CN1166770C - 酵母中核黄素的过量生产 - Google Patents
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Abstract
本发明提供一种酵母菌株,此酵母菌株已用包含编码至少一种具核黄素生物合成活性的多肽并在适当寄主中表达的DNA序列的重组DNA序列转化,所述DNA序列在这种酵母菌株中与有功能的启动子可转录地相连。本发明也提供核黄素的生产方法,其特征在于在适当培养条件培养上述酵母菌株以及从培养基或酵母菌株分离合成的核黄素;以及食品或饲料组合物的生产方法,其特征在于将获得的核黄素与一种或多种适当食品或饲料成分混合。
Description
核黄素的衍生物〔黄素辅酶(flavocoenzyme)FMN和FAD〕是所有细胞生物中氧化还原反应普遍必需的。核黄素(维生素B2)由所有植物和许多微生物合成[Demain A.L.,核黄素的过量合成,微生物年鉴,1972,26,369]。核黄素不能在脊椎动物中合成,因此核黄素是动物和人必需的营养物。
核黄素可以通过化学方法合成,也可以使用芽孢杆菌属种类(例如枯草芽孢杆菌),子囊菌纲的棉阿舒囊霉和阿舒假囊酵母[DemainA.L.核黄素的过量合成,微生物学年鉴,1972,26,369和Mitsuda H,Nakaiima K.,8-氮鸟嘌呤对阿舒假囊酵母非生长细胞中核黄素合成和核苷酸库的影响,维生素学营养科学杂志(东京)1973;19(3):215-227],各种酵母菌株如季也蒙假丝酵母和Candida.famata[F.W.Tanner,Jr.C.Voinovich,J.M.Van Lanen,假丝酵母属种类的核黄素合成,自然,1945,101(2616):180-181]和相关菌株及其它微生物通过发酵方法合成。
酵母中核黄素生物合成途径如图1所示。维生素生物合成的前体是鸟嘌呤核苷三磷酸(GTP)和核酮糖5-磷酸。合成1摩尔核黄素需要1摩尔GTP和2摩尔核酮糖5-磷酸。
在酵母如酿酒酵母中,维生素生物合成至少需要6个基因,具体地RIB1,RIB2,RIB3,RIB4,RIB5和RIB7[Oltmanns O.,BacherA.,Lingens F.和Zimmermann F.K.,酿酒酵母的核黄素缺损突变体的生物化学和遗传分类,Mol.Gen.Genet.,1969,105,306]。已证明在季也蒙假丝酵母中核黄素的生物合成也需要6个基因的产物,具体是基因RIB1,RIB2,RIB3,RIB4,RIB5和RIB6(2)。这些季也蒙假丝酵母基因的特定酶和它们在生物合成中的作用已总结于图1中。与枯草芽孢杆菌中情况相反,真核生物酿酒酵母和季也蒙假丝酵母中核黄素生物合成基因不成簇存在。
生物合成的初始步骤是由GTP环化水解酶II催化的GTP咪唑环的打开。已有报道此酶的产物是2,5-二氨基-6-核糖氨基-4(3H)-嘧啶酮5’-磷酸。此中间产物通过一系列支链还原作用、环脱氨基作用和去磷酸化作用转化成5-氨基-6-核糖醇氨基-2,4(1H,3H)-嘧啶二酮。参与5-氨基-6-核糖醇氨基5’磷酸的去磷酸化作用的假想酶仍不清楚。5-氨基-6-核糖醇氨基-2,4(1H,3H)嘧啶二酮在6,7-二甲基-8-核糖醇基2,4-二氧四氢蝶啶合成酶的作用下转化为6,7-二甲基-8-核糖醇基2,4-二氧四氢蝶啶的反应需要第二种底物3,4-二羟基-2-丁酮4-磷酸,该底物是通过3,4-二羟基-2-丁酮-4-磷酸合成酶的催化作用从核酮糖5-磷酸得来的。最后,6,7-二甲基-8-核糖醇基2,4-二氧四氢蝶啶在核黄素合成酶的催化作用下经过歧化作用转变成核黄素。指导核黄素合成途径的初始酶,GTP环化水解酶II合成的RIB1基因序列已在酵母季也蒙假丝酵母中得到确定(4)。
例如在EP 405 370中已有描述用于发酵生产核黄素的枯草芽孢杆菌重组菌株。这些菌株带有在指导同类酶大量合成的强启动子的控制下的核黄素操纵子。在强启动子控制下的核黄素操纵子的基因构建体可以存在于枯草芽孢杆菌染色体的一个或几个不同位置上。已证明,在强启动子控制下的核黄素途径的其它基因在枯草芽孢杆菌染色体其它位置的插入可以增加通过发酵得到的核黄素产量,见EP 821 063。
尽管已有报道用酵母菌株如季也蒙假丝酵母生产核黄素,但到目前为止,DNA重组技术还未用于季也蒙假丝酵母或相关产黄素酵母中核黄素生物合成基因的过量表达。
因此,本发明的目的是提供能制备过量合成核黄素的酵母菌株的重组方法。更具体地,本发明的目的是提供一种酵母菌株,此酵母菌株已用包含编码至少一种具核黄素生物合成活性的多肽并在适当寄主中表达的DNA序列的重组DNA序列转化,所述DNA序列在这种酵母菌株中与有功能的启动子可转录地相连;甚至更具体地这种酵母菌株属于在铁离子缺失条件下能过量合成核黄素的产黄素酵母类,诸如选自以下类:许旺酵母属,优选地许旺酵母,德巴利酵母属,优选地克洛德巴利酵母,球拟酵母属,优选地白球拟酵母,或特别地假丝酵母属,优选地季也蒙假丝酵母或C.famata的酵母菌株〔Logvinenko等,Ukrainskii Biokhimicheskii Zhurnal 61(1),28-32,1989;Logvinenko等,Mikrobiologiya 57(2),181-186,1988以及Nakase和Suzuki,普通和应用微生物学报31(1):49-70(1985)〕。本发明的另一目的是提供其中编码多肽的DNA序列来自酵母,优选地来自在铁离子缺失条件下能过量合成核黄素的产黄素酵母,更优选地来自假丝酵母属如季也蒙假丝酵母或C.famata的酵母菌株。
本发明的目的也包括提供其中编码多肽的DNA序列编码具有GTP环化水解酶II活性的蛋白并选自以下DNA序列的酵母菌株:
a)图5所示的DNA序列或其互补链,
b)在标准条件下与a)中所定义的DNA序列的蛋白编码区域或其片段杂交的DNA序列。
c)除了遗传密码的兼并性,与a)和b)中定义的DNA杂交的DNA序列。
本发明另一目的是提供其中启动子是酿酒酵母TEF启动子的酵母菌株。
本发明另一目的是提供核黄素的生产方法,其特征在于在适当培养条件培养上述酵母菌株以及用本领域技术人员所知的方法从培养基或酵母菌株分离合成的核黄素;以及食品或饲料组合物的生产方法,其特征在于用本领域技术人员所知的方法将用这样的方法获得的核黄素与一种或多种适当的食品或饲料成分混合。
在本发明的实施中使用的所有季也蒙假丝酵母菌株都是来自美国典型培养物保藏中心(ATCC)的登记号为ATCC 9058的季也蒙假丝酵母的衍生种(1)。季也蒙假丝酵母(ATCC 9058)已根据布达佩斯条约于1998年4月1日重新保藏并得到登记号ATCC 74437。季也蒙假丝酵母是在铁离子缺失情况下可以过量合成核黄素(维生素B2)的酵母种类的代表性菌株。这一类还包括许旺酵母(也称为Debaryomycesoccidentalis)、克洛德巴利酵母、白球拟酵母和C.famata。后者已用于核黄素的工业生产。关于酵母种类分类命名,本领域技术人员知道这些命名由不同的作者不定地掌握,例如Candida famata-汉逊德巴利酵母-Torulaspora hansenii,季也蒙假丝酵母-季也蒙毕赤酵母-Yamadazyma guilliermondii作为同物异名使用。本领域技术人员知道可以用于本发明的实施作为寄主细胞或作为分离DNA序列来源的微生物可从保藏单位如美国典型培养物保藏中心(ATCC),真菌菌种保藏中心(CBS)或德意志微生物保藏中心(DSM)和其它列于“工业产权”杂志[(1991)1,29-40页]中任何保藏单位得到。
用于本发明实施并编码具有核黄素生物合成活性的多肽的DNA序列可以例如用本领域技术人员所掌握的方法或使用著名的PCR技术以基因组或cDNA序列的形式从任何已知合成核黄素的微生物(见上)中获得。White等已描述了多聚酶链式反应(PCR)方法的原理[遗传学动向,5,185-189(1989)],而改进方法也得到描述,例如参见Innis等[PCR方法:方法和应用指南,学术出版公司(1990)]。
设计PCR引物所需的序列信息可以从例如诸如基因库(Intelligenetics,加利福尼亚,美国),欧洲生物信息研究所(HinstonHall,剑桥,英国),NBRF(Georgetown大学医学中心,华盛顿,美国)和Vecbase(威士康星大学生物技术中心,Madison,威士康星,美国)的序列数据库得到。
一旦得到这样的DNA序列,它们就可以在任何目的寄主中表达,并且编码多肽的核黄素生物合成活性可以通过本领域技术人员所知的任何测定法测定,这些方法例如描述于Bacher A.,G.Richter,H.Ritz,S.Eberhardt,M.Fisher和Krieger,核黄素的生物合成:GTP环化水解酶II、脱氨酶和还原酶,酶学方法,1997;280:382-389;K.Kis,R.Volk和A.Bacher,核黄素的生物合成:对6,7-二甲基-8-核糖醇基2,4-二氧四氢蝶啶合成酶反应机制的研究,生物化学,1995,34,2883-2892;Logvineko EM,Shavlovskii GM,Zakal’skiiAE,Kontorovskaia Niu.,2,5-二氨基-4-氧-6-核糖基氨基嘧啶-5’-磷酸还原酶的特性,季也蒙毕赤酵母黄素发生(flavinogenesis)第二阶段的酶,Ukr Biokkhim Zh,1989年6月;61(4):47-54;G.Richter,M.Fischer,C.Krieger,S.Eberhardt,H.Lütgen,I.Gerstenschlaer和A.Bacher,核黄素生物合成:大肠杆菌和枯草芽孢杆菌的双功能脱氨酶/还原酶的鉴定,细菌学杂志,1997,179,2022-2028;K.Ritsert,D.Turk,R.Huber,R.Ladenstein,K.Schmidt-Base和A.Bacher,对枯草芽孢杆菌2,4-二氧四氢蝶啶合成酶/核黄素合成酶复合物的研究:再构建的二十面体β亚单位衣壳粒于2.4分辨率的结晶结构分析,分子生物学杂志,1995,253,151-167。
