｄ−エリスリトール非存在下では本遺伝子が細胞の生育に必須であることが明白となった。
【０２１４】
実施例１０ １−デオキシ−Ｄ−キシルロース５−リン酸レダクトイソメラーゼ阻害剤が菌の生育に及ぼす効果
１−デオキシ−Ｄ−キシルロース５−リン酸レダクトイソメラーゼ反応の産物である２−Ｃ−メチル−Ｄ−エリスリトール４−リン酸やこの酵素反応で想定される反応中間体とフォスミドマイシンは構造的に似ているため、フォスミドマイシンが該酵素を阻害する可能性があるとの推定の基に以下の実験をおこなった。
【０２１５】
実施例８に示した１−デオキシ−Ｄ−キシルロース５−リン酸レダクトイソメラーゼ活性測定系にフォスミドマイシンを添加し、酵素活性への影響を調べた。
フォスミドマイシンは〔Chem. Pharm. Bull., 30, 111-118 (1982)〕に記載の方法に従って合成した。
【０２１６】
実施例８（２）(iii)に示した反応系を０．２ｍｌにスケールダウンした系（各濃度は同じ）に各種濃度のフォスミドマイシンを添加し、３７℃でインキュベートし、ＮＡＤＰＨの増減をベンチマークマイクロプレートリーダー（バイオラド社製）を用いて測定した。
【０２１７】
図４に示したように、フォスミドマイシンは１−デオキシ−Ｄ−キシルロース５−リン酸レダクトイソメラーゼを阻害することが判った。
大腸菌W3110株をＬＢ寒天培地、フォスミドマイシンを３．１３ｍｇ／ｌ含むＬＢ寒天培地およびフォスミドマイシン３．１３ｍｇ／ｌと２−Ｃ−メチル−Ｄ−エリスリトール０．２５ｇ／ｌを含むＬＢ寒天培地にそれぞれ塗布し、３７℃で培養した。
【０２１８】
培養２日後において、ＬＢ寒天培地、フォスミドマイシンおよび２−Ｃ−メチル−Ｄ−エリスリトール０．２５ｇ／ｌを含むＬＢ寒天培地上の２種類の培地では菌は生育することができたが、フォスミドマイシンだけを添加したＬＢ寒天培地では菌は生育することができなかった。
【０２１９】
これらの結果から、フォスミドマイシンが１−デオキシ−Ｄ−キシルロース５−リン酸レダクトイソメラーゼを阻害することによって菌の生育を阻害することが明白となった。 以上のことから、ｙａｅＭ（１−デオキシ−Ｄ−キシルロース５−リン酸レダクトイソメラーゼ）の活性を阻害する物質は有効な抗菌剤あるいは除草剤となり得る。
【０２２０】
【発明の効果】
本発明によれば、心疾患、骨粗鬆症、止血、がん予防、免疫賦活等を目的とした医薬品、健康食品および貝類付着防止塗料等に有用なイソプレノイド化合物の生合成に関与するＤＮＡを１つ以上含むＤＮＡをベクターに組み込み、得られた組換え体ＤＮＡを原核生物由来の宿主細胞に導入し、得られた形質転換体を培地に培養し、培養物中にイソプレノイド化合物を生成蓄積させ、該培養物からイソプレノイド化合物を採取することを特徴とする、イソプレノイド化合物の製造方法、イソプレノイド化合物の生合成効率を向上させることのできる活性を有する蛋白質をコードするＤＮＡを１つ以上含むＤＮＡをベクターに組み込み、得られた組換え体ＤＮＡを宿主細胞に導入し、得られた形質転換体を培地に培養し、培養物中に該蛋白質を生成蓄積させ、該培養物から該蛋白質を採取することを特徴とする、該蛋白質の製造方法、および該蛋白質、ならびに１−デオキシ−Ｄ−キシルロース５−リン酸から２−Ｃ−メチル−Ｄ−エリスリトール４−リン酸を生じる反応を触媒する活性を有する新規な酵素蛋白質および該酵素を阻害する物質を探索することによる、抗菌およびまたは除草活性を有する化合物の探索方法を提供することができる。
【０２２１】
【配列表】

