CN103981156A - 来自ulkenia的PUFA-PKS基因 - Google Patents
来自ulkenia的PUFA-PKS基因 Download PDFInfo
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- CN103981156A CN103981156A CN201410175661.8A CN201410175661A CN103981156A CN 103981156 A CN103981156 A CN 103981156A CN 201410175661 A CN201410175661 A CN 201410175661A CN 103981156 A CN103981156 A CN 103981156A
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Abstract
本发明涉及来自ulkenia的PUFA-PKS基因。具体而言,本发明涉及编码特异于多酮化合物合酶(PKS)的序列的基因。由此合成的PKS特征在于其产生PUFAs(多不饱和脂肪酸)的酶能力。本发明还涉及鉴定相应的DNA序列,以及所述核苷酸序列用于产生重组和/或转基因生物的应用。
Description
本申请为国际申请PCT/EP2005/003701于2006年12月8日进入中国国家阶段、申请号为200580018878.7、发明名称为“来自ulkenia的PUFA-PKS基因”的分案申请。
技术领域
本发明描述了对于多酮化合物合酶(polyketide synthase)(PKS)特异性的基因编码序列。由它们合成的PKS的特征是具有产生PUFAs(多不饱和脂肪酸)的酶学能力。本发明另外包括相应DNA序列的鉴定以及所述核苷酸序列对于生产重组和/或转基因生物的用途。
背景技术
术语PUFAs(多不饱和脂肪酸)表示具有链长度>C12和至少两个双键的多重不饱和长链脂肪酸。有两个PUFA的主要家族,其根据相对于烷基末端,在ω-3和在ω-6脂肪酸中的第一个双键的位置而区别。它们是细胞膜的重要组分,在那里它们以脂质,特别是磷脂的形式存在。PUFAs还作为在人和在动物中的重要分子,例如,前列腺素,白三烯和环前列腺的初级阶段而起作用(A.P.Simopoulos,essential fattyacids in health and chronic disease,Am.J.Clin.Nutr.1999(70),pp.560-569)。ω-3脂肪酸族的重要代表是DHA(二十二碳六烯酸)和EPA(二十碳五烯酸),其可以在鱼油和在海洋微生物中发现。ω-6脂肪酸的重要代表是ARA(花生四烯酸),其出现在,例如,丝状真菌中,但是也可以从动物组织如肝和肾中分离。DHA和ARA在人母乳中彼此相接出现。
PUFAs对于人来说在适当发育方面,特别是对于发育脑,组织形成及其修复是必需的。因而,DHA是人细胞膜的重要组分,特别是神经的细胞膜。它在脑功能的成熟中发挥重要作用并且对于视力的发育是必需的。ω-3PUFAs如DHA和EPA被用作营养添加剂,因为具有DHA充分供应的平衡营养对于某些疾病的预防有利(A.P.Simopoulos,Essential fatty acids in health and chronic disease,AmericanJournal of Clinical Nutrition1999(70),pp.560-569)。例如,患有非胰岛素依赖型糖尿病的成人呈现与后来出现的心脏问题相关的DHA平衡的缺陷或者至少是失衡的DHA平衡。同样地,神经元疾病如,例如,阿尔茨海默病或精神分裂症伴随着低的DHA水平。
有大量的DHA商业提取物的来源,例如,来自海洋冷水鱼的油,蛋黄部分或海洋微生物。适于提取n-3PUFA的微生物发现于,例如,弧菌属(Vibrio)的细菌中(例如,海产弧菌(Vibrio marinus))或腰鞭毛虫(Dinophyta)中,其中特别是Crypthecodinium属,如C.cohnii或在Stramenopiles(或Labyrinthulomycota)中,如Pinguiophyceae如,例如,Glossomastix,Phaeomonas,Pinguiochrysis,Pinguiococcus和Polypodochrysis。其它生产PUFA的优选微生物特别属于Thraustochytriales目,(Thraustchytriidea)具有Japonochytrium属,Schizochytrium属,Thraustochytrium属,Althornia属,Labyrinthuloides属,Aplanochytrium属和Ulkenia属。
提取自商业上已知的PUFA来源如植物或动物的油的特征经常是非常不均匀的组成。以这种方式提取的油必须进行昂贵的纯化处理以便能够富集一种或几种PUFAs。另外,来自这些来源的PUFA的供应也会发生不可控制的波动。因而,疾病和天气影响能够减少动物也能够减少植物的产量。从鱼中提取PUFA出现季节波动并且甚至能够由于过度捕捞或气候变化(例如,厄尔尼诺现象)而暂时性地停止。动物油,特别是鱼油,可以通过食物链从环境中积聚有害物质。已知动物受有机氯化物,例如,多氯化联苯高度胁迫,特别是在商业性鱼场中,其抵消了鱼类消费的健康方面(Hites等,2004,Global assessmentof organic contaminants in farmed salmon,Science303,pp.226-229)。鱼产品质量的所得损失导致消费者对于鱼和鱼油作为ω-3PUFA来源的接受度下降。另外,从鱼浓缩DHA因为高度技术需要而相对昂贵。另一方面,DHA存在于少数海洋微生物,占细胞总脂肪组分的大约50%,并且它们能够在大的发酵罐中进行相对经济地培养。微生物的另一个优点是提取自它们的油的组成限于少数几种组分。
