JP6946183B2 - ステビオール配糖体の微生物による生成 - Google Patents
ステビオール配糖体の微生物による生成 Download PDFInfo
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- JP6946183B2 JP6946183B2 JP2017523897A JP2017523897A JP6946183B2 JP 6946183 B2 JP6946183 B2 JP 6946183B2 JP 2017523897 A JP2017523897 A JP 2017523897A JP 2017523897 A JP2017523897 A JP 2017523897A JP 6946183 B2 JP6946183 B2 JP 6946183B2
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Description
本願は、2014年11月5日に出願された米国仮出願第62/075,644号に対する優先権及び利益を主張するものであり、その参照によりその全体が本明細書に組み込まれる。
ステビオール配糖体はジテルペノイド誘導体であり、その生合成初期経路では、共通の中間体をジベレリン酸生合成と共有している(8)。全体の直線経路は、4部分、すなわち(1)グルコース由来の中心炭素代謝物から開始前駆体であるIPPとDMAPPとを形成させる部分、(2)最初に貢献する中間体であるカウレンを生成させる部分、(3)主要中間体であるステビオールを生合成させる部分、(4)各種のステビオール配糖体を形成させる部分にモジュール化される。さらなるモジュール(5)を独立して工学的に操作し、ステビオールのグリコシル化に必要な第2の基質であるUDP−グルコースの増量生成を支持する。5つのモジュールを図2に示す。
酵素に対して作用する自然選択では、十分な安定性及び生物学的機能の活性について選択しようとする。このプロセスで、酵素は特定の進化経路へ送られるが、これは産業用途に求められる安定性と活性の獲得に容易に適合しないことがある。一例として、特定の一基質に特化した酵素は、特化していなかった酵素よりも新しい基質の人工操作で問題が多くなりがちである。したがって、ドメインの接続を交換して酵素を「振り混ぜる」ことにより、タンパク質の折り畳みは同一であるが、折り返しの相互作用と折り込まれている相互作用とが新規である、新たに選択及び進化の対象となるであろう酵素が創作される可能性がある。言い換えれば、アミノ酸変異を何ら導入することなく、単純に配列をシャッフリングし、酵素をその本来の進化経路から移動させることによって、タンパク質の折り畳みから進化空間の別のポイントへと「飛ぶ」ことができる可能性がある。
円順列変異法(circular permutization)によって生じた酵素のシャッフリングと同じように、N末端の低分子糖受容体である結合ドメインまたはC末端の糖供与体である結合ドメインを、既知のUGT同士で交換することによって、グリコシルトランスフェラーゼ酵素をシャッフルする代替的方法を創作した。例えば、SrUGT85C2のN末端ドメインとSrUGT76G1のC末端ドメインとで構成されるキメラ酵素を創作した(図27)。このアプローチの背後にある根拠は、UGT酵素のドメインをシャッフリングするという概念にあるが、今回に限り特別に、各UGT間でドメインをシャッフリングするというニュアンスを加えた。その意図するところは、エネルギー全体像において親酵素が占めているポイントから離れて、新たに最適な酵素立体配置へ向けたさらなる進化が可能な、新規な配列の非最適化酵素を生成株に作製することにある。ここでも、この酵素はそれ自体が自然選択を受けた結果生じたものではないため、このキメラ法で生じたシャッフル酵素は、高い進化可能性/点変異から良好な利益を受ける大きな可能性(すなわち、より高い活性)を有していなければならない。さらに、この方法を使用して、折り畳み及び/または安定性が増強されたキメラタンパク質を作製することができる。
ステビア植物から明らかになっている、十分量で生じるステビオール配糖体は約20種のみであるが、生合成により生成され得る、グリコシル化パターンの異なった考えられるステビオール配糖体がさらにいくつかある。表10及び図29、30、31及び32には、本項に記載するステビオール配糖体として可能性の高い、既知のステビオール配糖体がまとめられており、それらは符号SG#で略称されている。これらのグリコシドには天然に存在するものもあれば、本明細書に記載の4つのグリコシル化反応(すなわち、C13−O−グリコシル化、C19−O−グリコシル化、1,2’−グリコシル化、及び1,3’−グリコシル化)を触媒するUGTを使用して生合成が可能なものもある。
22℃を上回る温度での最適下限を決定するためカウレンモジュールにおける酵素の性能を測定した。先の実施例に使用したGGPPS(ゲラニルゲラニル二リン酸)シンターゼ酵素及び二機能性コパリル二リン酸(CPP)/カウレンシンターゼ酵素の代替的酵素のクラスターを同定した。特に、E.coliにおいて細菌酵素のほうが植物酵素及び真菌酵素より良好に機能する場合があることから、細菌供給源に由来する代替的酵素を検討した。可能な場合は好熱性細菌由来の酵素を検討した。CPP合成酵素及びカウレン合成酵素の活性については、根圏細菌がその共生ライフスタイルからカウレン生成性であることが多いため、根圏細菌の遺伝子を同定した。
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UGT配列
>SrUGT85C2 gi|37993661|gb|AAR06916.1|UDP−グリコシルトランスフェラーゼ85C2[Stevia rebaudiana]
(配列番号1)
MDAMATTEKKPHVIFIPFPAQSHIKAMLKLAQLLHHKGLQITFVNTDFIHNQFLESSGPHCLDGAPGFRFETIPDGVSHSPEASIPIRESLLRSIETNFLDRFIDLVTKLPDPPTCIISDGFLSVFTIDAAKKLGIPVMMYWTLAACGFMGFYHIHSLIEKGFAPLKDASYLTNGYLDTVIDWVPGMEGIRLKDFPLDWSTDLNDKVLMFTTEAPQRSHKVSHHIFHTFDELEPSIIKTLSLRYNHIYTIGPLQLLLDQIPEEKKQTGITSLHGYSLVKEEPECFQWLQSKEPNSVVYVNFGSTTVMSLEDMTEFGWGLANSNHYFLWIIRSNLVIGENAVLPPELEEHIKKRGFIASWCSQEKVLKHPSVGGFLTHCGWGSTIESLSAGVPMICWPYSWDQLTNCRYICKEWEVGLEMGTKVKRDEVKRLVQELMGEGGHKMRNKAKDWKEKARIAIAPNGSSSLNIDKMVKEITVLARN
>SrUGT74G1 gi|37993669|gb|AAR06920.1|UDP−グリコシルトランスフェラーゼ74G1[Stevia rebaudiana]
(配列番号2)
MAEQQKIKKSPHVLLIPFPLQGHINPFIQFGKRLISKGVKTTLVTTIHTLNSTLNHSNTTTTSIEIQAISDGCDEGGFMSAGESYLETFKQVGSKSLADLIKKLQSEGTTIDAIIYDSMTEWVLDVAIEFGIDGGSFFTQACVVNSLYYHVHKGLISLPLGETVSVPGFPVLQRWETPLILQNHEQIQSPWSQMLFGQFANIDQARWVFTNSFYKLEEEVIEWTRKIWNLKVIGPTLPSMYLDKRLDDDKDNGFNLYKANHHECMNWLDDKPKESVVYVAFGSLVKHGPEQVEEITRALIDSDVNFLWVIKHKEEGKLPENLSEVIKTGKGLIVAWCKQLDVLAHESVGCFVTHCGFNSTLEAISLGVPVVAMPQFSDQTTNAKLLDEILGVGVRVKADENGIVRRGNLASCIKMIMEEERGVIIRKNAVKWKDLAKVAVHEGGSSDNDIVEFVSELIKA
>SrUGT76G1 gi|37993653|gb|AAR06912.1|UDP−グリコシルトランスフェラーゼ76G1[Stevia rebaudiana]
(配列番号3)
MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQILKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL
>SrUGT91D1
(配列番号4)
MYNVTYHQNSKAMATSDSIVDDRKQLHVATFPWLAFGHILPFLQLSKLIAEKGHKVSFLSTTRNIQRLSSHISPLINVVQLTLPRVQELPEDAEATTDVHPEDIQYLKKAVDGLQPEVTRFLEQHSPDWIIYDFTHYWLPSIAASLGISRAYFCVITPWTIAYLAPSSDAMINDSDGRTTVEDLTTPPKWFPFPTKVCWRKHDLARMEPYEAPGISDGYRMGMVFKGSDCLLFKCYHEFGTQWLPLLETLHQVPVVPVGLLPPEIPGDEKDETWVSIKKWLDGKQKGSVVYVALGSEALVSQTEVVELALGLELSGLPFVWAYRKPKGPAKSDSVELPDGFVERTRDRGLVWTSWAPQLRILSHESVCGFLTHCGSGSIVEGLMFGHPLIMLPIFCDQPLNARLLEDKQVGIEIPRNEEDGCLTKESVARSLRSVVVENEGEIYKANARALSKIYNDTKVEKEYVSQFVDYLEKNARAVAIDHES