【０２２２】

【０２２３】

【０２２４】

【０２２５】
【配列表フリーテキスト】
配列番号１２：合成ＤＮＡ
配列番号１３：合成ＤＮＡ
配列番号１４：合成ＤＮＡ
配列番号１５：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３２：合成ＤＮＡ
配列番号３３：合成ＤＮＡ
配列番号３４：合成ＤＮＡ
【図面の簡単な説明】
【図１】１−デオキシ−Ｄ−キシルロース５−リン酸レダクトイソメラーゼの活性に対する反応温度の影響を示した図である。
【図２】１−デオキシ−Ｄ−キシルロース５−リン酸レダクトイソメラーゼの活性に対する反応液ｐＨの影響を示した図である。１００ｍＭトリス塩酸緩衝液中の各ｐＨにおける活性を示した。ｐＨ８．０での活性を１００％として、各ｐＨにおける活性を相対活性として示した。
【図３】相同組換えを利用した染色体上のｙａｅＭ遺伝子破壊方法を示した図である。
【図４】１−デオキシ−Ｄ−キシルロース５−リン酸レダクトイソメラーゼ活性に及ぼすフォスミドマイシンの影響を示した図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing an isoprenoid compound using a prokaryotic-derived transformant, and a method for searching for an antibacterial or herbicidal active substance related to a non-mevalonic acid pathway.
[0002]
[Prior art]
The isoprenoid is a general term for compounds having a carbon number of 5 isoprene units as a basic skeleton, and is biosynthesized by polymerization of isopentenyl pyrophosphate (IPP).
There are a wide variety of isoprenoid compounds in nature, and many are useful to mankind.
For example, ubiquinone plays an important function in vivo as an essential component of an electron transport system, and is used as a pharmaceutical effective for heart disease. In addition, demand for health food is increasing in the United States and Europe.
[0003]
Vitamin K is an important vitamin involved in the blood coagulation system and is used as a hemostatic agent. Recently, it has been suggested to be involved in bone metabolism and is expected to be applied to the treatment of osteoporosis. Philoquinone and menaquinone are pharmaceutical products. As allowed.
Moreover, ubiquinone and vitamin K have an action of inhibiting the adhesion of shellfish, and application to shellfish adhesion preventing paints is expected.
[0004]
Furthermore, compounds based on a 40-carbon isoprene skeleton called carotenoids have an antioxidant action, and those that are expected to have cancer prevention and immunostimulatory activities such as β-carotene, astaxanthin, and cryptoxanthin. is there.
Thus, many useful substances are contained in the isoprenoid compound, and if these inexpensive production methods are established, it seems that there are great social and medical benefits.
[0005]
The production of isoprenoid compounds by fermentation has been studied for some time, and attempts have been made to study culture conditions, breed strains by mutation treatment, and further improve the production by genetic engineering techniques. However, the effect is limited to individual compound types, and no method effective for all isoprenoid compounds is known.
Isopentenyl pyrophosphate (IPP), the basic skeletal unit of isoprenoid compounds, has been proven to be biosynthesized from acetyl-CoA via mevalonic acid (mevalonic acid pathway) in eukaryotes such as animals and yeasts. .
[0006]
In the mevalonate pathway, 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase is considered to be rate-limiting [Mol. Biol. Cell,Five, 655 (1994)], an attempt has been made to increase the productivity of carotenoids by increasing the expression of HMG-CoA reductase in yeast [Misawa et al., Proceedings of the Carotenoid Research Conference (1997)].
[0007]
In prokaryotes, there is no knowledge that proves the existence of the mevalonate pathway, and there is another route, that is, via 1-deoxy-D-xylulose 5-phosphate produced by condensation of pyruvate and glyceraldehyde 3-phosphate. A non-mevalonate pathway in which IPP is biosynthesized has been discovered in many prokaryotes [Biochem. J.,295, 517 (1993)),¹³Experiments using C-labeled substrates suggest that 1-deoxy-D-xylulose 5-phosphate is converted to IPP via 2-C-methyl-D-erythritol 4-phosphate. [Tetrahedron Lett.38, 4769 (1997)].
[0008]
Encodes the enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS) that condenses pyruvate and glyceraldehyde 3-phosphate to biosynthesize 1-deoxy-D-xylulose 5-phosphate in E. coli [Proc. Natl. Acad. Sci. USA.,94, 12857 (1997)]. The gene is contained in an operon composed of four ORFs including ispA encoding farnesyl pyrophosphate synthase.
[0009]
Furthermore, it is known that E. coli has an activity to convert 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate [Tetrahedron Lett.39, 4509 (1998)].
At present, there is no description or suggestion regarding the manipulation of these genes contained in the operon to improve the productivity of isoprenoid compounds.
Although knowledge about the non-mevalonate pathway in prokaryotes is gradually accumulating, many of the enzymes involved and the genes that encode them are still unknown.
[0010]
A method for efficiently producing ubiquinone-10 by introducing a gene for ubiC, an enzyme that converts chorismate into 4-hydroxybenzoate (ubiC gene) and a gene for p-hydroxybenzoate transferase (ubiA) in photosynthetic bacteria is known. However, there is no example in which the productivity of isoprenoid compounds is improved by manipulating the enzyme gene of the non-mevalonate pathway.
Furthermore, there is no knowledge about how prokaryotes are affected by inhibiting reactions on the non-mevalonate pathway by mutation or drug treatment.
[0011]
[Problems to be solved by the invention]
An object of the present invention is to provide one or more DNAs involved in biosynthesis of isoprenoid compounds useful for pharmaceuticals, health foods, shellfish adhesion prevention paints, etc. for the purpose of heart disease, osteoporosis, hemostasis, cancer prevention, immunostimulation, etc. The contained DNA is incorporated into a vector, the obtained recombinant DNA is introduced into a prokaryotic host cell, the obtained transformant is cultured in a medium, and an isoprenoid compound is produced and accumulated in the culture. A method for producing an isoprenoid compound characterized by collecting an isoprenoid compound from a product, incorporating a DNA containing one or more DNAs encoding a protein having an activity capable of improving the biosynthesis efficiency of the isoprenoid compound, into a vector, The obtained recombinant DNA is introduced into a host cell, the obtained transformant is cultured in a medium, and the protein is produced in the culture. It was accumulated, and collecting the protein from the culture, a method of manufacturing a protein is to provide a DNA encoding the protein and the protein. Furthermore, the subject of this invention is providing the search method of an antibacterial and herbicidal active substance characterized by searching the substance which inhibits the enzyme reaction on a non-mevalonic acid pathway.
[0012]
[Means for Solving the Problems]
The present inventors have found that isoprenoid productivity can be improved by searching for DNA capable of improving isoprenoid productivity by prokaryotes and introducing the obtained DNA into prokaryotes. It came to be completed.
[0013]
That is, the first invention of the present application incorporates a DNA containing one or more DNAs selected from the following (a), (b), (c), (d), (e) and (f) into a vector, The obtained recombinant DNA is introduced into a prokaryotic host cell, the obtained transformant is cultured in a medium, and an isoprenoid compound is produced and accumulated in the culture, and the isoprenoid compound is collected from the culture. It is the manufacturing method of the isoprenoid compound characterized by the above-mentioned.
[0014]
(A) is a DNA encoding a protein having an activity of catalyzing a reaction for producing 1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde 3-phosphate;
(B) is a DNA encoding farnesyl pyrophosphate synthase,
(C) is a DNA encoding a protein having the amino acid sequence described in SEQ ID NO: 3, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein, and isoprenoid compound DNA encoding a protein having an activity capable of improving the biosynthesis efficiency of (1), (d) is a DNA encoding a protein having the amino acid sequence of SEQ ID NO: 4, or one or several amino acids in the amino acid sequence of the protein A DNA encoding a protein consisting of an amino acid sequence in which the amino acid is deleted, substituted or added, and having an activity capable of improving the biosynthesis efficiency of an isoprenoid compound, (e) is 1-deoxy-D-xylulose 5 The reaction that produces 2-C-methyl-D-erythritol 4-phosphate from -phosphate A DNA encoding a protein having an activity of: (f) hybridizing with a DNA selected from (a), (b), (c), (d) and (e) under stringent conditions; and DNA encoding a protein having substantially the same activity as that of the protein encoded by the encoded DNA.
[0015]
Deletion, substitution or addition of amino acids in the present specification can be performed by site-directed mutagenesis, which is a well-known technique before filing, and one or several amino acids are site-directed mutagenesis. It means a number such as 1 to 5 amino acids that can be deleted, substituted or added by the induction method.
[0016]
Proteins consisting of amino acid sequences in which one or several amino acids are deleted, substituted or added are described in Molecular Cloning, A laboratory manual, 2nd edition (Sambrook). , Fritsch, edited by Maniatis, Cold Spring Harbor Laboratory Press, 1989 (hereinafter abbreviated as Molecular Cloning 2nd edition)], Current Protocols in Molecular Biology , John Wiley & Sons (1987-1997), Nucleic Acids Research,Ten, 6487 (1982), Proc. Natl. Acad. Sci., USA,79, 6409 (1982), Gene,34, 315 (1985), Nucleic Acids Research,13, 4431 (1985), Proc. Natl. Acad. Sci USA,82, 488 (1985) and the like.
[0017]
In the above, DNA encoding a protein that catalyzes a reaction for producing 1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde 3-phosphate, for example, described in SEQ ID NO: 1, 26 or 28 DNA encoding a protein having the above amino acid sequence, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of these proteins, and pyruvic acid and glyceraldehyde 3-phosphate To DNA encoding a protein having an activity of catalyzing a reaction for producing 1-deoxy-D-xylulose 5-phosphate.
[0018]
Specific examples include DNA having the base sequence set forth in SEQ ID NO: 6, DNA having the base sequence set forth in SEQ ID NO: 27 or 29, and the like.
As DNA encoding farnesyl pyrophosphate synthase, for example, DNA encoding a protein having the amino acid sequence described in SEQ ID NO: 2, or one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein. And a DNA encoding a protein having an amino acid sequence and having farnesyl pyrophosphate synthase activity. Specific examples include DNA having the base sequence described in SEQ ID NO: 7.
[0019]
Specific examples of DNA encoding a protein having the amino acid sequence described in SEQ ID NO: 3 include DNA having the base sequence described in SEQ ID NO: 8, and the like.
Specific examples of DNA encoding a protein having the amino acid sequence described in SEQ ID NO: 4 include DNA having a base sequence described in SEQ ID NO: 9 and the like.
[0020]
As DNA which codes the protein which has the activity which catalyzes the reaction which produces | generates 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate, it describes in sequence number 5 or 30, for example DNA encoding a protein having the amino acid sequence of 1-deoxy-D-xylulose 5-phosphate consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein To DNA encoding a protein having an activity of catalyzing a reaction for producing 2-C-methyl-D-erythritol 4-phosphate.
[0021]
Specific examples of the DNA include DNA having the base sequence set forth in SEQ ID NO: 10 or 31.
The above-mentioned “DNA that hybridizes under stringent conditions” refers to colony hybridization method, plaque hybridization method, Southern blot hybridization method, etc. using the above DNA or a fragment of the DNA as a probe. Specifically, hybridization is performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques or a fragment of the DNA is immobilized. After washing, the filter is washed under a condition of 65 ° C. using an SSC solution of about 0.1 to 2 times (the composition of the SSC solution of 1 time concentration consists of 150 mM sodium chloride and 15 mM sodium citrate). DNAs that can be identified by
[0022]
Hybridization can be performed according to the method described in Molecular Cloning 2nd edition. Specifically, the DNA capable of hybridizing is a DNA having at least 70% homology with a base sequence selected from SEQ ID NOs: 1, 2, 3, 4 and 5, preferably a DNA having 90% or more homology. Can give.
[0023]
Examples of isoprenoid compounds include ubiquinone and vitamin K₂And carotenoids.
The second invention of the present application is a protein having an activity capable of improving the biosynthesis efficiency of an isoprenoid compound selected from the following (a), (b) and (c).
[0024]
(A) is a protein having the amino acid sequence set forth in SEQ ID NO: 3, or a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein,
(B) is a protein having the amino acid sequence shown in SEQ ID NO: 4, or a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein,
(C) is a protein having the amino acid sequence shown in SEQ ID NO: 5 or a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein.
[0025]
According to a third invention of the present application, a DNA encoding the protein described in the second invention is incorporated into a vector, the obtained recombinant DNA is introduced into a host cell, and the obtained transformant is used as a medium. A method for producing a protein having an activity capable of improving the biosynthesis efficiency of an isoprenoid compound, characterized by culturing, producing and accumulating the protein in the culture, and collecting the protein from the culture. .
In the above, as a transformant,EscherichiaMicroorganisms belonging to the genus,RhodobacterMicroorganisms belonging to the genus orErwiniaThe microorganisms belonging to the genus can be mentioned.
[0026]
4th invention of this application is DNA which codes the protein which has the activity which can improve the biosynthesis efficiency of the isoprenoid compound chosen from the following (a), (b), (c) and (d) .
(A) is a DNA encoding a protein having the amino acid sequence set forth in SEQ ID NO: 3,
(B) is a DNA encoding a protein having the amino acid sequence set forth in SEQ ID NO: 4,
(C) is a DNA encoding a protein having the amino acid sequence set forth in SEQ ID NO: 5,
(D) is a DNA having the base sequence set forth in SEQ ID NO: 8,
(E) is a DNA having the base sequence set forth in SEQ ID NO: 9,
(F) is a DNA having the base sequence set forth in SEQ ID NO: 10,
(G) is a DNA that hybridizes with the DNA of any one of (a) to (f) under stringent conditions.
[0027]
The fifth invention of the present application is the biosynthesis of 1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde triphosphate, and then the production of 2-C-methyl-D-erythritol 4-phosphate. It is characterized by searching for a substance that inhibits the reaction of a protein having an activity of an enzyme selected from enzymes existing on a non-mevalonate pathway for biosynthesis of isopentenyl pyrophosphate via synthesis. This is a method for searching for a substance having antibacterial activity.
[0028]
The sixth invention of the present application is the biosynthesis of 1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde 3-phosphate, and then the production of 2-C-methyl-D-erythritol 4-phosphate. It is characterized by searching for a substance that inhibits the reaction of a protein having an activity of an enzyme selected from enzymes existing on a non-mevalonate pathway for biosynthesis of isopentenyl pyrophosphate via synthesis. This is a method for searching for a substance having herbicidal activity.
[0029]
In the above inventions 5 and 6, examples of the protein include the following protein (a) or (b).
(A) is a protein having an activity of catalyzing a reaction for producing 1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde 3-phosphate,
(B) is a protein having an activity of catalyzing the reaction of producing 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate.
[0030]
In the above, as a protein that catalyzes a reaction for producing 1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde 3-phosphate, for example, a protein having the amino acid sequence described in SEQ ID NO: 1, or It consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence possessed by the protein, and 1-deoxy-D-xylulose 5-phosphate is converted from pyruvic acid and glyceraldehyde 3-phosphate. A protein having an activity of catalyzing a reaction to be generated can be exemplified.
[0031]
As a protein having an activity of catalyzing a reaction for producing 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate, for example, a protein having the amino acid sequence of SEQ ID NO: 5, Or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein, and 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol Examples thereof include proteins having an activity of catalyzing a reaction for producing 4-phosphate.
The seventh invention of the present invention is a substance having antibacterial activity obtained by the search method of the fifth invention. However, known substances obtained by the searching method do not enter the present invention.
[0032]
The present inventors have proposed 2-C-methyl-D-erythritol 4-phosphate, which is a product of 1-deoxy-D-xylulose 5-phosphate reductoisomerase reaction, and a reaction intermediate envisaged in this enzyme reaction. Focusing on the structural similarity with fosmidomycin [3- (N-formyl-N-hydroxyamino) propylphosphonic acid], fosmidomycin has 1-deoxy-D-xylulose 5-phosphate reductoisomerase inhibitory activity. In addition, based on the assumption that it has antibacterial activity, an experiment of the search method of the fifth invention described in Example 10 to be described later was conducted, and fosmidomycin had the inhibitory activity and antibacterial activity. While the substance was found to be an active substance and the validity of the search method of the fifth invention was proved, the known fosmidomycin is excluded from the present invention.
[0033]
The eighth invention of the present invention is a substance having herbicidal activity obtained by the search method of the sixth invention. As described above, known substances obtained by the searching method do not fall within the scope of the present invention.
Hereinafter, the present invention will be described in detail.
[0034]
DETAILED DESCRIPTION OF THE INVENTION
I. Acquisition of DNA encoding proteins involved in biosynthesis of isoprenoid compounds
(1) Acquisition of DNA encoding a protein involved in biosynthesis of isoprenoid compounds using the base sequence of DNA encoding DXS (DXS gene)
E. coli chromosome and DXS gene base sequence information already determined [Proc. Natl. Acad. Sci. USA.,94, 12857 (1997)], the DNA region of the gene containing the DXS gene from E. coli or in the vicinity of the DXS gene is analyzed by the PCR method [Science,230, 1350 (1985)].
[0035]
Examples of the base sequence information including the DXS gene include the base sequence described in SEQ ID NO: 11.
Specific examples of a method for obtaining a DNA region containing the DXS gene include the following methods.
[0036]
E. coli, for exampleE.coli XL1-Blue strain (available for purchase from Toyobo) is a medium suitable for Escherichia coli, for example, LB liquid medium [Bactotryptone (Difco) 10 g, Yeast extract (Difco) 5 g, NaCl 5 g in 1 liter of water, pH 7. Culture medium adjusted to 2] according to a conventional method.
After culturing, cells are obtained from the culture by centrifugation.
[0037]
Chromosomal DNA is isolated from the obtained microbial cells according to a known method (for example, molecular cloning second edition).
Using the nucleotide sequence information described in SEQ ID NO: 11, a sense primer and an antisense primer containing a DXS gene or containing a nucleotide sequence corresponding to the DNA region of a gene near the DXS gene are synthesized using a DNA synthesizer. To do.
[0038]
After amplification by the PCR method, an appropriate restriction enzyme site, for example, at the 5 ′ end of the sense primer and the antisense primer so that the amplified DNA fragment can be introduced into a plasmid.BamHI,EcoIt is preferable to add a restriction enzyme site such as RI.
[0039]
Examples of combinations of the sense primer and the antisense primer include SEQ ID NOs: 12 and 13, SEQ ID NOs: 14 and 15, SEQ ID NOs: 12 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 13, and SEQ ID NOs: 22 and 23. Examples thereof include DNA having a combined base sequence.
[0040]
These primers, TaKaRa LA-PCR, using chromosomal DNA as a template^TM Kit Ver.2 (Takara Shuzo) or Expand^TM PCR is performed with DNAThermal Cycler (Perkin Elmer Japan) using High-Fidelity PCR System (Boehringer Mannheim) or the like.
[0041]
As a condition of PCR, when the primer is a DNA fragment of 2 kb or less, DNA exceeding 2 kb with one reaction cycle consisting of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds to 1 minute, and 72 ° C. for 2 minutes. In the case of fragments, a reaction step consisting of 98 ° C. for 20 seconds and 68 ° C. for 3 minutes is defined as one cycle, and after 30 cycles, the reaction can be performed at 72 ° C. for 7 minutes.
[0042]