实施本发明所用的DNA序列包含至少一个编码具有核黄素生物合成活性的多肽的DNA序列。然而,本领域技术人员应该理解这种DNA序列不止一个,例如所有核黄素生物合成途径的酶都由这样的DNA序列编码,一种或一种以上的这些酶可以由不同种类来源的DNA序列编码,或者只要它们表现至少一种目的核黄素生物合成活性,可以是合成来源的部分或全部。这样编码GTP环化水解酶II的DNA序列的例子在图5中给出。而且,在标准条件下与此DNA序列杂交并编码这样的GTP环化水解酶的DNA序列也对本发明实施有用。
在本文中用于杂交的“标准条件”是本领域技术人员通常用于探测特定杂交信号的条件,并例如由Sambrook等,“分子克隆”,第二版,Cold Spring Harbor laboratory Press,1989,纽约描述,或者优选地所谓的严格杂交和非严格洗涤条件或更优选地所谓的严格杂交和严格洗涤条件也是本领域技术人员所熟知的并描述于Sambrook等(s.a.)。“DNA序列的片段”在本文中是指编码仍具有上述酶活性的多肽的片段。
为了过量表达本发明的DNA序列编码的蛋白,这些序列可以与目的酵母中有功能的启动子相连,并且这些启动子是例如酿酒酵母TEF启动子(见实施例2)或ph5启动子[Vogel等,分子细胞生物学,2050-2057(1989);Rudolf和Hinnen,美国国家科学院院报,84,1340-1344(1987)]或gap启动子或aoxl启动子[Koutz等,酵母,5,167-177(1989);Sreekrishna等,基础微生物学杂志,28,265-278(1988)]或FMD启动子[Hollenberg等,EP 299108]或MOS启动子[Ledeboer等,核酸研究,13,3063-3082(1985)]。
对本发明的实施有用的DNA序列也可包含有所谓的“ARS”元件(自主复制序列),如例如实施例1中所述。
实施例
如果没有特别的说明或通过参考文献提及,使用如例如Sambrook等,“分子克隆”(s.a)和Cregg,J.M.,K.J.Barriner,A.Y.Hessler和K.R.Madden(1985),巴士德毕赤酵母作为转化的宿主系统,分子细胞生物学,5,3376-3385等所述标准方法。
实施例1.质粒p19R1在季也蒙假丝酵母中的自主复制
已有报道质粒pFRI带有季也蒙假丝酵母的RIB1基因(Zakalsky等,Genetika,26,614-620,1990)。为了亚克隆RIB1基因,用限制性核酸酶SalI消化质粒pFR1。将产生的片段克隆入pUC19载体的SalI位点。将连接混合物转入带有导致核黄素缺陷的ribA基因突变的大肠杆菌突变菌株BSV821中。分离在核黄素缺失时生长的菌落并显示带有p19R1质粒。
p19R1质粒的序列得到测定,并表明在pUC19载体的SalI位点中含有2.18kb的季也蒙假丝酵母DNA的片段。此插入的序列如图2所示。
图2中所示的DNA序列带有季也蒙假丝酵母RIB1基因。质粒将RIB1基因缺陷的季也蒙假丝酵母突变体转变成为核黄素原养型并能在这种酵母中自主复制。
已表明复制是由于自主复制序列(ARS)的存在,ARS大约包含图2中的碱基对1542到1755并延伸至结构基因RIB1。
实施例2.用于超量表达季也蒙假丝酵母RIB1基因的质粒的构建酵母,酿酒酵母的TEF基因是指翻译延伸因子1-α。已知此基因在酿酒酵母中高效转录。
通过PCR扩增得到带有酿酒酵母TEF启动子和季也蒙假丝酵母RIB1基因5’部分的DNA片段。首先,位于从酿酒酵母TEF基因5’末端上游的DNA序列用引物ShBle_V和TEF1_H,使用酿酒酵母染色体DNA作为模板通过PCR扩增。扩增的DNA片段(随后称为TEF启动子)包含在EMBL登记号gb/U51033/YSCP9513下列出的序列的碱基对15984到16344。
另外,包含酿酒酵母GTP环化水解酶II结构基因的5’端部分的DNA片段通过用引物PGgtpCY_V和PGgtpCY_NCO并使用p19R1质粒作为模板的PCR得到。扩增的DNA序列(随后称为5’GTPcII)包含如图2所示的序列的碱基对460到1145。
将在上述两个PCR反应中得到的包含部分酿酒酵母TEF基因和5’GTPcII部分的DNA扩增产物混合。使用引物ShBle_V和PGgtpCY_nco进行第三次PCR扩增。此反应产生包含完整TEF启动子和季也蒙假丝酵母的RIB1基因5’部分的DNA片段。
在上述PCR反应中所用的引物列于表2中。图3表示最后扩增产物的序列。图6表示PCR扩增的2个步骤。
扩增产物包含通过引物ShBle_V导入的限制性核酸酶SphI的切割切点,扩增产物还包含RIB1基因本身的MscI位点。用SphI和MscI消化扩增产物(图6)。
质粒p19R1(上面已对其构建进行了描述)也用相同的酶消化。将PCR扩增产物连接入经消化的质粒中。
将连接混合物转入带有限定GTP环化水解酶II的Rib7基因的突变的命名为Rib7的大肠杆菌突变体。根据Invitrogene方法通过电穿孔进行转化(5)。
将大肠杆菌细胞在含有100mg/l氨苄青霉素但不含有核黄素的LB培养基平板上铺板,分离在此培养基上生长的菌落并表明菌落含有称为pTC2的质粒。
质粒pTC2的插入片段的序列如图3所示。
用XhoI和SalI限制性核酸内切酶消化质粒pTC2产生4.4kb和0.5kb片段。4.4kb片段用T4 DNA连接酶环化。将连接混合物转化入带有RibA突变的大肠杆菌Rib7突变体,用电穿孔完成转化。将这些细胞涂在不含核黄素但含有氨苄青霉素的LB培养基平板上(见上面)。分离在这些平板上生长的菌落并表明含有质粒pTCdXS2。此流程导致0.5kb的碱基对从质粒pTC2的去除。
pTCdXS2质粒的插入片段的序列如图4所示,并表示了季也蒙假丝酵母RIB1基因的开放阅读框架,也表示了代表酿酒酵母TEF基因启动子的DNA片段。
实施例3重组季也蒙假丝酵母菌株的构建
核黄素缺陷型突变体rh21是在对L2菌株进行化学诱变后获得的(2),rh21带有明显的RIB1基因缺陷,RIB1基因限定GTP环化水解酶II(2),而L2菌株先前已从季也蒙假丝酵母ATCC 9058得到。
通过LiCl方法,单独将pTCdXS2质粒转化入季也蒙假丝酵母RIB1突变菌株rh21中(6),将这些细胞在没加核黄素的YPD培养基上涂板。分离在没有核黄素时生长的菌落。如下所述检测GTP环化水解酶II活性和核黄素产量。
通过PCR分析检测原养型菌株中用质粒导入的DNA片段的存在。用引物ShBle_V和PGgtpCY_nco以及煮沸的季也蒙假丝酵母重组菌株细胞作为模板进行PCR。引物ShBle_V与TEF启动子互补,而引物PGgtpCY-nco与RIB1结构基因互补。从所有分离的转化子中都得到了预期长度(1175碱基对)的扩增产物。分离从XS-3菌株得到的扩增产物并用荧光双脱氧终止法进行测序。序列如图5所示。此序列与质粒pTCdXS2插入片段的碱基对1到1168相同。
重组转化子在遗传学上是稳定的。具体地,它们不分离核黄素缺陷的传代克隆。
实施例4.重组季也蒙假丝酵母菌株的GTP环化水解酶活性。
上述重组菌株的GTP环化水解酶II的活性水平如下述进行测定。
将季也蒙假丝酵母菌株重组细胞在加微量元素(科学,101,180,1954)但不加天冬酰胺的合成Bukholder培养基上于30℃有氧培养2-3天。在这些实验中,季也蒙假丝酵母L2菌株(野生型)作为对照。
通过离心(5000g,15分钟)收集对数生长期的细胞,并用含有1mMDTT和1mM MgCl2的20mM Tris-HCl溶液(PH8.2)洗2次。细胞储存于-20℃。将冷冻细胞团(1-3g)熔解于3-9ml洗涤缓冲液中。通过与直径为0.8mm的玻璃珠一起搅拌使细胞破碎。离心后,将细胞抽提物逆着100倍体积的洗涤缓冲液透析过夜。用劳里法测定蛋白浓度。
用于GTP环化水解酶测试的反应混合物含有pH为8.2的20mMTris-HCl,3mM DTT,2mM MgCl2,1mM GTP和蛋白(蛋白浓度为1-3mg/ml,总体积为4ml)。然后将混合物于37℃黑暗中温育20分钟。
温育后,取出2ml样品,加入2,3-丁二酮至终浓度0.5mg/ml。混合物在95℃温育30分钟。以相同方法但不加双乙酰基得到空白值。
测定两种样品特异荧光的不同并用于计算6,7-二甲基蝶呤的浓度和GTP环化水解酶II的活性。结果如表3所示。
rh21突变体从中衍生的L2菌株用作对照。L2菌株中的酶活性是2.9nmol mg-1h-1,而在带有RIB1基因突变的核黄素缺陷受体菌株rh-21中没有发现酶活性。通过用质粒pTCdXS2转化得到的重组菌株的酶水平在6.5至13.4nmol mg-1h-1之间。这样,与季也蒙假丝酵母L2菌株相比,重组菌株的酶活性水平提高了2.3-4.6倍。
重组菌株的核黄素合成酶活性也得到测定,核黄素合成酶的活性不受p19RI,pTC2和pTCdXS2质粒转化的影响。所有分析的菌株具有20nmol mg-1h-1范围的核黄素合成酶活性(表3)。
实施例5.重组季也蒙假丝酵母菌株的核黄素合成
