JP2021502054A - パチョロールおよびエレモールをならびに好ましくはポゴストールも産生するテルペンシンターゼ - Google Patents
パチョロールおよびエレモールをならびに好ましくはポゴストールも産生するテルペンシンターゼ Download PDFInfo
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- JP2021502054A JP2021502054A JP2020512424A JP2020512424A JP2021502054A JP 2021502054 A JP2021502054 A JP 2021502054A JP 2020512424 A JP2020512424 A JP 2020512424A JP 2020512424 A JP2020512424 A JP 2020512424A JP 2021502054 A JP2021502054 A JP 2021502054A
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Abstract
Description
ナルドスタキスジャタマンシのGC−MS分析
約20cmの高さのナルドスタキスジャタマンシ植物を、Poyntzfield Herb Nursery,Black Isle,By Dingwall IV7 8LX,Ross&Cromarty,Scotlandで購入した。その植物は、葉および根の材料が切り取られた。予め冷却されたガラス管に入れられた植物材料の重みは0.5gであり、2mLのジクロロメタンが加えられた。1分間吊り下げて回転され、超音波バス内で5分間、1500g、室温で超音波処理された。上澄みを集めて、1gの硫酸ナトリウムのコラム上にろ過された。約2マイクロリットルが、Cankar et al.(2015)により詳細に記載されているガスクロマトグラフを使用して、GC/MSにより分析された。パチョロールが、保持時間および増すスペクトルのパチョリ油5のそれらに対する比較(Sigma−Aldrich)によって同定された。
N.ジャタマンシの根は、パチョロール(Rt16.4min)およびアルファパチョレン(Rt13.9min)に対応する化合物を含むことが示された。したがって、この組織を、RNAの抽出のためにさらに採取した。
RNA抽出および分析
N.ジャタマンシの根の材料のRNAが以下のように単離された。約15mLの20抽出バッファ(ヘキサデシルトリメチルアンモニウムブロミドを2%、ポリビニルピロリドンK−30を2%、トリス塩酸(pH8.0)を100mM、EDTAを25mM、NaClを2.0M、スペルミジンを0.5g/Lおよびβ−メルカプトエタノールを2%)が65℃に温められ、その後、3gの地組織が加えられて混合された。混合物は、2回、等しい容積のクロロホルム:イソアミルアルコール(1:24)を用いて2回抽出され、10MのLiClの四分の一の容積が上澄みおよび混合物に加えられた。RNAは4℃で一晩中沈殿され、20分間10000gの遠心分離によって採取された。パレットが、500マイクロリットルのSSTE[1.0MのNaCl、0.5%のSDS、10mMのトリスHCl(pH8.0)、1mMのEDTA(pH 8.0)]に溶解されて、等しい容積のクロロホルム:イソアミルアルコールを用いて1回抽出された。エタノールの2つの容積が上澄みに加えられて、−20℃で少なくとも2時間培養され、13000gで遠心分離されて、上澄みが除去された。ペレットは、空気乾燥され、水中に再懸濁された。全RNA(60Vg)がVertis Biotechnology AG(フライジング郡、ドイツ)に発送された。PolyA+RNAが単離されて、ランダムにプライマー処理されたcDNAが、ランダム化されたN6アダプタープラーマーおよびM−MLV H−逆転写酵素を使用して合成された。cDNAはせん断されて、細分化され、サイズが500bpの5つの断片がさらなる分析のために使用された。cDNAキャリーは、Illuminaにより特定化されたアダプター配列AおおびBをそれらの5’および3’末端に取り付けられた。
全長オープンリーディングフレームはN.ジャタマンシのcDNAにより増幅された。表1に示すフォワードプライマーおよびリバースプライマーが、プラスミドpCDF−DUET−1(Novagen corporation)においてHis−6タグのC末端に対して読み出しフレームが溶融されたように、全オープンリーディングフレームを増幅するように、設計および使用された。5つの異なるテルペンシンターゼORFの全部がクローニングされた。プライマー27692−Fw(配列番号1)および27692−Re(配列番号2)を使用して、2つの異なる密接に関わるcDNAがクローニングされた。これら(pTS11−1)の1つは、上記のコンティグ27692によりコードされるタンパク質に対して、70個のアミノ酸を欠落しているタンパク質をコードした。他のクローン(pTS11−2)は、配列番号4のタンパク質をコードしたcDNA配列(配列番号3)を含んでいた。
酵素アッセイのために、ガラス管において混合物は、MOPSOバッファ(15mMのMOPSO(3−[N−モルホリノ]−2−ヒドロキシプロパンスルホン酸)pH=7.0、12.5%のグリセロール、1mMのMgCl2、0.1%のtween20、1mMのジチオトレイトール)、100マイクロリットルの純化酵素溶液、および5μLのファルネシル二リン酸またはゲラニル二リン酸(乾式蒸発されて、50%エタノール中に溶解された10mMのSigma FPP)および20μLのオルトバナジン酸ナトリウム250mMからなる。この混合物は、30℃で2時間穏やかな攪拌により培養された。続いて、水相が2mLの酢酸エチルにより抽出された。酢酸エチル相は、収集され、1200xgで遠心分離され、硫酸ナトリウム上で乾燥され、GC−MSにより分析された。
プロモーターSPppaの規則に基づいて異なる溶解度タグと組み合わされたNjPAT遺伝子の発現について、構築物SPppa−MBP、SPppa−Fh8、SPppa−SUMO、SPppa−NusA、SPppa−RsTRXおよびSPppa−GST、ならびにNjPAT遺伝子は、Genscript USA Inc.,(ピスカタウェイ、ニュージャージー州、米国)により合成されたものである。両方のNjPAT遺伝子(配列番号5)および溶解度タグについてコードするすべての配列は、R.スフェロイデスにおける発現について最適化されるコドンであった。
種培養が、100mg/Lのネオマイシンをおよびグリセロールストックのループを有する20mlのRS102培地を用いるバッフルのない100mlの振とうフラスコ内で実行された。フラスコは、110rpmで50mmの軌道で振とうインキュベータ内で30℃において72時間培養された。
培養物が、4500xgで20分遠心分離される、予め重量測定された50mlのPP管内での接種72時間後に収集された。n−ドデカン層は、後のGC分析のためにマイクロ遠心チューブに移行された。
ガスクロマトグラフ−は、Restek RTX−5Sil MSキャピラリコラム(30mx0.25mm、0.5μm)を備えたShimazu GC2010 Plusにおいて実行された。インジェクタおよびFID検出装置の温度は、それぞれ280℃および300℃に設定された。コラムを通るガス流は40mL/minに設定された。オーブンの内部温度は、160℃であり、2℃/minで180℃まで上げられ、50℃/minで300℃までさらに上げられ、3分間その温度に保持された。注入さる試料の容積は1:50の分割比において1μLであり、窒素補給流量は30ml/minであった。
R.スフェロイデス培養物からのドデカン抽出の原液は、ヘキサン内に1mg/mlで調製され、次いで、メタノール内に20μg/mlに希釈された。
プラスミドp−m−SPppa−MBP−NjPAT−mpmii altを宿すロドバクター株は、パチョロール、ポゴストールおよび驚くべきことに、エレモール間で、最も高いテルペン種の産生を示した(図7)。パチョロールとエレモールとの間の比は6.9:1であり、パチョロールとポゴストールとの間の比は5.6:1であり、最終的に、ポゴストールとエレモールとの間の比は1.3:1であった。Fh8タグのNjPATへの融合は、ファルネソール(FPPの脱リン酸化からもたらされる産生物)のみを産生し得る酵素を完全に非活性化した。溶解度タグSUMOを有するNjPATを発現する株を用いて得られる力価は、MBP株を用いて得られるものの74%であり、それは、NusA−NjPAT株からもたらされる73%の力価にかなり類似するものである。より低い力価は、NjPATがGSTまたはRsTRXと組み合わされるときに得られた(MBP−NjPATに比べてそれぞれ43%および7%)。