对于长链PUFA如二十二碳六烯酸(DHA;22:6,n-3)和二十碳五烯酸(EPA;20:5,n-3)的生物合成已知多种生物催化途径。在真核生物中生产长链PUFA的常规生物合成途径起始于亚油酸(LA;18:2,n-6)和α亚油酸的δ-6去饱和作用。它导致由亚油酸合成γ亚油酸(GLA;18:3,n-6)以及由α亚油酸合成十八碳四烯酸(OTA;18:4,n-3)。对于n-6以及n-3脂肪酸来说,此去饱和作用后接延伸步骤以及δ-5去饱和作用,致成花生四烯酸(ARA;20:4,n-6)和二十碳五烯酸(EPA;20:5,n-3)。起始自二十碳五烯酸(EPA;20:5,n-3)的二十二碳六烯酸(DHA;22:6,n-3)的合成随后能够通过两种不同的生物合成途径发生。在所谓的线性生物合成途径中,发生二十碳五烯酸(EPA;20:5,n-3)延伸另外两个碳单位,随后发生δ-4去饱和作用以形成二十二碳六烯酸(DHA;22:6,n-3)。这种生物合成途径的存在能够通过生物如破囊壶菌属(Thraustochytrium)和裸藻属(Euglena)中δ-4去饱和酶的存在而确证(Qiu,等,Identification of a delta4fatty acid desaturase fromThraustochytrium sp.involved in the biosynthesis of docosahexaenoic acidby heterologous expression in Saccharomyces cerevisiae and Brassicajuncea.,J.Biol.Chem.276(2001),pp.31561-31,566和Meyer等,Biosynthesis of docosahexaenoic acid in Euglena gracilis:Biochemicaland molecular evidence for the involvement of a delta4fatty acyl groupdesaturase.Biochemistry42(2003),pp.9779-9788)。起始自二十碳五烯酸(EPA;20:5,n-3)的二十二碳六烯酸(DHA;22:6,n-3)合成的第二条途径,所谓的Sprecher途径,独立于δ-4去饱和作用。它由两个连续延伸步骤,每步延伸2个碳单位至二十四碳五烯酸(24:5,n-3)以及随后δ-6去饱和作用至二十四碳六烯酸(24:6,n-3)组成。随后通过过氧化物酶体β氧化作用缩短两个碳单位而接着发生二十二碳六烯酸的形成(H.Sprecher,Metabolism of highly unsaturated n-3and n-6fatty acids.Biochimica et Biophysica Acta1486(2000),pp.219-231)。这一第二生物合成途径是在哺乳动物中占优势的DHA合成途径(Leonard等,Identification and expression of mammalian long-chain PUFA elongationenzymes.Lipids37(2002),pp.733-740)。对于C20PUFA形成的备选生物合成途径存在于少数缺δ-6变性酶活性的生物中。这些生物包括,例如,原生生物Acanthamoeba sp.和Euglena gracilis。在备选的C20PUFA合成中的第一步在于C18脂肪酸,亚油酸(LA;18:2,n-6)和α亚油酸(ALA;18:3,n-3)延伸两个碳单位。随后通过δ8去饱和作用和接下来的δ5去饱和作用将得到的脂肪酸二十碳二烯酸(20:2,n-6)和二十碳三烯酸(20:3,n-3)转化成花生四烯酸(ARA;20:4,n-6)和/或二十碳五烯酸(EPA;20:5,n-3)(Sayanova和Napier,Eicosapentaenoic acid:Biosynthetic routes and the potential for synthesis in transgenic plants.Phytochemistry65(2004),pp.147-158;Wallis和Browse;The delta-8desaturase of Euglena gracilis:An alternate pathway for synthesis of20-carbon polyunsaturated fatty acids.Arch.Biochem.Biophys.362(1999),pp.307-316)。
高等植物不具有由初级阶段合成C20PUFA的能力。它们通过各种去饱和酶起始自硬脂酸(18:0),形成油酸(C18:1;δ-9去饱和酶),亚油酸(18:2,n-6,δ12去饱和酶)和α亚油酸(18:3,n-3;δ15去饱和酶)。
不过,某些海洋微生物采取完全不同的生物合成途径来产生EPA和DHA。这些产生PUFA的微生物包括γ蛋白细菌的海洋代表以及少数几种cytophaga flavobacterium bacteroides族和到目前为止的真核性原生生物,Schizochytrium sp.ATCC20888(Metz等,2001,Productionof polyunsaturated fatty acids by polyketide synthases in both prokaryotesand eukaryotes.Science293:290-293)。它们通过所谓的多酮化合物合酶(PKS)来合成长链PUFA。这些PKSs代表催化由酮化合物(ketide)单位组成的次级代谢产物合成的大酶(G.W.Wallis,J.L.Watts和J.Browse,Polyunsaturated fatty acid synthesis:what will they think of next?Trendsin Biochemical Sciences27(9)(2000)pp.467-473)。多酮化合物的合成包含许多与脂肪酸合成类似的酶反应(Hopwood&Sherman Annu.Rev.Genet.24(1990)pp.37-66;Katz&Donadio Annu.Rev.of Microbiol.47(1993)pp.875-912)。