>SrUGT91D2
(配列番号5)
MATSDSIVDDRKQLHVATFPWLAFGHILPYLQLSKLIAEKGHKVSFLSTTRNIQRLSSHISPLINVVQLTLPRVQELPEDAEATTDVHPEDIPYLKKASDGLQPEVTRFLEQHSPDWIIYDYTHYWLPSIAASLGISRAHFSVTTPWAIAYMGPSADAMINGSDGRTTVEDLTTPPKWFPFPTKVCWRKHDLARLVPYKAPGISDGYRMGLVLKGSDCLLSKCYHEFGTQWLPLLETLHQVPVVPVGLLPPEVPGDEKDETWVSIKKWLDGKQKGSVVYVALGSEVLVSQTEVVELALGLELSGLPFVWAYRKPKGPAKSDSVELPDGFVERTRDRGLVWTSWAPQLRILSHESVCGFLTHCGSGSIVEGLMFGHPLIMLPIFGDQPLNARLLEDKQVGIEIPRNEEDGCLTKESVARSLRSVVVEKEGEIYKANARELSKIYNDTKVEKEYVSQFVDYLEKNTRAVAIDHES
>SrUGT91D2e
(配列番号6)
MATSDSIVDDRKQLHVATFPWLAFGHILPYLQLSKLIAEKGHKVSFLSTTRNIQRLSSHISPLINVVQLTLPRVQELPEDAEATTDVHPEDIPYLKKASDGLQPEVTRFLEQHSPDWIIYDYTHYWLPSIAASLGISRAHFSVTTPWAIAYMGPSADAMINGSDGRTTVEDLTTPPKWFPFPTKVCWRKHDLARLVPYKAPGISDGYRMGLVLKGSDCLLSKCYHEFGTQWLPLLETLHQVPVVPVGLLPPEIPGDEKDETWVSIKKWLDGKQKGSVVYVALGSEVLVSQTEVVELALGLELSGLPFVWAYRKPKGPAKSDSVELPDGFVERTRDRGLVWTSWAPQLRILSHESVCGFLTHCGSGSIVEGLMFGHPLIMLPIFGDQPLNARLLEDKQVGIEIPRNEEDGCLTKESVARSLRSVVVEKEGEIYKANARELSKIYNDTKVEKEYVSQFVDYLEKNARAVAIDHES
>OsUGT1−2(Q0DPB7_ORYSJ)
配列番号7
MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRTKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLLSTLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAVGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKD
UGTの変異体及び円順列変異体
>MbUGTC19
(配列番号8)
MAECMNWLDDKPKESVVYVAFGSLVKHGPEQVEEITRALIDSDVNFLWVIKHKEEGKLPENLSEVIKTGKGLIVAWCKQLDVLAHESVGCFVTHCGFNSTLEAISLGVPVVAMPQFSDQTTNAKLLDEILGVGVRVKADENGIVRRGNLASCIKMIMEEERGVIIRKNAVKWKDLAKVAVHEGGSSDNDIVEFVSELIKAGSGEQQKIKKSPHVLLIPFPLQGHINPFIQFGKRLISKGVKTTLVTTIHTLNSTLNHSNTTTTSIEIQAISDGCDEGGFMSAGESYLETFKQVGSKSLADLIKKLQSEGTTIDAIIYDSMTEWVLDVAIEFGIDGGSFFTQACVVNSLYYHVHKGLISLPLGETVSVPGFPVLQRWETPLILQNHEQIQSPWSQMLFGQFANIDQARWVFTNSFYKLEEEVIEWTRKIWNLKVIGPTLPSMYLDKRLDDDKDNGFNLYKANHH
>MbUGT1−2
(配列番号9)
MAGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLLSTLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAVGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRTK
>MbUGT1−3
(配列番号10)
MANWQILKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSLENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYS
>MbUGT1,2−2
(配列番号45)
MATKGSSGMSLAERFWLTLSRSSLVVGRSCVEFEPETVPLLSTLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAVGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAEMIASIADERLEHAETESPAAAGQGRPAAAPTFEVARMKLIR
>MbUGTC19−2
(配列番号46)
MANHHECMNWLDDKPKESVVYVAFGSLVKHGPEQVEEITRALIDSDVNFLWVIKHKEEGKLPENLSEVIKTGKGLIVAWCKQLDVLAHESVGCFVTHCGFNSTLEAISLGVPVVAMPQFSDQTTNAKLLDEILGVGVRVKADENGIVRRGNLASCIKMIMEEERGVIIRKNAVKWKDLAKVAVHEGGSSDNDIVEFVSELIKAGSGEQQKIKKSPHVLLIPFPLQGHINPFIQFGKRLISKGVKTTLVTTIHTLNSTLNHSNTTTTSIEIQAISDGCDEGGFMSAGESYLETFKQVGSKSLADLIKKLQSEGTTIDAIIYDSMTEWVLDVAIEFGIDGGSFFTQACVVNSLYYHVHKGLISLPLGETVSVPGFPVLQRWETPLILQNHEQIQSPWSQMLFGQFANIDQARWVFTNSFYKLEEEVIEWTRKIWNLKVIGPTLPSMYLDKRLDDDKDNGFNLYKA
>MbUGTC13
(配列番号51)
MADAMATTEKKPHVIFIPFPAQSHIKAMLKLAQLLHHKGLQITFVNTDFIHNQFLESSGPHCLDGAPGFRFETIPDGVSHSPEASIPIRESLLRSIETNFLDRFIDLVTKLPDPPTCIISDGFLSVFTIDAAKKLGIPVMMYWTLAACGFMGFYHIHSLIEKGFAPLKDASYLTNGYLDTVIDWVPGMEGIRLKDFPLDWSTDLNDKVLMFTTEATQRSHKVSHHIFHTFDELEPSIIKTLSLRYNHIYTIGPLQLLLDQIPEEKKQTGITSLHGYSLVKEEPECFQWLQSKEPNSVVYVNFGSTTVMSLEDMTEFGWGLANSNHYFLWIIRSNLVIGENAVLPPELEEHIKKRGFIASWCSQEKVLKHPSVGGFLTHCGWGSTIESLSAGVPMICWPYSWDQLTNCRYICKEWEVGLEMGTKVKRDEVKRLVQELMGEGGHKMRNKAKDWKEKARIAIAPNGSSSLNIDKMVKEITVLARN
ステビオール生合成の酵素
>TcGGPPS
(配列番号11)
MYTAMAAGTQSLQLRTVASYQECNSMRSCFKLTPFKSFHGVNFNVPSLGAANCEIMGHLKLGSLPYKQCSVSSKSTKTMAQLVDLAETEKAEGKDIEFDFNEYMKSKAVAVDAALDKAIPLEYPEKIHESMRYSLLAGGKRVRPALCIAACELVGGSQDLAMPTACAMEMIHTMSLIHDDLPCMDNDDFRRGKPTNHKVFGEDTAVLAGDALLSFAFEHIAVATSKTVPSDRTLRVISELGKTIGSQGLVGGQVVDITSEGDANVDLKTLEWIHIHKTAVLLECSVVSGGILGGATEDEIARIRRYARCVGLLFQVVDDILDVTKSSEELGKTAGKDLLTDKATYPKLMGLEKAKEFAAELATRAKEELSSFDQIKAAPLLGLADYIAFRQN
>TcGGPPS 98位で切断し角括弧で括った変異を有する
(配列番号12)
MADFNEYMKSKAVAVDAALDKAIPLEYPEKIHESMRYSLLAGGKRVRPALCIAACELVGGSQDLAMPTACAMEMIHTMSLIHDDLPCMDNDDFRRGKPTNHKVFGEDTAVLAGDALLSFAFEHIAVATSKTVPSDRTLRVI[C]ELGKTIGSQGLVGGQVVDITSEGDANVDLKTLEWIHIHKTAVLLECSVVSGGILG[D]ATEDEIARIRRYARCVGLLFQVVDDILDVTKSSEELGKTAGKDLLTDKATYPKLMGLEKAKEFAAELATRAKEELSSFDQIK[V]APLLGLADYIAFRQN