The amplified DNA fragment is cut at the same site as the restriction enzyme site to which an appropriate vector that can be amplified in E. coli is added with the above primers, and then the DNA fragment is obtained by agarose electrophoresis, sucrose density gradient ultracentrifugation or the like Fractionation and collection.
[0043]
  The recovered DNA fragment is obtained by a conventional method, for example, Molecular Cloning Second Edition, Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997), DNA Cloning 1: Core Techniques, A Practical Methods described in Approach, Second Edition, Oxford University Press (1995), etc.To the cloning vector according toE. coli, for exampleE.coli DH5α strain (available for purchase from Toyobo) is transformed.
[0044]
As a cloning vector for transforming the E. coli, any phage vector, plasmid vector, etc. can be used as long as it can autonomously replicate in E. coli K12. An E. coli expression vector may be used as the cloning vector. . Specifically, ZAP Express (Stratagies, made by Stratagene,Five, 58 (1992)), pBluescript II SK (+) [Nucleic Acids Research,17, 9494 (1989)], Lambda ZAP II (Stratagene), λgt10, λgt11 [DNA Cloning, A Practical Approach,1, 49 (1985)], λTriplEx (Clontech), λExCell (Pharmacia), pT7T318U (Pharmacia), pcD2 [H. Okayama and P. Berg; Mol. Cell. Biol.,Three, 280 (1983)], pMW218 (Wako Pure Chemical Industries), pUC118 (Takara Shuzo), pEG400 [J. Bac.,1722392 (1990)], pQE-30 (manufactured by QIAGEN) and the like.
[0045]
From the obtained transformant, a plasmid containing the target DNA is prepared in a conventional manner, for example, Molecular Cloning Second Edition, Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997), DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995).
[0046]
By this method, DNA encoding a protein having an activity of catalyzing the reaction for producing 1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde 3-phosphate, and farnesyl pyrophosphate synthase are encoded. DNA, a DNA encoding a protein having the amino acid sequence described in SEQ ID NO: 3, a plasmid having a DNA encoding a protein having the amino acid sequence described in SEQ ID NO: 4, and a plasmid containing one or more of these DNAs can be obtained .
[0047]
Examples of the plasmid include plasmid pADO-1 containing all of the above DNA, plasmid pDXS-1 or pQEDXS-1 containing DNA having the base sequence described in SEQ ID NO: 6, and plasmid containing DNA having the base sequence described in SEQ ID NO: 7 Examples thereof include pISP-1, plasmid pXSE-1 containing DNA having the base sequence shown in SEQ ID NO: 8, plasmid pTFE-1 containing DNA having the base sequence shown in SEQ ID NO: 9, and the like.
[0048]
Utilizing the base sequence of DNA fragments derived from E. coli inserted into these plasmids, other prokaryotes, for example,RhodobacterA homologue of the DNA can be obtained from a microorganism belonging to the genus by the same method as described above.
[0049]
(2) Obtaining a DNA (methylerythritol-requiring complementary gene) encoding a protein having an activity capable of complementing a methylerythritol-requiring mutant of Escherichia coli
(1) Acquisition of E. coli methylerythritol-requiring mutant
E. coli, for exampleE.coli The W3110 strain (ATCC14948) is cultured according to a conventional method.
[0050]
After culturing, microbial cells are obtained from the obtained culture solution by centrifugation.
After washing the cells with an appropriate buffer, for example, 0.05 M Tris-Maleate buffer (pH 6.0), the concentration of the cells is 10^Four-10^TenSuspend in the same buffer to give cells / ml.
[0051]
Mutation treatment is performed using this suspension by a conventional method. As a conventional method, for example, there can be mentioned a method in which NTG is added to the suspension so as to have a final concentration of 600 mg / l and kept at room temperature for 20 minutes for mutation treatment.
The mutation-treated suspension is cultured in a medium obtained by adding 0.05 to 0.5% methylerythritol to a minimal agar medium.
[0052]
As the minimum agar medium, for example, a medium in which agar is added to M9 medium (Molecular Cloning Second Edition) can be used.
Methylerythritol is a Tetrahedron Letters,38, 35, 6184 (1997), and those chemically synthesized can be used.
[0053]
After culturing, the colonies grown and formed are replicated on a minimal agar medium containing 0.05-0.5% of a minimal agar medium and methyl erythritol and exhibiting methyl erythritol requirement, ie, minimal agar containing methyl erythritol A strain that can grow on a medium but cannot grow on a minimal agar medium is selected as a target mutant.
The ME7 strain can be mentioned as a methylerythritol-requiring mutant strain obtained by this operation.
[0054]
(2) Acquisition of methylerythritol-requiring complementary gene
E. coli, for exampleE.coli Strain W3110 (ATCC14948) is inoculated into a culture medium, for example, LB liquid medium, and cultured until the logarithmic growth phase according to a conventional method.
After culturing, the resulting culture is centrifuged to recover the cells.
[0055]
Chromosomal DNA is isolated and purified from the obtained microbial cells according to a conventional method (for example, the method described in Molecular Cloning Second Edition). Chromosomal DNA obtained by the method described in (1) above can also be used as isolated and purified chromosomal DNA.
Appropriate amount of the chromosomal DNA is converted to an appropriate restriction enzyme, such asSauThe digested DNA fragment obtained by partial digestion with 3AI is subjected to size fractionation by a conventional method, for example, sucrose density gradient ultracentrifugation (26,000 rpm, 20 ° C., 20 hr).
[0056]
A chromosomal genomic library is prepared by ligating a DNA fragment having a size of 4 to 6 kb obtained by the fractionation to a vector digested with an appropriate restriction enzyme, for example, pMW118 (Nippon Gene).
Using the prepared chromosomal library, the methylerythritol-requiring mutant strain isolated in the above (1), for example, the ME7 strain, is transformed according to a conventional method (for example, the method described in Molecular Cloning Second Edition).
[0057]
The transformant is applied to a minimal agar medium supplemented with a drug corresponding to the drug resistance gene of the vector, for example, M9 agar medium containing 100 μg / l of ampicillin, and cultured at 37 ° C. overnight.
By this method, a transformant whose methylerythritol requirement has been recovered can be selected.
[0058]
A plasmid is extracted from the obtained transformant by a conventional method. Examples of plasmids that can restore the requirement for methylerythritol include pMEW73 and pQEDXR.
The base sequence of DNA introduced into the plasmid is determined.
[0059]
Examples of the base sequence determined by this method include a sequence containing the base sequence of the yaeM gene shown in SEQ ID NO: 10, and the like. Using the obtained base sequence information of the yaeM gene, the homologue of the yaeM gene can be obtained from other prokaryotic organisms or plants by the same method as described above.
[0060]
II. Production of protein having activity capable of improving biosynthesis efficiency of isoprenoid compound
In order to express the DNA obtained as described above in a host cell, first, the DNA fragment of interest is a restriction enzyme or a DNA degrading enzyme, and an appropriate length DNA containing the gene. After the fragmentation, it is inserted downstream of the promoter in the expression vector, and then the expression vector into which the DNA has been inserted is introduced into a host cell suitable for the expression vector.
[0061]
Any host cell that can express the target gene can be used. For example, bacteria belonging to the genus Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, Microbacteria, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Trichosporon, Sivaniomyces Examples include yeast, animal cells, insect cells and the like belonging to the genus.
[0062]
As the expression vector, a vector that can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter at a position where the target DNA can be transcribed is used.
When a bacterium or the like is used as a host cell, the expression vector for expressing the DNA is capable of autonomous replication in the bacterium, and at the same time, a recombination composed of a promoter, a ribosome binding sequence, the DNA and a transcription termination sequence. A vector is preferred. A gene that controls the promoter may also be included.
[0063]
Examples of expression vectors include pBTrp2, pBTac1, pBTac2 (all available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (QIAGEN), pQE-30 (QIAGEN), pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 [Agricultural Biological Chemistry,48, 669 (1984)], pLSA1 [Agric. Biol. Chem.,53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA,82, 4306 (1985)], pBluescriptII SK +, pBluescriptII SK (-) (Stratagene), pTrS30 (FERMBP-5407), pTrS32 (FERM BP-5408), pGEX (Pharmacia), pET-3 (Novagen) ), PTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pUC18 [gene,33, 103 (1985)], pUC19 (Gene,33, 103 (1985)], pSTV28 (Takara Shuzo), pSTV29 (Takara Shuzo), pUC118 (Takara Shuzo), pPA1 (JP-A 63-233798), pEG400 [J. Bacteriol.,1722392 (1990)], pQE-30 (manufactured by QIAGEN) and the like.
[0064]
Any promoter can be used as long as it can be expressed in the host cell. For example,trpPromoter (Ptrp),lacPromoter (Plac), P_LPromoter, P_RPromoter, P_SEExamples of the promoter include promoters derived from Escherichia coli and phages, SPO1 promoter, SPO2 promoter, penP promoter, and the like. PtrpTwo promoters in series (Ptrpx2),tacAn artificially designed and modified promoter such as a promoter, letI promoter, and lacT7 promoter can also be used.
[0065]
Any ribosome binding sequence can be used as long as it can be expressed in the host cell, but the distance between the Shine-Dalgarno sequence and the start codon is adjusted to an appropriate distance (eg, 6 to 18 bases). It is preferable to use a plasmid.
Although the transcription termination sequence is not necessarily required for the expression of the target DNA, it is preferable to arrange the transcription termination sequence immediately below the structural gene.
[0066]
As a host cell,EscherichiaGenus,CorynebacteriumGenus,BrevibacteriumGenus,BacillusGenus,MicrobacteriumGenus,SerratiaGenus,PseudomonasGenus,AgrobacteriumGenus,AlicyclobacillusGenus,AnabaenaGenus,AnacystisGenus,ArthrobacterGenus,AzobacterGenus,ChromatiumGenus,ErwiniaGenus,MethylobacteriumGenus,PhormidiumGenus,RhodobacterGenus,RhodopseudomonasGenus,RhodospirillumGenus,ScenedesmunGenus,StreptomycesGenus,SynnecoccusGenus,ZymomonasMicroorganisms belonging to the genus and the like, preferably,EscherichiaGenus,CorynebacteriumGenus,BrevibacteriumGenus,BacillusGenus,PseudomonasGenus,AgrobacteriumGenus,AlicyclobacillusGenus,AnabaenaGenus,AnacystisGenus,ArthrobacterGenus,AzobacterGenus,ChromatiumGenus,ErwiniaGenus,MethylobacteriumGenus,PhormidiumGenus,RhodobacterGenus,RhodopseudomonasGenus,RhodospirillumGenus,ScenedesmunGenus,StreptomycesGenus,SynnecoccusGenus,ZymomonasExamples include microorganisms belonging to the genus.
[0067]
  Specific examples of the microorganism include, for example,Escherichia coli XL1-Blue,Escherichia coli XL2-Blue,Escherichia coli DH1,Escherichia coli DH5α,Escherichia coli MC1000,Escherichia coli KY3276,Escherichia coli W1485,Escherichia coli JM109,Escherichia coli HB101,Escherichia coli No. 49,Escherichiacoli W3110,Escherichia coli NY49,Escherichia coli MP347,Escherichia coli NM522,Bacillus subtilis,Bacillus amyloliquefaciens,Brevibacterium ammoniagenes,Brevibacterium immariophilum ATCC14068,Brevibacterium saccharolyticum ATCC14066,Brevibacterium flavum ATCC14067,Brevibacterium lactofermentum ATCC13869,Corynebacterium glutamicum ATCC13032,Corynebacterium glutamicum ATCC14297,Corynebacterium acetoacidophilum ATCC13870,Microbacterium ammoniaphilum ATCC15354,Serratia ficaria,Serratia fonticola,Serratia liquefaciens,Serratia marcescens,Pseudomonas sp. D-0110,Agrobacterium radiobacter,Agrobacterium rhizogenes,Agrobacterium rubi,Anabaena cylindrica,Anabaena doliolum,Anabaena flos-aquae,Arthrobacter aurescens,Arthrobacter citreus,Arthrobacter globformis,Arthrobacter hydrocarboglutamicus,Arthrobacter mysorens,Arthrobacter nicotianae,Arthrobacter paraffineus,Arthrobacter protophormiae,Arthrobacter roseoparaffinus,Arthrobacter sulfureus,Arthrobacter ureafaciens,Chromatium buderi,Chromatium tepidum,Chromatium vinosum,Chromatium warmingii,Chromatium fluviatile,Erwinia uredovora,Erwinia carotovora,Erwinia ananas,Erwinia herbicola,Erwinia punctata,Erwinia terreus,Methylobacterium rhodesianum,Methylobacterium extorquens,Phormidium sp. ATCC29409,Rhodobacter capsulatus,Rhodobacter sphaeroides,Rhodopseudomonas blastica,Rhodopseudomonas marina,Rhodopseudomonas palustris,Rhodospirillum rubrum,Rhodospirillum salexigens,Rhodospirillum salinarum,Streptomyces ambofaciens,Streptomyces aureofaciens,Streptomyces aureus,Streptomyces fungicidicus,Streptomyces griseochromogenes,Streptomyces griseus,Streptomyces lividans,Streptomyces olivogriseus,Streptomyces rameus,Streptomyces tanashiensis,Streptomyces vinaceus,Zymomonas mobilisEtc.
[0068]
As a method for introducing the recombinant vector, any method can be used as long as it is a method for introducing DNA into the host cell, for example, a method using calcium ions [Proc. Natl. Acad. Sci. USA,69, 2110 (1972)], protoplast method (Japanese Patent Laid-Open No. 63-2483942), or Gene,17, 107 (1982) and Molecular & General Genetics,168, 111 (1979).
[0069]
When yeast is used as a host cell, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 and the like.
Any promoter can be used as long as it can be expressed in yeast, such as PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, CUP1 promoter, A promoter can be mentioned.
[0070]
Host cells include Saccharomyces cerevisiae (Saccharomyces cerevisae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Kluyveromyces lactis (Kluyveromyces lactis), Trichosporon Pulllance (Trichosporonpullulans), Schwaniomyces Albius
(Schwanniomycesalluvius) Etc.
[0071]
As a method for introducing the recombinant vector, any method for introducing DNA into yeast can be used. For example, the electroporation method [Methods. Enzymol.,194, 182 (1990), spheroplast method [Proc. Natl. Acad. Sci. USA,75, 1929 (1978)], lithium acetate method [J. Bacteriol.,153, 163 (1983)], Proc. Natl. Acad. Sci. USA,75, 1929 (1978).
[0072]
When animal cells are used as host cells, examples of expression vectors include pcDNAI, pcDM8 (commercially available from Funakoshi), pAGE107 [Japanese Patent Laid-Open No. 3-22979; Cytotechnology,Three, 133, (1990)], pAS3-3 (JP-A-2-27075), pCDM8 [Nature,329, 840, (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103 [J. Biochem.,1011307 (1987)], pAGE210 and the like.
[0073]
Any promoter can be used as long as it can be expressed in animal cells, such as cytomegalovirus (human CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter, Examples include metallothionein promoter, heat shock promoter, SRα promoter, and the like. In addition, an enhancer of human CMV IE gene may be used together with a promoter.
Examples of host cells include Namalva cells, HBT5637 (Japanese Patent Laid-Open No. 63-299), COS1 cells, COS7 cells, CHO cells, and the like.
[0074]
As a method for introducing a recombinant vector into animal cells, any method capable of introducing DNA into animal cells can be used. For example, electroporation [Cytotechnology,Three, 133 (1990)], calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), lipofection method [Proc. Natl. Acad. Sci., USA,84, 7413 (1987)), virology,52456 (1973) can be used. The transformant can be obtained and cultured according to the method described in JP-A-2-227075 or JP-A-2-257891.
[0075]
When insect cells are used as the host, for example, Baculovirus Expression Vectors, A Laboratory Manual, Current Protocols in Molecular Biology Supplement 1-38 (1987) -1997), Bio / Technology,6, 47 (1988) etc., proteins can be expressed.
[0076]
That is, the recombinant gene transfer vector and baculovirus are co-introduced into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into the insect cells to express the protein. it can.
Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacIII (both manufactured by Invitrogen) and the like.
[0077]
As the baculovirus, for example, Autographa californica nuclear polyhedrosis virus, which is a virus that infects the night stealing insects, can be used.
As an insect cell,SpodopterafrugiperdaSf9, Sf21 [Baculovirus Expression Vectors, A Laboratory Manual, W. H. Freeman and Company, New York, (1992)],TrichoplusianiHigh5 (manufactured by Invitrogen) and the like, which are ovarian cells of the above, can be used.
[0078]
Examples of the method for co-introducing the recombinant gene introduction vector and the baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method (JP-A-2-27075), the lipofection method [Proc. Natl. Acad Sci. USA,84, 7413 (1987)].
As a gene expression method, in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning Second Edition.
[0079]
When expressed in yeast, animal cells or insect cells, a protein with a sugar or sugar chain added can be obtained.
A transformant having the recombinant DNA incorporating the DNA is cultured in a medium, and a protein having an activity capable of improving the biosynthesis efficiency of the isoprenoid compound is produced and accumulated in the culture. By collecting the protein, a protein having an activity capable of improving the biosynthesis efficiency of the isoprenoid compound can be produced.
[0080]
The method for culturing a transformant for producing a protein having an activity capable of improving the biosynthesis efficiency of the isoprenoid compound of the present invention in a medium can be performed according to a usual method used for culturing a host.
When the transformant of the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the medium for culturing these microorganisms contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the microorganism. As long as the medium can efficiently culture the transformant, either a natural medium or a synthetic medium may be used.
[0081]
Any carbon source may be used as long as it can be assimilated by each microorganism. Glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, ethanol Alcohols such as propanol are used.
[0082]
As a nitrogen source, ammonium salts of various inorganic acids and organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof are used.
[0083]
As the inorganic substance, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used.
The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 ° C., and the culture time is usually 16 hours to 7 days. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.
[0084]
Moreover, you may add antibiotics, such as an ampicillin and a tetracycline, to a culture medium as needed during culture | cultivation.
When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example,lacWhen culturing a microorganism transformed with an expression vector using a promoter, isopropyl-β-D-thiogalactopyranoside (IPTG), etc.,trpWhen culturing a microorganism transformed with an expression vector using a promoter, indoleacrylic acid (IAA) or the like may be added to the medium.
[0085]
As a medium for culturing a transformant obtained using an animal cell as a host cell, a commonly used RPMI 1640 medium [The Journal of the American Medical Association,199519 (1967)], Eagle's MEM medium [Science,122, 501 (1952)], DMEM medium [Virology,8, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine,73, 1 (1950)] or a medium obtained by adding fetal calf serum or the like to these mediums.
[0086]
Culture is usually pH 6-8, 30-40 ° C., 5% CO₂Perform for 1-7 days under conditions such as presence.
Moreover, you may add antibiotics, such as kanamycin and penicillin, to a culture medium as needed during culture | cultivation.
[0087]
As a medium for culturing a transformant obtained using insect cells as host cells, commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Gibco BRL), ExCell400, ExCell405 (Both made by JRH Biosciences), Grace's Insect Medium (Grace, TCC, Nature,195, 788 (1962)] and the like.
[0088]
The culture is usually performed for 1 to 5 days under conditions of pH 6 to 7, 25 to 30 ° C, and the like.
Moreover, you may add antibiotics, such as a gentamicin, to a culture medium as needed during culture | cultivation.
In order to isolate and purify a protein having an activity capable of improving the biosynthesis efficiency of the isoprenoid compound of the present invention from the culture of the transformant of the present invention, an ordinary enzyme isolation and purification method can be used. Good.
[0089]
For example, when the protein of the present invention is expressed in a cell in a dissolved state, after culturing, the cells are collected by centrifugation, suspended in an aqueous buffer solution, an ultrasonic crusher, a French press, a Manton Gaurin homogenizer. Then, the cells are disrupted with dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary enzyme isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), cation exchange chromatography using resin such as S-Sepharose FF (Pharmacia) Methods such as electrophoresis, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectric focusing etc. A purified preparation can be obtained by using alone or in combination.
[0090]
