CN104520313A - 新的酶、酶组合物及其用途 - Google Patents
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- CN104520313A CN104520313A CN201380040953.4A CN201380040953A CN104520313A CN 104520313 A CN104520313 A CN 104520313A CN 201380040953 A CN201380040953 A CN 201380040953A CN 104520313 A CN104520313 A CN 104520313A
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- desaturase
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Abstract
本发明提供了编码来自金黄色破囊壶菌和Sphaeroforma arctica的新的脂肪酸去饱和酶、KCS、KCR和/或LACS的核酸分子。本发明还提供了含有所述核酸分子的重组表达载体、已经向其中引入表达载体的宿主细胞,和用于大规模产生长链多不饱和脂肪酸(LCPUFA),例如ARA、EPA和DHA和用于筛选δ-4去饱和酶的方法。
Description
本申请要求保护申请号为US 61/679100和EP 12179241.0的优先权,其全部并入作为参考。
本发明原则上涉及重组制造脂肪酸的领域。其提供编码溶血磷脂-辅酶A合酶(LACS)、去饱和酶、延伸酶和延伸酶组分的核酸分子。本发明还提供含有去饱和酶、KCS、KCR和/或LACS核酸分子的重组表达载体、已经向其中引入了表达载体的宿主细胞,和用于大规模产生长链多不饱和脂肪酸(LCPUFA),例如花生四烯酸(ARA,ω-6不饱和脂肪酸)、二十碳五烯酸(EPA,ω-3不饱和脂肪酸)和/或二十二碳五烯酸(DHA,ω-3不饱和脂肪酸)的方法。
脂肪酸是具有长链烃侧基的羧酸,其在许多生物过程中起主要作用。很少发现脂肪酸在自然中是游离的,而是作为脂类的主要成分以酯化形式存在。因此,脂类/脂肪酸是能量的来源(例如,b-氧化)。此外,脂类/脂肪酸是细胞膜的组成部分,并因而是处理生物学或生物化学信息所不可缺少的。
脂肪酸可以分为两类:单个碳键形成的饱和脂肪酸和含有一个或多个顺式构型的碳双键的不饱和脂肪酸。不饱和脂肪酸由属于非血红素-铁酶种类的末端去饱和酶产生。每种这样的酶都是电子转运系统的一部分,所述电子转运系统含有两种其他蛋白,即细胞色素b5和NADH-细胞色素b5还原酶。尤其是,此类酶例如通过催化脂肪酸的氧依赖性脱氢来催化脂肪酸分子的碳原子之间双键的形成(Sperling等,2003)。人和其他哺乳动物具有有限范围的为形成不饱和脂肪酸中特定双键所需要的去饱和酶并且因此具有合成必需脂肪酸,例如,长链多不饱和脂肪酸(LCPUFA)的有限能力。因此,人们不得不通过其膳食摄取某些脂肪酸。此类必需脂肪酸包括例如亚油酸(C18:2)和亚麻酸(C18:3)。相反,昆虫、微生物和植物能够合成种类多得多的不饱和脂肪酸及它们的衍生物。的确,脂肪酸的生物合成是植物和微生物的一项主要活动。
长链多不饱和脂肪酸(LCPUFA),如二十二碳六烯酸(DHA,22:6(4,7,10,13,16,19))是哺乳动物中多种组织和器官(神经、视网膜、脑和免疫细胞)的细胞膜的必需组分。例如,脑磷脂中超过30%的脂肪酸是22:6(n-3)和20:4(n-6)(Crawford,M.A.,等,(1997)Am.J.Clin.Nutr.66:1032S-1041S)。在视网膜中,DHA占据视杆外区(光受体细胞的光敏部分)多于60%的总脂肪酸(Giusto,N.M.,等(2000)Prog.Lipid Res.39:315-391)。临床研究已经显示,DHA对婴儿中脑的生长和发育和成人中正常脑功能的维持是至关重要的(Martinetz,M.(1992)J.Pediatr.120:S129-S138)。DHA还对参与信号转导过程的光受体功能、视紫红质激活和视杆与视锥发育具有显著的影响(Giusto,N.M.,等(2000)Prog.LipidRes.39:315-391)。此外,还发现DHA对疾病如高血压、关节炎、动脉粥样硬化、抑郁、血栓形成和癌症的一些积极影响(Horrocks,L.A.和Yeo,Y.K.(1999)Pharmacol.Res.40:211-215)。因而,脂肪酸的适当膳食供给对人健康是重要的。因为此类脂肪酸不能通过婴儿、幼儿和老年人有效合成,所以从膳食中充分摄取这些脂肪酸对于这些个体是特别重要的(Spector,A.A.(1999)Lipids 34:S1-S3)。
目前,DHA的主要来源是来自鱼类和藻类的油。鱼油是这种脂肪酸的主要和传统来源,然而,它到销售时一般已被氧化。此外,鱼油的供给是高度不定的,特别鉴于鱼群正在缩减。此外,油的藻类来源因低产量和高提取成本而昂贵。
LCPUFA的生物合成和LCPUFA掺入到膜脂或甘油三酯通过多条代谢途径进行(Abbadi 2001,European Journal of Lipid Science&Technology 103:106-113)。在细菌如弧菌(Vibrio),和微藻类,如裂殖壶菌(Schizochytrium)中,丙二酰-CoA通过LCPUFA产生聚酮合酶转化成LCPUFA(Metz 2001,Science 293:290-293;WO 00/42195;WO98/27203;WO 98/55625)。在微藻类,如褐指藻(Phaeodactylum),和苔藓,如剑叶藓(Physcomitrella)中,不饱和脂肪酸如亚油酸或亚麻酸在多个去饱和以及延伸步骤中转化以产生LCPUFA(Zank 2000,BiochemicalSociety Transactions 28:654-658)。去饱和发生在结合辅酶A(酰基-CoA)的酰基基团或膜脂的酰基基团上,而延伸在生物化学上受限于结合CoA的酰基链。在哺乳动物中,除去饱和以及延伸步骤外,DHA的生物合成包括通过β-氧化的链缩短。在微生物和低等植物中,LCPUFA仅仅以膜脂的形式存在(如在剑叶藓和褐指藻的情况下),或以膜脂和甘油三酯的形式存在(如在裂殖壶菌和被孢霉(Mortierella)的情况下)。LCPUFA向脂类和油中的掺入,以及脂肪酸部分(酰基基团)在脂类和其他分子种类如酰基-CoA之间的转移由多种转移酶,如酰基转移酶和转酰基酶催化。已知这些酶进行饱和与不饱和脂肪酸的掺入或交换(Slabas 2001,J.PlantPhysiology 158:505-513,Frentzen 1998,Fett/Lipid 100:161-166,Cases1998,Proc.Nat.Acad.Sci.USA 95:13018-13023,Lu等2009,Proc.Nat.Acad.Sci.USA第106卷:44号:18837-18842)。具有三种不同酶活性的一组酰基转移酶是“Kennedy途径”的酶,其定位在内质网(ER)的膜系统的胞质面。微粒体部分中的ER结合的酰基转移酶使用酰基-CoA作为脂肪酸的活性形式。甘油-3-磷酸酰基转移酶(GPAT)催化在甘油-3-磷酸的sn-1位置上掺入酰基基团。1-酰基甘油-3-磷酸酰基转移酶(也称为溶血磷脂酸酰基转移酶(LPAAT))催化在溶血磷脂酸(LPA)的sn-2位置上掺入酰基基团。通过磷脂酸磷酸酶(PAP)对磷脂酸去磷酸化后,二酰基甘油酰基转移酶(DGAT)催化在二酰基甘油的sn-3位置上掺入酰基基团。除了所述Kennedy途径的酶,直接参与TAG生物合成的其他酶是磷脂二酰甘油酰基转移酶(PDAT),其是将酰基基团从膜脂的sn-2位置上转移到二酰甘油的sn-3位置上的酶;二酰甘油二酰甘油转移酶(DDAT),其是将酰基基团从一个二酰甘油-分子的sn-2位置上转移到另一个二酰甘油-分子的sn-3位置上的酶,和磷脂酰胆碱:二酰基甘油胆碱磷酸转移酶(PDCT),其是将极性phosphatodyclcholine头部基团从多不饱和磷脂(例如含有18:2n-6或18:3n-3)的sn-3位置上转移到饱和(例如含有18:0)或单不饱和(例如,含有18:1n-9)二酰基甘油溶血磷脂酰基转移酶(LPLAT)的sn-3位置上的酶,代表能够将活化的酰基基团从酰基-CoA掺入到膜脂中,并还可能催化反向反应的一类酰基转移酶。更尤其是,LPLATs可以具有如溶血磷脂酰乙醇胺酰基转移酶(LPEAT)和溶血磷脂酰胆碱酰基转移酶(LPCAT)的活性。其他酶,如卵磷脂胆固醇酰基转移酶(LCAT)也可以参与酰基基团从膜脂转移到甘油三酯。一般地,细胞中的脂肪酸结合为硫酯。这些硫酯从游离脂肪酸的形成通过溶血磷脂辅酶A合酶(LACS)的作用发生。
EPA和ARA二者均是δ(d)5必需脂肪酸。它们形成人和动物的独特种类的食物和饲料成分。EPA属于酰基链中具有5个双键的n-3系列。EPA发现于海产品中并且在来自北大西洋的多油鱼类中丰富。ARA属于具有4个双键的n-6系列。与EPA中存在的那些性能相比,在ω-3位置上缺少双键赋予ARA不同的性能。从AA产生的类花生酸类具有强大的炎症和血小板聚集性能,而来自EPA的那些类花生酸类具有抗炎和抗血小板聚集性能。ARA可以从一些食物如肉、鱼和蛋类获得,但是浓度低。
γ-亚麻酸(GLA)是发现于哺乳动物中的另一种必需脂肪酸。GLA是极长链n-6脂肪酸和多种活性分子的代谢中间体。在哺乳动物中,长链多不饱和脂肪酸的形成受Δ6去饱和作用限速。已经显示许多生理学和病理学状况如老化、应激、糖尿病、湿疹和一些感染抑制Δ6去饱和步骤。此外,GLA因氧化和与某些病症(例如,癌症或炎症)相关的快速细胞分裂而易于分解代谢。因而,膳食补充GLA可以降低这些病症的风险。临床研究已经显示,膳食补充GLA在治疗一些病理学状况如特应性湿疹、经前期综合征、糖尿病、高胆固醇血症、炎性疾病和心血管疾病中有效。
虽然生物技术为产生特定的脂肪酸提供了有吸引力的途径,但是现有技术不能提供用于大规模产生不饱和脂肪酸的有效手段。因此,需要产生不饱和脂肪酸,如DHA、EPA和ARA的改进和有效方法。
由于DHA是特别重要的多不饱和脂肪酸,高度需要这种脂肪酸的有效产生。在DHA产生中特别重要的步骤是δ-4去饱和步骤。该步骤通过δ-4去饱和酶进行。这些酶利用结合ACP、CoA或在磷脂中的二十二碳五烯酸(DPA,22:5δ7,10,13,16,19)作为底物,产生结合ACP、CoA或在磷脂中的各DHA。DPA又由δ-5延伸酶产生。一般地,这些延伸酶延长酰基-CoA脂肪酸。已经观察到,如果使用与延伸过程中使用的那些结合相同骨架的脂肪酸作为底物的去饱和酶,那么可以极大地增加去饱和效率。尤其是,已经显示通过提供酰基-coA去饱和酶增加去饱和效率(Domergue等,Biochem.J.2005,483-490)。
因此需要当与δ-5延伸酶配对时具有高去饱和效率的δ-4去饱和酶。迄今为止,已知此类去饱和酶仅来自Pavlova/Rebecca物种(Uniprot标识Q6VPV2_PAVLU和A0PJ29_9EUKA,推测也是D6NST0_9EUKA)。这使得其难以筛选当与δ-5延伸酶配对时具有高去饱和效率的其他去饱和酶,由于Pavlova/Rebecca的δ-4去饱和酶的相似性,不可能确定这些去饱和酶中保守的氨基酸是否为其功能所需,或它们被保留是否仅因为没有经历足够的时间来产生其他突变。
本发明人现在提供了当与异源δ-5延伸酶配对时具有高去饱和效率的δ-4去饱和酶。该δ-4去饱和酶与上文提及的Pavlova/Rebecca去饱和酶具有非常低的序列同一性。本发明因此也提供了δ-4去饱和酶的可允许突变列表,从而仅很少这些突变会消除δ-4去饱和酶本身活性或当与δ-5延伸酶配对时的高去饱和效率。因此,本发明提供用于筛选当与异源δ-5延伸酶配对时具有高去饱和效率的其他δ-4去饱和酶的方法。
发明详述
尤其是,本发明提供与SEQ ID NO.79、86或85任一个的氨基酸序列具有至少60%,优选至少69%,甚至更优选至少74%并甚至更优选至少81%的序列同一性的δ-4去饱和酶,其中所述序列优选还包含
-选自“KHPGG”、“QHPGG”或“RHPGG”的基序,优选选自“KHPGGD”、“QHPGGD”或“RHPGGD”的基序,和
-选自“GLNIQHDANHG”或“HVVGHH”的基序,优选选自“AAIGLNIQHDANHG”或“QHVVGHH”的基序。
特别令人惊奇的是,可以提供当与异源δ-5去饱和酶配对时具有高去饱和效率的δ-4去饱和酶,其与上文提及的Pavlova/Rebecca的δ-4去饱和酶具有如此低的序列同一性。还特别令人惊奇的是,在Pavlova/Rebecca中保守的基序“EHPGG”中,第一个氨基酸G(其是酸性氨基酸)可以被K(其是碱性氨基酸)替换,而不消除高去饱和效率。因此可以推断,令人惊奇的还有“QHPGG”和“RHPGG”是替代“EHPGG”的有效基序。同样,令人惊奇的是,在Pavlova/Rebecca中保守的基序“HVVMHH”中,δ-4去饱和酶可以被“HVVGHH”替换,疏水性甲硫氨酸M被不起眼的甘氨酸G替换。并且令人惊奇的是,保守Pavlova/Rebecca基序“EHVVMHH”的第一个氨基酸E可以被Q替换,再次将酸性氨基酸交换成非酸性氨基酸。没有预料到的是,当与异源δ-5延伸酶比较时,保守基序中氨基酸性质的这种显著改变是可能的而不消除去饱和效率。
本发明的δ-4去饱和酶至少包含以下保守序列基序,其中“X”表示任何氨基酸:“HPGG”、“QDWIGG”、“NGGLN”、“QIEHHLFPR”和“IGLNIQHDXNHG”。如图2的比对中所示,这些序列为δ-4去饱和酶活性所需。
优选地,本发明的δ-4去饱和酶包含至少5个,更优选至少6个,甚至更优选至少7个并且最优选所有以下保守序列基序,其中“X”表示任何氨基酸:“DPDXK”、“HPGG”、“NGGLNXQIEHHLFPR”、“GYXQDWIGG”、“IGLNIQHDANHG”、“YLXFFF”、“HVVXHHXH”和“FXGXDAT”。这些较长的保守序列基序也发现于如图2中所示的Sphaeroforma、Pavlova或Rebecca属的δ-4去饱和酶中。
更优选地,本发明的δ-4去饱和酶包含至少5个,更优选至少6个,甚至更优选至少7个,甚至更优选至少8个,甚至更优选至少9个,甚至更优选至少10个,甚至更优选至少11个,甚至更优选至少12个,甚至更优选至少13个,甚至更优选至少14个并最优选所有以下保守序列基序:“DPD[VILMATQ]K”、“HPGG”、“NGGLN[FWY]QIEHHLFPR”、“GY[ASGM]QDWIGG”、“Y[FI]LP”、“IGLNIQHDANHG”、“YL[AG]FFF”、“HVV[GNASKDWM]HH[LIFT]H”、“AP[PA]S”、“F[WF][AVCGSPI]R”、“F[GP]G[RQ]DAT”、“T[LIMVFCA][LIVCA]C”、“P[LF][WA]L”、“KA[CA]A”和“I[VL][GADE]D[GA]”。在该列表中,“X”再次表示任何氨基酸。括号中的氨基酸列表表明,从列表成员中选择的一个成员存在于基序的相应位置上,其中每个单独的列表以降低优选性排序。例如,基序“PLWL”优于基序“PLAL”或“PFWL”或“PFAL”,并且这4个基序之一必须存在于本发明更优选的δ-4去饱和酶中。在如图2中所示的Sphaeroforma、Pavlova或Rebecca属的δ-4去饱和酶中没有发现列表中的一些氨基酸。然而,根据本发明,这些新的氨基酸与Sphaeroforma、Pavlova或Rebecca属的δ-4去饱和酶中相应位置上发现的那些氨基酸充分地相似,从而所述新的氨基酸一般不消除或严重降低δ-4去饱和酶效率,特别是当与用于从所述δ-5延伸酶的底物产生多不饱和脂肪酸的δ-5延伸酶组合时降低其去饱和的高效率。
最优选地,本发明的δ-4去饱和酶包含至少5个,更优选至少6个,甚至更优选至少7个,甚至更优选至少8个,甚至更优选至少9个,甚至更优选至少10个,甚至更优选至少11个,甚至更优选至少12个,甚至更优选至少13个,甚至更优选至少14个,并且最优选所有以下保守序列基序:“DPD[VQ]K”、“HPGG”、“NGGLN[YF]QIEHHLFPR”、“GY[SAM]QDWIGG”、“Y[FI]LP”、“IGLNIQHDANHG”、“YL[AG]FFF”、“HVV[GM]HH[LT]H”、“AP[PA]S”、“F[FW][AI]R”、“F[GP]G[RQ]DAT”、“T[LA][LA]C”、“P[LF][AW]L”、“KA[AC]A"和"I[VL][GE]D[GA]”。如上所述,括号中的氨基酸列表表明,从列表成员中选择的一个成员存在于基序的相应位置上,其中每个单独的列表以降低优选性排序。
根据本发明,进一步提供了具有氨基酸序列SEQ ID NO.85和任选地一个或多个以下突变的δ-4去饱和酶:H4S、H4Y、H4Q、H4E、H4N、A5S、A5G、A5C、A5P、A5T、A5V、A6R、A6G、A6K、A6S、A6N、T7K、T7S、T7R、T7N、T7A、T7G、K8E、K8D、K8R、K8Q、V9-、V9I、V9L、V9A、G10V、G10I、G10A、G10M、G10C、G11S、G11N、G11D、G11A、G11K、D12-、S13-、D14A、D14V、D14I、D14E、D14G、D14P、P15A、P15G、P15S、P15V、P15T、R18K、R18S、R18T、R18A、R18Q、D19A、D19G、D19P、D19S、L20-、K21-、M22A、M22L、M22I、M22V、E23K、E23R、E23Q、H24Y、H24F、H24I、H24M、H24L、H24Q、H24R、H24V、F25Y、F25W、F25I、F25A、S26T、S26A、S26C、S26G、S26N、Y27R、Y27F、E28T、E28D、E28N、E28S、R29K、R29E、R29Q、R29D、I30K、I30L、I30V、L31A、L31I、N32D、N32G、N32H、N32S、N32T、N32R、N32K、N32E、N32Q、N32A、N32V、D33H、D33N、D33T、E34T、E34N、E34D、E34Q、E34R、R35N、R35K、R35H、D36P、D36E、D36N、D36S、D37E、D37K、L38I、L38V、L38M、L38F、L38Y、C39T、C39A、C39S、C39V、V41L、V41I、V41M、V41T、G42A、G42D、G42E、G44A、A48S、A48C、A48L、A48R、A48K、T49S、T49V、T49N、T49P、T49K、T49A、A50C、A50N、A50G、D53E、D53N、K54Q、K54R、A59-、D60E、D60N、D60H、F61Y、F61W、F61V、F61I、V62L、V62I、V62M、V62A、V62T、D63S、D63N、D63E、L64I、L64F、L64M、G66P、R68Q、R68H、R68D、R68N、P72E、P72D、P72Q、H73G、H73A、H73N、H73S、F75W、F75M、F75Y、E76Q、E76M、E76H、Y77F、Y77W、Y77H、Y77L、R79K、R79Q、R79H、R80H、R80K、R80Q、R80Y、R80N、E81A、E81V、E81Q、E81L、E81P、W82G、W82S、P83D、P83V、P83K、P83A、P83T、P83E、P83Q、P83M、P83L、P84K、P84R、A85S、A85G、A85C、A85T、A85P、A85V、V86I、V86L、V86M、V86R、V86A、V86T、L87M、L87I、L87V、L87F、A88S、A88C、A88G、K89R、K89E、K89Q、K89P、K89N、Y90F、Y90W、K91F、K91G、K91Y、K91L、K91W、L95I、L95V、L95M、D96A、D96E、D96G、D96P、R97K、R97P、R97Q、R97H、R97E、R97A、D98E、D98N、D98S、D99E、D99Q、S100K、S100G、S100N、S100P、Y101F、Y101W、Y101H、Y101P、V102T、V102I、V102L、Q103H、Q103E、Q103R、Q103N、Q103K、Q103D、Q103Y、H104A、H104Y、H104V、H104I、H104F、H104R、D105E、D105P、D105T、S106P、S106E、S106A、S106D、S106N、S106T、G107L、G107I、G107M、G107A、Y108H、Y108Q、Y108W、Y108M、Y108V、L109K、L109M、L109I、L109V、R110Q、R110K、R110H、R110E、L111I、L111F、C112G、C112A、C112N、C112S、C112K、A113C、A113S、A113G、E114D、E114Q、E114P、E114S、E114K、E114A、N116R、N116D、N116H、G117A、G117K、G117R、G117S、I118L、I118V、L119I、L119M、K121R、K121M、K121Q、K121L、G122W、G122Q、G122N、S123E、S123D、S123N、S123G、G124W、G124A、G124N、G125-、W126F、P129A、P129G、W131Y、W132Y、I133L、I133V、C136A、C136G、C136S、L138I、L138V、L138M、L139I、L139V、L139M、V140A、V140I、V140L、V140C、V140M、A142T、A142P、A142S、A142G、L143I、L143V、Y144F、Y144H、Y144W、Y144S、Y144T、L145I、L145V、L145M、L145F、L145T、D146E、D146N、D146Q、Y147W、Y147F、Y147H、Y147G、Y148F、Y148H、Y148L、M149W、M149L、M149I、M149F、L150I、L150V、A151L、A151C、A151G、A151S、R152K、R152S、R152N、R152Q、R152D、P154K、P154E、P154R、I156L、I156V、L157F、L157I、L157M、L157Y、L157W、L157P、A159S、A159G、A159C、A159T、A159P、A159V、I160V、I160L、I161V、I161L、I161F、L162I、L162V、L162M、L162F、L162C、L162A、L162T、L162S、L165I、L165V、F166Y、W168G、W168A、W168C、A181S、A181G、A181C、A181T、A181P、A181V、L182I、L182V、L182M、N185H、N185Y、P186S、V187M、V187A、V187I、V187P、V188I、V188L、V188A、V188T、Y190R、Y190H、Y190W、L191C、L191I、F192L、F192I、A195S、A195G、A195M、A195Q、A195K、S202N、S202G、S202D、S202T、S202A、S202K、M203R、M203L、M203Q、M203I、M204L、M204V、M204I、M204Q、M204C、L207I、L207V、Q208R、Q208E、Q208K、Q209E、Q209K、Q209R、Q209D、Q209H、G213N、G213A、G213S、G213K、G213D、G213W、G213M、L216I、L216F、L216T、T218C、T218S、T218V、T218A、T218N、T218P、D220E、D220N、D220R、D220G、I221V、I221F、I221Y、I221H、D222N、D222Q、D222P、H223Y、H223F、H223Q、H223W、H223R、H223E、H223N、H223M、H223V、H223P、H223I、H223L、H223D、H223A、V227I、V227L、V227M、V227A、V227T、V227Q、G229A、G230H、G230N、G230D、G231S、G231N、G231A、A232V、A232T、A232I、A232G、L233I、L233V、R234K、R234Q、R234N、R234E、R234H、R234T、K236S、K236G、K236A、K236T、P237K、P237D、P237E、P237R、T238V、T238S、T238P、T238N、T238A、T238I、T238Y、D239S、D239G、D239E、G240I、G240L、G240F、G240C、G240M、G240S、W241F、W241Y、W241P、L242M、L242I、L242V、L242F、L242K、P243E、P243D、W244F、H246A、H246S、H246Y、H246G、H246N、L247I、L247V、L250I、L250V、L250M、L250F、L250Y、F252I、F252Y、L255G、L255M、L255A、L255K、L255C、L255F、E256D、E256Q、E256K、E256P、E256R、E256N、E256S、E256V、E256A、E256T、E256G、E256H、E256I、A257V、A257Q、A257L、A257C、A257P、A257E、L258M、L258I、Y259F、Y259W、Y259H、Y259L、G260A、G260C、K262Q、K262R、W263L、W263F、V264I、V264L、V264M、V264F、F265Y、F265W、F265H、F265S、D267G、L268A、L268I、L268V、H269N、H269Q、H269R、H269L、E270D、E270Q、E270K、L272I、L272F、E273D、E273A、E273G、E273P、W274F、W274Y、W274M、K275R、K275E、K275Q、W276Y、E277K、E277D、E277R、P280K、P280R、I281L、I281V、P282S、P282D、P282N、P282G、P283E、P283D、P283Q、L284I、L284M、L284V、L284C、L284A、L284T、L284S、A285Y、A285C、A285F、A285V、A285S、A285L、R286K、R286Q、R286H、R286E、R286L、R286M、R286V、R286A、P287K、P287D、P287E、P287G、P287Q、P287S、P287R、P287A、E288D、E288L、E288K、E288V、E288Q、F289Y、F289R、F289H、A290G、A290S、A290N、A290C、P291I、V293I、V293L、G294A、G294S、G294P、G294N、G294K、G294R、G294E、G294D、G294Q、G294T、G294H、G294C、G294W、G294M、G294Y、G294V、C295L、C295I、K296R、K296E、K296Q、K296N、K296P、L297I、L297V、G298F、G298A、G298W、G298S、W300F、A301V、A301C、A301G、A301S、A301P、A301I、F303Y、F303W、F303K、V304I、V304F、V304L、V304Y、A305V、A305I、A305L、A305T、A305C、L306I、L306V、L306M、L308F、L308I、W309A、W309G、H311Q、H311E、H311