CN116583534A - 前导肽和编码其的多核苷酸 - Google Patents
前导肽和编码其的多核苷酸 Download PDFInfo
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Abstract
本发明涉及前导肽、前导肽融合蛋白、信号肽、编码这些前导肽和信号肽的多核苷酸,并且涉及包含这些多核苷酸的核酸构建体、载体和宿主细胞,以及在宿主细胞中生产目的多肽的方法,这些宿主细胞表达与该目的多肽处于翻译融合的前导肽。
Description
序列表的引用
本申请含有计算机可读形式的序列表,其通过援引并入本文。
发明背景
技术领域
本发明涉及前导肽、前导肽融合蛋白、信号肽、编码这些前导肽和信号肽的多核苷酸,并且涉及包含这些多核苷酸的核酸构建体、载体和宿主细胞,以及在宿主细胞中生产目的多肽的方法,这些宿主细胞表达与该目的多肽处于翻译融合的前导肽。
背景技术
在真菌或细菌宿主中表达重组基因是生产重组蛋白的常用方法。在这样的宿主细胞系统中生产的重组蛋白是酶和其他有价值的蛋白质。例如,WO 2011127802描述了宿主细胞和用于生产葡糖淀粉酶的方法。在工业和商业目的中,所用细胞系统的生产率,即每个发酵单位的总蛋白质产量,是生产成本的一个重要因素。传统上,产率的增加是通过诱变和筛选产量增加的目的蛋白来实现的。然而,这种方法主要仅适用于在含有目的酶的分离物中过量生产内源蛋白。因此,对于每种新的蛋白质或酶产品,都需要一个漫长的菌株和工艺开发计划来实现生产率提高。
对于异源蛋白在真菌或细菌宿主细胞系统中的过量表达,生产过程被认为是一个复杂的多阶段和多组分过程。细胞生长和产物形成由多种参数决定,包括培养基的组成、发酵pH、发酵温度、溶解氧张力、剪应力和真菌形态。
已经在真菌和细菌中使用了多种改善表达和分泌的方法。对于异源基因的表达,密码子优化的合成基因可以提高转录率,而分泌伴侣(secretion chaperone)的过量表达用于保护异源蛋白不被降解。为了获得特定基因的高水平表达,一个成熟的程序是将重组基因构建体的多个拷贝靶向高表达内源基因的基因座。WO 2011/075677(诺维信公司(Novozymes A/S))中描述了通过破坏天然蛋白酶来提高蛋白质产率的另一策略。尽管提出了这些方法,但进一步提高真菌和细菌宿主细胞中的重组蛋白产量仍是持续令人感兴趣的。
本发明的目的是提供一种经修饰的宿主菌株和一种具有增加的重组蛋白生产率的蛋白质生产方法。
发明内容
本发明基于令人惊讶和创造性的发现,即与不存在所述前导肽的情况下异源蛋白的表达相比,在异源蛋白上游融合的合成前导肽可提供改进的异源蛋白的表达、活性和/或产率。此外,本发明人还令人惊讶地发现,作为不同信号肽的一部分或与不同信号肽组合的前导肽可提供改进的异源蛋白的表达、活性和/或产率。
经鉴定的前导肽用于增强在宿主细胞(如真菌宿主细胞)中生产的重组多肽的分泌的方法中。描述了编码新颖的前导肽的多核苷酸和使用所述多核苷酸生产异源蛋白的方法。通常,当与其野生型相比时,热稳定蛋白在工业规模上的生产更具挑战性,这主要是由于热稳定变体的表达水平降低。因此,对于这样的(稳定的)变体的蛋白质工程化(PE),发酵期间的低表达水平是取消选择工程化蛋白质变体候选物的主要原因,从而显著限制了PE工作。如实例中所述,本发明人已经对异源蛋白(AnPav498的葡糖淀粉酶)进行了PE工作,从而产生了具有增加的表达水平(N=16)和转化效率的延长的信号序列/另外的前导肽(JPO001)。由PE开发了anPav498的热稳定的变体(JPO变体),重点在于同时改善性能和产率。由主链分子(JPO001)产生的JPO051和JPO124提高了热稳定性,同时保留了足够高的表达水平,以用于异源酶的工业生产。本发明人还表明,可以在不同的菌株、不同的培养基中以及通过将前导肽融合到不同的信号肽(JSP035和JSP038)来获得高表达。因此,本发明的延长的信号序列/前导肽可在PE工作期间用作开发蛋白质变体(如热稳定的蛋白质变体)的工具。我们预计这些发现也适用于其他蛋白质,如其他糖蛋白,并且特别适用于其他葡糖淀粉酶。
因此,在第一方面,本发明涉及一种真菌宿主细胞,该真菌宿主细胞在其基因组中包含:
编码目的多肽的第一多核苷酸;和
在该第一多核苷酸的上游以翻译融合方式可操作地连接至该第一多核苷酸的第二多核苷酸,所述第二多核苷酸编码与SEQ ID NO:2(FARAPVAAR)具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性的前导肽。
在第二方面,本发明涉及一种用于生产目的多肽的方法,该方法包括:
(i)提供根据本发明的第一方面的真菌宿主细胞,
(ii)在有助于该目的多肽表达的条件下培养所述真菌宿主细胞;以及,任选地
(iii)回收该目的多肽。
在第三方面,本发明涉及一种核酸构建体,该核酸构建体包含编码目的多肽的第一多核苷酸和可操作地连接至该第一多核苷酸的第二多核苷酸,该第二多核苷酸编码与SEQ ID NO:2(FARAPVAAR)具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性的前导肽。
在第四和最后方面,本发明涉及一种表达载体,该表达载体包含根据第三方面的核酸构建体。
定义
根据此详细描述,以下定义适用。注意,单数形式“一种/个(a/an)”以及“该/这些(the)”包括复数个指示物,除非上下文中另外明确指明。
本文提及“约”值或参数包括针对该值或参数本身的方面。例如,提及“约X”的描述包括方面“X”。
除非另外定义或由上下文明确指示,否则本文所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。
催化结构域:术语“催化结构域”意指含有酶的催化机构的该酶的区域。
cDNA:术语“cDNA”意指可以通过从获得自真核或原核细胞的成熟的、剪接的mRNA分子进行反转录而制备的DNA分子。cDNA缺乏可以存在于对应基因组DNA中的内含子序列。初始的初级RNA转录物是mRNA的前体,通过一系列步骤(包括剪接)对前体进行加工,然后呈现为成熟的剪接的mRNA。
编码序列:术语“编码序列”意指直接指定多肽的氨基酸序列的多核苷酸。编码序列的边界通常由开放阅读框确定,该开放阅读框以起始密码子(如ATG、GTG或TTG)开始并且以终止密码子(如TAA、TAG或TGA)结束。编码序列可为基因组DNA、cDNA、合成DNA或其组合。
控制序列:术语“控制序列”意指对于表达编码本发明的多肽的多核苷酸所必需的核酸序列。每个控制序列对于编码多肽的多核苷酸而言可以是合成的、天然的(即,来自相同基因)或异源的(即,来自不同基因),或者相对于彼此是天然的或异源的。这样的控制序列包括但不限于前导肽、多腺苷酸化序列、前肽序列、启动子、信号肽序列、以及转录终止子。最少,这些控制序列包括启动子、以及转录和翻译终止信号。出于引入有利于将控制序列与编码多肽的多核苷酸的编码区连接的特异性限制位点的目的,这些控制序列可以提供有多个接头。
表达:术语“表达”意指涉及多肽产生的任何步骤,包括但不限于:转录、转录后修饰、翻译、翻译后修饰、以及分泌。
表达载体:术语“表达载体”意指直链或环状DNA分子,其包含编码多肽的多核苷酸并且可操作地连接至提供用于其表达的控制序列。
融合多肽:术语“融合多肽”是其中一种多肽在本发明多肽的N-末端或C-末端融合的多肽。通过将编码另一种多肽的多核苷酸与本发明的多核苷酸融合来生产融合多肽。用于产生融合多肽的技术是本领域已知的,并且包括连接编码多肽的编码序列使得它们符合读框,而且融合多肽的表达处于一个或多个相同的启动子和终止子的控制之下。还可以使用内含肽技术构建融合多肽,其中在翻译后产生融合多肽(Cooper等人,1993,EMBO J.[欧洲分子生物学学会杂志]12:2575-2583;Dawson等人,1994,Science[科学]266:776-779)。融合多肽可进一步包含两个多肽之间的切割位点。在融合蛋白分泌之时,位点被切割,从而释放出这两种多肽。切割位点的实例包括但不限于在以下文献中披露的位点:Martin等人,2003,J.Ind.Microbiol.Biotechnol.[工业微生物生物技术杂志]3:568-576;Svetina等人,2000,J.Biotechnol.[生物技术杂志]76:245-251;Rasmussen-Wilson等人,1997,Appl.Environ.Microbiol.[应用与环境微生物学]63:3488-3493;Ward等人,1995,Biotechnology[生物技术]13:498-503;和Contreras等人,1991,Biotechnology[生物技术]9:378-381;Eaton等人,1986,Biochemistry[生物化学]25:505-512;Collins-Racie等人,1995,Biotechnology[生物技术]13:982-987;Carter等人,1989,Proteins:Structure,Function,and Genetics[蛋白质:结构、功能以及遗传学]6:240-248;以及Stevens,2003,Drug Discovery World[药物发现世界]4:35-48。
葡糖淀粉酶:术语“葡糖淀粉酶”意指具有葡糖淀粉酶活性(EC编号3.2.1.3)的蛋白质,其催化末端(1->4)-连接的α-D-葡萄糖残基依次从链的非还原端水解并释放β-D-葡萄糖。出于本发明的目的,根据实例中所描述的程序确定葡糖淀粉酶活性。在一方面,本发明的多肽具有至少20%,例如至少至少40%、至少50%、至少60%、至少70%、至少80%、至少90%、至少95%或至少100%的SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51的成熟多肽的葡糖淀粉酶活性。术语“葡糖淀粉酶”可与术语“淀粉葡糖苷酶”、“葡聚糖1,4-α-葡糖苷酶”和/或“γ-淀粉酶”互换。
糖蛋白:术语“糖蛋白”意指其中非蛋白基团是碳水化合物的缀合蛋白。糖蛋白含有共价附接至多肽侧链的寡糖链/聚糖。碳水化合物在共翻译修饰和/或翻译后修饰期间附接至蛋白质。糖蛋白可以含有N-连接和/或O-连接的寡糖残基。糖蛋白的非限制性实例是α-葡糖苷酶,如SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49和SEQ ID NO:51的葡糖淀粉酶。
异源的:对于宿主细胞,术语“异源的”意指多肽或核酸不是宿主细胞中天然存在的。对于多肽或核酸,术语“异源的”意指控制序列(例如多肽或核酸的启动子或结构域)不与该多肽或核酸天然地相关联,即,控制序列是来自编码SEQ ID NO:15、SEQ ID NO:16、SEQID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51的成熟多肽的基因以外的基因。对于前导肽,术语“异源的”意指目的蛋白和/或信号肽不与该前导肽天然地相关联,即,前导肽是来自编码SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51的成熟多肽的基因以外的基因,和/或前导肽是来自编码SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52的信号肽的基因以外的基因。
宿主细胞:术语“宿主细胞”意指其中引入了包含本发明多核苷酸的核酸构建体或表达载体的任何微生物、真菌或植物细胞。引入方法包括但不限于原生质体融合、转染、转化、电穿孔、接合和转导。在一些实施例中,宿主细胞是分离的重组宿主细胞,其与至少一种其他组分(包括但不限于蛋白质、核酸、细胞等)部分或完全分离。
杂合多肽:术语“杂合多肽”意指包含来自两个或更多个多肽的结构域的多肽,例如,延长的信号肽模块(合成的或来自一个多肽)和来自另一个多肽的催化结构域。结构域可以在N-末端或C-末端融合。
杂交:术语“杂交”意指使用标准DNA印迹程序将核酸的基本上互补的链配对。杂交可以在中、中-高、高或非常高严格条件下进行。中严格条件意指在42℃下在5X SSPE、0.3%SDS、200微克/ml剪切并变性的鲑鱼精子DNA和35%甲酰胺中进行12至24小时的预杂交和杂交,随后在55℃下使用0.2X SSC、0.2% SDS洗涤三次,每次15分钟。中-高严格条件意指在42℃下在5X SSPE、0.3% SDS、200微克/ml剪切并变性的鲑鱼精子DNA和35%甲酰胺中进行12至24小时的预杂交和杂交,随后在60℃下使用0.2XSSC、0.2% SDS洗涤三次,每次15分钟。高严格条件意指在42℃下在5XSSPE、0.3% SDS、200微克/ml剪切并变性的鲑鱼精子DNA和50%甲酰胺中进行12至24小时的预杂交和杂交,随后在65℃下使用0.2X SSC、0.2%SDS洗涤三次,每次15分钟。非常高严格条件意指在42℃下在5X SSPE、0.3% SDS、200微克/ml剪切并变性的鲑鱼精子DNA和50%甲酰胺中进行12至24小时的预杂交和杂交,随后在70℃下使用0.2X SSC、0.2% SDS洗涤三次,每次15分钟。
分离的:术语“分离的”意指多肽、核酸、细胞或其他指定材料或组分与在自然界中发现的与其天然相关联的至少一种其他材料或组分(包括但不限于,例如,其他蛋白质、核酸、细胞等)分离。分离的多肽包括但不限于含有分泌的多肽的培养液。
前导肽:前体多肽典型地由N-末端前导肽和C-末端核心肽组成。前体肽是核糖体合成的,并经翻译后修饰成它们的活性结构。前导肽最常见的作用是分泌信号。成功的蛋白质分泌需要蛋白质有效易位穿过内质网-质膜或细胞膜。指定用于分泌的蛋白质经由它们各自的分泌信号靶向膜,这些分泌信号通常位于新生多肽的N-末端。经常被假设的第二个作用是翻译后修饰酶的识别基序。前导肽由可以在转录或翻译水平上调节基因表达的前导序列编码,如和Dal Degan(Leader sequences are not signal peptides[前导序列不是信号肽],Nature Biotechnology[自然生物技术]22,1502(2004))所述。在本发明的上下文中,前导肽从目的多肽上切除,留下目的成熟多肽。在一方面,编码前导肽的第二多核苷酸在第一多核苷酸的上游以翻译融合方式可操作地连接至编码目的多肽的第一多核苷酸,所述前导肽与SEQ ID NO:2(FARAPVAAR)具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性。在优选的实施例中,前导肽包含SEQ ID NO:2,基本上由其组成,或由其组成。
成熟多肽:术语“成熟多肽”意指在N-末端加工(例如,信号肽和/或前导肽的去除)后处于其成熟形式的多肽。在一方面,成熟多肽是SEQ ID NO:15、SEQ ID NO:16、SEQ IDNO:17和SEQ ID NO:18中的一个。
成熟多肽编码序列:术语“成熟多肽编码序列”意指编码具有生物活性的成熟多肽的多核苷酸。在一方面,成熟多肽编码序列是SEQ ID NO:9的核苷酸91至1878。
天然的:术语“天然的”意指天然存在于宿主细胞中的核酸或多肽。
核酸构建体:术语“核酸构建体”意指单链或双链的核酸分子,该核酸分子是从天然存在的基因中分离的,或以原本不存在于自然界中的方式被修饰成含有核酸的区段,或者是合成的,该核酸分子包含一个或多个控制序列。
可操作地连接:术语“可操作地连接”意指如下构型,在该构型中,控制序列被放置在相对于多核苷酸的编码序列适当的位置处,使得该控制序列指导该编码序列的表达。
纯化的:术语“纯化的”意指基本上不含其他组分的核酸或多肽,如通过本领域熟知的分析技术(例如,在电泳凝胶、色谱洗脱液和/或经过密度梯度离心的培养基中,纯化的多肽或核酸可形成离散的条带)所确定。纯化的核酸或多肽是至少约50%纯的,通常是至少约60%、约65%、约70%、约75%、约80%、约85%、约90%、约91%、约92%、约93%、约94%、约95%、约96%、约97%、约98%、约99%、约99.5%、约99.6%、约99.7%、约99.8%或更加纯的(例如,以摩尔计的重量百分比)。在相关意义上,当在应用纯化或富集技术之后存在分子的浓度的大幅度增加时,组合物富集了分子。术语“富集的”是指化合物、多肽、细胞、核酸、氨基酸或其他指定材料或组分以高于起始组合物的相对或绝对浓度存在于组合物中。
重组:当用于提及细胞、核酸、蛋白质或载体时,术语“重组”意指已经从其天然状态经修饰。因此,例如,重组细胞表达在天然(非重组)形式的细胞内未发现的基因,或与在自然界中发现的相比,以不同水平表达或在不同条件下表达天然基因。重组核酸与天然序列的差异在于一个或多个核苷酸和/或与异源序列(例如,表达载体中的异源启动子)可操作地连接。重组蛋白与天然序列的差异可以在于一个或多个氨基酸和/或与异源序列融合。包含编码多肽的核酸的载体是重组载体。术语“重组”与“遗传修饰的”和“转基因的”同义。
序列同一性:两个氨基酸序列之间或两个核苷酸序列之间的关联度通过参数“序列同一性”来描述。
出于本发明的目的,使用尼德曼-翁施算法(Needleman-Wunsch algorithm)(Needleman和Wunsch,1970,J.Mol.Biol.[分子生物学杂志]48:443-453)来确定两个氨基酸序列之间的序列同一性作为“最长同一性”的输出,该算法如EMBOSS软件包(EMBOSS:TheEuropean Molecular Biology Open Software Suite[欧洲分子生物学开放软件套件],Rice等人,2000,Trends Genet.[遗传学趋势]16:276-277)(优选6.6.0版本或更新版本)的尼德尔程序所实施的。使用的参数是空位开放罚分10、空位延伸罚分0.5以及EBLOSUM62(BLOSUM62的EMBOSS版本)取代矩阵。为了使尼德尔程序报告最长同一性,必须在命令行中指定非简化选项。尼德尔标记的“最长同一性”的输出计算如下:
(相同的残基x100)/(比对长度-比对中的空位总数)
出于本发明的目的,使用尼德曼-翁施算法(Needleman和Wunsch,1970,同上)来确定两个多核苷酸序列之间的序列同一性作为“最长同一性”的输出,该算法如EMBOSS软件包(EMBOSS:The European Molecular Biology Open Software Suite[欧洲分子生物学开放软件套件],Rice等人,2000,同上)(优选6.6.0版本或更新版本)的尼德尔程序所实施的。使用的参数是空位开放罚分10、空位延伸罚分0.5以及EDNAFULL(NCBI NUC4.4的EMBOSS版本)取代矩阵。为了使尼德尔程序报告最长同一性,必须在命令行中指定非简化选项。尼德尔标记的“最长同一性”的输出计算如下:
(相同的脱氧核糖核苷酸x 100)/(比对长度–比对中的空位总数)
信号肽:前体肽典型地由N-末端前导肽和C-末端核心肽组成。控制亚细胞定位的信号肽可以附接至前导肽的N-末端。在真核生物中,新生前体蛋白(前蛋白)的信号肽将核糖体引导至粗面内质网(ER)膜,并启动生长中的肽链(growing peptide chain)穿过其的转运。在本发明的一个实施例中,信号肽由第三多核苷酸编码,该第三多核苷酸在第二多核苷酸的上游以翻译融合方式可操作地连接至编码前导肽的第二多核苷酸;并且该信号肽与SEQ ID NO:4(MRLTLLSGVAGVLCAGQLTAA)、SEQ ID NO:41(MRLSTSSLFLSVSLLGKLALG)或SEQID NO:52(MGVSAVLLPLYLLSGVTFGLA)具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性。在优选的实施例中,信号肽包含SEQ IDNO:4、SEQ ID NO:41或SEQ ID NO:52,基本上由其组成,或由其组成。
根据术语,信号肽可包括前导肽,并由此被描述为延长的信号肽。因此,在一个实施例中,延长的信号肽由第三多核苷酸编码,该第三多核苷酸在第一多核苷酸的上游以翻译融合方式可操作地连接至编码目的多肽的第一多核苷酸;并且该延长的信号肽与SEQ IDNO:6(MRLTLLSGVAGVLCAGQLTAAFARAPVAAR)、SEQ ID NO:43(MRLSTSSLFLSVSLLGKLALGFARAPVAAR)或SEQ ID NO:45(MGVSAVLLPLYLLSGVTFGLAFARAPVAAR)具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性。
翻译融合:第一和第二多核苷酸以翻译融合方式可操作地连接。在本发明的上下文中,术语“以翻译融合方式可操作地连接”意指由第二多核苷酸编码的前导肽和由第一多核苷酸编码的目的多肽被框内编码(encoded in frame)并一起翻译为单个多肽。在翻译后,去除前导肽以提供目的成熟多肽。另外地或可替代地,编码信号肽的第三多核苷酸在第二多核苷酸的上游以翻译融合方式可操作地连接至该第二多核苷酸,所述第二多核苷酸以翻译融合方式可操作地连接至第一多核苷酸。在翻译后,去除信号肽和前导肽以提供目的成熟多肽。优选地,目的成熟多肽被分泌。
变体:术语“变体”意指具有葡糖淀粉酶活性的、在一个或多个(例如,几个)位置处包含人为突变(即取代、插入和/或缺失(例如截短))以改善表达和/或热稳定性的多肽。取代意指用不同的氨基酸替代占据某一位置的氨基酸;缺失意指去除占据某一位置的氨基酸;而插入意指在邻接并且紧随占据某一位置的氨基酸之后添加氨基酸。另外地或可替代地,术语“变体”意指具有生物活性的,包含前导肽、信号肽和延长的信号肽中的一种或多种的多肽。
野生型:提及氨基酸序列或核酸序列时术语“野生型”意指该氨基酸序列或核酸序列是天然或天然存在的序列。如本文所用,术语“天然存在的”是指在自然界中发现的任何物质(例如蛋白质、氨基酸或核酸序列)。相反,术语“非天然存在的”是指在自然界中未发现的任何物质(例如,在实验室中产生的重组核酸和蛋白质序列、或野生型序列的修饰)。
具体实施方式
宿主细胞
本发明还涉及重组宿主细胞,这些重组宿主细胞包含可操作地连接至一个或多个控制序列的本发明的多核苷酸,该一个或多个控制序列指导目的多肽的产生。将包含多核苷酸的构建体或载体引入宿主细胞中,这样使得该构建体或载体作为染色体整合体或作为自主复制的染色体外载体维持,如较早前所述。宿主细胞的选择将在很大程度上取决于编码多肽的基因及其来源。
在一些实施例中,多肽与重组宿主细胞是异源的。
在一些实施例中,一个或多个控制序列中的至少一个与编码目的多肽、信号肽和/或前导肽的多核苷酸是异源的。
在一些实施例中,重组宿主细胞包含本发明的多核苷酸的至少两个拷贝,例如三个、四个或五个拷贝。
宿主细胞可以是可用于重组产生目的多肽的任何微生物细胞,例如真菌宿主细胞。
宿主细胞可以是真菌细胞。如本文所用的“真菌”包括子囊菌门(Ascomycota)、担子菌门(Basidiomycota)、壶菌门(Chytridiomycota)和接合菌门(Zygomycota)以及卵菌门(Oomycota)和所有有丝分裂孢子真菌(如由Hawksworth等人在以下文献中所定义:Ainsworth and Bisby’s Dictionary of The Fungi[安斯沃思和拜斯比真菌字典],第8版,1995,CAB International[国际应用生物科学中心],University Press[大学出版社],Cambridge,UK[英国剑桥])。
真菌宿主细胞可以是酵母细胞。如本文所用的“酵母”包括产子囊孢子酵母(ascosporogenous yeast)(内孢霉目(Endomycetales))、产担子孢子酵母(basidiosporogenous yeast)和属于半知菌类(Fungi Imperfecti)(芽孢纲(Blastomycetes))的酵母。由于酵母的分类可能在将来变化,出于本发明的目的,酵母应当如Biology and Activities of Yeast[酵母的生物学与活性](Skinner,Passmore和Davenport编辑,Soc.App.Bacteriol.Symposium Series No.9[应用细菌学学会专题论文集系列9],1980)中所述的那样定义。
该酵母宿主细胞可以是假丝酵母属(Candida)、汉逊酵母属(Hansenula)、克鲁维酵母属(Kluyveromyces)、毕赤酵母属(Pichia)、酵母属(Saccharomyces)、裂殖酵母属(Schizosaccharomyces)或耶氏酵母属(Yarrowia)细胞,如乳酸克鲁维酵母(Kluyveromyces lactis)、卡尔酵母(Saccharomyces carlsbergensis)、酿酒酵母(Saccharomyces cerevisiae)、糖化酵母(Saccharomyces diastaticus)、道格拉氏酵母(Saccharomyces douglasii)、克鲁弗酵母(Saccharomyces kluyveri)、诺地酵母(Saccharomyces norbensis)、卵形酵母(Saccharomyces oviformis)或解脂耶氏酵母(Yarrowia lipolytica)细胞。
真菌宿主细胞可以是丝状真菌细胞。“丝状真菌”包括真菌门(Eumycota)和卵菌门细分的所有丝状形式(如由Hawksworth等人,1995,同上所定义)。丝状真菌一般的特征在于由几丁质、纤维素、葡聚糖、壳聚糖、甘露聚糖、和其他复杂多糖构成的菌丝体壁。营养生长是通过菌丝延伸来进行的,而碳分解代谢是专性需氧的。相反,酵母(如酿酒酵母)的营养生长是通过单细胞菌体的出芽(budding)来进行的,而碳分解代谢可以是发酵性的。
丝状真菌宿主细胞可以是枝顶孢霉属(Acremonium)、曲霉属(Aspergillus)、短梗霉属(Aureobasidium)、烟管菌属(Bjerkandera)、拟蜡菌属(Ceriporiopsis)、金孢子菌属(Chrysosporium)、鬼伞属(Coprinus)、革盖菌属(Coriolus)、隐球菌属(Cryptococcus)、线黑粉酵母属(Filibasidium)、镰孢属(Fusarium)、腐质霉属(Humicola)、梨孢菌属(Magnaporthe)、毛霉属(Mucor)、毁丝霉属(Myceliophthora)、新美鞭菌属(Neocallimastix)、脉孢菌属(Neurospora)、拟青霉属(Paecilomyces)、青霉属(Penicillium)、平革菌属(Phanerochaete)、射脉菌属(Phlebia)、瘤胃壶菌属(Piromyces)、侧耳属(Pleurotus)、裂褶菌属(Schizophyllum)、篮状菌属(Talaromyces)、嗜热子囊菌属(Thermoascus)、梭孢壳属(Thielavia)、弯颈霉属(Tolypocladium)、栓菌属(Trametes)、或木霉属(Trichoderma)细胞。
例如,丝状真菌宿主细胞可以是泡盛曲霉(Aspergillus awamori)、臭曲霉(Aspergillus foetidus)、烟曲霉(Aspergillus fumigatus)、日本曲霉(Aspergillusjaponicus)、构巢曲霉(Aspergillus nidulans)、黑曲霉(Aspergillus niger)、米曲霉(Aspergillus oryzae)、黑刺烟管菌(Bjerkandera adusta)、干拟蜡菌(Ceriporiopsisaneirina)、卡内基拟蜡菌(Ceriporiopsis caregiea)、浅黄拟蜡菌(Ceriporiopsisgilvescens)、潘诺希塔拟蜡菌(Ceriporiopsis pannocinta)、环带拟蜡菌(Ceriporiopsisrivulosa)、微红拟蜡菌(Ceriporiopsis subrufa)、虫拟蜡菌(Ceriporiopsissubvermispora)、狭边金孢子菌(Chrysosporium inops)、嗜角质金孢子菌(Chrysosporiumkeratinophilum)、卢克诺文思金孢子菌(Chrysosporium lucknowense)、粪状金孢子菌(Chrysosporiummerdarium)、毡金孢子菌(Chrysosporium pannicola)、昆士兰金孢子菌(Chrysosporium queenslandicum)、热带金孢子菌(Chrysosporium tropicum)、带纹金孢子菌(Chrysosporium zonatum)、灰盖鬼伞(Coprinus cinereus)、毛革盖菌(Coriolushirsutus)、杆孢状镰孢(Fusarium bactridioides)、谷类镰孢(Fusarium cerealis)、库威镰孢(Fusarium crookwellense)、大刀镰孢(Fusarium culmorum)、禾谷镰孢(Fusariumgraminearum)、禾赤镰孢(Fusarium graminum)、异孢镰孢(Fusarium heterosporum)、合欢木镰孢(Fusarium negundi)、尖孢镰孢(Fusarium oxysporum)、多枝镰孢(Fusariumreticulatum)、粉红镰孢(Fusarium roseum)、接骨木镰孢(Fusarium sambucinum)、肤色镰孢(Fusarium sarcochroum)、拟分枝孢镰孢(Fusarium sporotrichioides)、硫色镰孢(Fusarium sulphureum)、圆镰孢(Fusarium torulosum)、拟丝孢镰孢(Fusariumtorulosum)、镶片镰孢(Fusarium venenatum)、特异腐质霉(Humicola insolens)、柔毛腐质霉(Humicola lanuginosa)、米黑毛霉(Mucor miehei)、嗜热毁丝霉(Myceliophthorathermophila)、粗糙脉孢菌(Neurospora crassa)、产紫青霉(Penicilliumpurpurogenum)、黄孢原毛平革菌(Phanerochaete chrysosporium)、射脉菌(Phlebiaradiata)、刺芹侧耳(Pleurotus eryngii)、埃默森篮状菌(Talaromyces emersonii)、土生梭孢壳(Thielavia terrestris)、长域毛栓菌(Trametes villosa)、变色栓菌(Trametesversicolor)、哈茨木霉(Trichoderma harzianum)、康宁木霉(Trichoderma koningii)、长枝木霉(Trichoderma longibrachiatum)、里氏木霉(Trichoderma reesei)或绿色木霉(Trichoderma viride)细胞。
可以将真菌细胞通过涉及原生质体形成、原生质体转化、以及细胞壁再生的工艺以本身已知的方式转化。用于转化曲霉属和木霉属宿主细胞的合适程序在EP 238023、Yelton等人,1984,Proc.Natl.Acad.Sci.USA[美国科学院院刊]81:1470-1474和Christensen等人,1988,Bio/Technology[生物/技术]6:1419-1422中描述。用于转化镰孢属物种的合适方法由Malardier等人,1989,Gene[基因]78:147-156和WO 96/00787描述。可以使用由以下文献描述的程序转化酵母:Becker和Guarente,在Abelson,J.N.和Simon,M.I.编辑,Guide to Yeast Genetics and Molecular Biology[酵母遗传学与分子生物学指南],Methods in Enzymology[酶学方法],第194卷,第182-187页,Academic Press,Inc.[学术出版社有限公司],纽约;Ito等人,1983,J.Bacteriol.[细菌学杂志]153:163;以及Hinnen等人,1978,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]75:1920。
在第一方面,本发明涉及一种真菌宿主细胞,该真菌宿主细胞在其基因组中包含:
编码目的多肽的第一多核苷酸;和
在该第一多核苷酸的上游以翻译融合方式可操作地连接至该第一多核苷酸的第二多核苷酸,所述第二多核苷酸编码与SEQ ID NO:2(FARAPVAAR)具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性的前导肽。如在整个实例中所呈现的,具有可操作地连接至目的多肽的所述前导肽的宿主细胞令人惊讶地显示出增加的表达、产物产率和/或产物活性。
在第一方面的实施例中,前导肽包含SEQ ID NO:2,基本上由其组成,或由其组成。
在一个实施例中,前导肽是合成的。
在优选的实施例中,前导肽与目的多肽是异源的。
在另一个优选的实施例中,前导肽与信号肽是异源的。在另一个优选的实施例中,前导肽与信号肽和目的多肽是异源的。