>AgGGPPS gi|17352451|gb|AAL17614.2|AF425235_1 ゲラニルゲラニル二リン酸合成酵素[Abies grandis]
(配列番号13)
MAYSGMATSYHGLHFMNIATQECNLKRLSIPSRRFHGVSPSLWASNGFQGHLKRELSANSFLVSSSRYSNTIAKFTNLPEKVKEKVIEFDFKEYLRSKAMAVNEALDRAVPLRYPERIHEAMRYSLLAGGKRVRPVLCISACELVGGTEEVAMPTACAMEMIHTMSLIHDDLPCMDNDDFRRGKPTNHKVFGEGTAILAGDALLSFAFEHIAVSTSKSVGTDRILRVVSELGRTIGSQGLVGGQVADITSEGDASVDLDTLEWIHIHKTAVLLECSVMCGAIISGASDNEIERIQRYARSVGLLFQVVDDILDVTKSSKELGKTAGKDLISDKATYPKLMGLEKAKQFASDLLIRAKEDLSCFDPMKAAPLLGLADYIAFRQN
>AnGGPPS gi|259486923|tpe|CBF85177.1| TPA:保存された仮定上のタンパク質[Aspergillus nidulans FGSC A4}
(配列番号14)
MSPPLDSALEPLSEYKETAFPRTEKDPSQYKEHDLVTPEKEIQTGYFSPRGSHSSHGSHDSSASSNISLDDARMSDVNNSPNVFHDDPDTIDEKLSMYWKAANETVIREPYDYIAGIPGKEIRRKLLEAFNHWYKVDEQSCQAIATTVGMAHNASLLIDDIQDSSKLRRGVPCAHEVFGIAQTINSANYVYFLAQNQLFRLRSWPQAISVFNEEMVNLHRGQGMELFWRDNLLPPSMDDYLQMIANKTGGLFRMIVRLLQTSSRQVIDVEQLVDVLGLYFQILDDYKNIREEKMAAQKGFFEDLTEGKFSFPICHAIGEGAKNRTALLHMLRLKTDDMKIKQEAVCILDNAGSLDYTREVLYGLDRKARSLLREFKTPNPFMEALLDAMLSSLQACH
>SmGGPPS gi|260653869|dbj|BAI44337.1| ゲラニルゲラニル二リン酸合成酵素[Streptomyces melanosporofaciens]
配列番号15)
MTTPTLSPGRLDADTVRKSVDVVLEDFLTAKAHTTPQHHLPYLSGLLKDFLSGGKRIRPLLCVTGWQAVGGGEDTEPVFRVAACLEMFHAFALIHDDVMDDSDTRRGRPTIHRTLAALCATDRRPEQIERFGVSGAVLLGDLALTWSDELLHSAGLTPVQFDAVLPLLSEMRTEVMLGQYLDLQATGELTDDVEATLTVNRYKTAKYTIERPLHVGAAIAGAGPEAMEAFTAYALPLGEAFQLRDDLLGVYGDPESTGKSQLDDLRAGKNTTLIALALRGSDSTQAARLRSLIGNPLLDERDAATIQEIFAATTARDAVEQMIDDRRTQALRALDDAPFTADAVNALKQIARLATVRNS
>MbGGPPS[海洋細菌443]
(配列番号36)
MAENGLLDCEQYLEEAMAEHATAQCPPLLAQALNYAVFPGGARVRPKICKAVALANNSSDVGLANAAASAIELLHCASLVHDDLPCFDDATQRRGKPSVHAKFGERIAVLTGDALIVAAFQTLATHAIHAVRTERVPLVTAIVARGVGAPHGICAGQAWECERSVDLSRYHRAKTGALFVAATCAGAAAAGVDPGPWVNLGASIGEAYQVADDIKDAISDPETLGKPTGIDVKLDRPSAVRELGLDGAVTRLKQCLEAGLDSMPACAGQDLLQKIVRAQASRFVPEKIAQVAAVD
>PhGGPPS[Paracoccus haeundaensis]
(配列番号37)
MARRDVNPIHATLLQTRLEEIAQGFGAVSQPLGAAMSHGALSSGRRFRGMLMLLAAEASGGVCDTIVDAACAVEMVHAASLIFDDLPCMDDAGLRRGRPATHVAHGESRAVLGGIALITEAMALLAGARGASGTVRAQLVRILSRSLGPQGLCAGQDLDLHAAKNGAGVEQEQDLKTGVLFIAGLEMLAVIKEFDAEEQTQMIDFGRQLGRVFQSYDDLLDVVGDQAALGKDTGRDAAAPGPRRGLLAVSDLQNVSRHYEASRAQLDAMLRSKRLQAPEIAALLERVLPYAARAVD
>CtGGPPS[Clorobium tepidum TLS]
(配列番号38)
MASSPITQAQVESKYRQYHAKINEALAACFPKEKPATLYDPARYILEGKGKRIRPFLTLLAAEAVSGKSDNALGVALGIEVLHNFTLMHDDIMDQADLRHGRPTVHKQWNVNAAILSGDMMIAYAYELALKAISSRHAEIIHIFNDANITICEGQALDMELEQRKDVTIADYLDMISKKTGRLISAALEAGGVAGDGTPEQIAALVTFGEKIGRAFQIQDDYLDIMAGDGKSGKVPGGDVINGKKTWLLLRSLELAEGADRELLQSIFDNNGTSPDNVPAVKAIFEKCGVLNETRAKINEDTEAALAALDALPFEEGRGYLRGFANILMKRDFVD
>SsGGPPS[Synechoccus sp.JA−3−3Ab]
(配列番号39)
MAVAQTFNLDTYLSQRQQQVEEALSAALVPAYPERIYEAMRYSLLAGGKRLRPILCLAACELAGGSVEQAMPTACALEMIHTMSLIHDDLPAMDNDDFRRGKPTNHKVFGEDIAILAGDALLAYAFEHIASQTRGVPPQLVLQVIARIGHAVAATGLVGGQVVDLESEGKAISLETLEYIHSHKTGALLEASVVSGGILAGADEELLARLSHYARDIGLAFQIVDDILDVTATSEQLGKTAGKDQAAAKATYPSLLGLEASRQKAEELIQSAKEALRPYGSQAEPLLALADFITRRQHVD
>SsGGPPS2[Synechocystis sp.PCC6803]
(配列番号40)
MAVAQQTRTDFDLAQYLQVKKGVVEAALDSSLAIARPEKIYEAMRYSLLAGGKRLRPILCITACELCGGDEALALPTACALEMIHTMSLIHDDLPSMDNDDFRRGKPTNHKVYGEDIAILAGDGLLAYAFEYVVTHTPQADPQALLQVIARLGRTVGAAGLVGGQVLDLESEGRTDITPETLTFIHTHKTGALLEASVLTGAILAGATGEQQQRLARYAQNIGLAFQVVDDILDITATQEELGKTAGKDVKAQKATYPSLLGLEASRAQAQSLIDQAIVALEPFGPSAEPLQAIAEYIVARKYVD
>TmGGPPS[Thermotoga maritima HB8]
(配列番号41)
MAKKEKVEERIREILRPGWDLLTEEAMLYSATVGGKRIRPLLVLTLGEDLGVEEEKLLDVAVAVELFHTASLIHDDLPPIDNADFRRGKPSCHRTYGEDIALLAGDGLFFLAFSQISKIGNSKIFEEFSETAYKLLLGEAMDVEFERRKMEVSQEMVERMYAFKTGALFAFCFSAPFILKGKDHTKMKLLGEKFGVAFQIYDDLKDILGSFEKVGKDLGKDTEKVTLVKKVGIQKAREMADKYYEEVLKGIESEGLFRTLFLLKELKQMVEERVD
>CgGGPPS[Corynebacterium glutamicum]
(配列番号42)
MAKDVSLSSFDAHDLDLDKFPEVVRDRLTQFLDAQELTIADIGAPVTDAVAHLRSFVLNGGKRIRPLYAWAGFLAAQGHKNSSEKLESVLDAAASLEFIQACALIHDDIIDSSDTRRGAPTVHRAVEADHRANNFEGDPEHFGVSVSILAGDMALVWAEDMLQDSGLSAEALARTRDAWRGMRTEVIGGQLLDIYLESHANESVELADSVNRFKTAAYTIARPLHLGASIAGGSPQLIDALLHYGHDIGIAFQLRDDLLGVFGDPAITGKPAGDDIREGKRTVLLALALQRADKQSPEAATAIRAGVGKVTSPEDIAVITEHIRATGAEEEVEQRISQLTESGLAHLDDVDIPDEVRAQLRALAIRSTERRMVD
>TtGGPPS[Thermus thermophillus HB27]
(配列番号43)
MAVPAPEAIRQALQERLLARLDHPDPLYRDLLQDYPRRGGKMLRGLLTVYSALAHGAPLEAGLEAATALELFQNWVLVHDDIEDGSEERRGRPALHRLHPMPLALNAGDAMHAEMWGLLAEGLARGLFPPEVLLEFHEVVRRTAYGQHLDLLWTLGGTFDLRPEDYFRMVAHKAAYYTAVAPLRLGALLAGKTPPAAYEEGGLRLGTAFQIVDDVLNLEGGEAYGKERAGDLYEGKRTLILLRFLEEAPPEERARALALLALPREAKPEAEVGWLLERLLASRALAWAKAEAKRLQAEGLALLEAAFQDLPGKEALDHLRGLLAALVERRAVD