配列番号1
27692−fwヌクレオチド配列
atatgagctcaATGTCAATTATTATTGCAACAAACAGTACTGAGCATCC
配列番号2
27692−reヌクレオチド配列
atatgcggccgcTTATATGGATACGGGATCTACGAACAACGATATGATGTG
配列番号3
NjPATヌクレオチド配列
ATGTCAATTATTATTGCAACAAACAGTACTGAGCATCCAATTTTTCGTCCATTAGCAAATTTTCCACCAAGTTTATGGGGCAATCTTTTCACTTCATTCTCCATGGATAATCAGGCTAGGGAAATATATGCTAAAGAACATGAAGGTTTAAAAGAAAAAGTGAGAATGATGTTTTTAGATACAACAAATTACAAAATTTCAGAGAAAATCAATTTCATAAACACAGTGGAAAGATTAGGTGTATCATATCATTTTGAGAAAGAGATTGAAGAACTACTTCATCAAATGTTTGATGCTCATTCTAAACACCTAGATGATATTCAAGAATTTGATTTGTTCACTTTGGGAATTTACTTCAGGATTCTAAGGCAACATGGTTATAAAATCTCTTGTGATGTTTTCAATAAGTTGAAAGATAGCAATGGCGAATTCAAGGACGAACTTAAAGATGATGTGAATGGTATGCTAAGTTTCTATGAAGCAACACATGTAAGAACACATGGAGAAAATATTTTAGATGAAGCTCTCATTTATACAAAAGCTCAACTTGAATCCATGGCCGCTGCAAGTTTAAGCCCATTTCTCGCGAACCAAGTTAAGCATGCTTTGATGCAAGCTCTCCACAAAGGGATCCCAAGAATCGAAGCACGTAACTATATCTCTGTTTACGAAGAAGATCCTAACAAAAATGATTTGTTATTGAGGTTCTCAAAGATAGATTTCAATCTAGTACAAATGATTCACAAGCAAGAATTGTGCGATACCTTTAGgTGGTGGAAAGATTTGGAGTTCGAATCGAAACTATCTTTTGCAaGGAATAGAGTGGTGGAAGCCTACTTATGGACTCTTAGCGCGTACTACGAACCAAAATACTCTTCCGCTCGGATTATATTAGTCAAACTAATGGTTATAATATCTGTTACGGATGACACATATGATGCATATGGTACATTAGATGAACTTCAACTTTTTACAGATGCAATACAAAGGTTGGATATGAGTTCTATCAATCAACTTCCAGATTACATGAAGACCATCTATAAAGCTCTCCTAGATCTTTTTGACGAAATAGAAGATCGATTATCGAAGCATGAAACTGATCATTCTTACCGCGTTGCTTATGCGAAATATGTGTATAAAGAGATCGTTAGGTGCTACGATATGGAGTACAAATGGTTCAACAAAAATTACGTGCCGGCATTTGAAGAATATATGCAGAAAGCGTTAGTCACATCAGGTAACCGTTTGCTCATAACGTTTTCCTTTCTGGGAATGGACGAAGTCGCAACTATTCAAGCGTTCGAGTGGGTAAAAAGTAATGCCAAAATGATAGTCTCTTCCAATAAAGTATTACGACTTATTGATGACATAATGAGTCACGAGGAAGAGGATGAAAGGGGACATGTTGCAACAGGGATTGAATGCTTTGTAAAAGAACATGGACTAACTAGGGAAGAGGTTATCGTTGAATTTCATAAGAGGATTGATGATGCTTGGAAGGATATAAATGAGGAATTTATAACGCCAAATAATTTACCGATTGAGATACTTACGCGTGTTCTAAACCTTACAAGAATTGGAGATGTTGTTTACAAGTATGATGACGGGTATACTCATCCGGAGAAAGCGTTGAAAGATCACATCATATCGTTGTTCGTAGATCCCGTATCCATATAA
配列番号4
NjPATアミノ酸配列
MSIIIATNSTEHPIFRPLANFPPSLWGNLFTSFSMDNQAREIYAKEHEGLKEKVRMMFLDTTNYKISEKINFINTVERLGVSYHFEKEIEELLHQMFDAHSKHLDDIQEFDLFTLGIYFRILRQHGYKISCDVFNKLKDSNGEFKDELKDDVNGMLSFYEATHVRTHGENILDEALIYTKAQLESMAAASLSPFLANQVKHALMQALHKGIPRIEARNYISVYEEDPNKNDLLLRFSKIDFNLVQMIHKQELCDTFRWWKDLEFESKLSFARNRVVEAYLWTLSAYYEPKYSSARIILVKLMVIISVTDDTYDAYGTLDELQLFTDAIQRLDMSSINQLPDYMKTIYKALLDLFDEIEDRLSKHETDHSYRVAYAKYVYKEIVRCYDMEYKWFNKNYVPAFEEYMQKALVTSGNRLLITFSFLGMDEVATIQAFEWVKSNAKMIVSSNKVLRLIDDIMSHEEEDERGHVATGIECFVKEHGLTREEVIVEFHKRIDDAWKDINEEFITPNNLPIEILTRVLNLTRIGDVVYKYDDGYTHPEKALKDHIISLFVDPVSI
配列番号5
NjPATコドン最適化ヌクレオチド配列
ATGTCGATCATCATCGCCACCAACAGCACCGAGCATCCCATCTTCCGCCCGCTCGCCAACTTCCCGCCGTCGCTCTGGGGCAACCTGTTCACCTCGTTCAGCATGGACAACCAGGCGCGCGAGATCTACGCCAAGGAGCACGAGGGCCTCAAGGAGAAGGTCCGGATGATGTTCCTGGACACCACGAACTACAAGATCTCGGAGAAGATCAACTTCATCAACACCGTCGAGCGCCTGGGCGTGAGCTATCACTTCGAGAAGGAGATCGAGGAGCTGCTCCATCAGATGTTCGACGCCCACTCGAAGCATCTGGACGACATCCAGGAGTTCGACCTCTTCACGCTGGGCATCTACTTCCGCATCCTGCGGCAGCATGGCTATAAGATCTCGTGCGACGTCTTCAACAAGCTGAAGGACAGCAACGGCGAGTTCAAGGACGAGCTCAAGGACGACGTCAACGGCATGCTGTCGTTCTATGAGGCCACCCATGTGCGCACCCATGGCGAGAACATCCTCGACGAGGCGCTGATCTACACGAAGGCCCAGCTGGAGTCGATGGCGGCCGCCTCGCTCAGCCCGTTCCTGGCCAACCAGGTGAAGCACGCGCTCATGCAGGCCCTGCATAAGGGCATCCCGCGCATCGAGGCGCGGAACTACATCTCGGTCTATGAGGAAGACCCGAACAAGAACGACCTGCTCCTGCGCTTCAGCAAGATCGACTTCAACCTGGTGCAGATGATCCACAAGCAGGAGCTGTGCGACACCTTCCGGTGGTGGAAGGACCTGGAGTTCGAGTCGAAGCTGTCGTTCGCCCGCAACCGGGTGGTGGAGGCCTACCTCTGGACGCTGTCGGCGTACTATGAGCCGAAGTATTCGAGCGCCCGCATCATCCTCGTGAAGCTGATGGTCATCATCTCGGTGACCGACGACACGTACGACGCCTATGGCACCCTGGACGAGCTCCAGCTGTTCACGGACGCGATCCAGCGCCTCGACATGTCGAGCATCAACCAGCTGCCCGACTACATGAAGACCATCTATAAGGCGCTCCTGGACCTCTTCGACGAGATCGAGGACCGCCTGTCGAAGCACGAGACGGACCATAGCTACCGGGTGGCGTATGCCAAGTACGTCTATAAGGAGATCGTGCGCTGCTACGACATGGAGTATAAGTGGTTCAACAAGAACTACGTCCCCGCCTTCGAGGAGTATATGCAGAAGGCCCTGGTGACCTCGGGCAACCGGCTCCTGATCACGTTCAGCTTCCTGGGCATGGACGAGGTCGCGACCATCCAGGCCTTCGAGTGGGTGAAGTCGAACGCCAAGATGATCGTGTCGTCGAACAAGGTGCTCCGCCTGATCGACGACATCATGTCGCACGAGGAAGAGGACGAGCGCGGCCATGTGGCCACGGGCATCGAGTGCTTCGTGAAGGAGCACGGCCTGACCCGCGAGGAAGTGATCGTCGAGTTCCATAAGCGGATCGACGACGCGTGGAAGGACATCAACGAGGAGTTCATCACCCCGAACAACCTGCCGATCGAGATCCTGACCCGCGTGCTCAACCTGACCCGGATCGGCGACGTGGTCTACAAGTATGACGACGGCTACACGCACCCCGAGAAGGCCCTCAAGGACCATATCATCAGCCTGTTCGTGGACCCCGTCAGCATCTGA