R、H311Y、H311N、P312F、P312Y、S313T、S313N、S313G、S313A、W314F、W314L、W314Y、W314I、W314M、W314V、H315Y、L317I、L317M、L317V、L317F、L317C、L317A、L318I、L318V、L318C、L318A、V320I、V320T、V320P、V320L、C321Y、A322L、A322C、A322S、A322G、W323T、V324I、V324L、V324M、C325A、C325G、C325S、T326S、T326V、T326N、T326I、T326P、T326L、G327A、G327N、G327K、S328A、S328T、S328L、S328C、S328V、F329L、F329Y、A332G、A332C、A332S、A332V、A332P、A332T、I336V、I336F、I336L、L337I、L337F、L337M、I340N、I340L、I340V、I340M、I340F、I340T、I342V、I342D、I342L、I342C、V344I、V344L、V344A、V344M、K345G、K345R、K345A、I347V、I347L、G348P、P349D、P349E、D350E、D350N、D350Q、D350G、D350R、D350K、G351A、G351C、G351S、G351P、G351V、K352-、S353-、L354-、P355-、P355D、P355S、R356-、N357S、N357D、N357G、N357E、I358V、I358A、I358L、D359E、D359N、D359P、D359T、W360F、A361G、A361V、A361C、A361S、A361T、R362Q、R362K、R362H、R363H、R363K、R363N、I365V、I365L、T367S、T367N、T367P、T367V、T367A、T367D、G372C、G372A、G372N、G372S、E374D、E374K、E374R、E374N、E374Q、E374H、E374S、E374P、E374A、E374T、E374V、E374G、E374M、E374Y、W375K、W375G、G377A、G377W、H378Y、H378N、H378F、H378Q、H378R、H378M、H378I、H378E、H378W、H378D、H378V、L379I、L379M、L379S、L379V、L379A、L379T、F385W、F385Y、L395M、L395I、L395F、H396S、H396N、H396Y、H396Q、H396E、A398S、A398C、A398G、H399Y、A401S、A401G、A401N、K402T、K402P、K402S、K402R、K402E、K402Q、Q404E、Q404A、Q404R、Q404K、Q404H、Q404P、V406I、V406L、V406M、V406A、V406T、Q408R、Q408H、Q408E、Q408N、Q408M、K409R、K409Q、K409T、K409M、V410I、V410H、V410R、C411I、C411A、C411V、E413D、E413K、E413Q、E413R、E413P、E413N、E413S、E413H、E413A、N414M、N414D、N414L、N414G、N414S、V416I、V416F、V416L、V416Y、N417K、N417R、N417G、K419R、K419G、K419S、H420Y、H420Q、H420R、H420N、H420E、H420K、P422G、P422D、I424V、I424C、G425A、G425L、G425N、G425P、G425D、G429D、G429N、G429S、S430A、S430T、S430C、S430N、M431L、M431I、M431V、M431T、M431F、M431C、L432F、L432I、L432M、S433R、S433K、S433Q、S433A、S433T、S433N、H434Y、H434Q、H434N、H434R、L435I、L435M、L435V、L435F、G436K、G436S、G436P、A437G、A437K、A437P、A437S、L438I、L438M、L438V、G439A、G439N、G439S、G439D、A440S、A440C、A440T、A440V、R441V、R441I、R441L、P442A、P442V、T443V、T443I、T443A、T443G、T443L、W444-、W444Y、W444F、W444S、N445-、A446-、E447D、E447Q、E447K、F448Y、F448W、F448K、M449L、M449I、M449G、A450G、A450E、A450S、A450P、A450D、A450C、G451K、G451R、G451N、G451D、L452P、L452I、L452V、L452A、L452M、E453-、E453D、E453S、E454-、K455-、S456-、S457-、V458-、E459-、C460-、R461-、L462-、R463-、L464-、G465-、A466-、A467-、C468-、A469-、R470-、G471-、C472-、C472S、W473-、W473Q、C474-、C474A、S475-、D476-、A477-、A478-、S479-、L480-、I481-、S482-、W483-、L484-、G485-。优选地,所述氨基酸序列还包含
-选自“KHPGG”、“QHPGG”或“RHPGG”的基序,优选选自“KHPGGD”、“QHPGGD”或“RHPGGD”的基序,和
-选自“GLNIQHDANHG”或“HVVGHH”的基序,优选选自“AAIGLNIQHDANHG”或“QHVVGHH”的基序。
根据本发明,还提供了具有氨基酸序列SEQ ID NO.86(即,在比对中匹配序列并且不考虑由于下文中提及的突变的错配)和任选地一个或多个以下突变的δ-4去饱和酶:H4S、H4Y、H4Q、H4E、H4N、A5S、A5G、A5C、A5P、A5T、A5V、A6R、A6G、A6K、A6S、A6N、T7K、T7S、T7R、T7N、T7A、T7G、K8E、K8D、K8R、K8Q、G9V、G9I、G9A、G9M、G9C、G10S、G10N、G10D、G10A、G10K、D11A、D11V、D11I、D11E、D11G、D11P、P12A、P12G、P12S、P12V、P12T、R15K、R15S、R15T、R15A、R15Q、D16A、D16G、D16P、D16S、M17A、M17L、M17I、M17V、E18K、E18R、E18Q、H19Y、H19F、H19I、H19M、H19L、H19Q、H19R、H19V、F20Y、F20W、F20I、F20A、S21T、S21A、S21C、S21G、S21N、Y22R、Y22F、E23T、E23D、E23N、E23S、R24K、R24E、R24Q、R24D、I25K、I25L、I25V、L26A、L26I、N27D、N27G、N27H、N27S、N27T、N27R、N27K、N27E、N27Q、N27A、N27V、D28H、D28N、D28T、E29T、E29N、E29D、E29Q、E29R、R30N、R30K、R30H、D31P、D31E、D31N、D31S、D32E、D32K、L33I、L33V、L33M、L33F、L33Y、C34T、C34A、C34S、C34V、V36L、V36I、V36M、V36T、G37A、G37D、G37E、G39A、A43S、A43C、A43L、A43R、A43K、T44S、T44V、T44N、T44P、T44K、T44A、A45C、A45N、A45G、D48E、D48N、K49Q、K49R、D54E、D54N、D54H、F55Y、F55W、F55V、F55I、V56L、V56I、V56M、V56A、V56T、D57S、D57N、D57E、L58I、L58F、L58M、G60P、R62Q、R62H、R62D、R62N、P66E、P66D、P66Q、H67G、H67A、H67N、H67S、F69W、F69M、F69Y、E70Q、E70M、E70H、Y71F、Y71W、Y71H、Y71L、R73K、R73Q、R73H、R74H、R74K、R74Q、R74Y、R74N、E75A、E75V、E75Q、E75L、E75P、W76G、W76S、P77D、P77V、P77K、P77A、P77T、P77E、P77Q、P77M、P77L、P78K、P78R、A79S、A79G、A79C、A79T、A79P、A79V、V80I、V80L、V80M、V80R、V80A、V80T、L81M、L81I、L81V、L81F、A82S、A82C、A82G、K83R、K83E、K83Q、K83P、K83N、Y84F、Y84W、K85F、K85G、K85Y、K85L、K85W、L89I、L89V、L89M、D90A、D90E、D90G、D90P、R91K、R91P、R91Q、R91H、R91E、R91A、D92E、D92N、D92S、D93E、D93Q、S94K、S94G、S94N、S94P、Y95F、Y95W、Y95H、Y95P、V96T、V96I、V96L、Q97H、Q97E、Q97R、Q97N、Q97K、Q97D、Q97Y、H98A、H98Y、H98V、H98I、H98F、H98R、D99E、D99P、D99T、S100P、S100E、S100A、S100D、S100N、S100T、G101L、G101I、G101M、G101A、Y102H、Y102Q、Y102W、Y102M、Y102V、L103K、L103M、L103I、L103V、R104Q、R104K、R104H、R104E、L105I、L105F、C106G、C106A、C106N、C106S、C106K、A107C、A107S、A107G、E108D、E108Q、E108P、E108S、E108K、E108A、N110R、N110D、N110H、G111A、G111K、G111R、G111S、I112L、I112V、L113I、L113M、K115R、K115M、K115Q、K115L、G116W、G116Q、G116N、S117E、S117D、S117N、S117G、G118W、G118A、G118N、W119F、P122A、P122G、W124Y、W125Y、I126L、I126V、C129A、C129G、C129S、L131I、L131V、L131M、L132I、L132V、L132M、V133A、V133I、V133L、V133C、V133M、A135T、A135P、A135S、A135G、L136I、L136V、Y137F、Y137H、Y137W、Y137S、Y137T、L138I、L138V、L138M、L138F、L138T、D139E、D139N、D139Q、Y140W、Y140F、Y140H、Y140G、Y141F、Y141H、Y141L、M142W、M142L、M142I、M142F、L143I、L143V、A144L、A144C、A144G、A144S、R145K、R145S、R145N、R145Q、R145D、P147K、P147E、P147R、I149L、I149V、L150F、L150I、L150M、L150Y、L150W、L150P、A152S、A152G、A152C、A152T、A152P、A152V、I153V、I153L、I154V、I154L、I154F、L155I、L155V、L155M、L155F、L155C、L155A、L155T、L155S、L158I、L158V、F159Y、W161G、W161A、W161C、A174S、A174G、A174C、A174T、A174P、A174V、L175I、L175V、L175M、N178H、N178Y、P179S、V180M、V180A、V180I、V180P、V181I、V181L、V181A、V181T、Y183R、Y183H、Y183W、L184C、L184I、F185L、F185I、A188S、A188G、A188M、A188Q、A188K、S195N、S195G、S195D、S195T、S195A、S195K、M196R、M196L、M196Q、M196I、M197L、M197V、M197I、M197Q、M197C、L200I、L200V、Q201R、Q201E、Q201K、Q202E、Q202K、Q202R、Q202D、Q202H、G206N、G206A、G206S、G206K、G206D、G206W、G206M、L209I、L209F、L209T、T211C、T211S、T211V、T211A、T211N、T211P、D213E、D213N、D213R、D213G、I214V、I214F、I214Y、I214H、D215N、D215Q、D215P、H216Y、H216F、H216Q、H216W、H216R、H216E、H216N、H216M、H216V、H216P、H216I、H216L、H216D、H216A、V220I、V220L、V220M、V220A、V220T、V220Q、G222A、G223H、G223N、G223D、G224S、G224N、G224A、A225V、A225T、A225I、A225G、L226I、L226V、R227K、R227Q、R227N、R227E、R227H、R227T、K229S、K229G、K229A、K229T、P230K、P230D、P230E、P230R、T231V、T231S、T231P、T231N、T231A、T231I、T231Y、D232S、D232G、D232E、G233I、G233L、G233F、G233C、G233M、G233S、W234F、W234Y、W234P、L235M、L235I、L235V、L235F、L235K、P236E、P236D、W237F、H239A、H239S、H239Y、H239G、H239N、L240I、L240V、L243I、L243V、L243M、L243F、L243Y、F245I、F245Y、L248G、L248M、L248A、L248K、L248C、L248F、E249D、E249Q、E249K、E249P、E249R、E249N、E249S、E249V、E249A、E249T、E249G、E249H、E249I、A250V、A250Q、A250L、A250C、A250P、A250E、L251M、L251I、Y252F、Y252W、Y252H、Y252L、G253A、G253C、K255Q、K255R、W256L、W256F、V257I、V257L、V257M、V257F、F258Y、F258W、F258H、F258S、D260G、L261A、L261I、L261V、H262N、H262Q、H262R、H262L、E263D、E263Q、E263K、L265I、L265F、E266D、E266A、E266G、E266P、W267F、W267Y、W267M、K268R、K268E、K268Q、W269Y、E270K、E270D、E270R、P273K、P273R、I274L、I274V、P275S、P275D、P275N、P275G、P276E、P276D、P276Q、L277I、L277M、L277V、L277C、L277A、L277T、L277S、A278Y、A278C、A278F、A278V、A278S、A278L、R279K、R279Q、R279H、R279E、R279L、R279M、R279V、R279A、P280K、P280D、P280E、P280G、P280Q、P280S、P280R、P280A、E281D、E281L、E281K、E281V、E281Q、F282Y、F282R、F282H、A283G、A283S、A283N、A283C、P284I、V286I、V286L、G287A、G287S、G287P、G287N、G287K、G287R、G287E、G287D、G287Q、G287T、G287H、G287C、G287W、G287M、G287Y、G287V、C288L、C288I、K289R、K289E、K289Q、K289N、K289P、L290I、L290V、G291F、G291A、G291W、G291S、W293F、A294V、A294C、A294G、A294S、A294P、A294I、F296Y、F296W、F296K、V297I、V297F、V297L、V297Y、A298V、A298I、A298L、A298T、A298C、L299I、L299V、L299M、L301F、L301I、W302A、W302G、H304Q、H304E、H304R、H304Y、H304N、P305F、P305Y、S306T、S306N、S306G、S306A、W307F、W307L、W307Y、W307I、W307M、W307V、H308Y、L310I、L310M、L310V、L310F、L310C、L310A、L311I、L311V、L311C、L311A、V313I、V313T、V313P、V313L、C314Y、A315L、A315C、A315S、A315G、W316T、V317I、V317L、V317M、C318A、C318G、C318S、T319S、T319V、T319N、T319I、T319P、T319L、G320A、G320N、G320K、S321A、S321T、S321L、S321C、S321V、F322L、F322Y、A325G、A325C、A325S、A325V、A325P、A325T、I329V、I329F、I329L、L330I、L330F、L330M、I333N、I333L、I333V、I333M、I333F、I333T、I335V、I335D、I335L、I335C、V337I、V337L、V337A、V337M、K338G、K338R、K338A、I340V、I340L、G341P、P342D、P342E、D343E、D343N、D343Q、D343G、D343R、D343K、G344A、G344C、G344S、G344P、G344V、N345S、N345D、N345G、N345E、I346V、I346A、I346L、D347E、D347N、D347P、D347T、W348F、A349G、A349V、A349C、A349S、A349T、R350Q、R350K、R350H、R351H、R351K、R351N、I353V、I353L、T355S、T355N、T355P、T355V、T355A、T355D、G360C、G360A、G360N、G360S、E362D、E362K、E362R、E362N、E362Q、E362H、E362S、E362P、E362A、E362T、E362V、E362G、E362M、E362Y、W363K、W363G、G365A、G365W、H366Y、H366N、H366F、H366Q、H366R、H366M、H366I、H366E、H366W、H366D、H366V、L367I、L367M、L367S、L367V、L367A、L367T、F373W、F373Y、L383M、L383I、L383F、H384S、H384N、H384Y、H384Q、H384E、A386S、A386C、A386G、H387Y、A389S、A389G、A389N、K390T、K390P、K390S、K390R、K390E、K390Q、Q392E、Q392A、Q392R、Q392K、Q392H、Q392P、V394I、V394L、V394M、V394A、V394T、Q396R、Q396H、Q396E、Q396N、Q396M、K397R、K397Q、K397T、K397M、V398I、V398H、V398R、C399I、C399A、C399V、E401D、E401K、E401Q、E401R、E401P、E401N、E401S、E401H、E401A、N402M、N402D、N402L、N402G、N402S、V404I、V404F、V404L、V404Y、N405K、N405R、N405G、K407R、K407G、K407S、H408Y、H408Q、H408R、H408N、H408E、H408K、P410G、P410D、I412V、I412C、G413A、G413L、G413N、G413P、G413D、G417D、G417N、G417S、S418A、S418T、S418C、S418N、M419L、M419I、M419V、M419T、M419F、M419C、L420F、L420I、L420M、S421R、S421K、S421Q、S421A、S421T、S421N、H422Y、H422Q、H422N、H422R、L423I、L423M、L423V、L423F、G424K、G424S、G424P、A425G、A425K、A425P、A425S、L426I、L426M、L426V、G427A、G427N、G427S、G427D、A428S、A428C、A428T、A428V、R429V、R429I、R429L、P430A、P430V、T431V、T431I、T431A、T431G、T431L、E432D、E432Q、E432K、F433Y、F433W、F433K、M434L、M434I、M434G、A435G、A435E、A435S、A435P、A435D、A435C、G436K、G436R、G436N、G436D、L437P、L437I、L437V、L437A、L437M。优选地,所述氨基酸序列还包含
-选自“KHPGG”、“QHPGG”或“RHPGG”的基序,优选选自“KHPGGD”、“QHPGGD”或“RHPGGD”的基序,和
-选自“GLNIQHDANHG”或“HVVGHH”的基序,优选选自“AAIGLNIQHDANHG”或“QHVVGHH”的基序。
特别优选的是具有SEQ ID NO.86的骨架和任选地一个或多个以下突变的δ-4去饱和酶:H4S、H4Y、H4Q、H4E、H4N、A5S、A5G、A5C、A5P、A5T、A5V、A6R、A6G、A6K、A6S、A6N、T7K、T7S、T7R、T7N、T7A、T7G、K8E、K8D、K8R、K8Q、G9V、G9I、G9A、G9M、G9C、G10S、G10N、G10D、G10A、G10K、D11A、D11V、D11I、D11E、D11G、D11P、P12A、P12G、P12S、P12V、P12T、R15K、R15S、R15T、R15A、R15Q、D16A、D16G、D16P、D16S、M17A、M17L、M17I、M17V、E18K、E18R、E18Q、H19Y、H19F、H19I、F20Y、F20W、S21T、S21A、S21C、S21G、S21N、Y22R、Y22F、E23T、E23D、E23N、E23S、R24K、R24E、R24Q、R24D、I25K、I25L、I25V、L26A、L26I、N27D、N27G、N27H、N27S、N27T、N27R、D28H、D28N、D28T、E29T、E29N、E29D、E29Q、E29R、R30N、R30K、R30H、D31P、D31E、D31N、D31S、D32E、D32K、L33I、L33V、L33M、L33F、C34T、C34A、C34S、C34V、V36L、V36I、V36M、V36T、G37A、G37D、G37E、G39A、A43S、A43C、A43L、A43R、A43K、T44S、T44V、T44N、T44P、T44K、T44A、A45C、A45N、A45G、D48E、D48N、K49Q、K49R、D54E、D54N、D54H、F55Y、F55W、F55V、F55I、V56L、V56I、V56M、V56A、V56T、D57S、D57N、D57E、L58I、L58F、L58M、G60P、R62Q、R62H、R62D、R62N、P66E、P66D、P66Q、H67G、H67A、H67N、H67S、F69W、F69M、F69Y、E70Q、E70M、E70H、Y71F、Y71W、Y71H、Y71L、R73K、R73Q、R73H、R74H、R74K、R74Q、R74Y、R74N、E75A、E75V、E75Q、E75L、E75P、W76G、P77D、P77V、P77K、P78K、P78R、A79S、A79G、A79C、A79T、A79P、A79V、V80I、V80L、V80M、V80R、V80A、V80T、L81M、L81I、L81V、L81F、A82S、A82C、A82G、K83R、K83E、K83Q、K83P、K83N、Y84F、Y84W、K85F、K85G、K85Y、K85L、K85W、L89I、L89V、L89M、D90A、D90E、D90G、D90P、R91K、R91P、D92E、D92N、D92S、D93E、D93Q、S94K、S94G、S94N、S94P、Y95F、Y95W、Y95H、V96T、V96I、V96L、Q97H、Q97E、Q97R、Q97N、H98A、H98Y、H98V、H98I、H98F、H98R、D99E、D99P、D99T、S100P、S100E、S100A、S100D、S100N、S100T、G101L、G101I、G101M、G101A、Y102H、Y102Q、Y102W、Y102M、Y102V、L103K、L103M、L103I、L103V、R104Q、R104K、R104H、R104E、L105I、L105F、C106G、C106A、A107C、A107S、A107G、E108D、E108Q、E108P、E108S、N110R、N110D、N110H、G111A、G111K、G111R、G111S、I112L、I112V、L113I、L113M、K115R、K115M、K115Q、K115L、G116W、G116Q、G116N、S117E、S117D、S117N、S117G、G118W、G118A、G118N、W119F、P122A、P122G、W124Y、W125Y、I126L、I126V、C129A、C129G、C129S、L131I、L131V、L131M、L132I、L132V、L132M、V133A、V133I、V133L、V133C、V133M、A135T、A135P、A135S、A135G、L136I、L136V、Y137F、Y137H、Y137W、Y137S、Y137T、L138I、L138V、D139E、D139N、D139Q、Y140W、Y140F、Y140H、Y141F、Y141H、M142W、M142L、M142I、M142F、L143I、L143V、A144L、A144C、A144G、A144S、R145K、R145S、R145N、R145Q、R145D、P147K、P147E、P147R、I149L、I149V、L150F、L150I、L150M、L150Y、A152S、A152G、A152C、A152T、A152P、A152V、I153V、I153L、I154V、I154L、L155I、L155V、L155M、L155F、L158I、L158V、F159Y、W161G、W161A、W161C、A174S、A174G、A174C、A174T、A174P、A174V、L175I、L175V、L175M、N178H、N178Y、V180M、V180A、V180I、V180P、V181I、V181L、V181A、V181T、Y183R、Y183H、Y183W、L184C、L184I、F185L、F185I、A188S、A188G、A188M、A188Q、A188K、S195N、S195G、S195D、S195T、S195A、S195K、M196R、M196L、M196Q、M196I、M197L、M197V、M197I、M197Q、M197C、L200I、L200V、Q201R、Q201E、Q201K、