In addition, when the protein is expressed by forming an insoluble substance in the cell, the protein is recovered by a usual method from the precipitate fraction obtained by crushing and then centrifuging the cell. Thereafter, the insoluble material of the protein is solubilized with a protein denaturant. The solubilized solution is diluted or dialyzed into a solution that does not contain a protein denaturing agent or is so diluted that the concentration of the protein denaturing agent does not denature the protein, and the protein is formed into a normal three-dimensional structure. A purified sample can be obtained by the same isolation and purification method.
[0091]
When the protein of the present invention or a derivative such as a sugar modification product thereof is secreted extracellularly, the protein or a derivative such as a sugar chain adduct can be recovered in the culture supernatant. That is, a soluble fraction is obtained by treating the culture by a technique such as centrifugation as described above, and a purified preparation is obtained from the soluble fraction by using the same isolation and purification method as described above. be able to.
Examples of the protein thus obtained include a protein having an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 1 to 5.
[0092]
The protein expressed by the above method can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). Also, Kuwawa Trading (US Advanced chemTech), Perkin Elmer Jaban (Perkin-Elmer), Pharmacia Biotech (Studen Pharmacia Biotech), Aloka (Protein Technology Instrument), Kurabo (Synthecell- Vega), Nippon Perceptive Limited (US PerSeptive), Shimadzu Corporation and other peptide synthesizers can be used for synthesis.
[0093]
III. Production of isoprenoid compounds
II. The transformant obtained in the above II. The isoprenoid compound can be produced by culturing according to the above method, producing and accumulating the isoprenoid compound in the culture, and collecting the isoprenoid compound from the culture.
[0094]
By the cultivation, ubiquinone, vitamin K₂Isoprenoid compounds such as carotenoids can be produced. As a specific example, for example,EscherichiaProduction of ubiquinone-8 and menaquinone-8 using microorganisms belonging to the genus as transformants,RhodobacterProduction of ubiquinone-10 using a microorganism belonging to the genus as a transformant,ArthrobacterVitamin K using microorganisms belonging to the genus as transformants₂Manufacturing of,AgrobacteriumProduction of astaxanthin using a microorganism belonging to the genus as a transformant,ErwiniaExamples thereof include production of lycopene, β-carotene, and zeaxanthin using microorganisms belonging to the genus as transformants.
[0095]
After completion of the culture, an isoprenoid compound is extracted by adding an appropriate solvent to the culture solution, and after removing the precipitate by centrifugation or the like, the isoprenoid compound can be isolated and purified by performing various chromatography.
[0096]
IV. Search for substances that inhibit enzyme activity on the non-mevalonate pathway
(1) Measurement of enzyme activity on non-mevalonate pathway
The enzyme activity on the non-mevalonate pathway can be measured according to a normal enzyme activity measurement method.
That is, the pH of the buffer used for the reaction solution for activity measurement may be in the PH range that does not inhibit the activity of the target enzyme, and is preferably in the range including the optimum PH.
[0097]
For example, in 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the pH is 5 to 10, preferably 6 to 9.
As the buffer solution, any buffer solution can be used as long as the pH can be achieved without inhibiting the enzyme activity. As the buffer solution, Tris-HCl buffer solution, phosphate buffer solution, borate buffer solution, HEPES buffer solution, MOPS buffer solution, bicarbonate buffer solution and the like can be used. In 1-deoxy-D-xylulose 5-phosphate reductoisomerase, for example, a Tris-HCl buffer is preferably used.
[0098]
The concentration of the buffer can be used as long as it does not inhibit the enzyme activity, but it is preferably 1 mM to 1 M.
If the target enzyme requires a coenzyme, the coenzyme is added to the reaction solution. For example, in 1-deoxy-D-xylulose 5-phosphate reductoisomerase, NADPH, NADH or other electron donors can be used, preferably NADPH.
[0099]
The concentration of the coenzyme to be added can be any concentration as long as it does not inhibit the reaction, but it is preferably a concentration of 0.01 mM to 100 mM, more preferably 0.1 mM to 10 mM.
You may add a metal ion to a reaction liquid as needed. Any metal ion can be added as long as it does not inhibit the reaction.²⁺, Mg²⁺, Mn²⁺Etc.
[0100]
A metal ion can be added as a metal salt, and for example, it can be added as a chloride, sulfate, carbonate, phosphate or the like.
The concentration of the metal ion to be added can be any concentration as long as the reaction is not inhibited, but is preferably 0 mM to 100 mM, more preferably 0.1 mM to 10 mM.
[0101]
A substrate for the target enzyme is added to the reaction solution. For example, in 1-deoxy-D-xylulose 5-phosphate reductoisomerase, 1-deoxy-D-xylulose 5-phosphate is added.
Any concentration of the substrate can be used as long as it does not interfere with the reaction, but the concentration in the reaction solution is preferably 0.01 mM to 0.2 M.
[0102]
The enzyme concentration used in the reaction is not particularly limited, but the reaction is usually performed in a concentration range of 0.01 mg / ml to 100 mg / ml.
The enzyme to be used does not necessarily have to be purified to a single one, and may be a standard in which other contaminating proteins are mixed as long as the reaction is not disturbed. In the search of (2) below, a cell extract containing the enzyme activity or a cell having the enzyme activity can also be used.
[0103]
The reaction temperature may be in the temperature range that does not inhibit the activity of the target enzyme, and is preferably in the range including the optimum temperature. That is, the reaction temperature is 10 ° C to 60 ° C, more preferably 30 ° C to 40 ° C.
The detection of the activity can be performed by using a method capable of measuring the substrate or the reaction product to decrease the substrate accompanying the reaction or increase the reaction product.
[0104]
Examples of the method include a method of separating and quantifying the target substance by high performance liquid chromatography (HPLC) as necessary. When NADH or NADPH increases or decreases with the progress of the reaction, the activity can be directly measured by measuring the absorbance of the reaction solution at 340 nm. For example, in 1-deoxy-D-xylulose 5-phosphate reductoisomerase, NADPH that decreases as the reaction proceeds is quantified by measuring the decrease in absorbance at 340 nm with a spectrophotometer, and the activity is detected. Can do.
[0105]
(2) Search for substances that inhibit enzyme activity on the non-mevalonate pathway
The search for substances that inhibit the enzyme activity on the non-mevalonate pathway is performed by adding the substance to be searched to the enzyme activity measurement system of (1) above and reacting in the same manner, and suppressing the decrease in the amount of substrate compared to the absence of addition. Or it can carry out by searching for the substance which suppresses the production amount of a reaction product.
[0106]
Search methods include tracking the amount of substrate decrease or reaction product increase over time, measuring the amount of substrate decrease or reaction product increase after a certain time of reaction, etc. Can give.
In the method of tracking the decrease amount of the substrate or the increase amount of the reaction product over time, it is preferable to measure the decrease amount of the substrate or the increase amount of the reaction product at intervals of about 15 seconds to 20 minutes during the reaction. It is more preferable to measure at intervals of 1 to 3 minutes.
[0107]
In the method of measuring the decrease amount of the substrate or the increase amount of the reaction product after the reaction for a certain time, the reaction time is preferably 10 minutes to 1 day, more preferably 30 minutes to 2 hours.
A substance that inhibits enzyme activity on the non-mevalonate pathway inhibits the growth of microorganisms and plants having the non-mevalonate pathway. We have found for the first time that the substance inhibits the growth of the microorganisms and plants.
[0108]
Since the non-mevalonate pathway exists in microorganisms and plants and does not exist in animals and humans, substances that inhibit the enzyme activity on the non-mevalonate pathway that do not affect humans and animals are obtained by the above search method. can do.
The substance can be an effective antibacterial or herbicide.
Examples of the present invention are shown below, but the present invention is not limited to these Examples. The gene recombination experiments shown in the Examples were performed using the method described in Molecular Cloning 2nd edition (hereinafter referred to as a conventional method) unless otherwise specified.
[0109]
【Example】
Example 1 Acquisition of DNA encoding a protein involved in biosynthesis of isoprenoid compounds
(1) Acquisition of DNA encoding a protein involved in biosynthesis of isoprenoid compounds using the E. coli DXS gene base sequence
E.coli XL1-Blue strain (purchased from Toyobo) was inoculated into one platinum loop, 10 ml of LB liquid medium, and cultured at 37 ° C. overnight.
[0110]
After culturing, cells were obtained from the obtained culture broth by centrifugation.
Chromosomal DNA was isolated and purified from the cells according to a conventional method.
At the 5 ′ end having a combination of base sequences of SEQ ID NOs: 12 and 13, SEQ ID NOs: 14 and 15, SEQ ID NOs: 12 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 13BamHI andEcoSense primer and antisense primer having RI restriction enzyme cleavage sites, respectively, at the 5 ′ end having a combination of the nucleotide sequences of SEQ ID NOs: 22 and 23BamA sense primer and an antisense primer each having a HI restriction enzyme cleavage site were synthesized using a DNA synthesizer.
[0111]
Using chromosomal DNA as a template, these primers and TaKaRa LA-PCR^TM Kit Ver.2 (Takara Shuzo), Expand^TM PCR was performed with DNA Thermal Cycler (Perkin Elmer Japan) using High-Fidelity PCR System (Boehringer Mannheim) or Taq DNA polymerase (Boelinnger).
[0112]
PCR is a DNA fragment of 2 kb or less for 30 seconds at 94 ° C, a reaction step consisting of 30 seconds to 1 minute at 55 ° C and 2 minutes at 72 ° C, and a DNA fragment exceeding 2 kb for 20 seconds at 98 ° C, The reaction process consisting of 3 minutes at 68 ° C. was defined as one cycle, and after 30 cycles, the reaction was carried out at 72 ° C. for 7 minutes.
[0113]
Among DNA fragments amplified by PCR, at the 5 ′ endBamHI andEc oDNA fragments amplified using a sense primer and an antisense primer each having an RI restriction enzyme cleavage site areBamHI andEcoDigested with RI and at the 5 'endBamA DNA fragment amplified using a sense primer and an antisense primer each having a HI restriction enzyme cleavage site is a restriction enzymeBamDigested with HI.
[0114]
After digestion, these restriction enzyme-treated DNA fragments are subjected to agarose gel electrophoresis,BamHI-EcoRI-treated DNA fragments andBamA HI-treated DNA fragment was obtained.
A broad host range vector pEG400 with lac promoter [J. Bac.,172, 2392 (1990)), the restriction enzymeBamHI andEcoAfter digestion with RI, perform agarose gel electrophoresis,BamHI-EcoRI-treated pEG400 fragment was obtained.
[0115]
pUC118 (Takara Shuzo) is a restriction enzymeBamAfter digestion with HI, perform agarose gel electrophoresisBamHI-treated pUC118 fragment was obtained.
Obtained aboveBamHI-EcoAbout each RI-treated DNA fragmentBamHI-EcoAfter mixing with the RI-treated pEG400 fragment, ethanol precipitation was performed, the obtained DNA precipitate was dissolved in 5 μl of distilled water, and a ligation reaction was performed to obtain each recombinant DNA.
[0116]
Using the recombinant DNA,E.coli(Purchased from Toyobo) After transforming the DH5α strain according to a conventional method, the transformant was applied to an LB agar medium containing 100 μg / ml of spectinomycin and cultured at 37 ° C. overnight.
Several colonies of spectinomycin-resistant transformants that had grown were subjected to shaking culture at 37 ° C. for 16 hours in 10 ml of LB liquid medium containing 100 μg / ml of spectinomycin.
[0117]
The cells were obtained by centrifuging the obtained culture solution.
A plasmid was isolated from the cells according to a conventional method.
The plasmid isolated by this method was cleaved with various restriction enzymes, the structure was examined, and the nucleotide sequence was determined to confirm that the plasmid had the target DNA fragment inserted.
[0118]
A plasmid comprising DNA having the base sequence set forth in SEQ ID NO: 6, DNA having the base sequence set forth in SEQ ID NO: 7, DNA having the base sequence set forth in SEQ ID NO: 8 and DNA having the base sequence set forth in SEQ ID NO: 9 PDXS-1 is a plasmid containing DNA having the base sequence described in SEQ ID NO: 6, pISP-1 is a plasmid containing DNA having the base sequence described in SEQ ID NO: 7, and plasmid containing DNA having the base sequence described in SEQ ID NO: 8 PXSE-1, and a plasmid containing DNA having the base sequence shown in SEQ ID NO: 9 was named pTFE-1.
[0119]
Also obtained aboveBamHI-treated DNA fragments andBamAfter mixing the HI-treated pUC118 fragment, ethanol precipitation was performed, the obtained DNA precipitate was dissolved in 5 μl of distilled water, and a ligation reaction was performed to obtain recombinant DNA. Thereafter, E. coli was transformed by the same method as described above, and a plasmid was isolated from the E. coli.
[0120]
As described above, the plasmid isolated by this method was cleaved with various restriction enzymes, the structure was examined, and the nucleotide sequence was determined to confirm that the plasmid had the target DNA fragment inserted.
The plasmidBamAfter HI treatment, the target DNA fragment was recovered by the same method as described above, and subcloned into the expression vector pQE30 (manufactured by Qiagen) by a conventional method.
The plasmid having the base sequence described in SEQ ID NO: 6 obtained by the subcloning was named pQEDXS-1.
[0121]
(2) Acquisition of methylerythritol-requiring complementary genes
(1) Acquisition of E. coli methylerythritol-requiring mutant
E.coli The W3110 strain (ATCC14948) was inoculated into an LB liquid medium and cultured until the logarithmic growth phase.
After culturing, cells were obtained from the obtained culture broth by centrifugation.
After washing the cells with 0.05 M Tris-Maleate buffer (pH 6.0), the cell concentration was 10⁹It was suspended in the same buffer so that it might become a cell / ml.
[0122]
To this suspension, NTG was added so as to have a final concentration of 600 mg / l, and mutation treatment was carried out by maintaining at room temperature for 20 minutes.
The obtained mutant-treated cells were applied to M9 minimal agar medium [Molecular Cloning Second Edition] plate containing 0.1% methylerythritol and cultured.
Methylerythritol is a Tetrahedron Letters,38, 35, 6184 (1997).
[0123]
A colony that has grown on an M9 minimal agar medium containing 0.1% methylerythritol is replicated on an M9 minimal agar medium and an M9 minimal agar medium containing 0.1% methylerythritol, and exhibits methyl erythritol requirement. A strain capable of growing on an M9 minimal agar medium containing 0.1% methylerythritol but not growing on an M9 minimal agar medium was selected as a target mutant.
The methylerythritol-requiring mutant ME7 obtained by the selection was used in the following experiment.
[0124]
(2) Acquisition of methylerythritol-requiring complementary gene
E.coli The W3110 strain (ATCC14948) was inoculated into an LB liquid medium, cultured to the logarithmic growth phase, and then centrifuged to recover the cells.
Chromosomal DNA was isolated and purified from the obtained bacterial cells according to a conventional method.
[0125]
200 μg of the chromosomal DNA is used as a restriction enzymeSauThe digested DNA fragment obtained by partial digestion with 3AI was subjected to size fractionation by sucrose density gradient ultracentrifugation (26,000 rpm, 20 ° C., 20 hr).
A DNA fragment having a size of 4 to 6 kb obtained by the fractionation is converted into a restriction enzyme.BamA chromosomal genomic library was prepared by ligation to the vector pMW118 (Nippon Gene) digested with HI.
[0126]
Using the prepared chromosome library, the ME7 strain isolated in (1) above was transformed according to a conventional method.
The obtained transformant was applied to an LB agar medium containing 100 μg / l of ampicillin and cultured at 37 ° C. overnight.
A plasmid was extracted from a plurality of colonies grown in the culture, and the nucleotide sequence was determined.
[0127]
The clone whose base sequence was determined had a sequence containing the base sequence shown in SEQ ID NO: 10. These plasmids were named pMEW41 and pMEW73.
A plasmid extracted from one strain of the clone having the sequence was designated as pMEW73.
pMEW73HindIII andSacDouble-digested with I and having the base sequence shown in SEQ ID NO: 10 obtainedHindIII-SacThe I-treated DNA fragment was transferred to the broad host range vector pEG400 [J. Bac.,172, 2392 (1990)] to produce pEGYM1.
[0128]
the aboveHindIII-SacI-treated DNA fragment was transformed into vector pUC19 [Gene,33, 103 (1985))HindIII-SacPUCYM-1 was prepared by ligation to the I site.
From the chromosome base sequence information of E. coli based on the Genbank database, it was found that the DNA fragment inserted into the vector contained the yaeM gene.
[0129]
A recombinant vector that sufficiently expresses the yaeM gene is expressed by the PCR method [Science,230, 1350 (1985)].
A sense primer having the sequence shown in SEQ ID NO: 20 and an antisense primer having the sequence shown in SEQ ID NO: 21 were synthesized using a DNA synthesizer.
[0130]
At the 5 ′ end of the sense primer and antisense primer,B amAn HI restriction enzyme site was added.
Using chromosomal DNA as a template, these primers andTaqThe yaeM gene was amplified by PCR using DNA polymerase (manufactured by Boelinnger) and DNA Thermal Cycler (manufactured by PerkinElmer Japan).
[0131]
PCR was performed under the condition that a reaction step consisting of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 2 minutes was performed for 1 minute and 30 cycles and then reacted at 72 ° C. for 7 minutes.
Restriction enzyme from amplified DNA fragment and pUC118 (Takara Shuzo)BamAfter digestion with HI, each DNA fragment was purified by agarose gel electrophoresis.
[0132]
These purified fragments were mixed and then subjected to ethanol precipitation. The obtained DNA precipitate was dissolved in 5 μl of distilled water, and a ligation reaction was performed to obtain recombinant DNA.
After confirming that the recombinant DNA was the yaeM gene by determining the DNA sequence, it was subcloned into the expression vector pQE30 (manufactured by Qiagen).
The obtained recombinant DNA was named pQEYM1.
[0133]
After transforming the ME7 strain using pQEYM1 according to a conventional method, the transformant was applied to an LB agar medium containing 100 μg / ml of ampicillin and cultured at 37 ° C. overnight.
The transformed strain was confirmed to form colonies at a growth rate comparable to that of the wild type strain, and it was found that the mutation of ME7 strain was complemented by the yaeM gene.
[0134]
Example 2 Production of ubiquinone-8 (CoQ8) by recombinant Escherichia coli
(1) Plasmid pADO-1, pDXS-1, pXSE-1 obtained in Example 1 or pEG400 as a controlE.coli Transformants introduced into the DH5α strain and resistant to spectinomycin at a concentration of 100 μg / mlE.coli DH5α / pADO-1,E.coli DH5α / pDXS-1,E.coli DH5α / pXSE-1 andE.coli DH5α / pEG400 was obtained respectively.
[0135]
Thiamine and vitamin B₆These transformants were inoculated into a test tube containing 10 ml of LB medium supplemented with 100 mg / l of p-hydroxybenzoic acid, 50 mg / l of p-hydroxybenzoic acid and 100 μg / ml of spectinomycin, and cultured with shaking at 30 ° C. for 72 hours.
After completion of the culture, each culture solution was concentrated 10 times.
[0136]
300 μl of 2-butanol and 300 μl of glass beads were added to 300 μl of each concentrated solution, and the isoprenoid compound was subjected to solvent extraction while crushing the cells with a multi-bead shocker MB-200 (manufactured by Yasui Kikai Co., Ltd.) for 5 minutes, followed by centrifugation. The 2-butanol layer was collected by
The amount of CoQ8 produced by the transformant was calculated by quantitatively analyzing CoQ8 in the butanol layer by high performance liquid chromatography (LC-10A, manufactured by Shimadzu Corporation).
[0137]
The column was Develosil ODS-HG-5 (Nomura Chemical), and a solution of methanol: n-hexane = 8: 2 was used as a mobile phase, and analysis was performed under conditions of a flow rate of 1 ml / min and a measurement wavelength of 275 nm.
The results are shown in Table 1.
[0138]
[Table 1]