配列番号6
Pppa_Fwヌクレオチド配列
actggcctcagaattcAAAtttatttgctttgtgagcggataac
配列番号7
MBP_NjPAT_Rvヌクレオチド配列
GGCGATGATGATCGACATGATCTTG
配列番号8
MBP_NjPAT_Fwヌクレオチド配列
CAAGATCATGTCGATCATCATCGC
配列番号9
NjPAT_Rvヌクレオチド配列
tttatgatttggatcCTCAGATGCTGACGGGGT
配列番号10
SPppa−MBP−NjPATヌクレオチド配列
AAATTTATTTGCTTTGTGAGCGGATAACAATTATTAGATTCACCGGCGAGCCAGCAGGAATTTCACTCTAGATGACAGGAGGGACATATGAAGATCGAGGAAGGCAAGCTCGTGATCTGGATCAACGGCGACAAGGGCTACAACGGCCTGGCCGAGGTGGGCAAGAAGTTCGAGAAGGACACCGGCATCAAGGTGACGGTGGAGCACCCGGACAAGCTCGAGGAGAAGTTCCCGCAGGTGGCGGCCACGGGCGACGGCCCGGACATCATCTTCTGGGCCCATGACCGCTTCGGCGGCTACGCCCAGTCGGGCCTGCTGGCCGAGATCACCCCGGACAAGGCGTTCCAGGACAAGCTCTATCCCTTCACGTGGGACGCCGTGCGCTACAACGGCAAGCTGATCGCGTATCCCATCGCGGTGGAGGCCCTGTCGCTCATCTATAACAAGGACCTGCTCCCGAACCCGCCCAAGACCTGGGAGGAGATCCCCGCCCTCGACAAGGAGCTGAAGGCCAAGGGCAAGTCGGCGCTCATGTTCAACCTGCAGGAGCCGTACTTCACCTGGCCCCTGATCGCGGCCGACGGCGGCTACGCGTTCAAGTATGAGAACGGCAAGTATGACATCAAGGACGTGGGCGTGGACAACGCGGGCGCCAAGGCCGGCCTGACCTTCCTCGTGGACCTGATCAAGAACAAGCACATGAACGCCGACACGGACTACTCGATCGCGGAGGCCGCGTTCAACAAGGGCGAGACCGCCATGACGATCAACGGCCCGTGGGCGTGGTCGAACATCGACACCTCGAAGGTGAACTATGGCGTGACCGTGCTCCCCACGTTCAAGGGCCAGCCCTCGAAGCCCTTCGTGGGCGTGCTGTCGGCGGGCATCAACGCCGCGTCGCCGAACAAGGAGCTCGCGAAGGAGTTCCTGGAGAACTACCTGCTCACCGACGAGGGCCTGGAGGCCGTGAACAAGGACAAGCCCCTGGGCGCCGTGGCCCTGAAGTCGTATGAGGAAGAGCTGGTGAAGGACCCGCGCATCGCGGCCACCATGGAGAACGCGCAGAAGGGCGAGATCATGCCGAACATCCCCCAGATGTCGGCCTTCTGGTATGCGGTGCGCACCGCCGTGATCAACGCGGCCTCGGGCCGCCAGACCGTGGACGAGGCCCTCAAGGACGCCCAGACCGGCGACGACGACGACAAGATCATGTCGATCATCATCGCCACCAACAGCACCGAGCATCCCATCTTCCGCCCGCTCGCCAACTTCCCGCCGTCGCTCTGGGGCAACCTGTTCACCTCGTTCAGCATGGACAACCAGGCGCGCGAGATCTACGCCAAGGAGCACGAGGGCCTCAAGGAGAAGGTCCGGATGATGTTCCTGGACACCACGAACTACAAGATCTCGGAGAAGATCAACTTCATCAACACCGTCGAGCGCCTGGGCGTGAGCTATCACTTCGAGAAGGAGATCGAGGAGCTGCTCCATCAGATGTTCGACGCCCACTCGAAGCATCTGGACGACATCCAGGAGTTCGACCTCTTCACGCTGGGCATCTACTTCCGCATCCTGCGGCAGCATGGCTATAAGATCTCGTGCGACGTCTTCAACAAGCTGAAGGACAGCAACGGCGAGTTCAAGGACGAGCTCAAGGACGACGTCAACGGCATGCTGTCGTTCTATGAGGCCACCCATGTGCGCACCCATGGCGAGAACATCCTCGACGAGGCGCTGATCTACACGAAGGCCCAGCTGGAGTCGATGGCGGCCGCCTCGCTCAGCCCGTTCCTGGCCAACCAGGTGAAGCACGCGCTCATGCAGGCCCTGCATAAGGGCATCCCGCGCATCGAGGCGCGGAACTACATCTCGGTCTATGAGGAAGACCCGAACAAGAACGACCTGCTCCTGCGCTTCAGCAAGATCGACTTCAACCTGGTGCAGATGATCCACAAGCAGGAGCTGTGCGACACCTTCCGGTGGTGGAAGGACCTGGAGTTCGAGTCGAAGCTGTCGTTCGCCCGCAACCGGGTGGTGGAGGCCTACCTCTGGACGCTGTCGGCGTACTATGAGCCGAAGTATTCGAGCGCCCGCATCATCCTCGTGAAGCTGATGGTCATCATCTCGGTGACCGACGACACGTACGACGCCTATGGCACCCTGGACGAGCTCCAGCTGTTCACGGACGCGATCCAGCGCCTCGACATGTCGAGCATCAACCAGCTGCCCGACTACATGAAGACCATCTATAAGGCGCTCCTGGACCTCTTCGACGAGATCGAGGACCGCCTGTCGAAGCACGAGACGGACCATAGCTACCGGGTGGCGTATGCCAAGTACGTCTATAAGGAGATCGTGCGCTGCTACGACATGGAGTATAAGTGGTTCAACAAGAACTACGTCCCCGCCTTCGAGGAGTATATGCAGAAGGCCCTGGTGACCTCGGGCAACCGGCTCCTGATCACGTTCAGCTTCCTGGGCATGGACGAGGTCGCGACCATCCAGGCCTTCGAGTGGGTGAAGTCGAACGCCAAGATGATCGTGTCGTCGAACAAGGTGCTCCGCCTGATCGACGACATCATGTCGCACGAGGAAGAGGACGAGCGCGGCCATGTGGCCACGGGCATCGAGTGCTTCGTGAAGGAGCACGGCCTGACCCGCGAGGAAGTGATCGTCGAGTTCCATAAGCGGATCGACGACGCGTGGAAGGACATCAACGAGGAGTTCATCACCCCGAACAACCTGCCGATCGAGATCCTGACCCGCGTGCTCAACCTGACCCGGATCGGCGACGTGGTCTACAAGTATGACGACGGCTACACGCACCCCGAGAAGGCCCTCAAGGACCATATCATCAGCCTGTTCGTGGACCCCGTCAGCATCTGA
イタリック体の配列はSPppaプロモーターであり、アンダーライン付きシーケンスはコボン最適化MBPであり、通常フォントの配列はコボン最適化NjPATである。
配列番号11
MBP−NjPATアミノ酸配列
MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGDDDDKIMSIIIATNSTEHPIFRPLANFPPSLWGNLFTSFSMDNQAREIYAKEHEGLKEKVRMMFLDTTNYKISEKINFINTVERLGVSYHFEKEIEELLHQMFDAHSKHLDDIQEFDLFTLGIYFRILRQHGYKISCDVFNKLKDSNGEFKDELKDDVNGMLSFYEATHVRTHGENILDEALIYTKAQLESMAAASLSPFLANQVKHALMQALHKGIPRIEARNYISVYEEDPNKNDLLLRFSKIDFNLVQMIHKQELCDTFRWWKDLEFESKLSFARNRVVEAYLWTLSAYYEPKYSSARIILVKLMVIISVTDDTYDAYGTLDELQLFTDAIQRLDMSSINQLPDYMKTIYKALLDLFDEIEDRLSKHETDHSYRVAYAKYVYKEIVRCYDMEYKWFNKNYVPAFEEYMQKALVTSGNRLLITFSFLGMDEVATIQAFEWVKSNAKMIVSSNKVLRLIDDIMSHEEEDERGHVATGIECFVKEHGLTREEVIVEFHKRIDDAWKDINEEFITPNNLPIEILTRVLNLTRIGDVVYKYDDGYTHPEKALKDHIISLFVDPVSI
アンダーライン付き配列はMBPタグである。
配列番号12
Fh8_NjPAT_Rvヌクレオチド配列
GATCGACATCGACGAGAGGATCGAG
配列番号13
Fh8_NjPAT_Fwヌクレオチド配列
CTCGTCGATGTCGATCATCATCGCC
配列番号14
SPppa−Fh8−NjPATヌクレオチド配列
AAATTTATTTGCTTTGTGAGCGGATAACAATTATTAGATTCACCGGCGAGCCAGCAGGAATTTCACTCTAGATGACAGGAGGGACATATGCCCTCGGTGCAAGAGGTGGAGAAGCTGCTGCATGTGCTGGACCGGAACGGCGACGGCAAGGTGTCGGCGGAGGAGCTGAAGGCGTTCGCCGACGACTCGAAGTGCCCGCTGGACTCGAACAAGATCAAGGCGTTCATCAAGGAGCATGACAAGAACAAGGACGGCAAGCTGGACCTCAAGGAGCTGGTCTCGATCCTCTCGTCGATGTCGATCATCATCGCCACCAACAGCACCGAGCATCCCATCTTCCGCCCGCTCGCCAACTTCCCGCCGTCGCTCTGGGGCAACCTGTTCACCTCGTTCAGCATGGACAACCAGGCGCGCGAGATCTACGCCAAGGAGCACGAGGGCCTCAAGGAGAAGGTCCGGATGATGTTCCTGGACACCACGAACTACAAGATCTCGGAGAAGATCAACTTCATCAACACCGTCGAGCGCCTGGGCGTGAGCTATCACTTCGAGAAGGAGATCGAGGAGCTGCTCCATCAGATGTTCGACGCCCACTCGAAGCATCTGGACGACATCCAGGAGTTCGACCTCTTCACGCTGGGCATCTACTTCCGCATCCTGCGGCAGCATGGCTATAAGATCTCGTGCGACGTCTTCAACAAGCTGAAGGACAGCAACGGCGAGTTCAAGGACGAGCTCAAGGACGACGTCAACGGCATGCTGTCGTTCTATGAGGCCACCCATGTGCGCACCCATGGCGAGAACATCCTCGACGAGGCGCTGATCTACACGAAGGCCCAGCTGGAGTCGATGGCGGCCGCCTCGCTCAGCCCGTTCCTGGCCAACCAGGTGAAGCACGCGCTCATGCAGGCCCTGCATAAGGGCATCCCGCGCATCGAGGCGCGGAACTACATCTCGGTCTATGAGGAAGACCCGAACAAGAACGACCTGCTCCTGCGCTTCAGCAAGATCGACTTCAACCTGGTGCAGATGATCCACAAGCAGGAGCTGTGCGACACCTTCCGGTGGTGGAAGGACCTGGAGTTCGAGTCGAAGCTGTCGTTCGCCCGCAACCGGGTGGTGGAGGCCTACCTCTGGACGCTGTCGGCGTACTATGAGCCGAAGTATTCGAGCGCCCGCATCATCCTCGTGAAGCTGATGGTCATCATCTCGGTGACCGACGACACGTACGACGCCTATGGCACCCTGGACGAGCTCCAGCTGTTCACGGACGCGATCCAGCGCCTCGACATGTCGAGCATCAACCAGCTGCCCGACTACATGAAGACCATCTATAAGGCGCTCCTGGACCTCTTCGACGAGATCGAGGACCGCCTGTCGAAGCACGAGACGGACCATAGCTACCGGGTGGCGTATGCCAAGTACGTCTATAAGGAGATCGTGCGCTGCTACGACATGGAGTATAAGTGGTTCAACAAGAACTACGTCCCCGCCTTCGAGGAGTATATGCAGAAGGCCCTGGTGACCTCGGGCAACCGGCTCCTGATCACGTTCAGCTTCCTGGGCATGGACGAGGTCGCGACCATCCAGGCCTTCGAGTGGGTGAAGTCGAACGCCAAGATGATCGTGTCGTCGAACAAGGTGCTCCGCCTGATCGACGACATCATGTCGCACGAGGAAGAGGACGAGCGCGGCCATGTGGCCACGGGCATCGAGTGCTTCGTGAAGGAGCACGGCCTGACCCGCGAGGAAGTGATCGTCGAGTTCCATAAGCGGATCGACGACGCGTGGAAGGACATCAACGAGGAGTTCATCACCCCGAACAACCTGCCGATCGAGATCCTGACCCGCGTGCTCAACCTGACCCGGATCGGCGACGTGGTCTACAAGTATGACGACGGCTACACGCACCCCGAGAAGGCCCTCAAGGACCATATCATCAGCCTGTTCGTGGACCCCGTCAGCATCTGA
イタリック体の配列はSPppaプロモーターであり、アンダーライン付きシーケンスはコボン最適化fh8であり、通常フォントの配列はコボン最適化NjPATである。
配列番号15
Fh8−NjPATアミノ酸配列
MPSVQEVEKLLHVLDRNGDGKVSAEELKAFADDSKCPLDSNKIKAFIKEHDKNKDGKLDLKELVSILSSMSIIIATNSTEHPIFRPLANFPPSLWGNLFTSFSMDNQAREIYAKEHEGLKEKVRMMFLDTTNYKISEKINFINTVERLGVSYHFEKEIEELLHQMFDAHSKHLDDIQEFDLFTLGIYFRILRQHGYKISCDVFNKLKDSNGEFKDELKDDVNGMLSFYEATHVRTHGENILDEALIYTKAQLESMAAASLSPFLANQVKHALMQALHKGIPRIEARNYISVYEEDPNKNDLLLRFSKIDFNLVQMIHKQELCDTFRWWKDLEFESKLSFARNRVVEAYLWTLSAYYEPKYSSARIILVKLMVIISVTDDTYDAYGTLDELQLFTDAIQRLDMSSINQLPDYMKTIYKALLDLFDEIEDRLSKHETDHSYRVAYAKYVYKEIVRCYDMEYKWFNKNYVPAFEEYMQKALVTSGNRLLITFSFLGMDEVATIQAFEWVKSNAKMIVSSNKVLRLIDDIMSHEEEDERGHVATGIECFVKEHGLTREEVIVEFHKRIDDAWKDINEEFITPNNLPIEILTRVLNLTRIGDVVYKYDDGYTHPEKALKDHIISLFVDPVSI
アンダーライン付き配列はFh8Aタグである。
配列番号16
SUMO_NjPAT_Rvヌクレオチド配列
GATCGACATGCCGATCTGCTCGCG
配列番号17
SUMO_NjPAT_Fwヌクレオチド配列
GATCGGCATGTCGATCATCATCGCC
配列番号18
SPppa−SUMO−NjPATヌクレオチド配列