季也蒙假丝酵母菌株(野生型和重组菌株)在加微量元件但未加天冬酰胺的合成Burkholder培养基(科学,101,180页,1945)上于30℃有氧培养4天[F.W.Tanner,Jr.C.Vojnovich,J.M.VanLanen,假丝酵母属种类的核黄素合成,自然,1945,101(2616):180-181]。将悬浮液离心。对核黄素浓度进行荧光测定。结果如表4所示。
在所述条件下,野生型菌株L2合成1.2mg/l核黄素,而重组菌株XS3合成的核黄素水平提高了3倍(3.6mg/l)。
实施例6.核黄素的分离
将重组的季也蒙假丝酵母菌株XS-3接种于各装有0.5升加微量元件但未加天门冬酰胺的合成Burkholder培养基的2个2.5升三角瓶中。培养物在30℃振荡培养50小时。对溶液进行离心。使上清液以500ml/小时的速度通过Florisil柱(4ml柱床体积)。用7ml蒸馏水洗柱。用丙酮和1摩尔水相NH4OH洗脱核黄素。蒸发洗脱液至干燥状态。用光度法测定核黄素的产量。
参考文献
1.Sibirny,A.A.,Zharova,V.P.,Kshanovskaya,B.V.,Shavlovsky,G.M.能形成大量孢子的季也蒙毕赤酵母遗传株系的选择,Tstytologia i genetika,11,330-333,1977(俄语)。
2.Shavlovskyy,G.M.,Sibirnyy,A.A.,Kshanovs ka,B.V.,季也蒙假丝酵母核黄素营养缺陷型突变体的遗传学分类,Genetika,15,1561-1568,1979(俄语)。
3.Zakalsky,A.E.,Zlochevsky,M.L.,Stasiv,Y.Z.,Logvynenko,E.M.,Beburov,M.Y.,Shavlovsky,G.M.,编码GTP环化水解酶II-大肠杆菌细胞中黄素生成第一个酶的季也蒙毕赤酵母RIB1结构基因的克隆。Genetika,26,614-620,1990(俄语)。
4.Liauta-Teglivets,O.,Hasslacher,M.,Boretskyy,Y.,Kohlwein,S.D.,Shavlovskii,G.M.,GTP环化水解酶的分子克隆,参与核黄素生物合成的季也蒙毕赤酵母RIB1结构基因,酵母,11,945-952,1995。
5.零背景/Kan克隆试剂盒,A版本,151204,指导手册,INVITROGENE。
6.Logvinenko,E.M.,Stasiv,Yu.Z.,Zlochevsky,M.L.,Voronovsky,A.Ya.,Beburov,M.Yu.,Shavlovsky,G.M.,编码季也蒙毕赤酵母核黄素合成酶的RIB7基因的克隆,Genetika,29,922-927,1993(俄语)。
表1核黄素途径的酶和基因
酶 | 基因 | |||
酿酒酵母 | 季也蒙假丝酵母 | 大肠杆菌 | ||
A | GTP环化水解酶 | RIB1 | RIB1 | ribA |
B | 细菌脱氨酶 | ribD | ||
C | 酵母还原酶 | RIB7 | RIB2 | |
D | 酵母脱氨酶 | RIB2 | RIB3 | |
E | 细菌还原酶 | ribD | ||
F | 未知的磷酸酶 | |||
G | 2,4-二氧四氢蝶啶合成酶 | RIB4 | RIB5 | ribE |
H | 核黄素合成酶 | RIB5 | RIB7 | ribC |
I | 3,4-二羟基-2-丁酮4-磷酸合成酶 | RIB3 | RIB6 | ribB |
表2 所用引物的核苷酸序列
N | 引物 | 序列(5’-3’) |
1 | ShBle_V | GGGCATGCAATTCGAGCTCGGTACCCG |
2 | TEFl_H | CGACTCACTATAGGAGGAAGCTTGGCGC |
3 | PGgtpCY_V | AGGAGGAAGCTTGGCGCTATGGCATCGAAGG |
4 | PGgtpCY_nco | GCTGGTCGGTTAATGGGTGAAGCTGGG |
表3季也蒙假丝酵母重组菌株的GTP环化水解酶II和核黄素合成酶活性(生长时间:40-48小时)
N | 菌株 | 核黄素合成酶活性nmol mg-1h-1 | GTP环化水解酶II的活性nmol mg-1h-1 | 比率* |
1 | L2(野生型) | 21.6 | 2.88 | 1.00 |
2 | R1-1 | 未测定 | 10.08 | 3.50 |
3 | R1-2 | 未测定 | 4.20 | 1.46 |
4 | R1-3 | 20.4 | 8.76 | 3.04 |
5 | R1-4 | 19.8 | 7.80 | 2.70 |
6 | R1-5 | 21.6 | 7.80 | 2.70 |
7 | TC-1 | 20.4 | 9.60 | 3.33 |
8 | TC-2 | 未测定 | 8.40 | 2.92 |
9 | TC-3 | 未测定 | 7.56 | 2.63 |
10 | XS-1 | 22.8 | 13.38 | 4.60 |
11 | XS-2 | 未测定 | 12.60 | 4.37 |
12 | XS-3 | 未测定 | 6.60 | 2.29 |
*重组菌株的GTP环化水解酶活性/L2菌株的GTP环化水解酶活性
表4.重组季也蒙假丝酵母菌株核黄素的产量(生长时间:110小时;培养温度:30℃)
N | 菌株 | 核黄素的产量[mg/l] | 核黄素的相对产量 |
1 | L2(野生型) | 1.2 | 1.0 |
4 | R1-3 | 1.4 | 1.2 |
5 | R1-4 | 3.6 | 3.0 |
6 | R1-5 | 3.0 | 2.5 |
7 | TC-1 | 1.4 | 1.2 |
9 | TC-3 | 1.3 | 1.0 |
12 | XS-3 | 3.6 | 3.0 |
13 | XS-4 | 2.0 | 1.7 |
14 | XS-5 | 2.3 | 1.9 |
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gtcgactttc actccgaagg taggtgcggc tggaagacgt cgtcccaagt cgtatgcgtt 60
agctgagagc gacggaaacg aaagtgatga agattacatg ctggaataat ccatagctag 120
tgtacttgct aatacaaccg gtaaagctag ccaattgcag cgttattcac caccgccgtg 180
gatcgggtta gtcacgtgaa ctggccgttg ggtcctgcac gtcgcttcat tattcatata 240
ttagtgagag tcttcctata tcagtcagca gacgtatcgg ttgatttcag gtcaaaaaga 300
gaaaaggtgg tcttacaaaa gcgaaatagc tgatacattt ttactcacag cagcatcata 360
tttgtggaac ctttaaactt gacttttcat ttcaagcaag ttattttgaa attcaaatca 420
tttggaaatc aaaaaagaac atctaagttc tgaaaaattg tacgaacaac gct atg 476
Met
1
gca tcg aag gac ata gta cat ccg caa cca gag cgc cgg cac ggg tcg 524
Ala Ser Lys Asp Ile Val His Pro Gln Pro Glu Arg Arg His Gly Ser
5 10 15
gaa act cac gaa ttt acc atg cct ctc tta tct cct aca ttg aca cca 572
Glu Thr His Glu Phe Thr Met Pro Leu Leu Ser Pro Thr Leu Thr Pro
20 25 30
tcc cat att cca tcg caa acg cct caa att cct ccg gaa gtg cca gca 620
Ser His Ile Pro Ser Gln Thr Pro Gln Ile Pro Pro Glu Val Pro Ala
35 40 45
gaa gtc agg gat cgc ttg ccc ctt cct gaa acg ttg cct gtg gtg aaa 668
Glu Val Arg Asp Arg Leu Pro Leu Pro Glu Thr Leu Pro Val Val Lys
50 55 60 65
tgc atg gcg aga gct cgt ata ccg acc act cag ggg ccg gag ata ttt 716
Cys Met Ala Arg Ala Arg Ile Pro Thr Thr Gln Gly Pro Glu Ile Phe
70 75 80
ctc cat ttg tac gag aat aac gtt gac aat aaa gag cat ttg gct att 764
Leu His Leu Tyr Glu Asn Asn Val Asp Asn Lys Glu His Leu Ala Ile
85 90 95
gtt ttt ggg gaa gat gtg cgg tcg aaa acg ctc tat cag aaa cgt ccc 812
Val Phe Gly Glu Asp Val Arg Ser Lys Thr Leu Tyr Gln Lys Arg Pro