[0139]
The amount of CoQ8 produced was significantly higher in DH5α / pADO-1, DH5α / pDXS-1, and DH5α / pXSE-1 than in the control strain DH5α / pEG400. In particular, the highest productivity was obtained with DH5α / pADO-1 into which all of the DNA obtained in Example 1 was introduced.
[0140]
(2) Obtained in (1) above into a test tube containing 10 ml of M9 medium.E.coli DH5α / pDXS-1 orE.coli DH5α / pEG400 was inoculated and cultured with shaking at 30 ° C. for 72 hours.
After completion of the culture, the amount of CoQ8 produced by the transformant was calculated by the same method as in (1) above.
The results are shown in Table 2.
[0141]
[Table 2]

[0142]
The amount of CoQ8 produced was significantly higher in DH5α / pDXS-1 than in the control strain DH5α / pEG400.
(3) CoQ8 production by recombinant E. coli
Plasmid pEGYM1 obtained in Example 1 or pEG400 as a controlE.coli Transformant introduced into DH5α strain and resistant to spectinomycin at a concentration of 100 μg / mlE.coli DH5α / pEGYM1 andE.coli DH5α / pEG400 was obtained respectively.
[0143]
1% glucose, vitamin B₁100mg / l, vitamin B₆These transformants were inoculated into a test tube containing 10 ml of LB medium supplemented with 100 mg / l, p-hydroxybenzoic acid 50 mg / l, and cultured with shaking at 30 ° C. for 72 hours.
After completion of the culture, the amount of CoQ8 produced by the transformant was calculated by the same method as in (1) above.
The results are shown in Table 3.
[0144]
[Table 3]

