CN1303434A - 用于生产核黄素的遗传方法 - Google Patents
用于生产核黄素的遗传方法 Download PDFInfo
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Abstract
本发明涉及一种用于生产核黄素的遗传方法。通过在生物体中特别是在细菌、真菌、酵母和植物中特异性选择核黄素生物合成基因或其混合物并表达它们来提高生物体中核黄素的产量。本发明还涉及一种含有具有序列SEQ ID No.1、SEQ ID No.3或SEQ ID No.5的基因或其功能等价物的核酸片段;涉及含有所述核酸片段的表达载体并涉及含有至少一种核酸片段或至少一种载体的生物体。
Description
本发明涉及一种用于生产核黄素的遗传方法。在生物体中特别是在细菌、真菌、酵母和植物中特异性选择核黄素生物合成基因或其混合物并表达它们这一方法导致核黄素的产量在这些生物体中得到提高。
本发明进一步涉及一种含有具有序列SEQ ID No.1、SEQ ID No.3或SEQ ID No.5的基因或其功能等价物的核酸片段;涉及含有所述核酸片段的表达载体并涉及含有至少一种核酸片段或至少一种载体的生物体。
维生素B2、也称作核黄素由所有的植物和大量的微生物产生。它对于人和动物来说是必需的,因为它们不能合成核黄素。核黄素在代谢过程中起重要作用。因此,例如,它与碳水化合物的利用有关。缺乏维生素B2涉及口腔和咽喉粘膜的炎症、痒症和皮肤皱裂以及类似的皮肤损害、结膜炎症、角膜视敏度减小和混浊。在婴儿和儿童中可以存在生长的停滞和体重下降。维生素B2由此具有极高的经济上的重要性,例如作为用于维生素缺乏的维生素产品和作为作为动物的饲料添加剂。将它添加到大量的食品中。也将它用作例如蛋黄酱、冰淇淋、布丁等中的食品色素。
通过化学方法或微生物方法来生产维生素B2(参见,例如,Kurth等,1996,“ullman氏工业化学百科全书”(ullmann’s Encyclopediaof industrial chemistry)中的“核黄素”(Riboflavin),VCHWeinheim)。在化学生产方法中,核黄素通常作为多步法中的纯终产物获得,必须使用相对昂贵的原料诸如例如D-核糖。
另一种用于化学合成核黄素的方法是通过发酵微生物来生产维生素B2。在这种情况中,原料是可再生的粗原料诸如糖或植物油。通过发酵真菌诸如阿舒氏假囊酵母或棉桃阿舒氏囊霉来生产核黄素是的方法公知的(The Merck索引,Windholz等编辑,Merck&Co.,1183页,1983),而还将酵母诸如例如假丝酵母属、毕赤氏酵母属和糖酵母属或细菌诸如例如芽孢杆菌属、梭状芽孢杆菌属或棒状杆菌属描述为核黄素的生产者。EP-A-O 450 370和EP-A-O 821 063中描述了使用重组细菌菌株来生产核黄素的方法,所述的菌株通过用核黄素生物合成基因转化而从枯草芽孢杆菌中获得。
专利WO 95/26406或WO 93/03183和DE 44 20 785中描述了从真核生物体棉桃阿舒氏囊霉和啤酒糖酵母中克隆对核黄素生物合成具有特异性的基因、和已经用这些基因转化的微生物以及这类微生物用于核黄素合成的用途。
在两种生物体中,6种酶催化由鸟苷三磷酸(GTP)和由5-磷酸核酮糖来形成核黄素。该方法包括用GTP环水解酶-Ⅱ(ribl基因产品)将GTP转化成2,5-二氨基-6-(核糖基氨基)-4(3H)-5-磷酸嘧啶酮的步骤。然后用2,5-二氨基-6-(核糖基氨基)-4(3H)-5-磷酸嘧啶酮还原酶(rib7基因产品)将后一种化合物还原成2,5-二氨基-6-(核糖基氨基)-2,4(1H,3H)-5-磷酸嘧啶酮且随后将其用一种特异性脱胺酶(rib2基因产品)脱氨基而得到5-氨基-6-核糖基氨基-2,4(1H,3H)-5-磷酸嘧啶二酮。接着用一种非特异性磷酸酶来消去磷酸。
在核黄素生物合成的最后酶促步骤中,除GTP外,用3,4-二羟基-2-丁酮-4-磷酸合酶(rib3基因产品)将第二种原料5-磷酸核酮糖转化成3,4-二羟基-2-丁酮-4-磷酸(DBP)。
DBP和5-氨基-6-核糖基氨基-2,4(1H,3H)-嘧啶二酮是酶促合成6,7-二甲基-8-核糖基-2,4-二氧四氢蝶啶的前体。该反应由rib4基因产品(DMRL合酶)来催化。随后用核黄素合酶(rib5基因产品)将DMRL转化成核黄素(Bacher等(1993),《生物有机化学新领域》(Bioorg.Chem.Front.)第3卷,Springer Verlag)。
尽管在核黄素生产中有这些发展,但是仍然需要改进并提高维生素B2的生产能力以便满足增加的需求并使核黄素的生产效率更高。
本发明的一个目的是进一步改进维生素B2的生产能力。
我们已经发现通过一种使用能够合成核黄素的生物体来提高核黄素产量的方法可以实现这一目的,其中酶3,4-二羟基-2-丁酮-4-磷酸合酶、二甲基-8-核糖基-2,4-二氧四氢蝶啶合酶和核黄素合酶或其功能类似物在所述生物体中的活性得到了提高。使用一种能够合成核黄素且另外含有可编码酶3,4-二羟基-2-丁酮-4-磷酸合酶(=rib3基因产品)、二甲基-8-核糖基-2,4-二氧四氢蝶啶合酶(rib4基因产品)和核黄素合酶(rib5基因产品)或其功能类似物的基因混合物的生物体可有利地实施用于提高核黄素产量的方法。
另外有利的增加维生素B2生产能力的方法是将提高天然酶活性与引入上述基因混合物以增加基因表达两步骤结合起来。
用于本发明方法的合适的生物体或宿主生物体通常是能够合成核黄素的所有生物体。天然能够合成核黄素的生物体是优选的。然而,因引入完整维生素B2合成基因而能够合成核黄素的生物体也适合于本发明的方法。诸如细菌、酵母、真菌或植物这样的生物体适合于本发明的方法。可提及的实例是:真核生物体诸如《印度化学工程》(Indian Chem Engr.)B部第37卷第1,2期(1995)中的第15页、表6中所述的真菌诸如阿舒氏囊霉菌或假囊酵母属;酵母诸如假丝酵母属、糖酵母属或毕赤氏酵母属;或植物诸如拟南芥属、番茄、马铃薯、玉米、大豆、油菜子、大麦、小麦、黑麦、稻米、小米、棉花、制糖甜菜、向日葵、亚麻、大麻、canola、燕麦、烟草、苜蓿、莴苣或各种树木、坚果和藤本植物物种;或原核生物体诸如革兰氏阳性菌或革兰氏阴性菌例如棒状杆菌属、短杆菌属、芽孢杆菌属、梭状芽孢杆菌属、蓝细菌属(cyanobacter)、埃希氏杆菌属或克雷白氏杆菌属。优选的生物体选自:棒状杆菌属、短杆菌属、芽孢杆菌属、埃希氏杆菌属、阿舒氏囊霉菌、假囊酵母属、假丝酵母属或糖酵母属;或植物诸如玉米、大豆、油菜子、大麦、小麦、马铃薯或番茄。特别优选的生物体是下列属种:棉桃阿舒氏囊霉、阿舒氏假囊酵母、啤酒糖酵母、Candida flaveri、Candida famata、产氨棒杆菌或枯草芽孢杆菌。特别优选的植物是玉米、大豆、油菜子、大麦、小麦、马铃薯和番茄。
本发明将rib基因rib3、rib4和rib5混合和/或所述基因及其基因产物活性的增加会导致核黄素的生产能力显著提高。可以通过本领域技术人员所公知的所有方法将所述的基因引入通常使用的生物体中且可以通过转化、转染、电穿孔、使用所谓的基因枪或通过微量注射将所述的基因引入生物体或其细胞中。本领域技术人员可以在下列教科书中找到适合于微生物的方法:Sambrook,J.等(1989)《分子克隆》(Molecular cloning):实验室手册,Cold Spring HarborLaboratory Press;F.M.Ausubel等(1994)《分子生物学最新进展》(Current protocols in molecular biology);John Wiley和Sons;D.M.Glover等《DNA克隆》(DNACloning)第1卷(1995),IRL Press(ISBN 019-963476-9);Kaiser等(1994)《酵母遗传学方法》(Methods in Yeast Genetics),Cold Spring HarborLaboratory Press;或Guthrie等《酵母遗传学和分子生物学指南》(Guide to Yeast genetics and Molecular Biology)、《酶学方法》(Methods in Enzymology),1994,Academic Press。可提及的有利方法的实例是通过同源或异源重组例如借助于ura-3基因、特别是德国申请DE 19801120.2中所述的阿舒氏囊霉菌ura-3基因和/或通过如下所述的REMI法(=限制酶介导的整合)来引入DNA。
REMI技术以线性DNA构建体和限制性内切酶的在一个细胞内的共转化为基础,其中,构建体的两端用同一内切酶切割,而限制性内切酶则是进行该切割的内切酶。然后所述的限制性内切核酸酶切割已经一起引入所述DNA构建体与所述限制酶的生物体的基因组DNA。这将激活细胞自身修复机制。这些修复机制可修复由基因组DNA中的内切核酸酶导致的链的破坏,且这还与将所述共转化DNA构建体引入所述基因组的一定频率有关。通常的情况是在DNA两端保有限制切割位点。
这项技术是由Blker等描述(《分子基因遗传学》(Mol.Gen.Genet.)248,1995:547-552)的用于真菌插入诱变的技术。Schiestl和Petes(《美国国家科学院学报》(Proc.Nalt.Acad.Sci.USA)88,1991:7585-7589)使用了寻找异源重组是否发生在糖酵母属中的方法。该方法是由Brown等(《分子基因遗传学》(Mol.Gen.Genet.)251。1996:75-80)描述的用于稳定转化和可调型表达诱导型报道基因的方法。还未将该系统确定用作最佳代谢途径或商业上超报道蛋白质的遗传工程工具。
核黄素生物合成的实例已经证实通过REMI法可以将生物合成基因整合入上述生物体的基因组并由此能够使用于生产下列物质的初级或二级代谢、特别是生物合成途径的代谢产物的生产过程最佳化:例如,氨基酸诸如赖氨酸、甲硫氨酸、苏氨酸或色氨酸;维生素诸如维生素A、B2、B6;B12、C、D、E或F、S-腺苷甲硫氨酸、生物素、泛酸或叶酸;类胡萝卜素诸如β-胡萝卜素、番茄红素、角黄素、虾青素或玉米黄质;或蛋白质诸如水解酶例如脂酶类、酯酶类、酰胺酶类、腈水解酶类、蛋白酶类;介体诸如细胞因子,例如诸如MIF、MAF、TNF这样的淋巴因子、诸如白细胞介素1这样的白细胞介素、诸如γ-干扰素这样的干扰素、tPA、诸如蛋白激素、糖激素这样的激素、诸如血管升压素、内啡肽、内抑制素(endostatin)、制管张素、生长因子、红细胞生成素、转录因子这样的寡或多肽激素、诸如GPⅡb/Ⅲa或αvβⅢ这样的整联蛋白、诸如各种谷氨酸受体这样的受体、诸如血管紧张素这样的血管生成因子。
还可以将REMI法用于给本发明的核酸片段或其它上述基因在基因组中的转录活性位点处进行定位。
有利的是将所述的核酸彼此在至少一种报道基因处克隆入引入所述基因组中的DNA构建体。这种报道基因应通过一种生长、荧光、化学或生物发光测定法或通过一种光度测定法而使得可检测性变得容易。可提及的报道基因的实例是抗生素抗性基因;水解酶基因;过氧化物酶基因或生物合成基因诸如核黄素基因、荧光素酶基因、β-半乳糖苷酶基因、gfp基因、脂酶基因、酯酶基因、过氧化物酶基因、β-内酰胺酶基因、乙酰基、磷酸或腺苷酰转移酶基因。这些基因使得方便地测定和定量转录活性并由此表达所述基因成为可能。由此能够鉴定表现出高达2种因子的不同生产能力的基因组位点(参见附图1)。附图1表示在整合后获得的具有不同维生素B2(=核黄素)生产能力的克隆ITA-GS-15.2、ITA-GS-17.1和ITA-GS-01。
在生物合成基因自身使得可检测性变得方便的情况中,例如就核黄素而言,能够省去另外的报道基因。
如果将多个基因引入生物体,那么能够将所有的基因与一种单一载体中的报道基因一起或将每一个基因与一种载体中的一种报道基因引入生物体,能够同时或连续引入各种载体。也可以在REMI技术中使用编码特殊活性的基因片段。
一般来说,所有公知的限制酶适合于本发明将生物合成基因整合入生物体基因组中的方法。几乎不优选仅将4个碱基对识别为限制切割位点的限制酶,这是因为它们在经整合的基因组或载体中切割得过于频繁;优选的酶将6、7、8或8个以上的碱基对识别为切割位点,诸如BamHⅠ、EcoRⅠ、BglⅡ、SphⅠ、SpeⅠ、XbaⅠ、XhoⅠ、NcoⅠ、SalⅠ、ClaⅠ、KpnⅠ、HindⅢ、SacⅠ、PstⅠ、Bpnl、NotⅠ、SrfⅠ或SfiⅠ,这些仅是可提及的可能使用的酶中的几个。尽管所用的酶在所引入的DNA中没有另外的切割位点,但是有利的是它们可增加整合效率。一般来说,在REMI混合物中使用5-500U、优选使用10-250U、特别优选使用10-100U的酶。有利的是以水溶液的形式使用所述的酶,在所述的酶水溶液中含有:用于稳定渗透的物质诸如糖类诸如蔗糖、海藻糖或葡萄糖;多元醇类诸如甘油或聚乙二醇;有利缓冲范围在pH5-9、优选6-8、特别优选7-8的缓冲液诸如Tris、MOPS、HEPES、MES或PIPES;和/或用于稳定核酸的物质诸如Mg、Cu、Co、Fe、Mn或Mo的无机或有机盐。如果合适,在所述的酶水溶液中还可以含有其它物质,诸如EDTA、EDDA、DTT、β-巯基乙醇或核酸酶抑制剂。然而,不具有这些条件也能够实施REMI技术。
本发明的方法在5-80℃的温度范围进行,优选10-60℃,特别优选20-40℃。用于使细胞膜不稳定的所有公知方法均适合于本方法,诸如例如电穿孔法;与加载的囊泡融合的方法或使用各种碱金属或碱土金属盐诸如锂、铷或钙盐、优选锂盐的脱稳定法。
为了进行本发明的反应,可以在分离后直接或在纯化后使用核酸。
附图2和3以图解方式概括了按照本发明整合rib基因混合物的本发明方法。将酶SpeⅠ用于切割DNA,且同时将所述的DNA引入生物体。为了有利于选择,还将卡那霉素抗性基因导入位于TEF启动子序列两侧的片段(所谓的直接复制)。通过经这些序列的重组而再次删除所述的抗性基因(参见附图3)。
一般通过本领域技术人员所公知的所有方法可以将本发明的rib基因混合物引入植物。