在另一个实施例中,编码SEQ ID NO:2的前导肽的第二多核苷酸包含一个或多个突变,优选核苷酸取代、核苷酸缺失或核苷酸插入。所述一个或多个突变导致SEQ ID NO:2的前导肽的变体,例如包含以下的变体:(i)与SEQ ID NO:2相比一个或多个另外的氨基酸,(ii)与SEQ ID NO:2相比少至少一个氨基酸,例如总共3至8个氨基酸,(iii)或SEQ ID NO:2的至少一个氨基酸的氨基酸取代,如在对应于SEQ ID NO:2的位置1、2、3、4、5、6、7、8或9的位置处的氨基酸取代。
在另一实施例中,宿主细胞在其基因组中包含编码信号肽的第三多核苷酸,其中该第三多核苷酸在该第二多核苷酸的上游以翻译融合方式可操作地连接至该第二多核苷酸;并且其中该目的多肽被分泌。
在另一个实施例中,第三多核苷酸编码信号肽,该信号肽与SEQ ID NO:4(MRLTLLSGVAGVLCAGQLTAA)、SEQ ID NO:41(MRLSTSSLFLSVSLLGKLALG)或SEQ ID NO:52(MGVSAVLLPLYLLSGVTFGLA)具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性。在优选的实施例中,第三多核苷酸由SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52组成,基本上由它们组成,或包含它们。
在另一个实施例中,编码信号肽的第三多核苷酸包含一个或多个突变,优选核苷酸取代、核苷酸缺失或核苷酸插入。所述一个或多个突变导致SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52的信号肽的变体,例如包含以下的变体:(i)与SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52相比一个或多个另外的氨基酸,(ii)与SEQ ID NO:4、SEQ ID NO:41或SEQID NO:52相比少至少一个氨基酸,例如总共10至20个氨基酸,(iii)或SEQ ID NO:4、SEQ IDNO:41或SEQ ID NO:52的至少一个氨基酸的氨基酸取代,如在对应于SEQ ID NO:4、SEQ IDNO:41或SEQ ID NO:52的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或21的位置处的氨基酸取代。
在另一个实施例中,真菌宿主细胞是酵母宿主细胞,优选地,该酵母宿主细胞选自由以下组成的组:假丝酵母属、汉逊酵母属、克鲁维酵母属、毕赤酵母属(驹形氏酵母属)、酵母属、裂殖酵母属、和耶氏酵母属细胞;更优选地,该酵母宿主细胞选自由以下组成的组:乳酸克鲁维酵母、卡尔酵母、酿酒酵母、糖化酵母、道格拉氏酵母、克鲁弗酵母、诺地酵母、卵形酵母、和解脂耶氏酵母细胞,最优选地毕赤酵母(Pichia pastoris)(法夫驹形氏酵母(Komagataella phaffii))。
在一个实施例中,真菌宿主细胞是丝状真菌宿主细胞;优选地,该丝状真菌宿主细胞选自由以下组成的组:枝顶孢霉属、曲霉属、短梗霉属、烟管菌属、拟蜡菌属、金孢子菌属、鬼伞属、革盖菌属、隐球菌属、线黑粉酵母属、镰孢属、腐质霉属、梨孢菌属、毛霉属、毁丝霉属、新美鞭菌属、脉孢菌属、拟青霉属、青霉属、平革菌属、射脉菌属、瘤胃壶菌属、侧耳属、裂褶菌属、篮状菌属、嗜热子囊菌属、梭孢壳属、弯颈霉属、栓菌属、和木霉属细胞;更优选地,该丝状真菌宿主细胞选自由以下组成的组:泡盛曲霉、臭曲霉、烟曲霉、日本曲霉、构巢曲霉、黑曲霉、米曲霉、黑刺烟管菌、干拟蜡菌、卡内基拟蜡菌、浅黄拟蜡菌、潘诺希塔拟蜡菌、环带拟蜡菌、微红拟蜡菌、虫拟蜡菌、狭边金孢子菌、嗜角质金孢子菌、卢克诺文思金孢子菌、粪状金孢子菌、毡金孢子菌、昆士兰金孢子菌、热带金孢子菌、带纹金孢子菌、灰盖鬼伞、毛革盖菌、杆孢状镰孢、谷类镰孢、库威镰孢、大刀镰孢、禾谷镰孢、禾赤镰孢、异孢镰孢、合欢木镰孢、尖孢镰孢、多枝镰孢、粉红镰孢、接骨木镰孢、肤色镰孢、拟分枝孢镰孢、硫色镰孢、圆镰孢、拟丝孢镰孢、镶片镰孢、特异腐质霉、柔毛腐质霉、米黑毛霉、嗜热毁丝霉、粗糙脉孢菌、产紫青霉、黄孢原毛平革菌、射脉菌、刺芹侧耳、土生梭孢壳、长域毛栓菌、变色栓菌、哈茨木霉、康宁木霉、长枝木霉、里氏木霉、和绿色木霉细胞;甚至更优选地,该丝状宿主细胞选自由米曲霉、镶片镰孢和里氏木霉细胞组成的组;最优选地,该丝状真菌宿主细胞是黑曲霉细胞。在另一个优选的实施例中,丝状真菌宿主细胞是米曲霉细胞。在又一个优选的实施例中,丝状真菌宿主细胞是里氏木霉细胞。
在另一个优选的实施例中,目的多肽包含酶;优选地,该酶选自由以下组成的组:水解酶、异构酶、连接酶、裂解酶、氧化还原酶或转移酶;更优选地是氨肽酶、淀粉酶、糖酶、羧肽酶、过氧化氢酶、纤维二糖水解酶、纤维素酶、几丁质酶、角质酶、环糊精糖基转移酶、脱氧核糖核酸酶、内切葡聚糖酶、酯酶、α-半乳糖苷酶、β-半乳糖苷酶、α-葡糖苷酶、β-葡糖苷酶、转化酶、漆酶、脂肪酶、甘露糖苷酶、变聚糖酶、核酸酶、氧化酶、果胶分解酶、过氧化物酶、磷酸二酯酶、植酸酶、多酚氧化酶、蛋白水解酶、核糖核酸酶、转谷氨酰胺酶、木聚糖酶、和β-木糖苷酶。
在优选的实施例中,目的多肽是糖蛋白,优选地是α-葡糖苷酶;更优选地是1,4-α-葡糖苷酶;最优选地是葡糖淀粉酶,如与SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17或SEQID NO:18具有至少60%序列同一性的葡糖淀粉酶。
在一个实施例中,真菌宿主细胞包含多肽,所述多肽包含以翻译融合方式可操作地连接至目的多肽的前导肽,其中
(i)该前导肽与SEQ ID NO:2(FARAPVAAR)具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性;或
(ii)该前导肽包含SEQ ID NO:2,基本上由其组成,或由其组成。
另外地或可替代地,多肽还包含前导肽上游的信号肽,该信号肽与SEQ ID NO:4(MRLTLLSGVAGVLCAGQLTAA)、SEQ ID NO:41(MRLSTSSLFLSVSLLGKLALG)或SEQ ID NO:52(MGVSAVLLPLYLLSGVTFGLA)具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性。
在一个实施例中,前导肽上游的信号肽包含SEQ ID NO:4、SEQ ID NO:41或SEQ IDNO:52,基本上由其组成,或由其组成。
生产方法
在第二方面,本发明涉及一种用于生产目的多肽的方法,该方法包括:
(i)提供根据第一方面的真菌宿主细胞;
(ii)在有助于该目的多肽表达的条件下培养所述真菌宿主细胞;以及,任选地
(iii)回收该目的多肽。
宿主细胞是在适合于使用本领域已知和以下实例中所述的方法生产多肽的营养培养基中培养的。例如,可以通过摇瓶(SF)培养、或在实验室或工业发酵罐中小规模或大规模发酵(包括连续、分批、补料分批、或固态发酵)培养细胞,该培养在合适的培养基中并且在允许表达和/或分离多肽的条件下进行。使用本领域中已知的程序,培养发生在包含碳和氮来源及无机盐的合适的营养培养基中。合适的培养基可从商业供应商获得或可以根据公开的组成(例如,在美国典型培养物保藏中心的目录中)制备。如果多肽被分泌到该营养培养基中,那么可以直接从该培养基中回收该多肽。如果多肽不被分泌,则可以从细胞裂解物中将其回收。如整个实例中所示,本发明人令人惊讶地发现,可以通过在生产过程中使用不同的培养基实现目的多肽的表达、活性和/或产率的增加。
可以使用本领域已知的对于多肽是特异性的方法来检测多肽。这些检测方法包括但不限于:特异性抗体的使用、酶产物的形成或酶底物的消失。例如,可以使用酶测定来确定多肽的活性。
可以使用本领域已知的方法来回收多肽。例如,可通过常规程序,包括但不限于收集、离心、过滤、提取、喷雾干燥、蒸发或沉淀,从发酵培养基回收多肽。在一方面,回收包含多肽的全发酵液。
可以通过本领域已知的多种程序纯化多肽,包括但不限于色谱法(例如,离子交换色谱法、亲和色谱法、疏水色谱法、聚焦色谱法、和尺寸排阻色谱法)、电泳程序(例如,制备型等电聚焦电泳)、差异性溶解(例如,硫酸铵沉淀)、SDS-PAGE、或提取(参见例如,ProteinPurification[蛋白质纯化],Janson和Ryden编辑,VCH Publishers[VCH出版公司],纽约,1989),以便获得基本上纯的多肽。
具有葡糖淀粉酶活性的多肽
在一些实施例中,本发明涉及分离的或纯化的多肽,这些分离的或纯化的多肽与SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17或SEQ ID NO:18的成熟多肽具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少81%、至少82%、至少83%、至少84%、至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%序列同一性,这些分离的或纯化的多肽具有葡糖淀粉酶活性。在一方面,这些多肽与SEQ ID NO:15、SEQID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51的成熟多肽相差多达10个(例如,1、2、3、4、5、6、7、8、9、或10个)氨基酸。
该多肽优选地包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51或其成熟多肽的氨基酸序列,基本上由其组成,或由其组成;或是具有葡糖淀粉酶活性的其片段。在一方面,成熟多肽是SEQ ID NO:15。在另一方面,成熟多肽是SEQ ID NO:16。在另一方面,成熟多肽是SEQ ID NO:17。在又一方面,成熟多肽是SEQ ID NO:18。
在一些实施例中,本发明涉及由如下多核苷酸编码的、具有葡糖淀粉酶活性的分离的或纯化的多肽,这些多核苷酸在中严格条件、中-高严格条件、高严格条件、或非常高严格条件下与SEQ ID NO:7、9、11、13的成熟多肽编码序列的全长互补序列或其cDNA杂交(Sambrook等人,1989,Molecular Cloning,A Laboratory Manual[分子克隆:实验室手册],第2版,Cold Spring Harbor[冷泉港],纽约)。
可以将SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:46、SEQ ID NO:48或SEQ ID NO:50的多核苷酸或其子序列,以及SEQ ID NO:8、10、12、14、15、16、17、18的成熟多肽或其片段用于设计核酸探针以根据本领域熟知的方法来鉴定并克隆DNA,该DNA编码来自不同属或物种的菌株的具有葡糖淀粉酶活性的多肽。这样的探针可以用于遵循标准DNA印迹程序与目的细胞的基因组DNA或cDNA杂交,以便鉴定和分离其中相应的基因。这样的探针可明显短于完整序列,但是长度应为至少15个,例如至少25个、至少35个或至少70个核苷酸。优选地,核酸探针长度为至少100个核苷酸,例如,长度为至少200个核苷酸、至少300个核苷酸、至少400个核苷酸、至少500个核苷酸、至少600个核苷酸、至少700个核苷酸、至少800个核苷酸、或至少900个核苷酸。DNA和RNA探针两者都可使用。典型地将探针进行标记(例如,用32P、3H、35S、生物素、或抗生物素蛋白),用于检测相应基因。这样的探针涵盖于本发明中。
可以针对与上文所述的探针杂交并且编码具有葡糖淀粉酶活性的多肽的DNA来筛选由这样的其他菌株制备的基因组DNA或cDNA文库。来自这样的其他菌株的基因组DNA或其他DNA可通过琼脂糖或聚丙烯酰胺凝胶电泳或其他分离技术来分离。可将来自文库的DNA或分离的DNA转移到并固定在硝化纤维素或另一个合适的运载体材料上。为了鉴定与SEQ IDNO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:46、SEQ ID NO:48或SEQ IDNO:50或其子序列杂交的克隆或DNA,将运载体材料用于DNA印迹中。
出于本发明的目的,杂交表示多核苷酸在中至非常高严格条件下与对应于以下的标记的核酸探针杂交:(i)SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQID NO:46、SEQ ID NO:48或SEQ ID NO:50;(ii)SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:46、SEQ ID NO:48或SEQ ID NO:50的成熟多肽编码序列;(iii)其全长互补序列;或(iv)其子序列。可以使用例如X-射线胶片或本领域已知的任何其他检测手段来检测在这些条件下与核酸探针杂交的分子。
在一些实施例中,本发明涉及由如下多核苷酸编码的、具有葡糖淀粉酶活性的分离的多肽,这些多核苷酸与SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQID NO:46、SEQ ID NO:48或SEQ ID NO:50的成熟多肽编码序列具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少81%、至少82%、至少83%、至少84%、至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%序列同一性。
编码该多肽的多核苷酸优选地包含SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:46、SEQ ID NO:48或SEQ ID NO:50的核苷酸91至1878,基本上由其组成,或由其组成。
在一些实施例中,本发明涉及通过在SEQ ID NO:10或16的成熟多肽中取代、缺失或添加一个或几个氨基酸而从SEQ ID NO:10或16的成熟多肽衍生的多肽。在一些实施例中,本发明涉及在一个或多个(例如,几个)位置处包含取代、缺失、和/或插入的SEQ ID NO:10或16的成熟多肽的变体。在一方面,引入SEQ ID NO:10或16的成熟多肽中的氨基酸取代、缺失和/或插入的数目多达10个,例如1、2、3、4、5、6、7、8、9、或10个。在实施例中,多肽具有1-10个氨基酸,例如1、2、3、4、5、6、7、8、9或10个氨基酸的N-末端延伸和/或C-末端延伸。氨基酸改变可以具有微小性质,即,不会显著地影响蛋白质的折叠和/或活性的保守氨基酸取代或插入;典型地为1-30个氨基酸的小缺失;小的氨基末端或羧基末端延伸,如氨基末端的甲硫氨酸残基;多达20-25个残基的小接头肽;或小的延伸,其通过改变净电荷或另一功能(如聚组氨酸段、抗原表位或结合模块)来促进纯化。
在一些实施例中,本发明涉及通过在SEQ ID NO:12或17的成熟多肽中取代、缺失或添加一个或几个氨基酸而从SEQ ID NO:12或17的成熟多肽衍生的多肽。在一些实施例中,本发明涉及在一个或多个(例如,几个)位置处包含取代、缺失、和/或插入的SEQ ID NO:12或17的成熟多肽的变体。在一方面,引入SEQ ID NO:12或17的成熟多肽中的氨基酸取代、缺失和/或插入的数目多达10个,例如1、2、3、4、5、6、7、8、9、或10个。在实施例中,多肽具有1-10个氨基酸,例如1、2、3、4、5、6、7、8、9或10个氨基酸的N-末端延伸和/或C-末端延伸。氨基酸改变可以具有微小性质,即,不会显著地影响蛋白质的折叠和/或活性的保守氨基酸取代或插入;典型地为1-30个氨基酸的小缺失;小的氨基末端或羧基末端延伸,如氨基末端的甲硫氨酸残基;多达20-25个残基的小接头肽;或小的延伸,其通过改变净电荷或另一功能(如聚组氨酸段、抗原表位或结合模块)来促进纯化。
在一些实施例中,本发明涉及通过在SEQ ID NO:14或18的成熟多肽中取代、缺失或添加一个或几个氨基酸而从SEQ ID NO:14或18的成熟多肽衍生的多肽。在一些实施例中,本发明涉及在一个或多个(例如,几个)位置处包含取代、缺失、和/或插入的SEQ ID NO:14或18的成熟多肽的变体。在一方面,引入SEQ ID NO:14或18的成熟多肽中的氨基酸取代、缺失和/或插入的数目多达10个,例如1、2、3、4、5、6、7、8、9、或10个。在实施例中,多肽具有1-10个氨基酸,例如1、2、3、4、5、6、7、8、9或10个氨基酸的N-末端延伸和/或C-末端延伸。氨基酸改变可以具有微小性质,即,不会显著地影响蛋白质的折叠和/或活性的保守氨基酸取代或插入;典型地为1-30个氨基酸的小缺失;小的氨基末端或羧基末端延伸,如氨基末端的甲硫氨酸残基;多达20-25个残基的小接头肽;或小的延伸,其通过改变净电荷或另一功能(如聚组氨酸段、抗原表位或结合模块)来促进纯化。
在一些实施例中,本发明涉及通过在SEQ ID NO:16的成熟多肽中取代一个或几个氨基酸而从SEQ ID NO:16的成熟多肽衍生的多肽。在一些实施例中,本发明涉及在一个或多个(例如,几个)位置处包含取代、缺失、和/或插入的SEQ ID NO:16的成熟多肽的变体。引入SEQ ID NO:16的成熟多肽中的氨基酸取代、缺失和/或插入的数目多达20个,例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个。在一些实施例中,取代选自对应于SEQ ID NO:16的位置6、7、31、34、103、132、445、447、481、566、568、594或595的位置处的取代。在一些实施例中,取代选自对应于SEQ ID NO:16的位置6、7、31、34、103、132、445、447、481、566、568、594或595的位置处的取代,其中这些取代是G6S、G7T、R31F、K34Y、S103N、A132P、D445N、V447S、S481P、D566T、T568V、Q594R或F595S中的一个或多个。在一个实施例中,SEQ ID NO:16的变体多肽是包含SEQ ID NO:17,基本上由其组成,或由其组成的多肽。
在一些实施例中,取代选自对应于SEQ ID NO:16的位置6、7、31、34、50、103、132、445、447、481、484、501、539、566、568、594或595的位置处的取代。在一些实施例中,取代选自对应于SEQ ID NO:16的6、7、31、34、50、103、132、445、447、481、484、501、539、566、568、594或595的位置处的取代,其中这些取代是G6S、G7T、R31F、K34Y、E50R、S103N、A132P、D445N、V447S、S481P、T484P、E501A、N539P、D566T、T568V、Q594R或F595中的一个或多个。在一个实施例中,SEQ ID NO:16的变体多肽是包含SEQ ID NO:18,基本上由其组成,或由其组成的多肽。
可以根据本领域中已知的程序,如定点诱变或丙氨酸扫描诱变(Cunningham和Wells,1989,Science[科学]244:1081-1085)来鉴定多肽中的必需氨基酸。在后一项技术中,在该分子中的每个残基处引入单个丙氨酸突变,并且测试所得分子的葡糖淀粉酶活性以鉴定对于该分子的活性关键的氨基酸残基。还参见,Hilton等人,1996,J.Biol.Chem.[生物化学杂志]271:4699-4708。酶或其他生物学相互作用的活性位点还可以通过对结构的物理分析来确定,如由诸如以下技术确定:核磁共振、晶体学(crystallography)、电子衍射、或光亲和标记,连同对推定的接触位点氨基酸进行突变。参见例如,de Vos等人,1992,Science[科学]255:306-312;Smith等人,1992,J.Mol.Biol.[分子生物学杂志]224:899-904;Wlodaver等人,1992,FEBS Lett.[欧洲生化学会联合会快报]309:59-64。还可以从与相关多肽的比对来推断必需氨基酸的身份。关于热稳定性和/或酶活性,SEQ ID NO:16的氨基酸1至595序列中的必需氨基酸位于位置6、7、31、34、50、103、132、445、447、481、484、501、539、566、568、594或595处。
可以使用已知的诱变、重组和/或混编方法,随后进行相关的筛选程序做出单氨基酸或多氨基酸取代、缺失和/或插入并对其进行测试,该相关的筛选程序例如由Reidhaar-Olson和Sauer,1988,Science[科学]241:53-57;Bowie和Sauer,1989,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]86:2152-2156;WO 95/17413;或WO 95/22625披露的那些。其他可以使用的方法包括易错PCR、噬菌体展示(例如Lowman等人,1991,Biochemistry[生物化学]30:10832-10837;美国专利号5,223,409;WO 92/06204)以及区域定向诱变(Derbyshire等人,1986,Gene[基因]46:145;Ner等人,1988,DNA 7:127)。
诱变/混编方法可以与高通量、自动化的筛选方法组合以检测由宿主细胞表达的克隆的、诱变的多肽的活性(Ness等人,1999,Nature Biotechnology[自然生物技术]17:893-896)。可从宿主细胞回收编码活性多肽的诱变的DNA分子,并使用本领域的标准方法快速测序。这些方法允许快速确定多肽中单个氨基酸残基的重要性。
在一些实施例中,多肽是含有SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51的成熟多肽的至少100个氨基酸残基,SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ IDNO:49或SEQ ID NO:51的成熟多肽的至少300个氨基酸残基,或SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51的成熟多肽的至少400个氨基酸残基的片段。
该多肽可以是杂合多肽或融合多肽。
本发明的多肽具有改进的热稳定性和改进的在真菌宿主细胞中的表达。
多核苷酸
本发明还涉及编码本发明的目的多肽、信号肽、延长的信号肽或前导肽的分离的多核苷酸,如本文所述。
用于分离或克隆多核苷酸的技术是本领域已知的且包括从基因组DNA或cDNA或其组合进行分离。来自基因组DNA的多核苷酸的克隆可以例如通过使用聚合酶链式反应(PCR)或用以对具有共有的结构特征的克隆的DNA片段进行检测的表达文库抗体筛选来实现。参见例如,Innis等人,1990,PCR:A Guide to Methods and Application[PCR:方法和应用指南],Academic Press[学术出版社],纽约。可以使用其他核酸扩增程序如连接酶链式反应(LCR)、连接激活的转录(LAT)和基于多核苷酸的扩增(NASBA)。这些多核苷酸可以克隆自黑曲霉、草酸青霉(Penicillum oxalicum)、埃默森罗萨氏菌(Rasamsonia emersonii)的菌株或相关生物,并且因此,例如可以是该多核苷酸的多肽编码区的物种变体。
编码本发明的多肽的多核苷酸的修饰对于合成基本上类似于该多肽的多肽可以是必需的。术语“基本上类似”于该多肽是指多肽的非天然存在的形式。这些多肽可以因某种工程化方式而与从其天然来源分离的多肽不同,例如在比活性、热稳定性、最适pH等方面不同的变体。这些变体可以基于以SEQ ID NO:1、3、5、9的成熟多肽编码序列(例如,其子序列)形式呈现的多核苷酸,和/或通过引入不会改变多肽的氨基酸序列,但对应于旨在用于产生酶的宿主生物的密码子使用的核苷酸取代,或通过引入可能产生不同氨基酸序列的核苷酸取代来构建。对于核苷酸取代的一般描述,参见例如Ford等人,1991,ProteinExpression and Purification[蛋白质表达与纯化]2:95-107。
核酸构建体
本发明还涉及包含本发明的多核苷酸的核酸构建体,其中该多核苷酸可操作地连接至一个或多个控制序列,在与控制序列相容的条件下,该一个或多个控制序列指导该编码序列在合适的宿主细胞中的表达。
在第三方面,本发明涉及一种核酸构建体,该核酸构建体包含编码目的多肽的第一多核苷酸和可操作地连接至该第一多核苷酸的第二多核苷酸,该第二多核苷酸编码与SEQ ID NO:2(FARAPVAAR)具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性的前导肽。
在第三方面的一个实施例中,前导肽包含SEQ ID NO:2,基本上由其组成,或由其组成。
在一个实施例中,前导肽是合成的。
在优选的实施例中,前导肽与目的多肽是异源的。
在另一个优选的实施例中,前导肽与信号肽是异源的。在另一个优选的实施例中,前导肽与信号肽和目的多肽是异源的。
在另一个实施例中,编码SEQ ID NO:2的前导肽的第二多核苷酸包含一个或多个突变,优选核苷酸取代、核苷酸缺失或核苷酸插入。所述一个或多个突变导致SEQ ID NO:2的前导肽的变体,例如包含以下的变体:(i)与SEQ ID NO:2相比一个或多个另外的氨基酸,(ii)与SEQ ID NO:2相比少至少一个氨基酸,例如总共4至8个氨基酸,(iii)或SEQ ID NO:2的至少一个氨基酸的氨基酸取代,如在对应于SEQ ID NO:2的位置1、2、3、4、5、6、7、8或9的位置处的氨基酸取代。
在另一实施例中,第二多核苷酸可操作地连接至指导多肽在表达宿主中的产生的一个或多个控制序列。
在另一个实施例中,核酸构建体另外地或可替代地包含编码信号肽的第三多核苷酸,
其中该第三多核苷酸在该第二多核苷酸的上游以翻译融合方式可操作地连接至该第二多核苷酸;并且
该信号肽与SEQ ID NO:4(MRLTLLSGVAGVLCAGQLTAA)、SEQ ID NO:41或SEQ ID NO:52具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性。在优选的实施例中,信号肽由SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52组成,基本上由它们组成,或包含它们。在又一个优选的实施例中,第三多核苷酸可操作地连接至指导多肽在表达宿主中的产生的一个或多个控制序列。
在另一个实施例中,编码信号肽的第三多核苷酸包含一个或多个突变,优选核苷酸取代、核苷酸缺失或核苷酸插入。所述一个或多个突变导致SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52的信号肽的变体,例如包含以下的变体:(i)与SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52相比一个或多个另外的氨基酸,(ii)与SEQ ID NO:4、SEQ ID NO:41或SEQID NO:52相比少至少一个氨基酸,例如总共10至20个氨基酸,(iii)或SEQ ID NO:4、SEQ IDNO:41或SEQ ID NO:52的至少一个氨基酸的氨基酸取代,如在对应于SEQ ID NO:4、SEQ IDNO:41或SEQ ID NO:52的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或21的位置处的氨基酸取代。
可用多种方式操纵多核苷酸以提供多肽的表达。取决于表达载体,在多核苷酸插入载体之前对其进行操纵可能是希望的或必需的。利用重组DNA方法修饰多核苷酸的技术是本领域熟知的。
控制序列可为启动子,即,被宿主细胞识别用于表达编码本发明的多肽的多核苷酸的多核苷酸。启动子含有介导具有前导肽的多肽的表达的转录控制序列。启动子可以是在宿主细胞中显示转录活性的任何多核苷酸,包括突变启动子、截短启动子和杂合启动子,并且可以获得自编码与宿主细胞同源或异源的胞外或胞内多肽的基因。
用于在细菌宿主细胞中指导本发明多核苷酸的转录的合适启动子的实例是从以下基因中获得的启动子:解淀粉芽孢杆菌(Bacillus amyloliquefaciens)α-淀粉酶基因(amyQ)、地衣芽孢杆菌(Bacillus licheniformis)α-淀粉酶基因(amyL)、地衣芽孢杆菌青霉素酶基因(penP)、嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)产麦芽糖淀粉酶基因(amyM)、枯草芽孢杆菌(Bacillus subtilis)果聚糖蔗糖酶基因(sacB)、枯草芽孢杆菌xylA和xylB基因、苏云金芽孢杆菌(Bacillus thuringiensis)cryIIIA基因(Agaisse和Lereclus,1994,Molecular Microbiology[分子微生物学]13:97-107)、大肠杆菌lac操纵子、大肠杆菌trc启动子(Egon等人,1988,Gene[基因]69:301-315)、天蓝链霉菌(Streptomyces coelicolor)琼脂水解酶基因(dagA)和原核β-内酰胺酶基因(Villa-Kamaroff等人,1978,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]75:3727-3731)以及tac启动子(DeBoer等人,1983,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]80:21-25)。另外的启动子描述于Gilbert等人,1980,Scientific American[科学美国人]242:74-94的“Useful proteins from recombinant bacteria[来自重组细菌的有用蛋白质]”;和Sambrook等人,1989,同上中。串联启动子的实例披露于WO 99/43835中。
用于在丝状真菌宿主细胞中指导本发明多核苷酸的转录的合适启动子的实例是从以下基因中获得的启动子:构巢曲霉乙酰胺酶、黑曲霉中性α-淀粉酶、黑曲霉酸稳定性α-淀粉酶、黑曲霉或泡盛曲霉葡糖淀粉酶(glaA)、米曲霉TAKA淀粉酶、米曲霉碱性蛋白酶、米曲霉丙糖磷酸异构酶、尖孢镰孢胰蛋白酶样蛋白酶(WO 96/00787)、镶片镰孢淀粉葡糖苷酶(WO 00/56900)、镶片镰孢Daria(WO 00/56900)、镶片镰孢Quinn(WO 00/56900)、米黑根毛霉(Rhizomucor miehei)脂肪酶、米黑根毛霉天冬氨酸蛋白酶、里氏木霉β-葡糖苷酶、里氏木霉纤维二糖水解酶I、里氏木霉纤维二糖水解酶II、里氏木霉内切葡聚糖酶I、里氏木霉内切葡聚糖酶II、里氏木霉内切葡聚糖酶III、里氏木霉内切葡聚糖酶V、里氏木霉木聚糖酶I、里氏木霉木聚糖酶II、里氏木霉木聚糖酶III、里氏木霉β-木糖苷酶以及里氏木霉翻译延伸因子,连同NA2-tpi启动子(来自曲霉属中性α-淀粉酶基因的经修饰的启动子,其中已经用来自曲霉属丙糖磷酸异构酶基因的未翻译的前导序列替代未翻译的前导序列;非限制性实例包括来自黑曲霉中性α-淀粉酶基因的经修饰的启动子,其中已经用来自构巢曲霉或米曲霉丙糖磷酸异构酶基因的未翻译的前导序列替代未翻译的前导序列);及其突变启动子、截短启动子和杂合启动子。其他启动子在美国专利号6,011,147中描述。
在酵母宿主中,有用的启动子从以下的基因中获得:酿酒酵母烯醇酶(ENO-1)、酿酒酵母半乳糖激酶(GAL1)、酿酒酵母醇脱氢酶/甘油醛-3-磷酸脱氢酶(ADH1、ADH2/GAP)、酿酒酵母丙糖磷酸异构酶(TPI)、酿酒酵母金属硫蛋白(CUP1)、以及酿酒酵母3-磷酸甘油酸激酶。酵母宿主细胞的其他有用的启动子由Romanos等人,1992,Yeast[酵母]8:423-488描述。
控制序列也可为由宿主细胞识别以终止转录的转录终止子。终止子可操作地连接至编码多肽的多核苷酸的3'末端。在宿主细胞中有功能的任何终止子可用于本发明中。
细菌宿主细胞的优选终止子从以下的基因中获得:克劳氏芽孢杆菌(Bacillusclausii)碱性蛋白酶(aprH)、地衣芽孢杆菌α-淀粉酶(amyL)、和大肠杆菌(Escherichiacoli)核糖体RNA(rrnB)。
丝状真菌宿主细胞的优选终止子从以下的基因中获得:构巢曲霉乙酰胺酶、构巢曲霉邻氨基苯甲酸合酶、黑曲霉葡糖淀粉酶、黑曲霉α-葡糖苷酶、米曲霉TAKA淀粉酶、尖孢镰孢胰蛋白酶样蛋白酶、里氏木霉β-葡糖苷酶、里氏木霉纤维二糖水解酶I、里氏木霉纤维二糖水解酶II、里氏木霉内切葡聚糖酶I、里氏木霉内切葡聚糖酶II、里氏木霉内切葡聚糖酶III、里氏木霉内切葡聚糖酶V、里氏木霉木聚糖酶I、里氏木霉木聚糖酶II、里氏木霉木聚糖酶III、里氏木霉β-木糖苷酶和里氏木霉翻译延伸因子。
酵母宿主细胞的优选终止子从以下的基因中获得:酿酒酵母烯醇酶、酿酒酵母细胞色素C(CYC1)以及酿酒酵母甘油醛-3-磷酸脱氢酶。酵母宿主细胞的其他有用的终止子由Romanos等人(1992,同上)描述。
控制序列还可以是启动子下游和基因的编码序列上游的mRNA稳定子区域,其增加该基因的表达。