AAATTTATTTGCTTTGTGAGCGGATAACAATTATTAGATTCACCGGCGAGCCAGCAGGAATTTCACTCTAGATGACAGGAGGGACATATGTCGGACAGCGAGGTGAACCAGGAAGCCAAGCCCGAGGTGAAGCCCGAGGTGAAGCCGGAGACCCACATCAACCTGAAGGTGTCGGACGGCTCGTCGGAGATCTTCTTCAAGATCAAGAAGACCACGCCCCTGCGCCGCCTCATGGAGGCCTTCGCCAAGCGCCAGGGCAAGGAGATGGACTCGCTGCGCTTCCTCTACGACGGCATCCGCATCCAGGCGGACCAGACGCCGGAGGACCTCGACATGGAGGACAACGACATCATCGAGGCGCATCGCGAGCAGATCGGCATGTCGATCATCATCGCCACCAACAGCACCGAGCATCCCATCTTCCGCCCGCTCGCCAACTTCCCGCCGTCGCTCTGGGGCAACCTGTTCACCTCGTTCAGCATGGACAACCAGGCGCGCGAGATCTACGCCAAGGAGCACGAGGGCCTCAAGGAGAAGGTCCGGATGATGTTCCTGGACACCACGAACTACAAGATCTCGGAGAAGATCAACTTCATCAACACCGTCGAGCGCCTGGGCGTGAGCTATCACTTCGAGAAGGAGATCGAGGAGCTGCTCCATCAGATGTTCGACGCCCACTCGAAGCATCTGGACGACATCCAGGAGTTCGACCTCTTCACGCTGGGCATCTACTTCCGCATCCTGCGGCAGCATGGCTATAAGATCTCGTGCGACGTCTTCAACAAGCTGAAGGACAGCAACGGCGAGTTCAAGGACGAGCTCAAGGACGACGTCAACGGCATGCTGTCGTTCTATGAGGCCACCCATGTGCGCACCCATGGCGAGAACATCCTCGACGAGGCGCTGATCTACACGAAGGCCCAGCTGGAGTCGATGGCGGCCGCCTCGCTCAGCCCGTTCCTGGCCAACCAGGTGAAGCACGCGCTCATGCAGGCCCTGCATAAGGGCATCCCGCGCATCGAGGCGCGGAACTACATCTCGGTCTATGAGGAAGACCCGAACAAGAACGACCTGCTCCTGCGCTTCAGCAAGATCGACTTCAACCTGGTGCAGATGATCCACAAGCAGGAGCTGTGCGACACCTTCCGGTGGTGGAAGGACCTGGAGTTCGAGTCGAAGCTGTCGTTCGCCCGCAACCGGGTGGTGGAGGCCTACCTCTGGACGCTGTCGGCGTACTATGAGCCGAAGTATTCGAGCGCCCGCATCATCCTCGTGAAGCTGATGGTCATCATCTCGGTGACCGACGACACGTACGACGCCTATGGCACCCTGGACGAGCTCCAGCTGTTCACGGACGCGATCCAGCGCCTCGACATGTCGAGCATCAACCAGCTGCCCGACTACATGAAGACCATCTATAAGGCGCTCCTGGACCTCTTCGACGAGATCGAGGACCGCCTGTCGAAGCACGAGACGGACCATAGCTACCGGGTGGCGTATGCCAAGTACGTCTATAAGGAGATCGTGCGCTGCTACGACATGGAGTATAAGTGGTTCAACAAGAACTACGTCCCCGCCTTCGAGGAGTATATGCAGAAGGCCCTGGTGACCTCGGGCAACCGGCTCCTGATCACGTTCAGCTTCCTGGGCATGGACGAGGTCGCGACCATCCAGGCCTTCGAGTGGGTGAAGTCGAACGCCAAGATGATCGTGTCGTCGAACAAGGTGCTCCGCCTGATCGACGACATCATGTCGCACGAGGAAGAGGACGAGCGCGGCCATGTGGCCACGGGCATCGAGTGCTTCGTGAAGGAGCACGGCCTGACCCGCGAGGAAGTGATCGTCGAGTTCCATAAGCGGATCGACGACGCGTGGAAGGACATCAACGAGGAGTTCATCACCCCGAACAACCTGCCGATCGAGATCCTGACCCGCGTGCTCAACCTGACCCGGATCGGCGACGTGGTCTACAAGTATGACGACGGCTACACGCACCCCGAGAAGGCCCTCAAGGACCATATCATCAGCCTGTTCGTGGACCCCGTCAGCATCTGA
イタリック体の配列はSPppaプロモーターであり、アンダーライン付きシーケンスはコボン最適化SUMOであり、通常フォントの配列はコボン最適化NjPATである。
配列番号19
SUMO−NjPATアミノ酸配列
MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGMSIIIATNSTEHPIFRPLANFPPSLWGNLFTSFSMDNQAREIYAKEHEGLKEKVRMMFLDTTNYKISEKINFINTVERLGVSYHFEKEIEELLHQMFDAHSKHLDDIQEFDLFTLGIYFRILRQHGYKISCDVFNKLKDSNGEFKDELKDDVNGMLSFYEATHVRTHGENILDEALIYTKAQLESMAAASLSPFLANQVKHALMQALHKGIPRIEARNYISVYEEDPNKNDLLLRFSKIDFNLVQMIHKQELCDTFRWWKDLEFESKLSFARNRVVEAYLWTLSAYYEPKYSSARIILVKLMVIISVTDDTYDAYGTLDELQLFTDAIQRLDMSSINQLPDYMKTIYKALLDLFDEIEDRLSKHETDHSYRVAYAKYVYKEIVRCYDMEYKWFNKNYVPAFEEYMQKALVTSGNRLLITFSFLGMDEVATIQAFEWVKSNAKMIVSSNKVLRLIDDIMSHEEEDERGHVATGIECFVKEHGLTREEVIVEFHKRIDDAWKDINEEFITPNNLPIEILTRVLNLTRIGDVVYKYDDGYTHPEKALKDHIISLFVDPVSI
アンダーライン付き配列はSUMOタグである。
配列番号20
NusA_NjPAT_Rvヌクレオチド配列
TGATCGACATCGCCTCGTCGCCGAAC
配列番号21
NusA_NjPAT_Fwヌクレオチド配列
CGAGGCGATGTCGATCATCATCGCC
配列番号22
SPppa−NusA−NjPATヌクレオチド配列
AAATTTATTTGCTTTGTGAGCGGATAACAATTATTAGATTCACCGGCGAGCCAGCAGGAATTTCACTCTAGATGACAGGAGGGACAT
ATGAACAAGGAGATCCTCGCGGTGGTGGAGGCGGTGTCGAACGAGAAGGCGCTGCCCCGCGAGAAGATCTTCGAGGCCCTGGAGTCGGCCCTGGCCACCGCGACCAAGAAGAAGTACGAGCAGGAGATCGACGTGCGCGTGCAGATCGACCGCAAGTCGGGCGACTTCGACACGTTCCGCCGCTGGCTCGTGGTGGACGAGGTGACCCAGCCCACGAAGGAGATCACCCTGGAGGCGGCCCGCTATGAGGACGAGTCGCTGAACCTCGGCGACTATGTGGAGGACCAGATCGAGTCGGTGACCTTCGACCGCATCACCACGCAGACGGCGAAGCAGGTGATCGTGCAGAAGGTGCGCGAGGCCGAGCGCGCCATGGTGGTGGACCAGTTCCGCGAGCACGAGGGCGAGATCATCACCGGCGTGGTGAAGAAGGTGAACCGCGACAACATCTCGCTGGACCTGGGCAACAACGCGGAGGCCGTGATCCTGCGCGAGGACATGCTCCCGCGCGAGAACTTCCGCCCGGGCGACCGCGTGCGCGGCGTGCTCTATTCGGTGCGCCCCGAGGCCCGTGGCGCCCAGCTGTTCGTGACCCGCTCGAAGCCGGAGATGCTGATCGAGCTCTTCCGCATCGAGGTGCCCGAGATCGGCGAGGAAGTGATCGAGATCAAGGCGGCCGCCCGCGACCCGGGCTCGCGCGCGAAGATCGCCGTGAAGACCAACGACAAGCGCATCGACCCCGTGGGCGCCTGCGTGGGCATGCGTGGCGCCCGCGTGCAGGCCGTGTCGACCGAGCTCGGCGGCGAGCGCATCGACATCGTGCTGTGGGACGACAACCCGGCGCAGTTCGTGATCAACGCCATGGCCCCGGCGGACGTGGCCTCGATCGTGGTGGACGAGGACAAGCATACCATGGACATCGCCGTGGAGGCGGGCAACCTGGCCCAGGCCATCGGCCGCAACGGCCAGAACGTGCGCCTGGCCTCGCAGCTCTCGGGCTGGGAGCTGAACGTGATGACGGTGGACGACCTGCAGGCCAAGCATCAGGCCGAGGCCCATGCCGCCATCGACACCTTCACGAAGTACCTCGACATCGACGAGGACTTCGCGACCGTGCTCGTGGAGGAAGGCTTCTCGACGCTGGAGGAGCTCGCCTATGTGCCGATGAAGGAGCTGCTCGAGATCGAGGGCCTGGACGAGCCGACGGTGGAGGCGCTCCGCGAGCGCGCCAAGAACGCCCTGGCCACCATCGCCCAGGCCCAGGAAGAGTCGCTGGGCGACAACAAGCCGGCCGACGACCTGCTCAACCTGGAGGGCGTGGACCGCGACCTGGCCTTCAAGCTCGCCGCCCGCGGCGTGTGCACGCTCGAGGACCTGGCCGAGCAGGGCATCGACGACCTGGCCGACATCGAGGGCCTCACCGACGAGAAGGCCGGCGCCCTGATCATGGCCGCCCGCAACATCTGCTGGTTCGGCGACGAGGCGATGTCGATCATCATCGCCACCAACAGCACCGAGCATCCCATCTTCCGCCCGCTCGCCAACTTCCCGCCGTCGCTCTGGGGCAACCTGTTCACCTCGTTCAGCATGGACAACCAGGCGCGCGAGATCTACGCCAAGGAGCACGAGGGCCTCAAGGAGAAGGTCCGGATGATGTTCCTGGACACCACGAACTACAAGATCTCGGAGAAGATCAACTTCATCAACACCGTCGAGCGCCTGGGCGTGAGCTATCACTTCGAGAAGGAGATCGAGGAGCTGCTCCATCAGATGTTCGACGCCCACTCGAAGCATCTGGACGACATCCAGGAGTTCGACCTCTTCACGCTGGGCATCTACTTCCGCATCCTGCGGCAGCATGGCTATAAGATCTCGTGCGACGTCTTCAACAAGCTGAAGGACAGCAACGGCGAGTTCAAGGACGAGCTCAAGGACGACGTCAACGGCATGCTGTCGTTCTATGAGGCCACCCATGTGCGCACCCATGGCGAGAACATCCTCGACGAGGCGCTGATCTACACGAAGGCCCAGCTGGAGTCGATGGCGGCCGCCTCGCTCAGCCCGTTCCTGGCCAACCAGGTGAAGCACGCGCTCATGCAGGCCCTGCATAAGGGCATCCCGCGCATCGAGGCGCGGAACTACATCTCGGTCTATGAGGAAGACCCGAACAAGAACGACCTGCTCCTGCGCTTCAGCAAGATCGACTTCAACCTGGTGCAGATGATCCACAAGCAGGAGCTGTGCGACACCTTCCGGTGGTGGAAGGACCTGGAGTTCGAGTCGAAGCTGTCGTTCGCCCGCAACCGGGTGGTGGAGGCCTACCTCTGGACGCTGTCGGCGTACTATGAGCCGAAGTATTCGAGCGCCCGCATCATCCTCGTGAAGCTGATGGTCATCATCTCGGTGACCGACGACACGTACGACGCCTATGGCACCCTGGACGAGCTCCAGCTGTTCACGGACGCGATCCAGCGCCTCGACATGTCGAGCATCAACCAGCTGCCCGACTACATGAAGACCATCTATAAGGCGCTCCTGGACCTCTTCGACGAGATCGAGGACCGCCTGTCGAAGCACGAGACGGACCATAGCTACCGGGTGGCGTATGCCAAGTACGTCTATAAGGAGATCGTGCGCTGCTACGACATGGAGTATAAGTGGTTCAACAAGAACTACGTCCCCGCCTTCGAGGAGTATATGCAGAAGGCCCTGGTGACCTCGGGCAACCGGCTCCTGATCACGTTCAGCTTCCTGGGCATGGACGAGGTCGCGACCATCCAGGCCTTCGAGTGGGTGAAGTCGAACGCCAAGATGATCGTGTCGTCGAACAAGGTGCTCCGCCTGATCGACGACATCATGTCGCACGAGGAAGAGGACGAGCGCGGCCATGTGGCCACGGGCATCGAGTGCTTCGTGAAGGAGCACGGCCTGACCCGCGAGGAAGTGATCGTCGAGTTCCATAAGCGGATCGACGACGCGTGGAAGGACATCAACGAGGAGTTCATCACCCCGAACAACCTGCCGATCGAGATCCTGACCCGCGTGCTCAACCTGACCCGGATCGGCGACGTGGTCTACAAGTATGACGACGGCTACACGCACCCCGAGAAGGCCCTCAAGGACCATATCATCAGCCTGTTCGTGGACCCCGTCAGCATCTGA
イタリック体の配列はSPppaプロモーターであり、アンダーライン付きシーケンスはコボン最適化NusAであり、通常フォントの配列はコボン最適化NjPATである。
配列番号23
NusA−NjPATTアミノ酸配列
MNKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDVRVQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDRITTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVILREDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIEIKAAARDPGSRAKIAVKTNDKRIDPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPAQFVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMTVDDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLDEPTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLEDLAEQGIDDLADIEGLTDEKAGALIMAARNICWFGDEAMSIIIATNSTEHPIFRPLANFPPSLWGNLFTSFSMDNQAREIYAKEHEGLKEKVRMMFLDTTNYKISEKINFINTVERLGVSYHFEKEIEELLHQMFDAHSKHLDDIQEFDLFTLGIYFRILRQHGYKISCDVFNKLKDSNGEFKDELKDDVNGMLSFYEATHVRTHGENILDEALIYTKAQLESMAAASLSPFLANQVKHALMQALHKGIPRIEARNYISVYEEDPNKNDLLLRFSKIDFNLVQMIHKQELCDTFRWWKDLEFESKLSFARNRVVEAYLWTLSAYYEPKYSSARIILVKLMVIISVTDDTYDAYGTLDELQLFTDAIQRLDMSSINQLPDYMKTIYKALLDLFDEIEDRLSKHETDHSYRVAYAKYVYKEIVRCYDMEYKWFNKNYVPAFEEYMQKALVTSGNRLLITFSFLGMDEVATIQAFEWVKSNAKMIVSSNKVLRLIDDIMSHEEEDERGHVATGIECFVKEHGLTREEVIVEFHKRIDDAWKDINEEFITPNNLPIEILTRVLNLTRIGDVVYKYDDGYTHPEKALKDHIISLFVDPVSI
アンダーライン付き配列はNusAタグである。
配列番号24
RsTRX_NjPAT_Rvヌクレオチド配列
TGATCGACATGAGCGCCGAGGCGATC
配列番号25
RsTRX_NjPAT_Fwヌクレオチド配列
ggcgctcATGTCGATCATCATCGCC
配列番号26
SPppa−RsTRX−NjPATヌクレオチド配列