将外源基因转入植物的基因组被称作转化。在这种情况中,将所述用于从植物组织或植物细胞中转化和再生植物的方法用于瞬时或稳定转化。合适的方法是经聚乙二醇诱导的DNA吸收的原生质体转化法、使用基因枪、电穿孔法、干胚胎在含DNA的溶液中的培养法、微量注射法和农杆菌属介导的基因转移法。例如,所述的方法在下列文献中有描述:B.Jenes等《基因转移技术》(Techniques for GeneTransfer);S.D.kung和R.Wu编辑的《转基因植物》(TransgenicPlants)第1卷中的“工程与利用”(Engineering and Utilization),Academic Press(1993)128-143;和Potrykus《植物生理学和植物分子生物学年鉴回顾》(Ann.Rev.Plant Physiol.Plant Molec.Biol.)42(1991)205-225。优选将所表达的构建体克隆入适合于转化根癌土壤杆菌例如pBin19(Bevan等《核酸研究》(Nucl.AcidsRes.)12(1984)8711)的载体中。例如,Hfgen和Willmitzer在《核酸研究》(Nucl.Acids Res.) (1988)16,9877中描述了使用根癌土壤杆菌转化植物的方法。
同样可以按照公知的方式例如通过在土壤杆菌属溶液中浸泡受伤的叶子或叶片且然后在合适的培养基中培养而将使用本发明的表达载体转化的土壤杆菌属用于转化植物,特别是农作物植物诸如谷类植物、玉米、大豆、稻米、棉花、制糖甜菜、canola、向日葵、亚麻、大麻、马铃薯、烟草、番茄、油菜子、苜蓿、莴苣和各种树木、坚果或藤本植物物种以及豆类。
通过本领域技术人员所公知的方法可以再生遗传修饰的植物细胞。合适的方法可以在上述S.D.Kung和R.Wu、Potrykus或Hfgen和Willmitzer的公开文献中找到。
在细胞中增加rib基因产物的酶活性具有极大的可能性。
一种可能性是以这样一种方式修饰内源性rib基因3、4和5即它们可编码与原始酶相比活性提高的分别具有rib3、4或5的酶。例如,通过改变催化中心而导致底物的转化率增加或通过消除酶抑制剂的作用可以使另一种酶的活性增加,这意味着它们具有增加的比活或其活性没有受到抑制。在另一个有利的实施方案中,还能够通过在细胞中增加酶的合成、例如通过消除阻抑酶合成的因子或通过增加可促进合成提高的因子或调节元件的活性或优选通过进一步引入基因拷贝来影响酶活性的增加。后一种方法可测定细胞中基因产物的总体活性的增加而不需改变比活。还能够将这些方法结合使用,这意味着比活和总体活性均得到了提高。一般能够使用本领域技术人员所公知的所有方法将这些改变引入所述基因、调节元件或其启动子的核酸序列。为了达到这一目的,例如,能够对所述的序列进行诸如位点定向诱变这样的诱变,正如D.M.Glover等在《 DNA克隆》(DNA Cloning)第1卷(1995),IRL Press(ISBN 019-963476-9)第6章第193页以及以下内容中所述。
Spee等(《核酸研究》(Nucl.Acids Res.)第21卷第3期,1993:777-778)描述了使用dITP用于随机诱变的PCR法。
Stemmer描述了用于分子评估的体外重组技术的应用(《美国国家科学院学报》(Proc.Natl.Acad.Sci.USA)第91卷,1994:10747-10751)。
Moore等(《自然生物技术》(Nature Biotechnology)第14卷,1996:458-467)描述了将PCR法与重组法结合起来的方法。
随后将修饰的核酸通过载体转入生物体中。
为了提高酶的活性,还能够将改变的启动子区置于天然基因前,使得所述基因的表达得到增加并由此最终提高活性。还能够在3’端引导例如可增加mRNA稳定性并由此使得增加翻译成为可能的序列。这同样可导致酶的活性提高。
优选进一步将rib基因3、4和5的拷贝一起引入细胞。可以使这些基因的拷贝进行自然调节或修饰调节、在这种情况中已经将自然调节区修饰成它们能够增加对所述基因的表达;否则可以使用外源基因乃至异种基因的调节序列。
将上述方法结合起来是特别有利的。
例如,功能类似物指的是rib基因或其酶活性即可催化与rib基因酶所催化的反应相同的酶的功能同系物。这些基因同样可有利地使形成的核黄素增加。也可以使这些功能类似物诱变或以上述方式对其进行有利的修饰并由此可以提高它们的活性。
有利的功能类似物是例如来源于真核生物体或原核生物体的基因或基因产物。可提及的实例是:真核生物体诸如《印度化学工程》(Indian Chem Engr.)B部第37卷第1,2期(1995)中的第15页、表6中所述的真菌,诸如假囊酵母属;酵母诸如假丝酵母属、糖酵母属或毕赤氏酵母属;或植物诸如拟南芥属、番茄、马铃薯、玉米、大豆、油菜子、大麦、小麦、黑麦、稻米、小米、棉花、制糖甜菜、向日葵、亚麻、大麻、canola、燕麦、烟草、苜蓿、莴苣或各种树木、坚果或藤本植物物种;或原核生物体诸如革兰氏阳性菌或革兰氏阴性菌例如棒状杆菌属、短杆菌属、芽孢杆菌属、梭状芽孢杆菌属、蓝细菌属或埃希氏杆菌属。功能类似物有利地来源于诸如假囊酵母属这样的真菌、诸如糖酵母或假丝酵母属这样的酵母、诸如芽孢杆菌属或梭状芽孢杆菌属这样的革兰氏阳性菌或诸如大肠杆菌这样的革兰氏阴性菌。优选的功能类似物是阿舒氏假囊酵母、啤酒糖酵母、Candidaflaveri、Candida famata、大肠杆菌、产氨棒杆菌或枯草芽孢杆菌属种的生物体。
具有序列SEQ ID No.1、SEQ ID No.3或SEQ ID No.5的基因或其功能等价物的混合物在本发明的方法中是有利的。
用于本发明混合物中并具有序列SEQ ID No.1、SEQ ID)No.3或SEQ ID No.5的基因的功能等价物指的是例如在所衍生的氨基酸水平上具有至少35%同源性、优选至少40%同源性、特别优选至少45%同源性、极为优选50%同源性的等位变体。来源于所述核酸的氨基酸序列在序列SEQ ID No.2、SEQ ID No.4和SEQ ID No.6中被发现。等位变体特别包括通过缺失、插入或置换SEQ ID No.1、SEQ ID No.3和SEQ ID No.5中所描绘序列中的核苷酸而获得的功能变体,同时保留了所衍生的合成蛋白质的酶活性。
例如使用常用杂交法或PCR技术从其它真核细胞或原核细胞而不是棉桃阿舒氏囊霉中可以如上所述由SEQ ID No.1、SEQ ID No.3和SEQ ID No.5所述的DNA序列或这些序列中的一部分分离这类DNA序列。这些DNA序列在标准条件下与所述的序列杂交。有利的是用于对可以通过用一种为本领域技术人员所公知的方式与来自大肠杆菌和枯草芽孢杆菌的相应基因比较而鉴定的保守区中的短寡核苷酸进行杂交。
例如,标准条件指的是在42-58℃的温度、在浓度为0.1-5×SSC(1×SSC=0.1 5M NaCl,15mM柠檬酸钠,pH 7.2)的缓冲水溶液中或另外在有50%甲酰胺存在的条件下;诸如例如在42℃、5×SSC和在由50%甲酰胺存在的条件下。用于DNA杂交的实验条件如相关遗传学教科书中所述,诸如例如Sambrook等《分子克隆》(MolecularCloning),Cold Spring harbor Laboratory,1989。
例如,序列SEQ ID No.1、SEQ ID No.3和SEQ ID No.5的同系物还指真核或原核同系物、截短序列或单链DNA。
序列SEQ ID No.1、SEQ ID No.3和SEQ ID No.5的同系物另外指的是诸如例如启动子变体这样的衍生物。所述核苷酸序列的启动子上游可以通过一种或多种核苷酸交换、通过插入和/或缺失来彼此或单独修饰,不过,这不会对所述启动子的功能性和活性产生不利影响。另外的可能性是通过改变其序列来增加活性或用甚至来自异源生物体的更具有活性的启动子来完全替代它们。
有利的是衍生物还指起始密码子前的核苷酸序列已经被改变而使基因表达和/或蛋白质表达得到改变、优选得到增加的变体。
能够且优选从下列属的微生物或植物中分离序列SEQ ID No.1、SEQ ID No.3和SEQ ID No.5或其功能等价物:梭状芽孢杆菌属、棒状杆菌属、短杆菌属、蓝细菌属、芽孢杆菌属、假囊酵母属、埃希氏杆菌属、毕赤氏酵母属、阿舒氏囊霉菌或假丝酵母属;特别优选从产氨棒杆菌、大肠杆菌、Candida flaveri、Candida famata或《印度化学工程》(Indian Chem Engr.)B部第37卷第1,2期(1995)中的第15页、表6中所述的真菌,诸如例如阿舒氏假囊酵母或棉桃阿舒氏囊霉;特别优选阿舒氏假囊酵母或棉桃阿舒氏囊霉属种的微生物。因此,例如,可以将来自枯草芽孢杆菌的rib3、4、5-同源基因ribA、ribH和ribB或这些基因片段或来自大肠杆菌的rib3、4、5-同源基因ribB、ribE和ribC或这些基因片段有利地用于原核系统中以便提高本发明方法中核黄素的产量。
为了在生物体中使异源基因的表达最佳化,有利的是修饰核酸序列以便与所述生物体中使用的特异性密码子的用途相一致。可以通过在相关生物体中对其它公知基因进行计算机分析而方便地确定所述密码子的用途。
通过增加rib3、4和5基因的拷贝数量和/或通过增加对rib3、4和5基因表达具有有益作用的调节因子可以有利地增加对rib基因3、4和5的表达。因此,通过使用较强的转录信号诸如启动子和增强子可以更好地增强调节元件的转录水平。不过,也能够通过例如改进rib3、4和5 mRMA的稳定性或增加这种mRNA在所述核糖体上的读码效率来促进翻译。
为了增加所述基因的拷贝数量,可以将rib基因3、4和5或同源基因引入核酸片段例如引入优选含有配属相应rib基因调节基因序列或带有类似作用的启动子活性的载体中。
所用的调节序列特另是那些可促进基因表达的序列。另一方面,对于所述的各个基因来说,还能够将它们放入单一载体中或转入特殊的生产者生物体中。
本发明的核酸片段指的是rib基因序列SEQ ID No.1、SEQ ID No.3和SEQ ID No.5或其与一种或多种调节信号功能连接以便有利地增加基因表达的功能性等价物。例如,这些调节序列是诱导物或阻抑物与之结合并由此调节所述核酸表达的序列。除这些新型调节序列或取代这些序列外,还能够使这些序列的天然调节序列,这些序列存在于实际结构基因前,且如果合适对这些序列进行遗传修饰以便切断自然调节并增加基因表达。然而,基因构建体也可以具有更为简单的结构即在序列SEQ ID No.1、SEQ ID No.3或SEQ ID No.5或其功能等价物之前没有插入另外的调节信号,且不再缺失了用其调节的天然启动子。代之以不再发生调节的方式使天然调节序列发生突变并使基因表达得到增加。还可以将这些修饰的启动子置于独立的天然基因前以提高活性。还有利的是使所述的基因构建体另外含有一种或多种所谓的与启动子功能连接的增强子序列,使得提高对核酸序列的表达成为可能。还可以将另外有利的序列插入DNA序列、诸如另外调节元件或终止子的3’端。rib基因可以以一种或多种拷贝的形式存在于所述的基因构建体中。
例如,用于本发明方法的有利的调节序列存在于启动子诸如cos、tac、tet、trp-tet、lpp、lac、lpp-1ac、1acIq、T7、T5、T3、gal、trc、ara、SP6或λ-PR启动子中或λ-PL启动子中,将它们有利地用于革兰氏阴性菌。例如。另外有利的序列存在于革兰氏阳性启动子amy和SP02中、存在于酵母或真菌启动子ADCl、MFα、AC、P-60、CYC1、GAPDH、TEF、rp28、ADH中或存在于植物启动子CaMV/35S[Franck等《细胞》(Cell)21(1980)285-294]、PRPl[Ward等《植物分子生物学》(Plant.Mol.Biol.)22(1993)]、SSU、OCS、Lib4、usp、STLSl、B33、LEB4、nos中或存在于遍在蛋白质或菜豆蛋白启动子中。在这方面另外有利的是丙酮酸脱氢酶启动子和来自例如汉逊氏酵母属的甲醇氧化酶。进一步有利的植物启动子是例如苯磺酰胺诱导型启动子(EP 388186)、四环素诱导型启动子(Gatz等(1992)《植物杂志》(Plant J.)2,397-404)、脱落酸诱导型启动子(EP335528)或乙醇一或环己酮诱导型启动子(WO9321334)。特别有利的植物启动子是那些确保可进行嘌呤或其前体生物合成的植物组织或植物部位中的表达的启动子。特别应包括可确保叶特异性表达的启动子。应包括来自马铃薯的胞质FBPase启动子或来自马铃薯的ST-LSI启动子(Stockhaus等,《 欧洲分子生物学协会杂志》(EMBO J.)8(1989)2445-245)。还能够且有利的是使用来自甘氨酸max的磷酸核糖焦磷酸转酰胺酶的启动子(还参见基因库登记号U87999)或另一种如EP249676中所述的节特异性启动子。
通常能够使用带有类似于上述用于本发明方法的那些调节序列的所有天然启动子。也能够有利地使用合成启动子。
如上所述,核酸片段(=基因构建体)还可以含有引入生物体的其它基因。这些基因可以处于单独调节中或处于与rib基因相同的调节区中。例如,这些基因是进一步使得合成增加成为可能的生物合成基因。
为了进行表达,用一种载体有利地将核酸片段插入上述宿主生物体中,所述的载体诸如例如质粒、噬菌体或其它DNA,它们可使所述基因能够在宿主中的表达达到最佳化。合适的质粒的实例是大肠杆菌中的pLG338、pACYCl84、pBR322、pUC18、pUC19、pKC30、pRep4、pHS1、pHS2、pPLc236、pMBL24、pLG200、pUR290、pIN-Ⅲ113-B1、λgt11或pBdCⅠ;链霉菌属中的pIJ101、pIJ364、pIJ702或pIJ361;芽孢杆菌属中的pUB110、pC194或pBD214;棒状杆菌属中的pSA77或pAJ667;真菌中的pALS1、pIL2或pBB116;酵母中的2μm pAJ-1、YEp6、YEp13或pEMBLYe23或植物中的pLGV23、pGHlac+、pBIN19、pAK2004或pDH51或上述质粒的衍生物。所述的质粒代表了对可能质粒的小范围选择。另外的质粒对于本领域技术人员来说是众所周知的并可以在例如《克隆载体》(Cloning Vectors)一书(PouwelsP.H.等编辑,Elsevier,Amsterdam-New York-Oxford,1985,ISBN 0 444904018)中找到。在“植物分子生物学和生物技术方法”(“Methodsin Plant Molecular Biology and Biotechnology”) (CRC Press)第6/7章第71-119页中特别描述了合适的植物载体。