[0145]
The amount of CoQ8 produced was significantly higher in DH5a / pEGYM1 than in the control strain DH5a / pEG400.
[0146]
Example 3 Production of menaquinone-8 (MK-8) by recombinant Escherichia coli
(1) TB medium supplemented with 100 μg / ml of spectinomycin [12 g of Bactotryptone (Difco), 24 g of yeast extract (Difco) and 5 g of glycerol were dissolved in 900 ml of water, and KH₂PO_Four0.17M, K₂HPO_FourIn a test tube containing 10 ml of a medium prepared by adding 100 ml of an aqueous solution containing 0.72 M], obtained in Example 2 (1),E.coli DH5α / pADO-1 orE.coli DH5α / pEG400 was inoculated and cultured with shaking at 30 ° C. for 72 hours.
[0147]
After completion of the culture, MK-8 was quantified by the same method as the CoQ8 quantification method of Example 2 (1), and the amount of MK-8 produced by the transformant was calculated.
The results are shown in Table 4.
[0148]
[Table 4]

[0149]
MK-8 production was significantly higher in DH5α / pADO-1 compared to control strain DH5α / pEG400.
(2) Obtained in Example 2 (1)E.coli DH5α / pDXS-1 orE.coli DH5α / pEG400 was cultured in the same manner as in (1) above, and the amount of MK-8 produced by the transformant was calculated.
The results are shown in Table 5.
[0150]
[Table 5]

[0151]
The production amount of MK-8 was significantly higher in DH5α / pDXS-1 than in the control strain DH5α / pEG400.
[0152]
Example 4Erwinia carotovoraProduction of CoQ8
Plasmid pDXS-1 obtained in Example 1 or pEG400 as a controlErwinia carotovora Transformants IFO-3380 / pDXS-1 and IFO-3380 / pEG400 that were introduced into the IFO-3380 strain and showed resistance to spectinomycin at a concentration of 100 μg / ml were obtained.
[0153]
These transformants were inoculated into a test tube containing 10 ml of LB medium supplemented with 100 μg / ml of spectinomycin, and cultured with shaking at 30 ° C. for 72 hours.
After completion of the culture, the amount of CoQ8 produced by the transformant was calculated by the same method as in Example 2 (1).
The results are shown in Table 6.
[0154]
[Table 6]

[0155]
The amount of CoQ8 produced was significantly higher in IFO-3380 / pDXS-1 than in the control strain IFO-3380 / pEG400.
[0156]
Example 5:Erwinia uredovoraProduction of ubiquinone and carotenoids
The plasmids pUCYM-1, pQEDXS-1, pQEYM-1 obtained in Example 1 or pUC19 and pQE30 as controls were electroporated.Erwinia uredovor aTransformant introduced into strain DSM-30080 and resistant to ampicillin at a concentration of 100 μg / mlE.uredovoraDSM-30080 / pUCYM-1,E.uredovoraDSM-30080 / pQEDXS-1,E.uredovoraDSM-30080 / pQEYM-1,E.uredovoraDSM-30080 / pUC19 andE.uredovoraDSM-30080 / pQE30 was obtained.
[0157]
Ampicillin 100 μg / ml, glucose 1%, vitamin B₁100mg / l, vitamin B₆These transformants were inoculated into a test tube containing 10 ml of LB medium supplemented with 100 mg / l, p-hydroxybenzoic acid 50 mg / l, and cultured with shaking at 30 ° C. for 72 hours.
[0158]
After completion of the culture, the amount of CoQ8 produced by the transformant was calculated by the same method as in Example 2 (1).
The production amount of the carotenoid pigment was calculated by measuring the absorbance at 450 nm using a spectrophotometer for the 2-butanol layer obtained by the same method as in Example 2 (1).
The results are shown in Table 7.
[0159]
[Table 7]

[0160]
Both CoQ8 production and carotenoid pigment production were significantly higher in DSM-30080 / pUCYM-1 than in control strain DSM-30080 / pUC19.
Similarly, both CoQ8 production and carotenoid pigment production were significantly higher in DSM-30080 / pQEYM-1 and DSM-30080 / pQEDXS-1 than in control strains DSM-30080 / pQE30.
[0161]
Example 6 Photosynthetic bacteriaRhodobacter sphaeroidesOf DNA encoding proteins involved in biosynthesis of isoprenoid compounds from sea urchin
(1)R.sphaeroidesOf DXS gene from
Using DXS gene sequences found in E. coli, DXS homologs conserved in other species were searched from genbank. as a result,Haemophilus influenzae(P45205),Rhodobacter capsulatus(P26242),Bacillussubtilis(P54523),Synechocystissp. PCC6803 (P73067) andMycobacteriun tuberculosis(O07184) etc. found a homologue. These sequences were compared to select highly conserved amino acid sequences. The nucleotide sequence corresponding to the conserved amino acid sequenceR.sphaeroidesA DNA synthesizer was used in consideration of the codon usage frequency of the DNA fragments, and the DNA fragments having the nucleotide sequences set forth in SEQ ID NO: 32 and SEQ ID NO: 33 as sense primers and the DNA fragments having SEQ ID NO: 34 as antisense primers were used. And synthesized.
[0162]
R.sphaeroidesUsing the chromosomal DNA of KY4113 strain (FERM-P4675) as a template, the above primers and Expand^TM PCR was performed with DNA Thermal Cycler (Perkin Elmer Japan) using High-Fidelity PCR System (Boehringer Mannheim).
PCR is carried out under the condition that the reaction step consisting of 94 ° C. for 40 seconds, 60 ° C. for 40 seconds, and 72 ° C. for 1 minute is performed for 30 cycles, and then the reaction is performed at 72 ° C. for 7 minutes. A fragment was obtained. This DNA fragment was DIG-labeled using a DIG DNA Labeling Kit (Boehringer Mannheim).
[0163]
R.sphaeroidesIn order to obtain the full length DXS gene, a genomic library of KY4113 strain was prepared. The KY4113 strain was cultured overnight in LB medium, and chromosomal DNA was extracted. Restriction enzymeSauA 4-6 kb DNA fragment was purified by partial digestion with 3AI and sucrose density gradient ultracentrifugation. The DNA fragment andBamThe HI digested vector pUC19 was ligated using Ligation Pack (Nippon Gene) and transformed into E. coli DH5α. The transformant was applied to an LB plate containing 100 μg / ml of ampicillin to obtain about 10,000 colonies.
[0164]
  When screening was performed by colony hybridization using the DIG-labeled DNA fragment obtained by the above method as a probe, two types of DNA fragments were detected. When each was sequenced, an ORF having high homology with a known DXS gene of another species was found from each DNA fragment. The amino acid sequence shown in SEQ ID NO: 26 is DXS1, SEQ ID NO:28The amino acid sequence shown in FIG.
[0165]
(2) Complementarity confirmation using E. coli DXS gene-deficient strain
(1) Acquisition of E. coli DXS gene deficient strain
E.coli The W3110 strain (ATCC14948) was inoculated into an LB liquid medium and cultured until the logarithmic growth phase. After culturing, cells were obtained from the obtained culture broth by centrifugation.
After washing the cells with 0.05 M Tris-Maleate buffer (pH 6.0), the cell concentration was 10⁹It was suspended in the same buffer so that it might become a cell / ml.
[0166]
To this suspension, NTG was added so as to have a final concentration of 600 mg / l, and mutation treatment was carried out by maintaining at room temperature for 20 minutes.
The obtained mutant-treated cells were applied to a M9 minimal agar medium [Molecular Cloning Second Edition] plate containing 0.1% of 1-deoxyxylulose and cultured. 1-deoxyxylulose is a product of J. C. S. Perkin TransI, 2131-2137 (1982).
[0167]
Colonies grown on M9 minimal agar medium containing 0.1% 1-deoxyxylulose were replicated on M9 minimal agar medium and M9 minimal agar medium containing 0.1% 1-deoxyxylulose. A strain exhibiting the requirement for 1-deoxyxylulose, which can grow on the M9 minimal agar medium containing 0.1% xylulose but cannot grow on the M9 minimal agar medium, was selected as a target mutant.
[0168]
The mutant strain selected and obtained by this method was named ME1 strain.
When pDXS-1 was introduced into the ME1 strain, it was determined that the ME1 strain was deficient in the DXS gene because it complemented the 1-deoxyxylulose requirement.
[0169]
(3) Complementarity test of DXS1 and DXS2
Plasmids were constructed in which a DNA fragment encoding DXS1 consisting of SEQ ID NO: 27 and a DNA fragment encoding DXS2 consisting of SEQ ID NO: 29 from the KY4113 strain were each ligated downstream of the lac promoter of vector pUC19.
[0170]
When each of these constructed plasmids was introduced into the ME1 strain, both DXS1 and DXS2 complemented the 1-deoxyxylulose requirement of the ME1 strain.
From the above,R.sphaeroidesWas found to have two DXS genes, DXS1 and DXS2, that have the activity of catalyzing the reaction of producing 1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde 3-phosphate.
[0171]
(4)R.sphaeroidesObtaining methyl erythritol-requiring complementary gene from Example 1 (2) Logarithmic growth of E. coli methyl erythritol-requiring mutant ME7 obtained in Example 1 in an LB liquid medium containing 0.1% methyl erythritol After culturing to the end, the cells were collected by centrifugation.
[0172]
The cells were washed twice with a 1 mM HEPES aqueous solution containing 10% glycerol to remove as much medium components as possible.
The washed cells were prepared in Example 6 (1).R.sphaeroidesThe plasmid extracted from the genomic library of KY4113 strain was introduced by electroporation according to a conventional method.
[0173]
After the introduction, it was applied to an LB agar medium containing 100 μg / l of ampicillin and cultured at 37 ° C. overnight.
The grown colonies were fished, inoculated and cultured in an LB liquid medium, and a plasmid was extracted from the cultured cells.
[0174]
When the extracted plasmid was reintroduced into the ME7 strain, it grew in a medium containing no methylerythritol.R.sphaeroidesIt was confirmed that a DNA fragment complementary to the derived methylerythritol requirement was included.
As a result of determining the base sequence of this DNA fragment, a DNA sequence consisting of SEQ ID NO: 31 and encoding an amino acid sequence having high homology with E. coli yaeM was found.
[0175]
Example 7 Production of ubiquinone-10 (CoQ10) by recombinant photosynthetic bacteria
The plasmid prepared by ligating the glnB promoter derived from the KY4113 strain upstream of the DNA fragment DXS1 consisting of SEQ ID NO: 27 and the DNA fragment DXS2 consisting of SEQ ID NO: 29 obtained in Example 6 and inserting it into the broad host range vector pEG400 They were named pRSDX-1 and pRSDX-2. In addition, a plasmid in which yaeM and DXS1 were ligated in tandem and ligated downstream of the glnB promoter was constructed and named pRSYMDX1. These plasmids were electroporated (Bio-Rad)R.sphaeroidesIt was introduced into the KY4113 strain.
[0176]
After the introduction, the mixture was applied to an LB agar medium containing spectinomycin at a concentration of 100 μg / ml and cultured at 30 ° C. for 3 days.
The grown colonies were inoculated into an LB medium containing 100 μg / ml spectinomycin and cultured overnight, and the cultured cells were collected by centrifugation.
[0177]
Plasmid extraction (manufactured by Qiagen) was performed from the obtained bacterial cells, and it was confirmed that each introduced plasmid was retained. The transformants thus obtained were named KY4113 / pRSDX-1, KY4113 / pRSDX-2, KY4113 / pRSYMDX1, and KY4113 / pEG400.
Each transformant was inoculated into a test tube containing 5 ml of seed medium (glucose 2%, peptone 1%, yeast extract 1%, NaCl 0.5% (pH 7.2, adjusted with NaOH)). The cells were cultured at 24 ° C. for 24 hours.
[0178]
0.5 ml of the obtained culture broth was added to a ubiquinone-10 production medium (4% waste molasses, 2.7% glucose, 4% corn steep liquor, 0.8% ammonium sulfate, 0.05% potassium phosphate 1%, phosphoric acid 2 Medium containing 0.05% potassium, 0.025% magnesium sulfate heptahydrate, 3 mg / l ferrous sulfate heptahydrate, 8 mg / l thiamine, 8 mg / l nicotinic acid, 1 ml / l trace element The solution was adjusted to pH 9 and then autoclaved by adding 1% calcium carbonate) to a test tube containing 5 ml, and cultured with shaking at 30 ° C. for 5 days.
After completion of the culture, the production amount of CoQ10 by the transformant was calculated by the same method as the CoQ8 quantification method of Example 2 (1). The results are shown in Table 8.
[0179]
[Table 8]

