JP2012529277A - 新規の脂肪酸伸長成分およびその使用 - Google Patents
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Abstract
Description
a) 配列番号1または3に示すヌクレオチド配列を有する核酸配列、
b) 配列番号2または4に示すアミノ酸配列を有するポリペプチドをコードする核酸配列、
c) a)またはb)の核酸配列と少なくとも50%が同一である核酸配列であって、デヒドラターゼ/エノイル−CoAレダクターゼ(nECR)活性を有するポリペプチドをコードする核酸配列、
d) nECR活性を有し、かつa)〜c)のいずれか1つのアミノ酸配列と少なくとも50%が同一であるアミノ酸配列を有するポリペプチドをコードする核酸配列、および
e) a)〜d)のいずれか1つにストリンジェントな条件下でハイブリダイズすることができる核酸配列であって、nECR活性を有するポリペプチドをコードする核酸配列、
からなる群から選択された核酸配列を含むポリヌクレオチドに関する。
a)前記ポリペプチドの産生を可能にする条件下で、本発明の宿主細胞を培養するステップと、
b)ステップa)の宿主細胞からポリペプチドを得るステップと、
を含む方法にも関連する。
またはRicinus communis[トウゴマ](属・種)、Fabaceae、例えばPisum属、Albizia属、Cathormion属、Feuillea属、Inga属、Pithecolobium属、Acacia属、Mimosa属、Medicajo属、Glycine属、Dolichos属、Phaseolus属、Soja属、例えばPisum sativum、Pisum arvense、Pisum humile[エンドウマメ]、Albizia berteriana、Albizia julibrissin、Albizia lebbeck、Acacia berteriana、Acacia littoralis、Aibizia berteriana、Albizzia berteriana、Cathormion berteriana、Feuillea berteriana、Inga fragrans、Pithecellobium berterianum、Pithecellobium fragrans、Pithecolobium berterianum、Pseudalbizzia berteriana、Acacia julibrissin、Acacia nemu、Albizia nemu、Feuilleea julibrissin、Mimosa julibrissin、Mimosa speciosa、Sericanrda julibrissin、Acacia lebbeck、Acacia macrophylla、Albizia lebbek、Feuilleea lebbeck、Mimosa lebbeck、Mimosa speciosa[ネムノキ]、Medicago sativa、Medicago falcata、Medicago varia[ムラサキウマゴヤシ]、Glycine max Dolichos soja、Glycine gracilis、Glycine hispida、Phaseolus max、Soja hispida、またはSoja max[大豆](属・種)、Funariaceae、例えばAphanorrhegma属、Entosthodon属、Funaria属、Physcomitrella属、Physcomitrium属、例えばAphanorrhegma serratum、Entosthodon attenuatus、Entosthodon bolanderi、Entosthodon bonplandii、Entosthodon californicus、Entosthodon drummondii、Entosthodon jamesonii、Entosthodon leibergii、Entosthodon neoscoticus、Entosthodon rubrisetus、Entosthodon spathulifolius、Entosthodon tucsoni、Funaria americana、Funaria bolanderi、Funaria calcarea、Funaria californica、Funaria calvescens、Funaria convoluta、Funaria flavicans、Funaria groutiana、Funaria hygrometrica、Funaria hygrometrica var. arctica、Funaria hygrometrica var. calvescens、Funaria hygrometrica var. convoluta、Funaria hygrometrica var. muralis、Funaria hygrometrica var. utahensis、Funaria microstoma、Funaria microstoma