为了表达存在的其它基因,所述的核酸片段有利地另外含有用于增加表达的3’和/或5’末端调节序列,为了使表达最佳化,根据宿主生物体和所选择的基因或多种基因来选择这些调节序列。
这些调节序列用来使基因的特异性表达和蛋白质表达成为可能。例如,根据宿主生物体的不同,这意味着仅在诱导后表达和/或超表达基因或立即表达和/或超表达基因。
为了这一目的,所述的调节序列或因子可以优选对所引入基因的表达具有有益作用,且由此增加对它们的表达。因此,通过使用强转录信号诸如启动子和/或增强子可以有利地增强调节元件的转录水平。然而,也能够通过例如改进mRNA的稳定性来促进翻译。
在载体的另一个实施方案中,还可以将本发明的基因构建体以线性DNA的形式有利地引入微生物并通过异源或同源重组将所述的基因构建体整合入宿主生物体的基因组。这种线性DNA可以由线性化质粒组成或仅作为载体的核酸片段组成。
也能够使用可在细胞中进行自主复制的任何合适的质粒(而特别是隐藏来自啤酒糖酵母的2μm质粒的复制起点的质粒)以及如上所述的整合入所述的宿主基因组中的线性DNA片段作为载体。这种整合过程可以通过异源或同源重组、而优选如上所述通过同源重组来进行(Steiner等《遗传学》(Genetics)第140卷,1995:973-987)。此外,能够使rib3、rib4和rib5基因单独存在于基因组中不同位点上或不同载体上或共同存在于所述基因组中或一种载体上。
本发明方法中所用的含有rib基因3、4和5或其功能等价物的混合物的生物体表现出可增加核黄素产量的能力。
用本发明的方法培养用于生产核黄素的生物体,该过程在使得这些生物体生长的培养基中进行。该培养基可以是合成的或天然的培养基。根据生物体的不同使用的培养基是本领域技术人员所公知的。为了使微生物生长,所用的培养基中含有碳源、氮源、无机盐和(如果合适)少量的维生素和微量元素。
例如,有利的碳源是:糖类诸如单一、二-或多糖类,诸如葡萄糖、果糖、甘露糖、木糖、半乳糖、核糖、山梨糖、核酮糖、乳糖、麦芽糖、蔗糖、棉子糖、淀粉或纤维素;复合糖源诸如糖蜜;糖磷酸诸如果糖-1,6-二磷酸;糖醇类诸如甘露糖醇;多元醇类诸如甘油;醇类诸如甲醇或乙醇;羧酸类诸如柠檬酸、乳酸或乙酸;脂肪诸如大豆油或菜油;氨基酸诸如氨基酸混合物,例如所谓的酪蛋白氨基酸(Difco)或单个氨基酸诸如甘氨酸或天冬氨酸;或氨基糖类,且后者可以用作氮源。
有利的氮源是有机或无机氮化合物或含有这些化合物的物质。实例是诸如NH4Cl或(NH4)SO4这样的铵盐、柠檬酸盐、脲或复合氮源诸如玉米浆、酿造酵母或tolysate、大豆粉、面筋、酵母提取物、肉膏、酪蛋白水解物、酵母或马铃薯蛋白,通常它们也可以用作碳源。
无机盐的实例是钙、镁、钠、钴、钼、锰、钾、锌、铜和铁盐。在这些盐中特别提及的阴离子是氯离子、硫酸根离子或磷酸根离子。用于提高本发明方法生产能力的重要因素是控制Fe2+或Fe3+离子在生产培养基中的浓度。
此外,如果合适,可以将生长因子加入到营养培养基中,诸如:例如维生素或生长启动子诸如生物素、核黄素、硫胺素、叶酸、烟酸、泛酸盐或吡多醇;氨基酸诸如丙氨酸、半胱氨酸、脯氨酸、天冬氨酸、谷氨酸、丝氨酸、苯丙氨酸、鸟氨酸或缬氨酸;羧酸类诸如柠檬酸、甲酸、庚二酸或乳酸;或诸如二硫苏糖醇这样的物质。
所述营养物的混合比例取决于发酵的类型并在各种情况中确定。如果必要,培养基成分可以在单独灭菌或混合灭菌后的发酵开始阶段存在,否则随后就根据需要在发酵过程中连续或间断地加入。
确定培养条件使得生物体以最佳方式生长并获得能够达到的最佳产量。优选的培养温度是15℃-40℃。温度在25℃-37℃之间是特别有利的。优选将pH保持在3-9的范围。pH值在5-8之间是特别有利的。一般几小时至几天的培养时问是足够的,优选8小时至21天,特别优选4小时至14天。在此期间,在培养基中累积了最大量的产物。
本领域技术人员例如在教科书《应用微生物生理学》(AppliedMicrobiol Physiology)的“实用方法”(A Practical Approach)(P.M.Rhodes.P.F.Stanbury编辑,IRL-Press,1997,53-73页,ISBN 0 19 963577 3)中可以发现有利于培养基最佳化的可能性。例如,在公开文献EP-A-0 405 370、特别是实施例9中可以找到芽孢杆菌属和其它生物体的有利培养基和培养条件;在公开文献WO88/09822、特别是表3中可以找到假丝酵母属的有利培养基和培养条件;且在Schmit等(《微生物学》(Microbiology)142,1996:419-426)的公开文献中可以找到阿舒氏囊霉菌的有利培养基和培养条件。
可以按照批量或补料批量程序连续或间歇式进行本发明的方法。
无论所用生物体的最初生产能力如何,通过本发明的方法可以使核黄素的产量以可改变的程度增加。在与初始生物体相比的各种情况中,该产量通常有利地增加至少5%、优选增加至少10%、特另优选增加20%、极为优选增加至少100%。
实施例:
专利WO 95/26406和WO 93/03183中且特别是实施例中描述了从棉桃阿舒氏囊霉和啤酒糖酵母中分离rib基因1、2、3、4、5和7的方法并相应地进行该过程。将这些公开文献的内容引入本文作为参考。
序列1表示除转化所必需的选择标记外还隐藏了rib3、rib4和rib5基因片段的DNA构建体。
除非另有说明,按照Sambrook等(1989)(Cold Spring harborLaboratory Press:ISBN 0-87969-309-6)中所述实施一般的核酸方法诸如例如克隆法、限制性切割法、琼脂糖凝胶电泳法、DNA片段连接法、微生物转化法、细菌培养法和重组DNA的序列分析法。
使用Sanger的方法(Sanger等(1977)《美国国家科学院学报》(Proc.Natl.Acad.Sci.USA)74,5463-5467)、应用来自ABI的激光荧光DNA测序仪来进行重组DNA分子的测序。对聚合酶链反应产生的片段进行测序并检测以便避免在所表达的构建体中的聚合酶误差。
实施例1含有rib3、rib4和rib5基因拷贝的DNA构建体的克隆(载体Tef-
G418 rib3、4、5)
rib基因的表达构建体:rib3(载体pJR874)、rib4(载体pJR762)和rib5(载体pJR739)如WO95/26406中所述。将载体pAG-110(Steiner和Philipsen(1994)《 分子基因遗传学》(Mol.Gen.Genet.)242;263-271)用DraIII切割;用克列诺聚合酶和脱氧核苷酸培养(补平末端);沉淀且然后用SalI切割。将含有Tef启动子和卡那霉素抗性基因的DNA片段与HindⅢ-和SalⅠ-切割的载体Bluescript KS(Stratagene)连接,已经将其HindⅢ末端用克列诺聚合酶补平。得到载体pBS Tef-G418。
在第二个克隆步骤中用PvuII和SalI切割pJR874。然后将rib3基因片段与SalⅠ-切割并去磷酸化的载体pBS Tef-G418连接。由于仅可以连接所述片段和载体的SalⅠ端,所以用克列诺聚合酶补平不相容的PvuⅡ和SalⅠ端并连接它们。将所得的载体称作下面的Tef-G418-rib3。
为了将rib5基因亚克隆入载体Tef-G418-rib3,用NcoⅠ和NotⅠ切割载体pJR739。用克列诺聚合酶补平末端。然后将rib5基因片段亚克隆入SalⅠ-切割的载体Tef-G418-rib3中,其末端同样已经被补平。得到载体Tef-G418-rib3、5。
最后的克隆步骤通过使用下列引物的PCR而由载体pJR762产生rib4基因片段:5’GATCGATCGATCGCTAGCTGGGAGGATATGTTCTGGG3’5’TCCAAGCTTGCTAGCATCTCAAATAAGTGATTAGAAGGACAAGCTGCAAG3’。
将PCR片段用NheⅠ切割并亚克隆入NheⅠ-切割并经碱性磷酸酶处理的载体Tef-G418-rib3、5中。
所得的DNA构建体是载体Tef-G418-rib3、4、5。
实施例2
将DNA构建体转入真菌棉桃阿舒氏囊霉
用限制酶SpeⅠ完整切割实施例1中所述的DNA构建体(载体Tef-G418-rib3、4、5)并通过琼脂糖凝胶分级分离来纯化隐藏rib基因序列的插入片段。将用于转化的插入片段描述为SEQ ID NO.7。在序列SEQ ID NO.8(=rib3)、SEQ ID NO.9(=rib5)和SEQ IDNO.10(=rib4)中发现存在于所述插入片段的rib基因3、4和5的衍生氨基酸序列。
给MA2-培养基(10g/l细菌胨,1g/l酵母提取物,0.3g/l肌醇和10g/l D-葡萄糖)中接种棉桃阿舒氏囊霉孢子。在4℃下将培养物培养12小时且然后在28℃下振摇13小时。将细胞悬浮液离心并将细胞沉淀溶于5ml、50mM的磷酸钾缓冲液(pH 7.5)即25mM DTT。在28℃下加热处理30分钟后,再次将细胞离心并溶于25ml的STM缓冲液(270mM蔗糖、10mM TRIS-HCl pH 7.5、1mM MgCl2)。然后将0.5ml的该悬浮液与约3μg的上述纯化的插入片段和40U的SpeⅠ酶混合并在Biorad Gene Pulser(100Ω,20μF,1.5kV)中电穿孔。在电穿孔后,将细胞与1ml的MA2培养基混合并在MA2琼脂培养平板上划线。为了进行抗生素选择,在28℃下给平板接种5小时后,将它们用一层5ml含有抗生素G418(200μg/ml)的低熔点琼脂糖覆盖。通过微量操作使转化体进行克隆纯化(Steiner和Philipsen(1995)《遗传学》(Genetics),140;973-987)。通过对所述转化体的基因组DNA进行PCR分析检定所述构建体的成功整合。如Carle和0lson(《美国国家科学院学报》(Proc.Natl.Acad.Sci.)1985,82,3756-3760)以及Wright和Philipsen(《基因》(Gene)1991,109,99-105)所述进行所述基因组DNA的分离。使用对所述构建体具有特异性的引物进行的PCR由R.SaiKi(《PCR方案》(PCR Protocols),1990,Academic Press,13-20)来进行。通过在琼脂糖凝胶上的分级分离来对PCR片段进行分析。使用下列引物将DNA成功地整合入所述转化体的基因组:
引物A:5’-TCCCTTAATCATTGTCACTGC-3’;
引物B:5’-CCAAGCTTGCTAGCATCTC-3’;
引物C:5’-CTGCCTGAGAAGCTGGAAAGC-3’;
引物D:5’-TGTGAATTAGTAAGCGAAAGG-3’;
引物E:5’-TAAGGGATTAGGCGAAGTTGA-3’;
引物F:5’-GCTGCCACCCCTCTGATTCAC-3’;
引物G:5’-ATAAGCTTTTGCCATTCTCAC-3’;
引物H:5’-CTTTTGCTTTGCCACGGAACG-3’。
对于所有转化体来说,成功整合入所述基因组是可检测到的。
实施例3
在重组棉桃阿舒氏囊霉克隆中的核黄素测定
在28℃下,将棉桃阿舒氏囊霉Ita-GS-01(Schmidt,G.,Stahmann,K.-P.,Kaesler,B.,&Sahm,H.(1996)《微生物学》(Microbiology)142,419-426)和通过用实施例1中所述构建体转化而来源于其中的菌株Ita-GS-01#17.1在琼脂培养基上培养4天。将该平板用于接种3个装有10ml培养基(27.5g/l酵母提取物、0.5g/l MgSO4、50ml/l大豆油,pH 7.0)的100ml锥瓶(17.1-1、17.1-2、17.1-3)。在28℃下和180rpm的振动器上培养40小时后,将1ml的培养肉汤转入装有20ml YPD培养基(10g/l酵母提取物、20g/l细菌胨、20g/l葡萄糖)的250ml锥瓶中。在28℃2下以300rpm培养。在190小时后,从各瓶中取出1ml样品并使其与1ml的1M高氯酸混合。将所述样品过滤并通过HPLC分析测定核黄素含量。对所测定的核黄素含量用核黄素标准品(10mg/l、20mg/l、30mg/l、40mg/l、50mg/l)进行校准。
用于核黄素测定的HPLC方法的参数:
柱 ODS Hypersil 5mm 200X 2.1mm(HP)
洗脱液A 用340ml的H3PO4(89%)调水至pH 2.3
洗脱液B 100%乙腈
梯度 0-6分钟:2%B-50%B
6-6.5分钟:50%B-2%B
终止时间 10分钟
流速 0.5ml/分钟
检测 280nm
温度 40℃
注射 2-10μl
含有另外rib3、4和5基因拷贝的所有三批克隆17表现出比初始菌株显著提高的核黄素生产能力(附图4)。
附图4表示来自不同克隆的核黄素产量。
与未修饰菌株相比,引入rib3、4和5基因可使核黄素产率能够增加到150%。
序列表(1)一般信息:
(ⅰ)申请人:
(A)姓名:BASF Aktiengesellschaft
(B)街:Carl-Bosch-Strasse 38
(C)城市:Ludwigshafen
(D)州:Rhineland-Palatinate
(E)国家:德意志联邦共和国
(F)邮政编码:D-67056
(ⅱ)申请名称:用于生产核黄素的遗传方法
(ⅲ)序列数:10
(ⅳ)计算机可读形式:
(A)媒体类型:软盘
(B)计算机:IBM PC兼容机
(C)操作系统:PC-DOS/MS-DOS
(D)软件:PatentIn Release#1.0,#1.25版本(EPO)(2)SEQ ID NO:l的信息:
(ⅰ)序列特征:
(A)长度:655个碱基对
(B)类型:核酸
(C)链:单链
(D)拓扑结构:线性
(ⅱ)分子类型:DNA(基因组)
(ⅲ)假拟结构:无(ⅳ)反义:无(ⅵ)原始来源:(A)生物体:棉桃阿舒氏囊霉(ⅶ)直接来源:(B)克隆:ITA17(ⅸ)特征:(A)名称/关键词:CDS(B)位置:11..649(xi)序列描述:SEQ ID NO:1ACCAAGCAAC ATG ACA AGC CCA TGC ACT GAT ATC GGT ACC GCT ATA GAG 49