[0180]
The amount of CoQ10 produced was significantly higher in KY4113 / pRSDX-1, KY4113 / pRSDX-2 and KY4113 / pRSYMDX1 than in the control strain KY4113 / pEG400.
[0181]
Example 8 Activity measurement of enzyme encoded by yaeM gene
(1) High expression of yaeM gene
A recombinant plasmid that sufficiently expresses the yaeM gene was obtained by PCR [Science,230, 1350 (1985)].
A sense primer having the sequence shown in SEQ ID NO: 24 and an antisense primer having the sequence shown in SEQ ID NO: 25 were synthesized using a DNA synthesizer.
[0182]
At the 5 ′ end of the sense primer and antisense primer,BamAn HI restriction enzyme site was added.
Using chromosomal DNA as a template, these primers andTaqThe yaeM gene was amplified by performing PCR using DNA polymerase (manufactured by Boehringer) and DNA Thermal Cycler (manufactured by PerkinElmer Japan).
[0183]
PCR was performed under the condition that a reaction step consisting of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 2 minutes was performed for 1 minute and 30 cycles and then reacted at 72 ° C. for 7 minutes.
Restriction enzyme from amplified DNA fragment and pUC118 (Takara Shuzo)BamAfter digestion with HI, each DNA fragment was purified by agarose gel electrophoresis.
[0184]
These purified fragments were mixed and then subjected to ethanol precipitation. The obtained DNA precipitate was dissolved in 5 μl of distilled water, and a ligation reaction was performed to obtain recombinant DNA.
It was confirmed by determining the DNA sequence that the recombinant DNA was the yaeM gene.
[0185]
A plasmid is extracted from the recombinant, and a restriction enzyme restriction enzyme is extracted.BamAfter digestion with HI, perform agarose gel electrophoresisBamA DNA fragment containing HI-treated yaeM gene was obtained.
pQE30 (QIAGEN) is a restriction enzymeBamAfter digestion with HI, perform agarose gel electrophoresisBamA HI-treated pQE30 fragment was obtained.
[0186]
Obtained aboveBamHI-treated yaeM gene-containing DNA fragmentBamAfter mixing with the HI-digested pQE30 fragment, ethanol precipitation was performed, and the resulting DNA precipitate was dissolved in 5 μl of distilled water, and a ligation reaction was performed to obtain recombinant DNA.
Using the recombinant DNA,E.Coli After the JM109 strain was transformed according to a conventional method, the transformant was applied to an LB agar medium containing 100 μg / ml of ampicillin and cultured at 37 ° C. overnight.
[0187]
A plasmid was isolated from the E. coli by the same method as described above.
As described above, the plasmid isolated by this method was cleaved with various restriction enzymes, the structure was examined, and the nucleotide sequence was determined to confirm that the plasmid had the target DNA fragment inserted. This plasmid was named pQEDXR.
[0188]
(2) Activity measurement of yaeM gene product
(i)Purification of the yaeM gene product
  PQEDXR created in (1) has pREP4 in the usual wayE.coli M15 / pREP4 + pQEDXR strains resistant to ampicillin 200 μg / ml and kanamycin 25 μg / ml were obtained by introduction into M15 strain (manufactured by QIAGEN).
[0189]
The M15 / pREP4 + pQEDXR strain was cultured at 37 ° C. in 100 ml of LB liquid medium containing 200 μg / ml of ampicillin and 25 μg / ml of kanamycin. Added to 2 mM. After further culturing at 37 ° C. for 5 hours, the culture supernatant was removed by centrifugation (3000 rpm, 10 minutes). The cells were suspended in 6 ml of 100 mM Tris-HCl buffer (pH 8.0), and crushed using an ultrasonic crusher (SONIFIER, BRANSON) while cooling with ice. The obtained bacterial cell disruption solution was centrifuged (10,000 rpm, 20 minutes, 4 ° C.), and the supernatant was collected. The cell extract centrifugation supernatant was passed through a Ni-NTA resin column (QIAGEN) and washed with 20 ml of a washing buffer (100 mM Tris-HCl (pH 8.0), 50 mM imidazole, 0.5% Tween 20). Subsequently, 10 ml of elution buffer [100 mM Tris-HCl (pH 8.0), 200 mM imidazole] was passed through, and the eluate was fractionated by 1 ml.
The protein amount of each fraction was measured (using a protein quantification kit manufactured by BioRad), and the fraction containing the protein was used as a purified protein fraction.
[0190]
  (ii) Preparation of substrate 1-deoxy-D-xylulose 5-phosphate
  The reaction substrate, 1-deoxy-D-xylulose 5-phosphate, was prepared as follows. 1-deoxy-D-xylulose 5-phosphate was detected by HPLC [column: Senshu pak NH2-1251-N (4.6 × 250 mm, manufactured by Senshu)], moving bed: 100 mM KH.₂PO_Four(PH 3.5)] by the method of measuring the absorbance at 195 nm.
[0191]
  Plasmid pQDXS-1, which highly expresses the dxs gene of Escherichia coliE.coliThis was introduced into the M15 / pREP4 strain to obtain the M15 / pREP4 + pQDXS-1 strain. This strain was obtained in Example 8 (2).(i)The dxs enzyme protein was purified using a Ni-NTA resin column.
[0192]
20 ml of the purified dxs protein [100 mM Tris-HCl (pH 7.5), 10 mM sodium pyruvate, 30 mM DL-glyceraldehyde-3-phosphate, 1.5 mM thiamine pyrophosphate, 10 mM MgCl₂1 mM DL-dithiothreitol] and incubated at 37 ° C.
[0193]
After reacting for 12 hours, the reaction solution was diluted to 300 ml with water, passed through an activated carbon column (2.2 × 8 cm), passed through Dowex 1-X8 (Cl− type, 3.5 × 25 cm), and 1% saline Eluted with. The elution fraction was concentrated, passed through Sephadex G-10 (1.8 × 100 cm), and eluted with water. The fraction containing 1-deoxy-D-xylulose 5-phosphate was lyophilized to obtain about 50 mg of white powder.
It was confirmed by NMR analysis (A-500, manufactured by JEOL Ltd.) that the powder was 1-deoxy-D-xylulose 5-phosphate.
[0194]
  (iii) Enzyme activity measurement of yaeM gene product
  100 mM Tris-HCl (pH 7.5), 1 mM MnCl ₂ 0.3 mM NADPH and Example 8 (2)(i)1 mM of the 1-deoxy-D-xylulose 5-phosphate synthesized as described above (final concentration) was added to 1 ml of the reaction solution containing the yaeM gene product obtained in the above and incubated at 37 ° C. Increase or decrease NADPH during incubation by 340nmAs a result, it was found that NADPH decreased with the passage of time using a spectrophotometer (UV-160, manufactured by Shimadzu Corporation).
[0195]
In order to confirm the structure of the reaction product, the reaction was performed with the scale increased as follows, and the product was isolated. 200 ml of a reaction solution having the same composition as described above except that the concentration of 1-deoxy-D-xylulose 5-phosphate was 0.15 mM was similarly incubated at 37 ° C. for 30 minutes, and then the entire amount was passed through an activated carbon column. The passing liquid was diluted to 1 L with water and then passed through a Dowex 1-X8 (Cl-type, 3.5 × 20 cm) column.
[0196]
It was eluted with 400 ml of 1% saline, passed through Sephadex G-10 (1.8 × 100 cm) and eluted with water. The reaction product was isolated by lyophilizing the eluted fraction.
The molecular formula of the reaction product isolated from the HR-FABMS analysis is C_FiveH₁₂O₇P [m / z 215.0276 (M-H)^-, Δ-4.5 mmu].¹H and¹³The following chemical shifts were obtained from CNMR analysis.
[0197]
¹H NMR (D₂O, 500 MHz): δ4.03 (ddd, J = 11.5, 6.5, 2.5 Hz, 1H), 3.84 (ddd, J = 11.5, 8.0, 6.5 Hz, 1H), 3.78 (dd, J = 80, 2.5 Hz , 1H), 3.60 (d, J = 12.0 Hz, 1H), 3.50 (d, J = 12.0 Hz, 1H), 1.15 (s,^ThreeH);¹³C NMR (D₂O, 125 MHz): δ75.1 (C-2), 74.8 (C-3), 67.4 (C-1), 65.9 (C-4), 19.4 (2-Me)
A compound obtained by treating this reaction product with alkaline phosphatase (Takara Shuzo)¹H and¹³The chemical shift obtained by C NMR analysis is Tetrahedron Letter,38, 6184 (1997), and was completely consistent with the chemical shift obtained by NMR analysis of 2-C-methyl-D-erythritol synthesized by the method described in US Pat.
[0198]
Furthermore, the former optical rotation is [α]._D ^{twenty one}= + 6.0 (c = 0.050, H₂O), [Tetrahedron Letter,38, 6184 (1997)] Optical rotation of 2-C-methyl-D-erythritol [α]_D ^{twenty five}= + 7.0 (c = 0.13, H₂O).
[0199]
These results revealed that the reaction product of the yaeM gene product was 2-C-methyl-D-erythritol 4-phosphate. That is, it was found that the yaeM gene product has an activity of generating 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate with consumption of NADPH. Based on this catalytic activity, this enzyme was named 1-deoxy-D-xylulose 5-phosphate reductoisomerase.
[0200]
  (iv) Properties of 1-deoxy-D-xylulose 5-phosphate reductoisomerase
  Example 8 (2)(iii)The enzymatic properties of 1-deoxy-D-xylulose 5-phosphate reductoisomerase were examined using the 1 ml reaction system described in 1. above. Note that 1u is defined as an activity that oxidizes 1 mmol of NADPH per minute. When NADPH was replaced with NADH, the activity decreased to 1/100 or less.
[0201]
When 1-deoxy-D-xylulose was used instead of 1-deoxy-D-xylulose 5-phosphate, no reaction occurred.
From the SDS-PAGE analysis, it was found that this enzyme is composed of a 42 kDa polypeptide.
Table 9 shows the effect of metal addition to the reaction system.
[0202]
[Table 9]