var. obtusifolia、Funaria muhlenbergii、Funaria orcuttii、Funaria plano−convexa、Funaria polaris、Funaria ravenelii、Funaria rubriseta、Funaria serrata、Funaria sonorae、Funaria sublimbatus、Funaria tucsoni、Physcomitrella californica、Physcomitrella patens、Physcomitrella readeri、Physcomitrium australe、Physcomitrium californicum、Physcomitrium collenchymatum、Physcomitrium coloradense、Physcomitrium cupuliferum、Physcomitrium drummondii、Physcomitrium eurystomum、Physcomitrium flexifolium、Physcomitrium hookeri、Physcomitrium hookeri var. serratum、Physcomitrium immersum、Physcomitrium kellermanii、Physcomitrium megalocarpum、Physcomitrium pyriforme、Physcomitrium pyriforme var. serratum、Physcomitrium rufipes、Physcomitrium sandbergii、Physcomitrium subsphaericum、Physcomitrium washingtoniense(属・種)、Geraniaceae、例えばPelargonium属、Cocos属、Oleum属、例えばCocos nucifera、Pelargonium grossularioides、またはOleum cocois[ココヤシ](属・種)、Gramineae、例えばSaccharum属、例えばSaccharum officinarum(属・種)、Juglandaceae、例えばJuglans属、Wallia属、例えばJuglans regia、Juglans ailanthifolia、Juglans sieboldiana、Juglans cinerea、Wallia cinerea、Juglans bixbyi、Juglans californica、Juglans hindsii、Juglans intermedia、Juglans jamaicensis、Juglans major、Juglans microcarpa、Juglans nigra、またはWallia nigra[クルミ](属・種)、Lauraceae、例えばPersea属、Laurus属、例えばLaurus nobilis[月桂樹]、Persea americana、Persea gratissima、またはPersea persea[アボカド](属・種)、Leguminosae、例えばArachis属、例えばArachis hypogaea[ピーナッツ](属・種)、Linaceae、例えばLinum属、Adenolinum属、例えばLinum usitatissimum、Linum humile、Linum austriacum、Linum bienne、Linum angustifolium、Linum catharticum、Linum flavum、Linum grandiflorum、Adenolinum grandiflorum、Linum lewisii、Linum narbonense、Linum perenne、Linum perenne var. lewisii、Linum pratense、またはLinum trigynum[亜麻仁](属・種)、Lythrarieae、例えばPunica属、例えばPunica granatum[ザクロ](属・種)、Malvaceae、例えばGossypium属、例えばGossypium hirsutum、Gossypium arboreum、Gossypium barbadense、Gossypium herbaceum、またはGossypium thurberi[綿](属・種)、Marchantiaceae、例えばMarchantia属、例えばMarchantia berteroana、Marchantia foliacea、Marchantia macropora(属・種)、Musaceae、例えばMusa属、例えばMusa nana、Musa acuminata、Musa paradisiaca、Musa spp.