Met Thr Ser Pro Cys Thr Asp Ile Gly Thr Ala Ile Glu
1 5 10CAG TTC AAG CAA AAT AAG ATG ATC ATC GTC ATG GAC CAC ATC TCG AGA 97Gln Phe Lys Gln Asn Lys Met Ile Ile Val Met Asp His Ile Ser Arg
15 20 25GAA AAC GAG GCC GAT CTA ATA TGT GCA GCA GCG CAC ATG ACT GCC GAG 145Glu Asn Glu Ala Asp Leu Ile Cys Ala Ala Ala His Met Thr Ala Glu30 35 40 45CAA ATG GCA TTT ATG ATT CGG TAT TCC TCG GGC TAC GTT TGC GCT CCA 193Gln Met Ala Phe Met Ile Arg Tyr Ser Ser Gly Tyr Val Cys Ala Pro
50 55 60ATG ACC AAT GCG ATT GCC GAT AAG CTA GAC CTA CCG CTC ATG AAC ACA 241Met Thr Asn Ala Ile Ala Asp Lys Leu Asp Leu Pro Leu Met Asn Thr
65 70 75TTG AAA TGC AAG GCT TTC TCC GAT GAC AGA CAC AGC ACT GCG TAT ACA 289Leu Lys Cys Lys Ala Phe Ser Asp ASp Arg His Ser Thr Ala Tyr Thr
80 85 90ATC ACC TGT GAC TAT GCG CAC GGG ACG ACG ACA GGT ATC TCC GCA CGT 337Ile Thr Cys Asp Tyr Ala His Gly Thr Thr Thr Gly Ile Ser Ala Arg
95 100 105GAC CGG GCG TTG ACC TGT AAT CAG TTG GCG AAC CCG GAG TCC AAG GCT 385Asp Arg Ala Leu Thr Cys Asn Gln Leu Ala Asn Pro Glu Ser Lys Ala110 115 120 125ACC GAC TTC ACG AAG CCA GGC CAC ATT GTG CCA TTG CGT GCC CGT GAC 433Thr Asp Phe Thr Lys Pro Gly His Ile Val Pro Leu Arg Ala Arg Asp
130 135 140GGC GGC GTG CTC GAG CGT GAC GGG CAC ACC GAA GCG GCG CTC GAC TTG 481Gly Gly Val Leu Glu Arg Asp Gly His Thr Glu Ala Ala Leu Asp Leu
145 150 155TGC AGA CTA GCG GGT GTG CCA GAG GTC GCT GCT ATT TGT GAA TTA GTA 529Cys Arg Leu Ala Gly Val Pro Glu Val Ala Ala Ile Cys Glu Leu Val
160 165 170AGC GAA AGG GAC GTC GGG CTG ATG ATG ACT TTG GAT GAG TGT ATA GAA 577Ser Glu Arg Asp Val Gly Leu Met Met Thr Leu Asp Glu Cys Ile Glu
175 180 185TTC AGC AAG AAG CAC GGT CTT GCC CTC ATC ACC GTC GAT GAC CTG AAG 625Phe Ser Lys Lys His Gly Leu Ala Leu Ile Thr Val Asp Asp Leu Lys190 195 200 205GCT GCA GTT GCC GCC AAG CAG TAGACGGCA 655Ala Ala Val Ala Ala Lys Gln
210(2)SEQ ID NO:2的信息:
(ⅰ)序列特征:
(A)长度:212个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ⅱ)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:2Met Thr Ser Pro Cys Thr Asp Ile Gly Thr Ala Ile Glu Gln Phe Lys1 5 10 15Gln Asn Lys Met Ile Ile Val Met Asp His Ile Ser Arg Glu Asn Glu
20 25 30Ala Asp Leu Ile Cys Ala Ala Ala His Met Thr Ala Glu Gln Met Ala
35 40 45Phe Met Ile Arg Tyr Ser ser Gly Tyr Val Cys Ala Pro Met Thr Asn
50 55 60 Ala Ile Ala Asp Lys Leu Asp Leu Pro Leu Met Asn Thr Leu Lys Cys
65 70 75 80Lys Ala Phe Ser Asp Asp Arg His Ser Thr Ala Tyr Thr Ile Thr Cys
85 90 95Asp Tyr Ala His Gly Thr Thr Thr Gly Ile Ser Ala Arg Asp Arg Ala
100 105 110Leu Thr Cys Asn Gln Leu Ala Asn Pro Glu Ser Lys Ala Thr Asp Phe
115 120 125Thr Lys Pro Gly His Ile Val Pro Leu Arg Ala Arg Asp Gly Gly Val
130 135 140Leu Glu Arg ASp Gly His Thr Glu Ala Ala Leu Asp Leu Cys Arg Leu145 150 155 160Ala Gly Val Pro Glu Val Ala Ala Ile Cys Glu Leu Val Ser Glu Arg
165 170 175Asp Val Gly Leu Met Met Thr Leu Asp Glu Cys Ile Glu Phe Ser Lys
180 185 190Lys His Gly Leu Ala Leu Ile Thr Val Asp Asp Leu Lys Ala Ala Val
195 200 205Ala Ala Lys Gln
210(2)SEQ ID NO:3的信息:
(ⅰ)序列特征:
(A)长度:529个碱基对
(B)类型:核酸
(C)链:单链
(D)拓扑结构:线性
(ⅱ)分子类型:DNA(基因组)
(ⅲ)假拟结构:无
(ⅳ)反义:无
(ⅵ)原始来源:
(A)生物体:棉桃阿舒氏囊霉(ⅶ)直接来源:(B)克隆:ITA17(ⅸ)特征:(A)名称/关键词:CDS(B)位置:8..526(xi)序列描述:SEQ ID NO:3AGTGACA ATG ATT AAG GGA TTA GGC GAA GTT GAT CAA ACC TAC GAT GCG 49
Met Ile Lys Gly Leu Gly Glu Val Asp Gln Thr Tyr Asp Ala
1 5 10AGC TCT GTC AAG GTT GGC ATT GTC CAC GCG AGA TGG AAC AAG ACT GTC 97Ser Ser Val Lys VaI Gly Ile Vai His Ala Arg Trp Asn Lys Thr Val15 20 25 30ATT GAC GCT CTC GTC CAA GGT GCA ATT GAG AAA CTG CTT GCT ATG GGA 145Ile Asp Ala Leu Val Gln Gly Ala Ile Glu Lys Leu Leu Ala Met Gly
35 40 45GTG AAG GAG AAG AAT ATC ACT GTA AGC ACC GTT CCA GGT GCG TTT GAA 193Val Lys Glu Lys Asn Ile Thr Val Ser Thr Val Pro Gly Ala Phe Glu
50 55 60CTA CCA TTT GGC ACT CAG CGG TTT GCC GAG CTG ACC AAG GCA AGT GGC 241Leu Pro Phe Gly Thr Gln Arg Phe Ala Glu Leu Thr Lys Ala Ser Gly
65 70 75AAG CAT TTG GAC GTG GTC ATC CCA ATT GGA GTC CTG ATC AAA GGC GAC 289Lys His Leu Asp Val Val Ile Pro Ile Gly Val Leu Ile Lys Gly Asp
80 85 90TCA ATG CAC TTT GAA TAT ATA TCA GAC TCT GTG ACT CAT GCC TTA ATG 337Ser Met His Phe Glu Tyr Ile Ser Asp Ser Val Thr His Ala Leu Met95 100 105 110AAC CTA CAG AAG AAG ATT CGT CTT CCT GTC ATT TTT GGT TTG CTA ACG 385Asn Leu Gln Lys Lys Ile Arg Leu Pro Val Ile Phe Giy Leu Leu Thr
115 120 125TGT CTA ACA GAG GAA CAA GCG TTG ACA CGT GCA GGC CTC GGT GAA TCT 433Cys Leu Thr Glu Glu Gln Ala Leu Thr Arg Ala Gly Leu Gly Glu Ser
130 135 140GAA GGC AAG CAC AAC CAC GGT GAA GAC TGG GGT GCT GCT GCC GTG GAG 481Glu Gly Lys His Asn His Gly Glu Asp Trp Gly Ala Ala Ala Val Glu
145 150 155ATG GCT GTA AAG TTT GGC CCA CGC GCC GAA CAA ATG AAG AAG TGAATA 529Met Ala Val Lys Phe Gly Pro Arg Ala Glu Gln Met Lys Lys
160 165 170(2)SEQ ID NO:4的信息:(ⅰ)序列特征:(A)长度:172个氨基酸(B)类型:氨基酸(D)拓扑结构:线性(ⅱ)分子类型:蛋白质(xi)序列描述:SEQ ID NO:4Met Ile Lys Gly Leu Gly Glu Val Asp Gln Thr Tyr Asp Ala Ser Ser1 5 10 15Val Lys Val Gly Ile Val His Ala Arg Trp Asn Lys Thr Val Ile Asp
20 25 30Ala Leu Val Gln Gly Ala Ile Glu Lys Leu Leu Ala Met Gly Val Lys
35 40 45Glu Lys Asn Ile Thr Val Ser Thr Val Pro Gly Ala Phe Glu Leu Pro
50 55 60Phe Gly Thr Gln Arg Phe Ala Glu Leu Thr Lys Ala Ser Gly Lys His65 70 75 80Leu Asp Val Val Ile Pro Ile Gly Val Leu Ile Lys Gly Asp Ser Met
85 90 95His Phe Glu Tyr Ile Ser Asp Ser Val Thr His Ala Leu Met Asn Leu
100 105 110Gln Lys Lys Ile Arg Leu Pro Val Ile Phe Gly Leu Leu Thr Cys Leu
115 120 125Thr Glu Glu Gln Ala Leu Thr Arg Ala Gly Leu Gly Glu Ser Glu Gly
130 135 140Lys His Asn His Gly Glu ASp Trp Gly Ala Ala Ala Val Glu Met Ala145 150 155 160Val Lys Phe Gly Pro Arg Ala Glu Gln Met Lys Lys
165 170(2)SEQ ID NO:5的信息:
(ⅰ)序列特征:
(A)长度:712个碱基对
(B)类型:核酸
(C)链:单链
(D)拓扑结构:线性
(ⅱ)分子类型:DNA(基因组)(ⅲ)假拟结构:无(ⅳ)反义:无(ⅵ)原始来源:(A)生物体:棉桃阿舒氏囊霉(ⅶ)直接来源:(B)克隆:ITA17(ⅸ)特征:(A)名称/关键词:CDS(B)位置:5..712(xi)序列描述:SEQ ID NO:5TAGG ATG TTT ACC GGT ATA GTG GAA CAC ATT GGC ACT GTT GCT GAG TAC 49
Met Phe Thr Gly Ile Val Glu His Ile Gly Thr Val Ala Glu Tyr
1 5 10 15TTG GAG AAC GAT GCC AGC GAG GCA GGC GGC AAC GGT GTG TCA GTC CTT 97Leu Glu Asn Asp Ala Ser Glu Ala Gly Gly Asn Gly Val Ser Val Leu