合适的mRNA稳定子区域的实例是从以下获得的:苏云金芽孢杆菌cryIIIA基因(WO94/25612)和枯草芽孢杆菌SP82基因(Hue等人,1995,J.Bacteriol.[细菌学杂志]177:3465-3471)。
控制序列还可以是多腺苷酸化序列,一种可操作地连接至多核苷酸的3’末端并且当转录时由宿主细胞识别为将多腺苷酸残基添加至所转录的mRNA的信号的序列。可以使用在宿主细胞中有功能的任何多腺苷酸化序列。
丝状真菌宿主细胞的优选多腺苷酸化序列从以下的基因中获得:构巢曲霉邻氨基苯甲酸合酶、黑曲霉葡糖淀粉酶、黑曲霉α-葡糖苷酶、米曲霉TAKA淀粉酶以及尖孢镰孢胰蛋白酶样蛋白酶。
酵母宿主细胞的有用的多腺苷酸化序列由Guo和Sherman,1995,Mol.CellularBiol.[分子细胞生物学]15:5983-5990描述。
控制序列还可以是编码与多肽的N-末端连接的信号肽并指导多肽进入细胞的分泌途径的信号肽编码区。多核苷酸的编码序列的5'端可固有地含有信号肽编码序列,该信号肽编码序列在翻译阅读框中与编码多肽的编码序列的区段天然地连接,如SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52的信号肽,或SEQ ID NO:6、SEQ ID NO 43或SEQ ID NO:45的延长的信号肽。可替代地,编码序列的5’端可以含有对于该编码序列是异源的信号肽编码序列。在编码序列天然地不含有信号肽编码序列的情况下,可能需要异源信号肽编码序列。可替代地,异源信号肽编码序列可以单纯地替代天然信号肽编码序列以便增强多肽的分泌。然而,可以使用指导已表达多肽进入宿主细胞的分泌途径的任何信号肽编码序列。在优选的实施例中,信号肽包含SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52,基本上由其组成,或由其组成。在另一个优选的实施例中,信号肽包含SEQ ID NO:6、SEQ ID NO:43或SEQ IDNO:45,基本上由其组成,或由其组成。可替代地,信号肽与SEQ ID NO:4、SEQ ID NO:6、SEQID NO:41、SEQ ID NO:43、SEQ ID NO:45或SEQ ID NO:52具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性。
细菌宿主细胞的有效信号肽编码序列是从以下的基因中获得的信号肽编码序列:芽孢杆菌属NCIB 11837产麦芽糖淀粉酶、地衣芽孢杆菌枯草杆菌蛋白酶、地衣芽孢杆菌β-内酰胺酶、嗜热脂肪芽孢杆菌α-淀粉酶、嗜热脂肪芽孢杆菌中性蛋白酶(nprT、nprS、nprM)和枯草芽孢杆菌prsA。另外的信号肽由Simonen和Palva,1993,Microbiol.Rev.[微生物评论]57:109-137描述。
丝状真菌宿主细胞的有效信号肽编码序列是从以下的基因中获得的信号肽编码序列:黑曲霉中性淀粉酶、黑曲霉葡糖淀粉酶、米曲霉TAKA淀粉酶、特异腐质霉纤维素酶、特异腐质霉内切葡聚糖酶V、柔毛腐质霉脂肪酶和米黑根毛霉天冬氨酸蛋白酶。
酵母宿主细胞的有用的信号肽从酿酒酵母α-因子和酿酒酵母转化酶的基因中获得。其他的有用的信号肽编码序列由Romanos等人(1992,同上)描述。
控制序列还可以是编码位于多肽的N-末端的前肽的前肽编码序列。所得的多肽被称为前体酶(proenzyme)或多肽原(或在一些情况下被称为酶原(zymogen))。多肽原通常是无活性的并且可通过催化切割或自身催化切割来自多肽原的前肽而转化为活性多肽。前肽编码序列可以从以下的基因中获得:枯草芽孢杆菌碱性蛋白酶(aprE)、枯草芽孢杆菌中性蛋白酶(nprT)、嗜热毁丝霉漆酶(WO 95/33836)、米黑根毛霉天冬氨酸蛋白酶和酿酒酵母α-因子。
在信号肽序列和前肽序列二者都存在的情况下,该前肽序列位于紧邻多肽的N-末端且该信号肽序列位于紧邻前肽序列的N-末端。在优选的实施例中,前肽是具有SEQ IDNO:2的前导肽。可替代地,前肽是前导肽,该前导肽与SEQ ID NO:2具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性。
还可能希望的是添加调节序列,这些调节序列相对于宿主细胞的生长调节多肽的表达。调节序列的实例是响应于化学或物理刺激而引起基因的表达开启或关闭的那些,包括调节化合物的存在。原核系统中的调节序列包括lac、tac、和trp操纵子系统。在酵母中,可以使用ADH2系统或GAL1系统。在丝状真菌中,可以使用黑曲霉葡糖淀粉酶启动子、米曲霉TAKAα-淀粉酶启动子和米曲霉葡糖淀粉酶启动子、里氏木霉纤维二糖水解酶I启动子和里氏木霉纤维二糖水解酶II启动子。调节序列的其他实例是使基因扩增的那些序列。在真核系统中,这些调节序列包括在甲氨蝶呤存在下扩增的二氢叶酸还原酶基因以及用重金属扩增的金属硫蛋白基因。在这些情况中,编码多肽的多核苷酸会与调节序列可操作地连接。
表达载体
本发明还涉及包含本发明的多核苷酸、启动子、以及转录和翻译终止信号的重组表达载体。多个核苷酸和控制序列可连接在一起以产生重组表达载体,该重组表达载体可包括一个或多个便利的限制位点以允许编码目的多肽的多核苷酸在这样的位点处的插入或取代。可替代地,可以通过将多核苷酸或包含该多核苷酸的核酸构建体插入用于表达的适当载体中而表达该多核苷酸。在产生表达载体时,编码序列如此位于载体中,使得编码序列与用于表达的适当控制序列可操作地连接。
在第四方面,本发明涉及一种表达载体,该表达载体包含根据第三方面的核酸构建体。
重组表达载体可以是可以方便地经受重组DNA程序并且可以引起多核苷酸和前导肽一起表达的任何载体(例如,质粒或病毒)。载体的选择将典型地取决于载体与待引入载体的宿主细胞的相容性。载体可以是直链或闭合环状质粒。
载体可以是自主复制载体,即作为染色体外实体存在的载体,其复制独立于染色体复制,例如质粒、染色体外元件、微染色体或人工染色体。载体可以含有用于确保自我复制的任何手段。可替代地,载体可以是这样的载体,当将载体引入宿主细胞中时,载体被整合到基因组中并随已经整合入载体的一个或多个染色体一起复制。此外,可以使用单个载体或质粒或两个或更多个载体或质粒,其共同含有待引入宿主细胞基因组中的总DNA,或可以使用转座子。
载体优选地含有允许容易地选择转化细胞、转染细胞、转导细胞等细胞的一个或多个选择性标记。选择性标记是一种基因,其产物提供了杀生物剂抗性或病毒抗性、对重金属的抗性、相对于营养缺陷型的原养型(prototrophy to auxotrophs)等。
细菌选择性标记的实例是地衣芽孢杆菌或枯草芽孢杆菌dal基因、或赋予抗生素抗性(如氨苄青霉素、氯霉素、卡那霉素、新霉素、大观霉素、或四环素抗性)的标记。酵母宿主细胞的合适的标记包括但不限于:ADE2、HIS3、LEU2、LYS2、MET3、TRP1和URA3。用于在丝状真菌宿主细胞中使用的选择性标记包括但不限于adeA(磷酸核糖酰氨基咪唑-琥珀羧胺合酶)、adeB(磷酸核糖酰-氨基咪唑合酶)、amdS(乙酰胺酶)、argB(鸟氨酸氨甲酰基转移酶)、bar(草丁膦乙酰转移酶)、hph(潮霉素磷酸转移酶)、niaD(硝酸还原酶)、pyrG(乳清酸核苷-5'-磷酸脱羧酶)、sC(硫酸腺苷基转移酶)以及trpC(邻氨基苯甲酸合酶)连同其等同物。优选的用于在曲霉属细胞中使用的是构巢曲霉或米曲霉amdS和pyrG基因以及吸水链霉菌(Streptomyces hygroscopicus)bar基因。优选的用于在木霉属细胞中使用的是adeA、adeB、amdS、hph和pyrG基因。
选择性标记可以是如WO 2010/039889中描述的双选择性标记系统。在一方面,双选择性标记是hph-tk双选择性标记系统。
载体优选地含有允许载体整合到宿主细胞的基因组中或载体在细胞中独立于基因组自主复制的一个或多个元件。
对于整合到宿主细胞基因组中,载体可以依靠编码该多肽的多核苷酸序列或用于通过同源或非同源重组整合到该基因组中的该载体的任何其他元件。可替代地,载体可以含有用于指导通过同源重组而整合入宿主细胞基因组中的一个或多个染色体中的一个或多个精确位置处的另外的多核苷酸。为了提高在精确位置处整合的可能性,整合元件应当含有足够数目的核酸,如100至10,000个碱基对、400至10,000个碱基对、和800至10,000个碱基对,这些核酸与相对应的靶序列具有高度序列同一性以增强同源重组的概率。整合元件可以是与宿主细胞基因组内的靶序列同源的任何序列。此外,整合元件可以是非编码或编码的多核苷酸。另一方面,载体可以通过非同源重组整合入宿主细胞的基因组中。
为了自主复制,载体可以进一步包含复制起点,该复制起点使得载体能够在所讨论的宿主细胞中自主复制。复制起点可以是在细胞中发挥作用的介导自主复制的任何质粒复制子。术语“复制起点”或“质粒复制子”意指使质粒或载体能够在体内复制的多核苷酸。
细菌复制起点的实例是允许在大肠杆菌中复制的质粒pBR322、pUC19、pACYC177、和pACYC184的复制起点,以及允许在芽孢杆菌属中复制的pUB110、pE194、pTA1060和pAMβ1的复制起点。
用于在酵母宿主细胞中使用的复制起点的实例是2微米复制起点、ARS1、ARS4、ARS1与CEN3的组合、及ARS4与CEN6的组合。
在丝状真菌细胞中有用的复制起点的实例是AMA1和ANS1(Gems等人,1991,Gene[基因]98:61-67;Cullen等人,1987,Nucleic Acids Res.[核酸研究]15:9163-9175;WO00/24883)。可根据WO 00/24883中披露的方法完成AMA1基因的分离和包含该基因的质粒或载体的构建。
可以将本发明的多核苷酸的多于一个的拷贝插入宿主细胞中以增加目的多肽的产生。可以通过将序列的至少一个另外的拷贝整合到宿主细胞基因组中或者通过包括与多核苷酸一起的可扩增的选择性标记基因获得多核苷酸的增加的拷贝数目,其中可以通过在适当的选择性试剂的存在下培养细胞选择含有选择性标记基因的经扩增的拷贝以及由此该多核苷酸的另外的拷贝的细胞。
用于连接上述的元件以构建本发明的重组表达载体的程序是本领域的技术人员熟知的(参见例如,Sambrook等人,1989,同上)。
信号肽和前导肽
本发明还涉及编码信号肽的分离的多核苷酸,该信号肽包含SEQ ID NO:4的氨基酸1至21、SEQ ID NO:6的氨基酸1至21、或SEQ ID NO:10、SEQ ID NO:41或SEQ ID NO:52的氨基酸1至21,或由其组成。本发明还涉及编码合成前导肽的分离的多核苷酸,该合成前导肽包含SEQ ID NO:2的氨基酸1至9、SEQ ID NO:6的氨基酸22至30、SEQ ID NO:10的氨基酸22至30、SEQ ID NO:43的氨基酸22至30、或SEQ ID NO:45的氨基酸22至30,或由其组成。在一个实施例中,编码SEQ ID NO:2的前导肽的多核苷酸包含一个或多个突变,优选地核苷酸取代、核苷酸缺失或核苷酸插入。所述一个或多个突变导致SEQ ID NO:2的信号肽的变体,例如包含以下的变体:(i)与SEQ ID NO:2相比一个或多个另外的氨基酸,(ii)与SEQ IDNO:2相比少至少一个氨基酸,例如总共4至8个氨基酸,(iii)或SEQ ID NO:2的至少一个氨基酸的氨基酸取代,如在对应于SEQ ID NO:2的位置1、2、3、4、5、6、7、8或9的位置处的氨基酸取代。
在另一个实施例中,多核苷酸编码前导肽,该前导肽与SEQ ID NO:2(FARAPVAAR)具有至少60%,例如至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性。
本发明还涉及编码信号肽和前导肽的分离的多核苷酸,该信号肽和前导肽包含SEQ ID NO:6的氨基酸1至30、SEQ ID NO:10的氨基酸1至30、SEQ ID NO:43的氨基酸1至30、或SEQ ID NO:45的氨基酸1至30,或由其组成。优选地,多核苷酸编码信号肽和前导肽,该信号肽和前导肽与SEQ ID NO:6、SEQ ID NO:43或SEQ ID NO:45具有至少60%,例如至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性。
多核苷酸可以进一步包含编码蛋白质的基因,该蛋白质与信号肽和/或前导肽可操作地连接,如葡糖淀粉酶。优选地,该蛋白质对于该信号肽和/或前导肽来说是异源的。在一方面,编码信号肽的多核苷酸是SEQ ID NO:3、SEQ ID 42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48或SEQ ID NO:50的核苷酸1至63。在另一方面,编码前导肽的多核苷酸是SEQID NO:1的核苷酸1至27。在另一方面,编码信号肽和前导肽的多核苷酸是SEQ ID NO:5、SEQID 42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48或SEQ ID NO:50的核苷酸1至90。
本发明还涉及包含这样的多核苷酸的核酸构建体、表达载体和重组宿主细胞,特别是真菌宿主细胞。
本发明还涉及生产蛋白质的方法,这些方法包括:(a)培养包含这样的多核苷酸的重组宿主细胞;以及任选地(b)回收该蛋白质。
该蛋白质对于宿主细胞来说可以是天然的或异源的。术语“蛋白质”在本文不意指特定长度的编码产物,并且因此涵盖肽、寡肽和多肽。术语“蛋白质”还涵盖了组合形成编码产物的两个或更多个多肽。这些蛋白质还包括杂合多肽和融合多肽。
优选地,该蛋白质是激素、酶、受体或其部分、抗体或其部分,或报告基因。例如,该蛋白质可以是水解酶、异构酶、连接酶、裂解酶、氧化还原酶、或转移酶,例如α-半乳糖苷酶、α-葡糖苷酶、氨肽酶、淀粉酶、β-半乳糖苷酶、β-葡糖苷酶、β-木糖苷酶、糖酶、羧肽酶、过氧化氢酶、纤维二糖水解酶、纤维素酶、几丁质酶、角质酶、环糊精糖基转移酶、脱氧核糖核酸酶、内切葡聚糖酶、酯酶、葡糖淀粉酶、转化酶、漆酶、脂肪酶、甘露糖苷酶、变聚糖酶、氧化酶、果胶分解酶、过氧化物酶、植酸酶、多酚氧化酶、蛋白水解酶、核糖核酸酶、转谷氨酰胺酶、或木聚糖酶。优选地,该蛋白质是葡糖淀粉酶。
该基因可以从任何原核、真核或其他来源获得。
通过以下实例进一步描述本发明,这些实例不应理解为对本发明的范围进行限制。
实例
材料与方法
除非另外说明,否则使用如以下所述的分子生物学标准方法来进行DNA操纵和转化:Sambrook等人(1989)Molecular cloning:A laboratory manual[分子克隆:实验室手册],冷泉港实验室[Cold Spring Harbor lab.],冷泉港,纽约州;Ausubel,F.M.等人(编辑)“Current protocols in Molecular Biology[分子生物学现代方法]”,John Wileyand Sons[约翰威利父子出版公司],1995;Harwood,C.R.,和Cut-ting,S.M.(编辑)“Molecular Biological Methods for Bacillus[用于芽孢杆菌属的分子生物学方法]”.John Wiley and Sons[约翰威利父子出版公司],1990。
购买的材料(大肠杆菌和试剂盒)
使用Qiagen Plasmid试剂盒(凯杰公司(Qiagen))来回收扩增的质粒。根据制造商的说明书,用Rapid DNA Dephos&Ligation试剂盒(罗氏公司(Roche))或In-Fusion试剂盒(克隆技术实验室公司(Clontech Laboratories,Inc.))进行连接。用KOD-Plus系统(东洋公司(TOYOBO))来进行聚合酶链式反应(PCR)。通过使用Plant Direct PCR试剂盒(新英格兰生物实验室(New England Biolabs))进行真菌孢子PCR。QIAquickTM凝胶提取试剂盒(凯杰公司)用于纯化PCR片段并从琼脂糖凝胶中提取DNA片段。
酶
用于DNA操纵的酶(例如限制性内切核酸酶、连接酶等)可获得自新英格兰生物实验室公司(New England Biolabs,Inc.),并且根据制造商的说明书使用。
质粒
来自草酸青霉(Penicillium oxalicum)的淀粉葡糖苷酶的序列描述于WO2011/127802(SEQ ID NO:2)中。pHUda1511是AnPav498载体。来自微小根毛霉(Rhizomucorpusillus)的淀粉酶的序列描述于EP2527448-A1(SEQ ID 84)中。pJaL1470描述于WO2015144936A1中。
微生物菌株
如WO2012/160093中的实例14所述,表达宿主菌株黑曲霉M1396和M1412(pyrG-表型/尿苷营养缺陷型)由诺维信公司(Novozymes)分离并且为从土壤分离的黑曲霉NN049184的衍生物。C2446、C2661、C5502、C5503和C5553是可从草酸青霉产生葡糖淀粉酶(1,4-α-D-葡聚糖葡糖水解酶,EC 3.2.1.3)的菌株。
如WO2012/160093中的实例14所述,表达宿主菌株黑曲霉C2446、C2661、C5502、C5503和C5553(pyrG-表型/尿苷营养缺陷型)由诺维信公司分离并且为从土壤分离的黑曲霉NN049184的衍生物。C2578和M1328(C2578的pyrG-表型)是可从草酸青霉产生葡糖淀粉酶的菌株。
培养基
COVE痕量金属溶液由以下构成:0.04g NaB4O7·10H2O、0.4gCuSO4·5H2O、1.2gFeSO4·7H2O、0.7g MnSO4·H2O、0.8g Na2MoO2·2H20、10g ZnSO4·7H2O、以及补足至1升的去离子水。
50X COVE盐溶液由以下构成:26g KCl、26g MgSO4·7H2O、76gKH2PO4、50ml COVE痕量金属溶液、以及补足至1升的去离子水。
COVE培养基由以下构成:342.3g蔗糖、20ml 50X COVE盐溶液、10ml1M乙酰胺、10ml1.5M CsCl2、25g纯净琼脂、以及补足至1升的去离子水。
COVE-N-Gly板由以下构成:218g山梨醇、10g甘油、2.02g KNO3、50ml COVE盐溶液、25g纯净琼脂、以及补足至1升的去离子水。
COVE-N(tf)由以下构成:342.3g蔗糖、3g NaNO3、20ml COVE盐溶液、30g纯净琼脂、以及补足至1升的去离子水。
COVE-N顶层琼脂糖由以下构成:342.3g蔗糖、3g NaNO3、20ml COVE盐溶液、10g低熔点琼脂糖、以及补足至1升的去离子水。
COVE-N由以下构成:30g蔗糖、3g NaNO3、20ml COVE盐溶液、30g纯净琼脂、以及补足至1升的去离子水。
STC缓冲液由以下构成:0.8M山梨醇、25mM Tris pH 8、以及25mM CaCl2。
STPC缓冲液由以下构成:在STC缓冲液中的40% PEG 4000。
LB培养基由以下构成:10g胰蛋白胨、5g酵母提取物、5g氯化钠、以及补足至1升的去离子水。
LB加氨苄青霉素板由以下构成:10g胰蛋白胨、5g酵母提取物、5g氯化钠、15g细菌培养用琼脂、以100μg/ml氨苄青霉素、以及补足至1升的去离子水。
YPG培养基由以下构成:10g酵母提取物、20g细菌蛋白胨、20g葡萄糖、以及补足至1升的去离子水。
SOC培养基由以下构成:20g胰蛋白胨、5g酵母提取物、0.5g NaCl、10ml 250mMKCl、以及补足至1升的去离子水。
TAE缓冲液由以下构成:4.84g Tris碱、1.14ml冰乙酸、2ml 0.5M EDTA pH 8.0、以及补足至1升的去离子水。
-MSS由以下构成:70g蔗糖、100g大豆粉(pH 6.0)、补足至1升的水。
-MU-1由以下构成:260g麦芽糊精、3g MgSO4·7H2O、5g KH2PO4、6g K2SO4、淀粉糖苷酶痕量金属溶液0.5ml和尿素2g(pH 4.5)、补足至1升的水。
-MU-1glu由以下构成:260g葡萄糖、3g MgSO4·7H2O、5g KH2PO4、6g K2SO4、淀粉糖苷酶痕量金属溶液0.5ml和尿素2g(pH 4.5)、补足至1升的水。
CDM2培养基(pH 6.5)由以下构成:30g蔗糖、3g NaNO3、1g K2HPO4、0.5g MgSO47H2O、0.5g KCl、0.01g FeSO4 7H2O、20g麦芽糖H2O、20g琼脂、BA-10、以及补足至1升的去离子水。
普鲁兰多糖培养基由以下构成:0.2g普鲁兰多糖、1g NaNO3、1g琼脂、BA-10、0.1g叠氮化钠、5mL 1M醋酸盐缓冲液(pH4.3)、以及补足至100ml的去离子水。
黑曲霉的转化
曲霉属物种的转化可以使用用于酵母转化的一般方法实现。以下描述了用于本发明的优选程序。
将黑曲霉宿主菌株接种至100ml的补充有10mM尿苷的YPG培养基上,并且在32℃以80rpm孵育16小时。收集球粒并用0.6M KCl洗涤,并且重悬浮于含有商业β-葡聚糖酶产品(GLUCANEXTM,诺维信公司,鲍斯韦丹麦)的20ml 0.6M KCl(终浓度为20mg/ml)中。将悬浮液在32℃以80rpm孵育直到形成原生质体,然后用STC缓冲液洗涤两次。将这些原生质体用血细胞计数器计数,并且重悬浮于STC:STPC:DMSO的8:2:0.1溶液中并调整至终浓度为2.5x107个原生质体/ml。将大约4μg的质粒DNA添加到100μl原生质体悬浮液中,轻轻混合,并且在冰上孵育30分钟。添加1ml的SPTC,并且将原生质体悬浮液在37℃孵育20分钟。在添加10ml的50℃Cove或Cove-N顶层琼脂糖之后,将反应倾倒于Cove或Cove-N(tf)琼脂板上,并且将板于32℃孵育5天。
实例中的PCR扩增
聚合酶链式反应(PCR)是用PrimeSTAR Max DNA聚合酶[宝生物公司(TaKaRa)]进行的。
组分体积终浓度
2×PrimeSTAR Max DNA聚合酶混合物-25μl 1x
10pmol/μl引物#1 1.5μl 0.3μM
10pmol/μl引物#2 1.5μl 0.3μM
模板DNA
基因组DNA质粒DNA Xμl
10-200ng/50μl
1-50ng/50μl
PCR级水 Yμl
总反应体积 50μl
3步循环:
通过使用Plant Direct PCR试剂盒(新英格兰生物实验室)进行真菌孢子PCR。用1μl接种环从每种真菌菌株上挑取孢子,并将这些孢子悬浮于10μl稀释缓冲液(包含在试剂盒中)中。如下所示设置PCR混合物。
组分体积
无菌diH20(μL)7.1
植物PCR缓冲液(μL)10
模板(μL)0.5
10μM 5'-引物(μL)1
10μM 3'-引物(μL)1
热启动II聚合酶(μL)0.4
3步循环:
用于葡糖淀粉酶生产的摇瓶培养
将所选择的转化体的孢子接种进100ml的MSS培养基,并且在30℃下培养3天。将10%的种子培养物以实验室规模罐转移至MU-1培养基中,用适当量的葡萄糖和铵进行补料,并且在34℃下培养7天。通过离心获得上清液。
用于葡糖淀粉酶生产的实验室规模罐培养
按补料分批发酵进行发酵(H.Pedersen 2000,Appl Microbiol Biotechnol[应用微生物学与生物技术],53:272-277)。将所选择的菌株在液体培养基中预培养,然后将生长的菌丝体转移到罐中用于酶产生的进一步培养。在pH4.75下在34℃培养8天,用葡萄糖和铵进行补料而不过度给予(其阻止酶产生)。对于实例7至9,在pH 5.1下在34℃培养8天,用葡萄糖和铵进行补料而不过度给予(其阻止酶产生)。使用离心后的培养物上清液进行酶测定。
葡糖淀粉酶活性
葡糖淀粉酶活性通过RAG测定法(相对AG测定,pNPG法)确定。pNPG底物由以下构成:0.1g对硝基苯基-β-D-吡喃葡糖苷(Nacalai Tesque公司)、10ml 1M醋酸盐缓冲液(pH4.3)、以及补足至100ml的去离子水。从每个稀释的样品溶液中,一式两份地向孔中添加40ul作为“样品”。并向孔中添加40ul去离子水作为“空白”。并添加40ul的AG标准溶液作为“参考”。使用Multidrop(雷勃公司(Labsystem)),向每个孔中添加80ul的pNPG底物。在室温20分钟后,通过添加120ul的停止试剂(0.1M硼砂溶液)来停止该反应。通过酶标仪在400nm处(Power Wave X)或在405nm处(ELx808)测量OD值。
如下进行计算:
S=样品值 F=稀释因子
B=空白值 AGs=AG/ml的AG标准品。
Ss=AG标准品的值
Bs=AG标准品的空白
RAG=相对淀粉葡糖苷酶单位
实例1:JPO001、JPO002和JPO003的构建
如下构建葡糖淀粉酶变体JPO001、JPO002和JPO003。
使用反向PCR构建表达载体,这意指通过以下条件使用适当的模板质粒DNA(例如含有AnPav498基因的质粒DNA)通过反向定向引物扩增整个质粒DNA序列。通过QIAquick凝胶提取试剂盒[凯杰公司]来纯化所得PCR片段,然后将其引入大肠杆菌ECOS感受态大肠杆菌DH5α[立邦基因股份有限公司(NIPPON GENE CO.,LTD.)]中。通过MagExtractor质粒提取试剂盒[东洋公司]从大肠杆菌转化体中提取质粒DNA,然后将其引入黑曲霉感受态细胞(宿主:C2446、C2661、C5502和C5503)中。
信号肽和前导肽的序列(*粗体字符是目的成熟多肽的N-末端):
·根据SEQ ID NO:8的(AnPav498)
·根据SEQ ID NO:6的 (JPO001)
·根据SEQ ID NO:25的(JPO002)
·根据SEQ ID NO:26的(JPO003)
表1.引物
SEQ ID NO: | 引物名称 |
19 | JPO001_F 42 |
20 | JPO001 R 42 |
21 | JPO002_F 39 |
22 | JPO002 R 39 |
23 | JPO003_F 39 |
24 | JPO003_R 39 |
PCR反应混合物:
PrimeSTAR Max DNA聚合酶[宝生物公司]
总计25μl
1.0μl 模板DNA(1ng/μl)
9.5μl H2O
12.5μl 2x PrimeSTAR Max预混物
1.0μl 正向引物(5μM)
1.0μl 反向引物(5μM)
PCR程序:
98℃/2min
25x(98℃/10sec,60℃/15sec,72℃/2min)
10℃/保持
实例2:通过使用96MTP培养物筛选更高的生产率
将如实例1中构建的转化体在含有COVE液体培养基(2.0g/L蔗糖、2.0g/L异麦芽糖、2.0g/L麦芽糖、4.9mg/L、0.2ml/L 5N NaOH、10ml/LCOVE盐、10ml/L 1M乙酰胺)、YPMAc(5g/L蔗糖、2.5g/L酵母提取物、5.0g/L蛋白胨、10.0g/L大豆粉、1.36g/L CH3COONa 3H2O)的96孔MTP(微量滴定板)中在32℃下发酵3天。然后,在几种温度下通过如下所述的pNPG测定来测量培养物上清液中的葡糖淀粉酶活性。表2和表3中列出了活性,作为相对于用作对照的AnPav498的相对活性(产率)。
pNPG测定
将含有所希望的酶的培养物上清液与相同体积的pH 5.0 200mM NaOAc缓冲液混合。将二十微升该混合物分配于96孔板或8联PCR管中。将那些样品与含有0.1%(w/v)pNPG[和光株式会社(wako)]的10μl底物溶液于pH 5.0 200mM NaOAc缓冲液中混合,并且在70℃下孵育20min以进行酶反应。反应后,添加60μl的0.1M硼砂缓冲液以停止反应。取出八十微升的反应上清液并且通过光度计读取其OD405值以评估酶活性。
表2.这些变体与C2446中的AnPav498相比的相对产率的列表,该C2446通过Cove-II液体1%异麦芽糖在96MTP中培养
表3.每种宿主中JPO001与它们的亲本(AnPav498)相比的相对产率的列表,这些亲本在Cove-II液体培养基和YPMAc培养基中于96MTP中培养
实例3:黑曲霉在SF中的发酵
将如实例1中构建的黑曲霉菌株在旋转摇床上在含有100ml MU1(260.0g/L麦芽糊精(MD-11)、3.0g/L MgSO4 7H2O、6.0g/L K2SO4、5.0mg/LKH2PO4、5ml/L COVE盐)和4ml50%尿素的500ml带挡板的烧瓶中在220rpm,30℃下发酵。将培养液离心(10,000x g,20min)并且将上清液小心地与沉淀物倾析分开。然后,在几种温度下通过如实例2中所述的pNPG测定来测量培养物上清液中的葡糖淀粉酶活性。从表4可以看出,与AnPav498对照的多肽产率相比,JPO001变体的多肽产率增加高达108%、135%和151%。
表4.这些变体与它们的亲本(C2661中的AnPav498)相比的相对产率的列表,这些亲本通过MU1培养基在带挡板的SF中培养
变体 | 宿主 | 相对于对照的相对葡糖淀粉酶活性 |
JPO001 | C2661 | 108% |
JPO001 | C5502 | 135% |
JPO001 | C5503 | 151% |
JPO002 | C2661 | 3% |
JPO002 | C5502 | 75% |
JPO002 | C5503 | 2% |
JPO003 | C2661 | 76% |
JPO003 | C5502 | 92% |
JPO003 | C5503 | 83% |
AnPav498 | C2661 | 100% |
实例4:葡糖淀粉酶的纯化
通过以下两个步骤对黑曲霉变体进行纯化:硫酸铵沉淀和阳离子交换色谱法。最终,使用离心过滤装置(Vivaspin Turbo 15,赛多利斯公司(Sartorius))将样品脱盐并用20mM醋酸钠缓冲液(pH 4.5)进行缓冲液交换。通过A280值确定酶浓度。
实例5:JPO变体在黑曲霉菌株中的表达
用具有FLP介导的3-4个JPO变体拷贝的整合的黑曲霉宿主C5553测试JPO变体的表达。如WO 2012/160093中所述,已经进行了FLP介导的整合。如WO 2012/160093中的实例14所述,表达宿主菌株C5553由诺维信公司分离并且为从土壤分离的黑曲霉NN049184的衍生物。
通过MTP对来自相同变体的总共9至10个克隆进行初步评估(表5)。与主链anPAV498相比,信号修饰的变体(JPO001)将多肽活性提高了6%。在通过SF进行的二次评估中(表6),与用anPAV498表达相比,所有变体显示出高达2414%的显著增加的活性(构建体JPO001,第6天)。
表5.MTP发酵中的相对葡糖淀粉酶活性(宿主:C5553)
表6.SF发酵中的相对葡糖淀粉酶活性
实例6:实验室罐中的JPO变体测试
将AnPav498和JPO001在实验室罐中在当前标准条件下分两批进行评估,以研究信号肽修饰的效果。如表7所呈现的结果,与AnPav498相比,JPO001在C3085中的滴度高15%,在C5553中的滴度高77%。
表7.5L罐中的相对葡糖淀粉酶活性
实例7:质粒pIhar234、pHiTe384和pHiTe387的构建
如下构建表达质粒,这些表达质粒包含与曲霉属启动子、信号序列JSP001(pIhar234)、JSP035(pHiTe384)和JSP038(pHiTe387)、和终止子连接的编码微小根毛霉α-淀粉酶的核苷酸序列的串联重复序列,并且进一步包含用于在曲霉属中进行amdS选择的amdS基因。用相应的引物对(本申请的SEQ ID:27和28)通过PCR从具有SEQ ID:NO 84的质粒(在EP2527448-A1中描述)中扩增淀粉酶基因的约1.8kb区域。
根据制造商的方案,通过HiFi DNA组装主混合物(新英格兰生物实验室)将获得的1.8kb DNA片段与pHiTe169(WO 2015144936A1中描述的pJaL1470的衍生物)的BamHI/PmlI消化物连接,以产生单个表达质粒。将所得质粒用NheI或NheI/SpeI消化。然后将衍生自相同质粒的这些片段通过凝胶提取试剂盒(凯杰公司)纯化,并通过连接试剂盒(罗氏公司)连接,得到串联表达质粒pIhar234。
对于信号变体JSP035和JSP038,用相应的DNA模板和引物,使用重叠延伸PCR产生具有信号肽和前导肽的α-淀粉酶的全长DNA。
表8.PCR扩增
3步循环:
<第1次PCR>
JSP035模板DNA1(HTJP-1053):SEQ ID NO:29
JSP035模板DNA2(HTJP-1149):SEQ ID NO:30
用于第1次PCR的正向引物(HTJP-1183):SEQ ID NO:31
用于第1次PCR的反向引物(HTJP-1184):SEQ ID NO:32
JSP038模板DNA1(HTJP-1112):SEQ ID NO:33
JSP038模板DNA2(HTJP-1151):SEQ ID NO:34
用于第1次PCR的正向引物(HTJP-1187):SEQ ID NO:35
用于第1次PCR的反向引物(HTJP-1184):SEQ ID NO:32
pIhar234用作以下PCR的DNA模板:
<JSP035>
用于第1次PCR的正向引物(HTJP-1185):SEQ ID NO:36