>PcGGPPS[Pyrobaculum calidifontis JCM11548]
(配列番号44)
MADVVSRLHQKYGAEVEKALVRYLSIGLAEDFREAVLYQVKTGGKRLRPLLTLAAAEAVSGQWRPALPAAAIVELIHNYSLIYDDIIDRGDVRRGLPTVRKAFGDNAAILVGIWYREAIEEAVLDTPKPTLFAKEVAEVIKAIDEGERLDILFEAAGRSDPYFVQARWREVTLDDYIKMVSLKTGALIAAAAKWGVLSVSDDRGLAEAAWNFGMAAGVAFQIIDDVLDIYGDPKKFGKEIGKDIKEHKRGNAVVAVALSHLGEGERRRLLEILAREVVEEADVREAVALLDSVGAREEALRLAARYREEAERHLAKIPNNGTLKELLDFIVAREY
>SrCPPS[Stevia rebaudiana]
(配列番号16)
MKTGFISPATVFHHRISPATTFRHHLSPATTNSTGIVALRDINFRCKAVSKEYSDLLQKDEASFTKWDDDKVKDHLDTNKNLYPNDEIKEFVESVKAMFGSMNDGEINVSAYDTAWVALVQDVDGSGSPQFPSSLEWIANNQLSDGSWGDHLLFSAHDRIINTLACVIALTSWNVHPSKCEKGLNFLRENICKLEDENAEHMPIGFEVTFPSLIDIAKKLNIEVPEDTPALKEIYARRDIKLTKIPMEVLHKVPTTLLHSLEGMPDLEWEKLLKLQCKDGSFLFSPSSTAFALMQTKDEKCLQYLTNIVTKFNGGVPNVYPVDLFEHIWVVDRLQRLGIARYFKSEIKDCVEYINKYWTKNGICWARNTHVQDIDDTAMGFRVLRAHGYDVTPDVFRQFEKDGKFVCFAGQSTQAVTGMFNVYRASQMLFPGERILEDAKKFSYNYLKEKQSTNELLDKWIIAKDLPGEVGYALDIPWYASLPRLETRYYLEQYGGEDDVWIGKTLYRMGYVSNNTYLEMAKLDYNNYVAVLQLEWYTIQQWYVDIGIEKFESDNIKSVLVSYYLAAASIFEPERSKERIAWAKTTILVDKITSIFDSSQSSKEDITAFIDKFRNKSSSKKHSINGEPWHEVMVALKKTLHGFALDALMTHSQDIHPQLHQAWEMWLTKLQDGVDVTAELMVQMINMTAGRWVSKELLTHPQYQRLSTVTNSVCHDITKLHNFKENSTTVDSKVQELVQLVFSDTPDDLDQDMKQTFLTVMKTFYYKAWCDPNTINDHISKVFEIVI
>SrKS[Stevia rebaudiana]
(配列番号17)
MNLSLCIASPLLTKSNRPAALSAIHTASTSHGGQTNPTNLIIDTTKERIQKQFKNVEISVSSYDTAWVAMVPSPNSPKSPCFPECLNWLINNQLNDGSWGLVNHTHNHNHPLLKDSLSSTLACIVALKRWNVGEDQINKGLSFIESNLASATEKSQPSPIGFDIIFPGLLEYAKNLDINLLSKQTDFSLMLHKRELEQKRCHSNEMDGYLAYISEGLGNLYDWNMVKKYQMKNGSVFNSPSATAAAFINHQNPGCLNYLNSLLDKFGNAVPTVYPHDLFIRLSMVDTIERLGISHHFRVEIKNVLDETYRCWVERDEQIFMDVVTCALAFRLLRINGYEVSPDPLAEITNELALKDEYAALETYHASHILYQEDLSSGKQILKSADFLKEIISTDSNRLSKLIHKEVENALKFPINTGLERINTRRNIQLYNVDNTRILKTTYHSSNISNTDYLRLAVEDFYTCQSIYREELKGLERWVVENKLDQLKFARQKTAYCYFSVAATLSSPELSDARISWAKNGILTTVVDDFFDIGGTIDELTNLIQCVEKWNVDVDKDCCSEHVRILFLALKDAICWIGDEAFKWQARDVTSHVIQTWLELMNSMLREAIWTRDAYVPTLNEYMENAYVSFALGPIVKPAIYFVGPKLSEEIVESSEYHNLFKLMSTQGRLLNDIHSFKREFKEGKLNAVALHLSNGESGKVEEEVVEEMMMMIKNKRKELMKLIFEENGSIVPRACKDAFWNMCHVLNFFYANDDGFTGNTILDTVKDIIYNPLVLVNENEEQR
>GfCPPS/KS[Gibberella fujikuroi]
(配列番号18)
MPGKIENGTPKDLKTGNDFVSAAKSLLDRAFKSHHSYYGLCSTSCQVYDTAWVAMIPKTRDNVKQWLFPECFHYLLKTQAADGSWGSLPTTQTAGILDTASAVLALLCHAQEPLQILDVSPDEMGLRIEHGVTSLKRQLAVWNDVEDTNHIGVEFIIPALLSMLEKELDVPSFEFPCRSILERMHGEKLGHFDLEQVYGKPSSLLHSLEAFLGKLDFDRLSHHLYHGSMMASPSSTAAYLIGATKWDDEAEDYLRHVMRNGAGHGNGGISGTFPTTHFECSWIIATLLKVGFTLKQIDGDGLRGLSTILLEALRDENGVIGFAPRTADVDDTAKALLALSLVNQPVSPDIMIKVFEGKDHFTTFGSERDPSLTSNLHVLLSLLKQSNLSQYHPQILKTTLFTCRWWWGSDHCVKDKWNLSHLYPTMLLVEAFTEVLHLIDGGELSSLFDESFKCKIGLSIFQAVLRIILTQDNDGSWRGYREQTCYAILALVQARHVCFFTHMVDRLQSCVDRGFSWLKSCSFHSQDLTWTSKTAYEVGFVAEAYKLAALQSASLEVPAATIGHSVTSAVPSSDLEKYMRLVRKTALFSPLDEWGLMASIIESSFFVPLLQAQRVEIYPRDNIKVDEDKYLSIIPFTWVGCNNRSRTFASNRWLYDMMYLSLLGYQTDEYMEAVAGPVFGDVSLLHQTIDKVIDNTMGNLARANGTVHSGNGHQHESPNIGQVEDTLTRFTNSVLNHKDVLNSSSSDQDTLRREFRTFMHAHITQIEDNSRFSKQASSDAFSSPEQSYFQWVNSTGGSHVACAYSFAFSNCLMSANLLQGKDAFPSGTQKYLISSVMRHATNMCRMYNDFGSIARDNAERNVNSIHFPEFTLCNGTSQNLDERKERLLKIATYEQGYLDRALEALERQSRDDAGDRAGSKDMRKLKIVKLFCDVTDLYDQLYVIKDLSSSMK
>PpCPPS/KS[Physcomitrella patens]
(配列番号19)
MASSTLIQNRSCGVTSSMSSFQIFRGQPLRFPGTRTPAAVQCLKKRRCLRPTESVLESSPGSGSYRIVTGPSGINPSSNGHLQEGSLTHRLPIPMEKSIDNFQSTLYVSDIWSETLQRTECLLQVTENVQMNEWIEEIRMYFRNMTLGEISMSPYDTAWVARVPALDGSHGPQFHRSLQWIIDNQLPDGDWGEPSLFLGYDRVCNTLACVIALKTWGVGAQNVERGIQFLQSNIYKMEEDDANHMPIGFEIVFPAMMEDAKALGLDLPYDATILQQISAEREKKMKKIPMAMVYKYPTTLLHSLEGLHREVDWNKLLQLQSENGSFLYSPASTACALMYTKDVKCFDYLNQLLIKFDHACPNVYPVDLFERLWMVDRLQRLGISRYFEREIRDCLQYVYRYWKDCGIGWASNSSVQDVDDTAMAFRLLRTHGFDVKEDCFRQFFKDGEFFCFAGQSSQAVTGMFNLSRASQTLFPGESLLKKARTFSRNFLRTKHENNECFDKWIITKDLAGEVEYNLTFPWYASLPRLEHRTYLDQYGIDDIWIGKSLYKMPAVTNEVFLKLAKADFNMCQALHKKELEQVIKWNASCQFRDLEFARQKSVECYFAGAATMFEPEMVQARLVWARCCVLTTVLDDYFDHGTPVEELRVFVQAVRTWNPELINGLPEQAKILFMGLYKTVNTIAEEAFMAQKRDVHHHLKHYWDKLITSALKEAEWAESGYVPTFDEYMEVAEISVALEPIVCSTLFFAGHRLDEDVLDSYDYHLVMHLVNRVGRILNDIQGMKREASQGKISSVQIYMEEHPSVPSEAMAIAHLQELVDNSMQQLTYEVLRFTAVPKSCKRIHLNMAKIMHAFYKDTDGFSSLTAMTGFVKKVLFEPVPE
>PsCPPS/KS[Phaeosphaeria sp.]