100 105 110
aat gag acc cag caa gat aga atg act cgt ggt gct tat gtg ggc aga 860
Asn Glu Thr Gln Gln Asp Arg Met Thr Arg Gly Ala Tyr Val Gly Arg
115 120 125
ttg ttt cct gga aga acc gag gca gac tat gac agt gag tct aat ttg 908
Leu Phe Pro Gly Arg Thr Glu Ala AsP Tyr Asp Ser Glu Ser Asn Leu
130 135 140 145
aga ttg aat ttc gat gaa aat ggc caa ctt atc aga gat ccg agt acc 956
Arg Leu Asn Phe Asp Glu Asn Gly Gln Leu Ile Arg Asp Pro Ser Thr
150 155 160
acc tgt agt ggt gag ccc att ttg gcc cgt att cat tcg gaa tgt tat 1004
Thr Cys Ser Gly Glu Pro Ile Leu Ala Arg Ile His Ser Glu Cys Tyr
165 170 175
acg ggg gaa acc gca tgg agt gct cgt tgc gat tgt gga gaa caa ttc 1052
Thr Gly Glu Thr Ala Trp Ser Ala Arg Cys Asp Cys Gly Glu Gln Phe
180 185 190
gat gaa gct ggt cgg tta atg ggt gaa gct ggg cac ggg tgt atc gtg 1100
Asp Glu Ala Gly Arg Leu Met Gly Glu Ala Gly His Gly Cys Ile Val
195 200 205
tac ctt cgt cag gaa ggt cgt gga att gga ctt ggg gaa aag ttg aag 1148
Tyr Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Gly Glu Lys Leu Lys
210 215 220 225
gct tat aat ttg caa gac ttg gga gcg gat acc gtc cag gcc aat ttg 1196
Ala Tyr Asn Leu Gln Asp Leu Gly Ala Asp Thr Val Gln Ala Asn Leu
230 235 240
atg tta cga cat cct gct gat gcg aga tct ttt tcg ctc gct aca gcc 1244
Met Leu Arg His Pro Ala Asp Ala Arg Ser Phe Ser Leu Ala Thr Ala
245 250 255
ata ctc ttg gac ttg ggg ctc aac gag atc aag ttg ttg acc aac aat 1292
Ile Leu Leu Asp Leu Gly Leu Asn Glu Ile Lys Leu Leu Thr Asn Asn
260 265 270
ccc gat aaa att gct gca gtt gag gga aga aac aga gag gtc aag gta 1340
Pro Asp Lys Ile Ala Ala Val Glu Gly Arg Asn Arg Glu Val Lys Val
275 280 285
gtg gaa cgg gtg cct atg gtg ccg ttg gca tgg aga agt gag aat gga 1388
Val Glu Arg Val Pro Met Val Pro Leu Ala Trp Arg Ser Glu Asn Gly
290 295 300 305
atc aag tca aaa gag ata gag ggc tac ttg agt gct aag att gaa agg 1436
Ile Lys Ser Lys Glu Ile Glu Gly Tyr Leu Ser Ala Lys Ile Glu Arg
310 315 320
atg ggg cac ttg ctt gaa aag cca ctc aag ata tga tagaagagat 1482
Met Gly His Leu Leu Glu Lys Pro Leu Lys Ile
325 330
gaagttaagg acttaagaaa taaatgatga attaaatgac gcaaatgtca ctactcgatt 1542
agagaaatag ctataatgaa gaattttgca tttcgcaaaa tttaagataa atgcaaaaat 1602
tgcaaattac gaaatatgca tatgatacaa gacaagaaaa gactactaaa agtctctcga 1662
gaagaatact gggtaacctt catctcttga ttatgcactg gggctattca tatgcagatt 1722
cgcacgccga ggtgcagcgt ttaggcgcgg ctcaacggaa gccaacggcc gccacaaatt 1782
gtccggaaag tcgccgaaac tgatccactg gtaccacagc cccataagaa ccccctttaa 1842
tattaaaaac cgttcttcag ccacttttga tcacattgtt tgcagccgcc cgttgctgcc 1902
atccaaacac cacgcgtccc ccgcaccttt tacggtgccc actgcattgg aatttgcata 1962
aaacagcctc acgaagtgga ttaattttta gagcactcaa gtcatcatgc tgcaatctct 2022
gcatcatgaa atgactcccg ttgatacagg gaactcagac cgcaagcggc gaagagtcac 2082
aagagcgtgt gatgtgtgtc gactctagag atccccgggt accgagctcg aattcactgg 2142
ccgtcgtttt acaacgtcgt gactgggaaa accctggcg 2181
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Met Ala Ser Lys Asp Ile Val His Pro Gln Pro Glu Arg Arg His Gly
1 5 10 15
Ser Glu Thr His Glu Phe Thr Met Pro Leu Leu Ser Pro Thr Leu Thr
20 25 30
Pro Ser His Ile Pro Ser Gln Thr Pro Gln Ile Pro Pro Glu Val Pro
35 40 45
Ala Glu Val Arg Asp Arg Leu Pro Leu Pro Glu Thr Leu Pro Val Val
50 55 60
Lys Cys Met Ala Arg Ala Arg Ile Pro Thr Thr Gln Gly Pro Glu Ile
65 70 75 80
Phe Leu His Leu Tyr Glu Asn Asn Val Asp Asn Lys Glu His Leu Ala
85 90 95
Ile Val Phe Gly Glu Asp Val Arg Ser Lys Thr Leu Tyr Gln Lys Arg
100 105 110
Pro Asn Glu Thr Gln Gln Asp Arg Met Thr Arg Gly Ala Tyr Val Gly
115 120 125
Arg Leu Phe Pro Gly Arg Thr Glu Ala Asp Tyr Asp Ser Glu Ser Asn
130 135 140
Leu Arg Leu Asn Phe Asp Glu Asn Gly Gln Leu Ile Arg Asp Pro Ser
145 150 155 160
Thr Thr Cys Ser Gly Glu Pro Ile Leu Ala Arg Ile His Ser Glu Cys
165 170 175
Tyr Thr Gly Glu Thr Ala Trp Ser Ala Arg Cys Asp Cys Gly Glu Gln
180 185 190
Phe Asp Glu Ala Gly Arg Leu Met Gly Glu Ala Gly His Gly Cys Ile
195 200 205
Val Tyr Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Gly Glu Lys Leu
210 215 220
Lys Ala Tyr Asn Leu Gln Asp Leu Gly Ala Asp Thr Val Gln Ala Asn
225 230 235 240