用于第1次PCR的反向引物(HTJP-1049):SEQ ID NO:28
<JSP038>
用于第1次PCR的正向引物(HTJP-1186):SEQ ID NO:37
用于第1次PCR的反向引物(HTJP-1049):SEQ ID NO:28
用相应的引物对(SEQ ID:28和31)通过重叠延伸PCR从第1次PCR片段中扩增具有JSP035的淀粉酶基因的约1.9kb区域。
<第2次PCR>
用于第2次PCR的正向引物(HTJP-1183):SEQ ID NO:31
用于第2次PCR的反向引物(HTJP-1049):SEQ ID NO:28
用相应的引物对(SEQ ID:28和35)通过重叠延伸PCR从第1次PCR片段中扩增具有JSP038的淀粉酶基因的约1.9kb区域。
<第2次PCR>
用于第2次PCR的正向引物(HTJP-1187):SEQ ID NO:35
用于第2次PCR的反向引物(HTJP-1049):SEQ ID NO:28
根据制造商的方案,通过HiFi DNA组装主混合物(新英格兰生物实验室)将获得的JSP035和JSP038二者的1.9kb DNA片段与pHiTe169的BamHI/PmlI消化物连接,以产生单个表达质粒。将所得质粒用NheI或NheI/SpeI消化。然后将衍生自相同质粒的这些片段通过凝胶提取试剂盒(凯杰公司)纯化,并通过连接试剂盒(罗氏公司)连接,得到串联表达质粒pHiTe384(JSP035)和pHiTe387(JSP038)。
显示了信号肽和前导肽的序列(*粗体字符是目的成熟多肽的N-末端):
根据SEQ ID NO:41的(JSP001,参考菌株)
根据SEQ ID NO:43的(JSP035)
根据SEQ ID NO:45的(JSP038)
实例8:α-淀粉酶在黑曲霉菌株中的表达
如WO 2012/160093中所述,将具有amdS选择性标记的微小根毛霉α-淀粉酶基因染色体插入到黑曲霉C5554中。通过flp重组酶应在四个预先指定的基因座处引入微小根毛霉α-淀粉酶表达质粒pIhar234、pHiTe384和pHiTe387,这些基因座是甘露糖基转移酶(alg2)、葡糖激酶(gukA)、酸稳定性淀粉酶(asaA)和多铜氧化酶(mcoH)。将菌株进行纯化,并且对其进行DNA印迹分析以确认是否将微小根毛霉α-淀粉酶基因正确引入mcoH、gukA、asaA和alg2基因座处。以下用以制备非放射性探针的引物组被用来分析所选择的转化体。
对于启动子区:
SEQ ID NO 38:HTJP-324AAGGGATGCAAGACCAAACC
SEQ ID NO 39:HTJP-325TGAAGAATTTGTGTTGTCTGAG
将从所选择的转化体提取的基因组DNA通过SpeI和HindIII消化,然后用启动子区进行探测。通过正确基因引入事件,如上所述探测观察通过SpeI和HindIII消化的11.0kb(alg2)、7.3kb(mcoH)、11.1kb(gukA)和7.8kb(asaA)大小的杂交信号。
实例9:实验室罐中α-淀粉酶菌株的评估
将来自C5554的每个信号肽和前导肽的一个菌株在实验室规模罐中发酵,并如下所述测量它们的酶活性(FAU(F)活性)。结果显示在下表中(表9)。在实验室发酵罐中,具有前导肽的菌株(JSP035和JSP038)显示出比没有前导序列的参考信号(JSP001)高约1.11-1.25倍的淀粉酶活性(表9)。
表9.实验室罐中的相对淀粉酶活性
从每个宿主菌株中选择的六个菌株的平均FAU(F)活性,其中将来自O73RGP的平均FAU(F)产率归一化为1.00。
相对于已知强度的酶标准品,以FAU(F)(真菌α-淀粉酶单位(Fungamyl))测量淀粉酶活性。Fungamyl是具有酶分类号EC 3.2.1.1的1,4α-D-葡聚糖水解酶。样品和试剂盒中的α-葡糖苷酶将底物(4,6-亚乙基(G7)-对硝基苯基(G1)-α,D-麦芽七糖苷(亚乙基-G7PNP))水解成葡萄糖和黄色对硝基苯酚。可以通过Konelab(赛默飞世尔科技公司(Thermo FisherScientific))观察对硝基苯酚的形成速率。
表10.反应条件.
反应缓冲液组成
87mM NaCl
52.4mM HEPES
12.6mM MgCl2
0.075mM CaCl2
>4kU/Lα-葡糖苷酶
底物组成
52.4mM HEPES
22mM亚乙基-G7PNP
从标准曲线读取稀释样品的酶活性。
如下进行计算:
S=标准曲线读数,以mFAU(F)/ml为单位
V=使用的容量瓶的体积,以mL为单位
F=稀释因子
W=样品的重量,以g为单位
表11.核苷酸和氨基酸序列的概述。
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本文描述和要求保护的本发明不限于本文披露的特定方面的范围,因为这些方面旨在作为本发明几个方面的说明。任何等同方面旨在处于本发明的范围之内。实际上,除了本文所示和描述的那些之外,本发明的各种修改因前述描述而对本领域的技术人员变得显而易见。这样的修改也旨在落入所附权利要求书的范围内。在冲突的情况下,以包括定义的本披露为准。
通过以下编号的段落进一步定义本发明:
1.一种真菌宿主细胞,该真菌宿主细胞在其基因组中包含:
a)编码目的多肽的第一多核苷酸;和
b)在该第一多核苷酸的上游以翻译融合方式可操作地连接至该第一多核苷酸的第二多核苷酸,所述第二多核苷酸编码与SEQ ID NO:2(FARAPVAAR)具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性的前导肽,优选地该前导肽是合成的,或与该目的多肽是异源的。
2.根据段落1所述的真菌宿主细胞,其中该前导肽包含SEQ ID NO:2,基本上由其组成,或由其组成。
3.根据段落1所述的真菌宿主细胞,其中该前导肽与SEQ ID NO:2的氨基酸序列相同。
4.根据前述段落中任一项所述的真菌宿主细胞,其中该宿主细胞在其基因组中包含编码信号肽的第三多核苷酸,其中该第三多核苷酸在该第二多核苷酸的上游以翻译融合方式可操作地连接至该第二多核苷酸;并且其中该目的多肽被分泌。
5.根据段落4所述的真菌宿主细胞,其中该第三多核苷酸编码与SEQ ID NO:4、SEQID NO:41或SEQ ID NO:52具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性的信号肽。
6.根据前述段落中任一项所述的真菌宿主细胞,其中该至少一个控制序列可操作地连接至该信号肽或该前导肽,并且其中所述控制序列指导该目的多肽的产生。
7.根据前述段落中任一项所述的真菌宿主细胞,其中该目的多肽与该宿主细胞是异源的。
8.根据段落6至7中任一项所述的真菌宿主细胞,其中该至少一个控制序列与编码该目的多肽、该信号肽和/或该前导肽的该多核苷酸是异源的。
9.根据前述段落中任一项所述的真菌宿主细胞,其中该宿主细胞包含该第一和第二多核苷酸的至少两个拷贝,如该第一和第二多核苷酸的两个、三个、四个、五个或六个拷贝。
10.根据前述段落中任一项所述的真菌宿主细胞,其中编码SEQ ID NO:2的前导肽的该第二多核苷酸包含一个或多个突变,优选地核苷酸取代、核苷酸缺失或核苷酸插入。所述一个或多个突变导致SEQ ID NO:2的前导肽的变体,例如包含以下的变体:(i)与SEQ IDNO:2相比一个或多个另外的氨基酸,(ii)与SEQ ID NO:2相比少至少一个氨基酸,例如总共3至8个氨基酸,(iii)或SEQ ID NO:2的至少一个氨基酸的氨基酸取代,如在对应于SEQ IDNO:2的位置1、2、3、4、5、6、7、8或9的位置处的氨基酸取代。
11.根据段落4至10中任一项所述的真菌宿主细胞,其中该第三多核苷酸编码与SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性的信号肽。
12.根据段落4至11中任一项所述的真菌宿主细胞,其中该第三多核苷酸基本上由SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52组成,由它们组成,或包含它们。
13.根据段落4至11中任一项所述的真菌宿主细胞,其中编码SEQ ID NO:4、SEQ IDNO:41或SEQ ID NO:52的信号肽的该第三多核苷酸包含一个或多个突变,优选地核苷酸取代、核苷酸缺失或核苷酸插入。所述一个或多个突变导致SEQ ID NO:4、SEQ ID NO:41或SEQID NO:52的信号肽的变体,例如包含以下的变体:(i)与SEQ ID NO:4、SEQ ID NO:41或SEQID NO:52相比一个或多个另外的氨基酸,(ii)与SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52相比少至少一个氨基酸,例如总共10至20个氨基酸,(iii)或SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52的至少一个氨基酸的氨基酸取代,如在对应于SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或21的位置处的氨基酸取代。
14.根据前述段落中任一项所述的真菌宿主细胞,其中该宿主细胞是酵母宿主细胞;优选地,该酵母宿主细胞选自由以下组成的组:假丝酵母属、汉逊酵母属、克鲁维酵母属、毕赤酵母属(驹形氏酵母属)、酵母属、裂殖酵母属、和耶氏酵母属细胞;更优选地,该酵母宿主细胞选自由以下组成的组:乳酸克鲁维酵母、卡尔酵母、酿酒酵母、糖化酵母、道格拉氏酵母、克鲁弗酵母、诺地酵母、卵形酵母、和解脂耶氏酵母细胞,最优选地毕赤酵母(法夫驹形氏酵母)。
15.根据段落1至13中任一项所述的真菌宿主细胞,其中该宿主细胞是丝状真菌宿主细胞;优选地,该丝状真菌宿主细胞选自由以下组成的组:枝顶孢霉属、曲霉属、短梗霉属、烟管菌属、拟蜡菌属、金孢子菌属、鬼伞属、革盖菌属、隐球菌属、线黑粉酵母属、镰孢属、腐质霉属、梨孢菌属、毛霉属、毁丝霉属、新美鞭菌属、脉孢菌属、拟青霉属、青霉属、平革菌属、射脉菌属、瘤胃壶菌属、侧耳属、裂褶菌属、篮状菌属、嗜热子囊菌属、梭孢壳属、弯颈霉属、栓菌属、和木霉属细胞;更优选地,该丝状真菌宿主细胞选自由以下组成的组:泡盛曲霉、臭曲霉、烟曲霉、日本曲霉、构巢曲霉、黑曲霉、米曲霉、黑刺烟管菌、干拟蜡菌、卡内基拟蜡菌、浅黄拟蜡菌、潘诺希塔拟蜡菌、环带拟蜡菌、微红拟蜡菌、虫拟蜡菌、狭边金孢子菌、嗜角质金孢子菌、卢克诺文思金孢子菌、粪状金孢子菌、毡金孢子菌、昆士兰金孢子菌、热带金孢子菌、带纹金孢子菌、灰盖鬼伞、毛革盖菌、杆孢状镰孢、谷类镰孢、库威镰孢、大刀镰孢、禾谷镰孢、禾赤镰孢、异孢镰孢、合欢木镰孢、尖孢镰孢、多枝镰孢、粉红镰孢、接骨木镰孢、肤色镰孢、拟分枝孢镰孢、硫色镰孢、圆镰孢、拟丝孢镰孢、镶片镰孢、特异腐质霉、柔毛腐质霉、米黑毛霉、嗜热毁丝霉、粗糙脉孢菌、产紫青霉、黄孢原毛平革菌、射脉菌、刺芹侧耳、土生梭孢壳、长域毛栓菌、变色栓菌、哈茨木霉、康宁木霉、长枝木霉、里氏木霉、和绿色木霉细胞;甚至更优选地,该丝状宿主细胞选自由米曲霉、镶片镰孢和里氏木霉细胞组成的组;最优选地,该丝状真菌宿主细胞是黑曲霉细胞。
16.根据段落15所述的真菌宿主细胞,其中该丝状宿主细胞是黑曲霉细胞。
17.根据段落15所述的真菌宿主细胞,其中该丝状宿主细胞是米曲霉细胞。
18.根据段落15所述的真菌宿主细胞,其中该丝状宿主细胞是里氏木霉细胞。
19.根据前述段落中任一项所述的真菌宿主细胞,其中该目的多肽包含酶;优选地,该酶选自由以下组成的组:水解酶、异构酶、连接酶、裂解酶、氧化还原酶或转移酶;更优选地是氨肽酶、淀粉酶、糖酶、羧肽酶、过氧化氢酶、纤维二糖水解酶、纤维素酶、几丁质酶、角质酶、环糊精糖基转移酶、脱氧核糖核酸酶、内切葡聚糖酶、酯酶、α-半乳糖苷酶、β-半乳糖苷酶、α-葡糖苷酶、β-葡糖苷酶、转化酶、漆酶、脂肪酶、甘露糖苷酶、变聚糖酶、核酸酶、氧化酶、果胶分解酶、过氧化物酶、磷酸二酯酶、植酸酶、多酚氧化酶、蛋白水解酶、核糖核酸酶、转谷氨酰胺酶、木聚糖酶、和β-木糖苷酶。
20.根据权利要求7所述的真菌宿主细胞,其中该目的多肽是糖蛋白,优选地是α-葡糖苷酶;更优选地是1,4-α-葡糖苷酶;最优选地是葡糖淀粉酶,如与SEQ ID NO:15、SEQID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51具有至少60%序列同一性的葡糖淀粉酶。
21.一种真菌宿主细胞,该真菌宿主细胞包含多肽,所述多肽包含以翻译融合方式可操作地连接至目的多肽的前导肽,其中该前导肽与SEQ ID NO:2(FARAPVAAR)具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性;或者其中该前导肽包含SEQ ID NO:2,基本上由其组成,或由其组成。
22.根据段落21所述的真菌宿主细胞,其中该多肽进一步包含在该前导肽的上游以翻译融合方式可操作地连接的信号肽,该信号肽与SEQ ID NO:4、SEQ ID NO:41或SEQ IDNO:52具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性。
23.根据段落21至22中任一项所述的真菌宿主细胞,其中该前导肽上游的该信号肽包含SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52,基本上由其组成,或由其组成。
24.一种用于生产目的多肽的方法,该方法包括:
i)提供根据段落1至23中任一项所述的真菌宿主细胞,
ii)在有助于该目的多肽表达的条件下培养所述真菌宿主细胞;以及,任选地
iii)回收该目的多肽。
25.一种分离的或纯化的多肽,该分离的或纯化的多肽与SEQ ID NO:15、SEQ IDNO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51的成熟多肽具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少81%、至少82%、至少83%、至少84%、至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%、或100%序列同一性。
26.根据段落25所述的分离的或纯化的多肽,其中该多肽具有葡糖淀粉酶活性。
27.根据段落25至26中任一项所述的分离的或纯化的多肽,其中该多肽与SEQ IDNO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQID NO:51的成熟多肽相差多达10个氨基酸,例如,1、2、3、4、5、6、7、8、9、或10个。
28.根据段落25至27中任一项所述的分离的或纯化的多肽,其中该多肽包含SEQID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51或其成熟多肽的氨基酸序列,基本上由其组成,或由其组成;或是其片段。
29.根据段落28所述的分离的或纯化的多肽,其中该成熟多肽与SEQ ID NO:15相同。
30.根据段落28所述的分离的或纯化的多肽,其中该成熟多肽与SEQ ID NO:16相同。
31.根据段落28所述的分离的或纯化的多肽,其中该成熟多肽与SEQ ID NO:17相同。
32.根据段落28所述的分离的或纯化的多肽,其中该成熟多肽与SEQ ID NO:18相同。
33.一种编码信号肽的分离的多核苷酸,该信号肽包含SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52的氨基酸1至21、SEQ ID NO:6的氨基酸1至21、或SEQ ID NO:10的氨基酸1至21,基本上由其组成,或由其组成。
34.一种编码合成前导肽的分离的多核苷酸,该合成前导肽包含SEQ ID NO:2的氨基酸1至9、SEQ ID NO:6的氨基酸22至30、或SEQ ID NO:10的氨基酸22至30,基本上由其组成,或由其组成。
35.根据段落34所述的分离的多核苷酸,其中编码SEQ ID NO:2的前导肽的该多核苷酸包含一个或多个突变,优选地核苷酸取代、核苷酸缺失或核苷酸插入。
36.根据段落35所述的分离的多核苷酸,其中所述一个或多个突变导致SEQ IDNO:2的信号肽的变体,例如包含以下的变体:(i)与SEQ ID NO:2相比一个或多个另外的氨基酸,(ii)与SEQ ID NO:2相比少至少一个氨基酸,例如总共4至8个氨基酸,(iii)或SEQ IDNO:2的至少一个氨基酸的氨基酸取代,如在对应于SEQ ID NO:2的位置1、2、3、4、5、6、7、8或9的位置处的氨基酸取代。
37.根据段落34至36中任一项所述的分离的多核苷酸,其中该多核苷酸编码前导肽,该前导肽与SEQ ID NO:2(FARAPVAAR)具有至少60%,例如至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性。
38.根据段落37所述的分离的多核苷酸,其中该前导肽与SEQ ID NO:2相同。
39.一种编码信号肽和前导肽的分离的多核苷酸,该信号肽和前导肽包含SEQ IDNO:6的氨基酸1至30或SEQ ID NO:10、SEQ ID NO:43或SEQ ID NO:45的氨基酸1至30,基本上由其组成,或由其组成。
40.根据段落39所述的分离的多核苷酸,其中该多核苷酸编码信号肽和前导肽,该信号肽和前导肽与以下具有至少60%,例如至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性:
SEQ ID NO:6(MRLTLLSGVAGVLCAGQLTAAFARAPVAAR),
SEQ ID NO:43(MRLSTSSLFLSVSLLGKLALGFARAPVAAR);或
SEQ ID NO:45(MGVSAVLLPLYLLSGVTFGLAFARAPVAAR)。
41.根据段落33至40中任一项所述的分离的多核苷酸,其中编码该信号肽或前导肽的该多核苷酸以翻译融合方式可操作地连接至编码蛋白质如葡糖淀粉酶的基因。
42.一种核酸构建体,该核酸构建体包含编码目的多肽的第一多核苷酸和可操作地连接至该第一多核苷酸的第二多核苷酸,该第二多核苷酸编码与SEQ ID NO:2(FARAPVAAR)具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性的前导肽,优选地该前导肽是合成的,或与该目的多肽是异源的。
43.根据段落42所述的核酸构建体,其中该前导肽包含SEQ ID NO:2,基本上由其组成,或由其组成。
44.根据段落43所述的核酸构建体,其中编码SEQ ID NO:2的前导肽的该第二多核苷酸包含一个或多个突变,优选地核苷酸取代、核苷酸缺失或核苷酸插入。
45.根据段落44所述的核酸构建体,其中所述一个或多个突变导致SEQ ID NO:2的前导肽的变体,例如包含以下的变体:(i)与SEQ ID NO:2相比一个或多个另外的氨基酸,(ii)与SEQ ID NO:2相比少至少一个氨基酸,例如总共4至8个氨基酸,(iii)或SEQ ID NO:2的至少一个氨基酸的氨基酸取代,如在对应于SEQ ID NO:2的位置1、2、3、4、5、6、7、8或9的位置处的氨基酸取代。
46.根据段落42至45中任一项所述的核酸构建体,其中该第二多核苷酸可操作地连接至指导该多肽在表达宿主中的产生的一个或多个控制序列。
47.根据段落42至46中任一项所述的核酸构建体,其中该核酸构建体包含编码信号肽的第三多核苷酸,其中该第三多核苷酸在该第二多核苷酸的上游以翻译融合方式可操作地连接至该第二多核苷酸;并且该信号肽与SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性。
48.根据段落47所述的核酸构建体,其中该信号肽由SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52组成,基本上由它们组成,或包含它们。
49.根据段落46至47中任一项所述的核酸构建体,其中该第三多核苷酸可操作地连接至指导该多肽在表达宿主中的产生的一个或多个控制序列。
50.根据段落47至49中任一项所述的核酸构建体,其中编码该信号肽的该第三多核苷酸包含一个或多个突变,优选地核苷酸取代、核苷酸缺失或核苷酸插入。
51.根据段落50所述的核酸构建体,其中所述一个或多个突变导致SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52的信号肽的变体,例如包含以下的变体:(i)与SEQ ID NO:4、SEQ ID NO:41或SEQ ID NO:52相比一个或多个另外的氨基酸,(ii)与SEQ ID NO:4、SEQ IDNO:41或SEQ ID NO:52相比少至少一个氨基酸,例如总共10至20个氨基酸,(iii)或SEQ IDNO:4、SEQ ID NO:41或SEQ ID NO:52的至少一个氨基酸的氨基酸取代,如在对应于SEQ IDNO:4、SEQ ID NO:41或SEQ ID NO:52的位置1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或21的位置处的氨基酸取代。
52.一种表达载体,该表达载体包含根据段落33至51中任一项所述的多核苷酸或核酸构建体。
53.一种真菌宿主细胞,该真菌宿主细胞包含根据段落33至52中任一项所述的多核苷酸、核酸构建体或表达载体。
54.一种具有葡糖淀粉酶活性的分离的或纯化的多肽,该分离的或纯化的多肽选自由以下组成的组:
(a)与SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51具有至少60%序列同一性的多肽;
(b)由以下多核苷酸编码的多肽,该多核苷酸在中严格条件下与SEQ ID NO:7、SEQID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:46、SEQ ID NO:48或SEQ ID NO:50的成熟多肽编码序列的全长互补序列杂交;
(c)由以下多核苷酸编码的多肽,该多核苷酸与SEQ ID NO:7、SEQ ID NO:9、SEQID NO:11、SEQ ID NO:13、SEQ ID NO:46、SEQ ID NO:48或SEQ ID NO:50的成熟多肽编码序列具有至少60%序列同一性;
(d)通过在SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ IDNO:47、SEQ ID NO:49或SEQ ID NO:51的成熟多肽中取代、缺失或添加一个或几个氨基酸而从SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51的成熟多肽衍生的多肽;以及
(e)(a)、(b)、(c)或(d)的多肽的片段,该片段具有葡糖淀粉酶活性。
55.一种具有葡糖淀粉酶活性的分离的或纯化的多肽,该分离的或纯化的多肽是:
(a)与SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51具有至少60%序列同一性的多肽;或
(b)(a)的多肽的片段,该片段具有葡糖淀粉酶活性。
56.根据段落54至55中任一项所述的多肽,该多肽与SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51具有至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%或100%序列同一性。
57.根据段落54-56中任一项所述的多肽,该多肽由以下多核苷酸编码,该多核苷酸在中严格条件、中-高严格条件、高严格条件或非常高严格条件下与SEQ ID NO:7、SEQ IDNO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:46、SEQ ID NO:48或SEQ ID NO:50的成熟多肽编码序列的全长互补序列杂交。
58.根据段落54-57中任一项所述的多肽,该多肽由以下多核苷酸编码,该多核苷酸与SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:46、SEQ ID NO:48或SEQ ID NO:50的成熟多肽编码序列具有至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%或100%序列同一性。
59.根据段落54-58中任一项所述的多肽,该多肽是SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51的变体,该变体在一个或多个位置处包含取代、缺失和/或插入。
60.根据段落54-59中任一项所述的多肽,该多肽包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51,基本上由其组成,或由其组成。
61.根据段落54-60中任一项所述的多肽,该多肽包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:47、SEQ ID NO:49或SEQ ID NO:51以及1-10个氨基酸,例如1、2、3、4、5、6、7、8、9或10个氨基酸的N-末端延伸和/或C-末端延伸。
62.根据段落61所述的多肽,该多肽包含与SEQ ID NO:2具有至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%或100%序列同一性的前导肽。
63.一种融合多肽,该融合多肽包含根据段落54至62中任一项所述的多肽和第二多肽。
64.一种颗粒,该颗粒包含:
(a)含有根据段落54至63中任一项所述的多肽的核心,以及任选地,
(b)由包围该核心的一个或多个层组成的包衣。
65.一种颗粒,该颗粒包含:
(a)核心,和
(b)由包围该核心的一个或多个层组成的包衣,其中该包衣包含根据段落54至63中任一项所述的多肽。
66.一种组合物,该组合物包含根据段落54至63中任一项所述的多肽或根据段落64或65所述的颗粒。
67.一种全培养液配制品或细胞培养组合物,该全培养液配制品或细胞培养组合物包含根据段落54至63中任一项所述的多肽。
68.一种分离的或纯化的多核苷酸,该分离的或纯化的多核苷酸编码根据段落54至63中任一项所述的多肽。
序列表
<110> 诺维信公司(NOVOZYMES A/S)
<120> 前导肽和编码其的多核苷酸
<130> 15217-WO-PCT
<160> 52
<170> PatentIn 3.5版
<210> 1
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> JPO001前导肽
<400> 1
ttcgcacgtg cacctgttgc tgctaga 27
<210> 2
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> JPO001前导肽
<400> 2
Phe Ala Arg Ala Pro Val Ala Ala Arg
1 5
<210> 3
<211> 63
<212> DNA
<213> 人工序列
<220>
<223> JPO001信号肽cDNA
<400> 3
atgcgtctca ctctattatc aggtgtagcc ggcgttctct gcgcaggaca gctgacggcg 60
gcg 63
<210> 4
<211> 21
<212> PRT
<213> 草酸青霉(Penicillium oxalicum)
<400> 4
Met Arg Leu Thr Leu Leu Ser Gly Val Ala Gly Val Leu Cys Ala Gly
1 5 10 15
Gln Leu Thr Ala Ala
20
<210> 5
<211> 90
<212> DNA
<213> 人工序列
<220>
<223> JPO001信号肽与前导肽
<400> 5
atgcgtctca ctctattatc aggtgtagcc ggcgttctct gcgcaggaca gctgacggcg 60
gcgttcgcac gtgcacctgt tgctgctaga 90
<210> 6
<211> 30
<212> PRT
<213> 人工序列
<220>
<223> JPO001信号肽与前导肽
<400> 6
Met Arg Leu Thr Leu Leu Ser Gly Val Ala Gly Val Leu Cys Ala Gly
1 5 10 15
Gln Leu Thr Ala Ala Phe Ala Arg Ala Pro Val Ala Ala Arg
20 25 30
<210> 7
<211> 1851
<212> DNA
<213> 人工序列
<220>
<223> AnPav498信号肽与葡糖淀粉酶cDNA
<400> 7
atgcgtctca ctctattatc aggtgtagcc ggcgttctct gcgcaggaca gctgacggcg 60
gcgcgtaacg attcgaaggg tgggaatctg acgttcttca tccacaaaga gggcgagcgg 120
tcgctccaag gcatcttgga caatctcggt gggcgaggta agaaaacacc cggcactgcc 180
gcagggttgt ttattgccag tccaaacaca gagaatccaa actattatta tacatggact 240
cgtgactcag ctttggccgc caagtgcttg atcgacctgt tcgaagactc tcgggcagtc 300
tttccaattg accgcaaata cttggaaaca ggaattcggg actacgtgtc gtcccaagca 360
atcctccaga gtgtgtctaa tccttctgga accctgaagg atggctctgg tctgggtgaa 420
cccaagtttg agattgacct gaatcccttt tcgggtgcct ggggtcggcc tcagcgggat 480
ggcccagcgc tgcgagcgac cgctatgatc acctacgcca actacctgat atcccatggt 540
cagaaatcgg atgtgtcaca ggtcatgtgg ccgattattg ccaatgatct agcatatgtt 600
ggtcaatact ggaataatac cggatttgac ctgtgggaag aggtggatgg gtcaagcttt 660
ttcacgattg cggtccagca ccgagccctt gttgaaggct cgcaactggc gaaaaagctc 720
ggcaagtcct gcgatgcctg tgattctcag cctccccaga tattgtgttt cctgcagagt 780
ttctggaacg gaaagtacat cacctccaac atcaacacgc aagcaagccg ctctggtatc 840
gacctggact ctgtcctggg aagcattcat acctttgatc ccgaagcagc ctgtgacgat 900
gcaactttcc agccttgttc tgcccgcgct ctggcgaacc acaaggtcta tgtggattcc 960
ttccgctcta tctacaagat taatgcgggt cttgcagagg gatcggctgc caacgttggc 1020
cgctaccccg aggatgttta cttcggaggc aatccatggt atctcgccac cctaggcgca 1080
tctgaattgc tttacgacgc cttgtaccag tgggacagac ttggcaaact tgaagtctcg 1140
gagacctcgt tgtcattctt caaagacttt gacgcgaccg tgaaaattgg ctcgtactcg 1200
aggaacagca agacctacaa gaaattgacc cagtccatca agtcgtacgc ggacgggttc 1260
atccagttag tgcagcagta cactccttct aatggatctc tggccgagca atacgatcgc 1320
aatacggctg ctcctctctc tgcaaacgat ctgacttggt catttgcctc tttcttgacg 1380
gctacgcaac gccgcgatgc cgtggttcct ccctcctggg gcgcaaagtc ggcaaacaaa 1440
gtcccaacca cttgttcagc ctcccctgtt gtgggtactt ataaggcgcc cacggcaact 1500