Q202E、Q202K、Q202R、Q202D、Q202H、G206N、G206A、G206S、G206K、L209I、L209F、L209T、T211C、T211S、T211V、T211A、T211N、T211P、D213E、D213N、D213R、D213G、I214V、I214F、I214Y、D215N、D215Q、D215P、H216Y、V220I、V220L、V220M、V220A、V220T、G223H、G223N、G223D、G224S、G224N、G224A、A225V、A225T、A225I、A225G、L226I、L226V、R227K、R227Q、R227N、R227E、R227H、K229S、K229G、K229A、K229T、P230K、P230D、P230E、P230R、T231V、T231S、T231P、T231N、T231A、D232S、D232G、D232E、G233I、G233L、G233F、W234F、L235M、L235I、P236E、P236D、H239A、H239S、H239Y、H239G、H239N、L240I、L240V、L243I、L243V、L243M、L243F、F245I、F245Y、L248G、L248M、L248A、L248K、L248C、L248F、E249D、E249Q、E249K、A250V、A250Q、A250L、A250C、A250P、A250E、L251M、L251I、Y252F、G253A、G253C、K255Q、K255R、W256L、W256F、V257I、V257L、V257M、V257F、F258Y、D260G、L261A、L261I、L261V、H262N、H262Q、H262R、E263D、E263Q、E263K、L265I、L265F、E266D、E266A、E266G、E266P、W267F、W267Y、K268R、K268E、K268Q、W269Y、E270K、E270D、E270R、P273K、P273R、I274L、I274V、P275S、P275D、P275N、P275G、P276E、P276D、L277I、L277M、L277V、L277C、A278Y、A278C、A278F、A278V、A278S、A278L、R279K、R279Q、P280K、P280D、P280E、E281D、E281L、E281K、E281V、E281Q、F282Y、F282R、F282H、A283G、A283S、A283N、A283C、P284I、V286I、V286L、G287A、G287S、G287P、C288L、C288I、K289R、K289E、K289Q、K289N、K289P、L290I、L290V、G291F、G291A、G291W、G291S、A294V、A294C、A294G、A294S、F296Y、F296W、F296K、V297I、V297F、V297L、V297Y、A298V、A298I、A298L、A298T、A298C、L299I、L299V、L299M、L301F、L301I、W302A、W302G、H304Q、H304E、H304R、H304Y、H304N、P305F、P305Y、S306T、S306N、S306G、S306A、W307F、W307L、H308Y、L310I、L310M、L310V、L310F、L311I、L311V、L311C、V313I、V313T、V313P、V313L、C314Y、A315L、A315C、A315S、A315G、W316T、V317I、V317L、C318A、C318G、C318S、T319S、T319V、T319N、T319I、T319P、T319L、G320A、G320N、G320K、S321A、S321T、S321L、S321C、S321V、F322L、F322Y、A325G、A325C、A325S、A325V、A325P、A325T、I329V、I329F、I329L、L330I、L330F、L330M、I333N、I333L、I333V、I333M、I333F、I333T、I335V、I335D、I335L、I335C、V337I、V337L、V337A、V337M、K338G、K338R、K338A、I340V、I340L、P342D、D343E、D343N、D343Q、D343G、G344A、G344C、G344S、G344P、G344V、N345S、N345D、N345G、N345E、I346V、I346A、I346L、D347E、D347N、D347P、D347T、W348F、A349G、A349V、A349C、A349S、A349T、R350Q、R350K、R350H、R351H、R351K、R351N、I353V、I353L、T355S、T355N、T355P、T355V、T355A、T355D、G360C、G360A、G360N、G360S、E362D、E362K、E362R、W363K、W363G、G365A、G365W、H366Y、H366N、H366F、H366Q、H366R、L367I、L367M、L367S、L367V、L367A、L367T、F373W、F373Y、L383M、L383I、L383F、H384S、H384N、H384Y、H384Q、H384E、A386S、A386C、A386G、H387Y、A389S、A389G、A389N、K390T、K390P、K390S、K390R、K390E、Q392E、Q392A、Q392R、Q392K、Q392H、Q392P、V394I、V394L、V394M、V394A、V394T、Q396R、Q396H、Q396E、Q396N、Q396M、K397R、K397Q、K397T、K397M、V398I、V398H、V398R、C399I、C399A、C399V、E401D、E401K、E401Q、E401R、E401P、N402M、N402D、N402L、N402G、N402S、V404I、V404F、V404L、V404Y、N405K、N405R、N405G、K407R、K407G、K407S、H408Y、H408Q、H408R、P410G、P410D、I412V、I412C、G413A、G413L、G413N、G413P、G413D、G417D、G417N、G417S、S418A、S418T、S418C、S418N、M419L、M419I、M419V、M419T、M419F、M419C、L420F、L420I、L420M、S421R、S421K、S421Q、S421A、S421T、S421N、H422Y、H422Q、H422N、H422R、L423I、L423M、L423V、L423F、G424K、G424S、G424P、A425G、A425K、A425P、A425S、L426I、L426M、L426V、G427A、G427N、G427S、G427D、A428S、A428C、A428T、A428V、R429V、R429I、R429L、P430A、P430V、T431V、T431I、T431A、T431G、T431L、E432D、E432Q、E432K、F433Y、F433W、F433K、M434L、M434I、M434G、A435G、A435E、A435S、A435P、A435D、A435C、G436K、G436R、G436N、G436D、L437P、L437I、L437V、L437A、L437M。优选地,所述氨基酸序列还包含
-选自“KHPGG”、“QHPGG”或“RHPGG”的基序,优选选自“KHPGGD”、“QHPGGD”或“RHPGGD”的基序,和
-选自“GLNIQHDANHG”或“HVVGHH”的基序,优选选自“AAIGLNIQHDANHG”或“QHVVGHH”的基序。
本发明还优选的是具有SEQ ID NO.86的骨架和任选地一个或多个以下突变的δ-4去饱和酶:H4S、H4Y、A5S、A5G、A6R、A6G、T7K、T7S、T7R、K8E、G9V、G10S、D11A、D11V、P12A、R15K、R15S、D16A、M17A、E18K、S21T、S21A、E23T、R24K、I25K、L26A、N27D、N27G、D28H、E29T、E29N、R30N、D31P、L33I、V36L、A43S、A43C、T44S、T44V、T44N、T44P、A45C、D54E、F55Y、V56L、V56I、D57S、L58I、R62Q、H67G、F69W、E70Q、Y71F、R73K、R74H、R74K、E75A、E75V、A79S、A79G、V80I、V80L、L81M、A82S、K83R、K83E、Y84F、K85F、K85G、L89I、D90A、D90E、S94K、Y95F、V96T、Q97H、H98A、H98Y、D99E、S100P、S100E、S100A、G101L、Y102H、Y102Q、L103K、R104Q、A107C、E108D、N110R、G111A、L113I、K115R、S117E、L131I、L132I、V133A、V133I、A135T、Y137F、Y137H、Y140W、M142W、A144L、R145K、R145S、L150F、A152S、A152G、L155I、A174S、A174G、L175I、V180M、V180A、Y183R、A188S、A188G、S195N、S195G、M196R、M197L、M197V、Q201R、Q202E、Q202K、G206N、L209I、T211C、T211S、D213E、I214V、V220I、V220L、A225V、R227K、R227Q、K229S、K229G、T231V、T231S、G233I、L235M、H239A、L243I、L248G、L248M、E249D、A250V、A250Q、K255Q、V257I、L261A、H262N、E266D、K268R、E270K、P275S、L277I、A278Y、A278C、E281D、E281L、F282Y、A283G、K289R、L290I、G291F、A294V、V297I、A298V、A298I、L299I、H304Q、H304E、S306T、L310I、L311I、V313I、A315L、T319S、T319V、S321A、S321T、A325G、A325C、I329V、I333N、I333L、I335V、V337I、K338G、D343E、G344A、N345S、I346V、D347E、A349G、A349V、T355S、T355N、G360C、E362D、H366Y、L367I、L367M、H384S、H384N、A386S、A389S、K390T、K390P、Q392E、Q392A、V394I、Q396R、K397R、V398I、E401D、E401K、N402M、N402D、V404I、N405K、G413A、S418A、M419L、M419I、L420F、S421R、S421K、S421Q、H422Y、L423I、A425G、L426I、G427A、A428S、R429V、T431V、T431I、F433Y、M434L、A435G、A435E、G436K、L437P、L437I。优选地,所述氨基酸序列还包含
-选自“KHPGG”、“QHPGG”或“RHPGG”的基序,优选选自“KHPGGD”、“QHPGGD”或“RHPGGD”的基序,和
-选自“GLNIQHDANHG”或“HVVGHH”的基序,优选选自“AAIGLNIQHDANHG”或“QHVVGHH”的基序。
甚至更优选地,本发明的δ-4去饱和酶具有SEQ ID NO.86的骨架和任选地一个或多个以下突变:H4S、A5S、A6R、T7K、T7S、K8E、K8D、G9V、G10S、D11A、D11V、P12A、R15S、D16A、M17A、E18K、S21T、Y22R、E23T、R24E、I25K、L26A、D28H、D28T、E29T、R30N、D31P、D31S、D32K、C34T、V36L、G37E、G39A、A43L、T44K、A45N、D48E、D54H、F55V、V56L、D57S、L58F、G60P、R62Q、P66E、H67A、E70M、Y71L、R73Q、R74Y、E75A、P78K、A79S、V80R、L81M、A82S、K83E、Y84F、K85F、L89V、D90A、R91P、D92S、D93E、S94K、V96T、H98A、H98V、D99T、S100P、S100E、G101L、G101A、Y102M、L103K、R104Q、L105I、A107S、N110R、G111A、G111K、L113M、K115M、G116Q、S117E、G118W、W119F、P122A、W124Y、W125Y、I126L、C129A、L131I、L132I、L132V、V133A、A135T、L136V、Y137S、Y137T、L138I、D139E、M142W、L143I、A144L、R145S、P147K、I149L、L150F、A152S、I153V、L158V、F159Y、W161A、A174S、L175V、N178H、N178Y、V180M、V180A、V181I、Y183R、L184C、F185L、A188S、A188M、S195N、M196R、M197V、L200I、Q201R、Q202E、L209T、T211C、D213R、D215Q、G223H、G224S、A225V、L226I、K229S、P230R、D232S、G233L、L235M、P236E、H239A、H239S、L240I、L243V、F245I、L248G、A250Q、L251M、G253A、K255Q、W256L、V257L、D260G、L261A、E263D、L265I、E266A、K268R、W269Y、E270K、P273K、I274L、P275S、P276E、A278Y、P280K、E281L、F282Y、F282R、A283N、P284I、V286I、G287A、C288L、K289R、L290V、G291F、G291A、F296K、V297F、A298V、L299V、L301F、W302A、H304Q、P305F、S306T、W307L、H308Y、C314Y、A315L、W316T、C318A、T319I、G320A、S321A、F322L、A325G、I329F、L330I、I333N、V337A、K338G、K338A、D343Q、G344A、N345S、I346A、D347T、W348F、A349V、A349T、R350Q、R351H、I353V、T355S、G360C、E362K、W363K、G365A、L367S、F373Y、L383M、H384S、A386S、H387Y、A389S、K390T、Q392A、V394L、Q396R、K397Q、K397T、V398H、V398R、C399I、E401K、N402M、N402L、V404F、V404Y、N405K、K407R、K407S、P410G、I412V、G413L、G417D、G417S、S418A、M419T、L420F、L420M、S421R、S421Q、H422Q、L423M、L423V、G424K、A425K、L426M、G427A、A428S、A428T、R429V、P430A、T431V、T431G、E432D、F433K、M434L、M434G、A435G、A435E、G436K、G436R、L437P、L437A。优选地,所述氨基酸序列还包含
-选自“KHPGG”、“QHPGG”或“RHPGG”的基序,优选选自“KHPGGD”、“QHPGGD”或“RHPGGD”的基序,和
-选自“GLNIQHDANHG”或“HVVGHH”的基序,优选选自“AAIGLNIQHDANHG”或“QHVVGHH”的基序。
并且甚至更优选地,本发明的δ-4去饱和酶具有SEQ ID NO.86的骨架和一个或多个以下突变:H4S、A5S、A6R、T7K、T7S、K8E、G9V、G10S、D11A、D11V、P12A、R15S、D16A、M17A、E18K、S21T、E23T、I25K、L26A、D28H、E29T、R30N、D31P、V36L、V56L、D57S、R62Q、E75A、A79S、L81M、A82S、K83E、Y84F、K85F、D90A、S94K、V96T、H98A、S100P、S100E、G101L、L103K、R104Q、N110R、G111A、S117E、L131I、L132I、V133A、A135T、M142W、A144L、R145S、L150F、A152S、A174S、V180M、V180A、Y183R、A188S、S195N、M196R、M197V、Q201R、Q202E、T211C、A225V、K229S、L235M、H239A、L248G、A250Q、K255Q、L261A、K268R、E270K、P275S、A278Y、E281L、F282Y、K289R、G291F、A298V、H304Q、S306T、A315L、S321A、A325G、I333N、K338G、G344A、N345S、A349V、T355S、G360C、H384S、A386S、A389S、K390T、Q392A、Q396R、E401K、N402M、N405K、S418A、L420F、S421R、S421Q、G427A、A428S、R429V、T431V、M434L、A435G、A435E、G436K、L437P。优选地,所述氨基酸序列还包含
-选自“KHPGG”、“QHPGG”或“RHPGG”的基序,优选选自“KHPGGD”、“QHPGGD”或“RHPGGD”的基序,和
-选自“GLNIQHDANHG”或“HVVGHH”,优选选自“AAIGLNIQHDANHG”或“QHVVGHH”的基序。
应理解,优选选择突变以增加突变序列与SEQ ID NO.79序列之间的序列同一性。照这样,特别制备非功能性突变体的危险降低。
因此,还优选的是本发明的δ-4去饱和酶具有SEQ ID NO.79的骨架,优选具有一个或多个以下突变:V9-、V10G、S11G、D12-、S13-、L20-、K21-、E29R、K37D、E42G、F63L、F63I、L76Y、L76F、L76W、Q78R、Y79R、S81W、L82P、S84A、E88K、Y89F、V94L、V94I、T104D、Q109R、K111C、A113E、R115N、W123G、W124F、W129Y、Y130W、I136L、L137I、D144E、Y145W、Y145F、W147M、I158V、S160L、Y164F、S179A、I205L、C216T、R218D、S229G、T232R、R235P、P239W、E241P、F242W、V262I、V262L、D268E、M272W、Y274W、K278P、L279I、K285P、I289P、I291V、R294K、V295L、V295I、F298W、V304L、V304I、H309Q、F310P、Y313H、A323C、A325G、L327F、G330A、A342V、A342I、P346G、E347P、A349G、K350-、W354F、S361T、C366G、K369W、I372H、Y379F、K414H、G416P、D423G、T425M、F426L、Q428H、V429L、V429I、A436P、Y438-、N439-、E440D。在这些突变中,更优选以下:V9-、V10G、S11G、D12-、S13-、L20-、K21-、E29R、F63L、L76Y、L76f、S81W、L82P、Y89F、V94L、T104D、K111C、W123G、W124F、Y130W、I136L、Y164F、R218D、S229G、T232R、R235P、P239W、E241P、F242W、V262i、M272W、Y274W、K285P、F298W、V304L、A325G、L327F、E347P、K350-、C366G、K369W、I372H、K414H、G416P、Q428H、V429L、A436P、Y438-、N439-。甚至更优选的是以下突变:V9-、D12-、S13-、L20-、K21-、L82P、W123G、Y130W、R235P、P239W、E241P、M272W、Y274W、K285P、F298W、K350-、C366G、G416P、Y438-、N439-。当与δ-5延伸酶组合时仍然产生高效δ-4去饱和酶的SEQ ID NO.79单独或组合的10个最优选突变是:V9-、D12-、S13-、L20-、K21-、Y130W、R235P、K350-、Y438-、N439-。在这些列表中,“-”表示缺失。
本发明还提供筛选δ-4去饱和酶基因的方法,其包括步骤
a)从属于Ichthyosporea或定鞭藻纲(Haptophyceae)的分类等级,优选鱼孢霉目(Ichthyophonida)或Pavlovales目,更优选Anurofeca、Creolimax、Ichthyophonus、帕金虫(Pseudoperkinsus)、胶孢子虫孢子(Psorospermium)或Sphaeroforma属的生物提取遗传物质,
b)将所述遗传物质与编码如上文或下文定义的本发明的δ-4去饱和酶的至少10个,优选至少20个连续氨基酸的核酸在严格条件下杂交,和
c)检测杂交或杂交的缺失。
杂交表明在步骤a)中提供遗传的生物确实包含δ-4去饱和酶的基因。
特别优选的是通过核酸扩增反应检测杂交或杂交的缺失。如果探针与充当扩增反应模板的遗传物质杂交,那么该反应将仅提供明显的产物。因此,本发明还提供筛选δ-4去饱和酶基因的方法,其包括步骤
a)从属于Ichthyosporea或定鞭藻纲的分类等级,优选鱼孢霉目或Pavlovales目,更优选Anurofeca、Creolimax、Ichthyophonus、帕金虫、胶孢子虫孢子或Sphaeroforma属的生物提取遗传物质,
b)提供用于核酸扩增反应的反应物,用于在严格条件下扩增编码如上文或下文定义的本发明的δ-4去饱和酶的至少10个,优选至少20个连续氨基酸的核酸,和
c)检测扩增或扩增的缺失。
扩增表明在步骤a)中提供遗传物质的生物确实包含δ-4去饱和酶的基因。
用于杂交的核酸或用于核酸扩增,特别是聚合酶链式反应的反应物优选针对编码保守氨基酸基序的核酸区段,其中所述基序优选包含
-选自“KHPGG”、“QHPGG”或“RHPGG”的基序,优选选自“KHPGGD”、“QHPGGD”或“RHPGGD”的基序,和
-选自“GLNIQHDXNHG”或“HVVGHH”的基序,优选选自“AAIGLNIQHDANHG”或“QHVVGHH”的基序。
下文描述了用于聚合酶链式反应的合适引物。
因此,本发明还涉及包含选自以下的核酸序列的多核苷酸:
a)具有如SEQ ID NOs:1、4、7、10、13、16、19、22、25、28、31、78或83中所示核苷酸序列的核酸序列,
b)编码具有如SEQ ID NOs:2、5、8、11、14、17、20、23、26、29、32、79、84、85或86中所示氨基酸序列的多肽,或上述δ-4去饱和酶的核酸序列,
c)与a)或b)的核酸序列具有至少70%同一性的核酸序列,其中所述核酸序列编码具有去饱和酶、酮酰基-CoA合酶(KCS)或酮酰基-CoA还原酶(KCR)或溶血磷脂-辅酶A合酶(LACS)活性的多肽,
d)编码具有去饱和酶、酮酰基-CoA合酶(KCS)、酮酰基-CoA还原酶(KCR)或溶血磷脂-辅酶A合酶(LACS)活性并具有与a)-c)任一项的氨基酸序列具有至少60%同一性的氨基酸序列的多肽的核酸序列;和
e)能够在严格条件下与a)-d)任一项杂交的核酸序列,其中所述核酸序列编码具有去饱和酶、酮酰基-CoA合酶(KCS)、酮酰基-CoA还原酶(KCR)或溶血磷脂-辅酶A合酶(LACS)活性的多肽。
根据本发明所用的术语“多核苷酸”指这样的多核苷酸,其包含编码具有去饱和酶、酮酰基-CoA合酶(KCS)、酮酰基-CoA还原酶(KCR)或溶血磷脂-辅酶A合酶(LACS)活性的多肽的核酸序列。优选地,本发明多核苷酸编码的分别具有去饱和酶、KCS、KCR或LACS活性的多肽在植物中表达时应该能够增加例如种子油或完整植物或其部分中PUFA的量,特别是LCPUFA的量。与产生LCPUFA的转基因对照植物相比时,这种增加优选地是统计学显著的,所述转基因对照植物表达为LCPUFA合成所需的最小集合的去饱和酶和延伸酶,但是不表达本发明的多核苷酸。可以通过本领域熟知的统计学检验法(包括例如斯氏t-检验)确定增加是否是显著的。更优选地,增加是与所述对照相比,含有LCPUFA的甘油三酯的量增加至少5%、至少10%、至少15%、至少20%或至少30%。优选地,之前所指的LCPUFA是具有C-20、C-22或C-24脂肪酸主体(body)的多不饱和脂肪酸,更优选是ARA、EPA或DHA。在所附实施例中描述用于测定之前所提及的活性的合适测定法。
如本文所用的术语“酰基转移酶活性”或“酰基转移酶”包括所有的酶活性和酶,其通过转酯过程能够转移或参与将PUFA,特别是LCPUFA从酰基-CoA池或膜脂转移到甘油三酯中,从酰基-CoA池转移到膜脂和从膜脂转移到酰基-CoA池。将理解的是,该酰基转移酶活性将导致例如种子油中酯化成甘油三酯的LCPUFA增加。特别地,设想这些酰基转移酶能够产生具有酯化的EPA或甚至DHA的甘油三酯,或设想这些酰基转移酶能够通过在酰基-CoA池(延伸位点)和膜脂(去饱和的主要位点)之间增加所需PUFA的特定中间物的流量而增加所需PUFA的合成。尤其是,如本文所用的酰基转移酶活性涉及溶血磷脂酰基转移酶(LPLAT)活性,优选地溶血磷脂酰胆碱酰基转移酶(LPCAT)或溶血磷脂酰乙醇胺酰基转移酶(LPEAT)活性、溶血磷脂酸酰基转移酶(LPAAT)活性、甘油-3-磷酸酰基转移酶(GPAT)活性或二酰甘油酰基转移酶(DGAT),并且更优选地涉及PLAT、LPAAT、DGAT或GPAT活性。
术语“去饱和酶”包括所有的酶活性和酶,其催化具有不同长度和不饱和碳原子双键数量的脂肪酸的去饱和作用。尤其是,这包括δ-4(d4)去饱和酶,其催化第4个和第5个碳原子的脱氢作用。δ-5(d5)去饱和酶,其催化第5个和第6个碳原子的脱氢作用。δ-6(d6)去饱和酶,其催化第6个和第7个碳原子的脱氢作用。δ-8(d8)去饱和酶,其催化第8个和第9个碳原子的脱氢作用。δ-9(d9)去饱和酶,其催化第9个和第10个碳原子的脱氢作用。δ-12(d12)去饱和酶,其催化第12个和第13个碳原子的脱氢作用。δ-15(d15)去饱和酶,其催化第15个和第16个碳原子的脱氢作用。应理解,脂肪酸可以结合酰基载体蛋白质(ACP)、辅酶A(CoA)或磷脂中,由此形成不同的池。去饱和酶一般展示对这些池之一的优选性。
术语“延伸酶”和“d5Elo、d6Elo或d9Elo”与KCS同义并且指酮酰基-CoA合酶酶活性,其允许在脂肪酸中引入两个碳原子,由此延长脂肪酸。尤其是,d5Elo、d6Elo或d9Elo催化将两个碳原子引入到在位置5、6或9上分别具有18个或20个碳原子和双键的脂肪酸中。
如本文所用的术语“KCR”指酮酰基-CoA还原酶活性,其在脂肪酸延伸过程中将酮酰基-CoA的酮基基团还原成羟基基团。
如本文所用的术语“DH”指脱水酶活性,其去除羟基基团,导致酰基-2-烯-CoA酯(δ-2-烯酰-CoA)和H2O在脂肪酸延伸过程中的形成。
如本文所用的术语“ECR”指烯酰-CoA还原酶活性,其在脂肪酸延伸过程中还原δ-2-烯酰-CoA的双键,产生延长的酰基-CoA酯。
脂肪酸延伸以4种酶代表的四个步骤催化:KCR(酮酰基-CoA合酶)、KCR(酮酰基-CoA还原酶)、DH(脱水酶)和ECR(烯酰-CoA还原酶)。在第一步中,脂肪酸-CoA酯与丙二酰-CoA缩合,产生延长了两个碳原子的酮酰基-CoA中间体和CO2。该中间体的酮基基团然后由KCR还原成羟基基团。在下一步中,DH切去羟基基团(产生H2O),形成酰基-2-烯-CoA酯(δ-2-烯酰-CoA)。在最后一步中,在第2、3位置上的双键由ECR还原,形成延长的酰基-CoA酯(Buchanan,Gruissem,Jones(2000)Biochemistry&Molecular biology of plants,American Society of Plant Physiologists)。