(配列番号20)
MFAKFDMLEEEARALVRKVGNAVDPIYGFSTTSCQIYDTAWAAMISKEEHGDKVWLFPESFKYLLEKQGEDGSWERHPRSKTVGVLNTAAACLALLRHVKNPLQLQDIAAQDIELRIQRGLRSLEEQLIAWDDVLDTNHIGVEMIVPALLDYLQAEDENVDFEFESHSLLMQMYKEKMARFSPESLYRARPSSALHNLEALIGKLDFDKVGHHLYNGSMMASPSSTAAFLMHASPWSHEAEAYLRHVFEAGTGKGSGGFPGTYPTTYFELNWVLSTLMKSGFTLSDLECDELSSIANTIAEGFECDHGVIGFAPRAVDVDDTAKGLLTLTLLGMDEGVSPAPMIAMFEAKDHFLTFLGERDPSFTSNCHVLLSLLHRTDLLQYLPQIRKTTTFLCEAWWACDGQIKDKWHLSHLYPTMLMVQAFAEILLKSAEGEPLHDAFDAATLSRVSICVFQACLRTLLAQSQDGSWHGQPEASCYAVLTLAESGRLVLLQALQPQIAAAMEKAADVMQAGRWSCSDHDCDWTSKTAYRVDLVAAAYRLAAMKASSNLTFTVDDNVSKRSNGFQQLVGRTDLFSGVPAWELQASFLESALFVPLLRNHRLDVFDRDDIKVSKDHYLDMIPFTWVGCNNRSRTYVSTSFLFDMMIISMLGYQIDEFFEAEAAPAFAQCIGQLHQVVDKVVDEVIDEVVDKVVGKVVGKVVGKVVDERVDSPTHEAIAICNIEASLRRFVDHVLHHQHVLHASQQEQDILWRELRAFLHAHVVQMADNSTLAPPGRTFFDWVRTTAADHVACAYSFAFACCITSATIGQGQSMFATVNELYLVQAAARHMTTMCRMCNDIGSVDRDFIEANINSVHFPEFSTLSLVADKKKALARLAAYEKSCLTHTLDQFENEVLQSPRVSSAASGDFRTRKVAVVRFFADVTDFYDQLYILRDLSSSLKHVGT
>EtCPPS[Erwina tracheiphila]
(配列番号32)
MAHALAENILTELNTLLSDMDDGGYVGPSVYDTAQLLRFHPNPPDRAGIYRWLIKQQHEDGGWGSPDFPLHRQVPTVAAILALHEAQPQPEGAAAALAAAAVYLAQERDLYADTIPDDAPIGAELILPQLCRQAAALFPHLAYPRYGALYEAEAARLGKVESLTAVPSGHPLLHSWESWGRSSTEVTPDVFGSIGISPSATAVWLGRACAENPACLPEHATRYLHNASRATGVGIDGVVPNVWPIDVFEPCWSLYSLHLAGLFSHPGLSTVVQNIATNIQAILTPLGLGPALSFASDADDTAIAAAVVQLSGHSLTCYPLHQFEKGDLFVTFPGERNPSLSTTIHAVHALSLLGTTAPDARAYIENSKSADGVWKNEKWHASWLYPTSHAVAALAHGMPSWRDNDVLYKILEAQHLSGGWGAGAAPTQEETAYALFALHVMNDRVNAPLREKLVSAVARAREWLLVRYQSNQLPITPLWIGKELYCPQRVVRVTELTGLWLALNWNPSHSDVSDTRTETPGERI
>EtKS[Erwina tracheiphila]
(配列番号33)
MATSHDDACQQVKVWGETLFGFLDEHAVEAVRGGQFILRHIRPELAAISARTGRDPDDEARELAFYQEMALLFWIDDCHDRGVMSPDDYAVVEGILVGRMPDAPTPSVGCSFLRHRLAQLASHKHDYSQLLADTQAYSTALRNGKRLASDPDRWSYSEHLRNGVDSIGYQNVFGCLSLLWGLDMPRWRTEPAFQNALSFLCAIGRLQNDLHGLANDRTLGEADNLAVQLERRYPTLDAVEFLQTEITGYERMLRPLLETANFDPVWVRLMETMLTVSDQYYATSTLRYRIDDTATTAPSCDTRHASGAVTGSGNETE
>SfCPPS[Sinorhizobium fredii]
(配列番号34)
MANALSEQILFELRHLLSEMSDGGSVGPSVYDTARALQFGGNVTGRQDAYAWLLAQQQADGGWGSADFPLFRHAPTWAALLALQRADPLPGAADAVQAATRFLERQADPYAHAVPEDAPIGAELILPQLCGEAASLLGGVAFPRHPALLPLRQACLVKLGAVATLPSGHPLLHSWEAWGTWPTAACPDDDGSIGISPAATAAWRAHAVTQGSTPQVGRADAYLQAASRATRSGIEGVVPNVWPINVFEPCWSLYTLHLAGLFAHPALDEAVRVIVAQLDARLGVRGLGPALHFAADADDTAVALCVLRLAGRDPAVDALRHFEIGELFVTFPGERNASVSTNIHALHALRLLGKPAAGTSAYVEANRNPHGLWDNEKWHVSWLYPTAHAVAALAQGKPQWRDERALAALLQAQRDDGGWGAGRASTFEETAYALFALHVMDGSEEPTGRRRIAQAVARALEWMLARHAAPALPQMPLWIGKELYCPIRVVRVAELAGLWLALRWGPRVPAEGAGAAP
>SfKS[Sinorhizobium fredii]
(配列番号35)
MAIPTERGLQQVLEWGRSLTGFADEHAAEAVRGGQYILQRIHPSLRDTSARTGRDPQDETLIVAFYRELALLFWLDDCNDLDLIAPEQLAAVEQALGQGVPCALPGFEGCAVLRASLAALAYDRRDYAQLLDDTRCYCAALRAGHAQAAGAAERWSYAEYLHNGIDSIAYANVFCCLSLLWGLDMATLRARPAFRQVLRLISAIGRLQNDLHGRDKDRSAGEADNAAILLLERYPAMPVVEFLNDELAGHTRMLHRVMAEERFPAPWGPLIEAMAAIRAHYYQTSTSRYRSDAAGGGQHAPA
>SrKO AAQ63464.1|ent−カウレンオキシダーゼCYP701A5[Stevia rebaudiana]
(配列番号21)
MDAVTGLLTVPATAITIGGTAVALAVALIFWYLKSYTSARRSQSNHLPRVPEVPGVPLLGNLLQLKEKKPYMTFTRWAATYGPIYSIKTGATSMVVVSSNEIAKEALVTRFQSISTRNLSKALKVLTADKTMVAMSDYDDYHKTVKRHILTAVLGPNAQKKHRIHRDIMMDNISTQLHEFVKNNPEQEEVDLRKIFQSELFGLAMRQALGKDVESLYVEDLKITMNRDEIFQVLVVDPMMGAIDVDWRDFFPYLKWVPNKKFENTIQQMYIRREAVMKSLIKEHKKRIASGEKLNSYIDYLLSEAQTLTDQQLLMSLWEPIIESSDTTMVTTEWAMYELAKNPKLQDRLYRDIKSVCGSEKITEEHLSQLPYITAIFHETLRRHSPVPIIPLRHVHEDTVLGGYHVPAGTELAVNIYGCNMDKNVWENPEEWNPERFMKENETIDFQKTMAFGGGKRVCAGSLQALLTASIGIGRMVQEFEWKLKDMTQEEVNTIGLTTQMLRPLRAIIKPRI
>t20−8RPSrKO
(配列番号22)
MALLLAVFAVALAVALIFWYLKSYTSARRSQSNHLPRVPEVPGVPLLGNLLQLKEKKPYMTFTRWAATYGPIYSIKTGATSMVVVSSNEIAKEALVTRFQSISTRNLSKALKVLTADKTMVAMSDYDDYHKTVKRHILTAVLGPNAQKKHRIHRDIMMDNISTQLHEFVKNNPEQEEVDLRKIFQSELFGLAMRQALGKDVESLYVEDLKITMNRDEIFQVLVVDPMMGAIDVDWRDFFPYLKWVPNKKFENTIQQMYIRREAVMKSLIKEHKKRIASGEKLNSYIDYLLSEAQTLTDQQLLMSLWEPIIESSDTTMVTTEWAMYELAKNPKLQDRLYRDIKSVCGSEKITEEHLSQLPYITAIFHETLRRHSPVPIIPLRHVHEDTVLGGYHVPAGTELAVNIYGCNMDKNVWENPEEWNPERFMKENETIDFQKTMAFGGGKRVCAGSLQALLTASIGIGRMVQEFEWKLKDMTQEEVNTIGLTTQMLRPLRAIIKPRI