ttctcatcca agactaagtg cgtccccgct aaagatattg tgcctatcac gttctacctg 1560
attgagaaca cttactatgg agagaacgtc ttcatgagtg gcaacattac tgcgctgggt 1620
aactgggacg ccaagaaagg cttcccactc accgcaaacc tctacacgca agatcaaaac 1680
ttgtggttcg ccagtgtcga gttcatccca gcaggcacac cctttgagta caagtactac 1740
aaggtcgagc ccaatggcga tattacttgg gagaagggtc ccaaccgggt gttcgtcgct 1800
cccacgggat gcccagttca gcctcactcc aacgacgtgt ggcagttttg a 1851
<210> 8
<211> 616
<212> PRT
<213> 草酸青霉(Penicillium oxalicum)
<400> 8
Met Arg Leu Thr Leu Leu Ser Gly Val Ala Gly Val Leu Cys Ala Gly
1 5 10 15
Gln Leu Thr Ala Ala Arg Asn Asp Ser Lys Gly Gly Asn Leu Thr Phe
20 25 30
Phe Ile His Lys Glu Gly Glu Arg Ser Leu Gln Gly Ile Leu Asp Asn
35 40 45
Leu Gly Gly Arg Gly Lys Lys Thr Pro Gly Thr Ala Ala Gly Leu Phe
50 55 60
Ile Ala Ser Pro Asn Thr Glu Asn Pro Asn Tyr Tyr Tyr Thr Trp Thr
65 70 75 80
Arg Asp Ser Ala Leu Ala Ala Lys Cys Leu Ile Asp Leu Phe Glu Asp
85 90 95
Ser Arg Ala Val Phe Pro Ile Asp Arg Lys Tyr Leu Glu Thr Gly Ile
100 105 110
Arg Asp Tyr Val Ser Ser Gln Ala Ile Leu Gln Ser Val Ser Asn Pro
115 120 125
Ser Gly Thr Leu Lys Asp Gly Ser Gly Leu Gly Glu Pro Lys Phe Glu
130 135 140
Ile Asp Leu Asn Pro Phe Ser Gly Ala Trp Gly Arg Pro Gln Arg Asp
145 150 155 160
Gly Pro Ala Leu Arg Ala Thr Ala Met Ile Thr Tyr Ala Asn Tyr Leu
165 170 175
Ile Ser His Gly Gln Lys Ser Asp Val Ser Gln Val Met Trp Pro Ile
180 185 190
Ile Ala Asn Asp Leu Ala Tyr Val Gly Gln Tyr Trp Asn Asn Thr Gly
195 200 205
Phe Asp Leu Trp Glu Glu Val Asp Gly Ser Ser Phe Phe Thr Ile Ala
210 215 220
Val Gln His Arg Ala Leu Val Glu Gly Ser Gln Leu Ala Lys Lys Leu
225 230 235 240
Gly Lys Ser Cys Asp Ala Cys Asp Ser Gln Pro Pro Gln Ile Leu Cys
245 250 255
Phe Leu Gln Ser Phe Trp Asn Gly Lys Tyr Ile Thr Ser Asn Ile Asn
260 265 270
Thr Gln Ala Ser Arg Ser Gly Ile Asp Leu Asp Ser Val Leu Gly Ser
275 280 285
Ile His Thr Phe Asp Pro Glu Ala Ala Cys Asp Asp Ala Thr Phe Gln
290 295 300
Pro Cys Ser Ala Arg Ala Leu Ala Asn His Lys Val Tyr Val Asp Ser
305 310 315 320
Phe Arg Ser Ile Tyr Lys Ile Asn Ala Gly Leu Ala Glu Gly Ser Ala
325 330 335
Ala Asn Val Gly Arg Tyr Pro Glu Asp Val Tyr Phe Gly Gly Asn Pro
340 345 350
Trp Tyr Leu Ala Thr Leu Gly Ala Ser Glu Leu Leu Tyr Asp Ala Leu
355 360 365
Tyr Gln Trp Asp Arg Leu Gly Lys Leu Glu Val Ser Glu Thr Ser Leu
370 375 380
Ser Phe Phe Lys Asp Phe Asp Ala Thr Val Lys Ile Gly Ser Tyr Ser
385 390 395 400
Arg Asn Ser Lys Thr Tyr Lys Lys Leu Thr Gln Ser Ile Lys Ser Tyr
405 410 415
Ala Asp Gly Phe Ile Gln Leu Val Gln Gln Tyr Thr Pro Ser Asn Gly
420 425 430
Ser Leu Ala Glu Gln Tyr Asp Arg Asn Thr Ala Ala Pro Leu Ser Ala
435 440 445
Asn Asp Leu Thr Trp Ser Phe Ala Ser Phe Leu Thr Ala Thr Gln Arg
450 455 460
Arg Asp Ala Val Val Pro Pro Ser Trp Gly Ala Lys Ser Ala Asn Lys
465 470 475 480
Val Pro Thr Thr Cys Ser Ala Ser Pro Val Val Gly Thr Tyr Lys Ala
485 490 495
Pro Thr Ala Thr Phe Ser Ser Lys Thr Lys Cys Val Pro Ala Lys Asp
500 505 510
Ile Val Pro Ile Thr Phe Tyr Leu Ile Glu Asn Thr Tyr Tyr Gly Glu
515 520 525
Asn Val Phe Met Ser Gly Asn Ile Thr Ala Leu Gly Asn Trp Asp Ala
530 535 540
Lys Lys Gly Phe Pro Leu Thr Ala Asn Leu Tyr Thr Gln Asp Gln Asn
545 550 555 560
Leu Trp Phe Ala Ser Val Glu Phe Ile Pro Ala Gly Thr Pro Phe Glu
565 570 575
Tyr Lys Tyr Tyr Lys Val Glu Pro Asn Gly Asp Ile Thr Trp Glu Lys
580 585 590
Gly Pro Asn Arg Val Phe Val Ala Pro Thr Gly Cys Pro Val Gln Pro
595 600 605
His Ser Asn Asp Val Trp Gln Phe
610 615
<210> 9
<211> 1878
<212> DNA
<213> 人工序列
<220>
<223> JPO001信号肽与前导肽和葡糖淀粉酶
<400> 9
atgcgtctca ctctattatc aggtgtagcc ggcgttctct gcgcaggaca gctgacggcg 60
gcgttcgcac gtgcacctgt tgctgctaga gccaacgatt cgaagggtgg gaatctgacg 120
ttcttcatcc acaaagaggg cgagcggtcg ctccaaggca tcttggacaa tctcggtggg 180
cgaggtaaga aaacacccgg cactgccgca gggttgttta ttgccagtcc aaacacagag 240
aatccaaact attattatac atggactcgt gactcagctt tggccgccaa gtgcttgatc 300
gacctgttcg aagactctcg ggcagtcttt ccaattgacc gcaaatactt ggaaacagga 360
attcgggact acgtgtcgtc ccaagcaatc ctccagagtg tgtctaatcc ttctggaacc 420
ctgaaggatg gctctggtct gggtgaaccc aagtttgaga ttgacctgaa tcccttttcg 480
ggtgcctggg gtcggcctca gcgggatggc ccagcgctgc gagcgaccgc tatgatcacc 540
tacgccaact acctgatatc ccatggtcag aaatcggatg tgtcacaggt catgtggccg 600
attattgcca atgatctagc atatgttggt caatactgga ataataccgg atttgacctg 660
tgggaagagg tggatgggtc aagctttttc acgattgcgg tccagcaccg agcccttgtt 720
gaaggctcgc aactggcgaa aaagctcggc aagtcctgcg atgcctgtga ttctcagcct 780
ccccagatat tgtgtttcct gcagagtttc tggaacggaa agtacatcac ctccaacatc 840
aacacgcaag caagccgctc tggtatcgac ctggactctg tcctgggaag cattcatacc 900
tttgatcccg aagcagcctg tgacgatgca actttccagc cttgttctgc ccgcgctctg 960
gcgaaccaca aggtctatgt ggattccttc cgctctatct acaagattaa tgcgggtctt 1020
gcagagggat cggctgccaa cgttggccgc taccccgagg atgtttactt cggaggcaat 1080
ccatggtatc tcgccaccct aggcgcatct gaattgcttt acgacgcctt gtaccagtgg 1140
gacagacttg gcaaacttga agtctcggag acctcgttgt cattcttcaa agactttgac 1200
gcgaccgtga aaattggctc gtactcgagg aacagcaaga cctacaagaa attgacccag 1260
tccatcaagt cgtacgcgga cgggttcatc cagttagtgc agcagtacac tccttctaat 1320
ggatctctgg ccgagcaata cgatcgcaat acggctgctc ctctctctgc aaacgatctg 1380
acttggtcat ttgcctcttt cttgacggct acgcaacgcc gcgatgccgt ggttcctccc 1440
tcctggggcg caaagtcggc aaacaaagtc ccaaccactt gttcagcctc ccctgttgtg 1500
ggtacttata aggcgcccac ggcaactttc tcatccaaga ctaagtgcgt ccccgctaaa 1560
gatattgtgc ctatcacgtt ctacctgatt gagaacactt actatggaga gaacgtcttc 1620
atgagtggca acattactgc gctgggtaac tgggacgcca agaaaggctt cccactcacc 1680
gcaaacctct acacgcaaga tcaaaacttg tggttcgcca gtgtcgagtt catcccagca 1740
ggcacaccct ttgagtacaa gtactacaag gtcgagccca atggcgatat tacttgggag 1800
aagggtccca accgggtgtt cgtcgctccc acgggatgcc cagttcagcc tcactccaac 1860
gacgtgtggc agttttga 1878
<210> 10
<211> 625
<212> PRT
<213> 人工序列
<220>
<223> JPO001信号肽与前导肽和葡糖淀粉酶
<400> 10
Met Arg Leu Thr Leu Leu Ser Gly Val Ala Gly Val Leu Cys Ala Gly
1 5 10 15
Gln Leu Thr Ala Ala Phe Ala Arg Ala Pro Val Ala Ala Arg Ala Asn
20 25 30
Asp Ser Lys Gly Gly Asn Leu Thr Phe Phe Ile His Lys Glu Gly Glu
35 40 45
Arg Ser Leu Gln Gly Ile Leu Asp Asn Leu Gly Gly Arg Gly Lys Lys
50 55 60
Thr Pro Gly Thr Ala Ala Gly Leu Phe Ile Ala Ser Pro Asn Thr Glu
65 70 75 80
Asn Pro Asn Tyr Tyr Tyr Thr Trp Thr Arg Asp Ser Ala Leu Ala Ala
85 90 95
Lys Cys Leu Ile Asp Leu Phe Glu Asp Ser Arg Ala Val Phe Pro Ile
100 105 110
Asp Arg Lys Tyr Leu Glu Thr Gly Ile Arg Asp Tyr Val Ser Ser Gln
115 120 125
Ala Ile Leu Gln Ser Val Ser Asn Pro Ser Gly Thr Leu Lys Asp Gly
130 135 140
Ser Gly Leu Gly Glu Pro Lys Phe Glu Ile Asp Leu Asn Pro Phe Ser
145 150 155 160
Gly Ala Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr
165 170 175
Ala Met Ile Thr Tyr Ala Asn Tyr Leu Ile Ser His Gly Gln Lys Ser
180 185 190
Asp Val Ser Gln Val Met Trp Pro Ile Ile Ala Asn Asp Leu Ala Tyr
195 200 205
Val Gly Gln Tyr Trp Asn Asn Thr Gly Phe Asp Leu Trp Glu Glu Val
210 215 220
Asp Gly Ser Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala Leu Val
225 230 235 240
Glu Gly Ser Gln Leu Ala Lys Lys Leu Gly Lys Ser Cys Asp Ala Cys
245 250 255
Asp Ser Gln Pro Pro Gln Ile Leu Cys Phe Leu Gln Ser Phe Trp Asn
260 265 270
Gly Lys Tyr Ile Thr Ser Asn Ile Asn Thr Gln Ala Ser Arg Ser Gly
275 280 285
Ile Asp Leu Asp Ser Val Leu Gly Ser Ile His Thr Phe Asp Pro Glu
290 295 300
Ala Ala Cys Asp Asp Ala Thr Phe Gln Pro Cys Ser Ala Arg Ala Leu
305 310 315 320
Ala Asn His Lys Val Tyr Val Asp Ser Phe Arg Ser Ile Tyr Lys Ile
325 330 335
Asn Ala Gly Leu Ala Glu Gly Ser Ala Ala Asn Val Gly Arg Tyr Pro
340 345 350
Glu Asp Val Tyr Phe Gly Gly Asn Pro Trp Tyr Leu Ala Thr Leu Gly
355 360 365
Ala Ser Glu Leu Leu Tyr Asp Ala Leu Tyr Gln Trp Asp Arg Leu Gly
370 375 380
Lys Leu Glu Val Ser Glu Thr Ser Leu Ser Phe Phe Lys Asp Phe Asp
385 390 395 400
Ala Thr Val Lys Ile Gly Ser Tyr Ser Arg Asn Ser Lys Thr Tyr Lys
405 410 415
Lys Leu Thr Gln Ser Ile Lys Ser Tyr Ala Asp Gly Phe Ile Gln Leu
420 425 430
Val Gln Gln Tyr Thr Pro Ser Asn Gly Ser Leu Ala Glu Gln Tyr Asp
435 440 445
Arg Asn Thr Ala Ala Pro Leu Ser Ala Asn Asp Leu Thr Trp Ser Phe
450 455 460
Ala Ser Phe Leu Thr Ala Thr Gln Arg Arg Asp Ala Val Val Pro Pro
465 470 475 480
Ser Trp Gly Ala Lys Ser Ala Asn Lys Val Pro Thr Thr Cys Ser Ala
485 490 495
Ser Pro Val Val Gly Thr Tyr Lys Ala Pro Thr Ala Thr Phe Ser Ser
500 505 510
Lys Thr Lys Cys Val Pro Ala Lys Asp Ile Val Pro Ile Thr Phe Tyr
515 520 525
Leu Ile Glu Asn Thr Tyr Tyr Gly Glu Asn Val Phe Met Ser Gly Asn
530 535 540
Ile Thr Ala Leu Gly Asn Trp Asp Ala Lys Lys Gly Phe Pro Leu Thr
545 550 555 560
Ala Asn Leu Tyr Thr Gln Asp Gln Asn Leu Trp Phe Ala Ser Val Glu
565 570 575
Phe Ile Pro Ala Gly Thr Pro Phe Glu Tyr Lys Tyr Tyr Lys Val Glu
580 585 590
Pro Asn Gly Asp Ile Thr Trp Glu Lys Gly Pro Asn Arg Val Phe Val
595 600 605
Ala Pro Thr Gly Cys Pro Val Gln Pro His Ser Asn Asp Val Trp Gln
610 615 620
Phe
625
<210> 11
<211> 1878
<212> DNA
<213> 人工序列
<220>
<223> JPO124信号肽与前导肽和葡糖淀粉酶
<400> 11
atgcgtctca ctctattatc aggtgtagcc ggcgttctct gcgcaggaca gctgacggcg 60
gcgttcgcac gtgcacctgt tgctgctaga gccaacgatt cgaagagtac gaatctgacg 120
ttcttcatcc acaaagaggg cgagcggtcg ctccaaggca tcttggacaa tctcggtggg 180
ttcggtaagt acacacccgg cactgccgca gggttgttta ttgccagtcc aaacacagag 240
aatccaaact attattatac atggactcgt gactcagctt tggccgccaa gtgcttgatc 300
gacctgttcg aagactctcg ggcagtcttt ccaattgacc gcaaatactt ggaaacagga 360
attcgggact acgtgtcgtc ccaagcaatc ctccagaacg tgtctaatcc ctctggaacc 420
ctgaaggatg gctctggtct gggtgaaccc aagtttgaga ttgacctgaa tcccttttcg 480
ggtccctggg gtcggcctca gcgggatggc ccagcgctgc gagcgaccgc tatgatcacc 540
tacgccaact acctgatatc ccatggtcag aaatcggatg tgtcacaggt catgtggccg 600
attattgcca atgatctagc atatgttggt caatactgga ataataccgg atttgacctg 660
tgggaagagg tggatgggtc aagctttttc acgattgcgg tccagcaccg agcccttgtt 720
gaaggctcgc aactggcgaa aaagctcggc aagtcctgcg atgcctgtga ttctcagcct 780
ccccagatat tgtgtttcct gcagagtttc tggaacggaa agtacatcac ctccaacatc 840
aacacgcaag caagccgctc tggtatcgac ctggactctg tcctgggaag cattcatacc 900
tttgatcccg aagcagcctg tgacgatgca actttccagc cttgttctgc ccgcgctctg 960
gcgaaccaca aggtctatgt ggattccttc cgctctatct acaagattaa tgcgggtctt 1020
gcagagggat cggctgccaa cgttggccgc taccccgagg atgtttactt cggaggcaat 1080
ccatggtatc tcgccaccct aggcgcatct gaattgcttt acgacgcctt gtaccagtgg 1140
gacagacttg gcaaacttga agtctcggag acctcgttgt cattcttcaa agactttgac 1200
gcgaccgtga aaattggctc gtactcgagg aacagcaaga cctacaagaa attgacccag 1260
tccatcaagt cgtacgcgga cgggttcatc cagttagtgc agcagtacac tccttctaat 1320
ggatctctgg ccgagcaata cgatcgcaat acggctgctc ctctctctgc aaacgatctg 1380
acttggtcat ttgcctcttt cttgacggct acgcaacgcc gcaatgcctc ggttcctccc 1440
tcctggggcg caaagtcggc aaacaaagtc ccaaccactt gttcagcctc ccctgttgtg 1500
ggtacttata aggcgcccac ggcaactttc ccatccaaga ctaagtgcgt ccccgctaaa 1560
gatattgtgc ctatcacgtt ctacctgatt gagaacactt actatggaga gaacgtcttc 1620
atgagtggca acattactgc gctgggtaac tgggacgcca agaaaggctt cccactcacc 1680
gcaaacctct acacgcaaga tcaaaacttg tggttcgcca gtgtcgagtt catcccagca 1740
ggcacaccct ttgagtacaa gtactacaag gtcgagccca atggcactat tgtttgggag 1800
aagggtccca accgggtgtt cgtcgctccc acgggatgcc cagttcagcc tcactccaac 1860
gacgtgtggc gctcctga 1878
<210> 12
<211> 625
<212> PRT
<213> 人工序列
<220>
<223> JPO124信号肽与前导肽和葡糖淀粉酶
<400> 12
Met Arg Leu Thr Leu Leu Ser Gly Val Ala Gly Val Leu Cys Ala Gly
1 5 10 15
Gln Leu Thr Ala Ala Phe Ala Arg Ala Pro Val Ala Ala Arg Ala Asn
20 25 30
Asp Ser Lys Ser Thr Asn Leu Thr Phe Phe Ile His Lys Glu Gly Glu
35 40 45
Arg Ser Leu Gln Gly Ile Leu Asp Asn Leu Gly Gly Phe Gly Lys Tyr
50 55 60
Thr Pro Gly Thr Ala Ala Gly Leu Phe Ile Ala Ser Pro Asn Thr Glu
65 70 75 80
Asn Pro Asn Tyr Tyr Tyr Thr Trp Thr Arg Asp Ser Ala Leu Ala Ala
85 90 95
Lys Cys Leu Ile Asp Leu Phe Glu Asp Ser Arg Ala Val Phe Pro Ile
100 105 110
Asp Arg Lys Tyr Leu Glu Thr Gly Ile Arg Asp Tyr Val Ser Ser Gln
115 120 125
Ala Ile Leu Gln Asn Val Ser Asn Pro Ser Gly Thr Leu Lys Asp Gly
130 135 140
Ser Gly Leu Gly Glu Pro Lys Phe Glu Ile Asp Leu Asn Pro Phe Ser
145 150 155 160
Gly Pro Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr
165 170 175
Ala Met Ile Thr Tyr Ala Asn Tyr Leu Ile Ser His Gly Gln Lys Ser
180 185 190
Asp Val Ser Gln Val Met Trp Pro Ile Ile Ala Asn Asp Leu Ala Tyr
195 200 205
Val Gly Gln Tyr Trp Asn Asn Thr Gly Phe Asp Leu Trp Glu Glu Val
210 215 220
Asp Gly Ser Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala Leu Val
225 230 235 240
Glu Gly Ser Gln Leu Ala Lys Lys Leu Gly Lys Ser Cys Asp Ala Cys
245 250 255
Asp Ser Gln Pro Pro Gln Ile Leu Cys Phe Leu Gln Ser Phe Trp Asn
260 265 270
Gly Lys Tyr Ile Thr Ser Asn Ile Asn Thr Gln Ala Ser Arg Ser Gly
275 280 285
Ile Asp Leu Asp Ser Val Leu Gly Ser Ile His Thr Phe Asp Pro Glu
290 295 300
Ala Ala Cys Asp Asp Ala Thr Phe Gln Pro Cys Ser Ala Arg Ala Leu
305 310 315 320
Ala Asn His Lys Val Tyr Val Asp Ser Phe Arg Ser Ile Tyr Lys Ile
325 330 335
Asn Ala Gly Leu Ala Glu Gly Ser Ala Ala Asn Val Gly Arg Tyr Pro
340 345 350
Glu Asp Val Tyr Phe Gly Gly Asn Pro Trp Tyr Leu Ala Thr Leu Gly
355 360 365
Ala Ser Glu Leu Leu Tyr Asp Ala Leu Tyr Gln Trp Asp Arg Leu Gly
370 375 380
Lys Leu Glu Val Ser Glu Thr Ser Leu Ser Phe Phe Lys Asp Phe Asp
385 390 395 400
Ala Thr Val Lys Ile Gly Ser Tyr Ser Arg Asn Ser Lys Thr Tyr Lys
405 410 415
Lys Leu Thr Gln Ser Ile Lys Ser Tyr Ala Asp Gly Phe Ile Gln Leu
420 425 430
Val Gln Gln Tyr Thr Pro Ser Asn Gly Ser Leu Ala Glu Gln Tyr Asp
435 440 445
Arg Asn Thr Ala Ala Pro Leu Ser Ala Asn Asp Leu Thr Trp Ser Phe
450 455 460
Ala Ser Phe Leu Thr Ala Thr Gln Arg Arg Asn Ala Ser Val Pro Pro
465 470 475 480
Ser Trp Gly Ala Lys Ser Ala Asn Lys Val Pro Thr Thr Cys Ser Ala
485 490 495
Ser Pro Val Val Gly Thr Tyr Lys Ala Pro Thr Ala Thr Phe Pro Ser
500 505 510
Lys Thr Lys Cys Val Pro Ala Lys Asp Ile Val Pro Ile Thr Phe Tyr
515 520 525
Leu Ile Glu Asn Thr Tyr Tyr Gly Glu Asn Val Phe Met Ser Gly Asn
530 535 540
Ile Thr Ala Leu Gly Asn Trp Asp Ala Lys Lys Gly Phe Pro Leu Thr
545 550 555 560
Ala Asn Leu Tyr Thr Gln Asp Gln Asn Leu Trp Phe Ala Ser Val Glu
565 570 575
Phe Ile Pro Ala Gly Thr Pro Phe Glu Tyr Lys Tyr Tyr Lys Val Glu
580 585 590
Pro Asn Gly Thr Ile Val Trp Glu Lys Gly Pro Asn Arg Val Phe Val
595 600 605
Ala Pro Thr Gly Cys Pro Val Gln Pro His Ser Asn Asp Val Trp Arg
610 615 620
Ser
625
<210> 13
<211> 1878
<212> DNA
<213> 人工序列
<220>
<223> JPO172信号肽与前导肽和葡糖淀粉酶