在本发明的研究中,已经提供了对产生PUFA具有优良去饱和酶、KCS、KCR、DH和ECR催化活性的酶。
更优选地,具有如在SEQ ID NO:1中所示的核酸序列的多核苷酸编码具有如在SEQ ID NO:2中所示的氨基酸序列的多肽或其变体,所述多肽或其变体优选地展示d6-去饱和酶活性。
具有如在SEQ ID NO:4中所示的核酸序列的多核苷酸编码优选地展示d9-去饱和酶活性的具有如在SEQ ID NO:5中所示的氨基酸序列的多肽或其变体。
具有如在SEQ ID NO:7和10中所示的核酸序列的多核苷酸编码优选地展示o3-去饱和酶活性的具有如在SEQ ID NO:8和11中所示的氨基酸序列的多肽或其变体。
具有如在SEQ ID NO:13中所示的核酸序列的多核苷酸编码优选地展示d12-去饱和酶活性的具有如在SEQ ID NO:14中所示的氨基酸序列的多肽或其变体。
具有如在SEQ ID NO:78中所示的核酸序列的多核苷酸编码优选地展示d4-去饱和酶活性的具有如在SEQ ID NO:79中所示的氨基酸序列的多肽或其变体。已经在上文详细描述了其他优选的δ-4去饱和酶多肽序列。
具有如在SEQ ID NO.83中所示的核酸序列的多核苷酸编码优选地展示溶血磷脂辅酶A合酶活性的具有如在SEQ ID NO.84中所示的氨基酸序列的多肽或其变体。
具有如在SEQ ID NOs:16、19、22、25或28中所示的核酸序列的多核苷酸编码优选地展示酮酰基-CoA合酶活性的具有如在SEQ ID NOs:17、20、23、26或29中所示的氨基酸序列的多肽或其变体。尤其是,SEQID NO.16编码展示d5-延伸酶活性的多肽SEQ ID NO 17;SEQ ID NO.19、22和25分别编码展示d6-延伸酶活性的多肽SEQ ID NO 20、23和26;SEQ ID NO.28编码展示d9-延伸酶活性的多肽SEQ ID NO 29。
具有如在SEQ ID NO:31中所示的核酸序列的多核苷酸编码优选地展示酮酰基-CoA还原酶活性的具有如在SEQ ID NO:32中所示的氨基酸序列的多肽或其变体。
根据本发明,已经优选地从金黄色破囊壶菌(Thraustochytriumaureum)和Sphaeroforma arctica获得如上定义的编码具有去饱和酶、KCS、KCR和LACS活性的多肽的多核苷酸。然而,可以从其他物种中鉴定直向同源物、旁系同源物或其他同系物。优选地,它们获自植物如藻类,例如等鞭金藻属(Isochrysis)、Mantoniella、Ostreococcus或Crypthecodinium,藻类/硅藻类,如褐指藻、海链藻(Thalassiosira)或破囊壶菌(Thraustochytrium),领鞭虫(choanoflagellates),如Monosiga,苔藓,如剑叶藓属或角齿藓属(Ceratodon)或高等植物,如报春花科(Primulaceae),如石栗(Aleuritia)、Calendula stellata、Osteospermumspinescens或Osteospermum hyoseroides,微生物,如真菌,如曲霉属(Aspergillus)、疫霉属(Phytophthora)、虫霉属(Entomophthora)、毛霉属(Mucor)或被孢霉属,细菌,如希瓦氏菌(Shewanella),酵母或动物。优选的动物是线虫,如新杆状线虫(Caenorhabditis),昆虫或脊椎动物。在脊椎动物中,核酸分子可以优选来自真骨类(Euteleostomi)、辐鳍鱼纲(Actinopterygii);新鳍类(Neopterygii);真骨鱼类(Teleostei);真真骨鱼(Euteleostei)、原棘鳍总目(Protacanthopterygii)、鲑目(Salmoniformes);鲑科(Salmonidae)或大麻哈鱼属(Oncorhynchus),更优选来自鲑目,最优选地,鲑科,如鲑属(Salmo),例如来自裂喉大麻哈鱼(Oncorhynchusmykiss)、Trutta trutta或河鲑(Salmo trutta fario)的属和种。此外,该核酸分子可以获自硅藻类,如海链藻属或褐指藻属。
因此,根据本发明所用的术语“多核苷酸”还包括上述特定多核苷酸的变体,其代表了本发明多核苷酸的直向同源物、旁系同源物或其他同系物。此外,本发明多核苷酸的变体也包括人工改造的突变蛋白。所述突变蛋白包括,例如通过诱变技术产生的并且显示出改进的或改变的底物特异性的酶,或密码子优化的多核苷酸。多核苷酸变体优选包含这样的核酸序列,所述核酸序列的特征在于,其序列可以通过编码具有如在SEQ ID NOs:2、5、8、11、14、17、20、23、26、29、32、79、84、85或86任一项所示的氨基酸序列的多肽的多核苷酸的至少一个核苷酸取代、添加和/或缺失衍生自SEQ ID NOs:1、4、7、10、13、16、19、22、25、28、31、78或83任一项所示的上述特定核酸序列,由此变体核酸序列应当仍编码具有如上所定义的去饱和酶、KCS、KCR和LACS活性的多肽。变体还包括这样的多核苷酸,其包含能够与上述特定核酸序列优选在严格条件下杂交的核酸序列。这些严格条件为本领域技术人员所知并可见于Current Protocolsin Molecular Biology,John Wiley&Sons,N.Y.(1989),6.3.1-6.3.6.中。严格杂交条件的优选实例是在约45℃的6×氯化钠/柠檬酸钠(=SSC)中的杂交条件,然后在50-65℃下在0.2×SSC,0.1%SDS中进行一次或多次洗涤步骤。技术人员知道根据核酸类型和例如当存在有机溶剂时,在温度和缓冲液浓度方面,杂交条件不同。例如,在“标准杂交条件”下,在浓度为0.1至5×SSC(pH 7.2)的水性缓冲溶液中,温度根据核酸类型而在42℃和58℃之间不同。如果上述缓冲液中存在有机溶剂,例如50%甲酰胺,则标准条件下的温度是大约42℃。对于DNA:DNA杂交体,杂交条件优选是0.1×SSC和20℃至45℃,优选在30℃至45℃之间。对于DNA:RNA杂交体,杂交条件优选是0.1×SSC和30℃至55℃,优选在45℃至55℃之间。例如为长度约100bp(=碱基对),G+C含量为50%的核酸,在缺少甲酰胺的情况下确定上述杂交温度。技术人员通过参考教科书,例如上文提及的教科书或以下教科书知道如何确定所需的杂交条件:Sambrook等,“Molecular Cloning”,Cold Spring Harbor Laboratory,1989;Hames和Higgins(编辑)1985,“Nucleic Acids Hybridization:A PracticalApproach”,IRL Press at Oxford University Press,Oxford;Brown(编辑)1991,“Essential Molecular Biology:A Practical Approach”,IRL Press atOxford University Press,Oxford。或者,通过基于PCR的技术,如基于混合的寡核苷酸引物的DNA扩增,即,使用针对本发明多肽的保守结构域的简并引物,可以获得多核苷酸变体。可以通过序列比较本发明多核苷酸的核酸序列或多肽的氨基酸序列来鉴定本发明多肽的保守结构域。在所附实施例中描述了适合作为PCR引物的寡核苷酸以及合适的PCR条件。作为模板,可以使用来自细菌、真菌、植物或动物的DNA或cDNA。此外,变体包括这样的多核苷酸,所述多核苷酸包含与SEQ ID NO:1、4、7、10、13、16、19、22、25、28、31、78或83任一项中所示的核酸序列具有至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少98%或至少99%同一性的核酸序列,优选地,其编码保留了如上所指定的去饱和酶、KCS、KCR和LACS活性的多肽。此外,还包括这样的多核苷酸,其包含编码具有与SEQ ID NO:2、5、8、11、14、17、20、23、26、29、32、79、84、85或86任一项中所示的氨基酸序列具有至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少98%或至少99%同一性的氨基酸序列的多肽的核酸序列,其中所述多肽优选保留了如上所指定的去饱和酶、KCS、KCR和LACS。优选在整个氨基酸或核酸序列区域上计算百分比同一性值。本领域技术人员可获得基于多种算法的一系列程序用于比较不同的序列。在优选的实施方案中,使用已经整合到EMBOSS软件包(EMBOSS:The EuropeanMolecular Biology Open Software Suite,Rice,P.,Longden,I.,和Bleasby,A,Trends in Genetics 16(6),276-277,2000)中的needle程序中的Needleman和Wunsch算法(Needleman 1970,J.Mol.Biol.(48):444-453),对远缘相关的蛋白质,使用BLOSUM 45或PAM250评分矩阵,或对近缘相关的蛋白质,使用BLOSUM 62或PAM160评分矩阵,和16、14、12、10、8、6或4的空位开放罚分和0.5、1、2、3、4、5或6的空位延伸罚分确定两条氨基酸序列之间的百分比同一性。可以在http://emboss.sourceforge.net发现EMBOSS包的本地安装以及与WEB-Services连接的指导。使用needle程序用于比对两条氨基酸序列的参数的优选非限制性实例是默认参数,包括EBLOSUM62评分矩阵、10的空位开放罚分和0.5的空位延伸罚分。在又一优选的实施方案中,使用EMBOSS软件包中的needle程序(EMBOSS:The European Molecular Biology Open Software Suite,Rice,P.,Longden,I.,和Bleasby,A,Trends in Genetics 16(6),276-277,2000),使用EDNAFULL评分矩阵和16、14、12、10、8、6或4的空位开放罚分和0.5、1、2、3、4、5或6的空位延伸罚分确定两条核苷酸序列之间的百分比同一性。使用needle程序用于组合比对两条核酸序列的参数的优选非限制性实例是默认参数,包括EDNAFULL评分矩阵、10的空位开放罚分和0.5的空位延伸罚分。可以将本发明的核酸和蛋白质序列进一步用作为“查询序列”以对公共数据库进行检索,以例如鉴定其他家族成员或相关序列。可以使用Altschul等(Altschul 1990,J.Mol.Biol.215:403-10)的BLAST系列程序(版本2.2)进行此类检索。可以利用BLASTn、BLASTx或tBLASTx程序使用默认参数进行使用本发明的去饱和酶、KCS、KCR和LACS核酸序列作为查询序列的BLAST,以获得与本发明的去饱和酶、KCS、KCR和LACS序列同源的核苷酸序列(BLASTn、tBLASTx)或氨基酸序列(BLASTx)。可以利用BLASTp或tBLASTn程序使用默认参数进行使用本发明的去饱和酶、KCS、KCR和LACS蛋白质序列作为查询序列的BLAST,以获得与本发明的去饱和酶、KCS、KCR和LACS序列同源的氨基酸序列(BLASTp)或核酸序列(tBLASTn)。为了获得用于比较目的的空位比对,如在Altschul等(Altschul 1997,Nucleic Acids Res.25(17):3389-3402)中所述,可以利用使用默认参数的空位BLAST。
表1:序列类型的关系:用于多种BLAST程序的查询和命中序列的DNA或PRT(蛋白质)
包含上述核酸序列任何一条的片段的多核苷酸也包括为本发明的多核苷酸。该片段应当编码仍具有如上所指定的去饱和酶、KCS、KCR或LACS活性的多肽。因此,该多肽可以包含赋予所述生物学活性的本发明多肽的结构域或由上述结构域组成。本文中所指的片段,优选包含上述核酸序列的任何一条的至少50、至少100、至少250或至少500个连续的核苷酸,或编码包含上述氨基酸序列任何一条的至少20、至少30、至少50、至少80、至少100或至少150个连续氨基酸的氨基酸序列。
上文所指变体多核苷酸或片段优选编码这样的多肽,其保留了显著程度的去饱和酶、KCS、KCR或LACS活性,优选是至少10%、至少20%、至少30%、至少40%、至少50%、至少60%、至少70%、至少80%或至少90%的饱和酶、KCS、KCR或LACS活性,所述去饱和酶、KCS、KCR或LACS活性由SEQ ID NOs:2、5、8、11、14、17、20、23、26、29、32、79、84、85或86任一项中所示的任何多肽展示。可以如在所附实施例中所述测试活性。
本发明的多核苷酸基本上由上述核酸序列组成或包含上述核酸序列。因此,它们也可以含有其他核酸序列。优选地,除可读框外,本发明的多核苷酸可以在编码基因区的3’和5’末端包含其他非翻译序列:编码区5’末端上游序列的至少500,优选200,更优选100个核苷酸和编码基因区3’末端下游序列的至少100,优选50,更优选20个核苷酸。此外,本发明的多核苷酸可以编码融合蛋白质,其中所述融合蛋白质的一个配偶体是由上文说明的核酸序列编码的多肽。此类融合蛋白质可以包含脂肪酸或PUFA生物合成途径的其他酶、用于监测表达的多肽(例如绿色、黄色、蓝色或红色荧光蛋白质、碱性磷酸酶等)或可以充当可检测标志物或辅助测定用于纯化目的的所谓的“标签”作为额外部分。用于不同目的的标签为本领域所熟知并包含FLAG-标签、6-组氨酸标签、MYC-标签等。
应该优选地以分离的多核苷酸(即纯化或至少从其天然背景,如其天然基因座中分离出来)或以遗传修饰或外源(即人工)操纵的形式提供本发明的多核苷酸。分离的多核苷酸可以例如包含少于约5kb、4kb、3kb、2kb、1kb、0.5kb或0.1kb的核苷酸序列,所述核苷酸序列天然地位于该核酸分子所来源的细胞的基因组DNA中该核酸分子的侧翼。优选地以双链或单链分子形式提供所述多核苷酸。应理解,本发明所提及的本发明的任何前述多核苷酸也指前述特定序列或其变体的互补链或反向互补链。多核苷酸包括DNA,包括cDNA和基因组DNA或RNA多核苷酸。
然而,本发明也涉及来自本发明的多核苷酸并能干扰本发明多核苷酸的转录或翻译的多核苷酸变体。此类变体多核苷酸包括反义核酸、核酶、siRNA分子、吗啉代核酸(磷酰二胺吗啉代寡核苷酸(phosphorodiamidatemorpholino oligos))、形成三螺旋的寡核苷酸、抑制性寡核苷酸或microRNA分子,由于存在互补的或基本上互补的序列,所述全部变体多核苷酸都应该特异识别本发明的多核苷酸。这些技术为本领域技术人员所熟知。基于本发明多核苷酸的结构,可以容易地设计前述种类的合适变体多核苷酸。
此外,还包含的是化学修饰的多核苷酸,包括天然存在的修饰多核苷酸,如糖基化的或甲基化的多核苷酸或人工修饰的一类,如生物素化的多核苷酸。
在本发明的研究中,有利地,其中所鉴定的多核苷酸编码来自金黄色破囊壶菌和Sphaeroforma arctica的去饱和酶、酮酰基-CoA合酶、酮酰基-CoA还原酶和溶血磷脂辅酶A合酶。具体而言,金黄色破囊壶菌d6-去饱和酶(pd6Des(Ta)_c3318)、d9-去饱和酶(pd9Des(Ta)_c4008)、o3-去饱和酶(po3Des(Ta)_c959、po3Des(Ta)_c1830)、d12-去饱和酶(pd12Des(Ta)_c1219)酮酰基-CoA-合酶(pd5Elo(Ta)_c1、pd6Elo(Ta)_c231、pd6Elo(Ta)_c752、pd6Elo(Ta)_c4696、pd9Elo(Ta)_c4589)酮酰基-CoA-还原酶(pKR(Ta)_c1703)和Sphaeroforma arctica d4-去饱和酶d4Des(Sa)和溶血磷脂辅酶A合酶(LACS)。本发明的多核苷酸特别适合用于重组制造LCPUFA,并且具体而言,花生四烯酸(ARA)、二十碳五烯酸(EPA)和/或二十二碳五烯酸(DHA)。
在本发明多核苷酸的优选实施方案中,所述多核苷酸进一步包含与所述核酸序列有效连接的表达控制序列。
如本文中所用的术语“表达控制序列”指能支配,即起始和控制目的核酸序列(在当前情况下为上文说明核酸序列)的转录的核酸序列。该序列一般包含或由启动子或启动子和增强子序列的组合组成。多核苷酸的表达优选地包含核酸分子转录成可翻译的mRNA。额外的调节元件可以包括转录以及翻译增强子。以下启动子和表达控制序列可以优选地用于本发明的表达载体。cos、tac、trp、tet、trp-tet、lpp、lac、lpp-lac、lacIq、T7、T5、T3、gal、trc、ara、SP6、λ-PR或λ-PL启动子优选用于革兰氏阴性菌。对于革兰氏阳性菌,可以使用启动子amy和SPO2。对于酵母或真菌,优选使用启动子ADC1、AOX1r、GAL1、MFα、AC、P-60、CYC1、GAPDH、TEF、rp28、ADH。对于动物细胞或生物表达,优选使用启动子CMV-、SV40-、RSV-启动子(劳氏肉瘤病毒)、CMV-增强子、SV40-增强子。对于植物,使用启动子CaMV/35S(Franck 1980,Cell 21:285-294]、PRP1(Ward1993,Plant.Mol.Biol.22)、SSU、OCS、lib4、usp、STLS1、B33、nos或泛素或菜豆蛋白启动子。在本上下文中还优选的是诱导型启动子,如在EP0 388 186 A1中描述的启动子(即苄基磺酰胺-诱导型启动子)、Gatz 1992,Plant J.2:397-404(即四环素-诱导型启动子)、EP0 335 528 A1(即脱落酸-诱导型启动子)或WO 93/21334(即乙醇-或环己烯醇-诱导型启动子)。其他合适的植物启动子是来自马铃薯的细胞质FBP酶启动子或ST-LSI启动子(Stockhaus 1989,EMBO J.8,2445)、来自大豆的磷酸核糖焦磷酸酰氨基转移酶启动子(Genbank登录号U87999)或在EP 0 249 676 A1中所述的结节特异性启动子。特别优选的是能在参与脂肪酸生物合成的组织中表达的启动子。根据实践,还特别优选的是种子特异性启动子,如USP启动子,还有其他启动子如LeB4、DC3、菜豆蛋白或油菜籽蛋白启动子。其他尤其优选的启动子是种子特异性启动子,其可用于单子叶或双子叶植物,并描述在US 5,608,152(来自油菜的油菜籽蛋白启动子)、WO 98/45461(来自拟南芥的油质蛋白启动子)、US5,504,200(来自菜豆(Phaseolus vulgaris)的菜豆蛋白启动子)、WO 91/13980(来自芸苔的Bce4启动子)、Baeumlein等人,PlantJ.,2,2,1992:233-239(来自豆科植物的LeB4启动子)中,这些启动子适于双子叶植物。以下启动子适合于单子叶植物:来自大麦的lpt-2或lpt-1启动子(WO 95/15389和WO 95/23230)、WO 99/16890中描述的合适的来自大麦的醇溶蛋白启动子和其他合适的启动子。原则上,对于新方法,可以使用所有的天然启动子及其调控序列,例如上文提及的那些。同样,可以并且有利地额外或单独地使用合成启动子,尤其是当它们介导种子特异性表达时,例如在WO 99/16890中所述。在特定的实施方案中,利用种子特异性启动子,以增强想要的PUFA或LCPUFA的产生。
如本文中所用的术语“有效连接”表示表达控制序列和目的核酸是连接的,从而所述目的核酸的表达可以受所述表达控制序列支配,即表达控制序列应当与待表达的所述核酸序列功能性连接。因此,表达控制序列和待表达的核酸序列可以彼此物理连接,例如,通过将表达控制序列插入到待表达核酸序列的5’末端。或者,表达控制序列和待表达的核酸可以仅仅是物理邻近的,从而表达控制序列能够支配至少一种目的核酸序列的表达。表达控制序列和待表达的核酸优选地相隔不多于500bp、300bp、100bp、80bp、60bp、40bp、20bp、10bp或5bp。
在本发明多核苷酸的其他优选实施方案中,所述多核苷酸进一步包含与所述核酸序列有效连接的终止序列。
如本文中所用的术语“终止子”指能够终止转录的核酸序列。这些序列会引起转录机器从待转录的核酸序列上解离。优选地,终止子应当是在植物中和,特别地在植物种子中有活性的。合适的终止子为本领域所知,并优选包括多腺苷酸化信号,如SV40聚腺苷酸位点或tk聚腺苷酸位点或在Loke等(Loke 2005,Plant Physiol 138,第1457-1468页)中指出的植物特异性信号之一,其位于待表达的核酸序列的下游。
本发明还涉及包含本发明的多核苷酸的载体。
术语“载体”或“构建体”优选包括噬菌体、质粒、病毒载体以及人工染色体,如细菌或酵母人工染色体。此外,该术语还指靶向构建体,其允许靶向构建体随机或位点定向整合到基因组DNA中。此类靶向构建体优选包含足够长度的DNA用于下文详细描述的同源或异源重组。包括本发明的多核苷酸的载体优选进一步包含选择标志物用于在宿主中的繁殖和/或选择。可以通过本领域所熟知的多种技术将载体掺入到宿主细胞中。如果引入到宿主细胞中,载体可以定居在细胞质中或者可以掺入到基因组中。在后者的情况下,应当理解,该载体可以进一步包含允许同源重组或者异源插入的核酸序列。通过常规转化或转染技术,可以将载体引入到原核或真核细胞中。如本文上下文使用的,术语“转化”和“转染”、接合和转导旨在包含用于向宿主细胞引入外源核酸(例如DNA)的多种现有技术方法,包括磷酸钙、氯化铷或氯化钙共沉淀、DEAE-葡聚糖-介导的转染、脂转染、天然感受态、基于碳的簇(carbon-based cluster)、化学介导的转移、电穿孔或粒子轰击。用于转化或转染宿主细胞(包括植物细胞)的合适方法可见于Sambrook等(Molecular Cloning:A Laboratory Manual,第2版,ColdSpring Harbor Laboratory,Cold Spring Harbor Laboratory Press,ColdSpring Harbor,NY,1989)和其他的实验室手册,如Molecular Biology,1995,第44卷,Agrobacterium protocols,编辑:Gartland和Davey,Humana Press,Totowa,New Jersey中的方法。或者,质粒载体可以通过热激或电穿孔技术来引入。如果载体是病毒,那么其可以在应用于宿主细胞之前使用合适的包装细胞系在体外进行包装。
优选地,本文中提及的载体适合用作克隆载体,即在微生物系统中是可复制的。此类载体确保在细菌并优选在酵母或真菌中有效克隆且可能稳定转化植物。具体而言,必须提及的那些载体是适用于T-DNA介导的转化的多种二元和共整合载体系统。此类载体系统的特征通常是它们至少含有为农杆菌介导的转化所需的vir基因和划界T-DNA的序列(T-DNA边界)。这些载体系统还优选包含其他顺式调节区域,如启动子和终止子和/或选择标志物,通过所述选择标志物可以鉴定合适的转化的宿主细胞或生物。尽管共整合载体系统具有在相同载体上排列的vir基因和T-DNA序列,但二元系统以至少两个载体为基础,其中一个载体携带vir基因但没有T-DNA,然而第二个载体携带T-DNA而无vir基因。因此,最后提及的载体相对较小,容易在大肠杆菌(E.coli)和农杆菌中操作和复制。这些二元载体包括来自pBIB-HYG、pPZP、pBecks、pGreen系列的载体。根据本发明优选使用的是Bin19、pBI101、pBinAR、pGPTV和pCAMBIA。二元载体及其用途的综述可见于Hellens等,Trends in Plant Science(2000)5,446–451。此外,通过使用适当的克隆载体,可以将多核苷酸引入宿主细胞或生物体内,如植物或动物,并因而用于转化植物,如在以下中公开和引用的那些:Plant Molecular Biology and Biotechnology(CRC Press,BocaRaton,Florida),第6/7章,第71-119页(1993);F.F.White,Vectors forGene Transfer in Higher Plants;Transgenic Plants,第1卷,Engineeringand Utilization,编辑:Kung和R.Wu,Academic Press,1993,15-38;B.Jenes等,Techniques for Gene Transfer,Transgenic Plants,第1卷,Engineering and Utilization,编辑:Kung和R.Wu,Academic Press(1993),128-143;Potrykus 1991,Annu.Rev.Plant Physiol.Plant Molec.Biol.42,205-225。
更优选地,本发明的载体是表达载体。在该表达载体,即包含本发明的多核苷酸的载体,其具有与表达控制序列有效连接的核酸序列(也称为“表达盒”),允许在原核或真核细胞或其分离的部分中表达。合适的表达载体为本领域所知,如Okayama-Berg cDNA表达载体pcDV1(Pharmacia)、pCDM8、pRc/CMV、pcDNA1、pcDNA3(Invitrogene)或pSPORT1(GIBCOBRL)。典型的融合表达载体的其他实例为pGEX(Pharmacia Biotech Inc;Smith 1988,Gene 67:31-40)、pMAL(New England Biolabs,Beverly,MA)和pRIT5(Pharmacia,Piscataway,NJ),其中谷胱甘肽S-转移酶(GST)、麦芽糖E结合蛋白和蛋白A分别与重组靶蛋白融合。合适的诱导型非融合大肠杆菌表达载体的实例尤其为pTrc(Amann 1988,Gene 69:301-315)和pET11d(Studier 1990,Methods in Enzymology 185,60-89)。pTrc载体的靶基因表达是基于通过宿主RNA聚合酶的从杂合trp-lac融合启动子的转录。来自pET 11d载体的靶基因表达是基于经共表达的病毒RNA聚合酶(T7gn1)所介导的T7-gn10-lac融合启动子的转录。该病毒聚合酶由宿主菌株BL21(DE3)或HMS174(DE3)从定居λ-原噬菌体提供,所述定居λ-原噬菌体携带受lacUV 5启动子转录控制的T7gn1基因。技术人员熟悉适用于原核生物的其他载体;这些载体是例如大肠杆菌中的pLG338、pACYC184、pBR系列如pBR322、pUC系列如pUC18或pUC19、M113mp系列、pKC30、pRep4、pHS1、pHS2、pPLc236、pMBL24、pLG200、pUR290、pIN-III113-B1、λgt11或pBdCl,链霉菌中的plJ101、plJ364、plJ702或plJ361,芽孢杆菌中的pUB110、pC194或pBD214,棒状杆菌中的pSA77或pAJ667。用于在酿酒酵母(S.cerevisiae)中表达的载体的实例包含pYepSec1(Baldari 1987,Embo J.6:229-234)、pMFa(Kurjan 1982,Cell30:933-943)、pJRY88(Schultz 1987,Gene 54:113-123)和pYES2(InvitrogenCorporation,San Diego,CA)。适用于其他真菌(如丝状真菌)的载体和用于载体构建的方法包括在以下中详细描述的那些:van den Hondel,C.A.M.J.J.,&Punt,P.J.(1991)“Gene transfer systems and vectordevelopment for filamentous fungi,in:Applied Molecular Genetics of fungi,J.F.Peberdy等,编辑,第1-28页,Cambridge University Press:Cambridge,或More Gene Manipulations in Fungi(J.W.Bennett&L.L.Lasure,编辑,第396-428页:Academic Press:San Diego)。其他合适的酵母载体是,例如pAG-1、YEp6、YEp13或pEMBLYe23。作为备选,本发明的多核苷酸还可以使用杆状病毒表达载体在昆虫细胞表达。可用于在培养的昆虫细胞(例如Sf9细胞)中表达蛋白质的杆状病毒载体包含pAc系列(Smith 1983,Mol.Cell Biol.3:2156-2165)和pVL系列(Lucklow 1989,Virology170:31-39)。