>AtKO[Arabidopsis thaliana]
(配列番号23)
MAFFSMISILLGFVISSFIFIFFFKKLLSFSRKNMSEVSTLPSVPVVPGFPVIGNLLQLKEKKPHKTFTRWSEIYGPIYSIKMGSSSLIVLNSTETAKEAMVTRFSSISTRKLSNALTVLTCDKSMVATSDYDDFHKLVKRCLLNGLLGANAQKRKRHYRDALIENVSSKLHAHARDHPQEPVNFRAIFEHELFGVALKQAFGKDVESIYVKELGVTLSKDEIFKVLVHDMMEGAIDVDWRDFFPYLKWIPNKSFEARIQQKHKRRLAVMNALIQDRLKQNGSESDDDCYLNFLMSEAKTLTKEQIAILVWETIIETADTTLVTTEWAIYELAKHPSVQDRLCKEIQNVCGGEKFKEEQLSQVPYLNGVFHETLRKYSPAPLVPIRYAHEDTQIGGYHVPAGSEIAINIYGCNMDKKRWERPEDWWPERFLDDGKYETSDLHKTMAFGAGKRVCAGALQASLMAGIAIGRLVQEFEWKLRDGEEENVDTYGLTSQKLYPLMAIINPRRS
>PpKO[Physcomitrella patens]
(配列番号24)
MAKHLATQLLQQWNEALKTMPPGFRTAGKILVWEELASNKVLITIALAWVLLFVARTCLRNKKRLPPAIPGGLPVLGNLLQLTEKKPHRTFTAWSKEHGPIFTIKVGSVPQAVVNNSEIAKEVLVTKFASISKRQMPMALRVLTRDKTMVAMSDYGEEHRMLKKLVMTNLLGPTTQNKNRSLRDDALIGMIEGVLAELKASPTSPKVVNVRDYVQRSLFPFALQQVFGYIPDQVEVLELGTCVSTWDMFDALVVAPLSAVINVDWRDFFPALRWIPNRSVEDLVRTVDFKRNSIMKALIRAQRMRLANLKEPPRCYADIALTEATHLTEKQLEMSLWEPIIESADTTLVTSEWAMYEIAKNPDCQDRLYREIVSVAGTERMVTEDDLPNMPYLGAIIKETLRKYTPVPLIPSRFVEEDITLGGYDIPKGYQILVNLFAIANDPAVWSNPEKWDPERMLANKKVDMGFRDFSLMPFGAGKRMCAGITQAMFIIPMNVAALVQHCEWRLSPQEISNINNKIEDVVYLTTHKLSPLSCEATPRISHRLP
>SrKAH1 gi|189418962|gb|ACD93722.1|ent−カウレン酸13−ヒドロキシラーゼ[Stevia rebaudiana]
(配列番号25)
MIQVLTPILLFLIFFVFWKVYKHQKTKINLPPGSFGWPFLGETLALLRAGWDSEPERFVRERIKKHGSPLVFKTSLFGDRFAVLCGPAGNKFLFCNENKLVASWWPVPVRKLFGKSLLTIRGDEAKWMRKMLLSYLGPDAFATHYAVTMDVVTRRHIDVHWRGKEEVNVFQTVKLYAFELACRLFMNLDDPNHIAKLGSLFNIFLKGIIELPIDVPGTRFYSSKKAAAAIRIELKKLIKARKLELKEGKASSSQDLLSHLLTSPDENGMFLTEEEIVDNILLLLFAGHDTSALSITLLMKTLGEHSDVYDKVLKEQLEISKTKEAWESLKWEDIQKMKYSWSVICEVMRLNPPVIGTYREALVDIDYAGYTIPKGWKLHWSAVSTQRDEANFEDVTRFDPSRFEGAGPTPFTFVPFGGGPRMCLGKEFARLEVLAFLHNIVTNFKWDLLIPDEKIEYDPMATPAKGLPIRLHPHQV
>SrKAH2 特許US20080064063によるent−カウレン酸13−ヒドロキシラーゼ[Stevia rebaudiana]
(配列番号26)
MGLFPLEDSYALVFEGLAITLALYYLLSFIYKTSKKTCTPPKASGEHPITGHLNLLSGSSGLPHLALASLADRCGPIFTIRLGIRRVLVVSNWEIAKEIFTTHDLIVSNRPKYLAAKILGFNYVSFSFAPYGPYWVGIRKIIATKLMSSSRLQKLQFVRVFELENSMKSIRESWKEKKDEEGKVLVEMKKWFWELNMNIVLRTVAGKQYTGTVDDADAKRISELFREWFHYTGRFVVGDAFPFLGWLDLGGYKKTMELVASRLDSMVSKWLDEHRKKQANDDKKEDMDFMDIMISMTEANSPLEGYGTDTIIKTTCMTLIVSGVDTTSIVLTWALSLLLNNRDTLKKAQEELDMCVGKGRQVNESDLVNLIYLEAVLKEALRLYPAAFLGGPRAFLEDCTVAGYRIPKGTCLLINMWKLHRDPNIWSDPCEFKPERFLTPNQKDVDVIGMDFELIPFGAGRRYCPGTRLALQMLHIVLATLLQNFEMSTPNDAPVDMTASVGMTNAKASPLEVLLSPRVKWS
>AtKAH gi|332005993|gb|AED93376.1|シトクロムP450、ファミリー714、サブファミリーA、ポリペプチド2[Arabidopsis thaliana]
(配列番号27)
MESLVVHTVNAIWCIVIVGIFSVGYHVYGRAVVEQWRMRRSLKLQGVKGPPPSIFNGNVSEMQRIQSEAKHCSGDNIISHDYSSSLFPHFDHWRKQYGRIYTYSTGLKQHLYINHPEMVKELSQTNTLNLGRITHITKRLNPILGNGIITSNGPHWAHQRRIIAYEFTHDKIKGMVGLMVESAMPMLNKWEEMVKRGGEMGCDIRVDEDLKDVSADVIAKACFGSSFSKGKAIFSMIRDLLTAITKRSVLFRFNGFTDMVFGSKKHGDVDIDALEMELESSIWETVKEREIECKDTHKKDLMQLILEGAMRSCDGNLWDKSAYRRFVVDNCKSIYFAGHDSTAVSVSWCLMLLALNPSWQVKIRDEILSSCKNGIPDAESIPNLKTVTMVIQETMRLYPPAPIVGREASKDIRLGDLVVPKGVCIWTLIPALHRDPEIWGPDANDFKPERFSEGISKACKYPQSYIPFGLGPRTCVGKNFGMMEVKVLVSLIVSKFSFTLSPTYQHSPSHKLLVEPQHGVVIRVV
>t26−8RPAtKAH
(配列番号28)
MALLLAVFVYGRAVVEQWRMRRSLKLQGVKGPPPSIFNGNVSEMQRIQSEAKHCSGDNIISHDYSSSLFPHFDHWRKQYGRIYTYSTGLKQHLYINHPEMVKELSQTNTLNLGRITHITKRLNPILGNGIITSNGPHWAHQRRIIAYEFTHDKIKGMVGLMVESAMPMLNKWEEMVKRGGEMGCDIRVDEDLKDVSADVIAKACFGSSFSKGKAIFSMIRDLLTAITKRSVLFRFNGFTDMVFGSKKHGDVDIDALEMELESSIWETVKEREIECKDTHKKDLMQLILEGAMRSCDGNLWDKSAYRRFVVDNCKSIYFAGHDSTAVSVSWCLMLLALNPSWQVKIRDEILSSCKNGIPDAESIPNLKTVTMVIQETMRLYPPAPIVGREASKDIRLGDLVVPKGVCIWTLIPALHRDPEIWGPDANDFKPERFSEGISKACKYPQSYIPFGLGPRTCVGKNFGMMEVKVLVSLIVSKFSFTLSPTYQHSPSHKLLVEPQHGVVIRVV