AAATTTATTTGCTTTGTGAGCGGATAACAATTATTAGATTCACCGGCGAGCCAGCAGGAATTTCACTCTAGATGACAGGAGGGACATATGTCCACCGTTCCCGTGACGGACGCCACCTTCGACACCGAGGTGCGCAAGTCCGACGTGCCCGTCGTCGTCGATTTCTGGGCCGAATGGTGCGGCCCCTGCCGGCAGATCGGCCCGGCGCTCGAGGAGCTCTCGAAGGAATATGCCGGCAAGGTGAAGATCGTGAAGGTCAATGTCGACGAGAACCCCGAGAGCCCGGCGATGCTGGGCGTTCGCGGCATCCCGGCGCTGTTCCTGTTCAAGAACGGTCAGGTCGTGTCGAACAAGGTCGGCGCTGCGCCGAAGGCCGCGCTGGCCACCTGGATCGCCTCGGCGCTCATGTCGATCATCATCGCCACCAACAGCACCGAGCATCCCATCTTCCGCCCGCTCGCCAACTTCCCGCCGTCGCTCTGGGGCAACCTGTTCACCTCGTTCAGCATGGACAACCAGGCGCGCGAGATCTACGCCAAGGAGCACGAGGGCCTCAAGGAGAAGGTCCGGATGATGTTCCTGGACACCACGAACTACAAGATCTCGGAGAAGATCAACTTCATCAACACCGTCGAGCGCCTGGGCGTGAGCTATCACTTCGAGAAGGAGATCGAGGAGCTGCTCCATCAGATGTTCGACGCCCACTCGAAGCATCTGGACGACATCCAGGAGTTCGACCTCTTCACGCTGGGCATCTACTTCCGCATCCTGCGGCAGCATGGCTATAAGATCTCGTGCGACGTCTTCAACAAGCTGAAGGACAGCAACGGCGAGTTCAAGGACGAGCTCAAGGACGACGTCAACGGCATGCTGTCGTTCTATGAGGCCACCCATGTGCGCACCCATGGCGAGAACATCCTCGACGAGGCGCTGATCTACACGAAGGCCCAGCTGGAGTCGATGGCGGCCGCCTCGCTCAGCCCGTTCCTGGCCAACCAGGTGAAGCACGCGCTCATGCAGGCCCTGCATAAGGGCATCCCGCGCATCGAGGCGCGGAACTACATCTCGGTCTATGAGGAAGACCCGAACAAGAACGACCTGCTCCTGCGCTTCAGCAAGATCGACTTCAACCTGGTGCAGATGATCCACAAGCAGGAGCTGTGCGACACCTTCCGGTGGTGGAAGGACCTGGAGTTCGAGTCGAAGCTGTCGTTCGCCCGCAACCGGGTGGTGGAGGCCTACCTCTGGACGCTGTCGGCGTACTATGAGCCGAAGTATTCGAGCGCCCGCATCATCCTCGTGAAGCTGATGGTCATCATCTCGGTGACCGACGACACGTACGACGCCTATGGCACCCTGGACGAGCTCCAGCTGTTCACGGACGCGATCCAGCGCCTCGACATGTCGAGCATCAACCAGCTGCCCGACTACATGAAGACCATCTATAAGGCGCTCCTGGACCTCTTCGACGAGATCGAGGACCGCCTGTCGAAGCACGAGACGGACCATAGCTACCGGGTGGCGTATGCCAAGTACGTCTATAAGGAGATCGTGCGCTGCTACGACATGGAGTATAAGTGGTTCAACAAGAACTACGTCCCCGCCTTCGAGGAGTATATGCAGAAGGCCCTGGTGACCTCGGGCAACCGGCTCCTGATCACGTTCAGCTTCCTGGGCATGGACGAGGTCGCGACCATCCAGGCCTTCGAGTGGGTGAAGTCGAACGCCAAGATGATCGTGTCGTCGAACAAGGTGCTCCGCCTGATCGACGACATCATGTCGCACGAGGAAGAGGACGAGCGCGGCCATGTGGCCACGGGCATCGAGTGCTTCGTGAAGGAGCACGGCCTGACCCGCGAGGAAGTGATCGTCGAGTTCCATAAGCGGATCGACGACGCGTGGAAGGACATCAACGAGGAGTTCATCACCCCGAACAACCTGCCGATCGAGATCCTGACCCGCGTGCTCAACCTGACCCGGATCGGCGACGTGGTCTACAAGTATGACGACGGCTACACGCACCCCGAGAAGGCCCTCAAGGACCATATCATCAGCCTGTTCGTGGACCCCGTCAGCATCTGA
イタリック体の配列はSPppaプロモーターであり、アンダーライン付きシーケンスはコボン最適化NjPATであり、通常フォントの配列はコボン最適化NjPATである。
配列番号27
RsTRX−NjPATアミノ酸配列
MSTVPVTDATFDTEVRKSDVPVVVDFWAEWCGPCRQIGPALEELSKEYAGKVKIVKVNVDENPESPAMLGVRGIPALFLFKNGQVVSNKVGAAPKAALATWIASALMSIIIATNSTEHPIFRPLANFPPSLWGNLFTSFSMDNQAREIYAKEHEGLKEKVRMMFLDTTNYKISEKINFINTVERLGVSYHFEKEIEELLHQMFDAHSKHLDDIQEFDLFTLGIYFRILRQHGYKISCDVFNKLKDSNGEFKDELKDDVNGMLSFYEATHVRTHGENILDEALIYTKAQLESMAAASLSPFLANQVKHALMQALHKGIPRIEARNYISVYEEDPNKNDLLLRFSKIDFNLVQMIHKQELCDTFRWWKDLEFESKLSFARNRVVEAYLWTLSAYYEPKYSSARIILVKLMVIISVTDDTYDAYGTLDELQLFTDAIQRLDMSSINQLPDYMKTIYKALLDLFDEIEDRLSKHETDHSYRVAYAKYVYKEIVRCYDMEYKWFNKNYVPAFEEYMQKALVTSGNRLLITFSFLGMDEVATIQAFEWVKSNAKMIVSSNKVLRLIDDIMSHEEEDERGHVATGIECFVKEHGLTREEVIVEFHKRIDDAWKDINEEFITPNNLPIEILTRVLNLTRIGDVVYKYDDGYTHPEKALKDHIISLFVDPVSI
ヌクレオチド配列はRsTRXタグである。
配列番号28
GST_NjPAT_Rvヌクレオチド配列
ATCGACATCTTGGGCGGATGG
配列番号29
GST_NjPAT_Fwヌクレオチド配列
GCCCAAGATGTCGATCATCATCGCC
配列番号30
SPppa−GST−NjPATヌクレオチド配列
AAATTTATTTGCTTTGTGAGCGGATAACAATTATTAGATTCACCGGCGAGCCAGCAGGAATTTCACTCTAGATGACAGGAGGGACATATGTCGCCGATCCTGGGCTATTGGAAGATCAAGGGCCTCGTGCAGCCCACCCGCCTGCTCCTGGAGTACCTGGAGGAGAAGTATGAGGAGCACCTCTACGAGCGCGACGAGGGCGACAAGTGGCGCAACAAGAAGTTCGAGCTCGGCCTGGAGTTCCCGAACCTGCCCTACTATATCGACGGCGACGTGAAGCTCACGCAGTCGATGGCCATCATCCGCTACATCGCGGACAAGCATAACATGCTGGGCGGCTGCCCCAAGGAGCGCGCGGAGATCTCGATGCTGGAGGGCGCGGTGCTCGACATCCGCTATGGCGTGTCGCGCATCGCCTACTCGAAGGACTTCGAGACCCTGAAGGTGGACTTCCTCTCGAAGCTGCCGGAGATGCTCAAGATGTTCGAGGACCGCCTGTGCCACAAGACCTATCTCAACGGCGACCACGTGACGCATCCCGACTTCATGCTCTATGACGCGCTGGACGTGGTGCTCTACATGGACCCGATGTGCCTGGACGCCTTCCCCAAGCTCGTGTGCTTCAAGAAGCGCATCGAGGCGATCCCGCAGATCGACAAGTATCTGAAGTCGTCGAAGTACATCGCCTGGCCCCTCCAGGGCTGGCAGGCGACGTTCGGCGGCGGCGACCATCCGCCCAAGATGTCGATCATCATCGCCACCAACAGCACCGAGCATCCCATCTTCCGCCCGCTCGCCAACTTCCCGCCGTCGCTCTGGGGCAACCTGTTCACCTCGTTCAGCATGGACAACCAGGCGCGCGAGATCTACGCCAAGGAGCACGAGGGCCTCAAGGAGAAGGTCCGGATGATGTTCCTGGACACCACGAACTACAAGATCTCGGAGAAGATCAACTTCATCAACACCGTCGAGCGCCTGGGCGTGAGCTATCACTTCGAGAAGGAGATCGAGGAGCTGCTCCATCAGATGTTCGACGCCCACTCGAAGCATCTGGACGACATCCAGGAGTTCGACCTCTTCACGCTGGGCATCTACTTCCGCATCCTGCGGCAGCATGGCTATAAGATCTCGTGCGACGTCTTCAACAAGCTGAAGGACAGCAACGGCGAGTTCAAGGACGAGCTCAAGGACGACGTCAACGGCATGCTGTCGTTCTATGAGGCCACCCATGTGCGCACCCATGGCGAGAACATCCTCGACGAGGCGCTGATCTACACGAAGGCCCAGCTGGAGTCGATGGCGGCCGCCTCGCTCAGCCCGTTCCTGGCCAACCAGGTGAAGCACGCGCTCATGCAGGCCCTGCATAAGGGCATCCCGCGCATCGAGGCGCGGAACTACATCTCGGTCTATGAGGAAGACCCGAACAAGAACGACCTGCTCCTGCGCTTCAGCAAGATCGACTTCAACCTGGTGCAGATGATCCACAAGCAGGAGCTGTGCGACACCTTCCGGTGGTGGAAGGACCTGGAGTTCGAGTCGAAGCTGTCGTTCGCCCGCAACCGGGTGGTGGAGGCCTACCTCTGGACGCTGTCGGCGTACTATGAGCCGAAGTATTCGAGCGCCCGCATCATCCTCGTGAAGCTGATGGTCATCATCTCGGTGACCGACGACACGTACGACGCCTATGGCACCCTGGACGAGCTCCAGCTGTTCACGGACGCGATCCAGCGCCTCGACATGTCGAGCATCAACCAGCTGCCCGACTACATGAAGACCATCTATAAGGCGCTCCTGGACCTCTTCGACGAGATCGAGGACCGCCTGTCGAAGCACGAGACGGACCATAGCTACCGGGTGGCGTATGCCAAGTACGTCTATAAGGAGATCGTGCGCTGCTACGACATGGAGTATAAGTGGTTCAACAAGAACTACGTCCCCGCCTTCGAGGAGTATATGCAGAAGGCCCTGGTGACCTCGGGCAACCGGCTCCTGATCACGTTCAGCTTCCTGGGCATGGACGAGGTCGCGACCATCCAGGCCTTCGAGTGGGTGAAGTCGAACGCCAAGATGATCGTGTCGTCGAACAAGGTGCTCCGCCTGATCGACGACATCATGTCGCACGAGGAAGAGGACGAGCGCGGCCATGTGGCCACGGGCATCGAGTGCTTCGTGAAGGAGCACGGCCTGACCCGCGAGGAAGTGATCGTCGAGTTCCATAAGCGGATCGACGACGCGTGGAAGGACATCAACGAGGAGTTCATCACCCCGAACAACCTGCCGATCGAGATCCTGACCCGCGTGCTCAACCTGACCCGGATCGGCGACGTGGTCTACAAGTATGACGACGGCTACACGCACCCCGAGAAGGCCCTCAAGGACCATATCATCAGCCTGTTCGTGGACCCCGTCAGCATCTGA
イタリック体の配列はSPppaプロモーターであり、アンダーライン付きシーケンスはコボン最適化GSTであり、通常フォントの配列はコボン最適化NjPATである。
配列番号31
GST−NjPATアミノ酸配列
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKMSIIIATNSTEHPIFRPLANFPPSLWGNLFTSFSMDNQAREIYAKEHEGLKEKVRMMFLDTTNYKISEKINFINTVERLGVSYHFEKEIEELLHQMFDAHSKHLDDIQEFDLFTLGIYFRILRQHGYKISCDVFNKLKDSNGEFKDELKDDVNGMLSFYEATHVRTHGENILDEALIYTKAQLESMAAASLSPFLANQVKHALMQALHKGIPRIEARNYISVYEEDPNKNDLLLRFSKIDFNLVQMIHKQELCDTFRWWKDLEFESKLSFARNRVVEAYLWTLSAYYEPKYSSARIILVKLMVIISVTDDTYDAYGTLDELQLFTDAIQRLDMSSINQLPDYMKTIYKALLDLFDEIEDRLSKHETDHSYRVAYAKYVYKEIVRCYDMEYKWFNKNYVPAFEEYMQKALVTSGNRLLITFSFLGMDEVATIQAFEWVKSNAKMIVSSNKVLRLIDDIMSHEEEDERGHVATGIECFVKEHGLTREEVIVEFHKRIDDAWKDINEEFITPNNLPIEILTRVLNLTRIGDVVYKYDDGYTHPEKALKDHIISLFVDPVSI
アンダーライン付き配列はGSTタグである。
Claims (16)
- 配列番号4で示されるアミノ酸配列を含むパチョロールシンターゼ、またはその機能的相同体であって、前記相同体は、配列番号4との少なくとも80%の配列同一性を有するアミノ酸配列を含むパチョロールシンターゼである、パチョロールシンターゼ、またはその機能的相同体。
- 配列番号4との少なくとも85%、少なくとも90%、少なくとも95%、または少なくとも98%の配列同一性を有する、請求項1に記載のパチョロールシンターゼ。
- 請求項1または2に記載のパチョロールシンターゼをコードする核酸配列、またはその相補的配列を含む、核酸。
- 前記核酸は、配列番号3もしくは配列番号5で示される核酸配列、または配列番号3もしくは配列番号5で示される配列との少なくとも80%、より好ましくは少なくとも85%、より好ましくは少なくとも90%、より好ましくは少なくとも95%、最も好ましくは少なくとも98%の配列同一性を有する核酸配列、またはそれらの配列のいずれかの相補的配列を含む、請求項3に記載の核酸。
- 請求項3または4に記載の核酸を含む発現ベクター。
- それ自体が生物または多細胞生物の一部であってもよい宿主細胞であって、請求項3または4に記載の異種核酸配列を含む発現ベクターを含む、宿主細胞。
- 前記宿主細胞は、グラム陰性細菌の群、特に、ロドバクター、パラコッカスおよびエシェリキアの群から選択される細菌細胞である、請求項6に記載の宿主細胞。
- 前記宿主細胞は、アスペルギルス、ブラケスレア、ペニシリウム、ファフィア(キサントフィロマイセス)、ピキア、サッカロマイセス、およびヤロウイアの群から選択される真菌細胞である、請求項6に記載の宿主細胞。
- トランスジェニック植物細胞を含むトランスジェニック植物または培養物であって、前記植物または培養物は、請求項6に記載の宿主細胞を含み、前記宿主細胞は、タバコ属、ナス属、キクニガナ属、アキノノゲシ属、ハッカ属、クソニンジン属、塊茎形成植物、油料作物、液体培養植物、タバコBY2細胞、ヒメツリガネゴケおよび樹木から選択されるトランスジェニック植物のものである、トランスジェニック植物または培養物。
- トランスジェニックキノコ細胞を含むトランスジェニックキノコまたは培養物であって、前記キノコまたは培養物は請求項6に記載の宿主細胞を含み、前記宿主細胞は、スエヒロタケ、アガリクスおよびヒラタケから選択される、トランスジェニックキノコまたは培養物。
- パチョロールおよびエレモール、ならびに好ましくはポゴストールも調製する方法であって、請求項1または2に記載のパチョロールシンターゼの存在下でファルネシル二リン酸をパチョロールおよびエレモール、ならびに好ましくはポゴストールにも変換することを含む、方法。
- 前記パチョロールおよびエレモール、ならびに好ましくはポゴストールも、請求項6、7もしくは8に記載の宿主細胞、請求項9に記載の植物もしくは植物培養物、または請求項10に記載のキノコもしくはキノコ培養物において調製され、前記パチョロールシンターゼを発現する、請求項11に記載のパチョロールおよびエレモール、ならびに好ましくはポゴストールも調製する方法。
- 前記パチョロールおよび/またはポゴストールおよび/またはエレモールを単離することをさらに含む、請求項11または12に記載の方法。
- 比ポゴストールのパチョロールに対する比が1:5より高い、請求項11〜13のいずれか一項に記載の方法。
- エレモールのパチョロールに対する比が1:10より高い、請求項11〜14のいずれか一項に記載の方法。
- エレモールのポゴストールに対する比が1:3以上であり、1:2以上である、請求項11〜15のいずれか一項に記載の方法。
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US20210102224A1 (en) | 2021-04-08 |
EP3676382A1 (en) | 2020-07-08 |
KR102469382B1 (ko) | 2022-11-21 |
WO2019045568A1 (en) | 2019-03-07 |
CN111225979A (zh) | 2020-06-02 |
BR112020004093A2 (pt) | 2020-09-24 |
MX2020002329A (es) | 2020-07-13 |
KR20200043472A (ko) | 2020-04-27 |
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US11584941B2 (en) | 2023-02-21 |
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