可以通过使用植物表达载体,在单细胞植物细胞(如藻类)(参见Falciatore 1999,Marine Biotechnology 1(3):239-251及其中所引用的参考文献)和来自高等植物(例如种子植物(Spermatophyte),如农耕作物)的植物细胞中表达本发明的多核苷酸。植物表达载体的实例包含在以下中详细描述的那些:Becker 1992,Plant Mol.Biol.20:1195-1197;Bevan 1984,Nucl.Acids Res.12:8711-8721;Vectors for Gene Transfer in Higher Plants;Transgenic Plants,第1卷,Engineering and Utilization,编辑:Kung和R.Wu,Academic Press,1993,第15-38页。植物表达盒优选包含能够在植物细胞中控制基因表达并有效连接的调节序列,从而每一种序列都可以实现其功能,如转录终止功能,所述调节序列为例如多聚腺苷酸化信号。优选的多聚腺苷酸化信号是来自根癌农杆菌(Agrobacterium tumefaciens)T-DNA,如Ti质粒pTiACH5(Gielen 1984,EMBO J.3,835)的基因3的那些多聚腺苷酸化信号,已知所述基因3是章鱼碱合酶(Gielen 1984,EMBO J.3,835),或其功能性等同物,但是在植物中具有功能性活性的所有其他终止子也是合适的。由于植物基因表达通常不限于转录水平,植物表达盒优选包含其他功能连接的序列,如翻译增强子,例如超驱动序列,其包含增加蛋白质/RNA比的5’-非翻译烟草花叶病毒前导序列(Gallie1987,Nucl.Acids Research 15:8693-8711)。如上所述,植物基因表达必须与合适的启动子功能连接,所述启动子以适时细胞特异性或组织特异性方式进行基因的表达。可使用的启动子是组成型启动子(Benfey 1989,EMBOJ.8:2195-2202),如来自植物病毒如35S CAMV(Franck 1980,Cell21:285-294)、19S CaMV(参见US 5352605和WO 84/02913)的那些启动子,或植物启动子,如在US 4,962,028中描述的Rubisco小亚基的启动子。用于在植物基因表达盒中功能连接的其他优选序列是靶向序列,其为将基因产物靶向其相关的细胞区室中所需(对于综述,参见Kermode 1996,Crit.Rev.Plant Sci.15,4:285-423和其中引用的参考文献),例如靶向液泡、细胞核、所有类型质体如造粉体、叶绿体、色质体、胞外空间、线粒体、内质网、油质体、过氧化物酶体和植物细胞的其他区室。如上所述,还可以通过化学诱导型启动子促进植物基因表达(对于综述,参见Gatz 1997,Annu.Rev.Plant Physiol.Plant Mol.Biol.,48:89-108)。如果希望基因以时间特异性方式表达,那么化学诱导型启动子是特别合适的。此类启动子的实例是水杨酸诱导型启动子(WO 95/19443)、四环素诱导型启动子(Gatz1992,Plant J.2,397-404)和乙醇诱导型启动子。应答生物或非生物的胁迫条件的启动子也是合适的启动子,例如病原体诱导的PRP1基因启动子(Ward 1993,Plant Mol.Biol.22:361-366)、来自番茄的热诱导型hsp80启动子(US 5,187,267)、来自马铃薯的冷诱导型α淀粉酶启动子(WO 96/12814)或创伤诱导型pinII启动子(EP 0 375 091 A)。尤其优选的启动子是在发生脂肪酸、脂类和油生物合成的组织和器官中,在种子细胞中,如胚乳细胞和发育胚的细胞中引起基因表达的那些启动子。合适的启动子是来自油菜的油菜籽蛋白基因启动子(US 5,608,152)、来自蚕豆(Vicia faba)的USP启动子(Baeumlein 1991,Mol.Gen.Genet.225(3):459-67)、来自拟南芥的油质蛋白启动子(WO 98/45461)、来自菜豆的菜豆蛋白启动子(US 5,504,200)、来自芸苔属的Bce4启动子(WO 91/13980)或豆球蛋白B4启动子(LeB4;Baeumlein 1992,Plant Journal,2(2):233-9),以及在单子叶植物如玉米、大麦、小麦、黑麦、稻等中引起种子特异性表达的启动子。待考虑的合适启动子是来自大麦的lpt2或lpt1基因启动子(WO 95/15389和WO95/23230),或在WO 99/16890中描述的那些启动子(来自大麦的大麦醇溶蛋白基因、稻的谷蛋白基因、稻的水稻素基因、稻的谷醇溶蛋白基因、小麦的小麦醇溶蛋白基因、小麦的谷蛋白基因、玉米的玉米醇溶蛋白基因、燕麦的谷蛋白基因、高粱的kasirin基因、黑麦的裸麦醇溶蛋白基因的启动子)。由于质体是合成脂类生物合成的前体和某些终产物的区室,因此,同样尤其适合的是引起质体特异性表达的启动子。合适的启动子,如WO95/16783和WO 97/06250中描述的病毒RNA聚合酶启动子,和WO99/46394中描述的来自拟南芥的clpP启动子。
上述载体仅仅是对待根据本发明使用的载体的小型概述。其他载体为技术人员所知并描述于例如:Cloning Vectors(编辑,Pouwels,P.H.,等,Elsevier,Amsterdam-New York-Oxford,1985,ISBN 0444904018)。对于用于原核和真核细胞的其他合适表达系统,参见上述Sambrook的第16和17章。
由上可知,优选所述载体是表达载体。更优选地,本发明的所述多核苷酸处于在本发明的载体中的种子特异性启动子的控制下。本文中所指的优选的种子特异性启动子选自Conlinin 1、Conlinin 2、油菜籽蛋白、LuFad3、USP、LeB4、Arc、Fae、ACP、LuPXR和SBP。对于详细信息,参见例如US 2003-0159174。
此外,本发明涉及包含本发明的多核苷酸或载体的宿主细胞。
优选地,所述宿主细胞是植物细胞和,更优选地,植物细胞获自油料作物。更优选地,所述油料作物选自亚麻(亚麻属的种(Linum sp.))、油菜(芸苔属的种(Brassica sp.))、大豆(大豆属和黄豆属的种(Glycine和Soja sp.))、向日葵、(向日葵属的的种(Helianthus sp.))、棉花(棉属的种(Gossypiumsp.))、玉米(玉米(Zea mays))、橄榄(洋橄榄属的种(Olea sp.))、红花(红蓝花属的种(Carthamus sp.))、可可(可可树(Theobroma cacoa)、花生(落花生属的种(Arachis sp.))、大麻、亚麻荠属(camelina)、两节荠属(crambe)、油椰、椰子、落花生、芝麻籽、蓖麻籽、lesquerella、乌桕、sheanuts、油桐果(tungnuts)、木棉花果实(kapok fruit)、罂粟籽、西蒙德木籽(jojobaseeds)和紫苏属(perilla)。
还优选地,所述宿主细胞是微生物。更优选地,所述微生物是细菌、真菌或藻类。更优选地,其选自假丝酵母属(Candida)、隐球酵母属(Cryptococcus)、油脂酵母属(Lipomyces)、红冬孢酵母属(Rhodosporidium)、耶氏酵母属(Yarrowia)和裂殖壶菌属。
此外,本发明的宿主细胞也可以是动物细胞。优选地,所述动物宿主细胞是鱼的宿主细胞或者从其中获得的细胞系。更优选地,鱼宿主细胞来自鲱鱼、鲑鱼、沙丁鱼、雄鲑、鳗鲡、鲤鱼、鳟鱼、大比目鱼、鲭鱼、豹纹鱼或金枪鱼。
一般地,LCPUFA的产生,即长链不饱和脂肪酸生物合成途径的控制步骤由膜结合的脂肪酸延伸酶复合体催化。植物和大多数其他真核生物具有特化的延伸酶系统,用于延长超过C18原子的脂肪酸。这些延伸酶反应与脂肪酸合酶复合体(FAS)具有相同的几个重要特征。然而,延伸酶复合体与FAS复合体不同,因为该复合体定位在胞质中并且是膜结合的,ACP不参与并且延伸酶3-酮-酰基-CoA合酶催化丙二酰-CoA与酰基引物的缩合。延伸酶复合体由具有不同催化功能的4个组分组成:酮酰基-CoA合酶(KCS,丙二酰-CoA与酰基-CoA的缩合反应,产生2个C原子的较长酮酰基-CoA脂肪酸)、酮酰基-CoA还原酶(KCR,将3-酮基基团还原为3-羟基基团)、脱水酶(DH,脱水产生δ-2-烯酰-酰基-CoA脂肪酸)和烯酰-CoA还原酶(ECR,还原位置2处的双键,从复合体中释放)。对于包括ARA、EPA和/或DHA的LCPUFA的产生,延伸和去饱和反应是必需的。高等植物不具有用于产生LCPUFA(4个或更多个双键,20个或更多个C原子)所必需的酶。因而,需要赋予植物或植物细胞催化活性。LCPUFA生物合成方法中的重要步骤是脂肪酸从18个延伸到24个碳原子和碳原子的去饱和。本发明的多核苷酸令人惊奇地催化酮酰基-CoA合酶、酮酰基-CoA还原酶反应,并因而催化18碳原子脂肪酸的延长。本发明的多核苷酸令人惊奇地催化第4个、第9个、第12个、第15个和第17个脂肪酸碳原子键的去饱和。通过递送这些酶,产生增加水平的PUFA和LCPUFA。
然而,应当理解根据宿主细胞,可以例如通过重组技术进一步赋予宿主细胞酶活性。因此,本发明优选设想如下宿主细胞,根据所选择的宿主细胞,除本发明的多核苷酸外,其还包含根据需要编码此类去饱和酶和/或延伸酶的多核苷酸。应该在宿主细胞中存在的优选的去饱和酶和/或延伸酶是选自以下的至少一种酶:d4-去饱和酶、d5-去饱和酶、d5-延伸酶、d6-去饱和酶、d12-去饱和酶、d15-去饱和酶、ω3-去饱和酶和d-6-延伸酶或d-9-延伸酶。尤其优选的是来自卡氏棘阿米巴(Acanthamoeba castellanii)的双功能性d12d15-去饱和酶d12d15Des(Ac)(WO2007042510)、来自麦角菌(Claviceps purpurea)的d12d15Des(Cp)(WO2008006202)和来自Lottiagigantea的d12d15Des(Lg)1(WO2009016202)、来自金盏菊(Calendulaofficinalis)的d12-去饱和酶d12Des(Co)(WO200185968)、来自双色蜡蘑(Laccaria bicolor)的d12Des(Lb)(WO2009016202)、来自领鞭毛虫(Monosiga brevicollis)的d12Des(Mb)(WO2009016202)、来自禾生球腔菌(Mycosphaerella graminicola)的d12Des(Mg)(WO2009016202)、来自Nectria haematococca的d12Des(Nh)(WO2009016202)、来自Ostreococcuslucimarinus的d12Des(Ol)(WO2008040787)、来自布拉克须霉菌(Phycomyces blakesleeanus)的d12Des(Pb)(WO2009016202)、来自大豆疫霉(Phytophthora sojae)的d12Des(Ps)(WO2006100241)和来自假微型海链藻(Thalassiosira pseudonana)的d12Des(Tp)(WO2006069710)、来自Helobdella robusta的d15-去饱和酶d15Des(Hr)(WO2009016202)、来自微鞘藻(Microcoleus chthonoplastes)的d15Des(Mc)(WO2009016202)、来自香蕉黑条叶斑病菌(Mycosphaerella fijiensis)的d15Des(Mf)(WO2009016202)、来自禾生球腔菌的d15Des(Mg)(WO2009016202)和来自Nectria haematococca的d15Des(Nh)2(WO2009016202)、来自纤细裸藻(Euglena gracilis)的d4-去饱和酶d4Des(Eg)(WO2004090123)、来自破囊壶菌种(Thraustochytrium sp.)的d4Des(Tc)(WO2002026946)和来自假微型海链藻的d4Des(Tp)(WO2006069710)、来自Ostreococcus lucimarinus的d5-去饱和酶d5Des(Ol)2(WO2008040787)、来自展叶剑叶藓(Physcomitrella patens)的d5Des(Pp)(WO2004057001)、来自三角褐指藻(Phaeodactylumtricornutum)的d5Des(Pt)(WO2002057465)、来自破囊壶菌属的d5Des(Tc)(WO2002026946)、来自假微型海链藻的d5Des(Tp)(WO2006069710)和来自角齿藓(Ceratodon purpureus)的d6-去饱和酶d6Des(Cp)(WO2000075341)、来自Ostreococcus lucimarinus的d6Des(Ol)(WO2008040787)、来自Ostreococcus tauri的d6Des(Ot)(WO2006069710)、来自粉报春(Primula farinosa)的d6Des(Pf)(WO2003072784)、来自拉曼被孢霉菌(Pythium irregulare)的d6Des(Pir)_BO(WO2002026946)、来自拉曼被孢霉菌的d6Des(Pir)(WO2002026946)、来自Primula luteola的d6Des(Plu)(WO2003072784)、来自展叶剑叶藓的d6Des(Pp)(WO200102591)、来自三角褐指藻的d6Des(Pt)(WO2002057465)、来自高穗花报春(Primula vialii)的d6Des(Pv)(WO2003072784)和来自假微型海链藻的d6Des(Tp)(WO2006069710)、来自Acanthamoeba castellanii的d8-去饱和酶d8Des(Ac)(EP1790731)、来自纤细裸藻的d8Des(Eg)(WO200034439)和来自Perkinsus marinus的d8Des(Pm)(WO2007093776)、来自致病疫霉(Phytophthora infestans)的o3-去饱和酶o3Des(Pi)(WO2005083053)、来自拉曼被孢霉菌的o3Des(Pir)(WO2008022963)、来自拉曼被孢霉菌的o3Des(Pir)2(WO2008022963)和来自大豆疫霉菌的o3Des(Ps)(WO2006100241)、来自裂喉大麻哈鱼(Oncorhynchus mykiss)的双功能性d5d6-延伸酶d5d6Elo(Om)2(WO2005012316)、来自金黄色破囊壶菌的d5d6Elo(Ta)(WO2005012316)和来自破囊壶菌属的d5d6Elo(Tc)(WO2005012316)、来自拟南芥(Arabidopsis thaliana)的d5-延伸酶d5Elo(At)(WO2005012316)、来自拟南芥的d5Elo(At)2(WO2005012316)、来自玻璃海鞘(Ciona intestinalis)的d5Elo(Ci)(WO2005012316)、来自Ostreococcus lucimarinus的d5Elo(Ol)(WO2008040787)、来自Ostreococcus tauri的d5Elo(Ot)(WO2005012316)、来自假微型海链藻的d5Elo(Tp)(WO2005012316)和来自光滑爪蟾(Xenopus laevis)的d5Elo(Xl)(WO2005012316)、来自Ostreococcus lucimarinus的d6-延伸酶d6Elo(Ol)(WO2008040787)、来自Ostreococcus tauri的d6Elo(Ot)(WO2005012316)、来自致病疫霉的d6Elo(Pi)(WO2003064638)、来自拉曼被孢霉菌的d6Elo(Pir)(WO2009016208)、来自展叶剑叶藓的d6Elo(Pp)(WO2001059128)、来自大豆疫霉菌的d6Elo(Ps)(WO2006100241)、来自大豆疫霉菌的d6Elo(Ps)2(WO2006100241)、来自大豆疫霉菌的d6Elo(Ps)3(WO2006100241)、来自三角褐指藻的d6Elo(Pt)(WO2005012316)、来自破囊壶菌属的d6Elo(Tc)(WO2005012316)和来自假微型海链藻的d6Elo(Tp)(WO2005012316)、来自球等鞭金藻(Isochrysis galbana)的d9-延伸酶d9Elo(Ig)(WO2002077213)、来自Perkinsus marinus的d9Elo(Pm)(WO2007093776)和来自米根霉(Rhizopus oryzae)的d9Elo(Ro)(WO2009016208)。特别地,如果设想在高等植物中制造ARA,那么可以在宿主细胞中组合在下表5或6中描述的酶(即另外的d6-去饱和酶、d6-延伸酶、d5-去饱和酶和d12-去饱和酶)或具有基本上相同的活性的酶。如果设想在高等植物中制造EPA,那么可以在宿主细胞中组合在下表7中描述的酶(即另外的d6-去饱和酶、d6-延伸酶、d5-去饱和酶、d12-去饱和酶、ω3-去饱和酶和d15去饱和酶)或具有基本上相同的活性的酶。如果设想在高等植物中制造DHA,那么可以在宿主细胞中组合在下表8中描述的酶(即另外的d6-去饱和酶、d6-延伸酶、d5-去饱和酶、d12-去饱和酶、ω3-去饱和酶、d15去饱和酶、d5-延伸酶和d4-去饱和酶)或具有基本上相同的活性的酶。
本发明还涉及细胞,优选如上指定的宿主细胞或在本文中其他地方指定的非人类生物的细胞,所述细胞包含这样的多核苷酸,其通过点突变、截短、倒位、缺失、添加、取代和同源重组获自本发明的多核苷酸。如何对多核苷酸实施此类修饰为本领域技术人员所熟知并已经在本说明书中的其他地方进行了详细描述。
本发明还提供包含δ-4去饱和酶、酮酰基-CoA合酶(KCS)、酮酰基-CoA还原酶(KCR)或溶血磷脂辅酶A合酶(LACS)活性和/或相应的核酸的植物或植物部分,也提供相应的死植物或其部分,优选收获的材料,例如种子或叶,或废弃材料,例如麦秆、死叶和压榨饼。压榨饼是挤压植物种子或其他植物材料用于油提取后获得的物质。此类压榨饼一般仍包含高浓度的多不饱和脂肪酸并特别适合作为动物饲料,例如鱼饲料。本发明还提供包含植物材料,优选如上所述种子,和/或废弃材料的容器。
本发明此外涉及用于制造任何本发明的多核苷酸编码的多肽的方法,其包括
a)在允许所述多肽产生的条件下培育本发明的宿主细胞;和
b)从步骤a)的宿主细胞中获得多肽。
允许表达由宿主细胞包含的本发明多核苷酸的合适条件取决于宿主细胞以及用于支配所述多核苷酸表达的表达控制序列。这些条件以及如何选择它们为本领域技术人员所熟知。表达的多肽可以例如通过所有常规纯化技术获得,所述常规纯化技术包括亲和层析、大小排阻层析、高压液相层析(HPLC)和包括抗体沉淀的沉淀技术。应当理解,尽管是优选的,但是该方法可能并不必定产生多肽的基本纯制剂。应当理解,根据用于前述方法的宿主细胞,由此产生的多肽可以是翻译后修饰的或者另外加工的。
本发明包括由本发明的多核苷酸编码的或者由前述方法获得的多肽。
如本文中所用的术语“多肽”包括基本纯的多肽或此外包含其他蛋白质的多肽制剂。此外,该术语还涉及融合蛋白质或由上文所指的本发明多核苷酸至少部分编码的多肽片段。此外,其包括化学修饰的多肽。此类修饰可以是人工修饰或天然存在的修饰,例如磷酸化、糖基化、十四烷基化等(Mann 2003,Nat.Biotechnol.21,255–261中的综述,Huber 2004,Curr.Opin.Plant Biol.7,318-322中着重在植物上综述)。目前,已知超过300种的翻译后修饰(参见在http://www.abrf.org/index.cfm/dm.home中列出的full ABFRC Delta mass)。本发明的多肽应当展示出上文所指的去饱和酶、酮酰基-CoA合酶和酮酰基-CoA还原酶活性。
此外,本发明包括特异识别本发明多肽的抗体。
可以使用本发明的纯化多肽或其衍生的合适片段作为抗原,通过众所周知的方法制备针对本发明的多肽的抗体。可以通过本领域众所周知的抗原性决定算法鉴定适合作为抗原的片段。可以从本发明的多肽通过蛋白质水解消化获得此类片段或者此类片段可以是合成肽。优选地,本发明的抗体是单克隆抗体、多克隆抗体、单链抗体、嵌合抗体或任何这些抗体的片段,如Fab、Fv或scFv片段等。本发明还包含的抗体是双特异性抗体、合成抗体或任何前述抗体的化学修饰衍生物。本发明的抗体应当特异性结合本发明的多肽(即不与其他多肽或肽显著的交叉反应)。可以通过多种众所周知的技术检测特异性结合。抗体或其片段可以通过使用例如在Harlow和Lane"Antibodies,A Laboratory Manual",CSH Press,Cold SpringHarbor,1988中描述的方法获得。单克隆抗体可以通过最初在Nature 256,495,和Galfré1981,Meth.Enzymol.73,3中描述的技术制备,所述技术包括将小鼠骨髓瘤细胞与来源于免疫哺乳动物的脾细胞融合。可以将抗体例如,用于本发明多肽的免疫沉淀、免疫定位或纯化(例如,通过亲和层析),以及用于例如在重组生物中监测所述变体多肽的存在,和用于鉴定与本发明的蛋白质相互作用的蛋白质或化合物。
此外,本发明的抗体可以应用于鉴定本发明多肽的存在或缺失。优选地,抗体用于鉴定如本文其他地方指定的非人转基因生物,和优选地,包含本发明的多肽的转基因植物。为此,可以以允许鉴定非人转基因生物,和优选地包含本发明的多肽的转基因植物的试剂盒形式提供抗体。除了本发明的抗体外,试剂盒可以进一步包含用于检测本发明的抗体与本发明的多肽的复合体的检测试剂。
此外,本发明涵盖包含本发明的多核苷酸或载体的非人转基因生物。
优选地,非人转基因生物是植物、植物部分或植物种子。待用于引入本发明的多核苷酸或载体的优选植物是能够合成脂肪酸的植物,如所有的双子叶植物或单子叶植物、藻类或苔藓。应当理解来自植物的宿主细胞还可以用于产生本发明的植物。优选的植物选自植物Adelotheciaceae、漆树科(Anacardiaceae)、菊科(Asteraceae)、伞形科(Apiaceae)、桦木科(Betulaceae)、紫草科(Boraginaceae)、十字花科(Brassicaceae)、凤梨科(Bromeliaceae)、番木瓜科(Caricaceae)、大麻科(Cannabaceae)、旋花科(Convolvulaceae)、藜科(Chenopodiaceae)、隐甲藻科(Crypthecodiniaceae)、葫芦科(Cucurbitaceae)、牛毛藓科(Ditrichaceae)、胡颓子科(Elaeagnaceae)、杜鹃花科(Ericaceae)、大戟科(Euphorbiaceae)、蝶形花科(Fabaceae)、牻牛儿苗科(Geraniaceae)、禾本科(Gramineae)、胡桃科(Juglandaceae)、樟科(Lauraceae)、豆科(Leguminosae)、亚麻科(Linaceae)、草绿藻科(Prasinophyceae)或蔬菜植物或观赏植物,如万寿菊(Tagetes)。可以提及的实例是以下植物,其选自:Adelotheciaceae,如剑叶藓属,如展叶剑叶藓的属和种、漆树科,如黄连木属(Pistacia)、芒果属(Mangifera)、腰果属(Anacardium),例如阿月混子(Pistacia vera[阿月混子实])、芒果(Mangifer indica[芒果])或腰果(Anacardium occidentale[腰果])的属和种、菊科,如金盏花属(Calendula)、红蓝花属(Carthamus)、矢车菊属(Centaurea)、菊苣属(Cichorium)、菜蓟属(Cynara)、向日葵属(Helianthus)、莴苣属(Lactuca)、Locusta、万寿菊属、缬草属(Valeriana),例如金盏花(Calendula officinalis[普通金盏花])、红花(Carthamus tinctorius[红花])、矢车菊(Centaurea cyanus[矢车菊))、菊苣(Cichorium intybus[菊苣])、洋蓟(Cynara scolymus[朝鲜蓟))、向日葵(Helianthus annus[向日葵])、莴苣(Lactuca sativa)、皱叶莴苣(Lactuca crispa)、芋(Lactuca esculenta)、毒莴苣的某些种(Lactuca scariola L.ssp.sativa)、Lactuca scariola L.var.integrata、Lactuca scariola L.var.integrifolia、莴苣romana亚种(Lactucasativa subsp.Romana)、普通莴苣(Locusta communis)、莴苣缬草(Valeriana locusta[沙拉蔬菜])、香叶万寿菊(Tagetes lucida)、万寿菊(Tagetes erecta)或细叶万寿菊(Tagetes tenuifolia)[非洲或法国金盏花]的属和种、伞形科,如胡萝卜属(Daucus),例如胡萝卜(Daucus carota[胡萝卜])的属和种、桦木科,如榛属(Corylus),例如欧洲榛(Corylus avellana)或榛子(Corylus colurna[榛子])的属和种、紫草科,如琉璃苣(Borago),例如,Borago officinalis[琉璃苣]的属和种、十字花科,如芸苔属(Brassica)、Melanosinapis、芥属(Sinapis)、拟南芥属,例如欧洲油菜(Brassica napus)、芜青属某些种(Brassica rapa ssp.