[バナナ](属・種)、Onagraceae、例えばCamissonia属、Oenothera属、例えば Oenothera biennis、またはCamissonia brevipes[マツヨイグサ](属・種)、Palmae、例えばElacis属、例えばElaeis guineensis[アブラヤシ](属・種)、Papaveraceae、例えばPapaver属、例えばPapaver orientale、Papaver rhoeas、Papaver dublum[ケシ](属・種)、Pedaliaceae、例えばSesamum属、例えばSesamum indicum[ゴマ](属・種)、Piperaceae、例えばPiper属、Artanthe属、Peperomia属、Steffensia属、例えばPiper aduncum、Piper amalago、Piper angustifolium、Piper auritum、Piper betel、Piper cubeba、Piper longum、Piper nigrum、Piper retrofractum、Artanthe adunca、Artanthe elongata、Peperomia elongata、Piper elongatum、Steffensia elongata[トウガラシ](属・種)、Poaceae、例えばHordeum属、Secale属、Avena属、Sorghum属、Andropogon属、Holcus属、Panicum属、Oryza属、Zea(トウモロコシ)属、
Triticum属、例えばHordeum vulgare、Hordeum jubatum、Hordeum murinum、Hordeum secalinum、Hordeum distichon、Hordeum aegiceras、Hordeum hexastichon、Hordeum hexastichum、Hordeum irregulare、Hordeum sativum、Hordeum secalinum[オオムギ]、Secale cereale[ライムギ]、Avena sativa、Avena fatua、Avena byzantina、Avena fatua var. sativa、Avena hybrida[カラスムギ]、Sorghum bicolor、Sorghum halepense、Sorghum saccharatum、Sorghum vulgare、Andropogon drummondii、Holcus bicolor、Holcus sorghum、Sorghum aethiopicum、Sorghum arundinaceum、Sorghum caffrorum、Sorghum cernuum、Sorghum dochna、Sorghum drummondii、Sorghum durra、Sorghum guineense、Sorghum lanceolatum、Sorghum nervosum、Sorghum saccharatum、Sorghum subglabrescens、Sorghum verticilliflorum、Sorghum vulgare、Holcus halepensis、Sorghum miliaceum、Panicum militaceum[キビ]、Oryza sativa、Oryza latifolia[イネ]、Zea mays[トウモロコシ]、Triticum aestivum、Triticum durum、Triticum turgidum、Triticum hybernum、Triticum macha、Triticum sativum、またはTriticum vulgare[コムギ](属・種)、Porphyridiaceae、例えばChroothece属、Flintiella属、Petrovanella属、Porphyridium属、Rhodella属、Rhodosorus属、Vanhoeffenia属、例えばPorphyridium cruentum(属・種)、Proteaceae、例えばMacadamia属、例えばMacadamia intergrifolia[マカダミア](属・種)、Prasinophyceae、例えばNephroselmis属、Prasinococcus属、Scherffelia属、Tetraselmis属、Mantoniella属、Ostreococcus属、例えばNephroselmis olivacea、Prasinococcus capsulatus、Scherffelia dubia、Tetraselmis chui、Tetraselmis suecica、Mantoniella squamata、Ostreococcus tauri(属・種)、Rubiaceae、例えばCofea属、例えばCofea spp.