Leu Met Leu Arg His Pro Ala Asp Ala Arg Ser Phe Ser Leu Ala Thr
245 250 255
Ala Ile Leu Leu Asp Leu Gly Leu Asn Glu Ile Lys Leu Leu Thr Asn
260 265 270
Asn Pro Asp Lys Ile Ala Ala Val Glu Gly Arg Asn Arg Glu Val Lys
275 280 285
Val Val Glu Arg Val Pro Met Val Pro Leu Ala Trp Arg Ser Glu Asn
290 295 300
Gly Ile Lys Ser Lys Glu Ile Glu Gly Tyr Leu Ser Ala Lys Ile Glu
305 310 315 320
Arg Met Gly His Leu Leu Glu Lys Pro Leu Lys Ile
325 330
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caattcgagc tcggtacccg gggatccccc acacaccata gcttcaaaat gtttctactc 60
cttttttact cttccagatt ttctcggact ccgcgcatcg ccgtaccact tcaaaacacc 120
caagcacagc atactaaatt tccctctttc ttcctctagg gtgtcgttaa ttacccgtac 180
taaaggtttg gaaaagaaaa aagagaccgc ctcgtttctt tttcttcgtc gaaaaaggca 240
ataaaaattt ttatcacgtt tctttttctt gaaatttttt tttttgattt ttttctcttt 300
cgatgacctc ccattgatat ttaagtcaat aaacggtctt caatttctca agtttcagtt 360
tcatttttct tgttctatta caactttttt tacttcttgc tcattagaaa gaaagcatag 420
caatctaatc taagggcgag ctcgaattcg aactagtact gcagcacgtg accggcgcct 480
agtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga ctcactatag 540
gaggaagctt ggcgct atg gca tcg aag tac ata gta cat ccg caa cca gag 592
Met Ala Ser Lys Tyr Ile Val His Pro Gln Pro Glu
1 5 10
cgc cgg cac ggg tcg gaa act cac gaa ttt acc atg cct ctc tta tct 640
Arg Arg His Gly Ser Glu Thr His Glu Phe Thr Met Pro Leu Leu Ser
15 20 25
cct aca ttg aca cca tcc cat att cca tcg caa acg cct caa att cct 688
Pro Thr Leu Thr Pro Ser His Ile Pro Ser Gln Thr Pro Gln Ile Pro
30 35 40
ccg gaa gtg cca gca gaa gtc agg gat cgc ttg ccc ctt cct gaa acg 736
Pro Glu Val Pro Ala Glu Val Arg Asp Arg Leu Pro Leu Pro Glu Thr
45 50 55 60
ttg cct gtg gtg aaa tgc atg gcg aga gct cgt ata ccg acc act cag 784
Leu Pro Val Val Lys Cys Met Ala Arg Ala Arg Ile Pro Thr Thr Gln
65 70 75
ggg ccg gag ata ttt ctc cat ttg tac gag aat aac gtt gac aat aaa 832
Gly Pro Glu Ile Phe Leu His Leu Tyr Glu Asn Asn Val Asp Asn Lys
80 85 90
gag cat ttg gct att gtt ttt ggg gaa gat gtg cgg tcg aaa acg ctc 880
Glu His Leu Ala Ile Val Phe Gly Glu Asp Val Arg Ser Lys Thr Leu
95 100 105
tat cag aaa cgt ccc aat gag acc cag caa gat aga atg act cgt ggt 928
Tyr Gln Lys Arg Pro Asn Glu Thr Gln Gln Asp Arg Met Thr Arg Gly
110 115 120
gct tat gtg ggc aga ttg ttt cct gga aga acc gag gca gac tat gac 976
Ala Tyr Val Gly Arg Leu Phe Pro Gly Arg Thr Glu Ala Asp Tyr Asp
125 130 135 140
agt gag tct aat ttg aga ttg aat ttc gat gaa aat ggc caa ctt atc 1024
Ser Glu Ser Asn Leu Arg Leu Asn Phe Asp Glu Asn Gly Gln Leu Ile
145 150 155
aga gat ccg agt acc acc tgt agt ggt gag ccc att ttg gcc cgt att 1072
Arg Asp Pro Ser Thr Thr Cys Ser Gly Glu Pro Ile Leu Ala Arg Ile
160 165 170
cat tcg gaa 1080
His Ser Glu
175
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Ser Glu Thr His Glu Phe Thr Met Pro Leu Leu Ser Pro Thr Leu Thr
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Pro Ser His Ile Pro Ser Gln Thr Pro Gln Ile Pro Pro Glu Val Pro
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Ala Glu Val Arg Asp Arg Leu Pro Leu Pro Glu Thr Leu Pro Val Val
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Phe Leu His Leu Tyr Glu Asn Asn Val Asp Asn Lys Glu His Leu Ala
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Ile Val Phe Gly Glu Asp Val Arg Ser Lys Thr Leu Tyr Gln Lys Arg
100 105 110
Pro Asn Glu Thr Gln Gln Asp Arg Met Thr Arg Gly Ala Tyr Val Gly
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Arg Leu Phe Pro Gly Arg Thr Glu Ala Asp Tyr Asp Ser Glu Ser Asn
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caattcgagc tcggtacccg gggatccccc acacaccata gcttcaaaat gtttctactc 60
cttttttact cttccagatt ttctcggact ccgcgcatcg ccgtaccact tcaaaacacc 120
caagcacagc atactaaatt tccctctttc ttcctctagg gtgtcgttaa ttacccgtac 180
taaaggtttg gaaaagaaaa aagagaccgc ctcgtttctt tttcttcgtc gaaaaaggca 240
ataaaaattt ttatcacgtt tctttttctt gaaatttttt tttttgattt ttttctcttt 300
cgatgacctc ccattgatat ttaagtcaat aaacggtctt caatttctca agtttcagtt 360
tcatttttct tgttctatta caactttttt tacttcttgc tcattagaaa gaaagcatag 420
caatctaatc taagggcgag ctcgaattcg aactagtact gcagcacgtg accggcgcct 480
agtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga ctcactatag 540
gaggaagctt ggcgct atg gca tcg aag tac ata gta cat ccg caa cca gag 592
Met Ala Ser Lys Tyr Ile Val His Pro Gln Pro Glu
1 5 10
cgc cgg cac ggg tcg gaa act cac gaa ttt acc atg cct ctc tta tct 640
Arg Arg His Gly Ser Glu Thr His Glu Phe Thr Met Pro Leu Leu Ser
15 20 25
cct aca ttg aca cca tcc cat att cca tcg caa acg cct caa att cct 688
Pro Thr Leu Thr Pro Ser His Ile Pro Ser Gln Thr Pro Gln Ile Pro
30 35 40
ccg gaa gtg cca gca gaa gtc agg gat cgc ttg ccc ctt cct gaa acg 736
Pro Glu Val Pro Ala Glu Val Arg Asp Arg Leu Pro Leu Pro Glu Thr
45 50 55 60
ttg cct gtg gtg aaa tgc atg gcg aga gct cgt ata ccg acc act cag 784
Leu Pro Val Val Lys Cys Met Ala Arg Ala Arg Ile Pro Thr Thr Gln
65 70 75
ggg ccg gag ata ttt ctc cat ttg tac gag aat aac gtt gac aat aaa 832
Gly Pro Glu Ile Phe Leu His Leu Tyr Glu Asn Asn Val Asp Asn Lys
80 85 90
gag cat ttg gct att gtt ttt ggg gaa gat gtg cgg tcg aaa acg ctc 880
Glu His Leu Ala Ile Val Phe Gly Glu Asp Val Arg Ser Lys Thr Leu
95 100 105
tat cag aaa cgt ccc aat gag acc cag caa gat aga atg act cgt ggt 928
Tyr Gln Lys Arg Pro Asn Glu Thr Gln Gln Asp Arg Met Thr Arg Gly
110 115 120
gct tat gtg ggc aga ttg ttt cct gga aga acc gag gca gac tat gac 976
Ala Tyr Val Gly Arg Leu Phe Pro Gly Arg Thr Glu Ala Asp Tyr Asp
125 130 135 140
agt gag tct aat ttg aga ttg aat ttc gat gaa aat ggc caa ctt atc 1024
Ser Glu Ser Asn Leu Arg Leu Asn Phe Asp Glu Asn Gly Gln Leu Ile
145 150 155
aga gat ccg agt acc acc tgt agt ggt gag ccc att ttg gcc cgt att 1072
Arg Asp Pro Ser Thr Thr Cys Ser Gly Glu Pro Ile Leu Ala Arg Ile
160 165 170
cat tcg gaa tgt tat acg ggg gaa acc gca tgg agt gct cgt tgc gat 1120
His Ser Glu Cys Tyr Thr Gly Glu Thr Ala Trp Ser Ala Arg Cys Asp
175 180 185
tgt gga gaa caa ttc gat gaa gct ggt cgg tta atg ggt gaa gct ggg 1168
Cys Gly Glu Gln Phe Asp Glu Ala Gly Arg Leu Met Gly Glu Ala Gly
190 195 200
cac ggg tgt atc gtg tac ctt cgt cag gaa ggt cgt gga att gga ctt 1216
His Gly Cys Ile Val Tyr Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu
205 210 215 220
ggg gaa aag ttg aag gct tat aat ttg caa gac ttg gga gcg gat acc 1264
Gly Glu Lys Leu Lys Ala Tyr Asn Leu Gln Asp Leu Gly Ala Asp Thr
225 230 235
gtc cag gcc aat ttg atg tta cga cat cct gct gat gcg aga tct ttt 1312
Val Gln Ala Asn Leu Met Leu Arg His Pro Ala Asp Ala Arg Ser Phe
240 245 250
tcg ctc gct aca gcc ata ctc ttg gac ttg ggg ctc aac gag atc aag 1360
Ser Leu Ala Thr Ala Ile Leu Leu Asp Lau Gly Leu Asn Glu Ile Lys
255 260 265
ttg ttg acc aac aat ccc gat aaa att gct gca gtt gag gga aga aac 1408
Leu Leu Thr Asn Asn Pro Asp Lys Ile Ala Ala Val Glu Gly Arg Asn
270 275 280
aga gag gtc aag gta gtg gaa cgg gtg cct atg gtg ccg ttg gca tgg 1456
Arg Glu Val Lys Val Val Glu Arg Val Pro Met Val Pro Leu Ala Trp
285 290 295 300
aga agt aag aat gga atc aag tca aaa gag ata gag ggc tac ttg agt 1504
Arg Ser Lys Asn Gly Ile Lys Ser Lys Glu Ile Glu Gly Tyr Lau Ser
305 310 315
gct aag att gaa agg atg ggg cac ttg ctt gaa aag cca ctc aag ata 1552
Ala Lys Ile Glu Arg Met Gly His Leu Leu Glu Lys Pro Leu Lys Ile
320 325 330
tga tagaagagat gaagttaagg acttaagaaa taaatgatga attaaatgac 1605
gcaaatgtca ctactcgatt agagaaatag ctataatgaa gaattttgcat ttcgcaaaa 1665
tttaagataa atgcaaaaat tgcaaattac gaaatatgca tatgatacaa gacaagaaaa 1725
gactactaaa agtctct 1742
<210>6
<211>332
<212>PRT
<213>人工序列
<400>6
Met Ala Ser Lys Tyr Ile Val His Pro Gln Pro Glu Arg Arg His Gly
1 5 10 15
Ser Glu Thr His Glu Phe Thr Met Pro Leu Leu Ser Pro Thr Leu Thr
20 25 30
Pro Ser His Ile Pro Ser Gln Thr Pro Gln Ile Pro Pro Glu Val Pro
35 40 45
Ala Glu Val Arg Asp Arg Leu Pro Leu Pro Glu Thr Leu Pro Val Val
50 55 60
Lys Cys Met Ala Arg Ala Arg Ile Pro Thr Thr Gln Gly Pro Glu Ile
65 70 75 80
Phe Leu His Leu Tyr Glu Asn Asn Val Asp Asn Lys Glu His Leu Ala
85 90 95
Ile Val Phe Gly Glu Asp Val Arg Ser Lys Thr Leu Tyr Gln Lys Arg
100 105 110
Pro Asn Glu Thr Gln Gln Asp Arg Met Thr Arg Gly Ala Tyr Val Gly
115 120 125
Arg Leu Phe Pro Gly Arg Thr Glu Ala Asp Tyr Asp Ser Glu Ser Asn
130 135 140
Leu Arg Leu Asn Phe Asp Glu Asn Gly Gln Leu Ile Arg Asp Pro Ser