<400> 13
atgcgtctca ctctattatc aggtgtagcc ggcgttctct gcgcaggaca gctgacggcg 60
gcgttcgcac gtgcacctgt tgctgctaga gccaacgatt cgaagagtac gaatctgacg 120
ttcttcatcc acaaagaggg cgagcggtcg ctccaaggca tcttggacaa tctcggtggg 180
ttcggtaagt acacacccgg cactgccgca gggttgttta ttgccagtcc aaacacacgg 240
aatccaaact attattatac atggactcgt gactcagctt tggccgccaa gtgcttgatc 300
gacctgttcg aagactctcg ggcagtcttt ccaattgacc gcaaatactt ggaaacagga 360
attcgggact acgtgtcgtc ccaagcaatc ctccagaacg tgtctaatcc ctctggaacc 420
ctgaaggatg gctctggtct gggtgaaccc aagtttgaga ttgacctgaa tcccttttcg 480
ggtccctggg gtcggcctca gcgggatggc ccagcgctgc gagcgaccgc tatgatcacc 540
tacgccaact acctgatatc ccatggtcag aaatcggatg tgtcacaggt catgtggccg 600
attattgcca atgatctagc atatgttggt caatactgga ataataccgg atttgacctg 660
tgggaagagg tggatgggtc aagctttttc acgattgcgg tccagcaccg agcccttgtt 720
gaaggctcgc aactggcgaa aaagctcggc aagtcctgcg atgcctgtga ttctcagcct 780
ccccagatat tgtgtttcct gcagagtttc tggaacggaa agtacatcac ctccaacatc 840
aacacgcaag caagccgctc tggtatcgac ctggactctg tcctgggaag cattcatacc 900
tttgatcccg aagcagcctg tgacgatgca actttccagc cttgttctgc ccgcgctctg 960
gcgaaccaca aggtctatgt ggattccttc cgctctatct acaagattaa tgcgggtctt 1020
gcagagggat cggctgccaa cgttggccgc taccccgagg atgtttactt cggaggcaat 1080
ccatggtatc tcgccaccct aggcgcatct gaattgcttt acgacgcctt gtaccagtgg 1140
gacagacttg gcaaacttga agtctcggag acctcgttgt cattcttcaa agactttgac 1200
gcgaccgtga aaattggctc gtactcgagg aacagcaaga cctacaagaa attgacccag 1260
tccatcaagt cgtacgcgga cgggttcatc cagttagtgc agcagtacac tccttctaat 1320
ggatctctgg ccgagcaata cgatcgcaat acggctgctc ctctctctgc aaacgatctg 1380
acttggtcat ttgcctcttt cttgacggct acgcaacgcc gcaatgcctc ggttcctccc 1440
tcctggggcg caaagtcggc aaacaaagtc ccaaccactt gttcagcctc ccctgttgtg 1500
ggtacttata aggcgcccac ggcaactttc ccatccaagc ctaagtgcgt ccccgctaaa 1560
gatattgtgc ctatcacgtt ctacctgatt gccaacactt actatggaga gaacgtcttc 1620
atgagtggca acattactgc gctgggtaac tgggacgcca agaaaggctt cccactcacc 1680
gcaaacctct acacgcaaga tcaacccttg tggttcgcca gtgtcgagtt catcccagca 1740
ggcacaccct ttgagtacaa gtactacaag gtcgagccca atggcactat tgtttgggag 1800
aagggtccca accgggtgtt cgtcgctccc acgggatgcc cagttcagcc tcactccaac 1860
gacgtgtggc gctcctga 1878
<210> 14
<211> 625
<212> PRT
<213> 人工序列
<220>
<223> JPO172信号肽与前导肽和葡糖淀粉酶
<400> 14
Met Arg Leu Thr Leu Leu Ser Gly Val Ala Gly Val Leu Cys Ala Gly
1 5 10 15
Gln Leu Thr Ala Ala Phe Ala Arg Ala Pro Val Ala Ala Arg Ala Asn
20 25 30
Asp Ser Lys Ser Thr Asn Leu Thr Phe Phe Ile His Lys Glu Gly Glu
35 40 45
Arg Ser Leu Gln Gly Ile Leu Asp Asn Leu Gly Gly Phe Gly Lys Tyr
50 55 60
Thr Pro Gly Thr Ala Ala Gly Leu Phe Ile Ala Ser Pro Asn Thr Arg
65 70 75 80
Asn Pro Asn Tyr Tyr Tyr Thr Trp Thr Arg Asp Ser Ala Leu Ala Ala
85 90 95
Lys Cys Leu Ile Asp Leu Phe Glu Asp Ser Arg Ala Val Phe Pro Ile
100 105 110
Asp Arg Lys Tyr Leu Glu Thr Gly Ile Arg Asp Tyr Val Ser Ser Gln
115 120 125
Ala Ile Leu Gln Asn Val Ser Asn Pro Ser Gly Thr Leu Lys Asp Gly
130 135 140
Ser Gly Leu Gly Glu Pro Lys Phe Glu Ile Asp Leu Asn Pro Phe Ser
145 150 155 160
Gly Pro Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr
165 170 175
Ala Met Ile Thr Tyr Ala Asn Tyr Leu Ile Ser His Gly Gln Lys Ser
180 185 190
Asp Val Ser Gln Val Met Trp Pro Ile Ile Ala Asn Asp Leu Ala Tyr
195 200 205
Val Gly Gln Tyr Trp Asn Asn Thr Gly Phe Asp Leu Trp Glu Glu Val
210 215 220
Asp Gly Ser Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala Leu Val
225 230 235 240
Glu Gly Ser Gln Leu Ala Lys Lys Leu Gly Lys Ser Cys Asp Ala Cys
245 250 255
Asp Ser Gln Pro Pro Gln Ile Leu Cys Phe Leu Gln Ser Phe Trp Asn
260 265 270
Gly Lys Tyr Ile Thr Ser Asn Ile Asn Thr Gln Ala Ser Arg Ser Gly
275 280 285
Ile Asp Leu Asp Ser Val Leu Gly Ser Ile His Thr Phe Asp Pro Glu
290 295 300
Ala Ala Cys Asp Asp Ala Thr Phe Gln Pro Cys Ser Ala Arg Ala Leu
305 310 315 320
Ala Asn His Lys Val Tyr Val Asp Ser Phe Arg Ser Ile Tyr Lys Ile
325 330 335
Asn Ala Gly Leu Ala Glu Gly Ser Ala Ala Asn Val Gly Arg Tyr Pro
340 345 350
Glu Asp Val Tyr Phe Gly Gly Asn Pro Trp Tyr Leu Ala Thr Leu Gly
355 360 365
Ala Ser Glu Leu Leu Tyr Asp Ala Leu Tyr Gln Trp Asp Arg Leu Gly
370 375 380
Lys Leu Glu Val Ser Glu Thr Ser Leu Ser Phe Phe Lys Asp Phe Asp
385 390 395 400
Ala Thr Val Lys Ile Gly Ser Tyr Ser Arg Asn Ser Lys Thr Tyr Lys
405 410 415
Lys Leu Thr Gln Ser Ile Lys Ser Tyr Ala Asp Gly Phe Ile Gln Leu
420 425 430
Val Gln Gln Tyr Thr Pro Ser Asn Gly Ser Leu Ala Glu Gln Tyr Asp
435 440 445
Arg Asn Thr Ala Ala Pro Leu Ser Ala Asn Asp Leu Thr Trp Ser Phe
450 455 460
Ala Ser Phe Leu Thr Ala Thr Gln Arg Arg Asn Ala Ser Val Pro Pro
465 470 475 480
Ser Trp Gly Ala Lys Ser Ala Asn Lys Val Pro Thr Thr Cys Ser Ala
485 490 495
Ser Pro Val Val Gly Thr Tyr Lys Ala Pro Thr Ala Thr Phe Pro Ser
500 505 510
Lys Pro Lys Cys Val Pro Ala Lys Asp Ile Val Pro Ile Thr Phe Tyr
515 520 525
Leu Ile Ala Asn Thr Tyr Tyr Gly Glu Asn Val Phe Met Ser Gly Asn
530 535 540
Ile Thr Ala Leu Gly Asn Trp Asp Ala Lys Lys Gly Phe Pro Leu Thr
545 550 555 560
Ala Asn Leu Tyr Thr Gln Asp Gln Pro Leu Trp Phe Ala Ser Val Glu
565 570 575
Phe Ile Pro Ala Gly Thr Pro Phe Glu Tyr Lys Tyr Tyr Lys Val Glu
580 585 590
Pro Asn Gly Thr Ile Val Trp Glu Lys Gly Pro Asn Arg Val Phe Val
595 600 605
Ala Pro Thr Gly Cys Pro Val Gln Pro His Ser Asn Asp Val Trp Arg
610 615 620
Ser
625
<210> 15
<211> 595
<212> PRT
<213> 草酸青霉(Penicillium oxalicum)
<400> 15
Arg Asn Asp Ser Lys Gly Gly Asn Leu Thr Phe Phe Ile His Lys Glu
1 5 10 15
Gly Glu Arg Ser Leu Gln Gly Ile Leu Asp Asn Leu Gly Gly Arg Gly
20 25 30
Lys Lys Thr Pro Gly Thr Ala Ala Gly Leu Phe Ile Ala Ser Pro Asn
35 40 45
Thr Glu Asn Pro Asn Tyr Tyr Tyr Thr Trp Thr Arg Asp Ser Ala Leu
50 55 60
Ala Ala Lys Cys Leu Ile Asp Leu Phe Glu Asp Ser Arg Ala Val Phe
65 70 75 80
Pro Ile Asp Arg Lys Tyr Leu Glu Thr Gly Ile Arg Asp Tyr Val Ser
85 90 95
Ser Gln Ala Ile Leu Gln Ser Val Ser Asn Pro Ser Gly Thr Leu Lys
100 105 110
Asp Gly Ser Gly Leu Gly Glu Pro Lys Phe Glu Ile Asp Leu Asn Pro
115 120 125
Phe Ser Gly Ala Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg
130 135 140
Ala Thr Ala Met Ile Thr Tyr Ala Asn Tyr Leu Ile Ser His Gly Gln
145 150 155 160
Lys Ser Asp Val Ser Gln Val Met Trp Pro Ile Ile Ala Asn Asp Leu
165 170 175
Ala Tyr Val Gly Gln Tyr Trp Asn Asn Thr Gly Phe Asp Leu Trp Glu
180 185 190
Glu Val Asp Gly Ser Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala
195 200 205
Leu Val Glu Gly Ser Gln Leu Ala Lys Lys Leu Gly Lys Ser Cys Asp
210 215 220
Ala Cys Asp Ser Gln Pro Pro Gln Ile Leu Cys Phe Leu Gln Ser Phe
225 230 235 240
Trp Asn Gly Lys Tyr Ile Thr Ser Asn Ile Asn Thr Gln Ala Ser Arg
245 250 255
Ser Gly Ile Asp Leu Asp Ser Val Leu Gly Ser Ile His Thr Phe Asp
260 265 270
Pro Glu Ala Ala Cys Asp Asp Ala Thr Phe Gln Pro Cys Ser Ala Arg
275 280 285
Ala Leu Ala Asn His Lys Val Tyr Val Asp Ser Phe Arg Ser Ile Tyr
290 295 300
Lys Ile Asn Ala Gly Leu Ala Glu Gly Ser Ala Ala Asn Val Gly Arg
305 310 315 320
Tyr Pro Glu Asp Val Tyr Phe Gly Gly Asn Pro Trp Tyr Leu Ala Thr
325 330 335
Leu Gly Ala Ser Glu Leu Leu Tyr Asp Ala Leu Tyr Gln Trp Asp Arg
340 345 350
Leu Gly Lys Leu Glu Val Ser Glu Thr Ser Leu Ser Phe Phe Lys Asp
355 360 365
Phe Asp Ala Thr Val Lys Ile Gly Ser Tyr Ser Arg Asn Ser Lys Thr
370 375 380
Tyr Lys Lys Leu Thr Gln Ser Ile Lys Ser Tyr Ala Asp Gly Phe Ile
385 390 395 400
Gln Leu Val Gln Gln Tyr Thr Pro Ser Asn Gly Ser Leu Ala Glu Gln
405 410 415
Tyr Asp Arg Asn Thr Ala Ala Pro Leu Ser Ala Asn Asp Leu Thr Trp
420 425 430
Ser Phe Ala Ser Phe Leu Thr Ala Thr Gln Arg Arg Asp Ala Val Val
435 440 445
Pro Pro Ser Trp Gly Ala Lys Ser Ala Asn Lys Val Pro Thr Thr Cys
450 455 460
Ser Ala Ser Pro Val Val Gly Thr Tyr Lys Ala Pro Thr Ala Thr Phe
465 470 475 480
Ser Ser Lys Thr Lys Cys Val Pro Ala Lys Asp Ile Val Pro Ile Thr
485 490 495
Phe Tyr Leu Ile Glu Asn Thr Tyr Tyr Gly Glu Asn Val Phe Met Ser
500 505 510
Gly Asn Ile Thr Ala Leu Gly Asn Trp Asp Ala Lys Lys Gly Phe Pro
515 520 525
Leu Thr Ala Asn Leu Tyr Thr Gln Asp Gln Asn Leu Trp Phe Ala Ser
530 535 540
Val Glu Phe Ile Pro Ala Gly Thr Pro Phe Glu Tyr Lys Tyr Tyr Lys
545 550 555 560
Val Glu Pro Asn Gly Asp Ile Thr Trp Glu Lys Gly Pro Asn Arg Val
565 570 575
Phe Val Ala Pro Thr Gly Cys Pro Val Gln Pro His Ser Asn Asp Val
580 585 590
Trp Gln Phe
595
<210> 16
<211> 595
<212> PRT
<213> 人工序列
<220>
<223> JPO001葡糖淀粉酶变体
<400> 16
Ala Asn Asp Ser Lys Gly Gly Asn Leu Thr Phe Phe Ile His Lys Glu
1 5 10 15
Gly Glu Arg Ser Leu Gln Gly Ile Leu Asp Asn Leu Gly Gly Arg Gly
20 25 30
Lys Lys Thr Pro Gly Thr Ala Ala Gly Leu Phe Ile Ala Ser Pro Asn
35 40 45
Thr Glu Asn Pro Asn Tyr Tyr Tyr Thr Trp Thr Arg Asp Ser Ala Leu
50 55 60
Ala Ala Lys Cys Leu Ile Asp Leu Phe Glu Asp Ser Arg Ala Val Phe
65 70 75 80
Pro Ile Asp Arg Lys Tyr Leu Glu Thr Gly Ile Arg Asp Tyr Val Ser
85 90 95
Ser Gln Ala Ile Leu Gln Ser Val Ser Asn Pro Ser Gly Thr Leu Lys
100 105 110
Asp Gly Ser Gly Leu Gly Glu Pro Lys Phe Glu Ile Asp Leu Asn Pro
115 120 125
Phe Ser Gly Ala Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg
130 135 140
Ala Thr Ala Met Ile Thr Tyr Ala Asn Tyr Leu Ile Ser His Gly Gln
145 150 155 160
Lys Ser Asp Val Ser Gln Val Met Trp Pro Ile Ile Ala Asn Asp Leu
165 170 175
Ala Tyr Val Gly Gln Tyr Trp Asn Asn Thr Gly Phe Asp Leu Trp Glu
180 185 190
Glu Val Asp Gly Ser Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala
195 200 205
Leu Val Glu Gly Ser Gln Leu Ala Lys Lys Leu Gly Lys Ser Cys Asp
210 215 220
Ala Cys Asp Ser Gln Pro Pro Gln Ile Leu Cys Phe Leu Gln Ser Phe
225 230 235 240
Trp Asn Gly Lys Tyr Ile Thr Ser Asn Ile Asn Thr Gln Ala Ser Arg
245 250 255
Ser Gly Ile Asp Leu Asp Ser Val Leu Gly Ser Ile His Thr Phe Asp
260 265 270
Pro Glu Ala Ala Cys Asp Asp Ala Thr Phe Gln Pro Cys Ser Ala Arg
275 280 285
Ala Leu Ala Asn His Lys Val Tyr Val Asp Ser Phe Arg Ser Ile Tyr
290 295 300
Lys Ile Asn Ala Gly Leu Ala Glu Gly Ser Ala Ala Asn Val Gly Arg
305 310 315 320
Tyr Pro Glu Asp Val Tyr Phe Gly Gly Asn Pro Trp Tyr Leu Ala Thr
325 330 335
Leu Gly Ala Ser Glu Leu Leu Tyr Asp Ala Leu Tyr Gln Trp Asp Arg
340 345 350
Leu Gly Lys Leu Glu Val Ser Glu Thr Ser Leu Ser Phe Phe Lys Asp
355 360 365
Phe Asp Ala Thr Val Lys Ile Gly Ser Tyr Ser Arg Asn Ser Lys Thr
370 375 380
Tyr Lys Lys Leu Thr Gln Ser Ile Lys Ser Tyr Ala Asp Gly Phe Ile
385 390 395 400
Gln Leu Val Gln Gln Tyr Thr Pro Ser Asn Gly Ser Leu Ala Glu Gln
405 410 415
Tyr Asp Arg Asn Thr Ala Ala Pro Leu Ser Ala Asn Asp Leu Thr Trp
420 425 430
Ser Phe Ala Ser Phe Leu Thr Ala Thr Gln Arg Arg Asp Ala Val Val
435 440 445
Pro Pro Ser Trp Gly Ala Lys Ser Ala Asn Lys Val Pro Thr Thr Cys
450 455 460
Ser Ala Ser Pro Val Val Gly Thr Tyr Lys Ala Pro Thr Ala Thr Phe
465 470 475 480
Ser Ser Lys Thr Lys Cys Val Pro Ala Lys Asp Ile Val Pro Ile Thr
485 490 495
Phe Tyr Leu Ile Glu Asn Thr Tyr Tyr Gly Glu Asn Val Phe Met Ser
500 505 510
Gly Asn Ile Thr Ala Leu Gly Asn Trp Asp Ala Lys Lys Gly Phe Pro
515 520 525
Leu Thr Ala Asn Leu Tyr Thr Gln Asp Gln Asn Leu Trp Phe Ala Ser
530 535 540
Val Glu Phe Ile Pro Ala Gly Thr Pro Phe Glu Tyr Lys Tyr Tyr Lys
545 550 555 560
Val Glu Pro Asn Gly Asp Ile Thr Trp Glu Lys Gly Pro Asn Arg Val
565 570 575
Phe Val Ala Pro Thr Gly Cys Pro Val Gln Pro His Ser Asn Asp Val
580 585 590
Trp Gln Phe
595
<210> 17
<211> 595
<212> PRT
<213> 人工序列
<220>
<223> JPO124葡糖淀粉酶变体
<400> 17
Ala Asn Asp Ser Lys Ser Thr Asn Leu Thr Phe Phe Ile His Lys Glu
1 5 10 15
Gly Glu Arg Ser Leu Gln Gly Ile Leu Asp Asn Leu Gly Gly Phe Gly
20 25 30
Lys Tyr Thr Pro Gly Thr Ala Ala Gly Leu Phe Ile Ala Ser Pro Asn
35 40 45
Thr Glu Asn Pro Asn Tyr Tyr Tyr Thr Trp Thr Arg Asp Ser Ala Leu
50 55 60
Ala Ala Lys Cys Leu Ile Asp Leu Phe Glu Asp Ser Arg Ala Val Phe
65 70 75 80
Pro Ile Asp Arg Lys Tyr Leu Glu Thr Gly Ile Arg Asp Tyr Val Ser
85 90 95
Ser Gln Ala Ile Leu Gln Asn Val Ser Asn Pro Ser Gly Thr Leu Lys
100 105 110
Asp Gly Ser Gly Leu Gly Glu Pro Lys Phe Glu Ile Asp Leu Asn Pro
115 120 125
Phe Ser Gly Pro Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg
130 135 140
Ala Thr Ala Met Ile Thr Tyr Ala Asn Tyr Leu Ile Ser His Gly Gln
145 150 155 160
Lys Ser Asp Val Ser Gln Val Met Trp Pro Ile Ile Ala Asn Asp Leu
165 170 175
Ala Tyr Val Gly Gln Tyr Trp Asn Asn Thr Gly Phe Asp Leu Trp Glu
180 185 190
Glu Val Asp Gly Ser Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala
195 200 205
Leu Val Glu Gly Ser Gln Leu Ala Lys Lys Leu Gly Lys Ser Cys Asp
210 215 220
Ala Cys Asp Ser Gln Pro Pro Gln Ile Leu Cys Phe Leu Gln Ser Phe
225 230 235 240
Trp Asn Gly Lys Tyr Ile Thr Ser Asn Ile Asn Thr Gln Ala Ser Arg
245 250 255
Ser Gly Ile Asp Leu Asp Ser Val Leu Gly Ser Ile His Thr Phe Asp
260 265 270
Pro Glu Ala Ala Cys Asp Asp Ala Thr Phe Gln Pro Cys Ser Ala Arg
275 280 285
Ala Leu Ala Asn His Lys Val Tyr Val Asp Ser Phe Arg Ser Ile Tyr
290 295 300
Lys Ile Asn Ala Gly Leu Ala Glu Gly Ser Ala Ala Asn Val Gly Arg
305 310 315 320
Tyr Pro Glu Asp Val Tyr Phe Gly Gly Asn Pro Trp Tyr Leu Ala Thr
325 330 335
Leu Gly Ala Ser Glu Leu Leu Tyr Asp Ala Leu Tyr Gln Trp Asp Arg
340 345 350
Leu Gly Lys Leu Glu Val Ser Glu Thr Ser Leu Ser Phe Phe Lys Asp
355 360 365
Phe Asp Ala Thr Val Lys Ile Gly Ser Tyr Ser Arg Asn Ser Lys Thr
370 375 380
Tyr Lys Lys Leu Thr Gln Ser Ile Lys Ser Tyr Ala Asp Gly Phe Ile
385 390 395 400
Gln Leu Val Gln Gln Tyr Thr Pro Ser Asn Gly Ser Leu Ala Glu Gln
405 410 415
Tyr Asp Arg Asn Thr Ala Ala Pro Leu Ser Ala Asn Asp Leu Thr Trp
420 425 430
Ser Phe Ala Ser Phe Leu Thr Ala Thr Gln Arg Arg Asn Ala Ser Val
435 440 445
Pro Pro Ser Trp Gly Ala Lys Ser Ala Asn Lys Val Pro Thr Thr Cys
450 455 460
Ser Ala Ser Pro Val Val Gly Thr Tyr Lys Ala Pro Thr Ala Thr Phe
465 470 475 480
Pro Ser Lys Thr Lys Cys Val Pro Ala Lys Asp Ile Val Pro Ile Thr
485 490 495
Phe Tyr Leu Ile Glu Asn Thr Tyr Tyr Gly Glu Asn Val Phe Met Ser
500 505 510
Gly Asn Ile Thr Ala Leu Gly Asn Trp Asp Ala Lys Lys Gly Phe Pro
515 520 525
Leu Thr Ala Asn Leu Tyr Thr Gln Asp Gln Asn Leu Trp Phe Ala Ser
530 535 540
Val Glu Phe Ile Pro Ala Gly Thr Pro Phe Glu Tyr Lys Tyr Tyr Lys
545 550 555 560
Val Glu Pro Asn Gly Thr Ile Val Trp Glu Lys Gly Pro Asn Arg Val
565 570 575
Phe Val Ala Pro Thr Gly Cys Pro Val Gln Pro His Ser Asn Asp Val
580 585 590
Trp Arg Ser
595
<210> 18
<211> 595
<212> PRT
<213> 人工序列
<220>
<223> JPO172葡糖淀粉酶变体
<400> 18
Ala Asn Asp Ser Lys Ser Thr Asn Leu Thr Phe Phe Ile His Lys Glu
1 5 10 15