20 25 30ATC AAG GAT GCG GCT CCG ATA CTG GCG GAT TGC CAC ATC GGT GAC TCG 145Ile Lys Asp Ala Ala Pro Ile Leu Ala Asp Cys His Ile Gly Asp Ser
35 40 45ATT GCA TGC AAT GGT ATC TGC CTG ACG GTG ACG GAG TTC ACG GCC GAT 193Ile Ala Cys Asn Gly Ile Cys Leu Thr Val Thr Glu Phe Thr Ala Asp
50 55 60AGC TTC AAG GTC GGG ATC GCA CCA GAA ACA GTT TAT CGG ACG GAA GTC 241Ser Phe Lys Val Gly Ile Ala Pro Glu Thr Val Tyr Arg Thr Glu Val
65 70 75AGC AGC TGG AAA GCT GGC TCC AAG ATC AAC CTA GAA AGG GCC ATC TCG 289Ser Ser Trp Lys Ala Gly Ser Lys Ile Asn Leu Glu Arg Ala Ile Ser80 85 90 95 GAC GAC AGG CGC TAC GGC GGG CAC TAC GTG CAG GGC CAC GTC GAC TCG 337Asp Asp Arg Arg Tyr Gly Gly His Tyr Val Gln Gly His Val Asp Ser
100 105 110GTG GCC TCT ATT GTA TCC AGA GAG CAC GAC GGG AAC TCT ATC AAC TTT 385Val Ala Ser Ile Val Ssr Arg Glu His Asp Gly Asn Ser Ile Asn Phe
115 120 125AAG TTT AAA CTG CGC GAT CAA GAG TAC GAG AAG TAC GTA GTA GAA AAG 433Lys Phe Lys Leu Arg Asp Gln Glu Tyr Glu Lys Tyr Val Val Glu Lys
130 135 140GGT TTT GTG GCG ATC GAC GGT GTG TCG CTG ACT GTA AGC AAG ATG GAT 481Gly Phe Val Ala Ile Asp Gly Val Ser Leu Thr Val Ser Lys Met Asp
145 150 155CCA GAT GGC TGT TTC TAC ATC TCG ATG ATT GCA CAC ACG CAG ACC GCT 529Pro Asp Gly Cys Phe Tyr Ile Ser Met Ile Ala His Thr Gln Thr Ala160 165 170 175GTA GCC CTT CCA CTG AAG CCG GAC GGT GCC CTC GTG AAC ATA GAA ACG 577Val Ala Leu Pro Leu Lys Pro Asp Gly Ala Leu Val Asn Ile Glu Thr
180 185 190GAT GTT AAC GGC AAG CTA GTA GAG AAG CAG GTT GCA CAG TAC CTG AAT 625Asp Val Asn Gly Lys Leu Val Glu Lys Gln Val Ala Gln Tyr Leu Asn
195 200 205GCG CAG CTG GAA GGT GAG AGC TCG CCA TTG CAG CGC GTG CTC GAA AGG 673Ala Gln Leu Glu Gly Glu Ser Ser Pro Leu Gln Arg Val Leu Glu Arg
210 215 220ATT ATT GAA TCC AAG CTT GCT AGC ATC TCA AAT AAG TG 712Ile Ile Glu Ser Lys Leu Ala Ser Ile Ser Asn Lys
225 230 235(2)SEQ ID N0:6的信息:
(ⅰ)序列特征:
(A)长度:235个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ⅱ)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:6Met Phe Thr Gly Ile Val Glu His Ile Gly Thr Val Ala Glu Tyr Leu1 5 10 15Glu Asn Asp Ala Ser Glu Ala Gly Gly Asn Gly Val Ser Val Leu Ile
20 25 30Lys Asp Ala Ala Pro Ile Leu Ala Asp Cys His Ile Gly Asp Ser Ile
35 40 45Ala Cys Asn Gly Ile Cys Leu Thr Val Thr Glu Phe Thr Ala Asp Ser
50 55 60Phe Lys Val Gly Ile Ala Pro Glu Thr Val Tyr Ar9 Thr Glu Val Ser65 70 75 80Ser Trp Lys Ala Gly Ser Lys Ile Asn Leu Glu Arg Ala Ile ser Asp
85 90 95Asp Arg Arg Tyr Gly Gly His Tyr Val Gln Gly His Val Asp Ser Val
100 105 110Ala Ser Ile Val Ser Arg Glu His Asp Gly Asn Ser Ile Asn Phe Lys
115 120 125Phe Lys Leu Arg Asp Gln Glu Tyr Glu Lys Tyr Val Val Glu Lys Gly
130 135 140Phe Val Ala Ile Asp Gly Val Ser Leu Thr Val Ser Lys Met Asp Pro145 150 155 160Asp Gly Cys Phe Tyr Ile Ser Met Ile Ala His Thr Gln Thr Ala Val
165 170 175Ala Leu Pro Leu Lys Pro Asp Gly Ala Leu Val Asn Ile Glu Thr Asp
180 185 190Val Asn Gly Lys Leu Val Glu Lys Gln Val Ala Gln Tyr Leu Asn Ala
195 200 205Gln Leu Glu Gly Glu Ser Ser Pro Leu Gln Arg Val Leu Glu Arg Ile
210 215 220Ile Glu Ser Lys Leu Ala Ser Ile Ser Asn Lys225 230 235(2)SEQ ID NO:7的信息:
(ⅰ)序列特征:
(A)长度:6317个碱基对
(B)类型:核酸
(C)链:单链
(D)拓扑结构:线性
(ⅱ)分子类型:DNA(基因组)
(ⅲ)假拟结构:无(ⅳ)反义:无(ⅵ)原始来源:(A)生物体:棉桃阿舒氏囊霉(ⅶ)直接来源:(B)克隆:5(ⅸ)特征:(A)名称/关键词:CDS(B)位置:2306..2944(ⅸ)特征:(A)名称/关键词:CDS(B)位置:3575..4282(ⅸ)特征:(A)名称/关键词:CDS(B)位置:4717..5235(xi)序列描述:SEQ ID NO:7ACTAGTGGAT CCCCCGGGCT GCAGGAATTC GATATCAAGC TTGTCTCAAA ATCTCTGATG 60TTACATTGCA CAAGATAAAA ATATATCATC ATGAACAATA AAACTGTCTG CTTACATAAA 120CAGTAATACA AGGGGTGTTA TGAGCCATAT TCAACGGGAA ACGTCTTGCT CGAAGCTTGC 180CTCGTCCCGC CGGGTCACCC GGCCAGCGAC ATGGAGGCCC AGAATACCCT CCTTGACAGT 240CTTGACGTGC GCAGCTCAGG GGCATGATGT GACTGTCGCC CGTACATTTA GCCCATACAT 300CCCCATGTAT AATCATTTGC ATCCATACAT TTTGATGGCC GCACGGCGCG AACGAAAAAT 360TACGGCTCCT CGCTGCAGAC CTGCGAGCAG GGAAACGCTC CCCTCACAGA CGCGTTGAAT 420TCTCCCCACG GCGCGCCCCT GTAGAGAAAT ATAAAAGGTT AGGATTTGCC ACTGAGGTTC 480TTCTTTCATA TACTTCCTTT TAAAATCTTG CTAGGATACA GTTCTCACAT CACATCCGAA 540
CATAAACAAA AATGGGTAAG GAAAAGACTC ACGTTTCGAG GCCGCGATTA AATTCCAACA 600
TGGATGCTGA TTTATATGGG TATAAATGGG CTCGCGATAA TGTCGGGCAA TCAGGTGCGA 660
CAATCTATCG ATTGTATGGG AAGCCCGATG CGCCAGAGTT GTTTCTGAAA CATGGCAAAG 720
GTAGCGTTGC CAATGATGTT ACAGATGAGA TGGTCAGACT AAACTGGCTG ACGGAATTTA 780
TGCCTCTTCC GACCATCAAG CATTTTATCC GTACTCCTGA TGATGCATGG TTACTCACCA 840
CTGCGATCCC CGGGAAAACA GCATTCCAGG TATTAGAAGA ATATCCTGAT TCAGGTGAAA 900
ATATTGTTGA TGCGCTGGCA GTGTTCCTGC GCCGGTTGCA TTCGATTCCT GTTTGTAATT 960
GTCCTTTTAA CAGCGATCGC GTATTTCGTC TCGCTCAGGC GCAATCACGA ATGAATAACG 1020
GTTTGGTTGA TGCGAGTGAT TTTGATGACG AGCGTAATGG CTGGCCTGTT GAACAAGTCT 1080
GGAAAGAAAT GCATAAGCTT TTGCCATTCT CACCGGATTC AGTCGTCACT CATGGTGATT 1140
TCTCACTTGA TAACCTTATT TTTGACGAGG GGAAATTAAT AGGTTGTATT GATGTTGGAC 1200
GAGTCGGAAT CGCAGACCGA TACCAGGATC TTGCCATCCT ATGGAACTGC CTCGGTGAGT 1260
TTTCTCCTTC ATTACAGAAA CGGCTTTTTC AAAAATATGG TATTGATAAT CCTGATATGA 1320
ATAAATTGCA GTTTCATTTG ATGCTCGATG AGTTTTTCTA ATCAGAATTG GTTAATTGGT 1380
TGTAACACTG GCAGAGCATT ACGCTGACTT GACGGGACGG CGGCTTTGTT GAATAAATCG 1440
AACTTTTGCT GAGTTGAAGG ATCAGATCAC GCATCTTCCC GACAACGCAG ACCGTTCCGT 1500
GGCAAAGCAA AAGTTCAAAA TCACCAACTG GTCCACCTAC AACAAAGCTC TCATCAACCG 1560
TGGCTCCCTC ACTTTCTGGC TGGATGATGG GGCGATTCAG GCCTGGTATG AGTCAGCAAC 1620
ACCTTCTTCA CGAGGCAGAC CTCAGCGCTA TTCTGACCTT GCCATCACGA CTGTGCTGGT 1680
CATTAAACGC GTATTCAGGC TGACCCTGCG CGCTGCGCAG GGCTTTATTG ATTCCATTTT 1740
TACACTGATG AATGTTCCGT TGCGCTGCCC GGATTACAGC TGTAATTGAC AAGCCAGACA 1800
GAACAAAGGG ACTTGGCACT TGTAACAGAA ATTCCAAGTA AATAAGGGGA GTTATTCAAG 1860
AACGCCATTG CTACATTGGG TCACGATGTT CGAGCCGGAA TTCGCATTAT CCATTGAACA 1920
CAGCCGCCAA CATAACCGGA AAACTCACAC TTGATTGCAA AGGAACAGCA CATCCCAAGT 1980
CACTAGAAGA TCCCTTCTTG CACGGTCGTT TCTGAAACTC TACGATTAAT GGAACAATGA 2040
GTAAGTCCTC AAATGTACCA CCTATCTGTA GTTTACTATC GGATTTACTG GCTAAGAGCT 2100
GACCTGTTAG GCAAGTGAAA CATATCACAT CGCCAGCAGG TTGGGCTACC AAGGATAGTT 2160
GATGACTTCC ATCACCTATA AAAGCGGCTT GAGTGCTTTT GCAATGATTC TGTTCACATG 2220
ATGGACAAGA AATACGTACA AAAATTTCAA CGTTTTACAA GTTCCCAAGC TTAGTCAACT 2280
CATCACCAAC GACAAACCAA GCAAC ATG ACA AGC CCA TGC ACT GAT ATC GGT 2332