145 150 155 160
Thr Thr Cys Ser Gly Glu Pro Ile Leu Ala Arg Ile His Ser Glu Cys
165 170 175
Tyr Thr Gly Glu Thr Ala Trp Ser Ala Arg Cys Asp Cys Gly Glu Gln
180 185 190
Phe Asp Glu Ala Gly Arg Leu Met Gly Glu Ala Gly His Gly Cys Ile
195 200 205
Val Tyr Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Gly Glu Lys Leu
210 215 220
Lys Ala Tyr Asn Leu Gln Asp Leu Gly Ala Asp Thr Val Gln Ala Asn
225 230 235 240
Leu Met Leu Arg His Pro Ala Asp Ala Arg Ser Phe Ser Leu Ala Thr
245 250 255
Ala Ile Leu Leu Asp Leu Gly Leu Asn Glu Ile Lys Leu Leu Thr Asn
260 265 270
Asn Pro Asp Lys Ile Ala Ala Val Glu Gly Arg Asn Arg Glu Val Lys
275 280 285
Val Val Glu Arg Val Pro Met Val Pro Leu Ala Trp Arg Ser Lys Asn
290 295 300
Gly Ile Lys Ser Lys Glu Ile Glu Gly Tyr Leu Ser Ala Lys Ile Glu
305 310 315 320
Arg Met Gly His Leu Leu Glu Lys Pro Leu Lys Ile
325 330
<210>7
<211>1175
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:PCR片段,含有pTCdXS-2插入子的碱基对1-1168
<220>
<221>启动子
<222>(8)..(559)
<223>TEF启动子片段
<220>
<221>CDS
<222>(564)..(1172)
<223>部分翻译的RIB1基因部分
<400>7
gggcatgcaa ttcgagctcg gtacccgggg atcccccaca caccatagct tcaaaatgtt 60
tctactcctt ttttactctt ccagattttc tcggactccg cgcatcgccg taccacttca 120
aaacacccaa gcacagcata ctaaatttcc ctctttcttc ctctagggtg tcgttaatta 180
cccgtactaa aggtttggaa aagaaaaaag agaccgcctc gtttcttttt cttcgtcgaa 240
aaaggcaata aaaattttta tcacgtttct ttttcttgaa attttttttt ttgatttttt 300
tctctttcga tgacctccca ttgatattta agtcaataaa cggtcttcaa tttctcaagt 360
ttcagtttca tttttcttgt tctattacaa ctttttttac ttcttgctca ttagaaagaa 420
agcatagcaa tctaatctaa gggcgagctc gaattcgaac tagtactgca gcacgtgacc 480
ggcgcctagt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgactc 540
actataggag gaagcttggc gct atg gca tcg aag tac ata gta cat ccg caa 593
Met Ala Ser Lys Tyr Ile Val His Pro Gln
1 5 10
cca gag cgc cgg cac ggg tcg gaa act cac gaa ttt acc atg cct ctc 641
Pro Glu Arg Arg His Gly Ser Glu Thr His Glu Phe Thr Met Pro Leu
15 20 25
tta tct cct aca ttg aca cca tcc cat att cca tcg caa acg cct caa 689
Leu Ser Pro Thr Leu Thr Pro Ser His Ile Pro Ser Gln Thr Pro Gln
30 35 40
att cct ccg gaa gtg cca gca gaa gtc agg gat cgc ttg ccc ctt cct 737
Ile Pro Pro Glu Val Pro Ala Glu Val Arg Asp Arg Leu Pro Leu Pro
45 50 55
gaa acg ttg cct gtg gtg aaa tgc atg gcg aga gct cgt ata ccg acc 785
Glu Thr Leu Pro Val Val Lys Cys Met Ala Arg Ala Arg Ile Pro Thr
60 65 70
act cag ggg ccg gag ata ttt ctc cat ttg tac gag aat aac gtt gac 833
Thr Gln Gly Pro Glu Ile Phe Leu His Leu Iyr Glu Asn Asn Val Asp
75 80 85 90
aat aaa gag cat ttg gct att gtt ttt ggg gaa gat gtg cgg tcg aaa 881
Asn Lys Glu His Leu Ala Ile Val Phe Gly Glu Asp Val Arg Ser Lys
95 100 105
acg ctc tat cag aaa cgt ccc aat gag acc cag caa gat aga atg act 929
Thr Leu Tyr Gln Lys Arg Pro Asn Glu Thr Gln Gln Asp Arg Met Thr
110 115 120
cgt ggt gct tat gtg ggc aga ttg ttt cct gga aga acc gag gca gac 977
Arg Gly Ala Tyr Val Gly Arg Leu Phe Pro Gly Arg Thr Glu Ala Asp
125 130 135
tat gac agt gag tct aat ttg aga ttg aat ttc gat gaa aat ggc caa 1025
Tyr Asp Ser Glu Ser Asn Leu Arg Leu Asn Phe Asp Glu Asn Gly Gln
140 145 150
ctt atc aga gat ccg agt acc acc tgt agt ggt gag ccc att ttg gcc 1073
Leu Ile Arg Asp Pro Ser Thr Thr Cys Ser Gly Glu Pro Ile Leu Ala
155 160 165 170
cgt att cat tcg gaa tgt tat acg ggg gaa acc gca tgg agt gct cgt 1121
Arg Ile His Ser Glu Cys Tyr Thr Gly Glu Thr Ala Trp Ser Ala Arg
175 180 185
tgc gat tgt gga gaa caa ttc gat gaa gct ggt cgg tta atg ggt gaa 1169
Cys Asp Cys Gly Glu Gln Phe Asp Glu Ala Gly Arg Leu Met Gly Glu
190 195 200
gct ggg 1175
Ala Gly
<210>8
<211>203
<212>PRT
<213>人工序列
<400>8
Met Ala Ser Lys Tyr Ile Val His Pro Gln Pro Glu Arg Arg His Gly
1 5 10 15
Ser Glu Thr His Glu Phe Thr Met Pro Leu Leu Ser Pro Thr Leu Thr
20 25 30
Pro Ser His Ile Pro Ser Gln Thr Pro Gln Ile Pro Pro Glu Val Pro
35 40 45
Ala Glu Val Arg Asp Arg Leu Pro Leu Pro Glu Thr Leu Pro Val Val
50 55 60
Lys Cys Met Ala Arg Ala Arg Ile Pro Thr Thr Gln Gly Pro Glu Ile
65 70 75 80
Phe Leu His Leu Tyr Glu Asn Asn Val Asp Asn Lys Glu His Leu Ala
85 90 95
Ile Val Phe Gly Glu Asp Val Arg Ser Lys Thr Leu Tyr Gln Lys Arg