Gly Glu Arg Ser Leu Gln Gly Ile Leu Asp Asn Leu Gly Gly Phe Gly
20 25 30
Lys Tyr Thr Pro Gly Thr Ala Ala Gly Leu Phe Ile Ala Ser Pro Asn
35 40 45
Thr Arg Asn Pro Asn Tyr Tyr Tyr Thr Trp Thr Arg Asp Ser Ala Leu
50 55 60
Ala Ala Lys Cys Leu Ile Asp Leu Phe Glu Asp Ser Arg Ala Val Phe
65 70 75 80
Pro Ile Asp Arg Lys Tyr Leu Glu Thr Gly Ile Arg Asp Tyr Val Ser
85 90 95
Ser Gln Ala Ile Leu Gln Asn Val Ser Asn Pro Ser Gly Thr Leu Lys
100 105 110
Asp Gly Ser Gly Leu Gly Glu Pro Lys Phe Glu Ile Asp Leu Asn Pro
115 120 125
Phe Ser Gly Pro Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg
130 135 140
Ala Thr Ala Met Ile Thr Tyr Ala Asn Tyr Leu Ile Ser His Gly Gln
145 150 155 160
Lys Ser Asp Val Ser Gln Val Met Trp Pro Ile Ile Ala Asn Asp Leu
165 170 175
Ala Tyr Val Gly Gln Tyr Trp Asn Asn Thr Gly Phe Asp Leu Trp Glu
180 185 190
Glu Val Asp Gly Ser Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala
195 200 205
Leu Val Glu Gly Ser Gln Leu Ala Lys Lys Leu Gly Lys Ser Cys Asp
210 215 220
Ala Cys Asp Ser Gln Pro Pro Gln Ile Leu Cys Phe Leu Gln Ser Phe
225 230 235 240
Trp Asn Gly Lys Tyr Ile Thr Ser Asn Ile Asn Thr Gln Ala Ser Arg
245 250 255
Ser Gly Ile Asp Leu Asp Ser Val Leu Gly Ser Ile His Thr Phe Asp
260 265 270
Pro Glu Ala Ala Cys Asp Asp Ala Thr Phe Gln Pro Cys Ser Ala Arg
275 280 285
Ala Leu Ala Asn His Lys Val Tyr Val Asp Ser Phe Arg Ser Ile Tyr
290 295 300
Lys Ile Asn Ala Gly Leu Ala Glu Gly Ser Ala Ala Asn Val Gly Arg
305 310 315 320
Tyr Pro Glu Asp Val Tyr Phe Gly Gly Asn Pro Trp Tyr Leu Ala Thr
325 330 335
Leu Gly Ala Ser Glu Leu Leu Tyr Asp Ala Leu Tyr Gln Trp Asp Arg
340 345 350
Leu Gly Lys Leu Glu Val Ser Glu Thr Ser Leu Ser Phe Phe Lys Asp
355 360 365
Phe Asp Ala Thr Val Lys Ile Gly Ser Tyr Ser Arg Asn Ser Lys Thr
370 375 380
Tyr Lys Lys Leu Thr Gln Ser Ile Lys Ser Tyr Ala Asp Gly Phe Ile
385 390 395 400
Gln Leu Val Gln Gln Tyr Thr Pro Ser Asn Gly Ser Leu Ala Glu Gln
405 410 415
Tyr Asp Arg Asn Thr Ala Ala Pro Leu Ser Ala Asn Asp Leu Thr Trp
420 425 430
Ser Phe Ala Ser Phe Leu Thr Ala Thr Gln Arg Arg Asn Ala Ser Val
435 440 445
Pro Pro Ser Trp Gly Ala Lys Ser Ala Asn Lys Val Pro Thr Thr Cys
450 455 460
Ser Ala Ser Pro Val Val Gly Thr Tyr Lys Ala Pro Thr Ala Thr Phe
465 470 475 480
Pro Ser Lys Pro Lys Cys Val Pro Ala Lys Asp Ile Val Pro Ile Thr
485 490 495
Phe Tyr Leu Ile Ala Asn Thr Tyr Tyr Gly Glu Asn Val Phe Met Ser
500 505 510
Gly Asn Ile Thr Ala Leu Gly Asn Trp Asp Ala Lys Lys Gly Phe Pro
515 520 525
Leu Thr Ala Asn Leu Tyr Thr Gln Asp Gln Pro Leu Trp Phe Ala Ser
530 535 540
Val Glu Phe Ile Pro Ala Gly Thr Pro Phe Glu Tyr Lys Tyr Tyr Lys
545 550 555 560
Val Glu Pro Asn Gly Thr Ile Val Trp Glu Lys Gly Pro Asn Arg Val
565 570 575
Phe Val Ala Pro Thr Gly Cys Pro Val Gln Pro His Ser Asn Asp Val
580 585 590
Trp Arg Ser
595
<210> 19
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> 引物JPO001_F 42
<400> 19
cgtgcacctg ttgctgctag agccaacgat tcgaagggtg gg 42
<210> 20
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> 引物JPO001 R 42
<400> 20
agcagcaaca ggtgcacgtg cgaacgccgc cgtcagctgt cc 42
<210> 21
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物JPO002 F 39
<400> 21
aagagaaccg gattggctta tgcgcgtaac gattcgaag 39
<210> 22
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物JPO002 R 39
<400> 22
ataagccaat ccggttctct ttcctgcgca gagaacgcc 39
<210> 23
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物JPO003 F 39
<400> 23
cgtcccacct ccaagtctgc caatctgacg ttcttcatc 39
<210> 24
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物JPO003 R 39
<400> 24
agacttggag gtgggacgct tcgccgccgt cagctgtcc 39
<210> 25
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> JPO002信号肽
<400> 25
Met Arg Leu Thr Leu Leu Ser Gly Val Ala Gly Val Leu Cys Ala Gly
1 5 10 15
Lys Arg Thr Gly Leu
20
<210> 26
<211> 23
<212> PRT
<213> 人工序列
<220>
<223> JPO003信号肽
<400> 26
Met Arg Leu Thr Leu Leu Ser Gly Val Ala Gly Val Leu Cys Ala Gly
1 5 10 15
Gln Leu Thr Ala Ala Ala Lys
20
<210> 27
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> JSP001和淀粉酶的HTJP-1022正向引物
<400> 27
ttgaggattt agtcttgatc ggatccacca tgcggctctc cacatcctc 49
<210> 28
<211> 48
<212> DNA
<213> 人工序列
<220>
<223> JSP001和淀粉酶的HTJP-1049反向引物
<400> 28
ctatgcgtta tcgtacgcac cacgtgctac cgccaggtgt cagtcacc 48
<210> 29
<211> 88
<212> DNA
<213> 人工序列
<220>
<223> HTJP-1053,JSP035模板DNA1
<400> 29
attcttcaaa attgaggatt tagtcttgat cggatccacc atgcggctct ccacatcctc 60
cctcttcttg tccgtctcct tgctcgga 88
<210> 30
<211> 115
<212> DNA
<213> 人工序列
<220>
<223> HTJP-1149,JSP035模板DNA2
<400> 30
tccctcttct tgtccgtctc cttgctcgga aagttggcct tgggcttcgc acgtgcacct 60
gttgctgcta gagctgtaag taacatccac tctgttctag tgccatgctg agatt 115
<210> 31
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> HTJP-1183,用于第一次PCR的正向引物(JSP035)
<400> 31
gatttagtct tgatcggatc caccatgcgg ctctccacat cctcc 45
<210> 32
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> HTJP-1184,用于第一次PCR的反向引物(JSP035和JSP038)
<400> 32
ttccaatcgt ccgacgtcgc agctctagca gcaacaggtg 40
<210> 33
<211> 86
<212> DNA
<213> 人工序列
<220>
<223> HTJP-1112,JSP038模板DNA1
<400> 33
attcttcaaa attgaggatt tagtcttgat cggatccacc atgggtgtct ctgccgttct 60
acttcctttg tacctcctgt ccggag 86
<210> 34
<211> 117
<212> DNA
<213> 人工序列
<220>
<223> HTJP-1151,JSP038模板DNA2
<400> 34
ttctacttcc tttgtacctc ctgtccggag ttaccttcgg actggcattc gcacgtgcac 60
ctgttgctgc tagagctgta agtaacatcc actctgttct agtgccatgc tgagatt 117
<210> 35
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> HTJP-1187,用于第一次PCR的正向引物(JSP038)
<400> 35
gatttagtct tgatcggatc caccatgggt gtctctgccg ttctacttc 49
<210> 36
<211> 50
<212> DNA
<213> 人工序列
<220>
<223> HTJP-1185,用于第一次PCR的正向引物(JSP035)
<400> 36
ggcttcgcac gtgcacctgt tgctgctaga gctgcgacgt cggacgattg 50
<210> 37
<211> 50
<212> DNA
<213> 人工序列
<220>
<223> HTJP-1186,用于第一次PCR的正向引物(JSP038)
<400> 37
gtgcacctgt tgctgctaga gctgcgacgt cggacgattg gaagggtaag 50
<210> 38
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> HTJP-324,用于启动子区中的DNA印迹探针的引物
<400> 38
aagggatgca agaccaaacc 20
<210> 39
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> HTJP-325,用于启动子区中的DNA印迹探针的引物
<400> 39
tgaagaattt gtgttgtctg ag 22
<210> 40
<211> 63
<212> DNA
<213> 人工序列
<220>
<223> JSP001(对照),信号肽的核苷酸序列
<400> 40
atgcggctct ccacatcctc cctcttcttg tccgtctcct tgctcggaaa gttggccttg 60
ggc 63
<210> 41
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> JSP001,信号肽的氨基酸序列
<400> 41
Met Arg Leu Ser Thr Ser Ser Leu Phe Leu Ser Val Ser Leu Leu Gly
1 5 10 15
Lys Leu Ala Leu Gly
20
<210> 42
<211> 90
<212> DNA
<213> 人工序列
<220>
<223> JSP035,信号肽与前导肽的核苷酸序列
<400> 42
atgcggctct ccacatcctc cctcttcttg tccgtctcct tgctcggaaa gttggccttg 60
ggcttcgcac gtgcacctgt tgctgctaga 90
<210> 43
<211> 30
<212> PRT
<213> 人工序列
<220>
<223> JSP035,信号肽与前导肽的氨基酸序列
<400> 43
Met Arg Leu Ser Thr Ser Ser Leu Phe Leu Ser Val Ser Leu Leu Gly
1 5 10 15
Lys Leu Ala Leu Gly Phe Ala Arg Ala Pro Val Ala Ala Arg
20 25 30
<210> 44
<211> 90
<212> DNA
<213> 人工序列
<220>
<223> JSP038,信号肽与前导肽的核苷酸序列
<400> 44
atgggtgtct ctgccgttct acttcctttg tacctcctgt ccggagttac cttcggactg 60
gcattcgcac gtgcacctgt tgctgctaga 90
<210> 45
<211> 30
<212> PRT
<213> 人工序列
<220>
<223> JSP038,信号肽与前导肽的氨基酸序列
<400> 45
Met Gly Val Ser Ala Val Leu Leu Pro Leu Tyr Leu Leu Ser Gly Val
1 5 10 15
Thr Phe Gly Leu Ala Phe Ala Arg Ala Pro Val Ala Ala Arg
20 25 30
<210> 46
<211> 1815
<212> DNA
<213> 人工序列
<220>
<223> JSP001,信号肽与前导肽和α-淀粉酶的核苷酸序列
<400> 46
atgcggctct ccacatcctc cctcttcttg tccgtctcct tgctcggaaa gttggccttg 60
ggcgcgacgt cggacgattg gaagggtaag gccatttacc agttgctcac ggaccgattc 120
ggtcgcgcag atgactcgac ctcgaactgt tcgaacctct cgaactactg tggtggcact 180
tacgagggca tcactaaaca tctcgactac atctccggta tgggcttcga tgcaatttgg 240
atttcgccga tccctaagaa ctcggacggt ggataccacg gttactgggc cacagacttc 300
tatcagctca actcgaactt cggcgacgag tcgcagttga aagcgctcat ccaggcggcc 360
catgagcggg acatgtatgt catgctcgat gtggtggcaa accacgccgg cccgacttcg 420
aacggatact cgggttacac tttcgacgat gcctccctct accatccgaa atgtaccatc 480
gattacaaca accagacatc gatcgaacag tgttgggtcg ccgatgagtt gcccgatatc 540
gacaccgaaa actcggacaa cgtcgcaatc ctcaacgaca tcgtctccgg ctgggtgggt 600
aactactcgt tcgatggtat tcggatcgac accgtcaagc acatccgcaa ggacttctgg 660
acaggttacg ccgaagccgc gggtgtgttc gcgaccggag aggtgttcaa cggagacccc 720
gcatacgtgg gaccctatca gaaatacttg ccttccctca tcaactatcc catgtactac 780
gccctcaacg acgtcttcgt ctcgaagtcg aagggtttct ccaggatttc cgagatgttg 840
ggctcgaacc gtaacgcctt cgaagatact tccgtcctca ccacgttcgt ggacaaccac 900
gacaaccctc gattcttgaa ctcccagtcc gacaaagccc tcttcaagaa cgcgctcaca 960
tacgtgttgc tcggcgaagg aatccccatc gtctactatg gatcggaaca gggcttctcg 1020
ggcggtgcag accctgccaa ccgagaagtc ctctggacta cgaactacga cacgtcgtcg 1080
gatctctacc agttcatcaa gaccgtcaac tcggtgcgta tgaagtcgaa caaggcggtg 1140
tacatggaca tttacgtggg cgataacgcg tatgcattca agcatggaga cgccttggtg 1200
gtcctcaaca actacggctc gggttcgacc aaccaggtgt ccttctcggt gtcgggaaag 1260
ttcgactccg gcgcctccct catggatatc gtgtccaaca tcacaactac tgtctcctcg 1320
gatggcacag tcactttcaa cttgaaggat ggcctcccgg cgattttcac ctccgcaact 1380
ggcggcacca ctacgacggc tacccccact ggctccggca gcgtgacctc gaccagcaag 1440
accaccgcga ctgccagcaa gaccagcacc agtacgtcat caacctcctg taccactccc 1500
accgccgtgg ctgtgacttt cgatctgaca gctaccacca cctacggcga gaacatctac 1560
ctggtcggat cgatctctca gctgggtgac tgggaaacca gcgacggcat agctctgagt 1620
gctgacaagt acacttccag cgacccgctc tggtatgtca ctgtgactct gccggctggt 1680
gagtcgtttg agtacaagtt tatccgcatt gagagcgatg actccgtgga gtgggagagt 1740
gatcccaacc gagaatacac cgttcctcag gcgtgcggaa cgtcgaccgc gacggtgact 1800
gacacctggc ggtag 1815
<210> 47
<211> 604
<212> PRT
<213> 人工序列
<220>
<223> JSP001,信号肽与前导肽和α-淀粉酶的氨基酸序列
<400> 47
Met Arg Leu Ser Thr Ser Ser Leu Phe Leu Ser Val Ser Leu Leu Gly
1 5 10 15
Lys Leu Ala Leu Gly Ala Thr Ser Asp Asp Trp Lys Gly Lys Ala Ile
20 25 30
Tyr Gln Leu Leu Thr Asp Arg Phe Gly Arg Ala Asp Asp Ser Thr Ser
35 40 45
Asn Cys Ser Asn Leu Ser Asn Tyr Cys Gly Gly Thr Tyr Glu Gly Ile
50 55 60
Thr Lys His Leu Asp Tyr Ile Ser Gly Met Gly Phe Asp Ala Ile Trp
65 70 75 80
Ile Ser Pro Ile Pro Lys Asn Ser Asp Gly Gly Tyr His Gly Tyr Trp
85 90 95
Ala Thr Asp Phe Tyr Gln Leu Asn Ser Asn Phe Gly Asp Glu Ser Gln
100 105 110
Leu Lys Ala Leu Ile Gln Ala Ala His Glu Arg Asp Met Tyr Val Met
115 120 125
Leu Asp Val Val Ala Asn His Ala Gly Pro Thr Ser Asn Gly Tyr Ser
130 135 140
Gly Tyr Thr Phe Asp Asp Ala Ser Leu Tyr His Pro Lys Cys Thr Ile
145 150 155 160
Asp Tyr Asn Asn Gln Thr Ser Ile Glu Gln Cys Trp Val Ala Asp Glu
165 170 175
Leu Pro Asp Ile Asp Thr Glu Asn Ser Asp Asn Val Ala Ile Leu Asn
180 185 190
Asp Ile Val Ser Gly Trp Val Gly Asn Tyr Ser Phe Asp Gly Ile Arg
195 200 205
Ile Asp Thr Val Lys His Ile Arg Lys Asp Phe Trp Thr Gly Tyr Ala
210 215 220
Glu Ala Ala Gly Val Phe Ala Thr Gly Glu Val Phe Asn Gly Asp Pro
225 230 235 240
Ala Tyr Val Gly Pro Tyr Gln Lys Tyr Leu Pro Ser Leu Ile Asn Tyr
245 250 255
Pro Met Tyr Tyr Ala Leu Asn Asp Val Phe Val Ser Lys Ser Lys Gly
260 265 270
Phe Ser Arg Ile Ser Glu Met Leu Gly Ser Asn Arg Asn Ala Phe Glu
275 280 285
Asp Thr Ser Val Leu Thr Thr Phe Val Asp Asn His Asp Asn Pro Arg
290 295 300
Phe Leu Asn Ser Gln Ser Asp Lys Ala Leu Phe Lys Asn Ala Leu Thr
305 310 315 320
Tyr Val Leu Leu Gly Glu Gly Ile Pro Ile Val Tyr Tyr Gly Ser Glu
325 330 335
Gln Gly Phe Ser Gly Gly Ala Asp Pro Ala Asn Arg Glu Val Leu Trp
340 345 350
Thr Thr Asn Tyr Asp Thr Ser Ser Asp Leu Tyr Gln Phe Ile Lys Thr
355 360 365
Val Asn Ser Val Arg Met Lys Ser Asn Lys Ala Val Tyr Met Asp Ile
370 375 380
Tyr Val Gly Asp Asn Ala Tyr Ala Phe Lys His Gly Asp Ala Leu Val
385 390 395 400
Val Leu Asn Asn Tyr Gly Ser Gly Ser Thr Asn Gln Val Ser Phe Ser
405 410 415
Val Ser Gly Lys Phe Asp Ser Gly Ala Ser Leu Met Asp Ile Val Ser
420 425 430
Asn Ile Thr Thr Thr Val Ser Ser Asp Gly Thr Val Thr Phe Asn Leu
435 440 445
Lys Asp Gly Leu Pro Ala Ile Phe Thr Ser Ala Thr Gly Gly Thr Thr
450 455 460
Thr Thr Ala Thr Pro Thr Gly Ser Gly Ser Val Thr Ser Thr Ser Lys
465 470 475 480
Thr Thr Ala Thr Ala Ser Lys Thr Ser Thr Ser Thr Ser Ser Thr Ser
485 490 495
Cys Thr Thr Pro Thr Ala Val Ala Val Thr Phe Asp Leu Thr Ala Thr
500 505 510
Thr Thr Tyr Gly Glu Asn Ile Tyr Leu Val Gly Ser Ile Ser Gln Leu
515 520 525
Gly Asp Trp Glu Thr Ser Asp Gly Ile Ala Leu Ser Ala Asp Lys Tyr
530 535 540
Thr Ser Ser Asp Pro Leu Trp Tyr Val Thr Val Thr Leu Pro Ala Gly
545 550 555 560
Glu Ser Phe Glu Tyr Lys Phe Ile Arg Ile Glu Ser Asp Asp Ser Val
565 570 575
Glu Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val Pro Gln Ala Cys
580 585 590
Gly Thr Ser Thr Ala Thr Val Thr Asp Thr Trp Arg
595 600
<210> 48
<211> 1845
<212> DNA
<213> 人工序列
<220>
<223> JSP035,信号肽与前导肽和α-淀粉酶的核苷酸序列
<400> 48
atgcggctct ccacatcctc cctcttcttg tccgtctcct tgctcggaaa gttggccttg 60
ggcttcgcac gtgcacctgt tgctgctaga gctgcgacgt cggacgattg gaagggtaag 120
gccatttacc agttgctcac ggaccgattc ggtcgcgcag atgactcgac ctcgaactgt 180
tcgaacctct cgaactactg tggtggcact tacgagggca tcactaaaca tctcgactac 240
atctccggta tgggcttcga tgcaatttgg atttcgccga tccctaagaa ctcggacggt 300
ggataccacg gttactgggc cacagacttc tatcagctca actcgaactt cggcgacgag 360
tcgcagttga aagcgctcat ccaggcggcc catgagcggg acatgtatgt catgctcgat 420
gtggtggcaa accacgccgg cccgacttcg aacggatact cgggttacac tttcgatgat 480
gcctccctct accatccgaa atgtaccatc gattacaaca accagacatc gatcgaacag 540
tgttgggtcg ccgatgagtt gcccgatatc gacaccgaaa actcggacaa cgtcgcaatc 600
ctcaacgaca tcgtctccgg ctgggtgggt aactactcgt tcgatggtat tcggatcgac 660
accgtcaagc acatccgcaa ggacttctgg acaggttacg ccgaagccgc gggtgtgttc 720
gcgaccggag aggtgttcaa cggagacccc gcatacgtgg gaccctatca gaaatacttg 780
ccttccctca tcaactatcc catgtactac gccctcaacg acgtcttcgt ctcgaagtcg 840
aagggtttct ccaggatttc cgagatgttg ggctcgaacc gtaacgcctt cgaagatact 900
tccgtcctca ccacgttcgt ggacaaccac gacaaccctc gattcttgaa ctcccagtcc 960
gacaaagccc tcttcaagaa cgcgctcaca tacgtgttgc tcggcgaagg aatccccatc 1020
gtctactatg gatcggaaca gggcttctcg ggcggtgcag accctgccaa ccgagaagtc 1080
ctctggacta cgaactacga cacgtcgtcg gatctctacc agttcatcaa gaccgtcaac 1140
tcggtgcgta tgaagtcgaa caaggcggtg tacatggaca tttacgtggg cgataacgcg 1200
tatgcattca agcatggaga cgccttggtg gtcctcaaca actacggctc gggttcgacc 1260
aaccaggtgt ccttctcggt gtcgggaaag ttcgactccg gcgcctccct catggatatc 1320
gtgtccaaca tcacaactac tgtctcctcg gatggcacag tcactttcaa cttgaaggat 1380
ggcctcccgg cgattttcac ctccgcaact ggcggcacca ctacgacggc tacccccact 1440
ggctccggca gcgtgacctc gaccagcaag accaccgcga ctgccagcaa gaccagcacc 1500