已知不同PUFA-PKSs(PUFA-合成的PKSs)的基因序列。由此,从海洋细菌Shewanella sp.分离出38kb基因组片段含有生产EPA的信息。随后对这一片段的测序导致鉴定了8个开放阅读框(ORFs)(H.Takeyama等,Microbiology143(1997)pp.2725-2731)。来自Shewanella的这些开放阅读框,其中五个与多酮化合物合酶基因密切相关。同样,美国专利号5,798,259描述了来自Shewanella putrefaciens SCRC-2874的EPA基因簇。PUFA-PKS基因也发现于海洋原核生物Photobacteriumprofundum株SS9中(Allen和Bartlett,Microbiology2002,148pp.1903-1913)和Moritella marina株MP-1,早期的Vibrio marinus(Tanaka等,Biotechnol.Letters1999,21,pp.939-945)。类似的产生PUFA的PKS样ORFs也能够在真核性原生生物Schizochytrium中鉴定(Metz等,Science293(2001)pp.290-293,US专利No.6,556,583及WO02/083870A2)。在Schizochytrium中确定了三种ORFs,其与来自Shewanella的EPA基因簇呈现部分同一性。在少数原核生物和真核生物Schizochytrium中存在这些保守性PKS基因给出了暗示,PUFA-PKS基因可能在原核生物和真核生物之间进行了水平转移。
即使是使用正常情况下不产生PUFAs的微生物中分离的基因簇对PUFAs进行转基因生产也已经能够得以显示了。因而,存在于来自Shewanella sp.SCRC-2738的簇中的上述五种ORFs(开放阅读框)足以在非IPA生产者大肠杆菌(E.coli)和Synechoccus sp.中生产可测量量的EPA(Yazawa,Lipids1996,31,pp.297-300和Takayama等,Microbiology1997,143,pp.2725-2731)。
通常,对于大规模生产PUFAs的新的PUFA生产者总是存在需要。首先这种生产是否发生在,例如,原核生物,原生生物或在植物中并不重要。目标始终是尽可能经济地和以尽可能保护环境的方式大量生产高质量的PUFAs。本发明追求这一目标,因为它介绍了来自特别有效的PUFA生产者Ulkenia sp.的合适的PUFA-PKS基因。
发明内容
考虑到技术状态,所以本发明的任务是从生产DHA的微生物Ulkenia sp.中鉴定和分离另外的PUFA-PKS基因,其极适于生产PUFAs。此外,应当获得关于这些基因的位置和排列以及它们的调控元件的知识。由此获得的知识,特别是由此获得的核酸物质,应当使得PUFA-PKS基因在同系生物以及在转基因生物中的加强表达成为可能。
通过本发明的权利要求书中所定义的主题解决了这些任务以及其它未曾被明确地说明但可以从本文件初始讨论的联系中轻易得到或总结的其它任务。
1.PUFA-PKS,其特征是它们
a.包括在SEQ ID No.6(ORF1),7(ORF2),8和/或80(ORF3)中所示氨基酸序列的至少其中一种,以及具有与它们有至少70%,优选80%,特别优选至少90%和更加特别优选至少99%和最优选100%序列同源性的同源序列,其具有PUFA-PKS的至少一个结构域的生物学活性,或
b.包括在SEQ ID No.32,34,45,58,59,60,61,72,74和/或77中所示氨基酸序列的至少其中一种,以及具有与它们有至少70%,优选80%,特别优选至少90%和更加特别优选至少99%和最优选100%序列同源性的同源序列,其具有PUFA-PKS的至少一个结构域的生物学活性。
2.具有10个或更多ACP结构域的根据权利要求1的分离的PUFA-PKS。
另外,本发明在优选的方面涉及这样一种PUFA-PKS,其包含与序列SEQ ID No.6(ORF1),7(ORF2)和/或8和/或80(ORF3)的至少500个直接连续氨基酸具有至少70%,优选至少80%,特别优选至少90%和更加特别优选至少99%同一性的至少一种氨基酸序列。
在另一个优选的方面,本发明涉及分离的DNA分子,其编码根据任一项在前权利要求的PUFA-PKS。
后者优选特征为它编码与序列SEQ ID No.6(ORF1),7(ORF2)和/或8和/或80(ORF3)的至少500个直接连续氨基酸具有至少70%同一性的氨基酸序列。
另外,本发明涉及这样的分离DNA分子,其与来自序列SEQ ID No.3,4,5和/或9的至少500个连续核苷酸具有至少70%,优选至少80%,特别优选至少90%和更加特别优选至少95%的同一性。
在另一个优选的方面,本发明涉及一种重组DNA分子,其包含与控制转录的至少一种DNA序列功能性连接的先前所述DNA分子的其中之一,优选选自SEQ ID No.3,4和5和/或9或其至少500个核苷酸的部分以及它们的功能性变体。
在又一个优选的方面,本发明涉及包含前述重组DNA分子的重组宿主细胞。
在又一个优选的视点下,本发明涉及内源性表达具有至少10个ACP结构域的根据本发明的PUFA-PKS的重组宿主细胞。
另外,在又一个优选的方面,本发明涉及一种生产含有PUFA的油的方法,包括培养这种重组宿主细胞,以及涉及以此方式生产的油。
另外,在又一个优选的方面,本发明涉及一种生产含有PUFA,优选DHA的生物质量的方法,包括培养这种重组宿主细胞,以及涉及以此方式生产的生物质量。
所以,在又一个优选的方面,本发明还涉及根据权利要求15的重组生物质量,其包含根据权利要求8的核酸和/或根据权利要求1的氨基酸序列或与它同源的至少500个连续氨基酸的部分。
本发明在又一个优选的方面还涉及SEQ ID No.32,33,34,45,58,59,60,61,72,74和/或77中所示、来自包含SEQ ID No.6,7,8和/或80的PUFA-PKS的个别酶结构域的用途,用于生产人工多酮化合物,例如,多酮化合物抗生素和/或新的,变化的脂肪酸。
根据本发明,有关核酸的同一性指示在待比较链的特定位置上的相同碱基对。不过,缺口是有可能的。以%计算同一性值的可能性由程序blastn和fasta代表。
就氨基酸而言,概念同源性也包含,例如氨基酸序列的保守性交换,其丝毫不影响蛋白质的功能和/或结构。甚至是这些同源性值也通过本领域熟练技术人员已知的程序,例如,blastp,Matrix PAM30,GapPenalties:9,Extension:1进行计算(Altschul等,NAR25,3389-3402)。
来自Ulkenia sp.的PUFA-PKS基因的序列信息可由SEQ ID No.3-5和/或9中所定义的核酸序列和氨基酸序列获得。SEQ ID No.1和2代表目前分离的两种粘粒上的完整基因组DNA序列(见实施例2和3)。后者对其部分包含PUFA合成所必需的三种相关开放阅读框ORFs1-3的信息以及它们的侧翼调控序列。另外,作为其结果提出了能够源自基因组序列的蛋白质序列。