Met Thr Ser Pro Cys Thr Asp Ile Gly
1 5
ACC GCT ATA GAG CAG TTC AAG CAA AAT AAG ATG ATC ATC GTC ATG GAC 2380
Thr Ala Ile Glu Gln Phe Lys Gln Asn Lys Met Ile Ile Val Met Asp
10 15 20 25
CAC ATC TCG AGA GAA AAC GAG GCC GAT CTA ATA TGT GCA GCA GCG CAC 2428
His Ile Ser Arg Glu Asn Glu Ala Asp Leu Ile Cys Ala Ala Ala His
30 35 40
ATG ACT GCC GAG CAA ATG GCA TTT ATG ATT CGG TAT TCC TCG GGC TAC 2476
Met Thr Ala Glu Gln Met Ala Phe Met Ile Arg Tyr Ser Ser Gly Tyr
45 50 55
GTT TGC GCT CCA ATG ACC AAT GCG ATT GCC GAT AAG CTA GAC CTA CCG 2524Val Cys Ala Pro Met Thr Asn Ala Ile Ala Asp Lys Leu Asp Leu Pro
60 65 70CTC ATG AAC ACA TTG AAA TGC AAG GCT TTC TCC GAT GAC AGA CAC AGC 2572Leu Met Asn Thr Leu Lys Cys Lys Ala Phe Ser Asp Asp Arg His Ser
75 80 85ACT GCG TAT ACA ATC ACC TGT GAC TAT GCG CAC GGG ACG ACG ACA GGT 2620Thr Ala Tyr Thr Ile Thr Cys Asp Tyr Ala His Gly Thr Thr Thr Gly90 95 100 105ATC TCC GCA CGT GAC CGG GCG TTG ACC TGT AAT CAG TTG GCG AAC CCG 2668Ile Ser Ala Arg Asp Arg Ala Leu Thr Cys Asn Gln Leu Ala Asn Pro
110 115 120GAG TCC AAG GCT ACC GAC TTC ACG AAG CCA GGC CAC ATT GTG CCA TTG 2716Glu Ser Lys Ala Thr Asp Phe Thr Lys Pro Gly His Ile Val Pro Leu
125 130 135CGT GCC CGT GAC GGC GGC GTG CTC GAG CGT GAC GGG CAC ACC GAA GCG 2764Arg Ala Arg Asp Gly Gly Val Leu Glu Arg Asp Gly His Thr Glu Ala
140 145 150GCG CTC GAC TTG TGC AGA CTA GCG GGT GTG CCA GAG GTC GCT GCT ATT 2812Ala Leu Asp Leu Cys Arg Leu Ala Gly Val Pro Glu Val Ala Ala Ile
155 160 165TGT GAA TTA GTA AGC GAA AGG GAC GTC GGG CTG ATG ATG ACT TTG GAT 2860Cys Glu Leu Val Ser Glu Arg Asp Val Gly Leu Met Met Thr Leu Asp170 175 180 185GAG TGT ATA GAA TTC AGC AAG AAG CAC GGT CTT GCC CTC ATC ACC GTC 2908Glu Cys Ile Glu Phe Ser Lys Lys His Gly Leu Ala Leu Ile Thr Val
190 195 200GAT GAC CTG AAG GCT GCA GTT GCC GCC AAG CAG TAGACGGCAA CGAGTTCTTT 2961Asp Asp Leu Lys Ala Ala Val Ala Ala Lys Gln
205 210AAGTCGGTGT TCATTTATGT AATATACCAT TTCGTCGAAA AAGTCAAATG GTATGAACTA 3021GATTTATCAA TAGTATCTAA GAGTTATGGT ATTCGCAAAA GCTTATCGAT ACCGTCGACA 3081TGGCGCGGGC GAATACCAAC CCACAGGAGC CAGATATAAG ACCAATCCCG GCGGGTGTGC 3141CAGCCGCCAT CAGAGACAGC GGGCCAGCAA GGCATGTGAA GTCAAAAGGC GCCAGCTCCT 3201TATCCGCTCC CGCACAAGCA GGACCGGCAT ATCCCGATGA GCGCGCCAGC ACCCAGACGC 3261TACACCACCA TTCGAAGTAG ACTTTAAAAG AGCGCTTTCC AGCTTCTCAG GCAGTTAGCT 3321CTACGACAAA GGAACCAAGT GATTTTCCCG ATAGACGCGA CTTGCTCAAC GATGTTTCTG 3381TGACCAGCGC AAGGAGAGAT AGTCCTAAAG TATAATCAGA TAGTTAGTCG TATCTTCTAG 3441TTTTATTAGT CAGCTACATG GCGAACCGCC ATTTCCTTAT GCATGTCTTA CGAGTTTAAA 3501AAGCTCGCGG TAGCAGAAAA GAAGATGCAT AGATGGCATA CCGAAGCCTA TATCGCCCAT 3561AGAAGTTGAT AGG ATG TTT ACC GGT ATA GTG GAA CAC ATT GGC ACT GTT 3610
Met Phe Thr Gly Ile Val Glu His Ile Gly Thr Val
1 5 10GCT GAG TAC TTG GAG AAC GAT GCC AGC GAG GCA GGC GGC AAC GGT GTG 3658Ala Glu Tyr Leu Glu Asn Asp Ala Ser Glu Ala Gly Gly Asn Gly Val
15 20 25TCA GTC CTT ATC AAG GAT GCG GCT CCG ATA CTG GCG GAT TGC CAC ATC 3706Set Val Leu Ile Lys Asp Ala Ala Pro Ile Leu Ala Asp Cys His Ile
30 35 40GGT GAC TCG ATT GCA TGC AAT GGT ATC TGC CTG ACG GTG ACG GAG TTC 3754Gly Asp Ser Ile Ala Cys Asn Gly Ile Cys Leu Thr Val Thr Glu Phe45 50 55 60ACG GCC GAT AGC TTC AAG GTC GGG ATC GCA CCA GAA ACA GTT TAT CGG 3802Thr Ala Asp Ser Phe Lys Val Gly Ile Ala Pro Glu Thr Val Tyr Arg
65 70 75ACG GAA GTC AGC AGC TGG AAA GCT GGC TCC AAG ATC AAC CTA GAA AGG 3850Thr Glu Val Ser Ser Trp Lys Ala Gly Ser Lys Ile Asn Leu Glu Arg
80 85 90GCC ATC TCG GAC GAC AGG CGC TAC GGC GGG CAC TAC GTG CAG GGC CAC 3898Ala Ile Ser Asp Asp Arg Arg Tyr Gly Gly His Tyr Val Gln Gly His
95 100 105GTC GAC TCG GTG GCC TCT ATT GTA TCC AGA GAG CAC GAC GGG AAC TCT 3946Val Asp Ser Val Ala Ser Ile Val Ser Arg Glu His ASp Gly Asn Ser110 115 120ATC AAC TTT AAG TTT AAA CTG CGC GAT CAA GAG TAC GAG AAG TAC GTA 3994Ile Asn Phe Lys Phe Lys Leu Arg Asp Gln Glu Tyr Glu Lys Tyr Val125 130 135 140GTA GAA AAG GGT TTT GTG GCG ATC GAC GGT GTG TCG CTG ACT GTA AGC 4042Val Glu Lys Gly Phe Val Ala Ile Asp Gly Val ser Leu Thr Val Ser
145 150 155AAG ATG GAT CCA GAT GGC TGT TTC TAC ATC TCG ATG ATT GCA CAC ACG 4090Lys Met Asp Pro Asp Gly Cys Phe Tyr Ile Ser Met Ile Ala His Thr
160 165 170CAG ACC GCT GTA GCC CTT CCA CTG AAG CCG GAC GGT GCC CTC GTG AAC 4138Gln Thr Ala Val Ala Leu Pro Leu Lys Pro Asp Gly Ala Leu Val Asn
175 180 185ATA GAA ACG GAT GTT AAC GGC AAG CTA GTA GAG AAG CAG GTT GCA CAG 4186Ile Glu Thr Asp Val Asn Gly Lys Leu Val Glu Lys Gln Val Ala Gln
190 195 200TAC CTG AAT GCG CAG CTG GAA GGT GAG AGC TCG CCA TTG CAG CGC GTG 4234Tyr Leu Asn Ala Gln Leu Glu Gly Glu Ser Ser Pro Leu Gln Arg Val205 210 215 220CTC GAA AGG ATT ATT GAA TCC AAG CTT GCT AGC ATC TCA AAT AAG TGATTAGAAG 4289Leu Glu Arg Ile Ile Glu Ser Lys Leu Ala Ser Ile Ser Asn Lys
225 230 235GACAAGCTGC AAGATAAGAA GCCCCCCGTT AACTTAGTGT AGGCAACCTT AGCCTTAGAT 4349TATCCGCTAA CGTCATTCTG TATTTTACTC ATATTATATG TAATATAGGG GGGTTATCCG 4409AGATACTAGA CTACTAGCGT ACTAGAGGAT TATACATGGT ATAATGATAC AGGAAGTGAA 4469AATCCGAAAG GTTCAGACGA TGAAAAGAGT TTGAGACGCA TCAATGATCA GCTTTGAGCT 4529ATATGTAAGT CTATTAATTG ATTACTAATA GCAATTTATG GTATCCTCTG TTCTGCATAT 4589CGACGGTTAC TCACGTGATG ATCAGCTTGA GGCTTCGCGG ATAAAGTTCC ATCGATTACT 4649ATAAAACCAT CACATTAAAC GTTCACTATA GGCATACACA CAGACTAAGT TCAAGTTAGC 4709AGTGACA ATG ATT AAG GGA TTA GGC GAA GTT GAT CAA ACC TAC GAT GCG 4758
Met Ile Lys Gly Leu Gly Glu Val Asp Gln Thr Tyr Asp Ala
1 5 10AGC TCT GTC AAG GTT GGC ATT GTC CAC GCG AGA TGG AAC AAG ACT GTC 4806Ser Ser Val Lys Val Gly Ile Val His Ala Arg Trp Asn Lys Thr Val15 20 25 30ATT GAC GCT CTC GTC CAA GGT GCA ATT GAG AAA CTG CTT GCT ATG GGA 4854Ile Asp Ala Leu Val Gln Gly Ala Ile Glu Lys Leu Leu Ala Met Gly
35 40 45GTG AAG GAG AAG AAT ATC ACT GTA AGC ACC GTT CCA GGT GCG TTT GAA 4902Val Lys Glu Lys Asn Ile Thr Val Ser Thr Val Pro Gly Ala Phe Glu
50 55 60CTA CCA TTT GGC ACT CAG CGG TTT GCC GAG CTG ACC AAG GCA AGT GGC 4950Leu Pro Phe Gly Thr Gln Arg Phe Ala Glu Leu Thr Lys Ala Ser Gly
65 70 75AAG CAT TTG GAC GTG GTC ATC CCA ATT GGA GTC CTG ATC AAA GGC GAC 4998Lys His Leu Asp Val Val Ile Pro Ile Gly Val Leu Ile Lys Gly Asp