[油菜])、野生欧白芥(Sinapis arvensis)、芥菜(Brassica juncea)、Brassica juncea var.juncea、皱叶芥菜(Brassicajuncea var.crispifolia)、大叶芥菜(Brassica juncea var.foliosa)、黑芥(Brassica nigra)、Brassica sinapioides、普通芥菜(Melanosinapiscommunis[芥菜]、甘蓝(Brassica oleracea[饲用甜菜])或拟南芥的属和种、凤梨科,如凤梨属(Anana)、Bromelia(菠萝),例如菠萝(Anana comosus)、Ananas ananas或Bromelia comosa[菠萝]的属和种、番木瓜科,如番木瓜属(Carica),如番木瓜(Carica papaya[番木瓜])的属和种、大麻科,如大麻属(Cannabis),如大麻(Cannabis sativa[大麻])的属和种、旋花科,如番薯属(Ipomea)、旋花属(Convolvulus),例如甘薯(Ipomoea batatus)、提琴叶牵牛花(Ipomoea pandurata)、Convolvulus batatas、Convolvulus tiliaceus、Ipomoea fastigiata、Ipomoea tiliacea、三裂叶薯(Ipomoea triloba)或Convolvulus panduratus[甜薯、番薯]的属和种、藜科,如甜菜属(Beta),如甜菜(Beta vulgaris)、Beta vulgaris var.altissima、甜菜普通变种(Betavulgaris var.Vulgaris)、Beta maritima、甜菜宿根变种(Beta vulgaris var.perennis)、Beta vulgaris var.Conditiva或Beta vulgaris var.esculenta[甜菜]的属和种、隐甲藻科,如隐甲藻科属,例如寇氏隐甲藻(Cryptecodiniumcohnii)的属和种、葫芦科,如南瓜属(Cucurbita),例如笋瓜(Cucurbitamaxima)、灰子南瓜(Cucurbita mixta)、西葫芦(Cucurbita pepo)或南瓜(Cucurbita moschata[西葫芦/南瓜])的属和种、桥弯藻科(Cymbellaceae),如双眉藻属(Amphora)、桥弯藻属(Cymbella)、Okedenia、褐指藻属、Reimeria,例如,三角褐指藻的属和种、牛毛藓科,如牛毛藓科、高地藓属(Astomiopsis)、角齿藓属、Chrysoblastella、牛毛藓属(Ditrichum)、对叶藓属(Distichium)、Eccremidium、Lophidion、Philibertiella、丛毛藓属(Pleuridium)、石缝藓属(Saelania)、毛齿藓属(Trichodon)、Skottsbergia,例如Ceratodon antarcticus、Ceratodon columbiae、Ceratodonheterophyllus、角齿藓、角齿藓、Ceratodon purpureus ssp.convolutus、Ceratodon、purpureus spp.stenocarpus、角齿藓圆叶变种(Ceratodonpurpureus var.rotundifolius)、Ceratodon ratodon、疣蒴角齿藓(Ceratodonstenocarpus)、Chrysoblastella chilensis、Ditrichum ambiguum、Ditrichumbrevisetum、皱波牛毛藓(Ditrichum crispatissimum)、卷叶牛毛藓(Ditrichum difficile)、Ditrichum falcifolium、扭叶牛毛藓(Ditrichumflexicaule)、Ditrichum giganteum、Ditrichum heteromallum、Ditrichumlineare、Ditrichum lineare、Ditrichum montanum、Ditrichum montanum、黄牛毛藓(Ditrichum pallidum)、Ditrichum punctulatum、细叶牛毛藓(Ditrichum pusillum)、细叶牛毛藓扭叶变种(Ditrichum pusillum var.Tortile)、Ditrichum rhynchostegium、Ditrichum schimperi、Ditrichumtortile、对叶藓(Distichium capillaceum)、小对叶藓(Distichium hagenii)、斜蒴对叶藓(Distichium inclinatum)、Distichium macounii、Eccremidiumfloridanum、Eccremidium whiteleggei、Lophidion strictus、尖叶丛毛藓(Pleuridium acuminatum)、Pleuridium alternifolium、Pleuridiumholdridgei、Pleuridium mexicanum、Pleuridium ravenelii、Pleuridiumsubulatum、Saelania glaucescens、Trichodon borealis、Trichodoncylindricus或Trichodon cylindricus var.oblongus、胡颓子科,如胡颓子属(Elaeagnus),例如油橄榄(Olea europaea[橄榄])的属和种、杜鹃花科,如山月桂属(Kalmia),例如阔叶山月桂(Kalmia latifolia)、狭叶山月桂(Kalmia angustifolia)、小叶山月桂(Kalmia microphylla)、沼泽山月桂(Kalmia polifolia)、西洋山月桂(Kalmia occidentalis)、Cistuschamaerhodendros或山月桂(Kalmia lucida[山月桂])的属和种、大戟科,如木薯属(Manihot)、Janipha、麻风树属(Jatropha)、蓖麻属(Ricinus),例如木薯(Manihot utilissima)、Janipha manihot、Jatropha manihot、Manihotaipil、甜木薯(Manihot dulcis)、Manihot manihot、Manihot melanobasis、木薯(Manihot esculenta[木薯])或蓖麻(Ricinus communis[蓖麻油植物])的属和种、蝶形花科,如豌豆属(Pisum)、合欢属(Albizia)、Cathormion、Feuillea、Inga、Pithecolobium、金合欢属(Acacia)、含羞草属(Mimosa)、Medicajo、大豆属(Glycine)、扁豆属(Dolichos)、菜豆属(Phaseolus)、大豆属(Soja),例如豌豆(Pisum sativum)、饲料豌豆(Pisum arvense)、Pisumhumile[豌豆)、Albizia berteriana、合欢(Albizia julibrissin)、阔荚合欢(Albizia lebbeck)、Acacia berteriana、Acacia littoralis、Albizia berteriana、Albizzia berteriana、Cathormion berteriana、Feuillea berteriana、Ingafragrans、Pithecellobium berterianum、Pithecellobium fragrans、Pithecolobium berterianum、Pseudalbizzia berteriana、Acacia julibrissin、Acacia nemu、Albizia nemu、Feuilleea julibrissin、Mimosa julibrissin、Mimosa speciosa、Sericanrda julibrissin、阔荚金合欢(Acacia lebbeck)、Acacia macrophylla、大叶合欢(Albizia lebbeck)、Feuilleea lebbeck、大叶含羞草(Mimosa lebbeck)、Mimosa speciosa[silk tree]、紫苜蓿(Medicagosativa)、野苜蓿(Medicago falcata)、杂交苜蓿(Medicago varia)[紫花苜蓿]、大豆(Glycine max)、镰扁豆(Dolichos soja)、宽叶蔓豆(Glycine gracilis)、大豆(Glycine hispida)、大菜豆(Phaseolus max)、Soja hispida或Soja max[大豆]的属和种、葫芦藓科,如Aphanorrhegma、Entosthodon、葫芦藓属(Funaria)、剑叶藓属、立碗藓属(Physcomitrium),例如Aphanorrhegmaserratum、Entosthodon attenuatus、Entosthodon bolanderi、Entosthodonbonplandii、Entosthodon californicus、Entosthodon drummondii、Entosthodon jamesonii、Entosthodon leibergii、Entosthodon neoscoticus、Entosthodon rubrisetus、Entosthodon spathulifolius、Entosthodon tucsoni、Funaria americana、Funaria bolanderi、齿叶葫芦藓(Funaria calcarea)、Funaria californica、Funaria calvescens、Funaria convoluta、Funariaflavicans、Funaria groutiana、葫芦藓(Funaria hygrometrica)、Funariahygrometrica var.arctica、葫芦藓暖地变种(Funaria hygrometrica var.Calvescens)、Funaria hygrometrica var.convoluta、Funaria hygrometricavar.muralis、Funaria hygrometrica var.utahensis、小口葫芦藓(Funariamicrostoma)、Funaria microstoma var.obtusifolia、Funaria muhlenbergii、Funaria orcuttii、Funaria plano-convexa、Funaria polaris、Funariaravenelii、Funaria rubriseta、Funaria serrata、Funaria sonorae、Funariasublimbatus、Funaria tucsoni、Physcomitrella californica、展叶剑叶藓、Physcomitrella readeri、Physcomitrium australe、Physcomitriumcalifornicum、Physcomitrium collenchymatum、Physcomitriumcoloradense、Physcomitrium cupuliferum、Physcomitrium drummondii、Physcomitrium eurystomum、Physcomitrium flexifolium、Physcomitriumhookeri、Physcomitrium hookeri var.serratum、Physcomitriumimmersum、Physcomitrium kellermanii、Physcomitrium megalocarpum、Physcomitrium pyriforme、Physcomitrium pyriforme var.serratum、Physcomitrium rufipes、Physcomitrium sandbergii、Physcomitriumsubsphaericum、Physcomitrium washingtoniense、牻牛儿苗科,如天竺葵属(Pelargonium)、椰子属(Cocos)、Oleum,例如椰子(Cocos nucifera)、茶麋子天竺葵(Pelargonium grossularioides)或椰子油(Oleum cocois[椰子])的属和种、禾本科,如甘蔗属(Saccharum),例如甘蔗(Saccharumofficinarum)的属和种、胡桃科,如胡桃属(Juglans)、Wallia,例如胡桃(Juglans regia)、Juglans ailanthifolia、山核桃(Juglans sieboldiana)、灰核桃(Juglans cinerea)、Wallia cinerea、Juglans bixbyi、加州黑核桃(Juglanscalifornica)、印度黑核桃(Juglans hindsii)、Juglans intermedia、Juglansjamaicensis、大核桃(Juglans major)、Juglans microcarpa、黑核桃(Juglansnigra)或胡桃(Wallia nigra)[胡桃]的属和种、樟科,如鳄梨属(Persea)、月桂属(Laurus),例如月桂(Laurus nobilis[bay])、鳄梨(Persea americana)、鳄梨油(Persea gratissima)或Persea persea[avocado]的属和种、豆科,如落花生属(Arachis),例如落花生(Arachis hypogaea[花生])的属和种、亚麻科,如亚麻属(Linum)、Adenolinum属,例如亚麻(Linum usitatissimum)、Linum humile、奥地利亚麻(Linum austriacum)、Linum bienne、窄叶亚麻(Linum angustifolium)、泻亚麻(Linum catharticum)、金黄亚麻(Linumflavum)、大花亚麻(Linum grandiflorum)、Adenolinum grandiflorum、刘易斯亚麻(Linum lewisii)、那旁亚麻(Linum narbonense)、宿根亚麻(Linumperenne)、宿根亚麻刘易斯变种(Linum perenne var.lewisii)、Linumpratense或亚麻子(Linum trigynum[亚麻子])的属和种、Lythrarieae,如石榴属(Punica),例如石榴(Punica granatum[pomegranate])的属和种、锦葵科(Malvaceae),如棉属(Gossypium),例如陆地棉(Gossypium hirsutum)、树棉(Gossypium arboreum)、海岛棉(Gossypium barbadense)、草棉(Gossypium herbaceum)或瑟伯棉(Gossypium thurberi[棉])的属和种、地钱科(Marchantiaceae),如地钱属(Marchantia),例如Marchantiaberteroana、Marchantia foliacea、大孢地钱(Marchantia macropora)的属和种、芭蕉科(Musaceae),如芭蕉属(Musa),例如香蕉(Musa nana)、小果野蕉(Musa acuminata)、大蕉(Musa paradisiaca)、芭蕉属的某些种(Musaspp.[香蕉])的属和种、柳叶菜科(Onagraceae),如Camissonia属、月见草属(Oenothera),例如月见草(Oenothera biennis)或夜来香(Camissoniabrevipes[月见草])的属和种、棕榈科(Palmae),如油棕属(Elacis),例如油棕(Elaeis guineensis[油棕榈])的属和种、罂粟科(Papaveraceae),如罂粟属(Papaver),,例如东方罂粟(Papaver orientale)、虞美人(Papaver rhoeas)、长荚罂粟(Papaver dubium[罂粟])的属和种、胡麻科(Pedaliaceae),如胡麻属(Sesamum),例如胡麻(Sesamum indicum[sesame])的属和种、胡椒科(Piperaceae),如胡椒属(Piper)、Artanthe、草胡椒属(Peperomia)、Steffensia,例如树胡椒(Piper aduncum)、Piper amalago、狭叶胡椒(Piperangustifolium)、大胡椒(Piper auritum)、萎叶(Piper betel)、荜澄茄(Pipercubeba)、荜菝(Piper longum)、胡椒(Piper nigrum)、假荜拔(Piperretrofractum)、Artanthe adunca、Artanthe elongata、长胡椒(Peperomiaelongata)、Piper elongatum、Steffensia elongata[cayenne pepper]的属和种、禾本科(Poaceae),如大麦属(Hordeum)、黑麦属(Secale)、燕麦属(Avena)、高梁属(Sorghum)、须芒草属(Andropogon)、绒毛草属(Holcus)、黍属(Panicum)、稻属(Oryza)、玉蜀黍属(Zea[玉米])、小麦属(Triticum),例如大麦(Hordeum vulgare)、芒颖大麦草(Hordeum jubatum)、鼠大麦草(Hordeum murinum)、短芒大麦草(Hordeum secalinum)、栽培二棱大麦(Hordeum distichon)、Hordeum aegiceras、六列大麦(Hordeumhexastichon)、六棱大麦(Hordeum hexastichum)、不规则大麦(Hordeumirregulare)、大麦(Hordeum sativum)、短芒大麦草(Hordeum secalinum)[大麦]、黑麦(Secale cereale[黑麦])、燕麦(Avena sativa)、野燕麦(Avena fatua)、地中海红燕麦(Avena byzantina)、Avena fatua var.sativa、杂种燕麦(Avenahybrida[燕麦])、二色高梁(Sorghum bicolor)、石茅高梁(Sorghumhalepense)、甜高梁(Sorghum saccharatum)、高梁(Sorghum vulgare)、Andropogon drummondii、二色绒毛草(Holcus bicolor)、Holcus sorghum、Sorghum aethiopicum、Sorghum arundinaceum、Sorghum caffrorum、垂穗高梁草(Sorghum cernuum)、工艺高梁(Sorghum dochna)、Sorghumdrummondii、硬高粱草(Sorghum durra)、Sorghum guineense、Sorghumlanceolatum、多脉高粱草(Sorghum nervosum)、甜高梁(Sorghumsaccharatum)、Sorghum subglabrescens、垂叶高梁草(Sorghumverticilliflorum)、高梁(Sorghum vulgare)、石茅高梁(Holcus halepensis)、Sorghum miliaceum、黍粟(Panicum militaceum[millet])、稻(Oryza sativa)、Oryza latifolia[稻]、玉米(Zea mays[玉米])、小麦(Triticum aestivum)、硬粒小麦(Triticum durum)、圆柱小麦(Triticum turgidum)、Triticumhybernum、马卡小麦(Triticum macha)、普通小麦(Triticum sativum)或Triticum vulgare[小麦]、紫球藻科(Porphyridiaceae),如Chroothece、Flintiella、Petrovanella、紫球藻属(Porphyridium)、Rhodella、Rhodosorus、Vanhoeffenia,例如紫球藻(Porphyridium cruentum)的属和种、山龙眼科(Proteaceae),如澳洲坚果属(Macadamia),例如全缘叶澳洲坚果(Macadamia intergrifolia[macadamia])的属和种,绿藻纲(Prasinophyceae),如肾藻属(Nephroselmis)、Prasinococcus、Scherffelia、扁藻属(Tetraselmis)、Mantoniella、Ostreococcus,例如绿肾藻(Nephroselmis olivacea)、Prasinococcus capsulatus、Scherffelia dubia、Tetraselmis chui、扁藻(Tetraselmis suecica)、Mantoniella squamata、Ostreococcus tauri、茜草科(Rubiaceae),如咖啡属[Coffea],例如咖啡属某些种、小果咖啡(Coffea arabica)、中果咖啡(Coffea canephora)或大果咖啡(Coffea liberica(咖啡))、玄参科(Scrophulariaceae),如毛蕊花属(Verbascum),例如毛瓣毛蕊花(Verbascum blattaria)、东方毛蕊花(Verbascum chaixii)、密叶毛蕊花(Verbascum densiflorum)、Verbascumlagurus、长叶毛蕊花(Verbascum longifolium)、Verbascum lychnitis、Verbascum nigrum、奥林匹克毛蕊花(Verbascum olympicum)、Verbascumphlomoides、紫毛蕊花(Verbascum phoenicum)、Verbascum pulverulentum或毛蕊花(Verbascum thapsus[mullein])的属和种、茄科(Solanaceae),如辣椒属(Capsicum)、烟草属(Nicotiana)、茄属(Solanum)、番茄属(Lycopersicon),例如辣椒(Capsicum annuum)、Capsicum annuum var.glabriusculum、五色椒(Capsicum frutescens[辣椒])、红椒(Capsicumannuum[红辣椒])、烟草(Nicotiana tabacum)、花烟草(Nicotiana alata)、长头烟草(Nicotiana attenuata)、光烟草(Nicotiana glauca)、Nicotianalangsdorffii、Nicotiana obtusifolia、Nicotiana quadrivalvis、Nicotianarepanda、黄花烟草(Nicotiana rustica)、Nicotiana sylvestris[烟草]、马铃薯(Solanum tuberosum[马铃薯])、茄(Solanum melongena[茄子])、番茄(Lycopersicon esculentum)、Lycopersicon lycopersicum、梨形番茄(Lycopersicon pyriforme)、红茄(Solanum integrifolium)或番茄(Solanumlycopersicum)[番茄]的属和种、梧桐科(Sterculiaceae),如可可树属(Theobroma),例如可可树(Theobroma cacao[可可])的属和种或山茶科(Theaceae),如山茶属(Camellia),例如大叶茶(Camellia sinensis[茶])的属和种。具体而言,待用作本发明的转基因植物的特定优选植物是包含大量脂类化合物的油料作物,如花生、油菜、芸苔、向日葵、红花、罂粟、芥菜、大麻、蓖麻、橄榄、芝麻、金盏花、石榴、月见草、毛蕊花、大蓟、野玫瑰、榛树、杏仁、澳洲坚果、鳄梨、月桂、西葫芦/南瓜、亚麻子、大豆、阿月混子、琉璃苣、树(油椰、椰子树、胡桃)或作物例如玉米、小麦、黑麦、燕麦、黑小麦、稻、大麦、棉、木薯、胡椒、万寿菊、茄科植物例如马铃薯、烟草、茄子和番茄、蚕豆属的种、豌豆、紫花苜蓿或灌木植物(咖啡、可可、茶)、柳属的种和多年生牧草及饲料作物。本发明的优选植物是油料植物如花生、油菜、芸苔、向日葵、红花、罂粟、芥菜、大麻、蓖麻、橄榄、金盏花、石榴、月见草、西葫芦/南瓜、亚麻子、大豆、琉璃苣、树(油椰、可可)。尤其优选的是向日葵、红花、烟草、毛蕊花、芝麻、棉、西葫芦/南瓜、罂粟、月见草、胡桃、亚麻子、大麻、大蓟或红花。非常尤其优选的植物是植物,如红花、向日葵、罂粟、月见草、胡桃、亚麻子或大麻。
优选的苔藓是剑叶藓属或角齿藓属植物。优选的藻类是等鞭金藻、Mantoniella、Ostreococcus或隐甲藻和藻类/硅藻,如褐指藻或破囊壶菌。更优选地,所述藻类或苔藓选自:希瓦氏菌、剑叶藓属、破囊壶菌、镰孢霉属(Fusarium)、疫霉属(Phytophthora)、角齿藓属、等鞭金藻属(Isochrysis)、Aleurita、Muscarioides、被孢霉属、褐指藻属、隐甲藻属,尤其来自以下属和种:假微型海链藻、细小裸藻(Euglena gracilis)、展叶剑叶藓、致病疫霉、禾谷镰孢霉(Fusarium graminaeum)、寇氏隐甲藻(Cryptocodinium cohnii)、角齿藓、球等鞭金藻、Aleurita farinosa、破囊壶菌属、Muscarioides viallii、高山被孢霉(Mortierella alpina)、三角褐指藻或秀丽隐杆线虫(Caenorhabditis elegans)或尤其有利地来自致病疫霉、假微型海链藻和寇氏隐甲藻。
可以通过本说明书中其他地方的转化技术获得转基因植物。优选地,可以通过T-DNA介导的转化获得转基因植物。此类载体系统的特征通常是它们至少含有为农杆菌介导的转化所需的vir基因和划界T-DNA的序列(T-DNA边界)。合适的载体在本说明书中其他地方详细描述。
还包括包含本发明载体或多核苷酸的转基因非人动物。本发明设想的优选的非人转基因动物是鱼类,如鲱鱼、鲑鱼、沙丁鱼、雄鲑、鳗鲡、鲤鱼、鳟鱼、大比目鱼、鲭鱼、豹纹鱼或金枪鱼
然而,应当理解取决于上文指定的非人转基因生物,可以例如通过重组技术进一步赋予所述生物酶活性。因此,本发明优选设想上文指定的非人转基因生物,根据所选择的宿主细胞,除本发明的多核苷酸外,其还包含根据需要编码此类去饱和酶和/或延伸酶的多核苷酸。应当存在于该生物中的优选去饱和酶和/或延伸酶是选自本说明书中其他地方特别说明的去饱和酶和/或延伸酶或它们的组合中的至少一种酶(参见上文和表5、6和7)。
此外,本发明包括用于制造多不饱和脂肪酸的方法,所述方法包括:
a)在允许在所述宿主细胞中产生多不饱和脂肪酸的条件下培育本发明的宿主细胞;和
b)从所述宿主细胞获得所述多不饱和脂肪酸。
如本文所用的术语“多不饱和脂肪酸(PUFA)”指包含至少2个,优选地3、4、5或6个双键的脂肪酸。此外,应当理解此类脂肪酸在脂肪酸链中优选包含18-24个碳原子。更优选地,该术语涉及在脂肪酸链中具有20-24个碳原子的长链PUFA(LCPUFA)。在本发明意义范围内的优选不饱和脂肪酸选自DGLA 20:3(8,11,14)、ARA 20:4(5,8,11,14)、iARA20:4(8,11,14,17)、EPA 20:5(5,8,11,14,17)、DPA 22:5(4,7,10,13,16)、DHA22:6(4,7,10,13,16,19)、20:4(8,11,14,17),更优选花生四烯酸(ARA)20:4(5,8,11,14)、二十碳五烯酸(EPA)20:5(5,8,11,14,17)和二十二碳六烯酸(DHA)22:6(4,7,10,13,16,19)。因此,应当理解最优选地,本发明提供的方法涉及ARA、EPA或DHA的制造。此外,还包括在合成过程出现的LCPUFA的中间体。此类中间体优选地由本发明多肽的去饱和酶、酮酰基-CoA合酶和酮酰基-CoA还原酶活性从底物形成。优选地,底物包括LA18:2(9,12)、GLA 18:3(6,9,12)、DGLA 20:3(8,11,14)、ARA 20:4(5,8,11,14)、二十碳二烯酸20:2(11,14)、二十碳四烯酸20:4(8,11,14,17)、二十碳五烯酸20:5(5,8,11,14,17)。