本发明另外包括用根据本发明的核酸对宿主生物进行同源和异源转化用来生产高纯PUFAs的方法。分离的开放阅读框优选导致在同系生物和转基因生物中生产PUFA,特别是DHA,EPA和DPA。
由此生产的PUFAs优选作为生物质量的组分或作为油而存在。
在本发明之前,只有真核生物,原生生物Schizochytrium的PUFA-PKS基因是已知的(美国专利号6,566,583,WO02/083870)。随后测定的序列数据部分源自cDNA和源自染色体DNA。在本发明中首次从染色体DNA完全描述了对于PUFA合成必需的真核性原生生物的所有PUFA-PKS基因。这不仅导致确定了以往未知的来自Ulkenia sp.的PUFA-PKS编码基因信息,还另外提供了关于侧翼调控元件如转录启动子和终止子的数据。此外,染色体序列信息使得深入了解个别PUFA-PKS基因的位置和排列成为可能。
这里完全令人吃惊的是簇同样地不再存在,因为以往知道它是来自原核性PUFA-PKS代表如Shewanella,Photobacterium或Moritella。鉴定的粘粒(Seq ID No.1)一开始显示个别ORFs的线性排列在Ulkenia中被打乱并且还显示个别ORFs的阅读方向是反向的(图1)。这可能是大段基因转座的结果。作为转座的结果,个别ORFs还清楚地呈现彼此的更大间隔。因而,两个ORFs1和2具有大约13kb的间隔。第三个ORF直到在另一个粘粒上才能够在此情况下得以鉴定(SeqID No.2)并且在两种粘粒之间(Seq ID No.1和2)没能发现部分同一性(图1)。这意味着来自Ulkenia sp.的ORF在空间上不再位于两种ORFs1和2附近。这作出结论,即PUFA基因簇,已知来自上述原核代表,不再存在于真核生物Ulkenia sp.中。已经部分测定了原生生物Schizochytrium的个别PUFA-PKS基因在基因组上的位置和排列(WO02/083870)并且还显示了两种ORFs A和B的相反方向。不过,它们彼此仅仅分离4224个碱基对。在专利申请WO02/083870中将这一序列片段讨论为具有双向启动子元件的基因间隔区。至少对于Ulkenia在同源性ORFs1和2之间的双向启动子元件似乎是不可能的,这是因为对于Ulkenia测定的12.95kb的间隔区。没有其它明显ORFs存在于来自Ulkenia的ORFs1和ORF2之间的12.95kb区域之内。表明区域中发生了大的重组和/或转座事件。转座酶样事件也能够基于少数重复序列重复而发生。
更加令人特别吃惊的是与EPA生产者Shewanella(6x ACP)和Photobacterium(5x ACP)的PUFA-PKS以及DHA生产者Moritella(5x ACP)和Schizochytrium(9x ACP)的PUFA-PKS相比,来自Ulkeniasp.的PUFA-PKS具有最大数目的酰基载体蛋白的重复,有10个ACP结构域(图3)。这意味着分离自Ulkenia sp.的PUFA-PKS相对于来自亲缘性原生生物Schizochytrium的PUFA-PKS不仅具有偏移性氨基酸序列,而且在结构上也是独特的。另一种特性是这样的事实,即来自Ulkenia sp.的第三个ORF相对于来自Schizochytrium的ORF C短了38个氨基酸并且另包含了丙氨酸富集的结构域,该结构域并不以此方式存在于Schizochytrium中(图6)中。令人感兴趣的是,这种序列类似存在于来自ORF1的个别ACT结构域之间的区域并且可能代表连接区。所述相似性在于序列长度以及丙氨酸连续仅被个别脯氨酸和缬氨酸打乱的事实。相对于Schizochytrium ORF C缺失的ORF3中氨基酸的最大部分是删除的结果,有30个氨基酸长,位于脱水酶/异构酶结构域之间(图6)。作为结果,这些结构域位于相应的蛋白质上,彼此相距短的间隔,这能够对于酶学活性具有影响。对于ORF3来说,即使其它的5’位置上的ATG密码子也可作为起始密码子,从而在理论上甚至是最大为1848个氨基酸长的ORF也能够存在(Seq ID No.9和80)。在此情况下甚至同时出现ORF3的变体也是可能的。
特别地,来自Ulkenia sp.的ORF1(Seq ID No.3和6)在一方面包含所谓的β酮酰基合成酶结构域(Seq ID No.14和32),其特征是靶标(motive)(DXAC)(Seq ID No.12和30)。Ulkenia ORF1中酶学结构域的活性中心的靶标能够以优选的形式扩展到17个氨基酸的范围(GMNCVVDAACASSLIAV)Seq ID No.11和29)。完整的β酮酰基合成酶结构域可以分成N末端(Seq ID No.10和28)和分成C末端(Seq ID No.13和31)部分。β酮酰基合成酶结构域的生物学功能是催化脂肪酸和/或PKS合成的缩合反应。进行延伸的酰基基团通过硫酯键结合到酶学结构域的活性中心的半胱氨酸基团并且以几个步骤转移到酰基载体蛋白上的丙二酰基团的碳原子2上,释放CO2。β酮酰基合成酶结构域后接丙二酰CoA-ACP转移酶结构域(Seq ID No.15和33)。此结构域催化丙二酰CoA转移到酰基载体蛋白(ACP)上的4’-phosphopantetheine基团。丙二酰CoA-ACP转移酶结构域也将甲基或乙基丙二酸酯转移到ACP上,期间它们将分枝导入其它的线性碳链上。随后将连接区域后接富含丙氨酸序列的部分(Seq ID No.16和34),该部分包含10个重复的酰基载体蛋白结构域(ACP结构域)(17-26和35-44)。这些ACP结构域对于它们的部分彼此通过连接区域相互分离,所述连接区域主要由丙氨酸和脯氨酸组成。每个ACP结构域的特征是4’-phosphopantetheine分子(LGXDS(L/I))的结合靶标。所述4’-phosphopantetheine分子在这里结合到靶标内的保守丝氨酸上。ACP结构域通过4’-phosphopantetheine基团作为载体起作用来生长脂肪酸和/或多酮化合物链。与酮还原酶具有部分同一性的序列(Seq ID No.27和45)随后接上。这些结构域的生物学功能在于3-酮酰基-ACP化合物的NADPH依赖型还原作用。它代表脂肪酸生物合成中的第一次还原反应。这种反应在多酮化合物合成中也经常发生(还参见图3)。