80 85 90TCA ATG CAC TTT GAA TAT ATA TCA GAC TCT GTG ACT CAT GCC TTA ATG 5046Ser Met His Phe Glu Tyr Ile Ser Asp Ser Val Thr His Ala Leu Met95 100 105 110AAC CTA CAG AAG AAG ATT CGT CTT CCT GTC ATT TTT GGT TTG CTA ACG 5094Asn Leu Gln Lys Lys Ile Arg Leu Pro Val Ile Phe Gly Leu Leu Thr
115 120 125TGT CTA ACA GAG GAA CAA GCG TTG ACA CGT GCA GGC CTC GGT GAA TCT 5142Cys Leu Thr Glu Glu Gln Ala Leu Thr Arg Ala Gly Leu Gly Glu Ser
130 135 140GAA GGC AAG CAC AAC CAC GGT GAA GAC TGG GGT GCT GCT GCC GTG GAG 5190Glu Gly Lys His Asn His Gly Glu Asp Trp Gly Ala Ala Ala Val Glu
145 150 155ATG GCT GTA AAG TTT GGC CCA CGC GCC GAA CAA ATG AAG AAG TGAATATTAA 5242Met Ala Val Lys Phe Gly Pro Arg Ala Glu Gln Met Lys Lys
160 165 170AAAATCACTA CTTAAAATTA ACGTTTTTAT TATGTCTATA TCAAATTCTT ACGTGATAAC 5302TTTTGATTTC GCTTCCTGGA TTGGCGCAAG GCCTCCCTGT GTCGCAGTTT TTGTTCACGG 5362GTCCACACAG CTCTGTTTTC CCAGAACATA TCCTCCCAGC TAGCATCTCA AATAAGTGAT 5422TATATTATCT TGGGTGCTGT ATATGTTATG TATGTCTTAC GACTGTGAAT CAGAGGGGTG 5482GCAGCTGGAA CACCAGCGAC ACACCTTCGT CTCCCGCGGT GATCAGCCTT CTGTTTTCCT 5542CAAGTAGTAC AAAGTCTAGG ACACCCATGT TGTGGCCAAC GCAAACATGG AGCTGCTGCC 5602CGTTACGCAC GTCGAACTCG TAGACCTTGC CGTCAATGCA CGAGGCGAAC AGGTGGAAAC 5662CGGTGGTCTT GTCAAACGCC AGCTTCGTGA CCGAGTCCGG CAGCACGAAC TTGTGTCTGA 5722CCTTCAACGT CACAGTGTCG TACAGCAGAA TGTCGCCCGA AACCAGGCCC ACGACCATGA 5782AGTTCATCCG AGACGAGAAT GCAATTGACT CTATCGAGGC GTCCATCTCC TCCTGGTCCT 5842CCTTGAGTTC GATCGTCTGC GTCAGGTGCG ACACTGCACC CTGCGCCGCG TTGATCACCG 5902CCAGCAGCCC GCTGTTCGAG CCGCACGCCA CGATGGGCGA ACCGCGGTTG ATCCCCAGCG 5962GGAGCGTGCT CAACGTTATC CACTGCGCCT CCTGGCCCTT GAGTTCAGAC GGCGTCACCT 6022TGAATACCTG TTGCCCGCTG TAGCAGTTCC AGCCAATTAT GGTCCCATCG AGGGAGCACG 6082TCACCAGTAT GACGTTTTCG TCGTCTGCCG CAGTCTCCAG GAACACACCC ATCGTACAGT 6142CCTGGGCGTG GGCCACCCCC GTCATCAGCA GCACAGGCGT GTTGTTCTGC GTGTCCATCT 6202CGTAGCACCA CACCGACCCG TCCACAGCGC CAATCGCAAA AATCCCAGCT CTGTGCGGGT 6262GCACCTTCAG CCAGGTCACC TCGTCCACCT CCTGCAGCCC GGGGGATCCA CTAGT 6317(2)SEQ ID NO:8的信息:
(ⅰ)序列特征:
(A)长度:212个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ⅱ)分子类型:蛋白质(xi)序列描述:SEQ ID NO:8Met Thr Ser Pro Cys Thr Asp Ile Gly Thr Ala Ile Glu Gln Phe Lys1 5 10 15Gln Asn Lys Met Ile IIe Val Met Asp Mis Ile Ser Arg Glu Asn Glu
20 25 30Ala Asp Leu Ile Cys Ala Ala Ala His Met Thr Ala Glu Gln Met Ala
35 40 45Phe Met Ile Arg Tyr Ser Ser Gly Tyr Val Cys Ala Pro Met Thr Asn
50 55 60Ala Ile Ala Asp Lys Leu Asp Leu Pro Leu Met Asn Thr Leu Lys Cys65 70 75 80Lys Ala Phe Ser Asp Asp Arg His Ser Thr Ala Tyr Thr Ile Thr Cys
85 90 95Asp Tyr Ala His Gly Thr Thr Thr Gly Ile Ser Ala Arg Asp Arg Ala
100 105 110Leu Thr Cys Asn Gln Leu Ala Asn Pro Glu Ser Lys Ala Thr Asp Phe
115 120 125Thr Lys Pro Gly His Ile Val Pro Leu Arg Ala Arg Asp Gly Gly Val
130 135 140Leu Glu Arg Asp Gly His Thr Glu Ala Ala Leu Asp Leu Cys Arg Leu145 150 155 160Ala Gly Val Pro Glu Val Ala Ala Ile Cys Glu Leu Val Ser Glu Arg
165 170 175Asp Val Gly Leu Met Met Thr Leu Asp Glu Cys Ile Glu Phe Ser Lys
180 185 190Lys His Gly Leu Ala Leu Ile Thr Val Asp Asp Leu Lys Ala Ala Val
195 200 205Ala Ala Lys Gln
210(2)SEQ ID NO:9的信息:
(ⅰ)序列特征:
(A)长度:235个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ⅱ)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:9Met Phe Thr Gly Ile Val Glu His Ile Gly Thr Val Ala Glu Tyr Leu1 5 10 15Glu Asn Asp Ala Ser Glu Ala Gly Gly Asn Gly Val Ser Val Leu Ile
20 25 30Lys Asp Ala Ala Pro Ile Leu Ala Asp Cys His Ile Gly Asp Ser Ile
35 40 45Ala Cys Asn Gly Ile Cys Leu Thr Val Thr Glu Phe Thr Ala Asp Ser
50 55 60Phe Lys Val Gly Ile Ala Pro Glu Thr Val Tyr Arg Thr Glu Val Ser65 70 75 80Ser Trp Lys Ala Gly ser Lys Ile Asn Leu Glu Arg Ala Ile Ser Asp
85 90 95Asp Arg Arg Tyr Gly Gly His Tyr Val Gln Gly His Val Asp Ser Val
100 105 110Ala Ser Ile Val Ser Arg Glu His Asp Gly Asn Ser Ile Asn Phe Lys
115 120 125Phe Lys Leu Arg Asp Gln Glu Tyr Glu Lys Tyr Val Val Glu Lys Gly
130 135 140Phe Val Ala Ile Asp Gly Val Ser Leu Thr Val Ser Lys Met Asp Pro145 150 155 160Asp Gly Cys Phe Tyr Ile Ser Mat Ile Ala His Thr Gln Thr Ala Val
165 170 175Ala Leu Pro Leu Lys Pro Asp Gly Ala Leu Val Asn Ile Glu Thr Asp
180 185 190
Val Asn Gly Lys Leu Val Glu Lys Gln Val Ala Gln Tyr Leu Asn Ala
195 200 205Gln Leu Glu Gly Glu Ser Ser Pro Leu Gln Arg Val Leu Glu Arg Ile
210 215 220Ile Glu Ser Lys Leu Ala Ser Ile Ser Asn Lys225 230 235(2)SEQ ID NO:10的信息:
(ⅰ)序列特征:
(A)长度:172个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ⅱ)分子类型:蛋白质
(ⅹⅰ)序列描述:SEQ ID NO:10Met Ile Lys Gly Leu Gly Glu Val Asp Gln Thr Tyr Asp Ala Ser Ser1 5 10 15Val Lys Val Gly Ile Val His Ala Arg Trp Asn Lys Thr Val Ile Asp
20 25 30Ala Leu Val Gln Gly Ala Ile Glu Lys Leu Leu Ala Met Gly Val Lys
35 40 45Glu Lys Asn Ile Thr Val Ser Thr Val Pro Gly Ala Phe Glu Leu Pro
50 55 60Phe Gly Thr Gln Arg Phe Ala Glu Leu Thr Lys Ala Ser Gly Lys His65 70 75 80Leu Asp Val Val Ile Pro Ile Gly Val Leu Ile Lys Gly Asp Ser Met
85 90 95His Phe Glu Tyr Ile Ser Asp Ser Val Thr His Ala Leu Met Asn Leu
100 105 110Gln Lys Lys Ile Arg Leu Pro Val Ile Phe Gly Leu Leu Thr Cys Leu
115 120 125Thr Glu Glu Gln Ala Leu Thr Arg Ala Gly Leu Gly Glu Ser Glu Gly
130 135 140Lys His Asn His Gly Glu Asp Trp Gly Ala Ala Ala Val Glu Met Ala145 150 155 160Val Lys Phe Gly Pro Arg Ala Glu Gln Met Lys Lys
165 170
Claims (17)
1.一种借助于能够合成核黄素的生物体提高核黄素产量的方法,其中在所述生物体中酶3,4-二羟基-2-丁酮-4-磷酸合酶、二甲基-8-核糖基-2,4-二氧四氢蝶啶合酶和核黄素合酶或其功能类似物的活性得到了提高。
2.一种如权利要求1中所述的方法,其中将可编码酶3,4-二羟基-2-丁酮-4-磷酸合酶、二甲基-8-核糖基-2,4-二氧四氢蝶啶合酶和核黄素合酶或其功能类似物的基因的混合物引入所述生物体以增加所述酶的活性。
3.一种如权利要求1或2中所述的方法,其中将细菌、酵母、真菌或植物用作所述的生物体。
4.一种如权利要求1-3中任意一项所述的方法,其中所述的生物体选自:芽孢杆菌属、梭状芽孢杆菌属、埃希氏杆菌属、毕赤氏酵母属、假丝酵母属、蓝细菌属、棒状杆菌属、短杆菌属、糖酵母属、假囊酵母属或阿舒氏囊霉菌;或使用植物诸如拟南芥属、番茄、马铃薯、玉米、油菜子、小麦、大麦、向日葵、小米、黑麦、燕麦、制糖甜菜、豆类品种或大豆。
5.一种如权利要求1-4中任意一项所述的方法,其中使用生物体,它们选自:芽孢杆菌属、棒状杆菌属、短杆菌属、埃希氏杆菌属、假丝酵母属、假囊酵母属或阿舒氏囊霉菌。
6.一种如权利要求1-5中任意一项所述的方法,其中使用具有序列SEQ ID No.1、SEQ ID No.3和SEQ ID No.5的基因或其功能等价物的基因。
7.一种如权利要求1-6中任意一项所述的方法,其中所述的等价物在由这些序列编码的衍生的氨基酸水平上具有35%的同源性。
8.一种如权利要求1-7中任意一项所述的方法,其中编码酶3,4-二羟基-2-丁酮-4-磷酸合酶、二甲基-8-核糖基-2,4-二氧四氢蝶啶合酶和核黄素合酶或其功能类似物的基因来源于真核生物体或原核生物体。
9.一种如权利要求1-8中任意一项所述的方法,其中所述的基因或其等价物来源于生物体,所述的生物体选自芽孢杆菌属、埃希氏杆菌属、梭状芽孢杆菌属、糖酵母属、假丝酵母属、假囊酵母属或阿舒氏囊霉菌。
10.一种如权利要求1-9中任意一项所述的方法,其中所述的基因或其等价物彼此或单独位于至少一种载体上或位于染色体中。