、Coffea arabica、Coffea canephora、またはCoffea liberica[コーヒー](属・種)、Scrophulariaceae、例えばVerbascum属、例えばVerbascum blattaria、Verbascum chaixii、Verbascum densiflorum、Verbascum lagurus、Verbascum longifolium、Verbascum lychnitis、Verbascum nigrum、Verbascum olympicum、Verbascum phlomoides、Verbascum phoenicum、Verbascum pulverulentum、またはVerbascum thapsus[モウズイカ](属・種)、Solanaceae、例えばCapsicum属、Nicotiana属、Solanum属、Lycopersicon属、例えばCapsicum annuum、Capsicum annuum var. glabriusculum、Capsicum frutescens[コショウ]、Capsicum annuum[パプリカ]、Nicotiana tabacum、Nicotiana alata、Nicotiana attenuata、Nicotiana glauca、Nicotiana langsdorffii、Nicotiana obtusifolia、Nicotiana quadrivalvis、Nicotiana repanda、Nicotiana rustica、Nicotiana sylvestris[タバコ]、Solanum tuberosum[ジャガイモ]、Solanum melongena[ナス]、Lycopersicon esculentum、Lycopersicon lycopersicum、Lycopersicon pyriforme、Solanum integrifoiium、またはSolanum lycopersicum[トマト](属・種)、Sterculiaceae、例えばTheobroma属、例えばTheobroma cacao [カカオ]、あるいはTheaceae、例えばCamellia属、例えばCamellia sinensis[チャ](属・種)。とりわけ、本発明に従ってトランスジェニック植物として使用すべき好適な植物は、多量の脂質化合物を含む油料果実作物、例えばピーナッツ、アブラナ、カノーラ、ヒマワリ、ベニバナ、ケシ、カラシ、麻、トウゴマ、オリーブ、ゴマ、キンセンカ、ザクロ、マツヨイグサ、モウズイカ、アザミ、野バラ、ヘーゼルナッツ、アーモンド、マカダミア、アボカド、月桂樹、カボチャ(pumpkin/squash)、亜麻仁、大豆、ピスタチオ、ルリヂサ、木(アブラヤシ、ココヤシ、クルミ)、または作物、例えばトウモロコシ、コムギ、ライムギ、カラスムギ、ライコムギ、イネ、オオムギ、綿、キャッサバ、コショウ、マンジュギク、ナス科植物、例えばジャガイモ、タバコ、ナス、およびトマト、Vicia属の種、エンドウマメ、ムラサキウマゴヤシ、または低木植物(コーヒー、カカオ、チャ)、Salix属の種、ならびに多年生草および飼料作物である。本発明による好適な植物は、油料作物植物、例えばピーナッツ、アブラナ、カノーラ、ヒマワリ、ベニバナ、ケシ、カラシ、麻、トウゴマ、オリーブ、キンセンカ、ザクロ、マツヨイグサ、カボチャ(pumpkin/squash)、亜麻仁、大豆、ルリヂサ、木(アブラヤシ、ココヤシ)である。特に好適なものは、ヒマワリ、ベニバナ、タバコ、モウズイカ、ゴマ、綿、カボチャ(pumpkin/squash)、ケシ、マツヨイグサ、クルミ、亜麻仁、麻、アザミ、またはベニバナである。極めて好適な植物は、ベニバナ、ヒマワリ、ケシ、マツヨイグサ、クルミ、亜麻仁、または麻などの植物である。
a)本発明の宿主細胞を、前記宿主細胞における多価不飽和脂肪酸の産生を可能にする条件下で培養するステップと、
b)前記宿主細胞から前記多価不飽和脂肪酸を得るステップと、
を含む方法を包含する。
a)本発明の非ヒトトランスジェニック生物を、前記宿主細胞における多価不飽和脂肪酸の産生を可能にする条件下で培養するステップと、
b)前記非ヒトトランスジェニック生物から前記多価不飽和脂肪酸を得るステップと、
を含む方法に関する。
実施例1:生物および培養条件
半数体を再生させるために、Pan et al 2004(Molecular Cell 16:487−496)に概説されている方法を使用した。簡潔に言うと、培養物をDOB−ウラシル中で一晩成長させ、その後OD500がおよそ25の各培養物を洗浄し、新鮮培地に再懸濁させ、3時間成長させた。次いで、細胞を胞子形成培地(1%酢酸カリウム、0.005%酢酸亜鉛)に懸濁させ、5日間胞子形成し、200mg/LのG418および60mg/Lのカナバニンを含有する半数体選択MagicMediaプレート(2%ガラクトース、アミノ酸混合物−ウラシル−ロイシン−ヒスチジン−アルギニン、アミノ酸または硫酸アンモニウムを含まない0.17%窒素塩基、0.1%グルタミン酸ナトリウム)に塗布した。
PUFA産生藻類をスクリーニングして、非PUFA産生株との差を明らかにした。これらのPUFA産生株に対して特異的な、ThalassiosiraおよびPhaeodactylumに由来する配列を得た(図1および2)。酵母(YJL097WおよびYDLO15C)由来の既知のデヒドラターゼ配列およびエノイル−CoAレダクターゼ配列とのアラインメントは、低い相同性を示した(図3および4)。したがって、この新しく生成されたnECRは、新規の酵素群に相当する。相補性アッセイ(実施例3)によって、これらの配列は、デヒドラターゼおよびエノイル−CoA−レダクターゼ活性を示し、したがってnECRと命名した。