来自Ulkenia sp.的ORF2(Seq ID No.4和7)也以β酮酰基合成酶结构域(Seq ID No.50和58)起始,其特征是靶标(DXAC)(Seq IDNo.48和56)。Ulkenia ORF2中酶学结构域的活性中心的这种靶标能够以优选的形式扩展到17个氨基酸的范围(PLHYSVDAACATALYVL)Seq ID No.47和55)。完整的β酮酰基合成酶结构域可以分成N末端(Seq ID No.46和54)和C末端(Seq ID No.49和57)部分。此结构域的生物学活性对应于ORF1中所述的β酮酰基合成酶结构域。Kethosynthases在延伸循环中发挥关键作用并且显示了比脂肪酸合成的其它酶更高的底物特异性。这再次后接与β酮酰基合成酶结构域具有较小部分同一性的序列片段。另外,这一结构域缺少用于活性中心的靶标DXAC。它具有来自II型PKS类似系统的所谓链长因子(CLF)的特性(Seq ID No.51和59)。CLF氨基酸序列与酮合成酶具有部分同一性,但是没有具有相应的半胱氨酸基团的特征性活性中心。PKS系统中的CLFs的部分目前正以争论方式进行讨论。最近的结果指出CLF结构的部分在于丙二酰ACP的脱羧作用。产生的乙酰基随后可以结合到β酮酰基合成酶结构域的活性中心上并且因而代表了起始缩合反应的所谓引动分子(priming molecule)。还发现CLF同源性序列作为分子PKS系统中的负载结构域。具有CLF序列特性的结构域存在于所有先前已知的PUFA-PKS系统。这后接酰基转移酶结构域(Seq ID No.52和60)。这种结构域催化许多酰基转移如从酰基转移到辅酶A或转移到ACP结构域。来自ORF2的终止结构域显示与氧化还原酶的部分同一性(Seq ID No.53和61)并且很可能代表了一种烯酰基还原酶结构域。烯酰基还原酶结构域的生物学活性存在于脂肪酸合成的第二次还原反应中。它催化脂肪酸酰基ACP的反式双键的还原(也参见图2)。
来自Ulkenia sp.的ORF3(Seq ID No.5和8)由两种脱水酶/异构酶结构域(Seq ID No.66,68,72和74)组成。两种结构域都包含“活性位点”组氨酸,直接相邻半胱氨酸(Seq ID No.67和73以及Seq ID No.69和75)。这些结构域的生物学功能是反式双键插入到脂肪酸或多酮化合物分子中,伴随着H2O的分解和双键随后转化成顺式异构形式。第二种脱水酶/异构酶结构域并入丙氨酸富集区(Seq ID No.70和76),所述丙氨酸富集区没有已知的功能但是可能代表连接区。这后接烯酰基还原酶结构域(Seq ID No.71和77),其与来自Ulkenia的已经存在于ORF2中的烯酰基还原酶结构域具有高度部分同一性。它的生物学功能对应于上面已经介绍过的烯酰基还原酶结构域(也参见图
优选在来自Ulkenia sp.的ORF1起始ATG密码子前面给出2000bp(Sequence ID No.62)作为启动子序列。它们特别优选1500bp,更加特别优选1000bp在起始密码子之前。
优选可以在终止密码子TAA之后给出2000bp(Sequence ID No.63)作为ORF1的终止序列。特别优选1500bp,更加特别优选1000bp在终止密码子之后。具有碱基序列AATAAA的ORF1的mRNA合成的潜在终止信号存在于终止密码子TAA之后的412bp。
优选在来自Ulkenia sp.的ORF2起始ATG密码子前面给出2000bp(Sequence ID No.64)作为启动子序列。它们特别优选1500bp,更加特别优选1000bp在起始密码子之前。
优选可以在终止密码子TAA之后给出2000bp(Sequence ID No.65)作为ORF2的终止序列。具有碱基序列AATAAA的ORF2的mRNA合成的潜在终止信号存在于终止密码子TAA之后的1650bp。
优选在来自Ulkenia sp.的ORF3起始ATG密码子前面给出2000bp(Sequence ID No.78)作为启动子序列。它们特别优选1500bp,更加特别优选1000bp在起始密码子之前。
优选可以在终止密码子TAA之后给出2000bp(Sequence ID No.79)作为ORF3的终止序列。具有碱基序列AATAAA的ORF3的mRNA合成的潜在终止信号存在于终止密码子TAA之后的4229bp。
PUFA,例如,DHA可以在Ulkenia sp.中进行同源生产,此外还可以在宿主,例如,大肠杆菌中利用本发明测定的序列信息进行异源生产。根据本发明的核酸序列可以用来提高PUFA的产量,其中它们被用来,例如,提高生产PUFA的生物中PUFA-PKS基因的数目。自然地,甚至是个别核酸片段,例如,编码ACP结构域的序列片段也可在同源或异源生产生物中进行扩增。特别地,ACP结构域呈现自己提高生产,因为辅因子4-phosphapantheteine的结合位点对于PUFA合成是必需的。自然地,即使是不同调控元件,例如,启动子,终止子和增强子元件的使用也能够导致经遗传修饰的PUFA生产者内产量的提高。在个别序列片段中的遗传修饰能够导致获得产物结构的变化并且因而导致不同PUFAs的生产。另外,PUFA合成酶与多酮化合物合酶的相似性使得混合系统的构建成为可能。这种所谓的组合性生物合成允许新的人工生物活性物质的生产。例如,通过PKS-和PUFA-PKS单位的混合系统在转基因微生物中生产的新型多酮化合物抗生素是有可能的。
适于这里给出的PUFA基因的异源表达的宿主除了大肠杆菌之外为,例如,酵母如酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia Pastoris)或者丝状真菌,例如,构巢曲霉(Aspergillus nidulans)和Acremonium chrysogenum。通过将根据本发明的基因导入,例如,大豆,油菜,向日葵,亚麻或其它的,优选富含油的植物中来生成生产PUFA的植物。为了PUFA基因的有效异源表达,甚至也可以使用其它的附属基因,例如,4-phosphopantheteine转移酶。另外,可以使用宿主特异性启动子/操纵系统进行加强的或可诱导的基因表达。