100 105 110
Pro Asn Glu Thr Gln Gln Asp Arg Met Thr Arg Gly Ala Tyr Val Gly
115 120 125
Arg Leu Phe Pro Gly Arg Thr Glu Ala Asp Tyr Asp Ser Glu Ser Asn
130 135 140
Leu Arg Leu Asn Phe Asp Glu Asn Gly Gln Leu Ile Arg Asp Pro Ser
145 150 155 160
Thr Thr Cys Ser Gly Glu Pro Ile Leu Ala ArgIle His Ser Glu Cys
165 170 175
Tyr Thr Gly Glu Thr Ala Trp Ser Ala Arg Cys Asp Cys Gly Glu Gln
180 185 190
Phe Asp Glu Ala Gly Arg Leu Met Gly Glu Ala Gly
195 200
<210>9
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:PGgtpCY_nco引物
<400>9
gctggtcggt taatgggtga agctggg 27
<210>10
<211>31
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:PGgtpCY_V引物
<400>10
aggaggaagc ttggcgctat ggcatcgaag g 31
<210>11
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:ShBle_v引物
<400>11
gggcatgcaa ttcgagctcg gtacccg 27
<210>12
<211>28
<212>DNA
<213>人工序列
<220>
<223>人工序列描述:TEF1_H引物
<400>12
cgactcacta taggaggaag cttggcgc 28
Claims (10)
1.酵母菌株,该菌株已用编码SEQ ID NO.6或8的多核苷酸序列转化。
2.根据权利要求1的酵母菌株,其中所述多核苷酸序列为SEQ ID NO.5或7。
3.根据权利要求1的酵母菌株,该菌株还包括与所述多核苷酸序列转录连接的启动子,其中所述启动子与所述多核苷酸序列不是天然连接的,而是在所述酵母菌株中功能性连接的。
4.根据权利要求3的酵母菌株,其中所述启动子是TEF酿酒酵母启动子。
5.权利要求1的酵母菌株,该菌株属于在铁离子缺失条件下能过量产生核黄素的产黄素酵母类。
6.权利要求5的酵母菌株,该菌株选自许旺酵母属、德巴利酵母属、球拟酵母属和假丝酵母属。
7.权利要求6的酵母菌株,该菌株选自许旺酵母、克洛德巴利酵母、白球拟酵母、季也蒙假丝酵母和无名假丝酵母。
8.生产核黄素的方法,该方法包括:
a)将表达盒插入编码SEQ ID NO.6或8的DNA序列中;
b)用所述表达盒转化选自下列的酵母细胞培养物:许旺酵母属、德巴利酵母属、球拟酵母属和假丝酵母属;
c)筛选超量产生核黄素的转化体;
d)在培养基中培养所述转化体;和
e)从所述转化体和/或培养基中回收核黄素。
9.根据权利要求8的方法,该方法还包括将回收的核黄素与食品或饲料成分混和以制备食品或饲料组合物。
10.纯化并分离的DNA分子,该分子包括编码SEQ ID NO.6或8的多核苷酸序列。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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EP98107380.2 | 1998-04-23 | ||
EP98107380 | 1998-04-23 |
Publications (2)
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CN1232872A CN1232872A (zh) | 1999-10-27 |
CN1166770C true CN1166770C (zh) | 2004-09-15 |
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CNB991050215A Expired - Fee Related CN1166770C (zh) | 1998-04-23 | 1999-04-23 | 酵母中核黄素的过量生产 |
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US (1) | US6376222B1 (zh) |
JP (1) | JP2000050883A (zh) |
KR (1) | KR100671593B1 (zh) |
CN (1) | CN1166770C (zh) |
AU (1) | AU2398499A (zh) |
BR (1) | BR9901399A (zh) |
CA (1) | CA2268382A1 (zh) |
ID (1) | ID23301A (zh) |
NO (1) | NO991924L (zh) |
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US7009045B2 (en) * | 2000-07-14 | 2006-03-07 | Archer-Daniels-Midland Company | Transformation systems for flavinogenic yeast |
AU2003270098A1 (en) * | 2002-09-06 | 2004-03-29 | Basf Aktiengesellschaft | Gtp cyclohydrolase ii as a target for fungicides |
ATE486944T1 (de) | 2005-11-16 | 2010-11-15 | Korea Res Inst Of Bioscience | Translationselongationsfaktor-promotor aus pichia pastoris und verfahren zur herstellung von rekombinantem protein unter verwendung davon |
KR20220062331A (ko) * | 2019-10-04 | 2022-05-16 | 코나겐 인크. | 알파-이오논 및 베타-이오논의 생합성 |
CN113801910A (zh) * | 2021-08-30 | 2021-12-17 | 湖北广济药业股份有限公司 | 一种纯天然核黄素的制备方法 |
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-
1999
- 1999-04-21 CA CA002268382A patent/CA2268382A1/en not_active Abandoned
- 1999-04-21 ID IDP990365D patent/ID23301A/id unknown
- 1999-04-22 NO NO991924A patent/NO991924L/no not_active Application Discontinuation
- 1999-04-22 KR KR1019990014369A patent/KR100671593B1/ko not_active IP Right Cessation
- 1999-04-23 US US09/299,041 patent/US6376222B1/en not_active Expired - Fee Related
- 1999-04-23 BR BR9901399-1A patent/BR9901399A/pt not_active Application Discontinuation
- 1999-04-23 CN CNB991050215A patent/CN1166770C/zh not_active Expired - Fee Related
- 1999-04-23 AU AU23984/99A patent/AU2398499A/en not_active Abandoned
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CA2268382A1 (en) | 1999-10-23 |
KR19990083401A (ko) | 1999-11-25 |
CN1232872A (zh) | 1999-10-27 |
NO991924L (no) | 1999-10-25 |
NO991924D0 (no) | 1999-04-22 |
US6376222B1 (en) | 2002-04-23 |
BR9901399A (pt) | 2000-03-28 |
JP2000050883A (ja) | 2000-02-22 |
AU2398499A (en) | 1999-11-04 |
KR100671593B1 (ko) | 2007-01-18 |
ID23301A (id) | 2000-04-05 |
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