如本文所用的术语“培育”指在允许细胞产生所述多不饱和脂肪酸(即上文所指的PUFA和/或LCPUFA)的培养条件下维持和培养宿主细胞。这暗含在宿主细胞中表达本发明的多核苷酸,从而存在去饱和酶、酮酰基-CoA合酶和酮酰基-CoA还原酶活性。在下文更详细地描述用于培育宿主细胞的合适培养条件。
如本文所用的术语“获得”包括提供细胞培养物,所述细胞培养物包括宿主细胞和培养基,以及提供纯化或部分地纯化的其制剂,所述制剂包含作为膜磷脂或作为三酰甘油酯以游离或-CoA结合形式的多不饱和脂肪酸,优选ARA、EPA、DHA。更优选地,PUFA和LCPUFA将作为甘油三酯形式例如以油形式获得。可以在下文其他地方找到关于纯化技术的更多细节。
根据宿主生物,以技术人员熟悉的方式培养或培育待在本发明方法中使用的宿主细胞。一般地,在液体培养基中,在0℃和100℃之间,优选在10℃和60℃之间的温度,根据生物类型在氧或无氧气氛下培育宿主细胞,所述液体培养基包含一般为糖形式的碳源、一般为有机氮源(如酵母提取物)形式的氮源或盐如硫酸铵、微量元素如铁、锰和镁的盐和适当时,维生素。液体培养基的pH可以保持恒定,即,在培育期间进行调节或不调节。培养物可以分批、半分批或连续地培养。营养物可以在发酵开始时提供或半连续地或连续地施用。可以通过技术人员已知的方法,例如通过提取、蒸馏、结晶、适当时用盐沉淀和/或层析,从如上文所述的宿主细胞分离产生的PUFA或LCPUFA。可能需要在纯化之前破坏宿主细胞。为此目的,可以事先破坏宿主细胞。待使用的培养基必须适当地满足所讨论宿主细胞的要求。可以在教科书“Manual of Methods for GeneralBacteriology”of the American Society for Bacteriology(Washington D.C.,USA,1981)中找到对于根据本发明能用作宿主的多种微生物的培养基的描述。也可以从多个商业供应者获得培养基。将全部培养基组分通过加热或过滤消毒法消毒。全部培养基组分可以在培育开始时存在,或根据需要,连续地或分批地添加。如果已经被引入宿主细胞中的本发明多核苷酸或载体还包含可表达的选择标志物,如抗生素耐药性基因,可能需要添加选择剂至所述培养物,如为维持所引入多核苷酸的稳定性的抗生素。继续培养直至想要的产物最多地形成。这通常在10-160小时内实现。发酵液可以直接使用或可以进一步加工。根据要求,生物量可以通过分离方法如例如离心、过滤、倾析或这些方法的组合完全或部分地从发酵液取出或完全留在所述发酵液中。通过本发明方法获得的包含作为甘油三酯的想要的PUFA或LCPUFA的脂肪酸制剂(例如油)还适合作为化学合成其他目的产物的起始材料。例如,它们可以彼此组合地或单独地用于药物或化妆品组合物、食物或动物饲料的制备。可以通过上文所述的方法制造包含想要的PUFA或LCPUFA的化学纯的甘油三酯。为此目的,进一步通过提取、蒸馏、结晶、层析或这些方法的组合纯化脂肪酸制剂。为了从甘油三酯释放脂肪酸部分,也可能需要水解。所述化学纯的甘油三酯或游离脂肪酸特别适用于在食品工业中应用或适于化妆品和药物组合物。
此外,本发明涉及用于制造多不饱和脂肪酸的方法,所述方法包括:
a)在允许在所述宿主细胞中产生多不饱和脂肪酸的条件下培育本发明的非人转基因生物;和
b)从所述非人转基因生物获得所述多不饱和脂肪酸。
此外,由上可知,本发明还设想用于制造油、脂类或脂肪酸组合物的方法,其包括前述方法的任意之一的步骤和将PUFA或LCPUFA配制为油、脂类或脂肪酸组合物的步骤。优选地,可以将所述油、脂类或脂肪酸组合物用于饲料、食品、化妆品或药物。因此,对于各个设想的产品,应当根据GMP标准实施PUFA或LCPUFA的配制。例如,可以通过榨油机从植物种子中获得油。然而,出于产品安全性原因,在适用的GMP标准下需要灭菌。应当将类似的标准应用于待在化妆品或药物组合物中应用的脂类或脂肪酸组合物。前述制造包含用于配制作为产品的油、脂类或脂肪酸组合物的所有这些措施。
对于ARA的产生,优选设想培育本发明的宿主细胞或包含本发明的多核苷酸组合的非人转基因生物。优选地,设想本发明的多核苷酸组合,其编码d12去饱和酶、d6去饱和酶、d6延伸酶、d5去饱和酶和KCR(也参见所附实施例中的表5)。
对于ARA的产生,或者但也优选设想培育本发明的宿主细胞或包含本发明的多核苷酸组合的非人转基因生物。优选地,设想本发明的多核苷酸组合,其编码d12去饱和酶、d9延伸酶、d8去饱和酶、d5去饱和酶和KCR(也参见所附实施例中的表6)。
对于EPA的产生,优选设想培育本发明的宿主细胞或包含本发明的多核苷酸组合的非人转基因生物。优选地,优选应用于上文指定ARA产生的多核苷酸的组合与编码d15去饱和酶的本发明的多核苷酸和编码ω-3去饱和酶的本发明的多核苷酸一起使用(即,在表7中涉及的活性与表5或表6中的活性的组合)。
对于DHA的产生,优选设想培育本发明的宿主细胞或包含本发明的多核苷酸组合的非人转基因生物。优选地,优选应用于上文指定EPA产生的多核苷酸的组合与编码d5延伸酶的本发明的多核苷酸和编码d4去饱和酶的本发明的多核苷酸一起使用(即,在表5和表7任一中涉及的活性与表8或表6和表7中的那些活性以及表8中的那些活性的组合)。
本发明也涉及包含通过前述方法可获得的多不饱和脂肪酸的油。
术语“油”指包含酯化成甘油三酯的不饱和和/或饱和脂肪酸的脂肪酸混合物。优选地,在本发明的油中的甘油三酯包含如上文所提及的PUFA或LCPUFA。酯化的PUFA和/或LCPUFA的量优选是大约30%,更优选50%的含量,甚至更优选60%、70%、80%或更高的含量。所述油可以进一步包含游离脂肪酸,优选地,上文所提及的PUFA和LCPUFA。为了分析,可以通过转酯反应将脂肪酸转化成甲酯后,例如通过GC分析确定脂肪酸含量。油或脂肪中多种脂肪酸的含量可以变化,尤其根据来源变化。然而,所述油应当在PUFA和/或LCPUFA组成和含量方面具有非天然存在的组成。已知植物油中大多数脂肪酸在甘油三酯中是酯化的。因此,在本发明的油中,PUFA和LCPUFA在甘油三酯中优选也以酯化形式存在。应当理解,在所述油的甘油三酯中PUFA和LCPUFA的这种独特油组成和独特酯化模式仅应当是可通过采用上文所述的本发明方法获得的。此外,本发明的油也可以包含其他分子种类。尤其是,它可以包含本发明多核苷酸或载体的少量杂质。然而,此类杂质仅可以通过高度灵敏的技术如PCR检测到。
本申请中引用的所有参考文献在其完整公开内容和本说明书中特定提及的公开内容方面并入作为参考。
图
图1显示了在转化有pYes-pd5Elo(Ta)_c1的酵母中d5延长的脂肪酸的产生。描述了利用不同脂肪酸喂养的转基因酵母的脂肪酸谱。A:利用20:5n-3喂养的对照pYes,B:利用20:5n-3喂养的pYes-pd5Elo(Ta)_c1。22:5n-3的形成证明了pd5Elo(Ta)_c1的d5-延伸酶活性。在表5中列出了所观察到的20:5n至3-22:5n-3的转化率。
图2显示了多条δ-4去饱和酶多肽序列的比对。“d4Des(Sa)”是本发明的序列SEQ ID NO.79;“d4des(Tc)”表示如本文所述破囊壶菌的δ-4去饱和酶。
图3显示了与SEQ ID NO.79的序列同一性降序的本发明的多条δ-4去饱和酶多肽序列的列表。通过突变SEQ ID NO.79的序列同时维持如上所述优选的保守序列基序获得序列。
本发明现在将通过以下实施例来阐明,然而所述实施例不应解释为限制本权利要求或本发明的范围。
实施例
实施例1:一般克隆方法
如Sambrook等(1989)(Cold Spring Harbor Laboratory Press:ISBN0-87965-309-6)中所述进行克隆方法,例如,使用限制性核酸内切酶在特定位点切割双链DNA、琼脂糖凝胶电泳、纯化DNA片段、转移核酸到硝酸纤维素膜和尼龙膜上、连接DNA片段、转化大肠杆菌细胞和培养细菌。
实施例2:重组DNA的序列分析
使用激光-荧光DNA测序仪(Applied Biosystems Inc,USA),采用sanger法(Sanger等(1977)Proc.Natl.Acad.Sci.USA 74,5463-5467)进行重组DNA分子的测序。将携带通过聚合酶链式反应获得的片段的表达构建体进行测序,以证实由启动子、待表达核酸分子和终止子组成的表达盒的正确性以避免可能因克隆过程中操作DNA引起的突变,例如,由于不正确的引物、因暴露于紫外线所致的突变或聚合酶的错误。
实施例3:通过同源重组克隆酵母表达构建体
可以在聚合酶链式反应中使用表2列出的引物分别扩增编码具有氨基酸序列SEQ ID NOs:2、5、8、11、14、17、20、23、26、29、32、79、84、85或86的多肽(其具有去饱和酶、延伸酶、KCR或LACS活性)的SEQ ID NOs:1、4、7、10、13、16、19、22、25、28、31、78和83中列出的可读框。通过这样操作,可读框5’融合到GAL1启动子序列3’末端的约60个核苷酸上,同时在融合位点之间引入Asc I和/或Nco I限制性位点,并且可读框3’融合到CYC1终止序列5’末端的约60个核苷酸上,同时引入Pac I限制性位点。为了通过同源重组将这些片段整合到半乳糖诱导型GAL1启动子下游的pYES2.1TOPO中,可以使用限制性核酸内切酶PvuII和Xba I消化载体pYES2.1(Invitrogen),并且可以利用5-20ng线性化的pYES2.1TOPO载体和20-100ng PCR产物/50μl感受态细胞,使用Schiestl等(Schiestl等(1989)Curr.Genet.16(5-6),第339-346页)所述的转化方法转化酿酒酵母(Saccharomyces cerevisiae),以在多种野生型酵母中获得pYes-pd6Des(Ta)_c3318、pYes-pd9Des(Ta)_c4008、pYes-po3Des(Ta)_c959、pYes-po3Des(Ta)_c1830、pYes-pd12Des(Ta)_c1219、pYes-pd5Elo(Ta)_c1、pYes-pd6Elo(Ta)_c231、pYes-pd6Elo(Ta)_c752、pYes-pd6Elo(Ta)_c4696、pYes-pd9Elo(Ta)_c4589、pYes-pKCR(Ta)_c1703和pYes-d4Des(Sa)。可以基于所选酿酒酵母菌株的URA营养缺陷型的互补选择阳性转化体。为了验证特定酵母克隆携带的表达构建体的正确性,可以如Current Protocols in Molecular Biology(Hoffmann,Curr.Protoc.Mol.Biol.2001May;第13章:第13.11章)中所述分离质粒,将其转化到大肠杆菌中用于扩增并如实施例2中所示进行表达盒的测序。
表2:用于克隆用于在酵母中表达的本发明的去饱和酶、酮酰基-CoA合酶、酮酰基-CoA还原酶、脱氢酶和烯酰-CoA还原酶的多核苷酸的引物序列
在表2中显示了所鉴定的全长编码序列的列表。
表3:来自本发明的金黄色破囊壶菌的去饱和酶、延伸酶或延伸酶组分的编码多核苷酸序列、其编码的氨基酸序列和表达序列(mRNA)。
实施例4:酵母中的活性测定
例如,可以在酵母中通过异源表达证实所鉴定多肽的优良活性。表4和5显示了利用以下转化的酵母的活性测定:pYes-pd6Des(Ta)_c3318(包含金黄色破囊壶菌δ-6-去饱和酶基因)、pYes-pd9Des(Ta)_c4008(包含金黄色破囊壶菌δ-9-去饱和酶基因)、pYes-po3Des(Ta)_c959(包含金黄色破囊壶菌ω-3-去饱和酶基因)、pYes-po3Des(Ta)_c1830(包含金黄色破囊壶菌ω-3-去饱和酶基因)、pYes-pd12Des(Ta)_c1219(包含金黄色破囊壶菌δ-12-去饱和酶基因)、pYes-pd5Elo(Ta)_c1(包含金黄色破囊壶菌δ-5-延伸酶基因)、pYes-pd6Elo(Ta)_c231(包含金黄色破囊壶菌δ-6-延伸酶基因)、pYes-pd6Elo(Ta)_c752(包含金黄色破囊壶菌δ-6-延伸酶基因)、pYes-pd6Elo(Ta)_c4696(包含金黄色破囊壶菌δ-6-延伸酶基因)、pYes-pd9Elo(Ta)_c4589(包含金黄色破囊壶菌δ-9-延伸酶基因)、pYes-pKCR(Ta)_c1703(包含金黄色破囊壶菌KCR基因)和pYes-d4Des(Sa)(包含Sphaeroforma arcticaδ-4-去饱和酶基因)构建体。将含有各质粒的酵母细胞在缺少尿嘧啶的液滴基础培养基(DOB-U培养基)中,28℃下200rpm温育12小时,随后在诱导培养基(DOB-U+2%(w/v)半乳糖+2%(w/v)棉子糖)中温育额外的12小时。向诱导培养基中加入250μM的各脂肪酸,以检查酶活性和特异性。此外,在表4和表5中指出了补料底物、预期的产物脂肪酸。
在转化有pYes-pd5Elo(Ta)_c1和用20:5n-3或20:4n-6喂养的酵母提取物的气相色谱中,检测到脂肪酸22:5n-3和22:4n-6(表5,图1)。该结果显示,pYes-pd5Elo(Ta)_c1具有d5-延伸酶活性并且展示令人惊奇的高转化率。在平行实验中的SEQ ID NO 78的基因d4Des(Sa)对SEQ ID NO76的d4Des(Tc)的直接比较中,与基因d4Des(Tc)仅10%的转化率相比,发现构建体pYes-d4Des(Sa)表达的d4Des(Sa)具有令人惊奇的22%的高转化率(转化效率)。根据本发明,相信Sphaeroforma arctica和破囊壶菌种的δ-4去饱和酶展示不同脂肪酸池,即对结合ACP、CoA或磷脂中的脂肪酸的优选性。
表4:酵母喂养实验设置
表5:酵母喂养实验结果。以总脂肪酸池的百分比给出底物和产物脂肪酸。
实施例5:去饱和酶、KCS和KCR在植物中的表达。
可以将来自金黄色破囊壶菌和Sphaeroforma arctica的新的去饱和酶、KCS和KCR克隆到如WO2003/093482、WO2005/083093或WO2007/093776中所述的植物转化载体中。
在表6、7、8和9中描述了基因的示例性合适组合用于优良产生ARA、EPA和/或DHA。
表6:用于产生花生四烯酸(ARA)的基因组合。具有d12-去饱和酶、d6-去饱和酶、d6-延伸酶和d5-去饱和酶活性的至少一种酶为花生四烯酸生物合成所需。多个生物合成步骤可以由本发明的金黄色破囊壶菌的酶催化。
可以通过涉及d9-延伸酶和d8-去饱和酶活性的备选途径产生花生四烯酸。表7显示了用于该途径的基因组合。
表7:用于产生花生四烯酸的备选途径的基因组合。若干生物合成步骤可以由本发明的金黄色破囊壶菌的酶催化。
为了产生EPA,将表8中列出的基因与表6或7中列出的基因组合。
表8:为了产生EPA,除了表6或7中列出的基因组合外,可以使用该表中列出的基因。
除了表6或7的基因外,表8和9中列出的基因可以用于生物合成DHA。这些基因允许EPA延长2个碳原子并且在第4个和第5个碳原子上脱氢,导致产生DHA。
表9:为了产生DHA,除了表6或7和8的基因外,还可以使用该表中列出的基因。
如Deblaere等(1984),(Nucl.Acids.Res.13,4777-4788)中所述产生转基因油菜籽株系并且如Qiu等(2001)(J.Biol.Chem.276,31561-31566)中所述分析转基因油菜籽植物的种子。
实施例6:利用LACS喂养酵母
为了研究该基因的作用,通过在培养物中包括该基因,所述培养物还携带延伸酶和去饱和酶基因对,即WO2011064181和WO2011064183中描述的D6ELO(SA)(即Sphaeroforma arctica的δ-6-延伸酶)和D5DES(SA)(即Sphaeroforma arctica的δ-5-去饱和酶)。当在存在GLA的情况下诱导培养物时,携带SA-LACS1(Sphaeroforma arctica LACS,SEQ ID NO.83/84)的培养物中至DGLA的第一次延长比在携带空pYES2.1/V5-His-TOPO的培养物中低,反映了由于在存在酰基CoA合酶的情况下更有效的GLA吸收导致可用底物的的水平更高,可能饱和的水平。然而,尽管DGLA在两个样品中均积累到相似的水平,但是去饱和成ARA在存在LACS基因的情况下高大约1.6倍。同样,DGLA进一步延长至22:3n-6和ARA进一步延长至22:4n-6在存在SA-LACS1的情况下更有效,其去饱和产物ARA的延长在存在SA-LACS1的情况下高约1.7倍。来自实验(其中用SDA补充培养物)的结果显示了相似的趋势,其中大约40%更多的20:4Δ8,11,14.17去饱和成EPA,并且25%更多的EPA延长成DHA。与使用单基因的实验相比,该实验的总体较低转化率反映了由于不同载体和大量基因表达而降低的效率。
表10.当在表达的酵母中共表达时,S.arctica LACS对延长和去饱和反应的影响。
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Claims (16)
1.δ-4去饱和酶多肽,其选自:
a)与SEQ ID NO.79、86或85任一个的氨基酸序列具有至少60%的序列同一性的δ-4去饱和酶多肽,其中所述序列优选还包含
-选自“KHPGG”、“QHPGG”或“RHPGG”的基序,优选选自“KHPGGD”、“QHPGGD”或“RHPGGD”的基序,和
-选自“GLNIQHDXNHG”或“HVVGHH”的基序,优选选自“AAIGLNIQHDANHG”或“QHVVGHH”的基序,或
b)具有氨基酸序列SEQ ID NO.85和任选地一个或多个以下突变的δ-4去饱和酶多肽:H4S、H4Y、H4Q、H4E、H4N、A5S、A5G、A5C、A5P、A5T、A5V、A6R、A6G、A6K、A6S、A6N、T7K、T7S、T7R、T7N、T7A、T7G、K8E、K8D、K8R、K8Q、V9-、V9I、V9L、V9A、G10V、G10I、G10A、G10M、G10C、G11S、G11N、G11D、G11A、G11K、D12-、S13-、D14A、D14V、D14I、D14E、D14G、D14P、P15A、P15G、P15S、P15V、P15T、R18K、R18S、R18T、R18A、R18Q、D19A、D19G、D19P、D19S、L20-、K21-、M22A、M22L、M22I、M22V、E23K、E23R、E23Q、H24Y、H24F、H24I、H24M、H24L、H24Q、H24R、H24V、F25Y、F25W、F25I、F25A、S26T、S26A、S26C、S26G、S26N、Y27R、Y27F、E28T、E28D、E28N、E28S、R29K、R29E、R29Q、R29D、I30K、I30L、I30V、L31A、L31I、N32D、N32G、N32H、N32S、N32T、N32R、N32K、N32E、N32Q、N32A、N32V、D33H、D33N、D33T、E34T、E34N、E34D、E34Q、E34R、R35N、R35K、R35H、D36P、D36E、D36N、D36S、D37E、D37K、L38I、L38V、L38M、L38F、L38Y、C39T、C39A、C39S、C39V、V41L、V41I、V41M、V41T、G42A、G42D、G42E、G44A、A48S、A48C、A48L、A48R、A48K、T49S、T49V、T49N、T49P、T49K、T49A、A50C、A50N、A50G、D53E、D53N、K54Q、K54R、A59-、D60E、D60N、D60H、F61Y、F61W、F61V、F61I、V62L、V62I、V62M、V62A、V62T、D63S、D63N、D63E、L64I、L64F、L64M、G66P、R68Q、R68H、R68D、R68N、P72E、P72D、P72Q、H73G、H73A、H73N、H73S、F75W、F75M、F75Y、E76Q、E76M、E76H、Y77F、Y77W、Y77H、Y77L、R79K、R79Q、R79H、R80H、R80K、R80Q、R80Y、R80N、E81A、E81V、E81Q、E81L、E81P、W82G、W82S、P83D、P83V、P83K、P83A、P83T、P83E、P83Q、P83M、P83L、P84K、P84R、A85S、A85G、A85C、A85T、A85P、A85V、V86I、V86L、V86M、V86R、V86A、V86T、L87M、L87I、L87V、L87F、A88S、A88C、A88G、K89R、K89E、K89Q、K89P、K89N、Y90F、Y90W、K91F、K91G、K91Y、K91L、K91W、L95I、L95V、L95M、D96A、D96E、D96G、D96P、R97K、R97P、R97Q、R97H、R97E、R97A、D98E、D98N、D98S、D99E、D99Q、S100K、S100G、S100N、S100P、Y101F、Y101W、Y101H、Y101P、V102T、V102I、V102L、Q103H、Q103E、Q103R、Q103N、Q103K、Q103D、Q103Y、H104A、H104Y、H104V、H104I、H104F、H104R、D105E、D105P、D105T、S106P、S106E、S106A、S106D、S106N、S106T、G107L、G107I、G107M、G107A、Y108H、Y108Q、Y108W、Y108M、Y108V、L109K、L109M、L109I、L109V、R110Q、R110K、R110H、R110E、L111I、L111F、C112G、C112A、C112N、C112S、C112K、A113C、A113S、A113G、E114D、E114Q、E114P、E114S、E114K、E114A、N116R、N116D、N116H、G117A、G117K、G117R、G117S、I118L、I118V、L119I、L119M、K121R、K121M、K121Q、K121L、G122W、G122Q、G122N、S123E、S123D、S123N、S123G、G124W、G124A、G124N、G125-、W126F、P129A、P129G、W131Y、W132Y、I133L、I133V、C136A、C136G、C136S、L138I、L138V、L138M、L139I、L139V、L139M、V140A、V140I、V140L、V140C、V140M、A142T、A142P、A142S、A142G、L143I、L143V、Y144F、Y144H、Y144W、Y144S、Y144T、L145I、L145V、L145M、L145F、L145T、D146E、D146N、D146Q、Y147W、Y147F、Y147H、Y147G、Y148F、Y148H、Y148L、M149W、M149L、M149I、M149F、L150I、L150V、A151L、A151C、A151G、A151S、R152K、R152S、R152N、R152Q、R152D、P154K、P154E、P154R、I156L、I156V、L157F、L157I、L157M、L157Y、L157W、L157P、A159S、A159G、A159C、A159T、A159P、A159V、I160V、I160L、I161V、I161L、I161F、L162I、L162V、L162M、L162F、L162C、L162A、L162T、L162S、L165I、L165V、F166Y、W168G、W168A、W168C、A181S、A181G、A181C、A181T、A181P、A181V、L182I、L182V、L182M、N185H、N185Y、P186S、V187M、V187A、V187I、V187P、V188I、V188L、V188A、V188T、Y190R、Y190H、Y190W、L191C、L191I、F192L、F192I、A195S、A195G、A195M、A195Q、A195K、S202N、S202G、S202D、S202T、S202A、S202K、M203R、M203L、M203Q、M203I、M204L、M204V、M204I、M204Q、M204C、L207I、L207V、Q208R、Q208E、Q208K、Q209E、Q209K、Q209R、Q209D、Q209H、G213N、G213A、G213S、G213K、G213D、G213W、G213M、L216I、L216F、L216T、T218C、T218S、T218V、T218A、T218N、T218P、D220E、D220N、D220R、D220G、I221V、I221F、I221Y、I221H、D222N、D222Q、D222P、H223Y、H223F、H223Q、H223W、H223R、H223E、H223N、H223M、H223V、H223P、H223I、H223L、H223D、H223A、V227I、V227L、V227M、V227A、V227T、V227Q、G229A、G230H、G230N、G230D、G231S、G231N、G231A、A232V、A232T、A232I、A232G、L233I、L233V、R234K、R234Q、R234N、R234E、R234H、R234T、K236S、K236G、K236A、K236T、P237K、P237D、P237E、P237R、T238V、T238S、T238P、T238N、T238A、T238I、T238Y、D239S、D239G、D239E、G240I、G240L、G240F、G240C、G240M、G240S、W241F、W241Y、W241P、L242M、L242I、L242V、L242F、L242K、P243E、P243D、W244F、H246A、H246S、H246Y、H246G、H246N、L247I、L247V、L250I、L250V、L250M、L250F、L250Y、F252I、F252Y、L255G、L255M、L255A、L255K、L255C、L255F、E256D、E256Q、E256K、E256P、E256R、E256N、E256S、E256V、E256A、E256T、E256G、E256H、E256I、A257V、A257Q、A257L、A257C、A257P、A257E、L258M、L258I、Y259F、Y259W、Y259H、Y259L、G260A、G260C、K262Q、K262R、W263L、W263F、V264I、V264L、V264M、V264F、F265Y、F265W、F265H、F265S、D267G、L268A、L268I、L268V、H269N、H269Q、H269R、H269L、E270D、E270Q、E270K、L272I、L272F、E273D、E273A、E273G、E273P、W274F、W274Y、W274M、K275R、K275E、K275Q、W276Y、E277K、E277D、E277R、P280K、P280R、I281L、I281V、P282S、P282D、P282N、P282G、P283E、P283D、P283Q、L284I、L284M、L284V、L284C、L284A、L284T、L284S、A285Y、A285C、A285F、A285V、A285S、A285L、R286K、R286Q、R286H、R286E