可以使用多种原核表达系统进行PUFA的异源生产。可以构建除了相应的PUFA基因之外还包含启动子,核糖体结合位点和转录终止子的表达载体。将大肠杆菌色氨酸生物合成的启动子/操纵子区和λ噬菌体的启动子引证作为大肠杆菌中这些调控元件的例子。同样地,可以将可选择的标记,例如,对氨苄青霉素、四环素或氯霉素的抗性用于合适的载体上。对于大肠杆菌的转化非常合适的载体为pBR322,pCQV2和pUC质粒以及它们的衍生物。这些质粒可包含病毒以及细菌元件。可以使用每种源自大肠杆菌K12的菌株,例如,JM101,JM109,RR1,HB101,DH1或AG1作为大肠杆菌宿主菌株。自然地,所有其它惯用的原核表达系统也可以用于异源PUFA生产(还参见Sambrook等)。还可以使用生油(oil-building)细菌作为宿主系统。
可以将哺乳动物、植物和昆虫细胞以及真菌,例如,酵母用作真核表达系统。对于酵母系统来说,可以使用来自于糖酵解酶基因的转录起始元件。这包括乙醇脱氢酶,甘油醛-3-磷酸脱氢酶,phosphoglukoisomerase,磷酸甘油酯激酶等的调控元件。不过,即使是来自基因如来自酸性磷酸酶,乳糖酶,金属硫蛋白或葡糖淀粉酶基因的调控元件也可以使用。这里还使用允许加强的或可诱导的表达的启动子。可由半乳糖诱导的启动子(GAL1,GAL7和GAL10)也是令人特别感兴趣的(Lue等,1987Mol.Cell.Biol.7,p.3446ff.和Johnston1987Mircobiol.Rev.51,p.458ff.)。3’终止序列还优选源自酵母。由于紧邻起始密码子(ATG)的核苷酸序列影响酵母中基因的表达,还优选来自酵母的有效翻译起始序列。在使用酵母质粒的情况下,它们包含来自酵母的复制起点并且包含选择标记。这种选择标记优选是营养缺陷型标记,例如,LEU,TRP或HIS。这种酵母质粒是所谓的YRps(酵母复制性质粒),YCps(酵母着丝点质粒)和YEps(酵母游离质粒)。没有复制起点的质粒是Yips(酵母整合质粒),其用于整合转化的DNA至基因组中。特别感兴趣的是质粒pYES2和pYX424以及pPICZ质粒。
如果将丝状真菌,例如,构巢曲霉用作异源PUFA生产者,也可以使用来自对应生物的启动子。可以将用于加强表达的gpdA启动子和用于可诱导表达的alcA启动子用作实例。优选使用酵母质粒如pHELP(D.J.Balance和G.Turner(1985)Development of ahigh-frequency transforming vector for Aspergillus nidulans.Gene36,321-331)和可选择标记如ura,bio或paba用于转化丝状真菌。甚至优选来自丝状真菌的3’调控元件。
通过杆状病毒表达系统可以在昆虫细胞中生产PUFA。这些表达系统可由,例如Clonetech或Invitrogen商购。
可以将载体,例如,来自土壤杆菌的Ti质粒或完整病毒如菜花样花叶病毒(CaMV),双粒病毒,番茄金黄花叶病毒或烟草花叶病毒(TMV)用于植物的转化。优选的启动子为,例如,CaMV的35S启动子。对于植物转化的其它可能性为磷酸钙法,聚乙二醇法,微注射,电穿孔或原生质体的脂染。还优选通过用DNA带电微粒轰击(基因枪)进行的转化。植物中备选的PUFA生产源自叶绿体的转化。例如,N末端引导肽使得蛋白质在叶绿体中的转运成为可能。优选的引导肽源自核酮糖双磷酸酯羧化酶的小亚基但是也可以使用其它chloroplastidary蛋白的引导肽。叶绿体基因组的稳定转化提供了另一种可能性。对此尤其可以考虑生物导弹法还可以考虑其它方法(Blowers等Plant Cell19891pp.123-132,Kline等.Nature1987327pp.70-73和Schrier等Embo J.4pp.25-32)。
对于哺乳动物细胞还可以使用可以商购的表达系统。其中,可以使用病毒性或非病毒性转化和表达系统,例如,慢病毒或腺病毒系统或Invitrogen的T-Rex系统等作为例子。同样,来自Invitrogen的Flp-In系统,可以用于哺乳动物细胞中DNA的目的性整合。
下面利用几个实施例介绍构成了根据本发明方法基础的核酸和氨基酸。不过,所述序列和本发明并不限于这些实施例。
附图说明
图1描述了来自Ulkenia sp.的PUFA-PKS基因在基因组上的位置。另外,显示了由这些基因编码的PUFA-PKS的个别结构域。KS:酮合成酶,MAT:丙二酰-CoA:ACP酰基转移酶,ACP:酰基载体蛋白,KR:酮还原酶,CLF:链长因子,AT:酰基转移酶,ER:烯酰基还原酶和DH:脱水酶/异构酶。
图2显示来自Ulkenia sp.的ORF2和ORF3与来自Moritellamarina(GenBank编号:AB025342.1),Photobacterium profundum SS9(GenBank编号:AF409100),Shewanella sp.SCRC-2783(GenBank编号:U73935.1)和Schizochytrium(GenBank编号:AF378327,AF378328,AF378329)的相应同源性ORFs的比较。在进化过程中个别ORFs之中和之间的基因转座也在结构域结构旁边指出。
图3显示来自Ulkenia sp.的ORF1与来自Moritella marina(GenBank编号:AB025342.1),Photobacterium profundum SS9(GenBank编号:AF409100),Shewanella sp.SCRC-2783(GenBank编号:U73935.1)和Schizochytrium(GenBank编号:AF378327,AF378328,AF378329)的相应同源性ORFs的比较。强调了ACP结构域和氨基酸连续LGIDSIKRVEIL重复的数目。
图4包含了来自Ulkenia sp.的ORF1与来自Schizochytrium的ORF A的序列比较。两种序列的部分同一性的程度为大约81.5%。
图5包含了来自Ulkenia sp.的ORF2与来自Schizochytrium的ORF B的序列比较。两种序列的部分同一性的程度为大约75.9%。
图6包含了来自Ulkenia sp.的ORF3与来自Schizochytrium的ORF C的序列比较。两种序列的部分同一性的程度为大约80.0%。
图7描述了由FASTAX进行的,实施例1中所述PCR产物与数据库序列(Swiss-PROT全文库)的序列比较。