>t14−8RPAtKAH
(配列番号29)
MALLLAVFIVIVGIFSVGYHVYGRAVVEQWRMRRSLKLQGVKGPPPSIFNGNVSEMQRIQSEAKHCSGDNIISHDYSSSLFPHFDHWRKQYGRIYTYSTGLKQHLYINHPEMVKELSQTNTLNLGRITHITKRLNPILGNGIITSNGPHWAHQRRIIAYEFTHDKIKGMVGLMVESAMPMLNKWEEMVKRGGEMGCDIRVDEDLKDVSADVIAKACFGSSFSKGKAIFSMIRDLLTAITKRSVLFRFNGFTDMVFGSKKHGDVDIDALEMELESSIWETVKEREIECKDTHKKDLMQLILEGAMRSCDGNLWDKSAYRRFVVDNCKSIYFAGHDSTAVSVSWCLMLLALNPSWQVKIRDEILSSCKNGIPDAESIPNLKTVTMVIQETMRLYPPAPIVGREASKDIRLGDLVVPKGVCIWTLIPALHRDPEIWGPDANDFKPERFSEGISKACKYPQSYIPFGLGPRTCVGKNFGMMEVKVLVSLIVSKFSFTLSPTYQHSPSHKLLVEPQHGVVIRVV
>SrCPR ABB88839.2|NADPHシトクロムP450レダクターゼ[Stevia rebaudiana]
(配列番号30)
MQSDSVKVSPFDLVSAAMNGKAMEKLNASESEDPTTLPALKMLVENRELLTLFTTSFAVLIGCLVFLMWRRSSSKKLVQDPVPQVIVVKKKEKESEVDDGKKKVSIFYGTQTGTAEGFAKALVEEAKVRYEKTSFKVIDLDDYAADDDEYEEKLKKESLAFFFLATYGDGEPTDNAANFYKWFTEGDDKGEWLKKLQYGVFGLGNRQYEHFNKIAIVVDDKLTEMGAKRLVPVGLGDDDQCIEDDFTAWKELVWPELDQLLRDEDDTSVTTPYTAAVLEYRVVYHDKPADSYAEDQTHTNGHVVHDAQHPSRSNVAFKKELHTSQSDRSCTHLEFDISHTGLSYETGDHVGVYSENLSEVVDEALKLLGLSPDTYFSVHADKEDGTPIGGASLPPPFPPCTLRDALTRYADVLSSPKKVALLALAAHASDPSEADRLKFLASPAGKDEYAQWIVANQRSLLEVMQSFPSAKPPLGVFFAAVAPRLQPRYYSISSSPKMSPNRIHVTCALVYETTPAGRIHRGLCSTWMKNAVPLTESPDCSQASIFVRTSNFRLPVDPKVPVIMIGPGTGLAPFRGFLQERLALKESGTELGSSIFFFGCRNRKVDFIYEDELNNFVETGALSELIVAFSREGTAKEYVQHKMSQKASDIWKLLSEGAYLYVCGDAKGMAKDVHRTLHTIVQEQGSLDSSKAELYVKNLQMSGRYLRDVW
>AtCPR[Arabidopsis thaliana]
(配列番号31)
MTSALYASDLFKQLKSIMGTDSLSDDVVLVIATTSLALVAGFVVLLWKKTTADRSGELKPLMIPKSLMAKDEDDDLDLGSGKTRVSIFFGTQTGTAEGFAKALSEEIKARYEKAAVKVIDLDDYAADDDQYEEKLKKETLAFFCVATYGDGEPTDNAARFSKWFTEENERDIKLQQLAYGVFALGNRQYEHFNKIGIVLDEELCKKGAKRLIEVGLGDDDQSIEDDFNAWKESLWSELDKLLKDEDDKSVATPYTAVIPEYRVVTHDPRFTTQKSMESNVANGNTTIDIHHPCRVDVAVQKELHTHESDRSCIHLEFDISRTGITYETGDHVGVYAENHVEIVEEAGKLLGHSLDLVFSIHADKEDGSPLESAVPPPFPGPCTLGTGLARYADLLNPPRKSALVALAAYATEPSEAEKLKHLTSPDGKDEYSQWIVASQRSLLEVMAAFPSAKPPLGVFFAAIAPRLQPRYYSISSCQDWAPSRVHVTSALVYGPTPTGRIHKGVCSTWMKNAVPAEKSHECSGAPIFIRASNFKLPSNPSTPIVMVGPGTGLAPFRGFLQERMALKEDGEELGSSLLFFGCRNRQMDFIYEDELNNFVDQGVISELIMAFSREGAQKEYVQHKMMEKAAQVWDLIKEEGYLYVCGDAKGMARDVHRTLHTIVQEQEGVSSSEAEAIVKKLQTEGRYLRDVW
Claims (43)
- レバウジオシドM(RebM)を作製する方法であって、
(1)ステビオールのC13−Oグリコシル化を触媒するウリジン二リン酸依存型グリコシルトランスフェラーゼ(UGT)酵素、
(2)ステビオールのC19−Oグリコシル化を触媒するUGT酵素、
(3)ステビオールのC13及びC19における1−2’グリコシル化を触媒するUGT酵素、並びに
(4)ステビオールのC13及びC19における1−3’グリコシル化を触媒するUGT酵素、
からなる4種類の酵素を全てを発現し、ステビオールからRebMを生成する宿主細胞を提供すること、ただし、該UGT酵素は、
(a)配列番号45のアミノ酸配列と少なくとも90%の配列同一性を有するアミノ酸配列を含み、配列番号45のアミノ酸配列を有するUGT酵素と少なくとも同等のレバウジオシドA(RebA)のC19及びステビオールモノシドのC13における1−2’グリコシル化活性を有するUGT酵素、及び
(b)配列番号3に対してL200A及びL200Gから選択される変異を有するアミノ酸配列を含むUGT酵素と少なくとも同等のレバウジオシドD(RebD)のC19及びステビオシドのC13における1−3’グリコシル化活性を有する、配列番号3のアミノ酸配列と少なくとも90%の配列同一性を有し、且つ、配列番号3に対してL200A及びL200Gから選択される変異を有するアミノ酸配列を含むUGT酵素、及び
(c)配列番号1に対してP215T置換を有するアミノ酸配列を含むUGT酵素と少なくとも同等のステビオールのC13−Oグリコシル化活性を有する、配列番号1と少なくとも90%の配列同一性を有し、且つ配列番号1に対してP215T置換を有するアミノ酸配列を含む、UGT酵素
のうち1つ以上を含んでおり、かつ
前記宿主細胞を前記RebMが生成される条件下で培養すること
を含む、
前記方法。 - 1−2’グリコシル化活性を有する前記UGT酵素は、配列番号45と少なくとも95%の配列同一性を有するアミノ酸配列を有する、請求項1に記載の方法。
- 1−3’グリコシル化活性を有する前記UGT酵素は、配列番号3と少なくとも95%の配列同一性を有し、配列番号3に対してL200A変異を有するアミノ酸配列を含む、請求項1に記載の方法。
- C13−Oグリコシル化活性を有する前記UGT酵素は、配列番号1と少なくとも95%の配列同一性を有し、且つ、配列番号1に対してP215T変異を有するアミノ酸配列を含む、請求項1に記載の方法。
- 前記宿主細胞は、カウレン合成酵素(KS)、カウレンオキシダーゼ(KO)、及びカウレン酸ヒドロキシラーゼ(KAH)を含む酵素経路を介してステビオール基質を産生し、かつ、前記KO酵素及びKAH酵素の1つ以上を再生するシトクロムP450レダクターゼ酵素(CPR)をさらに含む、請求項1〜4のいずれか一項に記載の方法。
- 前記宿主細胞はE.coliである、請求項5に記載の方法。
- 前記KAHは、Stevia rebaudiana、Arabidopsis thaliana、Vitis vinifera、もしくはMedicago trunculataのKAH、またはその誘導体である、請求項5に記載の方法。
- 前記KAHは、Arabidopsis thalianaのKAH(AtKAH)、またはその誘導体である、請求項7に記載の方法。
- 前記AtKAHは、カウレン酸ヒドロキシラーゼ活性の速度を高めるアミノ酸置換、アミノ酸挿入、及び/またはアミノ酸欠失を1つ以上有し、かつ/または、E.coliで機能発現するよう工学的に操作されたN末端を有する、請求項8に記載の方法。