、R286L、R286M、R286V、R286A、P287K、P287D、P287E、P287G、P287Q、P287S、P287R、P287A、E288D、E288L、E288K、E288V、E288Q、F289Y、F289R、F289H、A290G、A290S、A290N、A290C、P291I、V293I、V293L、G294A、G294S、G294P、G294N、G294K、G294R、G294E、G294D、G294Q、G294T、G294H、G294C、G294W、G294M、G294Y、G294V、C295L、C295I、K296R、K296E、K296Q、K296N、K296P、L297I、L297V、G298F、G298A、G298W、G298S、W300F、A301V、A301C、A301G、A301S、A301P、A301I、F303Y、F303W、F303K、V304I、V304F、V304L、V304Y、A305V、A305I、A305L、A305T、A305C、L306I、L306V、L306M、L308F、L308I、W309A、W309G、H311Q、H311E、H311R、H311Y、H311N、P312F、P312Y、S313T、S313N、S313G、S313A、W314F、W314L、W314Y、W314I、W314M、W314V、H315Y、L317I、L317M、L317V、L317F、L317C、L317A、L318I、L318V、L318C、L318A、V320I、V320T、V320P、V320L、C321Y、A322L、A322C、A322S、A322G、W323T、V324I、V324L、V324M、C325A、C325G、C325S、T326S、T326V、T326N、T326I、T326P、T326L、G327A、G327N、G327K、S328A、S328T、S328L、S328C、S328V、F329L、F329Y、A332G、A332C、A332S、A332V、A332P、A332T、I336V、I336F、I336L、L337I、L337F、L337M、I340N、I340L、I340V、I340M、I340F、I340T、I342V、I342D、I342L、I342C、V344I、V344L、V344A、V344M、K345G、K345R、K345A、I347V、I347L、G348P、P349D、P349E、D350E、D350N、D350Q、D350G、D350R、D350K、G351A、G351C、G351S、G351P、G351V、K352-、S353-、L354-、P355-、P355D、P355S、R356-、N357S、N357D、N357G、N357E、I358V、I358A、I358L、D359E、D359N、D359P、D359T、W360F、A361G、A361V、A361C、A361S、A361T、R362Q、R362K、R362H、R363H、R363K、R363N、I365V、I365L、T367S、T367N、T367P、T367V、T367A、T367D、G372C、G372A、G372N、G372S、E374D、E374K、E374R、E374N、E374Q、E374H、E374S、E374P、E374A、E374T、E374V、E374G、E374M、E374Y、W375K、W375G、G377A、G377W、H378Y、H378N、H378F、H378Q、H378R、H378M、H378I、H378E、H378W、H378D、H378V、L379I、L379M、L379S、L379V、L379A、L379T、F385W、F385Y、L395M、L395I、L395F、H396S、H396N、H396Y、H396Q、H396E、A398S、A398C、A398G、H399Y、A401S、A401G、A401N、K402T、K402P、K402S、K402R、K402E、K402Q、Q404E、Q404A、Q404R、Q404K、Q404H、Q404P、V406I、V406L、V406M、V406A、V406T、Q408R、Q408H、Q408E、Q408N、Q408M、K409R、K409Q、K409T、K409M、V410I、V410H、V410R、C411I、C411A、C411V、E413D、E413K、E413Q、E413R、E413P、E413N、E413S、E413H、E413A、N414M、N414D、N414L、N414G、N414S、V416I、V416F、V416L、V416Y、N417K、N417R、N417G、K419R、K419G、K419S、H420Y、H420Q、H420R、H420N、H420E、H420K、P422G、P422D、I424V、I424C、G425A、G425L、G425N、G425P、G425D、G429D、G429N、G429S、S430A、S430T、S430C、S430N、M431L、M431I、M431V、M431T、M431F、M431C、L432F、L432I、L432M、S433R、S433K、S433Q、S433A、S433T、S433N、H434Y、H434Q、H434N、H434R、L435I、L435M、L435V、L435F、G436K、G436S、G436P、A437G、A437K、A437P、A437S、L438I、L438M、L438V、G439A、G439N、G439S、G439D、A440S、A440C、A440T、A440V、R441V、R441I、R441L、P442A、P442V、T443V、T443I、T443A、T443G、T443L、W444-、W444Y、W444F、W444S、N445-、A446-、E447D、E447Q、E447K、F448Y、F448W、F448K、M449L、M449I、M449G、A450G、A450E、A450S、A450P、A450D、A450C、G451K、G451R、G451N、G451D、L452P、L452I、L452V、L452A、L452M、E453-、E453D、E453S、E454-、K455-、S456-、S457-、V458-、E459-、C460-、R461-、L462-、R463-、L464-、G465-、A466-、A467-、C468-、A469-、R470-、G471-、C472-、C472S、W473-、W473Q、C474-、C474A、S475-、D476-、A477-、A478-、S479-、L480-、I481-、S482-、W483-、L484-、G485-或
c)具有氨基酸序列SEQ ID NO.86和任选地一个或多个以下突变的δ-4去饱和酶多肽:H4S、H4Y、H4Q、H4E、H4N、A5S、A5G、A5C、A5P、A5T、A5V、A6R、A6G、A6K、A6S、A6N、T7K、T7S、T7R、T7N、T7A、T7G、K8E、K8D、K8R、K8Q、G9V、G9I、G9A、G9M、G9C、G10S、G10N、G10D、G10A、G10K、D11A、D11V、D11I、D11E、D11G、D11P、P12A、P12G、P12S、P12V、P12T、R15K、R15S、R15T、R15A、R15Q、D16A、D16G、D16P、D16S、M17A、M17L、M17I、M17V、E18K、E18R、E18Q、H19Y、H19F、H19I、H19M、H19L、H19Q、H19R、H19V、F20Y、F20W、F20I、F20A、S21T、S21A、S21C、S21G、S21N、Y22R、Y22F、E23T、E23D、E23N、E23S、R24K、R24E、R24Q、R24D、I25K、I25L、I25V、L26A、L26I、N27D、N27G、N27H、N27S、N27T、N27R、N27K、N27E、N27Q、N27A、N27V、D28H、D28N、D28T、E29T、E29N、E29D、E29Q、E29R、R30N、R30K、R30H、D31P、D31E、D31N、D31S、D32E、D32K、L33I、L33V、L33M、L33F、L33Y、C34T、C34A、C34S、C34V、V36L、V36I、V36M、V36T、G37A、G37D、G37E、G39A、A43S、A43C、A43L、A43R、A43K、T44S、T44V、T44N、T44P、T44K、T44A、A45C、A45N、A45G、D48E、D48N、K49Q、K49R、D54E、D54N、D54H、F55Y、F55W、F55V、F55I、V56L、V56I、V56M、V56A、V56T、D57S、D57N、D57E、L58I、L58F、L58M、G60P、R62Q、R62H、R62D、R62N、P66E、P66D、P66Q、H67G、H67A、H67N、H67S、F69W、F69M、F69Y、E70Q、E70M、E70H、Y71F、Y71W、Y71H、Y71L、R73K、R73Q、R73H、R74H、R74K、R74Q、R74Y、R74N、E75A、E75V、E75Q、E75L、E75P、W76G、W76S、P77D、P77V、P77K、P77A、P77T、P77E、P77Q、P77M、P77L、P78K、P78R、A79S、A79G、A79C、A79T、A79P、A79V、V80I、V80L、V80M、V80R、V80A、V80T、L81M、L81I、L81V、L81F、A82S、A82C、A82G、K83R、K83E、K83Q、K83P、K83N、Y84F、Y84W、K85F、K85G、K85Y、K85L、K85W、L89I、L89V、L89M、D90A、D90E、D90G、D90P、R91K、R91P、R91Q、R91H、R91E、R91A、D92E、D92N、D92S、D93E、D93Q、S94K、S94G、S94N、S94P、Y95F、Y95W、Y95H、Y95P、V96T、V96I、V96L、Q97H、Q97E、Q97R、Q97N、Q97K、Q97D、Q97Y、H98A、H98Y、H98V、H98I、H98F、H98R、D99E、D99P、D99T、S100P、S100E、S100A、S100D、S100N、S100T、G101L、G101I、G101M、G101A、Y102H、Y102Q、Y102W、Y102M、Y102V、L103K、L103M、L103I、L103V、R104Q、R104K、R104H、R104E、L105I、L105F、C106G、C106A、C106N、C106S、C106K、A107C、A107S、A107G、E108D、E108Q、E108P、E108S、E108K、E108A、N110R、N110D、N110H、G111A、G111K、G111R、G111S、I112L、I112V、L113I、L113M、K115R、K115M、K115Q、K115L、G116W、G116Q、G116N、S117E、S117D、S117N、S117G、G118W、G118A、G118N、W119F、P122A、P122G、W124Y、W125Y、I126L、I126V、C129A、C129G、C129S、L131I、L131V、L131M、L132I、L132V、L132M、V133A、V133I、V133L、V133C、V133M、A135T、A135P、A135S、A135G、L136I、L136V、Y137F、Y137H、Y137W、Y137S、Y137T、L138I、L138V、L138M、L138F、L138T、D139E、D139N、D139Q、Y140W、Y140F、Y140H、Y140G、Y141F、Y141H、Y141L、M142W、M142L、M142I、M142F、L143I、L143V、A144L、A144C、A144G、A144S、R145K、R145S、R145N、R145Q、R145D、P147K、P147E、P147R、I149L、I149V、L150F、L150I、L150M、L150Y、L150W、L150P、A152S、A152G、A152C、A152T、A152P、A152V、I153V、I153L、I154V、I154L、I154F、L155I、L155V、L155M、L155F、L155C、L155A、L155T、L155S、L158I、L158V、F159Y、W161G、W161A、W161C、A174S、A174G、A174C、A174T、A174P、A174V、L175I、L175V、L175M、N178H、N178Y、P179S、V180M、V180A、V180I、V180P、V181I、V181L、V181A、V181T、Y183R、Y183H、Y183W、L184C、L184I、F185L、F185I、A188S、A188G、A188M、A188Q、A188K、S195N、S195G、S195D、S195T、S195A、S195K、M196R、M196L、M196Q、M196I、M197L、M197V、M197I、M197Q、M197C、L200I、L200V、Q201R、Q201E、Q201K、Q202E、Q202K、Q202R、Q202D、Q202H、G206N、G206A、G206S、G206K、G206D、G206W、G206M、L209I、L209F、L209T、T211C、T211S、T211V、T211A、T211N、T211P、D213E、D213N、D213R、D213G、I214V、I214F、I214Y、I214H、D215N、D215Q、D215P、H216Y、H216F、H216Q、H216W、H216R、H216E、H216N、H216M、H216V、H216P、H216I、H216L、H216D、H216A、V220I、V220L、V220M、V220A、V220T、V220Q、G222A、G223H、G223N、G223D、G224S、G224N、G224A、A225V、A225T、A225I、A225G、L226I、L226V、R227K、R227Q、R227N、R227E、R227H、R227T、K229S、K229G、K229A、K229T、P230K、P230D、P230E、P230R、T231V、T231S、T231P、T231N、T231A、T231I、T231Y、D232S、D232G、D232E、G233I、G233L、G233F、G233C、G233M、G233S、W234F、W234Y、W234P、L235M、L235I、L235V、L235F、L235K、P236E、P236D、W237F、H239A、H239S、H239Y、H239G、H239N、L240I、L240V、L243I、L243V、L243M、L243F、L243Y、F245I、F245Y、L248G、L248M、L248A、L248K、L248C、L248F、E249D、E249Q、E249K、E249P、E249R、E249N、E249S、E249V、E249A、E249T、E249G、E249H、E249I、A250V、A250Q、A250L、A250C、A250P、A250E、L251M、L251I、Y252F、Y252W、Y252H、Y252L、G253A、G253C、K255Q、K255R、W256L、W256F、V257I、V257L、V257M、V257F、F258Y、F258W、F258H、F258S、D260G、L261A、L261I、L261V、H262N、H262Q、H262R、H262L、E263D、E263Q、E263K、L265I、L265F、E266D、E266A、E266G、E266P、W267F、W267Y、W267M、K268R、K268E、K268Q、W269Y、E270K、E270D、E270R、P273K、P273R、I274L、I274V、P275S、P275D、P275N、P275G、P276E、P276D、P276Q、L277I、L277M、L277V、L277C、L277A、L277T、L277S、A278Y、A278C、A278F、A278V、A278S、A278L、R279K、R279Q、R279H、R279E、R279L、R279M、R279V、R279A、P280K、P280D、P280E、P280G、P280Q、P280S、P280R、P280A、E281D、E281L、E281K、E281V、E281Q、F282Y、F282R、F282H、A283G、A283S、A283N、A283C、P284I、V286I、V286L、G287A、G287S、G287P、G287N、G287K、G287R、G287E、G287D、G287Q、G287T、G287H、G287C、G287W、G287M、G287Y、G287V、C288L、C288I、K289R、K289E、K289Q、K289N、K289P、L290I、L290V、G291F、G291A、G291W、G291S、W293F、A294V、A294C、A294G、A294S、A294P、A294I、F296Y、F296W、F296K、V297I、V297F、V297L、V297Y、A298V、A298I、A298L、A298T、A298C、L299I、L299V、L299M、L301F、L301I、W302A、W302G、H304Q、H304E、H304R、H304Y、H304N、P305F、P305Y、S306T、S306N、S306G、S306A、W307F、W307L、W307Y、W307I、W307M、W307V、H308Y、L310I、L310M、L310V、L310F、L310C、L310A、L311I、L311V、L311C、L311A、V313I、V313T、V313P、V313L、C314Y、A315L、A315C、A315S、A315G、W316T、V317I、V317L、V317M、C318A、C318G、C318S、T319S、T319V、T319N、T319I、T319P、T319L、G320A、G320N、G320K、S321A、S321T、S321L、S321C、S321V、F322L、F322Y、A325G、A325C、A325S、A325V、A325P、A325T、I329V、I329F、I329L、L330I、L330F、L330M、I333N、I333L、I333V、I333M、I333F、I333T、I335V、I335D、I335L、I335C、V337I、V337L、V337A、V337M、K338G、K338R、K338A、I340V、I340L、G341P、P342D、P342E、D343E、D343N、D343Q、D343G、D343R、D343K、G344A、G344C、G344S、G344P、G344V、N345S、N345D、N345G、N345E、I346V、I346A、I346L、D347E、D347N、D347P、D347T、W348F、A349G、A349V、A349C、A349S、A349T、R350Q、R350K、R350H、R351H、R351K、R351N、I353V、I353L、T355S、T355N、T355P、T355V、T355A、T355D、G360C、G360A、G360N、G360S、E362D、E362K、E362R、E362N、E362Q、E362H、E362S、E362P、E362A、E362T、E362V、E362G、E362M、E362Y、W363K、W363G、G365A、G365W、H366Y、H366N、H366F、H366Q、H366R、H366M、H366I、H366E、H366W、H366D、H366V、L367I、L367M、L367S、L367V、L367A、L367T、F373W、F373Y、L383M、L383I、L383F、H384S、H384N、H384Y、H384Q、H384E、A386S、A386C、A386G、H387Y、A389S、A389G、A389N、K390T、K390P、K390S、K390R、K390E、K390Q、Q392E、Q392A、Q392R、Q392K、Q392H、Q392P、V394I、V394L、V394M、V394A、V394T、Q396R、Q396H、Q396E、Q396N、Q396M、K397R、K397Q、K397T、K397M、V398I、V398H、V398R、C399I、C399A、C399V、E401D、E401K、E401Q、E401R、E401P、E401N、E401S、E401H、E401A、N402M、N402D、N402L、N402G、N402S、V404I、V404F、V404L、V404Y、N405K、N405R、N405G、K407R、K407G、K407S、H408Y、H408Q、H408R、H408N、H408E、H408K、P410G、P410D、I412V、I412C、G413A、G413L、G413N、G413P、G413D、G417D、G417N、G417S、S418A、S418T、S418C、S418N、M419L、M419I、M419V、M419T、M419F、M419C、L420F、L420I、L420M、S421R、S421K、S421Q、S421A、S421T、S421N、H422Y、H422Q、H422N、H422R、L423I、L423M、L423V、L423F、G424K、G424S、G424P、A425G、A425K、A425P、A425S、L426I、L426M、L426V、G427A、G427N、G427S、G427D、A428S、A428C、A428T、A428V、R429V、R429I、R429L、P430A、P430V、T431V、T431I、T431A、T431G、T431L、E432D、E432Q、E432K、F433Y、F433W、F433K、M434L、M434I、M434G、A435G、A435E、A435S、A435P、A435D、A435C、G436K、G436R、G436N、G436D、L437P、L437I、L437V、L437A、L437M或
d)具有氨基酸序列SEQ ID NO.79和任选地一个或多个以下突变的δ-4去饱和酶多肽:V9-、V10G、S11G、D12-、S13-、L20-、K21-、E29R、K37D、E42G、F63L、F63I、L76Y、L76F、L76W、Q78R、Y79R、S81W、L82P、S84A、E88K、Y89F、V94L、V94I、T104D、Q109R、K111C、A113E、R115N、W123G、W124F、W129Y、Y130W、I136L、L137I、D144E、Y145W、Y145F、W147M、I158V、S160L、Y164F、S179A、I205L、C216T、R218D、S229G、T232R、R235P、P239W、E241P、F242W、V262I、V262L、D268E、M272W、Y274W、K278P、L279I、K285P、I289P、I291V、R294K、V295L、V295I、F298W、V304L、V304I、H309Q、F310P、Y313H、A323C、A325G、L327F、G330A、A342V、A342I、P346G、E347P、A349G、K350-、W354F、S361T、C366G、K369W、I372H、Y379F、K414H、G416P、D423G、T425M、F426L、Q428H、V429L、V429I、A436P、Y438-、N439-、E440D。
2.多核苷酸,其包含选自以下的核酸序列:
a)具有如SEQ ID NOs:1、4、7、10、13、16、19、22、25、28、31、78或83中所示的核苷酸序列的核酸序列,
b)编码具有如SEQ ID NOs:2、5、8、11、14、17、20、23、26、29、32、79、84、85或86中所示的氨基酸序列的多肽或权利要求1的多肽的核酸序列,
c)与a)或b)的核酸序列具有至少70%同一性的核酸序列,其中所述核酸序列编码具有去饱和酶、酮酰基-CoA合酶(KCS)或酮酰基-CoA还原酶(KCR)或溶血磷脂-辅酶A合酶(LACS)活性的多肽,
d)核酸序列,其编码具有去饱和酶、酮酰基-CoA合酶(KCS)、酮酰基-CoA还原酶(KCR)或溶血磷脂-辅酶A合酶(LACS)活性并具有与a)-c)任一项的氨基酸序列具有至少60%同一性的氨基酸序列的多肽;和
e)能够在严格条件下与a)-d)任一项杂交的核酸序列,其中所述核酸序列编码具有去饱和酶、酮酰基-CoA合酶(KCS)、酮酰基-CoA还原酶(KCR)或溶血磷脂-辅酶A合酶(LACS)活性的多肽。
3.权利要求2的多核苷酸,其中所述多核苷酸还包含有效连接所述核酸序列的表达控制序列。
4.权利要求2或3的多核苷酸,其中所述多核苷酸还包含有效连接所述核酸序列的终止序列。
5.载体,其包含权利要求2-4任一项的多核苷酸。
6.宿主细胞,其包含权利要求2-4任一项的多核苷酸或权利要求5的载体或表达权利要求1的异源δ-4去饱和酶。
7.制造权利要求2-4任一项的多核苷酸编码的多肽的方法,其包括
a)在允许产生所述多肽的条件下培育权利要求5的宿主细胞;和
b)从步骤a)的宿主细胞获得所述多肽。
8.多肽,其由权利要求2-4任一项的多核苷酸编码或可通过权利要求7的方法获得。
9.非人转基因生物,其包含权利要求1-4任一项的多核苷酸或可通过权利要求7的方法获得。
10.权利要求9的非人转基因生物,其是植物、植物部分或植物种子。
11.用于制造多不饱和脂肪酸的方法,其包括:
a)在允许在所述宿主细胞中产生多不饱和脂肪酸的条件下培育权利要求6的宿主细胞;和
b)从所述宿主细胞获得所述多不饱和脂肪酸。
12.用于制造多不饱和脂肪酸的方法,其包括:
a)在允许在所述宿主细胞中产生多不饱和脂肪酸的条件下培育权利要求9或10的非人转基因生物;和
b)从所述非人转基因生物获得所述多不饱和脂肪酸。
13.权利要求11或12的方法,其中所述多不饱和脂肪酸是花生四烯酸(ARA)、二十碳五烯酸(EPA)或二十二碳六烯酸(DHA)。
14.用于制造油、脂类或脂肪酸组合物的方法,其包括权利要求11-13任一项的方法的步骤和将多不饱和脂肪酸配制为油、脂类或脂肪酸组合物的其他步骤,其中优选地所述油、脂类或脂肪酸组合物用于饲料、食品、化妆品或药物。
15.油,其包含可通过权利要求11-13任一项的方法获得的多不饱和脂肪酸。
16.筛选δ-4去饱和酶基因的方法,其包括步骤
a)从属于Ichthyosporea或定鞭藻纲的分类等级,优选鱼孢霉目或Pavlovales目,更优选Anurofeca、Creolimax、Ichthyophonus、Pseudoperkinsus、Psorospermium或Sphaeroforma属的生物提取遗传物质,
b)将所述遗传物质与编码如上文或下文定义的本发明的δ-4去饱和酶的至少10个,优选至少20个连续氨基酸的核酸在严格条件下杂交,和
c)检测杂交或杂交的缺失。
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CN112746066A (zh) * | 2021-01-25 | 2021-05-04 | 洛阳华荣生物技术有限公司 | 一种l-赖氨酸脱羧酶突变体及其应用 |
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AU2013298113A1 (en) | 2015-01-29 |
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