图8显示了用于生产来自实施例2的粘粒库的Cosmid SuperCosI(Stragagene)的载体图(card)。
图9描述了由BLASTX进行的,实施例3中所述PCR产物与数据库序列(Swiss-PROT全文库)的序列比较。
具体实施方式
实施例1:
从分离自Ulkenia sp.SAM2179的DNA扩增PUFA-PKS特异性序列
1.1包含编码PUFA-PKS的基因的基因组DNA的分离
在250ml带有阻流板的Erlenmeyer烧瓶中用Ulkenia sp.SAM2179接种50ml DH1培养基(50g/l葡萄糖;12.5g/l酵母提取物;16.65g/l Tropic Marin;pH6.0)并于28℃和150rpm培养48h。随后用灭菌自来水洗涤细胞,离心下去并将细胞沉淀物冷冻于-85℃中。为了进一步的检查(workup),随后将细胞沉淀物转移入研钵中并以研棒在液氮下粉碎成精细粉末。随后,将大约1/10研成粉末的细胞材料与2ml裂解缓冲液(50mM tris/Cl pH7.2;50mM EDTA;3%(v/v)SDA;0.01%(v/v)2-巯基乙醇)混合并于68℃温育1h。随后加入2ml苯酚/氯仿/异戊醇(25:24:1),搅动并于100000rpm离心20min。在除去上层水相后,将后者转移入两个新的反应容器中,每个600μl,并且分别再次与600μl苯酚/氯仿/异戊醇(25:24:1)混合,搅动并于13000rpm离心15min。随后将特定上层相每个400μl转移入新的反应容器中并在每种情况下加入1ml乙醇(100%)后倒转两到三次。随后,将沉淀的DNA缠绕在玻璃棒上,用70%乙醇洗涤,干燥并溶于50μl蒸馏水中。将以此方式提取的DNA与2μl RNase A混合并保存于4℃待用。
1.2利用靶标特异性寡核苷酸进行PCR反应
将PCR引物MOF1和MOR1用作靶标特异性寡核苷酸。
MOF1:5’–CTC GGC ATT GAC TCC ATC–3’(Seq ID No.81)MOR1:5’-GAG AAT CTC GAC ACG CTT–3’(Seq ID No.82)。将在上面1.1段中所述的来自Ulkenia sp.SAM2179的基因组DNA稀释1:100。随后将2μl的这种稀释液转移入50μl体积的PCR反应混合物中(1x缓冲液(Sigma);dNTPs(每种200μM);MOF1(20pmol),MOR1(20pmol)和2.5U Taq-DNA聚合酶(Sigma))。在下列条件下实施PCR:起始变性94℃3min,随后为30个循环,每个循环于94℃1min,55℃1min,72℃1min,和最后8min72℃。随后通过凝胶电泳分析PCR产物并通过T/A克隆(Invitrogen)将具有合适大小的片段插入载体pCR2.1TOPO中。在转化大肠杆菌TOP10F’之后,分离质粒DNA(Qiaprep Spin,QUAGEN)并进行测序。
将获得的序列数据与官方EMBL核苷酸序列数据库(http://www.ebi.ac.uk/embl/)相比较并进行评估。用FASTAX获得的序列比较对于来自Ulkenia sp.SAM2179的PCR主要产物与来自Schizochytrium sp.ATCC20888的PUFA-PKS(ORF A;ORF:开放阅读框)的酰基载体蛋白产生部分同一性,其在氨基酸水平上为大约90%(图7)。令人吃惊的是,为了确定在Ulkenia sp.SAM2179中的这种PUFA-PKS,仅须实施单次PCR实验。这说明所用寡核苷酸的特别高的效力。
实施例2:
由来自Ulkenia sp.SAM2179的基因组DNA生产基因组文库
在500μl体积中以2.5U Sau3AI于37℃2min将来自Ulkenia sp.SAM2179的50μg基因组DNA部分裂解并且接下着立即用相同体积的苯酚/氯仿进行沉淀,随后用乙醇沉淀并溶解于蒸馏水中。随后根据生产商的说明书用SAP(虾碱性磷酸酶;Roche)将Sau3AI裂解的基因组DNA去磷酸化。随后通过将该反应加热20分钟至65℃来进行酶的灭活。将粘粒Supercos I(Stratagene,图8)用作载体。将10μg Supercos I用XbaI于37℃完全裂解几小时。随后将酶于65℃加热灭活20min并且根据生产商的说明书用SAP(Roche)将剪切的粘粒去磷酸化。在这里也通过将该反应于65℃加热20分钟进行酶的灭活。随后用BamHI于37℃将XbaI裂解的和去磷酸化的Supercos I粘粒完全裂解几小时。随后将剪切的粘粒DNA用苯酚/氯仿进行沉淀,用乙醇沉淀并接下来溶解于蒸馏水中。为了进行连接,将1μg用XbaI和BamHI裂解的粘粒DNA,和3.5μl Sau3AI裂解的基因组DNA组合于20μl的体积中并用T4连接酶(Biolabs)根据生产商的说明书连接几小时。随后根据生产商的说明书利用Gigapack III XL Packaging Extract(Stratagene)将大约1/7的连接物包装在噬菌体中。随后将后者用于转染大肠杆菌XL1-Blue MR。随后以PCR筛选的形式由QIAGEN公司(Hilden,Germany)由基因文库中进行PUFA-PKS特异性粘粒的分离,所述PCR筛选利用Ulkenia-PKS-特异性寡核苷酸PSF2:5’–ATT ACT CCT CTCTGC ATC CGT–3’(Seq ID No.83)和PSR2:5’–GCC GAA GACAGC ATC AAA CTC–3’(Seq ID No.84)。随后对由此确定的粘粒克隆C19F09的粘粒DNA进行分离和测序(Seq ID No.1)。
实施例3:
来自Ulkenia sp.的ORF3的鉴定
为了鉴定来自Ulkenia sp.SAM2179的ORF,寡核苷酸源自不同PUFA-PKS的高度保守的序列片段。令人感兴趣的是,对于PCR扩增似乎合适的非常高的部分同一性出现在个别物种之间编码脱水酶/异构酶的序列片段区域。
3.1包含编码PUFA-PKS的基因的基因组DNA的分离
参见实施例1.1
3.2利用PUFA-PKS-特异性寡核苷酸进行的PCR反应
将下列PCR引物用作PUFA-PKS-特异性寡核苷酸:
CFOR1:5’–GTC GAG AGT GGC CAG TGC GAT–3’(Seq No.85)
CREV3:5’–AAA GTG GCA GGG AAA GTA CCA–3’(Seq IDNo.86).