- 前記AtKAHは、配列番号29の25位、79位、119位、137位、142位、155位、180位、193位、196位、197位、226位、331位、235位、238位、245位、272位、285位、287位、325位、330位、334位、339位、352位、373位、397位、470位、499位、506位、及び507位、に対応する位置のうち1つ以上の位置にアミノ酸置換を有する、請求項9に記載の方法。
- 前記AtKAHは、A25L、S79T、T119C、I137L、I142V、R155K、M180L、E193G、C196A、D197E、A226E、L235Q、I238M、F245L、F245V、L272I、I285R、C287S、C325I、C325M、F330L、D334E、S339T、S352E、E373D、I397F、V470L、Q499V、L506M、L507I、L507T、及びL507Vから選択される1つ以上のアミノ酸置換を有する、請求項10に記載の方法。
- 前記AtKAHは、C325Iを含む、請求項10又は11に記載の方法。
- 前記KSは、Stevia rebaudiana、Zea mays、Populus trichocarpa、もしくはArabidopsis thaliana、またはその誘導体に由来する、請求項5〜12のいずれか一項に記載の方法。
- 前記細胞は、Stevia rebaudiana、Erwina tracheiphila、Sinorhizobium fredii、Streptomyces clavuligerus、Bradyrhizobium japonicum、Zea mays、Arabidopsis thalianaに由来するコパリル二リン酸合成酵素(CPPS)、またはその誘導体を発現する、請求項5〜13のいずれか一項に記載の方法。
- 前記宿主細胞は、CPPSとKSとの二機能性酵素を発現する、請求項5〜13記載の方法。
- 前記宿主細胞は、Stevia rebaudiana、Arabidopsis thaliana、Physcomitrella patens、Gibberella fujikoroi、もしくはTrametes versicolor、または、その誘導体に由来するカウレン酸化酵素を発現する、請求項5〜15のいずれか一項に記載の方法。
- 前記カウレン酸化酵素は、E.coliに機能発現させるためにN末端で修飾される、請求項16に記載の方法。
- 前記CPRは、Stevia rebaudiana、Arabidopsis thaliana、もしくはGiberella fujikuroiの酵素、または、その誘導体である、請求項5〜17のいずれか一項に記載の方法。
- 前記CPRは、E.coliに機能発現させるためにN末端で修飾される、請求項18に記載の方法。
- 前記宿主細胞は、ゲラニルゲラニルピロリン酸合成酵素(GGPPS)を発現する、請求項1〜19のいずれか一項に記載の方法。
- 前記宿主細胞は、イソペンテニルピロリン酸(IPP)及びジメチルアリルピロリン酸(DMAPP)を生成する経路を発現する、請求項1〜20のいずれか一項に記載の方法。
- 前記経路は、メチルエリスリトールリン酸(MEP)経路及び/またはメバロン酸(MVA)経路である、請求項21に記載の方法。
- 前記宿主細胞は、原核細胞または真核細胞である、請求項21または22に記載の方法。
- 前記宿主細胞は、E.coli、Bacillus subtilis、またはPseudomonas putidaである、請求項23に記載の方法。
- 前記宿主細胞は、Saccharomyces cerevisiae、Pichia pastoris、及びYarrowia lipolyticaを含む、サッカロミセスの種、ピチアの種、またはヤロウィアの種である、請求項23に記載の方法。
- 前記宿主細胞は、dxs、idi、IspD、及びIspFの重複または過剰発現を有するE.coliである、請求項24に記載の方法。
- 前記E.coliは、UDP−グルコースの生成を高める遺伝子修飾を1つ以上有する、請求項24に記載の方法。
- 1つ以上の前記遺伝子修飾に、ΔgalE、ΔgalT、ΔgalK、ΔgalM、ΔushA、Δagp、Δpgm、E coliのGALUの重複、Bacillus subtilisのUGPAの発現、Bifidobacterium adolescentisのSPLの発現が含まれる、請求項27に記載の方法。
- 前記宿主細胞は、異種発現させた遺伝子である、
Taxus canadensisのGGPSまたはその誘導体、
Phaeosphaeria sp.のPsCK(またはその誘導体、
Stevia rebaudianaのKOまたはその誘導体、
Arabidopsis thalianaのKAHまたはその誘導体、
Stevia rebaudianaのCPRまたはその誘導体、
Stevia rebaudianaのUGT85C2またはその誘導体、
Stevia rebaudianaのUGT74G1もしくはその誘導体、またはMbUGTC19もしくはその誘導体、
Stevia rebaudianaのUGT76G1もしくはその誘導体、またはMbUGT1−3もしくはその誘導体、及び
Stevia rebaudianaのUGT91D2、またはOsUGT1−2、またはMbUGT1−2またはその誘導体
を含む、請求項1〜28のいずれか一項に記載の方法。 - 前記宿主細胞は、異種発現させた遺伝子である、
Corynebacterium glutamicumのGGPSまたはその誘導体、Erwina tracheiphilaのCPPSまたはその誘導体、
Erwina tracheiphilaのKSまたはその誘導体、
Stevia rebaudianaのKOまたはその誘導体、
Arabidopsis thalianaのKAHまたはその誘導体、
Stevia rebaudianaのCPRまたはその誘導体、
Stevia rebaudianaのUGT85C2またはその誘導体、
Stevia rebaudianaのUGT74G1もしくはその誘導体、またはMbUGTC19もしくはその誘導体、またはMbUGTC19−2もしくはその誘導体、Stevia rebaudianaのUGT76G1もしくはその誘導体、またはMbUGT1−3もしくはその誘導体、及び
Stevia rebaudianaのUGT91D2、またはOsUGT1−2、またはMbUGT1,2−2またはその誘導体
を含む、請求項1〜28のいずれか一項に記載の方法。 - 前記UGT85C2は、置換P215Tを有する誘導体である、請求項30に記載の方法。
- 前記宿主細胞は、22℃〜37℃で細胞培養されたE.coli宿主細胞である、請求項30又は31に記載の方法。
- 前記宿主細胞は、27℃〜37℃で細胞培養されたE.coli宿主細胞である、請求項32に記載の方法。
- 培地からRebMを回収することをさらに含む、請求項1〜33のいずれか一項に記載の方法。
- RebMを、濃度が少なくとも10mg/Lの培地で生成させる、請求項34に記載の方法。
- RebMとRebDとの比が1:1〜1:0である、請求項35に記載の方法。
- RebDを、RebM収率の20%未満で生成させる、請求項36に記載の方法。
- RebMが、前記ステビオールのグリコシル化産物の少なくとも25重量%を占める、請求項36に記載の方法。
- 請求項1〜38のいずれか一項に記載の方法にしたがってRebMを生成し、RebMを製品に組み込むことを含む、RebMを含む製品を製造する方法。
- 配列番号45と少なくとも95%同一なアミノ酸配列を含み、配列番号45のアミノ酸配列を有するUGT酵素と少なくとも同等のRebAのC19及びステビオールモノシドのC13における1−2’グリコシル化活性を有する、OsUGT1−2の円順列変異体。
- 配列番号1に対してP215T置換を含むアミノ酸配列を有するUGT酵素と少なくとも同等のステビオールC13−Oグリコシル化活性を有する、配列番号1に対して少なくとも95%同一なアミノ酸配列及びP215T置換を有するアミノ酸配列を含む、修飾されたStevia rebaudianaのUGT85C2。
- 請求項40又は41に記載の酵素をコードする、ポリヌクレオチド。
- 請求項42に記載のポリヌクレオチドを発現している、宿主細胞。
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