[0203]
MnCl₂The Km to 1-deoxy-D-xylulose 5-phosphate and NADPH in the presence was 249 μM and 7.4 μM, respectively.
The influence of the reaction temperature is shown in FIG. 1, and the influence of the reaction pH is shown in FIG.
[0204]
Example 9 Preparation and properties of yaeM-deficient mutant
(1) Creation of yaeM-deficient mutant
In order to investigate whether 1-deoxy-D-xylulose 5-phosphate reductoisomerase is essential for cell growth, a defective mutant thereof was prepared as follows.
A kanamycin resistance gene cassette for insertion into the yaeM gene was prepared as follows.
[0205]
The plasmid pMEW41 obtained in Example 1 (2) (2) was used as a restriction enzyme.BalAgarose gel electrophoresis after digestion with I,BaiII-treated DNA fragments were obtained.
Tn5 as a restriction enzymeHindIII andSamAfter digestion with I, the ends of the fragments were blunted using a DNA blunting kit (Takara Shuzo).
The resulting blunted DNA fragment was previously createdBaiIAfter mixing with the I-treated pMEW41 DNA fragment, ethanol precipitation was performed, the obtained DNA precipitate was dissolved in 5 μl of distilled water, and a ligation reaction was performed to obtain recombinant DNA.
[0206]
Using the recombinant DNA,E.coli The JM109 strain (purchased from Takara Shuzo) was transformed according to a conventional method, and the transformant was applied to an LB agar medium containing 100 μg / ml of ampicillin and 15 μg / ml of kanamycin and cultured at 37 ° C. overnight.
Several colonies of ampicillin-resistant transformants that had grown were subjected to shaking culture at 37 ° C. for 16 hours in 10 ml of LB liquid medium containing 100 μg / ml of ampicillin and 15 μg / ml of kanamycin.
The cells were obtained by centrifuging the obtained culture solution.
[0207]
A plasmid was isolated from the cells according to a conventional method.
The plasmid isolated by this method was cleaved with various restriction enzymes and the structure was examined to confirm that the plasmid had the target DNA fragment inserted therein. This plasmid was named pMEW41Km.
Using pMEW41Km, the yaeM gene on the chromosome was disrupted by homologous recombination. A schematic diagram of recombination is shown in FIG.
[0208]
pMEW41Km restriction enzymeHindIII andSacDigested with I and purified by agarose gel electrophoresis. Using this fragment, Escherichia coli FS1576 was transformed according to a conventional method. The FS1576 strain is available from the National Institute of Genetics as ME9019 strain. The transformant was applied to an LB agar medium containing 15 μg / ml of kanamycin and 1 g / l of 2-C-methyl-D-erythritol, and cultured at 37 ° C. overnight.
[0209]
Several kanamycin-resistant colonies that had grown were subjected to shaking culture at 37 ° C. for 16 hours in 10 ml of LB liquid medium containing 15 μg / ml of kanamycin and 1 g / l of 2-C-methyl-D-erythritol.
The cells were obtained by centrifuging the obtained culture solution.
Chromosomal DNA was isolated from the cells according to a conventional method.
[0210]
Chromosomal DNA restriction enzymeSmaI orPstDigested with I. The same procedure was applied to the chromosome of FS1576 strain. According to a conventional method, these restriction enzyme-treated DNAs were subjected to agarose gel electrophoresis and then subjected to Southern hybridization analysis using the kanamycin resistance gene and yaeM gene as probes. As a result, the chromosome of the kanamycin resistant colony had the structure shown in FIG. 3, and it was confirmed that the yaeM gene was disrupted by the kanamycin resistant gene as intended.
[0211]
(2) Properties of yaeM-deficient mutants
The yaeM deficient strain and its parent strain FS1576 prepared by the above procedure were applied to an LB agar medium and an LB agar medium containing 2-C-methyl-D-erythritol 1 g / l, and cultured at 37 ° C. The degree of growth after 2 days is shown in Table 10.
[0212]
[Table 10]

[0213]
Since the yaeM-deficient mutant cannot grow in a medium without addition of 2-C-methyl-D-erythritol, it is apparent that this gene is essential for cell growth in the absence of 2-C-methyl-D-erythritol. became.
[0214]
Example 10 Effect of 1-deoxy-D-xylulose 5-phosphate reductoisomerase inhibitor on bacterial growth
2-C-methyl-D-erythritol 4-phosphate, a product of 1-deoxy-D-xylulose 5-phosphate reductoisomerase reaction, a reaction intermediate assumed in this enzyme reaction, and fosmidomycin are structural Therefore, the following experiment was conducted based on the presumption that fosmidomycin may inhibit the enzyme.
[0215]
Fosmidomycin was added to the 1-deoxy-D-xylulose 5-phosphate reductoisomerase activity measurement system shown in Example 8, and the influence on enzyme activity was examined.
Fosmidomycin [Chem. Pharm. Bull.,30, 111-118 (1982)].
[0216]
  Example 8 (2)(iii)To the system scaled down to 0.2 ml (respective concentrations are the same), various concentrations of fosmidomycin were added, incubated at 37 ° C., and the increase or decrease of NADPH was measured with a benchmark microplate reader (manufactured by Bio-Rad). It measured using.
[0217]
As shown in FIG. 4, fosmidomycin was found to inhibit 1-deoxy-D-xylulose 5-phosphate reductoisomerase.
Escherichia coli W3110 strain is LB agar medium, LB agar medium containing 3.13 mg / l fosmidomycin and LB agar medium containing 3.13 mg / l fosmidomycin and 0.25 g / l 2-C-methyl-D-erythritol Each was applied and cultured at 37 ° C.
[0218]
After 2 days of culture, the bacteria could grow on two types of medium on LB agar medium containing LB agar medium, fosmidomycin and 2-C-methyl-D-erythritol 0.25 g / l. Bacteria could not grow on LB agar medium supplemented with only midomycin.
[0219]
From these results, it became clear that fosmidomycin inhibits the growth of bacteria by inhibiting 1-deoxy-D-xylulose 5-phosphate reductoisomerase. From the above, substances that inhibit the activity of yaeM (1-deoxy-D-xylulose 5-phosphate reductoisomerase) can be effective antibacterial agents or herbicides.
[0220]
【The invention's effect】
According to the present invention, one or more DNAs involved in biosynthesis of isoprenoid compounds useful for drugs, health foods, shellfish adhesion prevention paints, etc. for the purpose of heart disease, osteoporosis, hemostasis, cancer prevention, immunostimulation, etc. The contained DNA is incorporated into a vector, the obtained recombinant DNA is introduced into a prokaryotic host cell, the obtained transformant is cultured in a medium, and an isoprenoid compound is produced and accumulated in the culture. A method for producing an isoprenoid compound characterized in that the isoprenoid compound is collected from the product, and a DNA containing one or more DNAs encoding a protein having an activity capable of improving the biosynthesis efficiency of the isoprenoid compound, The obtained recombinant DNA is introduced into a host cell, the obtained transformant is cultured in a medium, and the protein is produced in the culture. A method for producing the protein, the protein, and 2-C-methyl-D-erythritol from 1-deoxy-D-xylulose 5-phosphate, characterized by accumulating and collecting the protein from the culture It is possible to provide a method for searching for a compound having antibacterial and / or herbicidal activity by searching for a novel enzyme protein having an activity of catalyzing a reaction that generates 4-phosphate and a substance that inhibits the enzyme.
[0221]
[Sequence Listing]

[0222]

[0223]

[0224]

[0225]
[Sequence Listing Free Text]
SEQ ID NO: 12: synthetic DNA
SEQ ID NO: 13: synthetic DNA
SEQ ID NO: 14: Synthetic DNA
SEQ ID NO: 15: synthetic DNA
SEQ ID NO: 16: synthetic DNA
Sequence number 17: Synthetic DNA
Sequence number 18: Synthetic DNA
Sequence number 19: Synthetic DNA
SEQ ID NO: 20: synthetic DNA
SEQ ID NO: 21: synthetic DNA
SEQ ID NO: 22: synthetic DNA
SEQ ID NO: 23: synthetic DNA
SEQ ID NO: 24: Synthetic DNA
SEQ ID NO: 25: synthetic DNA
SEQ ID NO: 32: synthetic DNA
SEQ ID NO: 33: synthetic DNA
SEQ ID NO: 34: Synthetic DNA
[Brief description of the drawings]
FIG. 1 shows the effect of reaction temperature on the activity of 1-deoxy-D-xylulose 5-phosphate reductoisomerase.
FIG. 2 is a graph showing the influence of reaction solution pH on the activity of 1-deoxy-D-xylulose 5-phosphate reductoisomerase. The activity at each pH in 100 mM Tris-HCl buffer was shown. The activity at pH 8.0 was taken as 100%, and the activity at each pH was shown as relative activity.
FIG. 3 is a diagram showing a method for disrupting a yaeM gene on a chromosome using homologous recombination.
FIG. 4 shows the effect of fosmidomycin on 1-deoxy-D-xylulose 5-phosphate reductoisomerase activity.

Claims (4)

A DNA comprising one or more DNAs encoding an enzyme that catalyzes a reaction on a non-mevalonate pathway selected from the following (a) to (d) is incorporated into a vector, and the resulting recombinant vector is a prokaryotic host A method for producing an isoprenoid compound, the method comprising culturing the obtained transformant in a medium, culturing the obtained transformant in a medium, producing and accumulating an isoprenoid compound in the culture, and collecting the isoprenoid compound from the culture.
(A) DNA encoding a protein having the amino acid sequence set forth in SEQ ID NO: 26 or 28, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein, and pyruvin DNA encoding a protein having an activity of catalyzing the reaction of producing 1-deoxy-D-xylulose 5-phosphate from acid and glyceraldehyde 3-phosphate
(B) Hybridizes under stringent conditions with DNA having the nucleotide sequence set forth in SEQ ID NO: 27 or 29, or DNA comprising the nucleotide sequence set forth in SEQ ID NO: 27 or 29, and pyruvate and glyceraldehyde 3 DNA encoding a protein having an activity of catalyzing a reaction for producing 1-deoxy-D-xylulose 5-phosphate from phosphate
(C) a DNA encoding a protein having the amino acid sequence set forth in SEQ ID NO: 5 or 30, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein; -DNA encoding a protein having an activity of catalyzing a reaction for producing 2-C-methyl-D-erythritol 4-phosphate from deoxy-D-xylulose 5-phosphate
(D) 1-deoxy-D-xylulose which hybridizes under stringent conditions with DNA having the base sequence described in SEQ ID NO: 10 or 31 or DNA consisting of the base sequence described in SEQ ID NO: 10 or 31 DNA encoding a protein having an activity of catalyzing a reaction for producing 2-C-methyl-D-erythritol 4-phosphate from 5-phosphate 2. The method according to claim 1, wherein the vector further comprises one or more DNAs selected from the following (e) to ( j ).
(E) DNA encoding a protein having the amino acid sequence described in SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the protein, and farnesyl pyrophosphate synthesis DNA encoding a protein having enzyme activity
(F) It encodes a protein having a farnesyl pyrophosphate synthase activity that hybridizes with DNA having the base sequence shown in SEQ ID NO: 7 or DNA having the base sequence shown in SEQ ID NO: 7 under stringent conditions DNA
(G) DNA encoding a protein having the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein, and isoprenoid compound DNA encoding a protein having activity capable of improving synthesis efficiency
(H) It is capable of hybridizing under stringent conditions with DNA having the base sequence shown in SEQ ID NO: 8 or DNA consisting of the base sequence shown in SEQ ID NO: 8 and improving the biosynthesis efficiency of the isoprenoid compound. DNA encoding a protein having activity
( I ) DNA encoding a protein having the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein, and pyruvic acid DNA encoding a protein having an activity of catalyzing a reaction for producing 1-deoxy-D-xylulose 5-phosphate from glyceraldehyde 3-phosphate
( J ) hybridizes under stringent conditions with DNA having the base sequence shown in SEQ ID NO: 6 or DNA consisting of the base sequence shown in SEQ ID NO: 6, and from pyruvic acid and glyceraldehyde 3-phosphate DNA encoding a protein having an activity of catalyzing a reaction for producing 1-deoxy-D-xylulose 5-phosphate The production method according to claim 1 or 2, wherein the isoprenoid compound is an isoprenoid compound selected from ubiquinone, vitamin K ₂ and carotenoid.   The production method according to any one of claims 1 to 3, wherein the transformant is a microorganism belonging to the genus Escherichia, a microorganism belonging to the genus Rhodobacter, or a microorganism belonging to the genus Erwinia.

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