将在上述3.1段所述的来自Ulkenia sp.2179的基因组DNA稀释到1:10的比例。随后将2μl这种稀释液转移入50μl体积的PCR反应混合物中(1x缓冲液(Sigma);dNTPs(每种200μM);CFOR1(20pmol),CREV3(20pmol)和2.5U Taq-DNA聚合酶(Sigma)。在下列条件下进行PCR:94℃初始变性3min,随后30个循环,每个循环于94℃1min,60℃1min,72℃1min,和最后8min72℃。随后通过凝胶电泳分析PCR产物并通过T/A克隆(Invitrogen)将合适大小的片段插入载体pCR2.1TOPO中。在转化大肠杆菌E.coli TOP10F’之后,分离质粒DNA(Qiaprep Spin,QUAGEN)并进行部分测序。
将获得的序列数据与官方EMBL核苷酸序列数据库(http://www.ebi.ac.uk/embl/)相比较并进行评估。用FASTAX获得的序列比较对于来自Ulkenia sp.SAM2179的PCR主要产物与来自Schizochytrium sp.ATCC20888的PUFA-PKS合成酶的ORF C产生部分同一性,其在氨基酸水平上为大约80%(图9)。令人吃惊的是,为了确定在Ulkenia sp.SAM2179中的这种PUFA-PKS,仅须实施单次PCR实验。这说明所用寡核苷酸的特别高的效力。随后以PCR筛选的形式通过QIAGEN公司(Hilden,Germany)由实施例2中所述基因文库中分离PUFA-PKS特异性粘粒,所述PCR筛选利用已经用于PCR的寡核苷酸CFOR1:5’–GTC GAG AGT GGC CAG TGC GAT–3’(Seq ID No.85)和CREV3:5’–AAA GTG GCA GGG AAA GTA CCA–3’(Seq IDNo.86)。随后对由此确定的粘粒克隆058G09的粘粒DNA进行分离和测序(Seq ID No.2)。
Claims (15)
1.具有PUFA-PKS酶活性的多肽,其包括:
a.具有β酮酰基合成酶的生物学活性的SEQ ID No.6(ORF1)所示的氨基酸序列,或具有β酮酰基合成酶的生物学活性的与SEQ IDNo.6(ORF1)有至少90%序列同一性的氨基酸序列,或
b.具有β酮酰基合成酶的生物学活性的SEQ ID No.32所示的氨基酸序列,或具有β酮酰基合成酶的生物学活性的与SEQ ID No.32有至少99%序列同一性的氨基酸序列,或
c.具有丙二酰CoA-ACP转移酶的生物学活性的SEQ ID No.33所示的氨基酸序列或具有丙二酰CoA-ACP转移酶的生物学活性的与SEQ ID No.33有至少90%序列同一性的氨基酸序列,具有富含丙氨酸序列的部分的生物学活性的SEQ ID No.34所示的氨基酸序列或具有富含丙氨酸序列的部分的生物学活性的与SEQ ID No.34有至少90%序列同一性的氨基酸序列,或具有3-酮酰基-ACP化合物的NADPH依赖型还原酶的生物学活性的SEQ ID No.45所示的氨基酸序列,或具有3-酮酰基-ACP化合物的NADPH依赖型还原酶的生物学活性的与SEQ IDNo.45有至少90%序列同一性的氨基酸序列。
2.具有10个或更多ACP结构域的根据权利要求1的具有PUFA-PKS酶活性的多肽。
3.根据任一项在前权利要求的具有PUFA-PKS酶活性的多肽,其特征是它包含与序列SEQ ID No.6(ORF1)的至少500个直接连续氨基酸具有至少90%,优选至少99%序列同源性的氨基酸序列,并且具有PUFA-PKS的至少一个结构域的生物学活性。
4.一种氨基酸序列,它与SEQ ID No.6(ORF1)的至少500个直接连续氨基酸具有至少90%,优选至少99%的同一性,并且具有PUFA-PKS的至少一个结构域的生物学活性。
5.一种分离的DNA分子,其编码根据任一项在前权利要求的氨基酸序列和与它完全互补的DNA。
6.根据权利要求5的分离的DNA分子,其特征是它与来自SEQ IDNo.3的至少500个直接连续核苷酸具有至少70%,优选至少80%,特别优选至少90%和更加特别优选至少95%的同一性。
7.包含根据权利要求5或6其中之一的DNA分子的重组DNA分子,其与至少一种控制转录的DNA序列功能性连接,所述DNA序列优选选自SEQ ID No.XX-YY(终止子/启动子),或其来自至少500个核苷酸的部分以及它们的功能性变体。
8.包含根据权利要求7的重组DNA分子的重组宿主细胞。
9.根据权利要求8的重组宿主细胞,其内源性表达具有至少另一种PUFA-PKS结构域活性的根据权利要求1的具有PUFA-PKS酶活性的多肽。
10.一种生产含有PUFA,优选DHA的油的方法,包括培养根据权利要求8或9的宿主细胞。
11.根据权利要求10的方法生产的油。
12.一种生产含有PUFA,优选DHA的生物质量的方法,包括培养根据权利要求8或9的宿主细胞。
13.根据权利要求12的方法生产的生物质量。
14.根据权利要求13的重组生物质量,其包含根据权利要求7的核酸和/或根据权利要求1的具有PUFA-PKS酶活性的多肽或与它同源的至少500个连续氨基酸的部分。
15.包含具有PUFA-PKS酶活性的多肽的来自SEQ ID No.6的个别酶结构域用于生产人工多酮化合物的用途。
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CN108753810B (zh) * | 2018-05-22 | 2021-06-18 | 昆明理工大学 | 一种转录调节蛋白基因orf2的用途 |
CN112601808A (zh) * | 2018-08-10 | 2021-04-02 | 协和发酵生化株式会社 | 生产二十碳五烯酸的微生物和二十碳五烯酸的制造方法 |
CN110577921A (zh) * | 2019-05-28 | 2019-12-17 | 浙江工业大学 | 产两性霉素b的重组结节链霉菌及其应用 |
CN110577921B (zh) * | 2019-05-28 | 2021-04-02 | 浙江工业大学 | 产两性霉素b的重组结节链霉菌及其应用 |
CN114107074A (zh) * | 2021-11-18 | 2022-03-01 | 厦门大学 | 一种过表达3-酮酰基合酶基因的裂殖壶菌基因工程菌株的构建方法及其应用 |
CN114107074B (zh) * | 2021-11-18 | 2024-04-09 | 厦门大学 | 一种过表达3-酮酰基合酶基因的裂殖壶菌基因工程菌株的构建方法及其应用 |
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DE102004017370A1 (de) | 2005-10-27 |
WO2005097982A3 (de) | 2007-04-05 |
KR20070056002A (ko) | 2007-05-31 |
AU2005231964A1 (en) | 2005-10-20 |
JP2007532104A (ja) | 2007-11-15 |
CN101087882A (zh) | 2007-12-12 |
IL178613A0 (en) | 2007-02-11 |
EP1733029A2 (de) | 2006-12-20 |
JP2012205595A (ja) | 2012-10-25 |
US7939305B2 (en) | 2011-05-10 |
AU2005231964B2 (en) | 2012-03-08 |
BRPI0509747A (pt) | 2007-09-25 |
US20090093033A1 (en) | 2009-04-09 |
KR20130114225A (ko) | 2013-10-16 |
CA2563427A1 (en) | 2005-10-20 |
KR101484097B1 (ko) | 2015-01-23 |
WO2005097982A2 (de) | 2005-10-20 |
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