S. C. EasyComp Transformation Kit(Invitrogen社、米国カリフォルニア州カールズバッド)を用いて、S. cerevisiae株YSC1021−674054(Open Biosystems社)を構築物pESC−nECR(Tp)、pESC−nECR(Pt)、およびpESCで形質転換し、ロイシン欠乏培地で選択した。デヒドラターゼ/エノイル−CoAレダクターゼ活性を評価するために、相補性試験を実施した。その目的で、ヘテロ接合体Magic Marker株YSC1021−674054(Open Biosystems社)を使用した。この株は、エノイル−CoAレダクターゼ活性を示さない。エノイル−CoAレダクターゼ活性は伸長脂肪酸を生じ、これらの脂肪酸は細胞の成長および分裂に必要とされるため、これらそれぞれの酵母菌株は、前記伸長脂肪酸を含有しない培地では成長しないことになる。
1.tsc13ΔTrp pTSC13 URA:機能性YDL015C遺伝子を含有するベクターpTSC13で形質転換した、エノイル−CoAレダクターゼの機能を有さない酵母突然変異体Δydt015C。
2.phsiΔKan pPHS1 URA:機能性YJL097W遺伝子を含有するベクターpPHS1で形質転換した、デヒドラターゼの機能を有さない酵母突然変異体Δyjl097w。
3.tsc13ΔTrp pESC−nECR(Tp):ベクターpESC−nECR(Tp)で形質転換した、エノイル−CoAレダクターゼの機能を有さない酵母突然変異体Δydl015C。
4.sc13ΔTrp pESC−nECR(Pt):ベクターpESC−nECR(Pt)で形質転換した、エノイル−CoAレダクターゼの機能を有さない酵母突然変異体Δydl015C。
植物における多価不飽和脂肪酸の産生において、例えば栄養補給食品用途でのnECR(Tp)の有用性を検討するために、PUFA−エロンガーゼ成分である、Isochrysis galbana由来のd9−エロンガーゼと組み合わせて遺伝子を発現させた(国際出願第2002/077213号)。この酵素は、リノール酸またはリノレン酸(18:2Δ9,12または18:3Δ9,12,15)の伸長を触媒する。実験の目的は、nECR(Tp)の付加が、I. gatbana由来のd9−エロンガーゼの生産性を向上させるかどうかを分析することであった。その目的で、pESC−nECR(Tp)で形質転換し、DOB(−ロイシン)プレートで成長させた酵母細胞(INVSC、Invitrogen社)を実施例1に記載のように、プラスミドpYES(Ura)−d9Elo(Ig)でさらに形質転換し、DOB(−ウラシル、−ロイシン)で成長させた。ベクターpYES(Ura)はInvitrogene社から取得したものであり、ウラシルの栄養要求性を媒介する。国際出願第2002/077213に記載されるd9Elo(Ig)のオープンリーディングフレームを、製品の条件に従ってpYES2にクローニングした。対照実験として、pYESベクターのみを含有する対照酵母菌株に、pYES−d9Elo(Ig)を形質転換した。
nECR(Tp)に対する抗体を製品の操作に従って産生しておいた(Eurogentec社、ベルギー;ペプチド抗体)。これらの抗体は、T. pseudonanaおよび酵母での異種発現におけるnECR(Tp)に対して極めて特異的である(図7および図8)。抗体を使用することにより、天然生物と異種発現との構造的な差を観察することができた(図7および図8)。
Biorad社製プレキャストゲルを用いて、Laemmli(1970)に従って、SDS−PAGEを実施した。ローディングバッファーとして、0.05M トリス/HCl(pH6.8)、0.1M DTT、2%(w/v) SDS、0.1% ブロモフェノールブルー、および10% グリセロールを使用した。次いで、Protean BA85 ニトロセルロース膜(Schleicher&Schuell社)を用いて、SDS−PAGEゲルをニトロセルロースにブロットした。膜への転写は、15mM Na2HPO4(pH7.2)、0.05%(w/v) SDS、20%(v/v) メタノールを含有する緩衝液を用いて、200mA、40Vで2時間実施した(Protean II、Biorad社)。免疫学的検査の目的で、5%(w/v)粉乳を含むPBS(0.14M NaCl、2.7mM KCl、10mM Na2HPO4、1.8mM KH2PO4(pH7.4))中で、この膜を1時間ブロッキングした。nECR(Tp)に対する抗体を含有する血清を濃度1:2000で添加し、4℃で一晩インキュベートした。抗体を検出するために、この膜をPBSで3回洗浄し、5%(w/v)粉乳を含むPBSで、30分間再びブロッキングした。1単位の二次抗体(Biorad社の抗ウサギセイヨウワサビペルオキシダーゼ)を添加し、さらに30分間インキュベートした。PBSで3回洗浄したあと、この膜をECL溶液に浸漬し、1分間インキュベートした。次いで、この溶液をティッシュペーパーで拭き取り、膜をサランラップで包んだ。BioIlluminator(LKB社)で化学発光の検出を行なった。