agtacgtcat caacctcctg taccactccc accgccgtgg ctgtgacttt cgatctgaca 1560
gctaccacca cctacggcga gaacatctac ctggtcggat cgatctctca gctgggtgac 1620
tgggaaacca gcgacggcat agctctgagt gctgacaagt acacttccag cgacccgctc 1680
tggtatgtca ctgtgactct gccggctggt gagtcgtttg agtacaagtt tatccgcatt 1740
gagagcgatg actccgtgga gtgggagagt gatcccaacc gagaatacac cgttcctcag 1800
gcgtgcggaa cgtcgaccgc gacggtgact gacacctggc ggtag 1845
<210> 49
<211> 614
<212> PRT
<213> 人工序列
<220>
<223> JSP035,信号肽与前导肽和α-淀粉酶的氨基酸序列
<400> 49
Met Arg Leu Ser Thr Ser Ser Leu Phe Leu Ser Val Ser Leu Leu Gly
1 5 10 15
Lys Leu Ala Leu Gly Phe Ala Arg Ala Pro Val Ala Ala Arg Ala Ala
20 25 30
Thr Ser Asp Asp Trp Lys Gly Lys Ala Ile Tyr Gln Leu Leu Thr Asp
35 40 45
Arg Phe Gly Arg Ala Asp Asp Ser Thr Ser Asn Cys Ser Asn Leu Ser
50 55 60
Asn Tyr Cys Gly Gly Thr Tyr Glu Gly Ile Thr Lys His Leu Asp Tyr
65 70 75 80
Ile Ser Gly Met Gly Phe Asp Ala Ile Trp Ile Ser Pro Ile Pro Lys
85 90 95
Asn Ser Asp Gly Gly Tyr His Gly Tyr Trp Ala Thr Asp Phe Tyr Gln
100 105 110
Leu Asn Ser Asn Phe Gly Asp Glu Ser Gln Leu Lys Ala Leu Ile Gln
115 120 125
Ala Ala His Glu Arg Asp Met Tyr Val Met Leu Asp Val Val Ala Asn
130 135 140
His Ala Gly Pro Thr Ser Asn Gly Tyr Ser Gly Tyr Thr Phe Asp Asp
145 150 155 160
Ala Ser Leu Tyr His Pro Lys Cys Thr Ile Asp Tyr Asn Asn Gln Thr
165 170 175
Ser Ile Glu Gln Cys Trp Val Ala Asp Glu Leu Pro Asp Ile Asp Thr
180 185 190
Glu Asn Ser Asp Asn Val Ala Ile Leu Asn Asp Ile Val Ser Gly Trp
195 200 205
Val Gly Asn Tyr Ser Phe Asp Gly Ile Arg Ile Asp Thr Val Lys His
210 215 220
Ile Arg Lys Asp Phe Trp Thr Gly Tyr Ala Glu Ala Ala Gly Val Phe
225 230 235 240
Ala Thr Gly Glu Val Phe Asn Gly Asp Pro Ala Tyr Val Gly Pro Tyr
245 250 255
Gln Lys Tyr Leu Pro Ser Leu Ile Asn Tyr Pro Met Tyr Tyr Ala Leu
260 265 270
Asn Asp Val Phe Val Ser Lys Ser Lys Gly Phe Ser Arg Ile Ser Glu
275 280 285
Met Leu Gly Ser Asn Arg Asn Ala Phe Glu Asp Thr Ser Val Leu Thr
290 295 300
Thr Phe Val Asp Asn His Asp Asn Pro Arg Phe Leu Asn Ser Gln Ser
305 310 315 320
Asp Lys Ala Leu Phe Lys Asn Ala Leu Thr Tyr Val Leu Leu Gly Glu
325 330 335
Gly Ile Pro Ile Val Tyr Tyr Gly Ser Glu Gln Gly Phe Ser Gly Gly
340 345 350
Ala Asp Pro Ala Asn Arg Glu Val Leu Trp Thr Thr Asn Tyr Asp Thr
355 360 365
Ser Ser Asp Leu Tyr Gln Phe Ile Lys Thr Val Asn Ser Val Arg Met
370 375 380
Lys Ser Asn Lys Ala Val Tyr Met Asp Ile Tyr Val Gly Asp Asn Ala
385 390 395 400
Tyr Ala Phe Lys His Gly Asp Ala Leu Val Val Leu Asn Asn Tyr Gly
405 410 415
Ser Gly Ser Thr Asn Gln Val Ser Phe Ser Val Ser Gly Lys Phe Asp
420 425 430
Ser Gly Ala Ser Leu Met Asp Ile Val Ser Asn Ile Thr Thr Thr Val
435 440 445
Ser Ser Asp Gly Thr Val Thr Phe Asn Leu Lys Asp Gly Leu Pro Ala
450 455 460
Ile Phe Thr Ser Ala Thr Gly Gly Thr Thr Thr Thr Ala Thr Pro Thr
465 470 475 480
Gly Ser Gly Ser Val Thr Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser
485 490 495
Lys Thr Ser Thr Ser Thr Ser Ser Thr Ser Cys Thr Thr Pro Thr Ala
500 505 510
Val Ala Val Thr Phe Asp Leu Thr Ala Thr Thr Thr Tyr Gly Glu Asn
515 520 525
Ile Tyr Leu Val Gly Ser Ile Ser Gln Leu Gly Asp Trp Glu Thr Ser
530 535 540
Asp Gly Ile Ala Leu Ser Ala Asp Lys Tyr Thr Ser Ser Asp Pro Leu
545 550 555 560
Trp Tyr Val Thr Val Thr Leu Pro Ala Gly Glu Ser Phe Glu Tyr Lys
565 570 575
Phe Ile Arg Ile Glu Ser Asp Asp Ser Val Glu Trp Glu Ser Asp Pro
580 585 590
Asn Arg Glu Tyr Thr Val Pro Gln Ala Cys Gly Thr Ser Thr Ala Thr
595 600 605
Val Thr Asp Thr Trp Arg
610
<210> 50
<211> 1845
<212> DNA
<213> 人工序列
<220>
<223> JSP038,信号肽与前导肽和α-淀粉酶的核苷酸序列
<400> 50
atgggtgtct ctgccgttct acttcctttg tacctcctgt ccggagttac cttcggactg 60
gcattcgcac gtgcacctgt tgctgctaga gctgcgacgt cggacgattg gaagggtaag 120
gccatttacc agttgctcac ggaccgattc ggtcgcgcag atgactcgac ctcgaactgt 180
tcgaacctct cgaactactg tggtggcact tacgagggca tcactaaaca tctcgactac 240
atctccggta tgggcttcga tgcaatttgg atttcgccga tccctaagaa ctcggacggt 300
ggataccacg gttactgggc cacagacttc tatcagctca actcgaactt cggcgacgag 360
tcgcagttga aagcgctcat ccaggcggcc catgagcggg acatgtatgt catgctcgat 420
gtggtggcaa accacgccgg cccgacttcg aacggatact cgggttacac tttcgatgat 480
gcctccctct accatccgaa atgtaccatc gattacaaca accagacatc gatcgaacag 540
tgttgggtcg ccgatgagtt gcccgatatc gacaccgaaa actcggacaa cgtcgcaatc 600
ctcaacgaca tcgtctccgg ctgggtgggt aactactcgt tcgatggtat tcggatcgac 660
accgtcaagc acatccgcaa ggacttctgg acaggttacg ccgaagccgc gggtgtgttc 720
gcgaccggag aggtgttcaa cggagacccc gcatacgtgg gaccctatca gaaatacttg 780
ccttccctca tcaactatcc catgtactac gccctcaacg acgtcttcgt ctcgaagtcg 840
aagggtttct ccaggatttc cgagatgttg ggctcgaacc gtaacgcctt cgaagatact 900
tccgtcctca ccacgttcgt ggacaaccac gacaaccctc gattcttgaa ctcccagtcc 960
gacaaagccc tcttcaagaa cgcgctcaca tacgtgttgc tcggcgaagg aatccccatc 1020
gtctactatg gatcggaaca gggcttctcg ggcggtgcag accctgccaa ccgagaagtc 1080
ctctggacta cgaactacga cacgtcgtcg gatctctacc agttcatcaa gaccgtcaac 1140
tcggtgcgta tgaagtcgaa caaggcggtg tacatggaca tttacgtggg cgataacgcg 1200
tatgcattca agcatggaga cgccttggtg gtcctcaaca actacggctc gggttcgacc 1260
aaccaggtgt ccttctcggt gtcgggaaag ttcgactccg gcgcctccct catggatatc 1320
gtgtccaaca tcacaactac tgtctcctcg gatggcacag tcactttcaa cttgaaggat 1380
ggcctcccgg cgattttcac ctccgcaact ggcggcacca ctacgacggc tacccccact 1440
ggctccggca gcgtgacctc gaccagcaag accaccgcga ctgccagcaa gaccagcacc 1500
agtacgtcat caacctcctg taccactccc accgccgtgg ctgtgacttt cgatctgaca 1560
gctaccacca cctacggcga gaacatctac ctggtcggat cgatctctca gctgggtgac 1620
tgggaaacca gcgacggcat agctctgagt gctgacaagt acacttccag cgacccgctc 1680
tggtatgtca ctgtgactct gccggctggt gagtcgtttg agtacaagtt tatccgcatt 1740
gagagcgatg actccgtgga gtgggagagt gatcccaacc gagaatacac cgttcctcag 1800
gcgtgcggaa cgtcgaccgc gacggtgact gacacctggc ggtag 1845
<210> 51
<211> 614
<212> PRT
<213> 人工序列
<220>
<223> JSP038,信号肽与前导肽和α-淀粉酶的氨基酸序列
<400> 51
Met Gly Val Ser Ala Val Leu Leu Pro Leu Tyr Leu Leu Ser Gly Val
1 5 10 15
Thr Phe Gly Leu Ala Phe Ala Arg Ala Pro Val Ala Ala Arg Ala Ala
20 25 30
Thr Ser Asp Asp Trp Lys Gly Lys Ala Ile Tyr Gln Leu Leu Thr Asp
35 40 45
Arg Phe Gly Arg Ala Asp Asp Ser Thr Ser Asn Cys Ser Asn Leu Ser
50 55 60
Asn Tyr Cys Gly Gly Thr Tyr Glu Gly Ile Thr Lys His Leu Asp Tyr
65 70 75 80
Ile Ser Gly Met Gly Phe Asp Ala Ile Trp Ile Ser Pro Ile Pro Lys
85 90 95
Asn Ser Asp Gly Gly Tyr His Gly Tyr Trp Ala Thr Asp Phe Tyr Gln
100 105 110
Leu Asn Ser Asn Phe Gly Asp Glu Ser Gln Leu Lys Ala Leu Ile Gln
115 120 125
Ala Ala His Glu Arg Asp Met Tyr Val Met Leu Asp Val Val Ala Asn
130 135 140
His Ala Gly Pro Thr Ser Asn Gly Tyr Ser Gly Tyr Thr Phe Asp Asp
145 150 155 160
Ala Ser Leu Tyr His Pro Lys Cys Thr Ile Asp Tyr Asn Asn Gln Thr
165 170 175
Ser Ile Glu Gln Cys Trp Val Ala Asp Glu Leu Pro Asp Ile Asp Thr
180 185 190
Glu Asn Ser Asp Asn Val Ala Ile Leu Asn Asp Ile Val Ser Gly Trp
195 200 205
Val Gly Asn Tyr Ser Phe Asp Gly Ile Arg Ile Asp Thr Val Lys His
210 215 220
Ile Arg Lys Asp Phe Trp Thr Gly Tyr Ala Glu Ala Ala Gly Val Phe
225 230 235 240
Ala Thr Gly Glu Val Phe Asn Gly Asp Pro Ala Tyr Val Gly Pro Tyr
245 250 255
Gln Lys Tyr Leu Pro Ser Leu Ile Asn Tyr Pro Met Tyr Tyr Ala Leu
260 265 270
Asn Asp Val Phe Val Ser Lys Ser Lys Gly Phe Ser Arg Ile Ser Glu
275 280 285
Met Leu Gly Ser Asn Arg Asn Ala Phe Glu Asp Thr Ser Val Leu Thr
290 295 300
Thr Phe Val Asp Asn His Asp Asn Pro Arg Phe Leu Asn Ser Gln Ser
305 310 315 320
Asp Lys Ala Leu Phe Lys Asn Ala Leu Thr Tyr Val Leu Leu Gly Glu
325 330 335
Gly Ile Pro Ile Val Tyr Tyr Gly Ser Glu Gln Gly Phe Ser Gly Gly
340 345 350
Ala Asp Pro Ala Asn Arg Glu Val Leu Trp Thr Thr Asn Tyr Asp Thr
355 360 365
Ser Ser Asp Leu Tyr Gln Phe Ile Lys Thr Val Asn Ser Val Arg Met
370 375 380
Lys Ser Asn Lys Ala Val Tyr Met Asp Ile Tyr Val Gly Asp Asn Ala
385 390 395 400
Tyr Ala Phe Lys His Gly Asp Ala Leu Val Val Leu Asn Asn Tyr Gly
405 410 415
Ser Gly Ser Thr Asn Gln Val Ser Phe Ser Val Ser Gly Lys Phe Asp
420 425 430
Ser Gly Ala Ser Leu Met Asp Ile Val Ser Asn Ile Thr Thr Thr Val
435 440 445
Ser Ser Asp Gly Thr Val Thr Phe Asn Leu Lys Asp Gly Leu Pro Ala
450 455 460
Ile Phe Thr Ser Ala Thr Gly Gly Thr Thr Thr Thr Ala Thr Pro Thr
465 470 475 480
Gly Ser Gly Ser Val Thr Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser
485 490 495
Lys Thr Ser Thr Ser Thr Ser Ser Thr Ser Cys Thr Thr Pro Thr Ala
500 505 510
Val Ala Val Thr Phe Asp Leu Thr Ala Thr Thr Thr Tyr Gly Glu Asn
515 520 525
Ile Tyr Leu Val Gly Ser Ile Ser Gln Leu Gly Asp Trp Glu Thr Ser
530 535 540
Asp Gly Ile Ala Leu Ser Ala Asp Lys Tyr Thr Ser Ser Asp Pro Leu
545 550 555 560
Trp Tyr Val Thr Val Thr Leu Pro Ala Gly Glu Ser Phe Glu Tyr Lys
565 570 575
Phe Ile Arg Ile Glu Ser Asp Asp Ser Val Glu Trp Glu Ser Asp Pro
580 585 590
Asn Arg Glu Tyr Thr Val Pro Gln Ala Cys Gly Thr Ser Thr Ala Thr
595 600 605
Val Thr Asp Thr Trp Arg
610
<210> 52
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> JSP038,信号肽的氨基酸序列
<400> 52
Met Gly Val Ser Ala Val Leu Leu Pro Leu Tyr Leu Leu Ser Gly Val
1 5 10 15
Thr Phe Gly Leu Ala
20
Claims (17)
1.一种真菌宿主细胞,该真菌宿主细胞在其基因组中包含:
a)编码目的多肽的第一多核苷酸;和
b)在该第一多核苷酸的上游以翻译融合方式可操作地连接至该第一多核苷酸的第二多核苷酸,所述第二多核苷酸编码与SEQ ID NO:2(FARAPVAAR)具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性的前导肽。
2.根据权利要求1所述的真菌宿主细胞,其中该前导肽是合成的,或与该目的多肽是异源的。
3.根据任一前述权利要求所述的真菌宿主细胞,其中该前导肽包含SEQ ID NO:2,基本上由其组成,或由其组成。
4.根据前述权利要求中任一项所述的真菌宿主细胞,其中该宿主细胞在其基因组中包含编码信号肽的第三多核苷酸,其中该第三多核苷酸在该第二多核苷酸的上游以翻译融合方式可操作地连接至该第二多核苷酸;并且其中该目的多肽被分泌。
5.根据权利要求4所述的真菌宿主细胞,其中该前导肽与该信号肽是异源的。
6.根据权利要求4至5中任一项所述的真菌宿主细胞,其中该第三多核苷酸编码与SEQID NO:4、SEQ ID NO:41或SEQ ID NO:52具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性的信号肽。
7.根据前述权利要求中任一项所述的真菌宿主细胞,其中该宿主细胞是酵母宿主细胞;优选地,该酵母宿主细胞选自由以下组成的组:假丝酵母属、汉逊酵母属、克鲁维酵母属、毕赤酵母属(驹形氏酵母属)、酵母属、裂殖酵母属、和耶氏酵母属细胞;更优选地,该酵母宿主细胞选自由以下组成的组:乳酸克鲁维酵母、卡尔酵母、酿酒酵母、糖化酵母、道格拉氏酵母、克鲁弗酵母、诺地酵母、卵形酵母、和解脂耶氏酵母细胞,最优选地,该酵母宿主细胞是毕赤酵母(法夫驹形氏酵母)。
8.根据权利要求1至6中任一项所述的真菌宿主细胞,其中该宿主细胞是丝状真菌宿主细胞;优选地,该丝状真菌宿主细胞选自由以下组成的组:枝顶孢霉属、曲霉属、短梗霉属、烟管菌属、拟蜡菌属、金孢子菌属、鬼伞属、革盖菌属、隐球菌属、线黑粉酵母属、镰孢属、腐质霉属、梨孢菌属、毛霉属、毁丝霉属、新美鞭菌属、脉孢菌属、拟青霉属、青霉属、平革菌属、射脉菌属、瘤胃壶菌属、侧耳属、裂褶菌属、篮状菌属、嗜热子囊菌属、梭孢壳属、弯颈霉属、栓菌属、和木霉属细胞;更优选地,该丝状真菌宿主细胞选自由以下组成的组:泡盛曲霉、臭曲霉、烟曲霉、日本曲霉、构巢曲霉、黑曲霉、米曲霉、黑刺烟管菌、干拟蜡菌、卡内基拟蜡菌、浅黄拟蜡菌、潘诺希塔拟蜡菌、环带拟蜡菌、微红拟蜡菌、虫拟蜡菌、狭边金孢子菌、嗜角质金孢子菌、卢克诺文思金孢子菌、粪状金孢子菌、毡金孢子菌、昆士兰金孢子菌、热带金孢子菌、带纹金孢子菌、灰盖鬼伞、毛革盖菌、杆孢状镰孢、谷类镰孢、库威镰孢、大刀镰孢、禾谷镰孢、禾赤镰孢、异孢镰孢、合欢木镰孢、尖孢镰孢、多枝镰孢、粉红镰孢、接骨木镰孢、肤色镰孢、拟分枝孢镰孢、硫色镰孢、圆镰孢、拟丝孢镰孢、镶片镰孢、特异腐质霉、柔毛腐质霉、米黑毛霉、嗜热毁丝霉、粗糙脉孢菌、产紫青霉、黄孢原毛平革菌、射脉菌、刺芹侧耳、土生梭孢壳、长域毛栓菌、变色栓菌、哈茨木霉、康宁木霉、长枝木霉、里氏木霉、和绿色木霉细胞;甚至更优选地,该丝状宿主细胞选自由米曲霉、镶片镰孢和里氏木霉细胞组成的组;最优选地,该丝状真菌宿主细胞是黑曲霉细胞。
9.根据前述权利要求中任一项所述的真菌宿主细胞,其中该目的多肽包含酶;优选地,该酶选自由以下组成的组:水解酶、异构酶、连接酶、裂解酶、氧化还原酶或转移酶;更优选地是氨肽酶、淀粉酶、糖酶、羧肽酶、过氧化氢酶、纤维二糖水解酶、纤维素酶、几丁质酶、角质酶、环糊精糖基转移酶、脱氧核糖核酸酶、内切葡聚糖酶、酯酶、α-半乳糖苷酶、β-半乳糖苷酶、α-葡糖苷酶、β-葡糖苷酶、转化酶、漆酶、脂肪酶、甘露糖苷酶、变聚糖酶、核酸酶、氧化酶、果胶分解酶、过氧化物酶、磷酸二酯酶、植酸酶、多酚氧化酶、蛋白水解酶、核糖核酸酶、转谷氨酰胺酶、木聚糖酶、和β-木糖苷酶。
10.根据权利要求9所述的真菌宿主细胞,其中该目的多肽是糖蛋白,优选地是α-葡糖苷酶;更优选地是1,4-α-葡糖苷酶;最优选地是葡糖淀粉酶,如与SEQ ID NO:15、SEQ IDNO:16、SEQ ID NO:17或SEQ ID NO:18具有至少60%序列同一性的葡糖淀粉酶。
11.一种用于生产目的多肽的方法,该方法包括:
i)提供根据权利要求1至10中任一项所述的真菌宿主细胞,
ii)在有助于该目的多肽表达的条件下培养所述真菌宿主细胞;以及,任选地
iii)回收该目的多肽。
12.一种核酸构建体,该核酸构建体包含编码目的多肽的第一多核苷酸和可操作地连接至该第一多核苷酸的第二多核苷酸,该第二多核苷酸编码与SEQ ID NO:2(FARAPVAAR)具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性的前导肽。
13.根据权利要求12所述的核酸构建体,其中该前导肽是合成的,或与该目的多肽是异源的。
14.根据权利要求12至13中任一项所述的核酸构建体,其中该前导肽包含SEQ ID NO:2,基本上由其组成,或由其组成。
15.根据权利要求12至14中任一项所述的核酸构建体,其中
该核酸构建体包含编码信号肽的第三多核苷酸;
该第三多核苷酸在该第二多核苷酸的上游以翻译融合方式可操作地连接至该第二多核苷酸;并且
该信号肽与SEQ ID NO:4、SEQ ID NO:41或SEQ ID 52具有至少60%,例如,至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、至少99%序列同一性。
16.根据权利要求15所述的核酸构建体,其中该前导肽与该信号肽是异源的。
17.一种表达载体,该表达载体包含根据权利要求12至16中任一项所述的核酸构建体。
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DKPA202001235 | 2020-11-02 | ||
DKPA202001235 | 2020-11-02 | ||
PCT/EP2021/080303 WO2022090555A1 (en) | 2020-11-02 | 2021-11-02 | Leader peptides and polynucleotides encoding the same |
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EP (1) | EP4237430A1 (zh) |
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DK122686D0 (da) | 1986-03-17 | 1986-03-17 | Novo Industri As | Fremstilling af proteiner |
US5989870A (en) | 1986-04-30 | 1999-11-23 | Rohm Enzyme Finland Oy | Method for cloning active promoters |
US5223409A (en) | 1988-09-02 | 1993-06-29 | Protein Engineering Corp. | Directed evolution of novel binding proteins |
IL99552A0 (en) | 1990-09-28 | 1992-08-18 | Ixsys Inc | Compositions containing procaryotic cells,a kit for the preparation of vectors useful for the coexpression of two or more dna sequences and methods for the use thereof |
FR2704860B1 (fr) | 1993-05-05 | 1995-07-13 | Pasteur Institut | Sequences de nucleotides du locus cryiiia pour le controle de l'expression de sequences d'adn dans un hote cellulaire. |
DE4343591A1 (de) | 1993-12-21 | 1995-06-22 | Evotec Biosystems Gmbh | Verfahren zum evolutiven Design und Synthese funktionaler Polymere auf der Basis von Formenelementen und Formencodes |
US5605793A (en) | 1994-02-17 | 1997-02-25 | Affymax Technologies N.V. | Methods for in vitro recombination |
AU694954B2 (en) | 1994-06-03 | 1998-08-06 | Novo Nordisk A/S | Purified myceliophthora laccases and nucleic acids encoding same |
CN1151762A (zh) | 1994-06-30 | 1997-06-11 | 诺沃诺尔迪斯克生物技术有限公司 | 非毒性、非产毒性、非致病性镰孢属表达系统及所用启动子和终止子 |
US5955310A (en) | 1998-02-26 | 1999-09-21 | Novo Nordisk Biotech, Inc. | Methods for producing a polypeptide in a bacillus cell |
CN1197965C (zh) | 1998-10-26 | 2005-04-20 | 诺维信公司 | 在丝状真菌细胞内构建和筛选目的dna文库 |
JP4620253B2 (ja) | 1999-03-22 | 2011-01-26 | ノボザイムス,インコーポレイティド | 菌類細胞中で遺伝子を発現させるためのプロモーター |
WO2010039889A2 (en) | 2008-09-30 | 2010-04-08 | Novozymes, Inc. | Methods for using positively and negatively selectable genes in a filamentous fungal cell |
EP3000871A1 (en) | 2009-12-18 | 2016-03-30 | Novozymes, Inc. | Methods for producing polypeptides in protease-deficient mutants of trichoderma |
WO2011127802A1 (en) | 2010-04-14 | 2011-10-20 | Novozymes A/S | Polypeptides having glucoamylase activity and polynucleotides encoding same |
EP2527448A1 (en) | 2011-05-23 | 2012-11-28 | Novozymes A/S | Simultaneous site-specific integrations of multiple gene-copies in filamentous fungi |
MX2016012260A (es) | 2014-03-28 | 2017-01-06 | Novozymes As | Resolubilizacion de cristales de proteina a bajo ph. |
CN107475219B (zh) * | 2017-09-29 | 2020-06-09 | 天津科技大学 | 三种重组糖化酶及其制备方法与应用 |
CA3093378A1 (en) * | 2018-03-09 | 2019-09-12 | Danisco Us Inc | Glucoamylases and methods of use thereof |
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