11.一种包括具有序列SEQ ID No.1、SEQ ID No.3和SEQ ID No.5的基因或其功能等价物的核酸片段,所述的基因或其等价物与一种或多种调节信号功能连接。
12.一种含有如权利要求11中所述核酸片段的表达载体。
13.一种如权利要求12中所述的表达载体,它是一种线性核酸。
14.一种含有至少一种如权利要求11中所述的核酸片段或至少一种如权利要求12中所述的载体的生物体。
15.可在能够合成核黄素的生物体中编码酶3,4-二羟基-2-丁酮-4-磷酸合酶、二甲基-8-核糖基-2,4-二氧四氢蝶啶合酶和核黄素合酶或其功能类似物的基因的混合物用于提高核黄素产量的用途。
16.如权利要求15中所述在棉桃阿舒氏囊霉中的用途。
17.一种用于将核酸整合入生物体基因组中的方法,该方法包括通过限制酶介导的整合而将至少一种核黄素合成基因引入所述生物体基因组的步骤。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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DE19823834.7 | 1998-05-28 | ||
DE19823834A DE19823834A1 (de) | 1998-05-28 | 1998-05-28 | Genetisches Verfahren zur Herstellung von Riboflavin |
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CN1303434A true CN1303434A (zh) | 2001-07-11 |
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CN99806749A Pending CN1303434A (zh) | 1998-05-28 | 1999-05-10 | 用于生产核黄素的遗传方法 |
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EP (1) | EP1082438A2 (zh) |
JP (1) | JP2002516108A (zh) |
KR (1) | KR20010043867A (zh) |
CN (1) | CN1303434A (zh) |
AU (1) | AU4140999A (zh) |
DE (1) | DE19823834A1 (zh) |
ID (1) | ID27073A (zh) |
RU (1) | RU2000133310A (zh) |
WO (1) | WO1999061623A2 (zh) |
Cited By (4)
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CN104531745A (zh) * | 2014-12-09 | 2015-04-22 | 江南大学 | 一种新型抗性质粒的构建及其在核黄素生产菌中的运用 |
CN113755551A (zh) * | 2021-09-30 | 2021-12-07 | 天津科技大学 | 一种提高核黄素产量的发酵方法 |
CN114317557A (zh) * | 2022-01-06 | 2022-04-12 | 河南农业大学 | 玉米ZmRIBA1基因在高赖氨酸玉米育种中的应用 |
CN116463305A (zh) * | 2023-06-15 | 2023-07-21 | 北京易醒生物科技有限公司 | 一种提高用于乙醇氧化的醇氧化酶表达量的方法、优化的核黄素生物合成基因 |
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EP1263963A2 (en) | 1999-06-25 | 2002-12-11 | Basf Aktiengesellschaft | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production |
TR200403465T2 (tr) | 1999-07-01 | 2005-03-21 | Basf Aktiengesellschaft | Corynebacterium glutamicum gen kodlayıcı fosfonolpiruvat:seker fosfotransferaz sistem proteini |
DE1246922T1 (de) | 1999-07-01 | 2003-03-20 | Basf Ag | Für proteine des phosphoenolpyruvat:zucker phosphotransferase systems kodierende gene aus corynebacterium glutamicum |
US7009045B2 (en) | 2000-07-14 | 2006-03-07 | Archer-Daniels-Midland Company | Transformation systems for flavinogenic yeast |
WO2002006448A2 (en) * | 2000-07-14 | 2002-01-24 | Archer-Daniels-Midland Company | Transformation systems for flavinogenic yeast |
DE10046870A1 (de) | 2000-09-20 | 2002-03-28 | Basf Ag | Verfahren zur Veränderung des Genoms von Corynebakterien |
DE60230971D1 (de) | 2001-04-04 | 2009-03-12 | Genencor Int | Entkoppelte anabole und katabole stoffwechselwege in wirtszellen |
DK1383903T3 (da) | 2001-04-04 | 2009-03-23 | Genencor Int | Fremgangsmåder til fremstilling af askorbinsyre-intermediater i vært-celler |
WO2003012101A1 (de) * | 2001-07-27 | 2003-02-13 | Basf Aktiengesellschaft | Neue genprodukte aus ashbya gossypii, die mit den mechanismen der signaltransduktion und isnbesondere mit der verbesserung der vitamin b2-produktion assoziiert sind |
KR20040027959A (ko) * | 2001-08-23 | 2004-04-01 | 바스프 악티엔게젤샤프트 | 아쉬비아 고쉬피로부터의 신규 대사-관련 유전자 산물 |
DE10159396A1 (de) * | 2001-12-04 | 2003-06-12 | Basf Ag | Genetische Stammoptimierung zur verbesserten Herstellung von Riboflavin |
DE10209363A1 (de) * | 2002-03-02 | 2003-09-11 | Basf Ag | Verfahren zur Herstellung von Riboflavin |
JP5565992B2 (ja) * | 2005-09-28 | 2014-08-06 | 興人ライフサイエンス株式会社 | 酵母キャンディダ・ユティリスの形質転換法 |
JP5498651B2 (ja) * | 2006-07-28 | 2014-05-21 | 花王株式会社 | ジピコリン酸又はその塩の製造方法 |
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CN1066486C (zh) * | 1989-06-22 | 2001-05-30 | 霍夫曼-拉罗奇有限公司 | 高产核黄素的细菌菌株 |
CA2111709A1 (en) * | 1991-08-22 | 1993-03-04 | Sabine Steiner | The specific genetic modification of ashbya gossypii |
ES2170058T3 (es) * | 1992-05-11 | 2002-08-01 | Basf Ag | Compuestos de adn y vectores de expresion de adn recombinantes, que codifican la actividad de riboflavinsintetasa de s. cerevisiae. |
DE4420785A1 (de) * | 1994-03-25 | 1995-10-05 | Basf Ag | Riboflavin-Biosynthese in Pilzen |
KR100414490B1 (ko) * | 1995-07-13 | 2004-04-30 | 바스프 악티엔게젤샤프트 | 이소시트레이트리아제활성이변성된미생물에의한리보플라빈의제조방법 |
JPH1084978A (ja) * | 1996-07-24 | 1998-04-07 | F Hoffmann La Roche Ag | 改良されたリボフラビン生産 |
-
1998
- 1998-05-28 DE DE19823834A patent/DE19823834A1/de not_active Withdrawn
-
1999
- 1999-05-10 WO PCT/EP1999/003196 patent/WO1999061623A2/de not_active Application Discontinuation
- 1999-05-10 KR KR1020007013352A patent/KR20010043867A/ko not_active Application Discontinuation
- 1999-05-10 AU AU41409/99A patent/AU4140999A/en not_active Abandoned
- 1999-05-10 CN CN99806749A patent/CN1303434A/zh active Pending
- 1999-05-10 EP EP99924924A patent/EP1082438A2/de not_active Withdrawn
- 1999-05-10 ID IDW20002426A patent/ID27073A/id unknown
- 1999-05-10 RU RU2000133310/13A patent/RU2000133310A/ru not_active Application Discontinuation
- 1999-05-10 JP JP2000551007A patent/JP2002516108A/ja not_active Withdrawn
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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CN104531745A (zh) * | 2014-12-09 | 2015-04-22 | 江南大学 | 一种新型抗性质粒的构建及其在核黄素生产菌中的运用 |
CN104531745B (zh) * | 2014-12-09 | 2018-05-04 | 江南大学 | 一种新型抗性质粒的构建及其在核黄素生产菌中的运用 |
CN113755551A (zh) * | 2021-09-30 | 2021-12-07 | 天津科技大学 | 一种提高核黄素产量的发酵方法 |
CN114317557A (zh) * | 2022-01-06 | 2022-04-12 | 河南农业大学 | 玉米ZmRIBA1基因在高赖氨酸玉米育种中的应用 |
CN114317557B (zh) * | 2022-01-06 | 2023-07-07 | 河南农业大学 | 玉米ZmRIBA1基因在高赖氨酸玉米育种中的应用 |
CN116463305A (zh) * | 2023-06-15 | 2023-07-21 | 北京易醒生物科技有限公司 | 一种提高用于乙醇氧化的醇氧化酶表达量的方法、优化的核黄素生物合成基因 |
CN116463305B (zh) * | 2023-06-15 | 2023-10-17 | 北京易醒生物科技有限公司 | 一种提高用于乙醇氧化的醇氧化酶表达量的方法、优化的核黄素生物合成基因 |
Also Published As
Publication number | Publication date |
---|---|
DE19823834A1 (de) | 1999-12-02 |
WO1999061623A3 (de) | 2000-01-27 |
JP2002516108A (ja) | 2002-06-04 |
AU4140999A (en) | 1999-12-13 |
ID27073A (id) | 2001-02-22 |
KR20010043867A (ko) | 2001-05-25 |
EP1082438A2 (de) | 2001-03-14 |
RU2000133310A (ru) | 2002-12-27 |
WO1999061623A2 (de) | 1999-12-02 |
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