T. pseudonanaおよびP. tricornutumに由来する新規のnECRを、国際出願第2003/093482号、国際出願第2005/083093号、または国際出願第2007/093776号に記載されているように、植物形質転換ベクターにクローニングする。例となる遺伝子の適切な組み合わせは、第3表、第4表、および第5表に記載する。
Claims (15)
- a)配列番号1または3に示すヌクレオチド配列を有する核酸配列、
b)配列番号2または4に示すアミノ酸配列を有するポリペプチドをコードする核酸配列、
c)a)またはb)の核酸配列と少なくとも50%が同一である核酸配列であって、nECR活性を有するポリペプチドをコードする前記核酸配列、
d)nECR活性を有し、かつa)〜c)のいずれか1つのアミノ酸配列と少なくとも50%が同一であるアミノ酸配列を有するポリペプチドをコードする核酸配列、および
e)a)〜d)のいずれか1つにストリンジェントな条件下でハイブリダイズすることができる核酸配列であって、nECR活性を有するポリペプチドをコードする前記核酸配列、
からなる群から選択された核酸配列を含むポリヌクレオチド。 - 前記ポリヌクレオチドが、前記核酸配列に作用可能に連結された発現制御配列をさらに含む、請求項1に記載のポリヌクレオチド。
- 前記ポリヌクレオチドが、前記核酸配列に作用可能に連結されたターミネーター配列をさらに含む、請求項1または2に記載のポリヌクレオチド。
- 請求項1から3のいずれか1つに記載のポリヌクレオチドを含む、ベクター。
- 請求項1から3のいずれか1つに記載のポリヌクレオチドまたは請求項4に記載のベクターを含む、宿主細胞。
- 請求項1から3のいずれか1つに記載のポリヌクレオチドによってコードされたポリペプチドの製造のための方法であって、
a)前記ポリペプチドの産生を可能にする条件下で、請求項5に記載の宿主細胞を培養するステップと、
b)ステップa)の宿主細胞からポリペプチドを得るステップと、
を含む方法。 - 請求項1から3のいずれか1つに記載のポリヌクレオチドによってコードされたポリペプチド、あるいは請求項6に記載の方法によって得られるポリペプチド。
- 請求項1から3のいずれか1つに記載のポリヌクレオチドまたは請求項4に記載のベクターを含む、非ヒトトランスジェニック生物。
- 植物、植物部位、または植物種子である、請求項8に記載の非ヒトトランスジェニック生物。
- 多価不飽和脂肪酸を製造するための方法であって、
a)請求項5に記載の宿主細胞を、前記宿主細胞における多価不飽和脂肪酸の産生を可能にする条件下で培養するステップと、
b)前記宿主細胞から前記多価不飽和脂肪酸を得るステップと、
を含む方法。 - 多価不飽和脂肪酸を製造するための方法であって、
a)請求項8または9に記載の非ヒトトランスジェニック生物を、前記宿主細胞における多価不飽和脂肪酸の産生を可能にする条件下で培養するステップと、
b)前記非ヒトトランスジェニック生物から、前記多価不飽和脂肪酸を得るステップと、
を含む方法。 - 前記多価不飽和脂肪酸が、アラキドン酸(ARA)、エイコサペンタエン酸(EPA)、またはドコサヘキサエン酸(DHA)である、請求項10または11に記載の方法。
- 請求項10から12のいずれか1つに記載の方法のステップと、前記多価不飽和脂肪酸を油、脂質、または脂肪酸組成物として配合するさらなるステップを含む、油、脂質、または脂肪酸組成物を製造するための方法。
- 前記油、脂質、または脂肪酸組成物が、飼料、食品、化粧品、または薬剤に使用される、請求項13に記載の方法。
- 請求項10から12のいずれか1つに記載の方法によって得られる多価不飽和脂肪酸を含む油。
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CL2011003104A1 (es) | 2012-06-01 |
CA2763107A1 (en) | 2010-12-16 |
DE112010002353T5 (de) | 2012-08-09 |
EP2440662B1 (en) | 2018-08-01 |
WO2010142522A3 (en) | 2011-04-14 |
US8993841B2 (en) | 2015-03-31 |
AU2010257729B2 (en) | 2016-03-24 |
EP2440662A2 (en) | 2012-04-18 |
CN102597242A (zh) | 2012-07-18 |
WO2010142522A2 (en) | 2010-12-16 |
BRPI1010614A2 (pt) | 2016-10-25 |
AU2010257729A1 (en) | 2012-01-12 |
US20120084889A1 (en) | 2012-04-05 |
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